Artículos

Artículos2021-07-26T11:32:01+02:00

Nuestro sistema CASA SCA® y consumibles son herramientas potentes de investigación y han sido ampliamente utilizadas en estudios de diferentes especies. A continuación, algunas de las publicaciones científicas y artículos publicados por nuestros clientes:

Association between self-reported mobile phone use and the semen quality of young men2023-10-31T12:57:25+01:00

Association between self-reported mobile phone use and the semen quality of young men

Rita Rahban, Alfred Senn, Serge Nef, Martin Rӧӧsli

Swiss Centre for Applied Human Toxicology (SCAHT), University of Geneva, Geneva, Switzerland; Department of Genetic Medicine and Development, University of Geneva, Geneva, Switzerland; Department of Epidemiology and Public Health, Swiss Tropical and Public Health Institute, Allschwill, Switzerland; University of Basel, Basel, Switzerland

Objectives: To investigate the association between mobile phone exposure and semen parameters.

Design: A nationwide cross-sectional study.

Setting: Andrology laboratories in close proximity to 6 army recruitment centers.

Patients: In total, 2886 men from the general Swiss population, 18–22 years old, were recruited between 2005 and 2018 during military conscription.

Intervention: Participants delivered a semen sample and completed a questionnaire on health and lifestyle, including the number of hours they spent using their mobile phones and where they placed them when not in use.

Main Outcome Measures: Using logistic and multiple linear regression models, adjusted odds ratios and β coefficients were determined, respectively. The association between mobile phone exposure and semen parameters such as volume, sperm concentration, total sperm count (TSC), motility, and morphology was then evaluated.

Results: A total of 2759 men answered the question concerning their mobile phone use, and 2764 gave details on the position of their mobile phone when not in use. In the adjusted linear model, a higher frequency of mobile phone use (>20 times per day) was associated with a lower sperm concentration (adjusted β: −0.152; 95% confidence interval: −0.316; 0.011) and a lower TSC (adjusted β: −0.271; 95% confidence interval: −0.515; −0.027). In the adjusted logistic regression model, this translates to a 30% and 21% increased risk for sperm concentration and TSC to be below the World Health Organization reference values for fertile men, respectively. This inverse association was found to be more pronounced in the first study period (2005–2007) and gradually decreased with time (2008–2011 and 2012–2018). No consistent associations were observed between mobile phone use and sperm motility or sperm morphology. Keeping a mobile phone in the pants pocket was not found to be associated with lower semen parameters.

Conclusion: This large population-based study suggests that higher mobile phone use is associated with lower sperm concentration and TSC. The observed time trend of decreasing association is in line with the transition to new technologies and the corresponding decrease in mobile phone output power. Prospective studies with improved exposure assessment are needed to confirm whether the observed associations are causal.

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Fertility and Sterility – https://doi.org/10.1016/j.fertnstert.2023.09.009
Received June 29, 2022; revised September 11, 2023; accepted September 15, 2023; Published October 31, 2023

Novel interpretation of sperm stress test and morphology for maturity assessment of young Norwegian Red bulls2023-05-19T12:23:03+02:00

Novel interpretation of sperm stress test and morphology for maturity assessment of young Norwegian Red bulls

Joanna Bremer, Bjørg Heringstad, Jane M. Morrell, Elisabeth Kommisrud

Department of Applied Ecology, Agricultural Sciences and Biotechnology, Inland Norway University of Applied Sciences, Hamar, Norway; Department of Animal and Aquacultural Sciences, Faculty of Biosciences, Norwegian University of Life Sciences, Ås, Norway; Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden

Abstract: The use of genomic selection significantly reduces the age of dairy bulls entering semen production compared to progeny testing. The study aimed to identify early indicators that could be used for screening bulls during their performance testing period and could give us insight into their future semen production performance, acceptance for the AI station, and prediction of their future fertility. The study population consisted of 142 young Norwegian Red bulls enrolled at the performance test station, followed until we received semen production data, semen doses, and, subsequently, non-return rates (NR56) from the AI station. A range of semen quality parameters were measured with computer-assisted sperm analysis and flow cytometry from ejaculates collected from 65 bulls (9–13 months). The population morphometry of normal spermatozoa was examined, showing that Norwegian Red bulls at 10 months of age have homogenous sperm morphometry. Norwegian Red bulls could be separated into 3 clusters according to their sperm’s reaction patterns to stress test and cryopreservation. Results of semi-automated morphology assessment of young Norwegian Red bulls showed that 42% of bulls rejected for the AI station and 18% of bulls accepted had ejaculates with abnormal morphology scores. For the youngest age group at 10 months, the mean (SD) proportion of spermatozoa with normal morphology was 77.5% (10.6). Using novel interpretation of sperm stress test combined with sperm morphology analysis and consecutive cryopreservation at a young age allowed identification of the candidate’s sperm quality status. This could help breeding companies introduce young bulls earlier to the AI stations.

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Animal Reproduction Science – https://doi.org/10.1016/j.anireprosci.2023.107261
Received 8 March 2023, Revised 16 May 2023, Accepted 17 May 2023, Available online 19 May 2023, Version of Record 31 May 2023

Machine learning and hypothesis driven optimization of bull semen cryopreservation media2022-12-25T15:32:07+01:00

Machine learning and hypothesis driven optimization of bull semen cryopreservation media

Frankie Tu, Maajid Bhat, Patrick Blondin, Patrick Vincent, Mohsen Sharafi, James D. Benson

Department of Computer Science, Memorial University of Newfoundland, St John’s, NL, Canada; Ro, Clinical Strategy, New York, NY, USA; Semex Alliance, Saint-Hyacinthe, Canada. Department of Biology; University of Saskatchewan, Saskatoon, Canada

Abstract: Cryopreservation provides a critical tool for dairy herd genetics management. Due to widely varying inter- and within-bull post thaw fertility, recent research on cryoprotectant extender medium has not dramatically improved suboptimal post-thaw recovery in industry. This progress is stymied by the interactions between samples and the many components of extender media and is often compounded by industry irrelevant sample sizes. To address these challenges, here we demonstrate blank-slate optimization of bull sperm cryopreservation media by supervised machine learning. We considered two supervised learning models: artificial neural networks and Gaussian process regression (GPR). Eleven media components and initial concentrations were identified from publications in bull semen cryopreservation, and an initial 200 extender-post-thaw motility pairs were used to train and 32 extender-post-thaw motility pairs to test the machine learning algorithms. The median post-thaw motility after coupling differential evolution with GPR the increased from 52.6 ± 6.9% to 68.3 ± 6.0% at generations 7 and 17 respectively, with several media performing dramatically better than control media counterparts. This is the first study in which machine learning was used to determine the best combination of constituents to optimize bull sperm cryopreservation media, and provides a template for optimization in other cell types.

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Nature.com/scientificreports (2022) 12:22328 – https://doi.org/10.1038/s41598-022-25104-6
Received: 23 August 2022; Accepted: 24 November 2022; Published: 25 December 2022

Influence of sperm cryopreservation on sperm motility and proAKAP4 concentration in mice2022-07-29T17:18:35+02:00

Influence of sperm cryopreservation on sperm motility and proAKAP4 concentration in mice

Auke Boersma, Jasmin Primus, Bettina Wagner, Veronika Broukal, Lill Andersen, Barbara Pachner, Maik Dahlhoff, Thomas Rülicke, Kerstin E. Auer

Institute of in vivo and in vitro Models, University of Veterinary Medicine Vienna, Vienna, Austria; Department of Radiology, Charité – Universitätsmedizin Berlin, Berlin, Germany

Abstract:

Background: The protein proAKAP4 is crucial for sperm motility and has been suggested as an indicator of male fertility. We determined the relationship between proAKAP4 concentration and sperm motility parameters in mice, and investigated the effects of cryopreservation on these variables.

Methods: Computer-assisted sperm analysis and ELISA were applied to determine sperm motility and proAKAP4 concentration in fresh and frozen-thawed epididymal sperm of SWISS, B6D2F1, C57BL/6N, and BALB/c mice.

Results: ProAKAP4 levels ranged between 12 and 89 ng/ml and did not differ between fresh and frozen-thawed samples, or between strains. We found a negative relationship between proAKAP4 levels and some sperm motility parameters. Sperm traits differed between strains, and cryopreservation negatively affected sperm velocity but not sperm direction parameters.

Conclusion: ProAKAP4 levels in epididymal mouse spermatozoa were unaffected by cryopreservation, highlighting the robustness of this parameter as a potentially time-independent marker for sperm motility and fertility. The high individual variation in proAKAP4 levels supports the potential role of proAKAP4 as a marker for sperm quality, though we found no positive, and even negative relationships between proAKAP4 levels and some sperm motility parameters. Future studies have to investigate the significance of proAKAP4 as an indicator for fertility in mice.

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Reprod Med Biol. 2022;21:e12480 – https://doi.org/10.1002/rmb2.12480
Received: 20 April 2022 / Accepted: 4 July 2022 / First published: 29 July 2022

The in-vitro effect of gonadotropin-releasing hormones, GnRH-I and GnRH-II, on the motility, vitality and acrosome integrity of Vervet monkey (Chlorocebus aethiops) spermatozoa2022-08-29T12:39:27+02:00

The in-vitro effect of gonadotropin-releasing hormones, GnRH-I and GnRH-II, on the motility, vitality and acrosome integrity of Vervet monkey (Chlorocebus aethiops) spermatozoa

Charon de Villiers, Liana Maree, Arieh A. Katz, Gerhard van der Horst

PUDAC-Delft Animal Facility, South African Medical Research Council, Cape Town, South Africa; Department of Medical Biosciences, University of the Western Cape, Bellville, South Africa; Department of Integrative Biomedical Sciences and Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa

Abstract: Two isoforms of the gonadotropin-releasing hormone (GnRH), GnRH-I and GnRH-II, are expressed in mammals, and the presence of one or more GnRH-like peptides has been demonstrated in the male reproductive tract. GnRH and its receptors (GnRHR) are present in human and non-human primate testis, prostate, epididymis, seminal vesicle, spermatozoa and seminal human plasma. GnRH-II is site-specific and acts directly in an inhibitory or stimulatory fashion. Previous studies speculated that GnRH-II could disrupt specific sperm processes, such as sperm motility or capacitation and could be utilized as an effective contraceptive agent. Our study aimed to investigate the in-vitro effects of GnRH-I and GnRH-II on Vervet monkey sperm function. Electro-ejaculated semen samples from 10 Vervet monkeys (Chlorocebus aethiops) were used to select motile sperm populations. Sperm aliquots were incubated with GnRH-I and GnRH-II at different concentrations for 1 h, where after sperm motility and kinematic parameters were assessed using the automated Sperm Class Analyser. Additional sperm aliquots were incubated with two 10-amino acid control peptides, a non-related peptide and an inactive peptide to exclude the possible influence on sperm motility from other peptides with a structure similar to GnRH. Additionally, a GnRHR-I antagonist (GnRHR-A), Cetrorelix, was tested to establish its antagonistic capability on GnRH. The effect of selected concentrations of GnRH-I and GnRH-II on sperm vitality and acrosome intactness was also evaluated after 10- and 60 min exposure. Analysis of the percentage total sperm motility revealed that different concentrations for GnRH-I and GnRH-II inhibited sperm motility significantly. While sperm progressiveness was also notably affected and a trend of decreased sperm kinematics were evident, no effect was found on sperm vitality or acrosome intactness. The non-related and inactive peptides had no impact on sperm motility. The GnRHR-A demonstrated no effect on sperm motility and effectively blocked the inhibitory outcome on the motility of both GnRH isoforms. While GnRH-I or GnRH-II at low-dose concentrations resulted in in-vitro inhibition of sperm motility, it appears to have no adverse effects on other sperm functional parameters evaluated. These collective observations possibly indicate an essential role for GnRH in the in-vivo process of sperm selection in the female reproductive tract.

Reprod Dom Anim. 2022;00:1–12. – https://doi.org/10.1111/rda.14216
Received: 17 June 2022 | Accepted: 23 July 2022 | First published: 25 July 2022

A new fluorescent method to determine honey bee sperm motility parameters with computer-aided sperm analysis2022-07-13T12:44:55+02:00

A new fluorescent method to determine honey bee sperm motility parameters with computer-aided sperm analysis

Janice Faith Murray, Gerhard van der Horst, Mike Allsopp, Retha Christina Magrietha Kotzé

Department of Medical Bioscience, University of the Western Cape, Bellville, South Africa; Honey bee research section, ARC-Plant Protection Research Institute, Stellenbosch, South Africa

Abstract: Honey bees are a keystone species, playing an important role in the food cycle. Improving honey bee reproduction will aid in the replacement of lost colonies. Fertility potential and reproductive health are dependent on semen and sperm quality. Current data on drone semen parameters are limited to semen volume, sperm concentration, sperm viability, and the assignment of sperm motility grade scores. The assessment of drone sperm motility is of importance to determine fertility potential and colony health. This study aimed to establish a quantitative and a qualitative method to measure drone sperm quality, and the fertility potential of Apis mellifera capensis subspecies of South Africa. Firstly, an improved five-point semi-quantitative manual motility index score was used to classify drone sperm motility. Secondly, it was possible to accurately analyse drone sperm motility qualitatively using a fluorescent technique in conjunction with a computer-aided sperm analysis (CASA) system. Manual motility index scores corresponded with total motility percentages as determined by using a CASA system. Furthermore, low values for motility kinematic parameters, particularly velocity parameters, were obtained in samples with both low motility index scores and low total motility percentages. Additionally, total sperm motility percentage and velocity parameters positively correlated with sperm total progressivity. This study provides in-depth data on honey bee drone sperm motility and motility kinematic parameters, which can serve as a reference for future studies on honey bee sperm and possibly related species.

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Journal of Apicultural Research – https://doi.org/10.1080/00218839.2022.2090729
Received 08 Oct 2021 / Accepted 02 Feb 2022 / Published online: 13 Jul 2022

New Approaches to Define The Functional Competency of Human Sperm Subpopulations and Its Relationship to Semen Quality2022-07-01T13:07:18+02:00

New Approaches to Define The Functional Competency of Human Sperm Subpopulations and Its Relationship to Semen Quality

Shannen Keyer, Gerhard Van der Horst, Liana Maree

Department of Medical Bioscience, Faculty of Natural Science, University of the Western Cape, Bellville, South Africa

Abstract:

Background: This study aimed at comparing a comprehensive set of functional and structural sperm characteristics between sperm motility fractions and correlating results to the standard semen parameters. By grouping related variables, our objective was to establish the predictive power of semen parameters and whether they accurately reflect the functionality of sperm motility fractions or merely a small set of parameters within individual fractions.

Materials and Methods: In this non-invasive experimental study, donor semen samples (n=55) were separated via
double density gradient centrifugation, isolating a high (HM) and low motile (LM) sperm fraction. Fractions were evaluated for percentage vitality, chromatin integrity, mature spermatozoa, motility and kinematic parameters, hyperactivation, positive reactive oxygen species, intact mitochondrial membrane potential (MMP) and acrosome reaction.

Results: HM fractions had significantly (P<0.001) enhanced percentages of induced acrosome reaction (HM, 55.6 ± 14.3%, LM, 25.0 ± 16.5%), motility and kinematic parameters, hyperactivation, vitality (HM, 70.4 ± 9.7%, LM, 47.9 ± 10.3%), mitochondrial membrane intactness (HM, 67.2 ± 10.4%, LM, 44.7 ± 15.0%) and mature spermatozoa (HM, 83.4 ± 10.0%, LM, 64.6 ± 8.2%) with intact chromatin (HM, 80.5 ± 8.1%, LM, 71.3 ± 8.0%). Various sperm morphology abnormalities correlated with LM fractions’ grouped motility parameters (range, 0.46 to 0.51; range -0.4 to -0.75), whereas combined semen traits of total motility, progressive motility, viscosity and mucus penetration (MPT) correlated with HM fractions’ grouped motility parameters (range, 0.44 to 0.84).

Conclusion: Collectively, total and progressive motility, viscosity and MPT may represent a reliable grouping of semen parameters for predicting the quality of HM sperm fractions. Separating the same donor semen samples into two significantly diverse motility sperm fractions could be a potential model in mimicking the qualities of fertile and sub-fertile males’ sperm populations and used for future research on the improvement of sperm subpopulations from males with different fertility statuses.

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Int J Fertil Steril. 2022; 16(3): 140-151. – doi: 10.22074/IJFS.2021.531517.1132.
Received: 03/June/2021, Accepted: 13/December/2021

The influence of SARS-CoV-2 on semen parameters of infected infertile male in comparison with those that noninfected2022-06-26T15:46:00+02:00

The influence of SARS-CoV-2 on semen parameters of infected infertile male in comparison with those that noninfected

Baida Rihan Ali, Saad A. Atiyah, Heba T. Yser, Ahmed M. Khelewe, Hameed N. Hameed

Department of Pathological Analysis, College of Science, University of Thi-Qar, Nasiriya, Iraq; Department of Microbiology, College of Medicine, University of Thi-Qar, Nasiriya, Iraq; Department of Biology, College of Pure Educational Science, University of Basrah, Basrah, Iraq; Thi-Qar Health Directorate, Ministry of Health/ Environment, Nasiriya, Iraq

Abstract:

Background: Coronavirus disease (COVID-19) is an infectious disease caused by SARS COV-2 that has spread globally, the virus can cause different pathological alterations in many organs, such as the lung, kidney, and testis. The study aimed to determine the effect of COVID-19 on the seminal fluid parameters of infected infertile males compared with those who are noninfected.

Methods: The study was performed in Al-Hussein Teaching Hospital during the period from September to November, 2021 and it involved 318 patients. The patients’ info included age, address, and vaccination. The sperm count, activity, and morphology were detected using Computer-assisted semen analysis CASA (Microptic-Spain) according to the WHO manual.

Results: There were high significant differences between the infertile males who were infected with COVID- 19 and those who were vaccinated (X2 = 12.509, p = 0.001). A high significant relation (p < 0.001) was recorded between types of infection severity and volume of semen (p < 0.001) and nonprogress life sperm (C) (p < 0.001). While significant differences were shown in the moderate progression sperm (B) (p = 0.012), and morphology (p = 0.02), respectively. High significant differences were reported between the types of infection severity (count of the sperm, presence of pus, B, C and D), (p < 0.001), while a significant difference was shown between severity types in relation to A and morphology of the sperms (p = 0.021 and 0.015), respectively.

Conclusion: The severity of COVID-19 has a significant impact on infertility and sperm parameters, particularly progression and sperm morphology, despite the fact that these parameters are unrelated to vaccination.

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J Clin Lab Anal. 2022;36:e24568. – https://doi.org/10.1002/jcla.24568
Received: 17 May 2022 / Revised: 8 June 2022 / Accepted: 9 June 202 / First published: 26 June 2022

ORF8 protein of SARS-CoV-2 reduces male fertility in mice2023-06-12T16:33:50+02:00

ORF8 protein of SARS-CoV-2 reduces male fertility in mice

Ting Yu, Qiao Ling, Mengxin Xu, Niu Wang, Lixia Wang, Hanwen Lin, Manqi Cao, Yong Ma, Yuanyuan Wang, Kuibiao Li, Liubing Du, Yunyun Jin, Ying Li, Deyin Guo, Xiaoxue Peng, Yao-qing Chen, Bo Zhao, Ji-An Pan

The Center for Infection and Immunity Study and Molecular Cancer Research Center, School of Medicine, Sun Yat‐sen University, Shenzhen, Guangdong, China; School of Public Health (Shenzhen), Sun Yat‐sen University, Shenzhen, Guangdong, China; Guangzhou Center for Diseases Control and Prevention, Guangzhou, Guangdong, China; Key Laboratory of Tropical Disease Control, Ministry of Education, Sun Yat‐sen University, Guangzhou, China

Abstract: As one of the most rapidly evolving proteins of the genus Betacoronavirus, open reading frames (ORF8’s) function and potential pathological consequence in vivo are still obscure. In this study, we show that the secretion of ORF8 is dependent on its N-terminal signal peptide sequence and can be inhibited by reactive oxygen species scavenger and endoplasmic reticulum-Golgi transportation inhibitor in cultured cells. To trace the effect of its possible in vivo secretion, we examined the plasma samples of coronavirus disease 2019 (COVID-19) convalescent patients and found that the patients aged from 40 to 60 had higher antibody titers than those under 40. To explore ORF8’s in vivo function, we administered the mice with ORF8 via tail-vein injection to simulate the circulating ORF8 in the patient. Although no apparent difference in body weight, food intake, and vitality was detected between vehicle- and ORF8-treated mice, the latter displayed morphological abnormalities of testes and epididymides, as indicated by the loss of the central ductal lumen accompanied by a decreased fertility in 5-week-old male mice. Furthermore, the analysis of gene expression in the testes between vehicle- and ORF8-treated mice identified a decreased expression of Col1a1, the loss of which is known to be associated with mice’s infertility. Although whether our observation in mice could be translated to humans remains unclear, our study provides a potential mouse model that can be used to investigate the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on the human reproductive system.

J Med Virol. 2022;1–13. – https://doi.org/10.1002/jmv.27855
Received: 27 April 2022 | Accepted: 10 May 2022 | First published: 15 May 2022

Mass Sperm Motility Is Correlated to Sperm Motility as Measured by Computer-Aided Sperm Analysis (CASA) Technology in Farmed Ostriches2022-04-25T17:20:49+02:00

Mass Sperm Motility Is Correlated to Sperm Motility as Measured by Computer-Aided Sperm Analysis (CASA) Technology in Farmed Ostriches

Pfunzo T. Muvhali, Maud Bonato, Irek A. Malecki, Schalk W. P. Cloete

Department of Animal Sciences, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa; Directorate Animal Sciences, Western Cape Department of Agriculture Elsenburg, Private Bag X1, Elsenburg 7607, South Africa; School of Agriculture and Environment, Faculty of Science, The University of Western Australia, 35 Stirling Highway, Crawley 6009, Australia

Abstract: Semen analyses have gained momentum in various livestock industries. However, in farmed ostriches, semen analysis is still in its experimental stage, and males are not screened for sperm quality before breeding. This study investigated the correlations between computer-aided sperm analysis (CASA) technology and the traditional, yet affordable, mass sperm motility score. Semen was collected from nine South African Black ostrich males (mean age ± SD: 5.25 ± 1.21 years), using the dummy female method for 5 consecutive days monthly, for 8 months. Mass sperm motility scores were recorded on a scale of 1–5 (1: little to no sperm movement; 5: rapid sperm movement). The CASA traits recorded were: total motility (MOT), progressive motility (PMOT), curve–linear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), amplitude of lateral head displacement (ALH), linearity (LIN), straightness (STR), wobble (WOB), and beat-cross frequency (BCF). The results revealed positive correlations between mass sperm motility and PMOT, MOT, VCL, and VAP ranging from 0.34 to 0.59 (p < 0.0001). In contrast, negative correlations were recorded between mass sperm motility and LIN, STR, and BCF, with correlations ranging from −0.20 to −0.39 (p < 0.0001). VSL, ALH, and WOB were not correlated to mass sperm motility (p > 0.05). Ostrich farmers may thus be able to evaluate sperm motility reliably and potentially select breeding males by using the affordable mass sperm motility scoring method. Determining the correlation between these methods and fertility after artificial insemination or natural mating is however needed.

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Animals 2022, 12(9), 1104; https://doi.org/10.3390/ani12091104
Received: 15 February 2022 / Revised: 18 March 2022 / Accepted: 1 April 2022 / Published: 25 April 2022
(This article belongs to the Special Issue Animal Reproduction: Semen Quality Assessment)

Recombinant Fsh and Lh therapy for spawning induction of previtellogenic and early spermatogenic arrested teleost, the flathead grey mullet (Mugil cephalus)2022-11-21T16:56:20+01:00

Recombinant Fsh and Lh therapy for spawning induction of previtellogenic and early spermatogenic arrested teleost, the flathead grey mullet (Mugil cephalus)

Sandra Ramos-Júdez, Ignacio Giménez, Josep Gumbau-Pous, Lucas Stephen Arnold-Cruañes, Alicia Estévez, Neil Duncan

Present address: S2AQUAcoLAB, Av. Do Parque Natural da Ria Formosa s/n, 8700-194, Olhão, Portugal; IRTA, Sant Carles de la Ràpita, Ctra. de Poble Nou km. 5.5, 43540, Sant Carles de la Ràpita, Tarragona, Spain; Rara Avis Biotec, S. L., Valencia, Spain

Abstract: With the expansion and diversification of global aquaculture, efforts continue to develop new bio-technologies for assisted reproduction in species that present reproductive dysfunctions. Flathead grey mullet (Mugil cephalus) males held in intensive conditions in the Mediterranean region do not produce fluent milt and most females are arrested at previtellogenesis. The weekly injections of recombinant follicle stimulating hormone (rFsh) and luteinizing hormone (rLh) induced and completed vitellogenesis in treated females (n = 21), and treated males produced fluent sperm (n = 9). The application of a priming dose of 30 µg kg−1 rLh and resolving dose of 40 mg kg−1 Progesterone, or priming and resolving doses of 30 µg kg−1 rLh, resulted in the induction of maturation, ovulation, and spontaneous spawns with a spawning success of the 85% (8 of 9 females) and 100% (n = 6), respectively. The eggs collected had 63 ± 21% fertilization with embryo development and 58 ± 23% hatching. In comparison, control individuals did not show advances in gonadal development and did not produce fluent sperm. The present results confirm the possibility of controlling oogenesis from previtellogenesis to the completion of maturation and fertilised tank spawning using exclusively rFsh and rLh in a teleost species.

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Nature, Scientific Reports (2022) 12:6563 – https://doi.org/10.1038/s41598-022-10371-0
Received: 30 November 2021; Accepted: 28 March 2022; Published online: 21 April 2022

Effect of Motility Factors D-Penicillamine, Hypotaurine and Epinephrine on the Performance of Spermatozoa from Five Hamster Species2022-03-30T15:02:56+02:00

Effect of Motility Factors D-Penicillamine, Hypotaurine and Epinephrine on the Performance of Spermatozoa from Five Hamster Species

Maximiliano Tourmente, Ana Sanchez-Rodriguez, Eduardo R. S. Roldan

Department of Biodiversity and Evolutionary Biology, Museo Nacional de Ciencias Naturales, Spanish Research Council (CSIC), 28006 Madrid, Spain; Centro de Biología Celular y Molecular, Facultad de Ciencias Exactas, Físicas y Naturales, Universidad Nacional de Córdoba, Cordoba X5016GCA, Argentina; Instituto de Investigaciones Biológicas y Tecnológicas (IIByT), Consejo Nacional de Investigaciones Científica y Técnicas (CONICET), Cordoba X5016GCA, Argentina

Abstract: Assessments of sperm performance are valuable tools for the analysis of sperm fertilizing potential and to understand determinants of male fertility. Hamster species constitute important animal models because they produce sperm cells in high quantities and of high quality. Sexual selection over evolutionary time in these species seems to have resulted in the largest mammalian spermatozoa, and high swimming and bioenergetic performances. Earlier studies showed that golden hamster sperm requires motility factors such as D-penicillamine, hypotaurine and epinephrine (PHE) to sustain survival over time, but it is unknown how they affect swimming kinetics or ATP levels and if other hamster species also require them. The objective of the present study was to examine the effect of PHE on spermatozoa of five hamster species (Mesocricetus auratus, Cricetulus griseus, Phodopus campbelli, P. sungorus, P. roborovskii). In sperm incubated for up to 4 h without or with PHE, we assessed motility, viability, acrosome integrity, sperm velocity and trajectory, and ATP content. The results showed differences in the effect of PHE among species. They had a significant positive effect on the maintenance of sperm quality in M. auratus and C. griseus, whereas there was no consistent effect on spermatozoa of the Phodopus species. Differences between species may be the result of varying underlying regulatory mechanisms of sperm performance and may be important to understand how they relate to successful fertilization.

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Biology 2022, 11(4), 526; https://doi.org/10.3390/biology11040526
Received: 3 February 2022 / Revised: 22 March 2022 / Accepted: 28 March 2022 / Published: 30 March 2022

Assessment of Sperm Viability and Computer-Assisted Motility Analysis in Budgerigars (Melopsittacus undulatus): Effect of Several In Vitro Processing Conditions2022-03-22T15:48:25+01:00

Assessment of Sperm Viability and Computer-Assisted Motility Analysis in Budgerigars (Melopsittacus undulatus): Effect of Several In Vitro Processing Conditions

Manuela Madeddu, Stefano Marelli, Ahmad Abdel Sayed, Fabio Mosca, Silvia Cerolini and Luisa Zaniboni

Department of Veterinary Medicine and Animal Sciences, University of Milan, via dell’Università 6, Lodi 26900, Italy

Abstract: In order to preserve endangered psittacine species, more basic and applied research in reproductive biology is required. Assisted reproductive technologies such as artificial insemination play an important role in parrots species conservation programs to overcome the problem of infertile eggs and male infertility. The aim of this study was to define an effective in vitro protocol in order to standardize the sperm quality evaluation in psittacines, studying Melopsittacus undulatus as model species. Semen was collected from twenty adult males by massage technique from May to June. Sperm concentration was measured by the spectrophotometric method. Sperm quality (sperm membrane integrity (SMI), motility, and kinetic parameters) was assessed on fresh semen. Three different experimental protocols were performed to compare the effects of various processing conditions on SMI, motility, and kinetic parameters. In protocol 1, test was performed by Lake extender with three different pH, 7.4 versus 8.2 versus 8.4, and two different equilibration temperatures after dilution of fresh semen (4°C versus 25°C). In protocol 2, two dilution rates of semen after collection were valuated, 1 : 3 versus 1 : 4, as well as three different semen storage temperatures (4°C versus 25°C versus 38°C) before sperm motility analysis with the computer-assisted sperm analysis (CASA). In protocol 3, two different Makler chamber temperatures (38 versus 41°C) during motility analysis were tested. A significant progressive improvement in spermatozoa motility and kinetic parameters was registered with pH 8.4. Progressive motility and all kinetic parameters were higher at 4°C equilibration temperature. Straightness (STR) kinetic parameter was better with 1 : 4 dilution rate. Total motile sperm was higher in 41°C Makler chamber. In this study, for the first time, the effects of different processing protocols on psittacines seminal quality analysis were investigated. Significant differences conditioning the effectiveness of analysis protocols have been described.

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Hindawi Veterinary Medicine International – https://doi.org/10.1155/2022/5997320
Received 4 January 2022; Accepted 23 February 2022; Published 22 March 2022

Origin, Migration, and Reproduction of Indigenous Domestic Animals with Special Reference to Their Sperm Quality2022-03-05T17:09:27+01:00

Origin, Migration, and Reproduction of Indigenous Domestic Animals with Special Reference to Their Sperm Quality

Gerhard van der Horst, Liana Maree

Comparative Spermatology Laboratory, Department of Medical Bioscience, University of the Western Cape, Bellville 7535, South Africa

Abstract: Indigenous domestic animals such as cattle, sheep, goats, pigs, and chickens have a natural resistance to endo- and ecto-parasites and are tolerant in terms of harsh environmental conditions. These species orginated from the Fertile Cresent between 12,000 and 10,000 BP before migrating into surrounding continents. In view of limited information on the reproductive status of indigenous breeds, it is important to examine their semen characteristics in order to select males to improve livestock production. We have largely relied on existing literature but also our published and ongoing research on sperm quality assessment of several indigenous breeds. The sperm quality of these breeds is similar to current commercial breeds and has been quantified using cutting-edge methods. In this context, we have presented sperm functional tests which provide a better estimate of semen quality than just a standard semen analysis. Initial results suggest that the indigenous breeds have a high sperm quality and sperm functionality similar to currently farmed exotic or crossbreeds. In the long-term, the importance of preserving the favorable traits of these breeds is a priority in view of crossbreeding with existing good meat and milk producers.

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Animals 2022, 12(5), 657; https://doi.org/10.3390/ani12050657
Received: 15 December 2021 / Revised: 17 February 2022 / Accepted: 20 February 2022 / Published: 5 March 2022

Comparison of two staining techniques on the manual and automated canine sperm morphology analysis2022-02-24T16:48:17+01:00

Comparison of two staining techniques on the manual and automated canine sperm morphology analysis

Paulina Surmacz, Anna Niwinska, Ewa Kautz, Slawomir Gizinski, Ricardo Faundez

Department of Large Animal Diseases with Clinic, Faculty of Veterinary Medicine, Warsaw University of Life Sciences WULS – SGGW, Warsaw, Poland; Institute of Genetics and Animal Biotechnology of the Polish Academy of Sciences, Warsaw, Poland; InviMed Fertility Clinics, Warsaw, Poland

Abstract: Detailed and direct analysis of semen, including sperm morphology, enables a diagnosis of male fertility. This study aimed to describe an economical and verified protocol for canine spermiograms and compare the effectiveness of Sperm Stain® and Sperm Blue® (Microptic, Spain) in veterinary practice. Sperm assessment was conducted manually, using a standard optical microscope, and via computerized semen analysis using the SCA® CASA (Sperm Class Analyzer® CASA System-MICROPTIC, Spain). This study showed that Sperm Blue® is a better solution for computerized sperm quality analysis of healthy dogs. At the same time, Sperm Stain® turned out to be more helpful in identifying specific morphological defects of sperm. Automated canine sperm morphology analysis worked better with Sperm Blue stain, but Sperm Stain simplified manual evaluation of various organelles’ defects. Standard, manual examination is more error-prone for an inexperienced andrology technician, but it seems to be still a gold standard technique for canine sperm assessment.

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Reprod Dom Anim. 2022;00:1–7. / https://doi.org/10.1111/rda.14100
Received: 11 January 2022 | Accepted: 18 February 2022 | First published: 24 February 2022

Sperm preparedness and adaptation to osmotic and pH stressors relate to functional competence of sperm in Bos taurus2022-11-21T16:52:44+01:00

Sperm preparedness and adaptation to osmotic and pH stressors relate to functional competence of sperm in Bos taurus

Maharajan Lavanya, Santhanahalli Siddalingappa Archana, Divakar Swathi, Laxman Ramya, Arunachalam Arangasamy, Balakrishnan Binsila, Arindam Dhali, Narayanan Krishnaswamy, Sanjay Kumar Singh, Harendra Kumar, Muniandy Sivaram, Sellappan Selvaraju

Reproductive Physiology Laboratory, Animal Physiology Division, ICAR-National Institute of Animal Nutrition and Physiology, Adugodi, Bengaluru 560030, India; Department of Animal Reproduction, Indian Veterinary Research Institute, Izatnagar, Bareilly 243122, India; Omics Laboratory, Bioenergetics and Environmental Sciences Division, ICAR-National Institute of Animal Nutrition and Physiology, Adugodi, Bengaluru 560030, India; Indian Veterinary Research Institute, Bangalore 560024, India; Southern Regional Station, ICAR-National Dairy Research Institute, Adugodi, Bengaluru 560030, India

Abstract: The adaptive ability of sperm in the female reproductive tract micromilieu signifies the successful fertilization process. The study aimed to analyze the preparedness of sperm to the prevailing osmotic and pH stressors in the female reproductive tract. Fresh bovine sperm were incubated in 290 (isosmotic-control), 355 (hyperosmotic-uterus and oviduct), and 420 (hyperosmotic-control) mOsm/kg and each with pH of 6.8 (uterus) and 7.4 (oviduct). During incubation, the changes in sperm functional attributes were studied. Sperm kinematics and head area decreased significantly (p < 0.05) immediately upon exposure to hyperosmotic stress at both pH. Proportion of sperm capacitated (%) in 355 mOsm/kg at 1 and 2 h of incubation were significantly (p < 0.05) higher than those in 290 mOsm media. The magnitude and duration of recovery of sperm progressive motility in 355 mOsm with pH 7.4 was correlated with the ejaculate rejection rate (R2 = 0.7). Using this information, the bulls were divided into good (n = 5) and poor (n = 5) osmo-adapters. The osmo-responsive genes such as NFAT5, HSP90AB1, SLC9C1, ADAM1B and GAPDH were upregulated (p < 0.05) in the sperm of good osmo-adapters. The study suggests that sperm are prepared for the osmotic and pH challenges in the female reproductive tract and the osmoadaptive ability is associated with ejaculate quality in bulls.

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Nature, Scientific Reports (2021) 11:22563 – https://doi.org/10.1038/s41598-021-01928-6
Published: 19 November 2021

Progesterone, Myo-Inositol, Dopamine and Prolactin Present in Follicular Fluid Have Differential Effects on Sperm Motility Subpopulations2021-11-19T12:14:49+01:00

Progesterone, Myo-Inositol, Dopamine and Prolactin Present in Follicular Fluid Have Differential Effects on Sperm Motility Subpopulations

Shannen Keyser, Gerhard van der Horst, Liana Maree

Comparative Spermatology Laboratory, Department of Medical Bioscience, Faculty of Natural Sciences, University of the Western Cape, Private Bag X17, Bellville 7535, South Africa

Abstract: Considering the challenges surrounding causative factors in male infertility, rather than relying on standard semen analysis, the assessment of sperm subpopulations and functional characteristics essential for fertilization is paramount. Furthermore, the diagnostic value of sperm interactions with biological components in the female reproductive tract may improve our understanding of subfertility and provide applications in assisted reproductive techniques. We investigated the response of two sperm motility subpopulations (mimicking the functionality of potentially fertile and sub-fertile semen samples) to biological substances present in the female reproductive tract. Donor semen was separated via double density gradient centrifugation, isolated into high (HM) and low motile (LM) sperm subpopulations and incubated in human tubal fluid (HTF), capacitating HTF, HD-C medium, progesterone, myo-inositol, dopamine and prolactin. Treated subpopulations were evaluated for vitality, motility percentages and kinematic parameters, hyperactivation, positive reactive oxygen species (ROS), intact mitochondrial membrane potential (MMP) and acrosome reaction (AR). While all media had a significantly positive effect on the LM subpopulation, dopamine appeared to significantly improve both subpopulations’ functional characteristics. HD-C, progesterone and myo-inositol resulted in increased motility, kinematic and hyperactivation parameters, whereas prolactin and myo-inositol improved the LM subpopulations’ MMP intactness and reduced ROS. Furthermore, progesterone, myo-inositol and dopamine improved the HM subpopulations’ motility parameters and AR. Our results suggest that treatment of sub-fertile semen samples with biological substances present in follicular fluid might assist the development of new strategies for IVF treatment.

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Life 2021, 11(11), 1250; https://doi.org/10.3390/life11111250
Received: 19 October 2021 / Revised: 9 November 2021 / Accepted: 11 November 2021 / Published: 17 November 2021

Cementing the relationship between conventional and advanced semen parameters2021-10-19T17:12:41+02:00

Cementing the relationship between conventional and advanced semen parameters

Bashir M. Ayad, Ibukun P. Oyeyipo, Gerhard Van der Horst, Stefan S. Du Plessis

Department of Physiology, Faculty of Medicine, Misurata University, Misurata, Libya; Division of Medical Physiology, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa; Department of Physiology, College of Health Sciences, Osun State University, Osogbo, Nigeria; Department of Basic Medical Sciences, Mohammed Bin Rashid University of Medicine and Health Sciences, Dubai, United Arab Emirates

Abstract: 

Background: Affordable conventional semen analysis remains a fundamental procedure to be performed routinely during the diagnosis of male infertility. Advanced semen analyses provide valuable clinical insights in treatment-related decision-making, but these are highly expensive and lack universal standardization. This study aimed at determining the relationship between conventional semen parameters, measured with assistance of computer-aided sperm analysis (CASA), and a set of advanced semen tests. Basic semen analysis (n = 124) was performed according to the World Health Organization (WHO) guidelines. Sperm DNA fragmentation and intracellular superoxide (O2−•) levels were assessed by flow cytometry. Seminal plasma thiobarbituric acid reactive substances (TBARS) levels as well as superoxide dismutase (SOD) and catalase (CAT) activity were measured by spectrophotometry. Spearman’s rank correlation coefficient was used, with significance set at p < 0.05.

Results: Semen pH correlated negatively with TBARS (p < 0.01). The proportions of total and progressively motile as well as rapid spermatozoa correlated positively with CAT activity (p < 0.05). Sperm viability correlated negatively with both O2−• (p < 0.05) and DNA fragmentation (p = 0.01), while normal morphology correlated negatively with O2−• levels (p < 0.05) and positively with CAT activity (p < 0.05). Straight-line velocity (VCL) and average-path velocity (VAP) correlated negatively with both O2−• (p < 0.01) and TBARS (p < 0.01). Amplitude of lateral head displacement (ALH) correlated negatively with O2−• (p < 0.01) and DNA fragmentation (p < 0.01), while its correlation with SOD activity was positive (p < 0.05).

Conclusion: The results obtained from this study support the validity of some CASA parameters as sensitive indicators of changes in sperm oxidative status and DNA integrity. Predicting advanced from conventional parameters through the building of linear regression models should be considered for future studies.

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Middle East Fertil Soc J 26, 39 (2021) – https://doi.org/10.1186/s43043-021-00086-z
Received: 22 May 2021 / Accepted: 10 October 2021 / Published: 19 October 2021

Decline in semen quality of North African men: a retrospective study of 20,958 sperm analyses of men from different North African countries tested in Tunisia over a period of 6 years (2013–2018)2021-09-07T12:50:38+02:00

Decline in semen quality of North African men: a retrospective study of 20,958 sperm analyses of men from different North African countries tested in Tunisia over a period of 6 years (2013–2018)

Hatem Bahri, Mustapha Ben Khalifa, Maroua Ben Rhouma, Zied Abidi, Emna Abbassi, Khémais Ben Rhouma, Moncef Benkhalifa

HB Clinical Laboratory for Medical Analyses, Tunis, Tunisia; Research laboratory LR99ES11, Department of Biochemistry, La Rabta Hospital, Faculty of Medicine of Tunis, University of Tunis El Manar, Tunis, Tunisia; Laboratory of Integrated Physiology, Faculty of Science of Bizerte, University of Carthage, Tunisia; Reproductive Medicine, Reproductive Biology & Genetics, University Hospital & School of Medicine Jules Verne, Amiens, France; Peritox Laboratory, CURS, Picardie University Jules Verne, Amiens, France

Abstract: 

Background: According to numerous studies from around the world, semen quality seems to have declined dramatically in recent times. However, the data available on male fertility status and semen quality in North Africa are limited.

Aim: To investigate the status of semen quality in North-African men and to understand its variations.

Subjects & methods: 20,958 sperm-analyses (Spermogram – Spermocytogram) of North-African men (19–77 years old) consulting for infertility, performed in a private laboratory of medical analyses (Tunis, Tunisia) over a period of 6 years (2013–2018), were investigated. All patients had at least 1 year of unprotected intercourse with their partners before the test. Statistical analyses were performed using SPSS 22.0 software for windows.

Results: Libyan men presented a clear decline in all sperm parameters. A continuous decline in sperm morphology quality was shown in Tunisian and Algerian men. Mauritanian men presented a significant increase in sperm vitality with pseudo-stability in the rest of the sperm parameters during the whole study period.

Conclusion: North-African men presented remarkable decreases in their semen quality over the last decade. This data could confirm possible global common-causes that need to be identified in order to limit their negative impact on sperm quality, and consequently on male-fertility.

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Annals of Human Biology, 2021, Vol. 48, No. 4, 350-359 – https://doi.org/10.1080/03014460.2021.1957501
Received 15 December 2020 / Revised 23 May 2021 / Accepted 12 July 2021 / Published online: 07 Sep 2021

P–043 Six years’ retrospective statistical study (2013 – 2018) investigating the impact of Sperm DNA fragmentation on sperm analysis parameters2021-08-06T16:30:21+02:00

P–043 Six years’ retrospective statistical study (2013 – 2018) investigating the impact of Sperm DNA fragmentation on sperm analysis parameters

H Bahri, W Zidi, M Benkhalifa

HB laboratory, Andrology / Reproductive Biology, Tunis, Tunisia; La Rabta Hospital- Faculty of Medicine of Tunis- University of Tunis El Manar- Tunis- Tunisia., Research laboratory LR99ES11 Department of Biochemistry, Tunis, Tunisia

Abstract: 

Study question: What is the relationship between Sperm DNA fragmentation (SDF) levels and sperm analysis (Spermocytogramme) parameters results?

Summary answer: SDF level of patients with pathological spermocytogramme presents negative correlations to total spermatozoa mobility, vitality and concentration, and positive correlation to sperm morphology defects.

What is known already: The relationship between SDF and Sperm analysis parameters and especially sperm morphology needs to be more studied since few studies over the last years were focused on this relationship. However, abnormalities in these two parameters are considered as the most important biological indicators of male infertility. The pathogenesis of Teratozoospermia (<4% morphologically normal sperm cells according to WHO 2010) is continuously increasing over the last decade according to several studies. In addition, SDF is also increasing over the years because of several factors such as pollution, stress and lifestyle changing.

Study design, size, duration: Retrospective study including 331 infertile patients undergoing SDF-index testing with Spermocytogramme from January 2013 – December 2018. Patients divided into two groups: 143 patients with normal-Spermocytogramme and 188 patients with pathological-Spermocytogramme. Each group includes patients with abnormal SDF levels (>30%). Statistical analyzes were performed using SPSS22.0 for Windows-software. Kolmogorov–Smirnov-test for normality analysis and comparisons by Student-t-test or Mann–Whitney U-test, as appropriate. Pearson/Spearman’ tests for correlations were used as appropriate, P-value<0.05 was considered as significant.

Participants/materials, setting, methods: 143 patients with normal Spermocytogramme (2.8% abnormal-SDF) vs 188 patients with pathological Spermocytogramme (10.6% abnormal-SDF). WHO–2010 instructions for sperm-analysis were used through Makler®-counting-chamber (Sefi-Medical Instrument Ltd) for sperm-concentration and motility-determination using Sperm-class-analyzer-software (CASA-system (Microptic®)) to detect sperm abnormalities. Normozoospermia was determined when sperm progressive-motility is ≥ 32%, sperm-concentration ≥15×106/mL, and sperm-morphology ≥4%. “Diff-Quick” staining-method for the coloration of the fixed-sperm-slides was used for Sperm-morphology analysis. GoldCyto Sperm®Kit (Goldcyto Biotech corp.) was used to analyze SDF.

Main results and the role of chance: SDF is significantly higher in pathological spermocytogramme’ patients than in normal spermocytogramme’ patients (17.02 ± 11.88 vs 12.16 ± 9.58 respectively). In patients with pathological spermocytogramme, SDF is negatively correlated to Progressive sperm motility (r= –0.137; p = 0.042), Total sperm motility (r= –0.153; p = 0.036), vitality (r=–0.140; p = 0.048) and concentration (r=–0.195; p = 0.007). In the other hand, SDF presented positive correlation with teratozoospermia and especially with sperm midpiece defects (r = 0.171; p = 0.02). However, SDF did not present any correlation with age, testosterone levels and total ejaculated sperm volume. However the latter was positively correlated to spermatozoa midpiece and head defects (r = 0.156; p = 0.034; r = 0.203; p = 0.006, respectively). These results are in accordance with García-Ferreyra et al. (2014) who found that men with abnormal spermatozoa morphology showed high levels of DNA fragmentation, Sá et al. (2015) who confirmed that semen with lower concentration, motility and morphology have higher levels of SDF and showed that sperm head staining patterns are correlated with the degree of SDF. In addition, recently the study of Jakubik-Uljaszstudy et al. (2020) could confirms our results when it concluded that detailed sperm structural defects coexist with abnormal nuclear sperm DNA dispersion and that men with teratozoospermia may have a higher risk for sperm DNA damage.

Limitations, reasons for caution: Our study is a retrospective statistical investigation that included patients attending to the laboratory for fertility diagnosis after a period of infertility. Meta-analyzes studies in addition to more prospective-randomized-controlled-trials with couples undergoing assisted-reproductive-treatments and in comparison with fertile men are needed to confirm the relationship between SDF and spermocytogramme defects.

Wider implications of the findings: These results should interest andrologists, reproductive science fundamentalists and embryologists who want to improve the investigations on the origin of infertility especially when it comes from male side.

Human Reproduction, Volume 36, Issue Supplement_1, July 2021, deab130.042, https://doi.org/10.1093/humrep/deab130.042
Published: 06 August 2021

Improved sperm function in human sperm subpopulations: a model for studying subfertility2021-07-28T17:10:12+02:00

Improved sperm function in human sperm subpopulations: a model for studying subfertility

Shannen Keyser, Gerhard van der Horst, Liana Maree

Department of Medical Bioscience, University of the Western Cape, Bellville, South Africa

Abstract: With many cases of male infertility being idiopathic or unknown, the necessity for in-depth functional and structural sperm analysis beyond routine basic semen analysis techniques has become crucial. Existence of subpopulations is often neglected in basic semen analysis thus sperm heterogeneity could interfere with an accurate evaluation of overall sperm quality. This study aimed to determine and compare various functional characteristics of two sperm subpopulations found within the same human semen samples before and after treatment with various capacitating media and chemicals. Normospermic donor semen samples were separated into two fractions via double density gradient centrifugation and further assessed for their sperm functional characteristics before and after treatment with capacitating media, progesterone (1.98, 3.96, 19.8 μM), and myoinositol (11 mM). High motile sperm fractions had significantly improved functional characteristics in contrast to the low motile fractions with a notable improvement in both sperm fractions’ functional characteristics after exposure to treatments. Selected sperm functional parameters in lower quality sperm subpopulations could be improved to the level of a higher quality sperm subpopulation, separated from the same semen samples. If the lower quality sperm subpopulation mimics the sperm population in subfertile males, this can be used as a model to study such subpopulations and possibly broaden the potential treatment regimens and sperm isolation procedures for ART.

Keyser S., van der Horst G., Maree L. (2021) Improved Sperm Function in Human Sperm Subpopulations: A Model for Studying Subfertility. In: Björndahl L., Flanagan J., Holmberg R., Kvist U. (eds) XIIIth International Symposium on Spermatology. Springer, Cham. https://doi.org/10.1007/978-3-030-66292-9_50
First Online: 23 July 2021

Proteomics and biomarker identification in improved sperm motility parameters after 4 h of ejaculatory abstinence2021-07-28T17:03:51+02:00

Proteomics and biomarker identification in improved sperm motility parameters after 4 h of ejaculatory abstinence

Dale M. Goss, Bashir Ayad, Maré Vlok, Suzél M. Hattingh, Gerhard van der Horst, Stefan S. du Plessis

Division of Medical Physiology, Stellenbosch University, Tygerberg, South Africa; Department of Biochemistry and LCMS-Central Analytical Facility, Stellenbosch University, Tygerberg, South Africa; Department of Basic Medical Sciences, College of Medicine, Mohammed Bin Rashid University of Medicine and Health Sciences, Dubai, UAE

Abstract: Various studies have sought to determine the optimal abstinence period after which semen samples should be collected, with many contradictory results reported. Several factors influence the semen microenvironment, and thus potentially sperm parameters. In this study, we focused on the secretions of the prostate, seminal vesicles and the epididymis. Semen samples were obtained from healthy normozoospermic males (n = 16) after 4 day and 4 h periods of ejaculatory abstinence (EA), and standard semen analysis was performed using computer-aided sperm analysis, whereas seminal plasma citric acid, neutral alpha-glucosidase and fructose concentrations were measured using assay kits. Proteomic analyses of seminal plasma proteins were performed using LC-MS/MS. There were significant decreases in total sperm count (P < 0.001), sperm concentration (P < 0.05) and semen volume (P < 0.05) after 4 h compared with 4 days ejaculatory abstinence. Furthermore, increases were observed in total sperm motility (P < 0.05), sperm progressive motility (P < 0.01), average path velocity (P < 0.001) and curvilinear velocity (P < 0.05) after a 4 h abstinence period, accompanied by significant reductions in citric acid (P < 0.05), neutral alpha-glucosidase (P < 0.01) and fructose (P < 0.01) concentrations. In addition, due to the decreased number of spermatozoa, these concentrations translated to a significant decrease in fructose (P < 0.05) per spermatozoon. A total of 2889 proteins were identified, with 24 proteins differentially expressed. These proteins have various roles in cellular processes and may influence the improvement of sperm motility after 4 h of EA. All these factors present in the seminal plasma indicate towards an intrinsic mechanism capitalising on alternative sources of energy for increased metabolic function and subsequent sperm motility.

Goss D.M., Ayad B., Vlok M., Hattingh S.M., van der Horst G., du Plessis S.S. (2021) Proteomics and Biomarker Identification in Improved Sperm Motility Parameters After 4 h of Ejaculatory Abstinence. In: Björndahl L., Flanagan J., Holmberg R., Kvist U. (eds) XIIIth International Symposium on Spermatology. Springer, Cham. https://doi.org/10.1007/978-3-030-66292-9_48
First Online: 23 July 2021

Have we conquered sperm morphology analysis in different mammalian species as analysed by CASMA?2022-05-04T15:56:08+02:00

Have we conquered sperm morphology analysis in different mammalian species as analysed by CASMA?

Gerhard van der Horst, Stefan S. du Plessis, Liana Maree

Department of Medical Biosciences, University of the Western Cape, Bellville, South Africa; Department of Basic Sciences, Mohammed Bin Rashid University of Medicine and Health Sciences, Dubai, United Arab Emirates; Division of Medical Physiology, Stellenbosch University, Tygerberg, South Africa

Abstract: Computer-aided sperm morphology analysis (CASMA) advanced to a level where it is possible to analyse sperm morphology of most mammalian laboratory animals (rat and mouse), domestic animals (bull, boar, ram, goat, horse, dog and cat) and wildlife species (e.g. African elephant, African white rhinoceros, African buffalo, leopard, cheetah, lion, wild dog, several antelope species). The Sperm Class Analyzer (SCA) CASMA is capable of analysing all major sperm components, including the acrosome, head, midpiece and tail, provided that the correct staining technique has been used. In this context, a stain which is isotonic and isosmotic as well as producing a clear background for analysis is preferred. In CASA systems such as the SCA, a large number of morphometric parameters are measured, including detailed facets such as roughness, smoothness and regularity. However, one of the major challenges faced with CASMA for animals is to provide cut-off values for so-called normal sperm morphology, for both diverse species and different breeds in the domestic market. Accordingly, a combination of CASMA morphometric parameters and visual analysis by experienced investigators has been used to define cut-off values for normal sperm morphology for a given species. In this investigation, sperm morphometry and the definition of normal sperm morphology will be discussed for a number of species, with emphasis on the methodology of performing such analyses quantitatively in Wistar rat, bull, Tankwa goat, hyrax and several other mammalian species. This paper provides invaluable information on what constitutes normal sperm morphology when assessed objectively and highlights the importance of sperm indices such as TZI (teratozoospermic index) as well as many other quantitative aspects.

van der Horst G., du Plessis S.S., Maree L. (2021) Have We Conquered Sperm Morphology Analysis in Different Mammalian Species as Analysed by CASMA?. In: Björndahl L., Flanagan J., Holmberg R., Kvist U. (eds) XIIIth International Symposium on Spermatology. Springer, Cham. https://doi.org/10.1007/978-3-030-66292-9_38
First Online: 23 July 2021

Processes and Data Management of Computer-Aided Sperm Analysis in Human and Animal Spermatozoa2022-05-04T15:58:23+02:00

Processes and Data Management of Computer-Aided Sperm Analysis in Human and Animal Spermatozoa

Gerhard van der Horst

Department of Medical Biosciences, University of the Western Cape, Bellville, South Africa

Abstract: Computer-Aided Sperm Analysis (CASA) has been revolutionized in the last two decades. It is now possible to measure a vast range of sperm characteristics such as sperm concentration, sperm motility parameters (including percentage of motility groupings, kinematics, subpopulations, mucous penetration, and hyperactivation), sperm morphology normality/types, sperm vitality, sperm DNA fragmentation, sperm acrosome reaction in many mammalian species but also in most vertebrate classes and some invertebrate phyla. While CASA methodology and analysis may be straightforward and performed within minutes, a major drawback is that during the analysis methodology variables (e.g., handling of semen sample, temperature control, correct optics, random field selection, and many more) are not observed/controlled in a manner that is consistent and repeatable. This paper emphasizes ways to control such variables. Advanced CASA systems can provide more than 100 quantitative parameters for sperm motility and morphology alone. Which of these sperm parameters are the most important in understanding sperm quality/physiology/potential to fertilize? What is the best way to analyze this large number of data points? In this regard, the paper recommends which parameters should be used (e.g., for motility subpopulations and hyperactivation) and which analysis techniques should be implemented, including aspects such as Bland and Altman plots, receiver-operating characteristic (ROC) curves, and various multivariate techniques including multivariate visualizations and principal component analysis.

van der Horst G. (2021) Processes and Data Management of Computer-Aided Sperm Analysis in Human and Animal Spermatozoa. In: Björndahl L., Flanagan J., Holmberg R., Kvist U. (eds) XIIIth International Symposium on Spermatology. Springer, Cham. https://doi.org/10.1007/978-3-030-66292-9_27
First Online: 23 July 2021

A possible highway system for the rapid delivery of sperm from the testis to the penis in the naked mole-rat, Heterocephalus glaber2022-05-04T15:57:45+02:00

A possible highway system for the rapid delivery of sperm from the testis to the penis in the naked mole-rat, Heterocephalus glaber

Gerhard van der Horst, Sanet Kotzè, Mannus Justin O’Riain, Nolan Muller, Liana Maree

Department of Medical, Biosciences, University of the Western Cape, Bellville, South Africa; Division of Clinical Anatomy, Department of
Biomedical Sciences, Stellenbosch University, Cape Town, South Africa; Ross University School of Veterinary Medicine, Basseterre, St. Kitts, West Indies; Department of Zoology, University of Cape Town, Cape Town, South Africa; National Health Laboratory Services,
Anatomical Pathology, Tygerberg Hospital, Parow, South Africa

Abstract: Gametogenesis is suppressed in most members of the eusocial naked mole-rat (NMR) colony, while the queen selects mainly one breeding male during her life span. Recently, it was reported that the NMR testicular organization seems to produce spermatozoa on demand after suppression of spermatogenesis during most of gestation. A Sertoli cell “pump” is then used to flush the spermatozoa into short tubuli recti and simplified rete testis to reach the excurrent duct system. We hypothesize that the components of this duct system are adapted for rapid delivery of spermatozoa to the penis and for numerous copulations with the queen. Therefore, the aim was to study the ultrastructure of the male NMR reproductive duct system using light microscopy and transmission electron microscopy. The NMR rete testis gives rise to six to eight efferent tubules joining the caput epididymis. The caput epididymis resembles that of other rodents but with less distinction in terms of histological zoning. The remainder of the epididymis is considerably reduced in length compared to other rodents. In contrast, the vas deferens epithelium is highly specialized in that a vast range of vesicles, often closely associated with the spermatozoa, were visible. The large ampulla is a factory for merocrine and apocrine secretions, producing even more diverse vesicles. The transitional epithelial cells of the bladder appear to secrete abundant mucous and the penis as well as its baculum is relatively small. We speculate that these modifications strongly suggest that the excurrent duct system has been simplified and adjusted to compensate for the absence of long maturation and storage of spermatozoa. We propose that these adaptations to the NMR reproductive tract are associated with a state of degenerative orthogenesis that was selected for due to the absence of sperm competition and apparently rapid delivery of spermatozoa from the testis.

Journal of Morphology. 2021;1–21. – DOI: 10.1002/jmor.21399
Received: 12 March 2021 / Revised: 8 July 2021 / Accepted: 17 July 2021 / Published: 23 July 2021

Seminal plasma metabolomics profiles following long (4-7 days) and short (2 hours) sexual abstinence periods2021-07-14T11:05:11+02:00

Seminal plasma metabolomics profiles following long (4-7 days) and short (2 hours) sexual abstinence periods

H. Alipour, R.K. Duus, R. Wimmer, F. Dardmeh, S.S. Du Plessis, N. Jørgensen, O.B. Christiansen, C. Hnida, H.I. Nielsen, G. Van Der Horst

Aalborg University, Department of Health Science and Technology, Biomedicine group, Aalborg, Denmark; Department of Chemistry and Bioscience, Aalborg University, Aalborg, Denmark; Department of Basic Medical Sciences, Mohammed Bin Rashid University of Medicine and Health Sciences, Dubai, United Arab Emirates; Division of Medical Physiology, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa; University Department of Growth and Reproduction and International Center for Research and Research Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC), Rigshospitalet, Copenhagen, Denmark; Aalborg University Hospital, Dept. of Obstetrics and Gynecology, Fertility unit, Aalborg, Denmark; Institute of Clinical Medicine, Aalborg University; University of the Western Cape, Department of Medical Biosciences, Cape Town, South Africa.

Abstract: 

Objective: Metabolomic profiling of seminal plasma has been suggested as a possible approach for a fast and non-invasive male infertility evaluation diagnosis. However, metabolomics profiles in normozoospermic men have not been thoroughly investigated, and the influence of ejaculation-abstinence has not been described. To provide interim reference values and find associations between the metabolomics profiles of human seminal plasma and length of ejaculation-abstinence period in normozoospermic men.

Study design: Semen samples collected after long (4-7 days) and short abstinence (2 hours) from 31 normozoospermic males were assessed for routine quality parameters before the seminal plasma was separated by centrifugation. Metabolomics profiles of the seminal plasma were then determined using untargeted Nuclear Magnetic Resonance Spectroscopy.

Results: In total, 30 metabolites were identified. Pyruvate showed a higher concentration, while fructose, acetate, choline, methanol, N-acetylglucosamine, O-acetylcarnitine, uridine, and sn-glycero-3-phosphocoline showed lower concentrations in samples collected after short abstinence (vs. long). All metabolites showed lower absolute amounts (volume x concentration) following shorter abstinence. However, the lower sperm concentration in samples collected after short abstinence resulted in higher absolute amounts of pyruvate and taurine per spermatozoa: pyruvate 1.92 (1.12-3.87) vs. 1.29 (0.83-2.62) (P<0.001) and taurine 0.58 (0.36-0.92) vs. 0.43 (0.28-0.95) (P<0.05) ng/106 spermatozoa. Simultaneously, there was a higher percentage of progressively motile spermatozoa in samples collected after the short abstinence.

Conclusion: The generally lower concentrations of seminal metabolites after short abstinence periods may be related to the shorter time available for secretion and collection of these metabolites by the accessory glands and the epididymides. The concomitant lower number of spermatozoa in the second ejaculate resulted in increased absolute amounts of pyruvate and taurine per spermatozoa, accompanied by increased spermatozoa motility in these samples. The simultaneous increase in percentages of motile spermatozoa and absolute amounts of pyruvate and taurine per spermatozoa after shorter abstinence might indicate that these two metabolites play a more critical role in sperm motility, which should be further investigated in future studies.

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European Journal of Obstetrics & Gynecology and Reproductive Biology; https://doi.org/10.1016/j.ejogrb.2021.07.024
Received Date: 29 March 2021 / Revised Date: 8 July 2021 / Accepted Date: 12 July 2021 / Published: July 14, 2021

Effects of Extender Type, Storage Time, and Temperature on Bull Semen Parameters2021-07-07T12:30:49+02:00

Effects of Extender Type, Storage Time, and Temperature on Bull Semen Parameters

Aitor Fernandez-Novo, Sergio Santos-Lopez, Clara Barrajon-Masa, Patricia Mozas, Eduardo de Mercado, Elisa Caceres, Aizic Garrafa, Juan Vicente Gonzalez-Martin, Natividad Perez-Villalobos, Agustin Oliet, Susana Astiz and Sonia Salome Perez-Garnelo

Department of Veterinary Medicine, School of Biomedical and Health Sciences, Universidad Europea de Madrid, Madrid, Spain; Animal Production Department, Veterinary Faculty, Complutense University of Madrid, Madrid, Spain; National Centre of Selection and Animal Reproduction (CENSYRA) of Colmenar Viejo, Madrid, Spain; Animal Reproduction Department, National Institute of Agronomic Research (INIA), Madrid, Spain; Department of Animal Medicine and Surgery, Veterinary Faculty, Complutense University of Madrid, Madrid, Spain

Abstract: Seminal parameters can be evaluated in situ, or samples can be delivered to a diagnostic centre. How storage conditions affect ejaculates up to evaluation is unclear. We assessed, in 25 commercial bulls electroejaculated in the field, the impact of time until evaluation (0–2 h, 4–6 h, and 24 h post-ejaculation), holding temperature (5 °C vs. room temperature), and extender (AndroMed®, BIOXcell® or INRA96®) on semen quality. Acrosome integrity, sperm viability and morphology, CASA-total and progressive motility, pH, and colony-forming units were assessed. Semen quality was preserved for up to 4–6 h post-ejaculation, except for INRA96® at 5 °C. Regardless of extender or temperature, motility decreased from 4 to 6 h up to 24 h, with the best values obtained with BIOXcell® at 5 °C. pH differed from 4 to 6 h up to 24 h, acidifying when stored at room temperature. Microbiological load was stable over time with AndroMed® and BIOXcell®, and increased at room temperature with INRA96®. Our results suggest that AndroMed® and BIOXcell® can preserve semen quality for up to 6 h, either at 5 °C or room temperature, while INRA96® only at room temperature. These results help to fix adequate protocols for short-term storage and shipment of bovine semen collected under field conditions.

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Biology 2021, 10(7), 630; https://doi.org/10.3390/biology10070630
Received: 6 June 2021 / Revised: 28 June 2021 / Accepted: 3 July 2021 / Published: 7 July 2021

Effects of quetiapine administration on sperm quality and testicular histology2022-05-04T15:58:15+02:00

Effects of quetiapine administration on sperm quality and testicular histology

Busra Korkut Celikates, Volkan Kilic, Ozlem Atli-Eklioglu, Merve Baysal, Gozde Aydogan-Kılıc, Seyda Ucarcan, Sinem Ilgin

Faculty of Pharmacy, Department of Pharmaceutical Toxicology, Anadolu University, Eskisehir, Turkey; Faculty of Science, Department of Biology, Eskisehir Technical University, Eskisehir, Turkey

Abstract: Quetiapine is one of the most commonly prescribed antipsychotics to treat schizophrenia in adults, in particular. In this study, quetiapine’s effects were assessed on healthy sperm production in rats at repeated-pharmacological doses. Additionally, the effects of quetiapine on oxidative status and hormonal balance were also evaluated in rats. Quetiapine was administered to rats orally at 10, 20, and 40 mg/kg body weight doses for 28 days. At the end of this period, body and organ weights were measured, sperm concentration, motility, and morphology were determined, sperm damage was assessed, and histopathological analysis of testicular tissue was performed. Additionally, serum FSH, LH, and testosterone levels as male reproductive hormones were measured. Catalase, superoxide dismutase, glutathione, and malondialdehyde levels were determined for evaluating the oxidative status of testicular tissue. The findings obtained in this study showed that relative epididymis weights and sperm concentration decreased and abnormal sperm morphology increased in quetiapine-administered rats. Irregularity of typical architecture of the seminiferous tubules and germinal cell disorganization was observed in testicular sections of 20 and 40 mg/kg quetiapine-administered rats. Further, serum LH and testosterone levels decreased in 20 and 40 mg/kg quetiapine-administered rats. Additionally, decreased catalase and superoxide dismutase activities in testicular tissue of quetiapine-administered rats and increased malondialdehyde levels in testicular tissue of 40 mg/kg quetiapine-administered rats were measured. In conclusion, quetiapine treatment decreased sperm quality, altered hormone levels, and induced oxidative stress may be considered potential contributors to this adverse effect.

DRUG AND CHEMICAL TOXICOLOGY; https://doi.org/10.1080/01480545.2021.1946558
Received 12 Aug 2020, Accepted 10 May 2021, Published online: 07 Jul 2021

Effect of Copper Sulphate and Cadmium Chloride on Non-Human Primate Sperm Function In Vitro2021-06-08T09:51:25+02:00

Effect of Copper Sulphate and Cadmium Chloride on Non-Human Primate Sperm Function In Vitro

Farren Hardneck, Charon de Villiers, Liana Maree

Department of Medical Bioscience, University of the Western Cape, Private Bag X17, Bellville 7535, South Africa; PUDAC-Delft Animal Facility, South African Medical Research Council, Cape Town 7505, South Africa

Abstract: In order to address the large percentage of unexplained male infertility in humans, more detailed investigations using sperm functional tests are needed to identify possible causes for compromised fertility. Since many environmental and lifestyle factors might be contributing to infertility, future studies aiming to elucidate the effect of such factors on male fertility will need the use of appropriate research models. The current study aimed to assess the effects of two heavy metals, namely copper sulphate, and cadmium chloride, on non-human primate (NHP) sperm function in order to establish the possibility of using these primate species as models for reproductive studies. Our combined results indicated that the functionality of NHP spermatozoa is inhibited by the two heavy metals investigated. After in vitro exposure, detrimental effects, and significant lowered values (p < 0.05) were obtained for sperm motility, viability and vitality, acrosome intactness, and hyperactivation. These metals, at the tested higher concentrations, therefore, have the ability to impair sperm quality thereby affecting sperm fertilizing capability in both humans and NHPs.

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Int. J. Environ. Res. Public Health 2021, 18(12), 6200; https://doi.org/10.3390/ijerph18126200
Received: 9 April 2021 / Revised: 18 May 2021 / Accepted: 21 May 2021 / Published: 8 June 2021

The validity and reliability of computer-aided semen analyzers in performing semen analysis: a systematic review2021-05-27T16:14:56+02:00

The validity and reliability of computer-aided semen analyzers in performing semen analysis: a systematic review

Renata Finelli, Kristian Leisegang, Samhita Tumallapalli, Ralf Henkel, Ashok Agarwal

American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, USA; School of Natural Medicine, Faculty of Community and Health Sciences, University of the Western Cape, Bellville, South Africa; Case Western University, Cleveland, Ohio, USA; Department of Metabolism, Digestion and Reproduction, Imperial College London, London, UK; Department of Medical Bioscience, University of the Western Cape, Bellville, South Africa; LogixX Pharma, Theale, Berkshire, UK

Abstract:

Background: Computer-aided sperm analyzers (CASA) are currently used worldwide for semen analysis. However, there are doubts about their reliability to fully substitute the human operator. Therefore, this study aimed to systematically review the current literature comparing results from semen evaluation by both CASA-based and manual approaches.

Methods: A systematic screening of the literature was performed based on the PRISMA guidelines and by searching on PubMed, Scopus, and Embase databases.

Results: A total of 14 studies were included. Our results showed a high degree of correlation for sperm concentration and motility when analysis was performed either manually or by using a CASA system. However, CASA results showed increased variability in low (<15 million/mL) and high (>60 million/mL) concentration specimens, while sperm motility assessment was inaccurate in samples with higher concentration or in the presence of non-sperm cells and debris. Morphology results showed the highest level of difference, due to the high amount of heterogeneity seen between the shapes of the spermatozoa either in one sample or across multiple samples from the same subject.

Conclusions: Overall, our study suggests CASA systems as a valid alternative for the evaluation of semen parameters in clinical practice, especially for sperm concentration and motility. However, further technological improvements are required before these devices can one day completely replace the human operator. Artificial intelligence-based CASA devices promise to offer higher efficiency of the analysis and improve the reliability of results.

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Translational Andrology and Urology; http://dx.doi.org/10.21037/tau-21-276
Submitted Apr 02, 2021. Accepted for publication May 27, 2021.

Status of Sperm Functionality Assessment in Wildlife Species: From Fish to Primates2021-06-10T09:57:00+02:00

Status of Sperm Functionality Assessment in Wildlife Species: From Fish to Primates

Gerhard van der Horst

Comparative Spermatology Laboratory, Department of Medical Bioscience, University of the Western Cape, Bellville, Cape Town 7535, South Africa

Abstract: (1) Background: in order to propagate wildlife species (covering the whole spectrum from species suitable for aquaculture to endangered species), it is important to have a good understanding of the quality of their sperm, oocytes and embryos. While sperm quality analyses have mainly used manual assessment in the past, such manual estimations are subjective and largely unreliable. Accordingly, quantitative and cutting-edge approaches are required to assess the various aspects of sperm quality. The purpose of this investigation was to illustrate the latest technology used in quantitative evaluation of sperm quality and the required cut-off points to distinguish the differential grades of fertility potential in a wide range of vertebrate species. (2) Methods: computer-aided sperm analysis (CASA) with an emphasis on sperm motility, 3D tracking and flagellar and sperm tracking analysis (FAST), as well as quantitative assessment of sperm morphology, vitality, acrosome status, fragmentation and many other complimentary technologies. (3) Results: Assessing sperm quality revealed a great deal of species specificity. For example, in freshwater fish like trout, sperm swam in a typical tight helical pattern, but in seawater species sperm motility was more progressive. In amphibian species, sperm velocity was slow, in contrast with some bird species (e.g., ostrich). Meanwhile, in African elephant and some antelope species, fast progressive sperm was evident. In most species, there was a high percentage of morphologically normal sperm, but generally, low percentages were observed for motility, vitality and normal morphology evident in monogamous species. (4) Conclusions: Sperm quality assessment using quantitative methodologies such as CASA motility, FAST analysis, morphology and vitality, as well as more progressive methodologies, assisted in better defining sperm quality—specifically, sperm functionality of high-quality sperm. This approach will assist in the propagation of wildlife species.

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Animals 2021, 11(6), 1491; https://doi.org/10.3390/ani11061491
Received: 8 March 2021 / Revised: 11 May 2021 / Accepted: 17 May 2021 / Published: 21 May 2021

Morphometric and structural analysis of Florida manatee spermatozoa2021-04-23T11:41:08+02:00

Morphometric and structural analysis of Florida manatee spermatozoa

Jonathan R. Cowart, Danielle M. Collins, Daniel L. Stanton, Gerhard van der Horst, Iskande V. Larkin

Aquatic Animal Health Program, College of Veterinary Medicine, University of Florida, Gainesville, Florida; Department of Animal Sciences, College of Agricultural and Life Sciences, University of Florida, Gainesville, Florida; University of Florida Institute of Food and Agricultural Sciences, Citrus Research and Education Center, Lake Alfred, Florida; Department of Medical Bioscience, University of the Western Cape, Bellville, South Africa

Abstract: Sperm characteristics, such as sperm morphology and sperm morphometry are important in assessing sperm quality. This is especially important for the management and conservation of endangered and exotic species, like the Florida manatee, where information of this nature is extremely limited. In this study, we fill this knowledge gap to better understand the reproductive physiology of Florida manatees by conducting the first extensive analysis of sperm morphometry and ultrastructure. Sperm were retrieved from the vas deferens of nine recently deceased Florida manatees. Computer-aided sperm morphology analysis (CASMA) was used for morphometric analysis and laser-scanning confocal microscopy and electron microscopy were used for structural and ultrastructural characterization. Our findings reveal new morphometric and structural data for the Florida manatee spermatozoon. Twelve morphometric features of Florida manatee sperm were quantified with some approximately 1.5 –2 times larger than those previously reported. Ultrastructurally, the Florida manatee spermatozoon followed a mammalian structural pattern with an ovate-shaped head, midpiece containing 84 –90 mitochondria, and a flagellum. However, unique ultrastructural features were identified. Distinct, rectangular like enlargement of four outer dense fibers surrounding the axoneme was evident, which may provide additional tensile strength to counteract the forces on sperm transiting the female reproductive tract. Likewise, strong localization of F-actin fibers within the midpiece may function to maintain sperm integrity within the female reproductive tract. These findings highlight the potential effects of sexual selective pressures on sperm size and structure in the Florida manatee and provide avenues for research on the occurrence of sperm competition in this species.

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Anat Rec. 2021;1 –16. – DOI: https://doi.org/10.1002/ar.24645
Received: 16 February 2021; Revised: 24 March 2021; Accepted: 29 March 2021; First published: 23 April 2021

Comparison of commercially available chamber slides for computer-aided analysis of human sperm2020-12-29T16:05:43+01:00

Comparison of commercially available chamber slides for computer-aided analysis of human sperm

Fereshteh Dardmeh, Mahmoud Heidari, Hiva Alipour

Laboratory of Regenerative Medicine, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark; Department of Biology, Islamic Azad University, Gorgan Branch, Gorgan, Iran

Abstract: Despite the increasing use of computer-aided sperm analysis (CASA) in clinical practice, there is still no golden standard for the type of slide to be used with these systems. Counting chamber depth and loading method, can profoundly influence motility and concentration estimates, thereby undermining the validity and accuracy of CASA. To contribute toward standardized sperm analysis, this study compared different commercially available capillary-filled slides including 10 and 20 µm deep Leja slides (Leja10 and Leja20); 10, 16 and 20 µm deep CellVision slides (CV10, CV16 and CV20); and drop-loaded slides including slide and coverslip (SCS) with a depth of 20.1 µm and the Makler chamber with a depth of 10 µm for sperm analysis when using CASA. The Sperm Class Analyzer (SCA) CASA system was used to assess concentration, motility, and detailed kinematic parameters of 20 normozoospermic human samples using the different chamber slides. Results were evaluated by the repeated measures ANOVA and Intraclass correlation coefficients. The Makler chamber showed significantly (P < 0.05) higher concentrations than other slides. However, there was no significant difference in the percentage of sperm in different motility groups among the slides. CV10, Leja10 and Makler showed significantly higher curvilinear-, average path- and straight-line velocity (VCL, VAP, VSL) values than other slides. In conclusion, despite the objectiveness of the assessments by CASA systems, there are still some discrepancies in the results of sperm concentration, motility and other kinematic parameters when using different commercially available slides. The possible negative influence of the sperm quality misdiagnosis on the selection of treatment strategy in a clinical setting, emphasizes the need for further standardization and quality control of the commercially available chamber slides for use with CASA. Furthermore, this study found more consistent results for capillary-filled chambers compared to drop-loaded slides, suggesting a superior method when using CASA.

Systems Biology in Reproductive Medicine – https://doi.org/10.1080/19396368.2020.1850907
Received 10 Jun 2020, Accepted 21 Oct 2020, Published online: 29 Dec 2020

Seasonal changes in reproductive anatomy and gonadal hormone concentrations of African penguins (Spheniscus demersus)2021-01-25T12:58:48+01:00

Seasonal changes in reproductive anatomy and gonadal hormone concentrations of African penguins (Spheniscus demersus)

Patrick Siyambulela Mafunda, Liana Maree, Andre Ganswindt, Antoinette Kotze, Gerhard van der Horst

Department of Medical Bioscience, University of the Western Cape, Bellville, 7535, South Africa; National Zoological Garden, South African National Biodiversity Institute, Pretoria, 0001, South Africa; Mammal Research Institute, Department of Zoology and Entomology, University of Pretoria, Hatfield, 0028, South Africa; Genetics Department, University of the Free State, Bloemfontein, 9300, South Africa

Abstract: Several standard descriptions of the avian male and female reproductive tract have been reported, including effects of age, stage of reproductive maturity and gonadal hormone concentrations. Limited information on penguin reproductive biology and a lack of information on the African penguin (Spheniscus demersus) necessitated a detailed description of salient structural features of this species and provided an opportunity to evaluate seasonal changes in gonadal steroid hormone concentrations. Tissues from 36 males (adults and juveniles) and 29 females (adults and juveniles) were used for macro-anatomical descriptions and histology of the testes and ovaries. In addition, concentrations of gonadal steroid hormones for eight captive African penguins (four females and four males) were quantified during two breeding and one non-breeding season. The testes were asymmetrical, with the right testis having smaller dimensions compared to the left testis. Marked spermatogenic cellular associations and spermatid developmental stages were present in adult testes only during the breeding season. There was variation in the dimensions of the single ovary during follicular development related to the age and breeding status of the females. Testosterone, dihydrotestosterone, and estradiol concentrations fluctuated during the breeding and non-breeding periods, with males and females having similar steroid concentrations. The results from this study confirm that the breeding status in African penguins can be deduced based on testicular and ovarian histological structures. The results from the present study focused on African penguin reproductive biology should be considered in management strategies for the conservation of the species.

Animal Reproduction Science Volume 224, January 2021, 106664 – https://doi.org/10.1016/j.anireprosci.2020.106664
Received 6 July 2020, Revised 21 November 2020, Accepted 23 November 2020, Available online 26 November 2020.

Quantitative sperm characteristics of Tankwa goats with special reference to hyperactivated motility2020-11-17T15:07:27+01:00

Quantitative sperm characteristics of Tankwa goats with special reference to hyperactivated motility

A. Ngcauzele, G. van der Horst, A. Kotze, T. Jonker & L. Maree

Department of Medical Bioscience, University of the Western Cape, P/Bag X17, Bellville, 7535, South Africa; National Zoological Garden, South African National Biodiversity Institute, PO Box 754, Pretoria, 0001, South Africa; Genetics Department, University of the Free Sate, PO Box 339, Bloemfontein 9300, South Africa; Northern Cape Department of Agriculture, Land Reform and Rural Development, Private Bag X9, Jan Kempdorp, 8550, South Africa

Abstract: Computer-assisted semen analysis (CASA) is an automated and objective method of evaluating structural (e.g. morphology) and functional sperm parameters (e.g. motility and hyperactivation). Sperm hyperactivation is essential for successful fertilization and is thus an important aspect in determining the fertility potential of a male. In the current study, CASA was used for standard semen analysis and for comparison of the ability of phosphate buffered saline (PBS), BO sperm wash (10 mM caffeine), 4% lignocaine, and 5 mM procaine hydrochloride to induce hyperactivation in Tankwa goat spermatozoa. Twenty-nine ejaculates were collected from randomly selected male goats by electroejaculation. Although none of the four media affected percentage total sperm motility, lignocaine caused a significant decrease (P >0.05) in percentage progressive motility. Exposure to procaine resulted in an increase in swimming speed (P ≤0.05) and star-spin motility tracks, which are typical of sperm hyperactivation. Using PBS and procaine motility data from individually selected spermatozoa, receiver operator characteristic curves were constructed to distinguish the kinematic parameters employed as cut-off values for sperm hyperactivation. PBS and BO sperm wash did not induce hyperactivation (0.1 ± 0.2% and 0.04 ± 0.2% respectively), while lignocaine induced little hyperactivation (3.4 ± 3.0%) and procaine hydrochloride had the highest percentage hyperactivation (25.3 ± 13.6%). The large variation in hyperactivation (0–54.5%) may reflect inter-individual differences in sperm quality among these males. This study indicated procaine hydrochloride was the most promising hyperactivation-inducing medium for Tankwa goat spermatozoa and should be considered for similar assessments in other animal species.

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South African Journal of Animal Science 2020, 50 (No. 5) – DOI: http://dx.doi.org/10.4314/sajas.v50i5.6
Submitted 28 January 2020; Accepted 4 July 2020; Published 26 October 2020

Sperm DNA fragmentation is a novel biomarker for early pregnancy loss2021-01-27T15:36:49+01:00

Sperm DNA fragmentation is a novel biomarker for early pregnancy loss

Lesley Haddock, Stephen Gordon, Sheena E.M. Lewis, Peter Larsen, Amjad Shehata, Hassan Shehata

Examenlab Ltd, Unit 18A, Block K, Weavers Court Business Park, Linfield Road, Belfast BT12 5GH, UK; Urology at Epsom and St Helier University Hospitals NHS Trust, Dorking Rd, Epsom KT18 7EG, UK; Cryos International, Vesterbro Torv I, Aarhus 8000, Denmark; Centre for Reproductive Immunology and Pregnancy, Bramshott House, 137/139 High Street, Epsom KT19 8EH, UK; Maternal Medicine at Epsom and St Helier University Hospitals NHS Trust, Dorking Road, Epsom KT18 7EG, UK

Abstract:

Research question: Spontaneous pregnancy loss affects 10–15% of couples, with 1–2% suffering recurrent pregnancy loss and 50% of miscarriages remaining unexplained. Male genomic integrity is essential for healthy offspring, meaning sperm DNA quality may be important in maintaining a pregnancy. Does sperm DNA fragmentation measured by alkaline Comet assay act as a biomarker for early pregnancy loss?

Design: Sperm DNA fragmentation was measured by alkaline Comet test in 76 fertile donors and 217 men whose partners had recently experienced miscarriage. Couples were divided into five groups for analysis: one miscarriage after spontaneous conception; two or more miscarriages after spontaneous conception; one miscarriage after fertility treatment; two or more miscarriages after fertility treatment and biochemical pregnancy.

Results: Receiver operator characteristic curve analysis was used to determine ability of the average Comet score (ACS), low Comet score (LCS) and high Comet score (HCS) to diagnose miscarriage and develop clinical thresholds comparing men whose partners have miscarried with men with recently proven fertility. Male partners of women who had miscarried had higher sperm DNA damage (ACS 33.32 ± 0.57%) than fertile men (ACS 14.87 ± 0.66%; P < 0.001). Average Comet score, HCS and LCS all have promise as being highly predictive of sporadic and recurrent miscarriage using clinical thresholds from comparisons with fertile men’s spermatozoa: receiver operating characteristic curve AUC for ACS ≥26%, 0.965; LCS ≤70%, 0.969; HCS ≥2%, 0.883; P <0.0001.

Conclusions: Sperm DNA damage measured by the alkaline Comet has promise as a robust biomarker for sporadic and recurrent miscarriage after spontaneous or assisted conception, and may provide novel diagnoses and guidance for future fertility pathways.

Reproductive Biomedicine Online (RBMO) – DOI: https://doi.org/10.1016/j.rbmo.2020.09.016
Published: September 21, 2020

Determination of the freezing of hair rams (Pelibuey-Blackbelly) according to sperm viability and kinetics2020-08-21T12:19:03+02:00

Determination of the freezing of hair rams (Pelibuey-Blackbelly) according to sperm viability and kinetics

Julio Cesar Gómez Vargas, Rosendo Cuicas Huerta, Isidro Jáuregui Plata, Isidro Gutiérrez Segura, Raúl Ulloa Arvizu, David Urban Duarte, Efrén Estrada Paqui

Facultad de Medicina Veterinaria y Zootecnia, Universidad Autónoma de Guerrero. Guerrero, México; Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México. Ciudad de México, México; Centro Nacional de Recursos Genéticos, Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias. Jalisco, México.

Abstract: The objective of this study was to categorize the freezing of sperm of 11 rams (PelibueyBlackbelly) aged 18 to 24 months based on total motility and sperm viability at thawing. The semen was diluted to 37 °C with Triladyl® and egg yolk, packed in 0.5 mL straws at a sperm concentration of 200×106. After 15 minutes after defrosting at 37 °C, sperm motility and kinetics were evaluated by means of the CASA system, and fluorescence viability with kit FluoVit® (Microptic, S.L., Barcelona, Spain). The freezing categorization of ram ejaculates was 27.2% and 72.7% for rams with high freezing (CAC) (n= 3) and rams with low freezing (CBC) (n= 8) respectively. Ejaculates from CAC showed significant differences (p<0.05) in kinetic speed parameters (VCL, VSL and VAP), which suggests that these ejaculates are more suitable for cryopreservation and would have a greater chance of fertilizing due to their faster and more progressive movements.

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SPERMOVA – DOI: 10.18548/aspe/0008.04
Received: 26-06-2020 / Accepted: 20-07-2020 / Published: 21-08-2020

Microgravity effects on frozen human sperm samples2020-07-18T11:22:14+02:00

Microgravity effects on frozen human sperm samples

M. Boada, A. Perez-Poch, M. Ballester, S. García-Monclús, D. V. González, S. García, P. N. Barri & A. Veiga

Women’s Health Dexeus, Department of Obstetrics, Gynaecology and Reproduction, Hospital Universitari Dexeus, Avinguda Carles III 71-75, 08028, Barcelona, Spain; Universitat Politècnica de Catalunya, UPC BarcelonaTech, EEBE Campus Diagonal-Besòs, C. E. Maristany 16, 08019, Barcelona, Spain; Aeroclub Barcelona-Sabadell, Sabadell Airport, Carretera de Bellaterra s/n, 08205 Sabadell, Barcelona, Spain; Women’s Health Dexeus, Unit of Biostatistics, Avinguda Carles III 71-75, 08028, Barcelona, Spain; Barcelona Stem Cell Bank, Centre of Regenerative Medicine in Barcelona, Hospital Duran i Reynals, Gran Via de l’Hospitalet 199, 08908 Hospitalet de Llobregat, Barcelona, Spain.

Abstract:

Purpose: Microgravity has severe effects on cellular and molecular structures as well as on metabolic interactions. The aim of this study is to investigate the effects of microgravity (μg) exposure on human frozen sperm samples.

Methods: Sibling samples from 15 normozoospermic healthy donors were frozen using glycerol as cryoprotectant and analyzed under microgravity and ground conditions. Microgravity was obtained by parabolic flights using a CAP10B plane. The plane executed 20 parabolic maneuvers with a mean of 8.5 s of microgravity for each parabola.

Results: Frozen sperm samples preserved in cryostraws and stored in a secure and specific nitrogen vapor cryoshipper do not suffer significant alterations after μg exposure. Comparing the study group (μg) and the control group (1 g), similar results were obtained in the main parameters studied: sperm motility (M/ml) 13.72 ± 12.57 vs 13.03 ± 12.13 (− 0.69 95% CI [− 2.9; 1.52]), progressive a + b sperm motility (%) 21.83 ± 11.69 vs 22.54 ± 12.83 (0.03 95% CI [− 0.08; 0.15]), sperm vitality (%) 46.42 ± 10.81 vs 44.62 ± 9.34 (− 0.04 95% CI [− 0.13; 0.05]), morphologically normal spermatozoa (%) 7.03 ± 2.61 vs 8.09 ± 3.61 (0.12 95% CI [0.01; 0.24]), DNA sperm fragmentation by SCD (%) 13.33 ± 5.12 vs 13.88 ± 6.14 (0.03 95% CI [− 0.09; 0.16]), and apoptotic spermatozoa by MACS (%) 15.47 ± 15.04 vs 23.80 ± 23.63 (− 0.20 95% CI [− 0.66; 1.05]).

Conclusion: The lack of differences obtained between frozen samples exposed to μg and those maintained in ground conditions provides the possibility of considering the safe transport of human male gametes to space. Nevertheless, further research is needed to validate the results and to consider the possibility of creating a human sperm bank outside the Earth.

Journal of Assisted Reproduction and Genetics – DOI: 10.1007/s10815-020-01877-5
Received: 16 April 2020 / Accepted: 29 June 2020 / Published: 18 July 2020

Covariation in superoxide, sperm telomere length and sperm velocity in a polymorphic reptile2020-06-03T17:10:32+02:00

Covariation in superoxide, sperm telomere length and sperm velocity in a polymorphic reptile

Christopher R Friesen, Nicky Rollings, Mark Wilson, Camilla M Whittington, Richard Shine, Mats Olsson

School Earth, Atmospheric and Life Sciences, University of Wollongong, Wollongong, NSW, 2522, Australia; Illawarra Health and Medical Research Institute (IHMRI), University of Wollongong, Wollongong, NSW, 2522, Australia; School of Life and Environmental Sciences, University of Sydney, Heydon-Laurence Building (A08), Camperdown, NSW, 2006, Australia; Department of Biological Sciences, Macquarie University, Sydney, NSW, 2109, Australia;

Abstract: Telomeres are DNA-protein caps at the ends of chromosomes that have been shown to be associated with male fertility may be heritable, reflect environmental influences and predict life span in some taxa. If heritable, paternal telomere length would be transmitted via sperm in the form of sperm telomere length (STL). We, therefore, investigated STL, sperm number and velocity in the Australian-painted dragon lizard, Ctenophorus pictus, an agamid lizard with distinct male colour morphs and related reproductive tactics. We measured telomere length in the sperm and blood as well as superoxide levels, as a measure for the potential for oxidative stress and ejaculate quality. We also noted a male’s head colour (red, orange, yellow or blue) and whether or not they had a yellow gular bib. Previous research has reported that yellow males outcompete red males in sperm competition; we found that yellow males had significantly shorter STL than red males. Males with bibs had greater STL than did males without bibs. Superoxide levels measured in the blood were negatively correlated with STL. Whole blood TL and body length were weakly but positively correlated with STL. Superoxide measurements were negatively correlated with progressive sperm motility and straight line sperm velocity across all males. The ejaculates of males with bibs had lower sperm counts and velocity than males without bibs. Our research adds to the growing research that indicates the importance of considering both somatic and gametic telomeres when assessing the interaction between telomere dynamics, life history and reproductive strategies.

Behavioral Ecology and Sociobiology (2020) 74: 74 – https://doi.org/10.1007/s00265-020-02855-8
Received: 19 February 2020 / Revised: 19 May 2020 / Accepted: 22 May 2020 / Published online: 3 June 2020

Effect of N-Methylacetamide Concentration and Thawing Rate on Chicken Sperm Quality after Cryopreservation2020-05-09T16:08:10+02:00

Effect of N-Methylacetamide Concentration and Thawing Rate on Chicken Sperm Quality after Cryopreservation

Fabio Mosca, Luisa Zaniboni, Ahmad Abdel Sayed, Nicolaia Iaffaldano, Dominga Soglia, Achille Schiavone and Silvia Cerolini

Dipartimento di Medicina Veterinaria, University of Milan, via dell’Università 6, 26900 Lodi, Italy; Dipartimento di Agricoltura, Ambiente e Alimenti, University of Molise, via De Sanctis, 86100 Campobasso, Italy; Dipartimento di Scienze Veterinarie, University of Turin, Largo Paolo Braccini 2, 10095 Grugliasco, Torino, Italy

Abstract: In seeking alternative cryoprotectants to glycerol for a reference chicken semen freezing procedure, the aim of the present study was to compare the effect of two concentrations of N-Methylacetamide (MA) and two thawing rates on the quality of frozen-thawed semen. Semen samples were diluted in Lake pre-freezing extender, including 0.1 M trehalose in presence of 6% or 9% MA, loaded into straws, frozen in nitrogen vapors, and stored in liquid nitrogen. The following thawing treatments were used: 5 °C for 100 s and 38 °C for 30 s. Sperm quality (cell membrane integrity, motility and kinetic parameters) was assessed before and after cryopreservation. The decrease of MA concentration from 9 to 6% improved sperm quality after freezing/thawing and this effect was dependent on thawing temperature. Decreasing the MA concentration from 9 to 6% improved the proportion of undamaged membrane, motile, and progressive motile sperm recovered after thawing at 5 °C for 100 s; in contrast, no effect of the MA concentration was observed thawing at 38 °C for 30 s. Therefore, the treatment with 6% MA and thawing at 5 °C for 100 s has given the best cryoprotective action. These results contribute to improve the efficacy of the current chicken semen cryopreservation procedures.

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Animals 2020, 10(5), 824; https://doi.org/10.3390/ani10050824
Received: 6 April 2020 / Revised: 3 May 2020 / Accepted: 7 May 2020 / Published: 9 May 2020

Novelty and emergent patterns in sperm: morphological diversity and evolution of spermatozoa and sperm conjugation in ground beetles (Coleoptera: Carabidae)2020-05-03T12:27:02+02:00

Novelty and emergent patterns in sperm: morphological diversity and evolution of spermatozoa and sperm conjugation in ground beetles (Coleoptera: Carabidae)

R. Antonio Gomez, David R. Maddison

Department of Integra tive Biology, Oregon; State University , Corvallis, Oregon

Abstract: The beetle family Carabidae, with about 40,000 species, exhibits enough diversity in sperm structure and behavior to be an excellent model system for studying patterns and processes of sperm evolution. We explore their potential, documenting sperm form in 177 species of ground beetles and collecting data on 1 qualitative and 7 quantitative sperm phenotypic traits. Our sampling captures 61% of the tribal-level diversity of ground beetles. These data highlight the notable morphological diversity of sperm in ground beetles and suggest that sperm in the group have dynamic evolutionary histories with much morphological innovation and convergence. Sperm vary among species in total length from 48-3,400μm and in length and width of the sperm head. Most ground beetles make filamentous sperm with visually indistinct heads, but some or all studied members of the genus Omophron, genus Trachypachus, and tribe Dyschiriini make broad-headed sperm that show morphological differences between species. Most ground beetles package their sperm into groups of sperm, termed conjugates, and ground beetles show variation in conjugate form and in the number and arrangement of sperm in a conjugate. Most ground beetles make sperm conjugates by embedding their sperm in a non-cellular rod or spermatostyle, but some Trechitae make conjugates without a spermatostyle. The spermatostyle is remarkably variable among species and varies in length from 17-41,000μm. Several unrelated groups of ground beetles make only singleton sperm, including Nebriinae, Cicindelinae, many Trechinae, and the tribe Paussini. Given current views about ground beetle relationships, we propose preliminary hypotheses on ground beetle sperm diversification. We hypothesize that spermatostyle and conjugate traits evolve faster than sperm traits and that head width evolves more slowly than head length and sperm length. We propose that conjugation with a spermatostyle evolved early within the history of Carabidae and that it has been lost independently at least three times.

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Journal of Morphology – DOI: 10.1002/jmor.21144
Received: 17 October 2019 / Revised: 30 April 2020 / Accepted: 3 May 2020

Optimization of Sperm Cryopreservation Protocol for Peregrine Falcon (Falco peregrinus)2020-04-16T15:46:12+02:00

Optimization of Sperm Cryopreservation Protocol for Peregrine Falcon (Falco peregrinus)

Beatriz Cardoso, Irene Sánchez-Ajofrín, Cristina Castaño, Olga García-Álvarez, Milagros Cristina Esteso, Alejandro Maroto-Morales, María Iniesta-Cuerda, José Julián Garde, Julián Santiago-Moreno and Ana Josefa Soler

SaBio IREC (CSIC-UCLM-JCCM), ETSIAM, 02071 Albacete, Spain; Department of Animal Reproduction, INIA, 28040 Madrid, Spain

Abstract: Sperm cryopreservation is a complex process that needs to be adapted to wild and domestic avian species to ensure proper efficiency. Because of its accessibility, the peregrine falcon may be used as a good model for studying other raptor species. To find the most optimal cryopreservation protocol for peregrine falcon ejaculates, sperm parameters such as motility, viability, DNA fragmentation, acrosome integrity, and mitochondrial activity were analyzed under different conditions by varying the freezing method (slow freezing in straws vs. ultrarapid freezing in pellets), thawing conditions (37 °C for 30 s vs. 5 °C for 1 min), type of cryoprotectant (DMA vs. DMSO), and concentration of DMSO (4% vs. 8%). Results show that slow cryopreservation in straws yielded greater percentages (p < 0.05) of motile spermatozoa (22.5% ± 4.4% vs. 0.0% ± 4.1%), viable spermatozoa with intact acrosomes (84.6% ± 4.3% vs. 77.4% ± 4.3%), and spermatozoa with active mitochondria (41.0% ± 6.7% vs.12.8% ± 6.7%), compared with those obtained by the ultrarapid freezing in pellets. However, no differences were found between different thawing conditions. Moreover, all sperm motility parameters were greater (p < 0.05) when DMSO was used during freezing compared with DMA, although the use of 3% and 8% DMSO produced similar results. In conclusion, these results represent important progress in the study of falcon semen cryopreservation protocol, highlighting the crucial steps of the process and the most suitable conditions.

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Animals 2020, 10(4), 691; https://doi.org/10.3390/ani10040691
Received: 25 March 2020 / Revised: 9 April 2020 / Accepted: 14 April 2020 / Published: 16 April 2020

Computer Aided Sperm Analysis (CASA) in domestic animals: Current status, three D tracking and flagellar analysis2021-01-27T17:45:10+01:00

Computer Aided Sperm Analysis (CASA) in domestic animals: Current status, three D tracking and flagellar analysis

Gerhard van der Horst

Department of Medical Bioscience, Comparative Spermatology Laboratory, University of the Western Cape, South Africa

Abstract: Computer Aided Sperm Analysis is currently well established in domestic animals. Apart from sperm concentration and sperm motility assessment (percentage groupings, kinematics groupings) sperm morphology, sperm viability, sperm fragmentation and the acrosome reaction are automated as part of modern CASA systems. This review cum new original research paper focuses on providing baseline data on sperm concentration and motility in common domestic species of animals of proven fertility including bull, boar, horse, ram, goat, dog, donkey, chicken. There is a great need to establish quantitative baseline values for sperm quality, breed differences and to develop and apply relevant sperm functional tests that relates to fertilization outcome. These approaches need to be standardized. Two new approaches are presented in this work that are complimentary to CASA and provide a whole range of new visualizations and parameters that may assist to define sperm function and quality better. The first new approach shows how Two-D analysis using X and Y coordinates of CASA can be converted to Three-dimensional (3D) tracks. This method shows how sperm movement can be visualized in 3D despite several shortcomings. The second approach of flagellar analysis through the use of the FAST programme (Flagellar and Sperm Tracking) of the University of Birmingham group represents a new development and provides several new quantitative measures such as flagellar speed and energy output (in Watts) expended by each sperm. Together with CASA and other sperm functional parameters, FAST may provide new and novel insights in sperm biology and assist in fertility assessment.

Animal Reproduction Science – DOI: 10.1016/j.anireprosci.2020.106350
Received 7 December 2019; Received in revised form 14 March 2020; Accepted 16 March 2020

Effect of semen extenders on viability of ISA Brown and Hubbard Flex roosters’ sperm stored for 24 h2020-03-13T16:46:07+01:00

Effect of semen extenders on viability of ISA Brown and Hubbard Flex roosters’ sperm stored for 24 h

Ewa Łukaszewicz, Anna Jerysz and Artur Kowalczyk

Division of Poultry Breeding, Institute of Animal Breeding, Wroclaw University of Environmental and Life Sciences, Wrocław, Poland

Abstract: Artificial insemination is used in almost 95% of turkey reproductive flocks and is becoming more important in chickens, particularly broiler breeders, as well as in assisted reproduction of wild birds kept in breeding centers. Diluted semen is recommended for artificial insemination. Pooled semen samples collected twice a week by dorso-abdominal massage from 2 chicken lines: laying—ISA Brown (ISA-B) and meat type—Hubbard Flex (H-F) were divided into 5 parts: neat semen and diluted in 1:2 ratio with 4 extenders: basic EK; EK + 1 μg/mL organic selenium and 8 μg/mL vitamin E; EK + 10 mg/mL of royal jelly; and EK + 0.25 g/mL of lyophilized bovine colostrum. Diluted semen samples were evaluated after 15 min and then 24 h storage at 4°C. Sperm concentration, motility, motility parameters (with Sperm Class Analyzer), and morphology were evaluated in the neat semen, whereas in diluted and stored samples, the last 3 traits were determined. In case of both lines, dilution did not affect (P > 0.05) the number of live normal cells (78.0–81.1% in ISA Brown and 73.8–68.7% in Hubbard Flex) in relation to neat semen; however, bovine colostrum addition increased (P < 0.05) the percentage of bulb head sperm (5.7 vs. 10.0% and 12.1 vs. 17.6%, for ISA and Hubbard, respectively) and decreased sperm motility (67.4 vs. 92.9% and 67.3 vs. 98.5% for ISA and Hubbard). The 24 h storage of neat semen and semen diluted with colostrum caused (P < 0.05) the unfavorable changes in all evaluated traits and both chicken lines, whereas semen dilution with remaining extenders decreased the percentage of live normal cells (by 18.8–23.4% ISA and by 20.9–25.5% Hubbard) but did not affect sperm motility (81.5–87.6% for ISA and 81.1–96.6% for Hubbard). Sperm motility and motility parameters depended both on the extender and chicken line.

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Poultry Science – https://doi.org/10.1016/j.psj.2019.12.055
Revised 11 December 2019, Accepted 12 December 2019, Available online 13 March 2020

The potential role of central obesity in male infertility: body mass index versus waist to hip ratio as they relate to selected semen parameters2020-03-12T16:36:59+01:00

The potential role of central obesity in male infertility: body mass index versus waist to hip ratio as they relate to selected semen parameters

Márton Keszthelyi, V. Anna Gyarmathy, András Kaposi and Zsolt Kopa

Department of Urology, Andrology Centre, Semmelweis University, Üllői út 78/b, Budapest, 1082 Hungary; EpiConsult LLC, 8 The Green, STE A, Dover, DE 19904 USA; Johns Hopkins Bloomberg School of Public Health, Baltimore, 615 N Wolfe St, Baltimore, MD 21205 USA; Department of Biophysics and Radiation Biology, Semmelweis University, Tűzoltó u. 37-47, Budapest, 1094 Hungary; Department of Urology, Andrology Centre, Semmelweis University, Üllői út 78/b, Budapest, 1082 Hungary

Abstract:

Background: Little is known about the potential role of central obesity among men. Our first aim was to confirm what is already known from prior research, namely that both BMI and WHR are inversely associated with selected semen parameters. Our second aim was to examine the potential role of central obesity by assessing if there was a difference between BMI and WHR regarding their relationships to these selected semen parameters.
Methods: In this cross-sectional study between January 2011 to January 2018, we analyzed semen samples from 1169 patients who visited an andrology clinic in Budapest for infertility reasons. Variables assessed were: body measurements (height, weight, waist circumference, and hip circumference), and the results of semen analysis (sperm concentration, total sperm count, progressive sperm motility, and normal sperm morphology).
Results: The mean height and weight were 180.6 cm and 87.3 kg, respectively – the mean BMI was 26.8. The mean waist and hip circumferences were 100.9 cm and 94.8 cm, respectively – the mean waist to hip ratio was 0.94. The mean sperm concentration, total sperm count, and percents of progressive motility and normal morphology were 48.7 M/ml, 165 million, 21.2, and 4.8%, respectively. Both BMI and WHR were significant correlates in all semen parameter regression models. When comparing the parameter estimates for BMI with those for WHR for each semen parameter, the parameter estimate for WHR was significantly lower (indicating a stronger negative association) than that for BMI for progressive motility and total sperm count, but not for normal morphology or concentration.
Conclusions: Our study is the first to examine, using a large patient sample, the potential role of central obesity by comparing the difference between BMI and WHR as they relate to selected semen parameters. Our findings indicate a potential role of central obesity for progressive motility and total sperm count, but not for normal morphology and concentration. Despite the limitations and the exploratory nature of this study, we can conclude that our results point to a potential role of central obesity in male infertility, but this finding should be confirmed and further explored in future research.

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BMC Public Health. 2020; 20: 307. – doi: 10.1186/s12889-020-8413-6
Received: 10 August 2019 Accepted: 26 February 2020 Published online 2020 Mar 12

Does single‐layer centrifugation with Bovicoll improve sperm quality of frozen‐thawed semen in Fleckvieh bulls?2020-03-30T13:25:08+02:00

Does single‐layer centrifugation with Bovicoll improve sperm quality of frozen‐thawed semen in Fleckvieh bulls?

Joanna Szlendak, Christine Adler, Jakob Scherzer, Anna Niwinska, Ewa Kautz, Ricardo Faundez

Department of Large Animal Diseases with Clinic, Faculty of Veterinary Medicine, Warsaw University of Life Sciences WULS –
SGGW, Warsaw, Poland; Bayern-Genetik GmbH, Kumhausen, Germany

Abstract: The aim of the present study was to evaluate the effect of sperm selection by single‐layer centrifugation (SLC) performed before freezing on sperm quality after thawing of Fleckvieh bull semen. Ejaculates from 22 bulls were collected by artificial vagina and divided into two aliquots. One aliquot (control sample) was diluted with Steridyl® and frozen over nitrogen vapour in a Digitcool freezer (IMV Technologies). Sperm from the second aliquot (SLC sample) was selected using the SLC technique with Bovicoll colloid and then frozen over nitrogen vapour in a Digitcool freezer. After thawing, both samples (control and SLC) were evaluated by computer‐aided sperm analysis (CASA; SCA 6.4 System; Microptic S.L) for sperm motility parameters. Integrity of the plasma membrane (viability), high mitochondrial membrane potential (HMMP) and acrosome integrity were assessed using a Guava® easyCyte flow cytometer (IMV Technologies). Morphological examination of spermatozoa was performed by Differential Interference Contrast microscopy (Leica DMi8). Morphological examination of live, immobilized spermatozoa was analysed under high magnification (≥6,600×). After thawing, the mean sperm viability of the control sample was 51.57%, compared to 40.37% for the SLC sample (p < .01). HMMP was higher (p < .01) in the control sample (40.37% versus 28.96%), and the mean of live spermatozoa with damaged acrosome was significantly higher (p < .03) in the SLC sample (1.63% versus 1.95%). The mean percentage of motile spermatozoa was 80.17% in the control sample, compared to 75.14% in the SLC sample (p < .0195), and rapid subpopulation reduced from 20.08% to 8.99% (p < .0001) after SLC. Percentage of hyperactivated sperm decreased from 12.23% to 4.28% (p < .0001) after SLC. Given the overall results, the sperm quality of thawed Fleckvieh bull semen was not improved when sperm were selected by SLC before freezing.

Reproduction in Domestic Animals – https://doi.org/10.1111/rda.13666
Received: 11 November 2019 / Accepted: 27 February 2020 / First published: 04 March 2020

Finding an Effective Freezing Protocol for Turkey Semen: Benefits of Ficoll as Non-Permeant Cryoprotectant and 1:4 as Dilution Rate2020-03-03T16:01:13+01:00

Finding an Effective Freezing Protocol for Turkey Semen: Benefits of Ficoll as Non-Permeant Cryoprotectant and 1:4 as Dilution Rate

Michele Di Iorio, Giusy Rusco, Roberta Iampietro, Maria Antonietta Colonna, Luisa Zaniboni, Silvia Cerolini and Nicolaia Iaffaldano 1,*

Department of Agricultural, Environmental and Food Sciences, University of Molise, 86100 Campobasso CB, Italy; Department of Agricultural and Environmental Science, University of Bari Aldo Moro, 70126 Bari BA, Italy; Department of Veterinary Medicine, University of Milan, 20122 Milano MI, Italy

Abstract: The present study aimed to find an effective cryopreservation protocol for turkey semen through the combined use of dimethylsulfoxide (DMSO) and three non-permeant cryoprotectants (NP-CPAs), sucrose, trehalose, and Ficoll 70. In addition, the action of two dilution rates (1:2 and 1:4) were also investigated. Semen was processed according to two final dilution rates and the following treatments: Tselutin extender (TE)/DMSO (control), TE/DMSO + sucrose or trehalose 50, 100, 200, or 400 mM, and TE/DMSO + Ficoll 0.5, 0.75, 1, or 1.5 mM. In total 26 different combinations treatments were achieved. The diluted semen was filled up into straws and frozen on liquid nitrogen vapor. The post-thawing sperm quality was assessed by analyzing motility, membrane integrity, osmotic resistance, and DNA integrity. The results obtained revealed a significant effect of NP-CPA concentration on total and progressive motility, on most of the kinetic parameters, on membrane integrity and DNA integrity, while the post-thaw quality was less affected by dilution rate. The highest post-thaw quality for all sperm quality parameters assessed except curvilinear velocity (VCL) and DNA integrity were found in semen frozen with 1 mM Ficoll/1:4 (p < 0.05). Our findings provide an important contribution for the identification of a reference procedure for turkey semen cryopreservation, in order to create the first national avian semen cryobank.

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Animals 2020, 10(3), 421; https://doi.org/10.3390/ani10030421
Received: 18 January 2020 / Revised: 25 February 2020 / Accepted: 1 March 2020 / Published: 3 March 2020

A Simple and Efficient Semen Cryopreservation Method to Increase the Genetic Variability of Endangered Mediterranean Brown Trout Inhabiting Molise Rivers2020-02-29T15:35:11+01:00

A Simple and Efficient Semen Cryopreservation Method to Increase the Genetic Variability of Endangered Mediterranean Brown Trout Inhabiting Molise Rivers

Giusy Rusco, Michele Di Iorio, Roberta Iampietro, Stefano Esposito, Pier Paolo Gibertoni, Maurizio Penserini, Alessandra Roncarati and Nicolaia Iaffaldano

Department of Agricultural, Environmental and Food Sciences, University of Molise, 86100 Campobasso CB, Italy; Mediterranean Trout Research Group “MTRG”, 42037 Collagna RE, Italy; School of Biosciences and Veterinary Medicine, University of Camerino, 62032 Camerino MC, Italy

Abstract: The aim of our study was to test the effectiveness of a simple semen cryopreservation procedure, developed for cultivated salmonid, on the wild salmonid of the Mediterranean area and to evaluate the effect of different thawing rates and sperm-to-egg ratios. The semen of five individual males was diluted into a final extender concentration of 0.15 M glucose and 7.5% methanol and loaded into 0.25 mL plastic straws, and a final sperm concentration of 3.0 × 109 sperm/mL was obtained. After equilibration, the straws were frozen by exposure to liquid nitrogen vapor at 3 cm above the liquid nitrogen level for 5 min. The semen was thawed at 40 °C/5 s or 10 °C/30 s. The sperm cryosurvival was evaluated by examining in vitro the sperm motility parameters using the CASA system, followed by fertilization trials in vivo, using three different sperm-to-egg ratios 6 × 105, 4.5 × 105 and 3 × 105:1. The applied cryopreservation procedure resulted in remarkably high (85.6%) post-thaw sperm total motility, when the semen was thawed at 40 °C/5 s, whilst the highest fertilization rate (53.1%) was recorded for a sperm-to-egg ratio of 4.5 × 105:1. According to these outcomes, the cryopreservation procedure that was tested turned out to be effective for the wild population of Mediterranean brown trout and practical for the creation of the first European semen cryobank foreseen as part of our “LIFE” Nat.Sal.Mo. project.

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Animals 2020, 10(3), 403; https://doi.org/10.3390/ani10030403
Received: 3 February 2020 / Revised: 19 February 2020 / Accepted: 26 February 2020 / Published: 29 February 2020

The effect of royal jelly on boar sperm viability and motility during liquid storage for 96 hours2020-01-28T16:51:17+01:00

The effect of royal jelly on boar sperm viability and motility during liquid storage for 96 hours

Aiste Iljenkaite, Sigita Kerziene, Agila Dauksiene, Zoja Mikniene, Henrikas Zilinskas, Neringa Sutkeviciene

Lithuanian University of Health Sciences, Veterinary Academy, Kaunas, Lithuania

Abstract: The current study was carried out to investigate the protective effects of royal jelly supplementation on sperm motility, viability and pH value during the liquid storage of boar semen at 16 °C and 4 °C, at various periods of time (0, 24, 48, 72 and 96 h). Semen samples were collected from 11 boars, diluted with a long-term extender and supplemented with different concentration of royal jelly (0%, 0.5%, 1% and 2%) at a final concentration of 50 × 10 ⁶ sperm/ml. In the laboratory, the semen was assessed for sperm morphology, viability (eosin-nigrosin staining), subjective motility and objective sperm motility by sperm class analyzer. In total, 396 tests for sperm viability and motility were performed. The longer storage time and the lower incubation temperature showed lower sperm motility and viability results. The results showed that royal jelly supplementation at 1% concentrations protected the functionality of the sperm plasma membrane during the liquid storage time of 96 h at 16 °C. Sperm subjective and objective motility results in samples stored at 4 °C decreased with higher royal jelly concentrations and longer storage time, and differ significantly from the results in samples stored at 16 °C ( P < 0.05). Our data showed that royal jelly supplementation at lower concentrations can improve boar semen motility and viability parameters during liquid storage at 16 °C for 96 h.

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Acta Veterinaria Brno 89(1):47-53 – DOI: 10.2754/avb202089010047
Received November 7, 2019 Accepted January 28, 2020

Sperm motility, kinematics, morphometry and morphology over two seasons in free-ranging African elephants (Loxodonta africana)2020-02-10T16:59:52+01:00

Sperm motility, kinematics, morphometry and morphology over two seasons in free-ranging African elephants (Loxodonta africana)

Ilse Luther, Liana Maree, Antoinette Kotze, Thomas Hildebrandt, Frank Göritz, Robert Hermes, Gerhard van der Horst

Department of Medical Bioscience, University of the Western Cape, Private Bag X17, Bellville, 7535, South Africa; National Zoological Garden, South African National Biodiversity Institute, PO Box 754, Pretoria, 0001, South Africa; GEOsperm, Wildlife Reproduction and Biotechnology, PO Box 3300, Brits, 0250, South Africa; Department of Genetics, University of the Free State, PO Box 339, Bloemfontein, 9300, South Africa; Department of Reproduction Management, Leibniz Institute for Zoo and Wildlife Research, Alfred-Kowalke-Straße 17, 10315, Berlin, Germany

Abstract: This study aimed to address the lack of information on quantitative semen and sperm characteristics of free-ranging African elephants. Nineteen ejaculates were collected from 12 elephant bulls by means of electroejaculation in spring (Season 1, end of dry season, n=7) and in autumn (Season 2, end of rainy season, n=12). While most elephant cows are in oestrus in the rainy season, it is not evident whether sperm quality also improves during this period. Semen samples were assessed using computer-aided sperm analysis (CASA), brightfield microscopy and transmission electron microscopy. Seasonal differences and individual variation in sperm quality of bulls were apparent, with ejaculates collected during Season 2 revealing higher percentages for total motility, progressive motility, rapid-swimming spermatozoa and kinematic parameters compared with Season 1 (P<0.05). Although normal sperm morphology percentage was similar over the two seasons, more sperm tail defects were found in Season 2 (P<0.05). The baseline reference data and multivariate sperm parameter associations reported in this study can be used to predict elephant bull sperm quality and potential to fertilise. It is clear that CASA can detect subtle differences in sperm quality of African elephant ejaculates and should be the approach for future investigations.

Reproduction, Fertility and Development – https://doi.org/10.1071/RD19182
Received 21 September 2018, accepted 25 July 2019, published online 24 January 2020

The occurrence of spermatozoa with acrosome reaction in semen of boars depending on staining method and storage duration2019-12-31T17:20:52+01:00

The occurrence of spermatozoa with acrosome reaction in semen of boars depending on staining method and storage duration

Anna Wysokińska, Angelika Chłopik

Siedlce University of Natural Sciences and Humanities, Faculty of Agrobioengineering and Animal Sciences, Prusa 14, 08-110 Siedlce, Poland

Abstract: The aim of the present study was to analyze the frequency of occurrence of acrosome-reacted spermatozoa in semen stained with two methods, depending on boar semen storage time. The studies were conducted on ejaculates collected from 10 Landrace boars used in artificial insemination. The smears were prepared by means of two staining methods: SpermBlue and eosin-gentian dye. It was concluded that the proportion of acrosome-reacted spermatozoa depends on the staining method and ejaculate storage duration. When the smears were stained with the SpermBlue method, the number of spermatozoa with head defects, including acrosome reaction, was greater compared with the smears stained with the eosin-gentian dye. With the passage of semen storage time, a change in sperm count with an acrosome reaction was observed. In the SpermBlue-stained smears, the proportion of acrosome-reacted spermatozoa was the highest in the 96th hour of storage and was about 2.5%. An increase in the number of acrosome-reacted spermatozoa was observed in the semen stained with the eosin-gentian dye method in the following semen storage hours.

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Acta Sci. Pol. Zootechnica – DOI: 10.21005/asp.2019.18.4.07
Received: 25.10.2019 / Accepted: 31.12.2019

Age‐related declines in ejaculate quality and sperm kinematics vary among strains of Japanese Quail (Coturnix japonica)2019-11-06T12:51:55+01:00

Age‐related declines in ejaculate quality and sperm kinematics vary among strains of Japanese Quail (Coturnix japonica)

Umar Farooq, Irek A. Malecki, Misbah Mahmood and Graeme B. Martin

School of Agriculture and Environment, Faculty of Science, The University of Western Australia, Crawley, Perth, Australia; UWA Institute of Agriculture, The University of Western Australia, Crawley, Perth, Australia; Department of Poultry Science, University of Agriculture Faisalabad, Sub Campus Toba Tek Singh, Toba Tek Singh, Pakistan; Department of Animal Sciences, University of Stellenbosch, Matieland, South Africa; Department of Mathematics, Government College University Faisalabad, Faisalabad, Pakistan

Abstract: For successful breeding programs, it is important to quantify the useful period of a male’s reproductive life and it is often done simply by measurement of semen quality. This information is lacking for Japanese quail so we tested whether there is a decline in ejaculate quality and sperm kinematics with age, and whether the decline varies among strains. Nine males (n = 9) from each of 5 strains (A, B, C, D and E) were subjected to 4 semen collections (n = 16 per male) at 8, 16, 26 and 36 weeks of age. Ejaculate volume, sperm concentration and total sperm per ejaculate were measured, and sperm kinematics were analysed using a Sperm Class Analyser (SCA®). There was a significant effect of age for ejaculate volume, total sperm per ejaculate and per cent medium sperm. The effect of the interaction between age and strain was significant for percent progressive motile sperm, percent rapid sperm, velocity curvilinear, velocity straight line, velocity average path, linearity, straightness and beat cross frequency. Ejaculate volume peaked at Week 26 in all strains, while peak values for sperm concentration and total sperm per ejaculate were observed at Week 16 for most strains. There were declines in percent motile sperm, progressive motile sperm and rapid sperm, and in velocity curvilinear velocity, velocity straight line and velocity average path, by Week 16 for most strains. Linearity declined by Week 26 in some strains, and all strains showed a significant decline in beat cross frequency by that age. In conclusion, the ability of CASA to detect age‐related changes in sperm kinematics makes it a valuable tool for identifying the best males and thus improving quail flock fertility. It is essential that breeders understand that age affects both sperm production and sperm kinematics, and that the changes vary with strain.

Reproduction in Domestic Animals – https://doi.org/10.1111/rda.13585
Published: 6 November 2019

Chlorogenic acid improves the quality of boar semen processed in Percoll2019-10-24T16:14:46+02:00

Chlorogenic acid improves the quality of boar semen processed in Percoll

Stenia Severo Rabelo; Carla Oliveira Resende; Thais Preisser Pontelo; Bruna Resende Chaves; Bárbara Azevedo Pereira; William Eduardo da Silva; Juliano Vogas Peixoto; Luciano José Pereira; Márcio Gilberto Zangeronimo

Departamento de Medicina Veterinária, Universidade Federal de Lavras, Lavras, MG, Brasil; Departamento de Ciências da Saúde, Universidade Federal de Lavras, Lavras, MG, Brasil

Abstract: This study aimed to evaluate if the addition of chlorogenic acid (ChA) to semen extenders improves the quality of cooled boar semen processed in Percoll. The experimental design was randomized blocks (ejaculates) in a 2×3 factorial (with or without Percoll, and three antioxidant systems: a negative control, without supplementation, a positive control – vitamin E, and ChA), totaling six treatments and 12 repetitions. ChA and vitamin E (VE) were added at 4.5 mg/ml and 400 μg/ml in extender, respectively. At 0, 48 and 72h of storage at 15ºC, 80 ml insemination doses each containing 2.0 billion sperm cells were submitted to centrifugation in Percoll. The use of Percoll impaired (P<0.01) all motility patterns but decreased (P<0.01) the number of abnormal cells at 0, 48 and 72h of storage. Both VE and ChA improved (P<0.05) the total motility after Percoll processing, but only in semen stored for 48h. The same effect was not observed (P>0.05) in semen stored for 72h. ChA improved (P<0.05) the total motility of the semen stored for 72h, but this effect was not observed (P>0.05) when the semen was processed in Percoll. The antioxidants had no effect (P>0.05) on the viability and integrity of the acrosome, but ChA reduced (P<0.05) the number of abnormal cells at 0h, while VE increased the number of abnormal cells in semen stored for 72h, independent of the use of Percoll. There was no effect (P>0.05) of antioxidants or Percoll on the concentration of malondialdehyde in seminal plasma. The use of Percoll had no effect (P>0.05) on the cholesterol efflux, but ChA increased (P<0.05) this parameter at 0h and reduced (P<0.05) in the semen stored for 72h not processed with Percoll. In conclusion, the addition of ChA to semen extenders improved the quality of boar semen processed or not in Percoll.

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Anim Reprod, vol.17, n1, e20190021, 2020 – http://dx.doi.org/10.21451/1984-3143-AR2019-0021
Received: February 27, 2019. Accepted: October 24, 2019

Testicular structure and spermatogenesis in the Naked Mole-Rat is unique (degenerate) and atypical compared to other mammals2022-05-04T15:57:38+02:00

Testicular structure and spermatogenesis in the Naked Mole-Rat is unique (degenerate) and atypical compared to other mammals

Gerhard van der Horst, Sanet H. Kotzé, M. Justin O’Riain and Liana Maree

Department of Medical Biosciences, University of the Western Cape, Bellville, South Africa; Division of Clinical Anatomy, Department of Biomedical Sciences, Stellenbosch University, Stellenbosch, South Africa; Institute for Communities and Wildlife in Africa, Department of Biological Sciences, University of Cape Town, Cape Town, South Africa

Abstract: The naked mole-rat (NMR) queen controls reproduction in her eusocial colony by usually selecting one male for reproduction and suppressing gametogenesis in all other males and females. Simplified, polymorphic and slow-swimming spermatozoa in the NMR seem to have been shaped by a low risk of sperm competition. We hypothesize that this unique mammalian social organization has had a dramatic influence on testicular features and spermatogenesis in the NMR. The testicular structure as well as spermatogenic cell types and its organization in breeding, subordinate and disperser males were studied using light microscopy and transmission electron microscopy. Even though the basic testicular design in NMRs is similar to most Afrotheria as well as some rodents with intra-abdominal testes, the Sertoli and spermatogenic cells have many atypical mammalian features. Seminiferous tubules are distended and contain large volumes of fluid while interstitial tissue cover about 50% of the testicular surface area and is mainly composed of Leydig cells. The Sertoli cell cytoplasm contains an extensive network of membranes and a variety of fluid-containing vesicles. Furthermore, Sertoli cells form numerous phagosomes that often appear as extensive accumulations of myelin. Another unusual feature of mature NMR Sertoli cells is mitotic division. While the main types of spermatogonia and spermatocytes are clearly identifiable, these cells are poorly organized and many spermatids without typical intercellular bridges are present. Spermatid heads appear to be malformed with disorganized chromatin, nuclear fragmentation and an ill-defined acrosome formed from star-like Golgi bodies. Rudimentary manchette development corresponds with the occurrence of abnormal sperm morphology. We also hypothesize that NMR testicular organization and spermiation are modified to produce spermatozoa on demand in a short period of time and subsequently use a Sertoli cell “pump” to flush the spermatozoa into short tubuli recti and simplified rete testis. Despite the difficulty in finding cellular associations during spermatogenesis, six spermatogenic stages could be described in the NMR. These numerous atypical and often simplified features of the NMR further supports the notion of degenerative orthogenesis that was selected for due to the absence of sperm competition.

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Frontiers in Cell and Developmental Biology – doi: 10.3389/fcell.2019.00234
Received 07 June 2019, Accepted 30 September 2019, Published: 16 October 2019.

Multigenerational exposure to uranium changes morphometric parameters and global DNA methylation in rat sperm2022-05-04T15:58:10+02:00

Multigenerational exposure to uranium changes morphometric parameters and global DNA methylation in rat sperm

Audrey Legendre, Ghada Elmhiri, Céline Gloaguen, Victor Magneron, Dimitri Kereselidze, Nawel Saci, Christelle Elie, Élodie Vaysset, Mohamedamine M. Benadjaoud, Karine Tack, Stéphane Grison, Maamar Souidi

Institut de radioprotection et de sûreté nucléaire (IRSN), PSE-SANTE, 92262 Fontenay-aux-Roses, France

Abstract: There is increasing evidence that environmental exposures early in fetal development influence phenotype and give rise to disease risk in the next generations. We previously found that lifelong exposure to uranium, an environmental contaminant, induced subtle testicular and hormonal defects; however, its impact on the reproductive system of multiple subsequent generations was unexplored. Herein, rats were exposed to a supra-environmental and non-nephrotoxic concentration of natural uranium (U, 40 mg·L−1 of drinking water) from postnatal life to adulthood (F0), during fetal life (F1), and only as the germ cells from the F1 generation (F2). General parameters (reproductive indices, epididymal weight) and sperm morphology were assessed in the three generations. In order to identify the epigenetic effects of U, we analyzed also the global DNA methylation profile and described for the first time the mRNA expression levels of markers involved in the (de)methylation system in rat epididymal spermatozoa. Our results showed that the F1 generation had a reduced pregnancy rate. Despite the sperm number being unmodified, sperm morphology was affected in the F0, F1 and F2 generations. Morphometric analysis for ten parameters was detailed for each generation. No common parameter was detected between the three generations, but the head and the middle-piece were always modified in the abnormal sperms. In the F1 U-exposed generation, the total number of abnormal sperm was significantly higher than in the F0 and F2 generations, suggesting that fetal exposure to uranium was more deleterious. This effect could be associated with the pregnancy rate to produce the F2 generation. Interestingly, global DNA methylation analysis showed also hypomethylation in the sperm DNA of the last F2 generation. In conclusion, our study demonstrates that uranium can induce morphological sperm defects and changes in the DNA methylation level after multigenerational exposure. The epigenetic transgenerational inheritance of U-induced reproductive defects should be assessed in further experiments.

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Comptes Rendus Biologies – https://doi.org/10.1016/j.crvi.2019.07.002
Received 22 November 2018, Accepted 19 July 2019, Available online 27 August 2019.

Prevalence of genital Mycoplasma and response to eradication treatment in patients undergoing assisted reproductive techniques2019-07-04T12:56:24+02:00

Prevalence of genital Mycoplasma and response to eradication treatment in patients undergoing assisted reproductive techniques

Ernesto Veiga, Mercedes Treviño, Ana Belén Romay, Daniel Navarro, Rocío Trastoy, Manuel Macía

Human Assisted Reproduction Unit. University Clinical Hospital of Santiago de Compostela, Spain; Clinical Microbiology Unit. University Clinical Hospital of Santiago de Compostela, Spain

Abstract: 

OBJECTIVE: Several studies have reported greater success of fertilisation by ART in couples who were not infected by Ureaplasma. Increased semen quality and better results have also been observed in couples who were treated with antibiotics to eradicate the infection. The aim of this study was to determine the prevalence of genital mycoplasmas in urine samples from male partners enrolled in the Assisted Reproduction Program (ARP) in our healthcare area so that, positive cases can be treated prior to the use of ART in order to increase the quality of semen, improve the embryo implantation rates and minimize the risk of adverse effects during pregnancy.

METHODS: This study included couples enrolled in the ARP during 2016. Mycoplasma detection was made using real-time PCR. In positive cases, both members of the couple were treated with antibiotics until eradication of the microorganism. The antibiotics used were: azithromycin, doxycycline, levofloxacin, moxifloxacin, and clindamycin.

RESULTS: Of the 205 men studied, 33 were positive: Ureaplasma urealyticum 15.1%, Mycoplasma hominis 3.9%. Eradication treatment with azithromycin failed in 50% compared to 10.2% for doxycycline. Of the 5 cases treated with levofloxacin, only 2 achieved elimination of U. urealyticum.

CONCLUSIONS: We consider that genital mycoplasma routine screening could be useful in order to increase the quality of semen which could simplify the in vitro fertilisation procedures and raise the success rate of embryo implantation and pregnancy, especially when fast, sensitive and specific technics as real time PCR are used.

Rev Esp Quimioter.
Received: 17 March 2019; Revision Requested: 14 April 2019; Revision Received: 19 April 2019; Accepted: 14 May 2019

Rosmarinus officinalis Essential Oil Preloaded in β-Cyclodextrin: Effect on Ram Spermatozoa Motility, Membrane Integrity and Oxidative Status During 4°C Storage2019-12-10T16:58:43+01:00

Rosmarinus officinalis Essential Oil Preloaded in β-Cyclodextrin: Effect on Ram Spermatozoa Motility, Membrane Integrity and Oxidative Status During 4°C Storage

Benberkane A, Khellouf A, Benhenia K, Fatmi S, Iguer-Ouada M.

Associated Laboratory in Marine Ecosystems and Aquaculture, Department of Biological Sciences of the Environment, Faculty of Nature and Life Sciences, Abderrahmane Mira University, Route de Targua Ouzemmour, Bejaia Algeria; Associated Laboratory in Marine Ecosystems and Aquaculture, Department of Biological Sciences of the Environment, Faculty of Nature and Life Sciences, Abderrahmane Mira University, Route de Targua Ouzemmour, 06000 Bejaia Algeria; National Center for Biotechnology Research (CRBt), Ali Mendjli Nouvelle Ville UV 03 BP E73, Constantine, Algeria; Pharmaceutical Laboratory, Department of Engineering Processes, Abderrahmane Mira University, Route de Targua Ouzemmour, Bejaia, Algeria; Associated Laboratory in Marine Ecosystems and Aquaculture, Department of Biological Sciences of the Environment, Faculty of Nature and Life Sciences, Abderrahmane Mira University, Route de Targua Ouzemmour, Bejaia Algeria

Abstract: 

BACKGROUND:
Rosmarinus officinalis essential oil (Rom) has been reported recently to be of interest for use in sperm cryopreservation. However, related to its lipophilic characteristics, encapsulation in cyclodextrin could enhance Rom positive effects by increasing its solubility in sperm extenders.

OBJECTIVE: To compare the effect of Rom preloaded in hydroxypropyl-β-cyclodextrin (Rom-cd) to Rom alone (Rom) on ram sperm conserved at 4°C.

MATERIALS AND METHODS: Ram epididymal sperm was collected from six testes. Each collected sperm was split into four equal aliquots. The control aliquot was diluted with Tris extender (Tris + citric acid + fructose + penicillin), two aliquots were treated with Rom at 0.5µl ml-1 and 1µl ml-1 respectively, and one aliquot treated with Rom-cd at 1µl ml-1. All sperm aliquots were analyzed for motility after 0, 2, 4, 24 and 48 h of storage at 4°C using a Computer Aided Semen Analysis (CASA). Membrane integrity and oxidative stress status were measured after 48 h of storage.

RESULTS: The results indicated that motility parameters were best preserved in the extender containing Rom-cd compared to the groups treated by Rom without cyclodextrin. Rom alone resulted to higher sperm motility than the control group. Lower oxidative stress and more cell membrane protection were observed in Rom treated samples, especially when using Rom-cd.

CONCLUSION: The ability of Rom to protect ram sperm against cryopreservation damages was improved after encapsulation in hydroxypropyl-β-cyclodextrin (HPβCD).

Cryo Letters
Published: July 2019

The importance of insect sperm: Sperm ultrastructure of Hermetia illucens (black soldier fly)2019-09-03T13:11:54+02:00

The importance of insect sperm: Sperm ultrastructure of Hermetia illucens (black soldier fly)

Retha C.M. Kotzé, Nolan Muller, Lizette du Plessis, Gerhard van der Horst

Department of Medical Bioscience, University of the Western Cape, Private Bag X17, Bellville, 7535, South Africa; National Health Laboratory Service, Anatomical Pathology, Tygerberg Hospital, Parow, 7505, South Africa; Electron Microscope Unit, Department of Anatomy and Physiology, University of Pretoria, Onderstepoort, 0110, South Africa

Abstract: Sperm structure and ultrastructure of Hermetia illucens was determined by light microscopy and transmission electron microscopy. The main sperm components were similar as for other Dipteran subspecies, while the ultrastructure revealed distinguishing features in the zone of overlap and anterior flagellar region. Sperm varied in size indicating sperm polymorphism. The head region is lacking an acrosome. The zone of overlap consisted of uniquely organized centriolar adjunct material, partly forming electron dense areas to finally form an outer ring separating the mitochondrial derivatives from the 9 + 9 + 2 axoneme. Accessory bodies arising from the zone of overlap are flanked by smaller to large mitochondrial derivatives into the anterior flagellum. This study confirms sperm structure diversity between brachyceran subspecies and support its relationship with nematoceran subspecies.

Tissue and Cell – https://doi.org/10.1016/j.tice.2019.06.002
Received 21 January 2019, Revised 18 June 2019, Accepted 21 June 2019, Available online 22 June 2019.

Computer‐aided sperm analysis, the new key player in routine sperm assessment2019-06-03T15:23:29+02:00

Computer‐aided sperm analysis, the new key player in routine sperm assessment

Benoit Schubert, Mélanie Badiou, André Force

Eurofins Biomnis – Institut Rhonalpin IVF Center, Clinique du Val d’Ouest – Medicentre, Ecully, France

Abstract: For sperm analysis, important inter-laboratory variations have been observed in manual analyses. In this study, a computer-aided sperm analysis (CASA) system was assessed versus manual technique, and specific software modifications were operated to fit the David’s classification already used in the laboratory. Four parameters were studied (concentration, motility, vitality and morphology), and at least 30 semen samples from 30 different patients have been tested. Manual and automated analyses were compared using a least-squares regression line analysis, Student’s t test, Bland-Altman plots and Passing-Bablok regressions. Repeatability was also assessed, and coefficients of variation (CV) were calculated. Both manual and automated methods gave similar results for sperm concentration (n = 150), motility (n = 30), vitality (n = 90) and morphology (n = 90). Repeatability always showed a decrease in the CV with automated analysis; for example in normal range of sperm values, CV for manual and CASA analyses were, respectively, 9.0% versus 4.4% for sperm concentration, 5.2% versus 4.1% for motility, 7.3% versus 4.2% for vitality and 11.4% versus 4.1% for morphology. All parameters were comparable between automated and manual analysis, and repeatability measures confirm the more reliable values of the SCA compared to those of manual analysis.

Andrologia – https://doi.org/10.1111/and.13417
Received 8 October 2018, Revised 17 May 2019, Accepted 3 June 2019

Evaluation of black glutinous rice (Oryza sativa L) extract as a novel nuclear stain for human sperm head assessment by microscopic examination2019-06-07T13:02:27+02:00

Evaluation of black glutinous rice (Oryza sativa L) extract as a novel nuclear stain for human sperm head assessment by microscopic examination

Sirinart Chomean, Tanawan Sukanto, Arreya Piemsup, Jirattikan Chaiya, Kolunya Saenguthai, Chollanot Kaset

Department of Medical Technology, Faculty of Allied Health Sciences, Thammasat University, Pathumthani; Thammasat Infertility Center and Medical laboratory, Thammasat University Hospital, Thammasat University, Pathumthani, Thailand

Abstract: 

Objective: To compare black rice (Oryza sativa L) extract with three different staining methods for human sperm head assessment.
Methods: Semen samples were collected from 34 volunteers. Four smears of each ejaculate were prepared for staining using the rapid Papanicolaou (PAP) stain, SpermBlue, DipQuick, and black rice extract. The percentage of defective sperm heads (mean±standard deviation) was compared.
Results: Black glutinous rice extract, a natural dye, was used instead of hematoxylin to stain the nuclei of the sperm heads. The percentage of defective sperm heads showed a significant difference between black rice extract and DipQuick (p=0.000). In contrast, black rice extract and rapid PAP showed no statistically significant difference (p=0.974). A strong correlation (r= 0.761) was found between the findings obtained using rapid PAP and black rice extract. In contrast, a weak correlation (r=0.248) was obtained between DipQuick and black rice extract for the percentage of defective sperm heads.
Conclusion: The results showed good agreement and a strong correlation between the rapid PAP and black rice extract stains. The advantages of black rice extract as a novel substitute for hematoxylin for nuclear staining include ease of preparation, local availability, and favorable nuclear staining properties. Further studies could also focus on comparing staining techniques in clinical samples

Full article here

Clinical and Experimental Reproductive Medicine – https://doi.org/10.5653/cerm.2019.46.2.60
Received: Feb 20, 2019 ∙ Revised: Apr 2, 2019 ∙ Accepted: Apr 2, 2019 ∙ Published: June 2019

Semen quality of young men in Switzerland: a nationwide cross‐sectional population‐based study2019-05-21T09:40:20+02:00

Semen quality of young men in Switzerland: a nationwide cross‐sectional population‐based study

R. Rahban, L. Priskorn, A. Senn, E. Stettler, F. Galli, J. Vargas, M. Van den Bergh, A. Fusconi, R. Garlantezec, T. K. Jensen, L. Multigner, N. E. Skakkebæk, M. Germond, N. Jørgensen, S. Nef

Department of Genetic Medicine and Development, University of Geneva, Geneva, Switzerland; Department of Growth and Reproduction, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark; Swiss Armed Forces Joint Staff, Medical Services, Ittigen, Switzerland; National Institute for Cancer Epidemiology and Registration (NICER), Zurich, Switzerland; Centre de Procreation Medicalement Assist ee SA, Fertas SA et Fondation FA BER, Lausanne, Switzerland; Kinderwunschzentrum, Kantonspital Baden, Baden, Switzerland; Centro Cantonale di Fertilita, Ospedale di Locarno La Carita, Locarno, Switzerland;  and Inserm, EHESP, Irset (Institut de recherche en sante, environnement et travail) – UMR_S 1085, Universite de Rennes, Rennes, France

Abstract: 

Background: Sperm counts have been steadily decreasing over the past five decades with regional differences in the Western world. The reasons behind these trends are complex, but numerous insights indicate that environmental and lifestyle factors are important players.
Objective: To evaluate semen quality and male reproductive health in Switzerland.
Materials and methods: A nationwide cross‐sectional study was conducted on 2523 young men coming from all regions of Switzerland, recruited during military conscription. Semen volume, sperm concentration, motility, and morphology were analyzed. Anatomy of the genital area and testicular volume was recorded. Testicular cancer incidence rates in the general population were retrieved from Swiss regional registries.
Results: Median sperm concentration adjusted for period of sexual abstinence was 48 million/mL. Comparing with the 5th percentile of the WHO reference values for fertile men, 17% of men had sperm concentration below 15 million/mL, 25% had less than 40% motile spermatozoa, and 43% had less than 4% normal forms. Disparities in semen quality among geographic regions, urbanization rates, and linguistic areas were limited. A larger proportion of men with poor semen quality had been exposed in utero to maternal smoking. Furthermore, testicular cancer incidence rates in the Swiss general population increased significantly between 1980 and 2014.
Discussion: For the first time, a systematic sampling among young men has confirmed that semen quality is affected on a national level. The median sperm concentration measured is among the lowest observed in Europe. No specific geographical differences could be identified. Further studies are needed to determine to what extent the fertility of Swiss men is compromised and to evaluate the impact of environmental and lifestyle factors.
Conclusion: A significant proportion of Swiss young men display suboptimal semen quality with only 38% having sperm concentration, motility, and morphology values that met WHO semen reference criteria.

Full article here

ANDROLOGY – https://doi.org/10.1111/andr.12645
First published: 21 May 2019

Can the Sperm Class Analyser (SCA) CASA-Mot system for human sperm motility analysis reduce imprecision and operator subjectivity and improve semen analysis?2019-05-06T11:07:20+02:00

Can the Sperm Class Analyser (SCA) CASA-Mot system for human sperm motility analysis reduce imprecision and operator subjectivity and improve semen analysis?

Chey Dearing, Channa Jayasena and Kevin Lindsay

School of Health & Sport Science and School of Nursing, Eastern Institute of Technology, Taradale Campus, Hawkes Bay, New Zealand; Andrology Laboratory, Hammersmith Hospital, Imperial College NHS Trust, London, UK; Andrology Laboratory, Hammersmith Hospital, Imperial College NHS Trust, London, UK

Abstract: Semen analysis (SA) is considered mandatory for suspected male infertility although its clinical value has recently become questionable. Sperm motility is an essential parameter for SA, but is limited by high measurement uncertainty, which includes operator subjectivity. Computer-assisted sperm analysis (CASA) can reduce measurement uncertainty compared with manual SA. The objective of this study was to determine whether the Sperm Class Analyser (SCA) CASA-Mot system could reduce specific components of sperm motility measurement uncertainty compared with the World Health Organization (WHO) manual method in a single laboratory undertaking routine diagnostic SA. The study examined: (i) operator subjectivity; (ii) precision, (iii) accuracy against internal and external quality standards; and (iv) a pilot sub-study examining the potential to predict an IVF fertilisation rate. Compared with the manual WHO method of SA on 4000 semen samples, SCA reduces but does not completely eliminate operator subjectivity. Study SCA and CASA-Mot are useful tools for well-trained staff that allow rapid, high-number sperm motility categorization with less analytical variance than the manual equivalent. Our initial data suggest that SCA motility may have superior predictive potential compared with the WHO manual method for predicating IVF fertilization.

Human Fertility – https://doi.org/10.1080/14647273.2019.1610581
Received 13 Dec 2018, Accepted 21 Mar 2019, Published online: 06 May 2019

Humanin levels in human seminal plasma and spermatozoa are related to sperm quality2019-03-28T16:59:37+01:00

Humanin levels in human seminal plasma and spermatozoa are related to sperm quality

M. Rao, Z. Wu, Y. Wen, R. Wang, S. Zhao, L. Tang

Department of Reproduction and Genetics, The First Affiliated Hospital of Kunming Medical University, Kunming, China

Abstract: 

Background: Humanin has reportedly been expressed in testis and spermatozoa, but no study has yet reported its presence in human seminal plasma (SP).

Objective: The aim of this study was to investigate the presence of humanin in human SP and to determine the correlation between humanin levels in SP/spermatozoa and sperm quality.

Materials and methods: Semen samples for SP/sperm humanin level measurement were collected from 164 patients who attended our andrology clinic for fertility evaluation. The localization of humanin in spermatozoa was evaluated using an immunofluorescence method, and SP/sperm humanin levels were measured with ELISA. Correlations between SP/sperm humanin levels and sperm parameters were analyzed.

Results: Humanin was expressed in the midpiece of the spermatozoa. Humanin concentrations in the SP ranged from 24.4 to 285.1 pg/mL, with a median of 89.7 pg/mL. The SP humanin concentrations in patients with normospermia were significantly higher than those in patients with oligospermia (p < 0.001), asthenospermia (p = 0.002), and oligoasthenospermia (p < 0.001). Spearman analysis showed a positive and significant correlation between SP humanin concentration and sperm concentration (r = 0.75, p < 0.001), and progressive sperm motility (r = 0.29, p < 0.001). Sperm humanin level was significantly and positively associated with progressive sperm motility (r = 0.70, p < 0.001). In addition, a significantly higher level of humanin was found in swim‐up spermatozoa than in non‐swim‐up spermatozoa (p = 0.03).

Conclusions: Seminal plasma and sperm humanin levels were significantly and positively correlated with sperm quality, especially sperm motility. Further studies of the origin of SP humanin and its role in spermatogenesis should be conducted.

Andrology – https://doi.org/10.1111/andr.12614
Received: 27-Aug-2018 / Revised: 26-Feb-20 19 / Accepted: 28-Feb-2019 / First published: 28 March 2019

Improved sperm motility after 4 h of ejaculatory abstinence: role of accessory sex gland secretions2019-03-26T16:49:05+01:00

Improved sperm motility after 4 h of ejaculatory abstinence: role of accessory sex gland secretions

Dale Goss, Bashir Ayad, Gerhard van der Horst, Bongekile Skosana and Stefan S. du Plessis

Division of Medical Physiology, Stellenbosch University, Private Bag X1, Matieland, 7602, Stellenbosch, South Africa

Abstract: Various studies have sought to determine the typical v. optimal abstinence period after which semen samples should be collected, with many contradictory results reported. Several factors influence the semen microenvironment, and thus sperm parameters. In this study we focused on the secretions of the prostate, seminal vesicles and the epididymis. Semen samples were obtained from healthy normozoospermic males (n = 16) after 4-day and 4-h periods of ejaculatory abstinence, and standard semen analysis was performed using computer-aided sperm analysis, whereas seminal plasma citric acid, neutral α-glucosidase and fructose concentrations were measured using assay kits. There were significant decreases in total sperm count (P < 0.001), sperm concentration (P < 0.05) and semen volume (P < 0.05) after 4 h compared with 4 days ejaculatory abstinence. Furthermore, increases were observed in total sperm motility (P < 0.05) and sperm progressive motility (P < 0.01) after a 4-h abstinence period, accompanied by significant reductions in citric acid (P < 0.05), α-glucosidase (P < 0.01) and fructose (P < 0.01) concentrations. In addition, due to the decreased number of spermatozoa, these concentrations translated to a significant decrease in fructose (P < 0.05) per spermatozoon, indicating an intrinsic mechanism capitalising on alternative sources of energy for increased metabolic function and subsequent sperm motility.

Reproduction, Fertility and Development – https://doi.org/10.1071/RD18135
Submitted: 11 April 2018  Accepted: 3 January 2019   Published online: 26 March 2019

The effect of the staining technique on morphological and morphometric parameters of boar sperm2019-03-25T09:43:57+01:00

The effect of the staining technique on morphological and morphometric parameters of boar sperm

Magdalena Czubaszek, Katarzyna Andraszek, Dorota Banaszewska, Renata Walczak-Jędrzejowska

Department of Animal Genetics and Horse Breeding, Siedlce University of Natural Sciences and Humanities, Siedlce, Poland; Department of Breeding Methods and Poultry Breeding, Siedlce University of Natural Sciences and Humanities, Siedlce, Poland; Department of Andrology and Repoductive Endocrinology, Medical University of Łodz, Łodz, Poland

Abstract: Sperm morphology and morphometry are important parameters in predicting fertility. Sperm are considered to be normal if the shape and size of the head, midpiece and tail fall within the classification for a given species. It is important to select the appropriate technique for staining the semen of a given species, because, as many authors have pointed out, some methods work well for one species but are not suitable for analysing another. The aim of the study was to assess the morphometric parameters of boar sperm following the use of different staining techniques and to verify the hypothesis that the staining technique affects the morphometric parameters of sperm. The staining method was found to significantly affect the dimensions of the boar sperm head. The semen stained by the SpermBlue technique had the closest morphometric sperm head parameters to those of the unstained sperm, so this technique, rather than the routinely used eosin and gentian complex, should be the leading technique in the evaluation of boar sperm morphometry. Silver nitrate staining reveals the structure of the sperm in the most detail; this method can be considered universal, and can be used independently or to supplement routine diagnostics. As the staining technique should interfere as little as possible with the structure of the sperm, while revealing its morphology in as much detail as possible, it is crucial to establish the natural dimensions of the unstained sperm head before determining the optimal technique and its reference values. The recommended or most commonly-used techniques are not always the best options for the staining and analysis of sperm of a given species.

Full article here

PLOS ONE – https://doi.org/10.1371/journal.pone.0214243
Published: March 25, 2019

155 Whole genome association analysis suggests an influence of inbreeding on bull sperm morphometry2019-01-14T15:29:51+01:00

155 Whole genome association analysis suggests an influence of inbreeding on bull sperm morphometry

F. Azcona, M. Sole, J. Dorado, P. Ross, E. Terán, S.E. Demyda-Peyrás

Instituto de Genética Veterinaria, La Plata, Buenos Aires, Argentina; Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata, La Plata, Buenos Aires, Argentina; Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden; Departamento de Medicina y Cirugía Animal, Universidad de Córdoba, Córdoba, España; University of California, Davis, Davis, California, USA

Abstract: Inbreeding depression, the genetic condition caused by mating related individuals, is particularly important in several cattle breeds with limited effective populations. This condition is often associated with decreases in performance, including fertility. Furthermore, sperm head morphometry was associated with fertility in several species. To our knowledge, the influence of inbreeding on sperm morphometry has not been widely reported in cattle. In this study, a Sperm Class Analyzer (SCA™, Microptic S.L., Barcelona, Spain) was used to characterise 7 sperm morphometry parameters in 59 Retinta bulls, including sperm head length, width, perimeter, ellipticity, elongation, regularity and rugosity. Two replicates of at least 100 sperm heads, from 2 frozen semen samples, were assessed per individual (n=200 sperm per bull). Additionally, all individuals were genotyped with the Axiom Bos1 high density SNP Array (Affymetrix, Santa Clara, CA, USA). The molecular-based inbreeding factor (FROH; mean 12.5%, range 1.75 to 33.0) had very low correlations with all sperm morphometry parameters. On average, sperm heads from bulls with higher FROH had a smaller area, but variability was high. Correlations between inbreeding and sperm shape were low and negative, length (r=−0.1449; P<0.01), width (r=−0.2494; P<0.01), and rugosity (r=−0.1086; P>0.01) being the most informative. Whole-genome association study (GWAS) analysis, performed using molecular inbreeding as co-factor, revealed 8 SNP, located on chromosomes BTA2, BTA5, BTA7, and BTA11, significantly associated with sperm head regularity and rugosity. Genomic analysis revealed that genes SLF1 and TMEFF2 are located close to enriched SNP. Gene SLF1 (SMC5-SMC6 complex localization factor 1) is associated with the regulation of protein complex and cytoskeleton assembly, whereas TMEFF2 (transmembrane protein with EGF like and 2 follistatin like domains 2) is associated with integral components of cell membrane and cell spreading and development. Therefore, we inferred that SLF1 and TMEFF2 may be involved in variations of sperm head shape. In this preliminary study, there was evidence of a potential influence of inbreeding on sperm morphometry in a beef cattle breed. However, additional studies, ideally including more individuals and additional breeds, are necessary to determine the validity of this potential association.

DOI: 10.1071/RDv31n1Ab155

Sperm macrocephaly syndrome in the ostrich Struthio camelus: morphological characteristics and implications for motility2018-11-21T15:38:36+01:00

Sperm macrocephaly syndrome in the ostrich Struthio camelus: morphological characteristics and implications for motility

L. du Plessis, M. Bonato, C. Durandt, S. W. P. Cloete and J. T. Soley

Electron Microscope Unit, Department of Anatomy and Physiology, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa; Department of Animal Sciences, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa; Department of Immunology, Institute for Cellular and Molecular Medicine, Faculty of Health Sciences, SAMRC Extramural Unit for Stem Cell Research and Therapy, University of Pretoria, South Africa; Directorate Animal Sciences: Elsenburg, Private Bag X1, Elsenburg 7607, South Africa; Department of Anatomy and Physiology, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa

Abstract: Sperm macrocephaly syndrome (SMS) is characterised by a high percentage of spermatozoa with enlarged heads and multiple tails, and is related to infertility. Although this multiple sperm defect has been described in other mammalian species, little is known about this anomaly in birds. Morphological examination of semen from nine South African black ostriches (Struthio camelus var. domesticus) involved in an AI trial revealed the variable presence of spermatozoa with large heads and multiple tails. Ultrastructural features of the defect were similar to those reported in mammals except that the multiple tails were collectively bound within the plasmalemma. The tails were of similar length and structure to those of normal spermatozoa, and the heads were 1.6-fold longer, emphasising the uniformity of the anomaly across vertebrate species. Flow cytometry identified these cells as diploid and computer-aided sperm analysis revealed that they swim slower but straighter than normal spermatozoa, probably due to the increased drag of the large head and constrained movement of the merged multiple tails. The high incidence of this defect in one male ostrich indicates that, although rare, SMS can occur in birds and may potentially have an adverse effect on breeding programs, particularly for endangered species.

Reproduction, Fertility and Development 31(4) 712-723 https://doi.org/10.1071/RD18242
Submitted: 26 June 2018 / Accepted: 23 October 2018 / Published: 21 November 2018

Super-resolution imaging of live sperm reveals dynamic changes of the actin cytoskeleton during acrosomal exocytosis2022-11-14T17:39:31+01:00

Super-resolution imaging of live sperm reveals dynamic changes of the actin cytoskeleton during acrosomal exocytosis

Ana Romarowski, Ángel G. Velasco Félix, Paulina Torres Rodríguez, María G. Gervasi, Xinran Xu, Guillermina M. Luque, Gastón Contreras-Jiménez, Claudia Sánchez-Cárdenas, Héctor V. Ramírez-Gómez, Diego Krapf, Pablo E. Visconti, Dario Krapf, Adán Guerrero, Alberto Darszon, Mariano G. Buffone

Instituto de Biología y Medicina Experimental (IBYME), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires C1428ADN, Argentina; Laboratorio Nacional de Microscopía Avanzada, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Cuernavaca, Morelos 62210, México; Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Cuernavaca, Morelos 62210, México; Department of Veterinary and Animal Science, Paige Labs, University of Massachusetts, Amherst, MA 01003, USA; Department of Electrical and Computer Engineering, School of Biomedical Engineering, 1301 Campus Delivery, Fort Collins, CO 80523, USA; Instituto de Biología Molecular y Celular de Rosario (CONICET-UNR), Rosario, Santa Fe S2000EZP, Argentina

Abstract: Filamentous actin (F-actin) is a key factor in exocytosis in many cell types. In mammalian sperm, acrosomal exocytosis (denoted the acrosome reaction or AR), a special type of controlled secretion, is regulated by multiple signaling pathways and the actin cytoskeleton. However, the dynamic changes of the actin cytoskeleton in live sperm are largely not understood. Here, we used the powerful properties of SiR-actin to examine actin dynamics in live mouse sperm at the onset of the AR. By using a combination of super-resolution microscopy techniques to image sperm loaded with SiR-actin or sperm from transgenic mice containing Lifeact-EGFP, six regions containing F-actin within the sperm head were revealed. The proportion of sperm possessing these structures changed upon capacitation. By performing live-cell imaging experiments, we report that dynamic changes of F-actin during the AR occur in specific regions of the sperm head. While certain F-actin regions undergo depolymerization prior to the initiation of the AR, others remain unaltered or are lost after exocytosis occurs. Our work emphasizes the utility of live-cell nanoscopy, which will undoubtedly impact the search for mechanisms that underlie basic sperm functions

See full article here

Journal of Cell Science – doi:10.1242/jcs.218958
Received 12 April 2018; Accepted 25 September 2018; Published 8 November 2018

Quantitative assessment of heavy metal effects on sperm function using computer-aided sperm analysis and cytotoxicity assays2018-10-29T17:40:03+01:00

Quantitative assessment of heavy metal effects on sperm function using computer-aided sperm analysis and cytotoxicity assays

Hardneck F, Israel G, Pool E, Maree L.

Department of Medical Bioscience, University of the Western Cape, Bellville, South Africa

Abstract: One known environmental risk factor impacting on human reproduction is heavy metal pollution. Although some metals (e.g., Cu, Se and Zn) have protective effects on the male reproductive system in low doses, heavy metals can accumulate to toxic levels and result in poor semen quality and decreased sperm function. We investigated the effect of CuSO4 and CdCl2 (10, 50, 100 and 250 µg/ml or 500 µg/ml) on human sperm motility and vitality by using computer-aided sperm analysis (CASA) and two cytotoxicity assays (WST-1 and XTT). Several sperm motility parameters were significantly reduced after 5 hr of exposure to the highest concentrations of CuSO4 (250 µg/ml) and CdCl2 (500 µg/ml). The WST-1 assay also revealed significantly lower absorbance values for 50, 100 and 250 µg/ml CuSO4 and for 500 µg/ml CdCl2 ; however, no significant effect was seen with XTT. The calculated average IC50 value was 50.31±  4.34 µg/ml for CuSO4 and 392.32  ±76.79 µg/ml for CdCl2 . The effects of these metals were confirmed with MgCl2 , a positive control. This study provides threshold concentrations for the harmful effect of CuSO4 and CdCl2 on human spermatozoa and recommends the use of WST-1 as vitality assay in future in vitro studies.

DOI: 10.1111/and.13141

Lysine acetylation modulates mouse sperm capacitation2019-01-14T15:16:10+01:00

Lysine acetylation modulates mouse sperm capacitation

Carla Ritagliati, Guillermina M. Luque, Cintia Stival, Carolina Baro Graf, Mariano G. Buffone & Dario Krapf

Laboratory of Cell Signal Transduction Networks, Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET-UNR, Rosario, 2000, Argentina; Laboratory of Cellular and Molecular Reproductive Biology, Instituto de Biología y Medicina Experimental (IBYME), CONICET, Buenos Aires, C1428ADN, Argentina

Abstract: Mammalian sperm are unable to fertilize the egg immediately after ejaculation. To gain fertilization competence, they need to undergo a series of modifications inside the female reproductive tract, known as capacitation. Capacitation involves several molecular events such as phosphorylation cascades, hyperpolarization of the plasma membrane and intracellular Ca2+ changes, which prepare the sperm to develop two essential features for fertilization competence: hyperactivation and acrosome reaction. Since sperm cells lack new protein biosynthesis, post-translational modification of existing proteins plays a crucial role to obtain full functionality. Here, we show the presence of acetylated proteins in murine sperm, which increase during capacitation. Pharmacological hyperacetylation of lysine residues in non-capacitated sperm induces activation of PKA, hyperpolarization of the sperm plasma membrane, CatSper opening and Ca2+ influx, all capacitation-associated molecular events. Furthermore, hyperacetylation of non-capacitated sperm promotes hyperactivation and prepares the sperm to undergo acrosome reaction. Together, these results indicate that acetylation could be involved in the acquisition of fertilization competence of mammalian sperm.

Full article here

https://doi.org/10.1038/s41598-018-31557-5

CRISPR-Cas9-mediated mutation revealed BSPH2 protein is dispensable for male fertility2018-10-29T17:38:28+01:00

CRISPR-Cas9-mediated mutation revealed BSPH2 protein is dispensable for male fertility

Eskandari-Shahraki M, Prud’homme B, Manjunath P

Maisonneuve-Rosemont Hospital Research Centre, Montreal, Canada; Departments of Pharmacology and Physiology, Faculty of Medicine, University of Montreal, Montreal, Canada; Department of Medicine, Faculty of Medicine, University of Montreal, Montreal, Canada.

Abstract: Members of the Binder of SPerm (BSP) superfamily have been identified in both human and mouse epididymis. These proteins are known to bind sperm membrane and promote sperm capacitation. Studies suggest that BSPH2 might play a different role in sperm functions from its counterparts; however, the role of BSPH2 remains mainly unexplored. To investigate whether the absence of one member of the BSP family could affect fertility, mice lacking Bsph2 expression were generated using clustered regularly interspaced short palindromic repeats (CRISPR) associated 9 (Cas9) technology. Knockout (KO) male mice were mated with wild-type (WT) females, and the number and weight of the pups were determined. Sperm motility in WT and KO was assessed using sperm class analyzer (SCA). Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used for protein identification. Fertility analysis of null Bsph2 mice did not reveal any phenotype. No differences were noticed on average litter size or average pup weight. Normal testis weight and morphology were observed in Bsph2+/- and Bsph2-/- compared to the WT. Quantitative polymerase chain reaction analyses revealed that Bsph1 messenger RNA expression was increased in mutant mice, whereas LC-MS/MS analysis displayed no increase in protein expression level. Taken together, we show the existence of redundant function for murine BSPH2 and the lack of BSPH2 itself does not lead to sterility.

DOI: 10.1002/mrd.23039

Sperm motility assessment using computer assisted semen analysis (CASA): a comparison of standard microscope slides and coverslips and the 20 µm MicroCell™2018-07-11T12:56:16+02:00

Sperm motility assessment using computer assisted semen analysis (CASA): a comparison of standard microscope slides and coverslips and the 20 µm MicroCell™

Callum Robinson, Peter Roberts, Phillip Matson

School of Medical and Health Sciences, Edith Cowan University, Joondalup, Western Australia 6027, Australia; Fertility North, Joondalup, Western Australia 6027, Australia

Abstract: Computer assisted semen analysis (CASA) uses instrumentation that makes precise measurements of sperm motility, but the values obtained can be influenced by a number of technical aspects. Motility of human sperm was measured using a Sperm Class Analyzer (Microptic S.L., Barcelona, Spain), and the effect of using different counting chamber/slide configurations was investigated. Results for 20μm MicroCell slides (Vitrolife Sweden AB, Göteborg, Sweden) were compared with microscope slides and 22mmx22mm coverslips loaded with either 5µl (CV.5µl) or 10µl (CV.10µl) semen. Operator-correction of readings for all slide configurations resulted in a significantly lower number of sperm assessed due to the elimination of non-sperm bodies. Following operator-correction, the MicroCell chamber and CV.10µl slide gave similar readings for both progressive motility and immotility for up to 5 minutes, whereas the CV.5µl had a progressive increase in immotile sperm. The interval to analysis was therefore standardised at 2 minutes prior to the measurement of kinetic parameters, and the MicroCell values were significantly different to the CV.10µl for curvilinear velocity (VCL), average path velocity (VAP) and straight line velocity (VSL), and the CV.5µl for VAP. It is concluded that the same configuration be used within the same study, and that care should be taken when comparing different studies that have used different slide/chamber configurations.

J Reprod Stem Cell Biotechnol 7:1-7

Effects of Cryopreservation on Sperm Subpopulations in Goats2018-06-22T17:30:45+02:00

Effects of Cryopreservation on Sperm Subpopulations in Goats

Leonardo Hernández Corredor, Omar Camargo Rodríguez, Albeiro Silva Torres, Juan David Montoya Páez, Armando Quintero Moreno

Unidad de Investigación en Producción Animal (UNIPA), Facultad de Ciencias Veterinarias (FCV), Universidad del Zulia (LUZ), Maracaibo, Venezuela; Universidad Nacional de Colombia, Medellín, Colombia; Universidad Francisco de Paula Santander-SENNOVA, SENA-Norte de Santander, Cúcuta, Colombia; Universidad de Pamplona, Pamplona, Colombia; Politécnico Colombiano Jaime Isaza Cadavid, Medellín, Colombia

Abstract: The aim of this study was to evaluate the effect of cryopreservation in the presence of sperm subpopulations, according to motility, in sperm ejaculates using the Computer Assisted Semen Analysis (CASA) system. The motility parameters were analysed with principal component analysis (PCA) where they showed the highest variance, thus reducing the number of variables. In the evaluation of 23 738 fresh spermatozoa, the subpopulation (Sp) 1 consisted of progressive spermatozoa and median progressive motility (18.34%), Sp 2 in high velocity and progressive spermatozoa (20.53%), Sp 3 in high-activity spermatozoa, but not progressive (46.79%) and Sp 4 in little activity and non-progressive spermatozoa (14.32%), showing significant differences in the distribution of the four Sp (p<0.001). The structure of the spermatozoa Sp was not maintained after freezing. When evaluating the same parameters in thawed samples in 36 450 motile sperm, the Sp 1 consisted of slow and linear slow progressive spermatozoa (38.43%), the Sp 2 in slowly active and slow spermatozoa (7.3%), the Sp 3 in spermatozoa with high activity and excellent progressive motility (11.65%) and Sp 4 in active spermatozoa, but not progressive (42.61%), showing significant differences in the distribution of the four Sp (p<0.001). Cryopreservation significantly modified both the specific parameters and the distribution of the spermatozoa within the subpopulations.

Full article here

Rev. investig. vet. Perú vol.29 no.3 Lima jul./set. 2018 – http://dx.doi.org/10.15381/rivep.v29i3.14169
Received 18 January 2018, Accepted 22 June 2018

Bottlenose dolphin (Tursiops truncatus) sperm revisited: Motility, morphology and ultrastructure of fresh sperm of consecutive ejaculates2018-08-20T15:55:28+02:00

Bottlenose dolphin (Tursiops truncatus) sperm revisited: Motility, morphology and ultrastructure of fresh sperm of consecutive ejaculates

Gerhard van der Horst, Katarina Medger, Daniela Steckler, Ilse Luther, Paul Bartels

University of the Western Cape, Private Bag X17, Bellville 7535, South Africa; National Zoological Garden, South African National Biodiversity Institute, PO Box 754, Pretoria 0001, South Africa; Department of Zoology and Entomology, University of Pretoria, Hatfield/Pretoria 0028, South Africa; Section of Reproduction, Faculty of Veterinary Science, University of Pretoria, Onderstepoort, Pretoria 0110, South Africa; GEOsperm, Brits 0250, South Africa; Department of Nature Conservation, Tshwane University of Technology, Pretoria-West 0001, South Africa.

Abstract: Computer aided sperm analysis systems allow detailed examination of sperm motility and morphology variables, which are important for the understanding of the spermatology of a species and the development of assisted reproductive techniques. Cetacean biology is too complex to study in the wild and data from captive individuals provide an important alternative for the conservation of these charismatic animals. The present study evaluates ejaculate and sperm characteristics, including sperm motility, kinematic variables and quantitative sperm morphology and ultrastructure, of consecutive ejaculates from Atlantic bottlenose dolphins (Tursiops truncatus). Sperm concentrations and total and progressive motilities were greater in the second than the first ejaculate, with all ejaculates being of very high quality (6.9–1127 × 106/ml sperm concentration, 75% to 91% total motility and 89% to 96% normal sperm). Most sperm in an ejaculate (≥84%) were highly (VCL>150 μm/s) and progressively motile with very few abnormal sperm. The sperm have small heads, a short but very bulky midpiece and a long tail. Detailed sperm morphometrics using CASA indicated there were similarities from one ejaculate to the next. The large mitochondria with extensive cristae mitochondriales are tightly packed in the midpiece resulting in a large midpiece volume. All the semen and sperm characteristics indicate high quality sperm and support the assumption that a multimale mating system is present in T. truncatus.

https://doi.org/10.1016/j.anireprosci.2018.06.009

CASA in invertebrates2018-04-09T11:48:25+02:00

CASA in invertebrates

Gerhard van der Horst, Monique Bennett and John D. D. Bishop

Department of Medical Biosciences, University of the Western Cape, Robert Sobukwe Road, Bellville, 7535, South Africa; Marine Biological Association of the United Kingdom, Citadel Hill Laboratory, Plymouth PL1 2PB, UK

Abstract: Sperm movement has been described in several phyla of invertebrates. Yet, sperm motility has only been quantified using computer-aided sperm analysis (CASA-Mot) in externally fertilising species (broadcast spawners) of two phyla, molluscs and echinoderms. In the present study we quantified in detail the nature of the sperm tracks, percentage motility groupings and detailed kinematics of rapid-, medium- and slow-swimming spermatozoa in the oyster Crassostrea gigas and four species never previously studied by CASA-Mot, namely the molluscs Choromytilus meridionalis, Donax serra and Haliotis midae and the echinoderm Parechinus angulosus. A feature common to all these species are the helical tracks, the diameter of which seems to be species specific. Using CASA-Mot, the behaviour of spermatozoa was also studied over time and in the presence of egg water and Ca2þ modulators such as caffeine and procaine hydrochloride. For the first time, we show that hyperactivation can be induced in all species in the presence of egg water (sea water that was mixed with mature eggs and then centrifuged) and/or caffeine, and these hyperactivated sperm tracks were characterised using CASA-Mot. We relate the different patterns of sperm motility and behaviour to reproductive strategies such as broadcast spawning and spermcasting, and briefly review studies using CASA-Mot on other invertebrates.

https://doi.org/10.1071/RD17470

Current perspectives of CASA applications in diverse mammalian spermatozoa2022-05-04T15:57:29+02:00

Current perspectives of CASA applications in diverse mammalian spermatozoa

Gerhard van der Horst, Liana Maree and Stefan S. du Plessis

Department of Medical Bioscience, University of the Western Cape, Private Bag X17, Bellville, 7535, South Africa; Division of Medical Physiology, Faculty of Medicine and Health Sciences, Stellenbosch University, PO Box 19063, Tygerberg, 7505, South Africa

Abstract:  Since the advent of computer-aided sperm analysis (CASA) some four decades ago, advances in computer technology and software algorithms have helped establish it as a research and diagnostic instrument for the analysis of spermatozoa. Despite mammalian spermatozoa being the most diverse cell type known, CASA is a great tool that has the capacity to provide rapid, reliable and objective quantitative assessment of sperm quality. This paper provides contemporary research findings illustrating the scientific and commercial applications of CASA and its ability to evaluate diverse mammalian spermatozoa (human, primates, rodents, domestic mammals, wildlife species) at both structural and functional levels. The potential of CASA to quantitatively measure essential aspects related to sperm subpopulations, hyperactivation, morphology and morphometry is also demonstrated. Furthermore, applications of CASA are provided for improved mammalian sperm quality assessment, evaluation of sperm functionality and the effect of different chemical substances or pathologies on sperm fertilising ability. It is clear that CASA has evolved significantly and is currently superior to many manual techniques in the research and clinical setting.

https://doi.org/10.1071/RD17468

The effects of gelatin supplementation prior to cooling on ram semen quality and fertility2018-03-01T17:15:49+01:00

The effects of gelatin supplementation prior to cooling on ram semen quality and fertility

Anderson Marques Pinto Bandeira; José Eduardo Matos; Alexandre Nízio Maria; Paulo César Falanghe Carneiro; Phillip H. Purdy; Hymerson Costa Azevedo

Universidade Federal de Sergipe, São Cristóvão, Brazil; Embrapa Tabuleiros Costeiros, Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA), Aracaju, SE, Brazil; Agricultural Research Service (ARS), United States Department of Agriculture (USDA), Fort Collins, United States of America

Abstract: The physical and chemical characteristics of gelatin have been used to justify its inclusion in extenders to preserve the sperm quality and improve results of cervical artificial insemination with cooled semen. The objective of this study was to evaluate the effects of gelatin supplementation in cooling extender on the quality and fertility of ram semen stored at 5°C. Semen samples (n = 24) of Santa Inês rams (n = 6) were diluted in Glycine-Yolk-Milk extender without (control) or with 1.5% of gelatin. The samples were loaded into 0.25 mL straws, cooled to 5°C and stored vertically for 48 and 72 hours. Sample quality was evaluated using straw homogeneity tests based on pH, osmolality and the proportion of spermatozoa (PS) in both upper and lower segments of straws (US and LS), analyses of sperm motility, plasma and acrosomal membrane integrity, and by fertility after artificial insemination. Differences between the US and LS of straws were found for pH and PS (%). They were significant only in the control group at both times: pH – 5.96 vs. 5.71 at 48 h and 6.13 vs. 5.89 at 72 h; PS – 21.66 vs. 78.34 at 48 h and 20.87 vs. 79.13 at 72 h. Storage in gelatin had very little, to no effect on the sperm kinetics or on the sperm membrane integrity evaluations. The addition of gelatin to the extender did not affect the pregnancy rate which ranged from 4.4 to 26.1%. We conclude that gelatin is effective in maintaining the physical and chemical homogeneity of the semen samples. Further research is needed in order to optimize the use of gelatin supplementation and elucidate any potential benefits.

Animal Reproduction – DOI: 10.21451/1984-3143-AR2017-0021
Jan./Mar. 2018

A novel flush technique to simulate natural dispersal of spermatozoa in the female reproductive tract and expedite motility assessment of fresh ejaculated Merino (Ovis aries) sperm2018-02-15T12:40:36+01:00

A novel flush technique to simulate natural dispersal of spermatozoa in the female reproductive tract and expedite motility assessment of fresh ejaculated Merino (Ovis aries) sperm

N. H. Boshoff, H. Lambrechts, L. Maree, S. W. P. Cloete, G. van der Horst

Department of Animal Sciences, Stellenbosch University, P/Bag X1, Matieland, 7602, South Africa; Department of Medical Biosciences, University of the Western Cape, P/Bag X17, Bellville, 7535, South Africa; Directorate Animal Sciences: Elsenburg, P/Bag X1, Elsenburg, 7607, South Africa

Abstract: Motility is an important criterion in the assessment and quantification of the quality of ejaculated and epididymal sperm samples. Ovine sperm spermatozoa are particularly susceptible to cryodamage, and shortening the interval from collection to cryostorage may potentially minimize the negative effects of cryopreservation, thereby improving the post-thaw viability of ram spermatozoa. The use of the swim-up technique (SUT) in quantifying spermatozoa motility is well documented, especially for the isolation of highly motile spermatozoa for assisted reproductive purposes. However, this technique is time consuming and involves a swim-up period of 10 minutes before the motility of a sample is recorded by using computerassisted sperm analysis (CASA) software such as the Sperm Class Analyser® (SCA®). The novel flush technique (FT) allows for capturing of sperm motility tracks via the SCA® system shortly after semen collection, that is, within a minute. This study compared fresh ejaculated sperm motility traits by using the SUT and FT. Motility evaluations were performed using 45 semen samples obtained from 15 adult Merino rams (Ovis aries) at weekly intervals. Motility recordings were captured at 100 frames per second, using a calibrated 20 µm deep Leja slide. The percentage total motile spermatozoa of samples subjected to the FT was 89.2%, which was significantly higher than that recorded by the SUT (83.9%). The results also indicated that the wobble (WOB) parameter showed significantly higher values when using the FT, and parameters curvilinear velocity (VCL) and amplitude of the lateral head displacement (ALH) indicated significantly higher values when using the SUT. Establishing the ideal spermatozoa concentration and analysis of sperm subpopulation motility characteristics would assist in the optimization of the FT, and its use in CASA motility analysis of ovine spermatozoa. Standardization of CASA analysis of ovine semen samples, which would enable the selection of quality spermatozoa samples for use in field insemination (fresh samples) or in vitro fertilization programs, and laparoscopic AI cryopreserved samples warrants further investigation.

http://dx.doi.org/10.4314/sajas.v48i3.7

Full article here

SA Journal of Animal Science website

Clinical application of the standardisation of computer-assisted sperm morphometry analysis with ASMA (assisted sperm morphometry analysis) using SCA2018-02-16T12:58:40+01:00

Clinical application of the standardisation of computer-assisted sperm morphometry analysis with ASMA (assisted sperm morphometry analysis) using a semen system analyser SCA 5.4 (Sperm Class Analyzer, Microptic)

Clara Ramírez, José Ramón Alonso, Pedro Jiménez, Jordi Ramis, Josep María Gris, Carlos Aulesa

Unidad de Andrología, Laboratorios Clínicos, Hospital Universitario Vall d’Hebron, Barcelona, Spain; Servicio de Reproducción Asistida, Hospital Universitario Vall d’Hebron, Barcelona, Spain

Abstract

Introduction: Use is made of computer-assisted sperm morphometry analysis (ASMA) in order to analyse sub-populations as a function of several pathologies.

Material and methods: A total of 703 patients were classified into 7 pathology groups, and their sperm sub-populations were analysed. Pathology groups were classified into infertility, male factor stress (anxiety, insomnia and/or depression), diabetes mellitus type 1 or 2, varicocele, urogenital infections, oncological patients, and other endocrine diseases. Morphological analysis was performed using the semen analyser system SCA 5.4 (Sperm Class Analyzer) (Microptic S.L., Barcelona, Spain). Pre-treatment and staining method used has been previously described by this group.

Results: Automatic human sperm morphology showed that the study of sperm sub-populations has a clinical usefulness in the diagnosis of varicocele. Higher values of 30% elongated sperms had a sensitivity of 37% and a specificity of 79% (1.54 odds ratio with a 95% confidence interval from 1.05 to 2.26). Furthermore, groups of infertility, stress, and diabetes showed new sperm sub-populations which had not been described previously, so they could become a new diagnostic tool.

Discussion: The coefficients of variation of sperm morphology were decreased using automation, enabling the study sperm subpopulations in different pathologies. There is a diagnostic usefulness for varicocele. There could be a prediction to choose the best assisted reproductive technique for infertility patients. However, for the rest of groups new sperm subpopulations could be identified that could make it a diagnostic tool, but requires a higher casuistry to conclude the study.

https://doi.org/10.1016/j.medre.2017.11.001

Revisiting the relationship between the ejaculatory abstinence period and semen characteristics2018-01-16T16:34:20+01:00

Revisiting the relationship between the ejaculatory abstinence period and semen characteristics

Bashir M Ayad, Gerhard Van der Horst, Stefan S Du Plessis

Division of Medical Physiology, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa.

Abstract:

Variation in the ejaculatory abstinence period suggested by different guidance bodies have resulted in a growing concern among researchers and clinicians over what the precise period of ejaculatory abstinence ought to be for an optimal semen sample. Several studies have thus been undertaken to examine the association between the length of sexual abstinence and semen characteristics. Not all studies, however, have arrived at the same conclusions. This study aims to review all existing literature published during the past few decades pertaining to the influence of ejaculatory abstinence on semen quality. For the purpose of this systematic review, all data related to sexual abstinence duration and seminal parameters were re-analysed to homogenize the current data. Thorough PubMed, MEDLINE and Google Scholar, a literature search was conducted using the keywords “sexual abstinence”, “ejaculatory abstinence”, “semen”, “spermatozoa”, “semen analysis”, “sperm parameters”, “motility”, “reactive oxygen species (ROS)” and “DNA fragmentation”. After carefully reviewing all the literature, 30 relevant papers, both written in English and published between January 1979 and December 2016, were included in this review. The weight of the evidence suggests that the decline in semen volume and sperm concentration with shorter abstinence periods is accompanied by a substantial improvement in sperm motility characteristics, especially progressive motility and velocity. Nevertheless, available data are insufficient to support definitive conclusions regarding the influence of the ejaculatory abstinence period on advanced semen parameters (ROS, DNA fragmentation and seminal plasma antioxidant capacity) and pregnancy rates. In conclusion, taking all data into account, shortening of the abstinence period may be beneficial to sperm quality. Furthermore, we recommend that the current guidelines regarding the prescribed abstinence period should be revisited.

doi:  10.22074/ijfs.2018.5192

Full article here

Cut-off values for normal sperm morphology and toxicology for automated analysis of rat sperm2022-05-04T15:58:03+02:00

Cut-off values for normal sperm morphology and toxicology for automated analysis of rat sperm morphology and morphometry

G van der Horst, B Skosana, A Legendre, P Oyeyipo & SS du Plessis

Department of Medical Bioscience, University of the Western Cape, Bellville; Division of Medical Physiology, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa; Laboratory of Experimental Toxicology, Fontenay aux Roses Cedex, France

Abstract:

We used automated sperm morphology analysis to investigate rat sperm morphometry and morphology in Sprague-Dawley and Wistar rats in three research centers to develop normal baseline values for sperm morphometry and to quantify the percentage of morphologically normal sperm in healthy rats. The participating centers were IRSN in Paris, France (Sprague-Dawley rats), University of the Western Cape, South Africa (Wistar rats) and Stellenbosch University (Wistar rats), South Africa. All three centers used identical sperm isolation techniques from the cauda epididymis, the same staining protocols, identical computer-aided sperm morphometry analysis (CASMA) software and microscopes with similar optics. With CASMA, fully automated analysis of the different parts of stained sperm, e.g., head, acrosome, mid-piece, can be performed, many sperm morphometric features can be measured accurately and eventually normal sperm morphology can be defined. We found that it is possible to distinguish sperm morphometric characteristics of Sprague-Dawley and Wistar rats. We also developed cut-off values for evaluating the percentage normal sperm in these two rat strains using the automatic analysis mode. Normal sperm morphology varied between 67 and 74% by contrast with previous findings of > 90%.

https://doi.org/10.1080/10520295.2017.1380842

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Effects of chronic musculoskeletal pain on fertility potential in lean and overweight male patients2017-12-24T16:57:29+01:00

Effects of chronic musculoskeletal pain on fertility potential in lean and overweight male patients

Fereshteh Dardmeh, Hiva Alipour, Hans Ingolf Nielsen, Sten Rasmussen and Parisa Gazerani

Department of Health Science and Technology, Biomedicine Group, The Faculty of Medicine, Aalborg University, Aalborg, Denmark; SMI®, Department of Health Science and Technology, The Faculty of Medicine, Aalborg University, Aalborg, Denmark; Department of Clinical Medicine, The Faculty of Medicine, Aalborg University Hospital, Aalborg, Denmark

Abstract: Both chronic pain and obesity are known to affect reproductive hormone profiles in male patients. However, the effect of these conditions, alone or in combination, on male fertility potential has received less attention. 20 chronic musculoskeletal pain patients and 20 healthy controls were divided into lean and overweight subgroups according to their BMI. Current level of chronic pain (visual analogue scale) and pressure pain thresholds (PPTs) in 16 predefined sites, classically described and tested as painful points on the lower body, were measured. Levels of reproductive hormone and lipid profiles were assessed by ELISA. Sperm concentration and motility parameters were analyzed using a computer-aided sperm analysis system. Sperm concentration, progressive motility, and percentage of hyperactivated sperm were generally lower in the chronic pain patients in both lean and overweight groups. The overweight control and the lean chronic pain groups demonstrated a significantly lower percentage of progressively motile sperm compared with the lean control group, suggesting that musculoskeletal chronic pain may have a negative influence on sperm quality in lean patients. However, due to the potential great negative influence of obesity on the sperm parameters, it is difficult to propose if musculoskeletal chronic pain also influenced sperm quality in overweight patients. Further research in chronic pain patients is required to test this hypothesis.

Pain Research and Management – https://doi.org/10.1155/2017/4628627
Received 30 August 2017; Revised 3 November 2017; Accepted 13 November 2017; Published 24 December 2017

Diabetes mellitus- induction: Effect of different streptozotocin doses on male reproductive parameters2022-05-04T15:57:18+02:00

Diabetes mellitus- induction: Effect of different streptozotocin doses on male reproductive parameters

Temidayo S. Omolaoye, Bongekile T. Skosana, Stefan S. du Plessis

Division of Medical Physiology, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa; Division of Medical Physiology, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa

Abstract: Diabetes mellitus (DM) is reported to be involved in male reproductive impairment, and its impact is evident in the increased prevalence of infertility. Various studies have reported that a single parenteral injection of <40 mg/kg Streptozotocin (STZ) is ineffective in ablating pancreatic β-cells and creating a rat model to investigate the effect of DM on the male reproductive system. This study therefore aims to validate these claims. Adult male Wistar rats received either a single intraperitoneal injection of STZ (30 mg/kg or 60 mg/kg) or saline (0.9%, Control). Diabetes was confirmed after 72 h if plasma glucose levels were ≥14 mmol/L. Body weight, glucose level, fluid and food intake were measured weekly. Animals were sacrificed after 8 weeks of treatment by an overdose of sodium pentobarbital (160 mg/kg body weight). The testis and epididymis were harvested and weighed prior to preparation for histological evaluation. Epididymal sperm morphology was analysed using computer aided sperm analysis (CASA). STZ60 animals presented with significantly lower body weights compared to both control and STZ30 groups. Animals in both STZ30 and STZ60 groups showed decreased normal sperm morphology compared to control. Histological evaluation of the testes showed a decrease in the number of spermatozoa in the seminiferous tubules of animals in the STZ30 and STZ60 groups compared to control. A complete absence of spermiogenesis was observed in the seminiferous tubules of STZ60 animals. These findings prove that an STZ concentration of 30 mg/kg, which is much lower than the reported 40 mg/kg, has adverse effects on the male reproductive system via its diabetogenic effect and can therefore be used to study the impact of DM on male fertility.

DOI:10.1016/j.acthis.2017.12.005

Grooves surrounding the micropyle decrease the inseminating dose in fish2017-10-10T15:54:09+02:00

Grooves surrounding the micropyle decrease the inseminating dose in fish

Matheus Pereira-Santos, Eduardo Shimoda, André Furugen Cesar de Andrade, Luciano Andrade Silva, Takafumi Fujimoto, José Augusto Senhorini, George Shigueki Yasui and Laura Satiko Okada Nakaghi

Aquaculture Center, São Paulo State University, Jaboticabal-SP, Brazil; Department of Pharmacy, Cândido Mendes University, Campos dos Goytacazes, Brazil; Department of Veterinary, University of São Paulo, Pirassununga, Brazil; Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Japan; and National Center for Research and Conservation of Continental Fish, Chico Mendes Institute of Biodiversity Conservation, Pirassununga, Brazil

Abstract: In fish with external fertilization, sperm must reach the oocyte through the micropyle to enter the cytoplasm. Fertilization success is then influenced by characteristics of oocytes or sperm. In this study, we evaluated oocyte morphology and sperm motility parameters and their effects on the inseminating dose in a teleost fish Astyanax altiparanae . Interestingly, we found one of the lowest yet described inseminating doses in teleosts (2390 spermatozoa oocyte ⁻¹ ml ⁻¹ ). Such a fertilization efficacy may be explained by the long duration of sperm motility (>75 s), the small oocyte diameter (695.119 µm), large micropyle diameter (7.57 µm), and the presence of grooves on the oocyte surface that guides spermatozoon to the fertilization area. Additionally, we have described for the first time a structure that combines grooves on the chorion surface and a ridge in the micropylar area.

DOI: 10.1017/S0967199417000624

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Lactobacillus rhamnosus PB01 supplementation affects markers of sperm kinematic parameters in a diet-induced obesity mice model2017-10-10T11:04:45+02:00

Lactobacillus rhamnosus PB01 (DSM 14870) supplementation affects markers of sperm kinematic parameters in a diet-induced obesity mice model

Fereshteh Dardmeh, Hiva Alipour, Parisa Gazerani, Gerhard van der Horst, Erik Brandsborg, Hans Ingolf Nielsen

Biomedicine Group, Department of Health Science and Technology, Faculty of Medicine, Aalborg University, Aalborg, Denmark; SMI®, Department of Health Science and Technology, Faculty of Medicine, Aalborg University, Aalborg, Denmark; Department of Medical Bioscience, University of the Western Cape, Bellville, South Africa; Bifodan A/S, Hundested, Denmark

Abstract:

Probiotics have been proposed as alternatives to pharmacological products in several medical conditions including the modulation of obesity, which is frequently associated with poor semen quality. However, effects of probiotics on male fertility have been less investigated. This study assessed the effect of Lactobacillus rhamnosus PB01 (DSM-14870) on sperm kinematic parameters in Normal-weight (NW) and diet-induced obese (DIO) models. NW and DIO C57BL/6NTac mice were divided into two subgroups with or without a single daily dose (1x109CFU) of L. rhamnosus for four weeks. Sperm motility and kinematics together with blood lipid profiles and reproductive hormone levels were assessed using the sperm class analyzer system. Probiotic supplementation increased serum testosterone, LH and FSH levels in both NW and DIO groups resulting in significantly (P<0.05) higher velocity (VSL, VCL and VAP) and percentages of progressively motile sperm and significantly lower percentages of immotile sperm. Other kinematic parameters (Lin, STR, ALH and BCF) were also increased in both probiotic supplemented DIO and NW groups at the 10% level of significance. Probiotic supplemented DIO mice demonstrated significantly higher percentages of progressively motile sperm versus DIO controls. This study demonstrated the potential of L. rhamnosus PB01 as a regulatory agent with positive effects on weight loss and reproductive-hormones, significantly improving sperm motility and kinematic parameters in male DIO models.

https://doi.org/10.1371/journal.pone.0185964

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Association between obesity and sperm quality2017-09-19T12:01:44+02:00

Association between obesity and sperm quality

G. A. Ramaraju, S. Teppala, K. Prathigudupu, M. Kalagara, S. Thota, M. Kota, R. Cheemakurthi

Center for Assisted Reproduction, Krishna IVF Clinic, Visakhapatnam, India

Summary:

There is awareness of likelihood of abnormal spermatozoa in obese men; however, results from previous studies are inconclusive. Advances in computer-aided sperm analysis (CASA) enable precise evaluation of sperm quality and include assessment of several parameters. We studied a retrospective cohort of 1285 men with CASA data from our infertility clinic during 2016. Obesity (BMI ≥30) was associated with lower (mean ± SE) volume (−0.28 ± 0.12, p-value = .04), sperm count (48.36 ± 16.51, p-value = .002), concentration (−15.83 ± 5.40, p-value = .01), progressive motility (−4.45 ± 1.92, p-value = .001), total motility (−5.50 ± 2.12, p-value = .002), average curve velocity (μm/s) (−2.09 ± 0.85, p-value = .001), average path velocity (μm/s) (−1.59 ± 0.75, p-value = .006), and higher per cent head defects (0.92 ± 0.81, p-value = .02), thin heads (1.12 ± 0.39, p-value = .007) and pyriform heads (1.36 ± 0.65, p-value = .02). Obese men were also more likely to have (odds ratio, 95% CI) oligospermia (1.67, 1.15–2.41, p-value = .007) and asthenospermia (1.82, 1.20–2.77, p-value = .005). This is the first report of abnormal sperm parameters in obese men based on CASA. Clinicians may need to factor in paternal obesity prior to assisted reproduction.

DOI: 10.1111/and.12888

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Effects of GnRH vaccination in wild and captive African Elephant bulls2017-09-18T17:14:49+02:00

Effects of GnRH vaccination in wild and captive African Elephant bulls (Loxodonta africana) on reproductive organs and semen quality

Imke Lueders, Debbie Young, Liana Maree, Gerhard van der Horst, Ilse Luther, Stephan Botha, Brendan Tindall, Geoffrey Fosgate, André Ganswindt, Henk J. Bertschinger

GEOlifes-Animal Fertility and Reproductive Research, Hamburg, Germany; Endocrine Research Laboratory, Department of Anatomy and Physiology, Faculty of Veterinary Science, University of Pretoria, Onderstepoort, South Africa; African Elephant Research Unit, Plettenberg Bay, South Africa; Department of Medical Biosciences, University of the Western Cape, Bellville, South Africa; Department of Research and Scientific Services, National Zoological Gardens of South Africa, Pretoria, South Africa; African Lion Safari, Cambridge, ON, Canada, 7 Robberg Veterinary Clinic, 56 Longships, Plettenberg Bay, South Africa; Department of Production Animal Studies, Faculty of Veterinary Science, University of Pretoria, Onderstepoort, South Africa

Abstract:

Objectives: Although the African elephant (Loxodonta africana) is classified as endangered by the International Union for Conservation of Nature (IUCN), in some isolated habitats in southern Africa, contraception is of major interest due to local overpopulation. GnRH vaccination has been promoted as a non-invasive contraceptive measure for population management of overabundant wildlife. We tested the efficacy of this treatment for fertility control in elephant bulls.

Methods: In total, 17 male African elephants that were treated with a GnRH vaccine were examined in two groups. In the prospective study group 1 (n = 11 bulls, ages: 8–36 years), semen quality, the testes, seminal vesicles, ampullae and prostate, which were all measured by means of transrectal ultrasound, and faecal androgen metabolite concentrations were monitored over a three-year period. Each bull in the prospective study received 5 ml of Improvac® (1000 μg GnRH conjugate) intramuscularly after the first examination, followed by a booster six weeks later and thereafter every 5–7 months. In a retrospective study group (group 2, n = 6, ages: 19–33 years), one examination was performed on bulls which had been treated with GnRH vaccine for 5–11 years.

Results: In all bulls of group 1, testicular and accessory sex gland sizes decreased significantly after the third vaccination. In six males examined prior to vaccination and again after more than five vaccinations, the testis size was reduced by 57.5%. Mean testicular height and length decreased from 13.3 ± 2.6 cm x 15.2 ± 2.8 cm at the beginning to 7.6 ± 2.1 cm x 10.2 ± 1.8 cm at the end of the study. Post pubertal bulls (>9 years, n = 6) examined prior to vaccination produced ejaculates with viable spermatozoa (volume: 8–175 ml, sperm concentration: 410-4000×106/ml, total motility: 0–90%), while after 5–8 injections, only 50% of these bulls produced ejaculates with a small number of immotile spermatozoa. The ejaculates of group 2 bulls (vaccinated >8 times) were devoid of spermatozoa. Faecal androgen metabolite concentrations measured in captive males decreased significantly after the fourth vaccination. None of the males entered musth during the treatment period.

Conclusions: Our results showed a marked decrease in semen quality, testicle and secondary sex gland sizes following repeated GnRH vaccinations. After 2–4 years of continuous treatment every 5–7 months, the effects were similar to surgical castration.

https://doi.org/10.1371/journal.pone.0178270

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Short abstinence: A potential strategy for the improvement of sperm quality2017-08-03T17:32:47+02:00

Short abstinence: A potential strategy for the improvement of sperm quality

Bashir M. Ayad, Gerhard Van der Horst, Stefan S. du Plessis

Division of Medical Physiology, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa

Abstract

Objective: To determine the effect of short (4 h) and long (4 days) abstinence periods on sperm quality based on functional and biochemical parameters in a population of normozoospermic men.

Methods: Two semen samples were collected in succession from potentially fertile, normozoospermic men (n = 100) after an abstinence period of 4 days and 4 h respectively. The mean values of semen volume, pH, viscosity, sperm concentration, percentage of total and progressively motile sperm, sperm kinematics/velocity, normal morphology, acrosome status, DNA fragmentation, intracellular superoxide (O2−•) levels and seminal antioxidant status were compared between the two abstinence duration groups.

Results: A significant increase in total and progressive motility and velocity parameter values were observed after short abstinence compared with long abstinence periods. Sperm DNA fragmentation and intracellular O2−• levels were not significantly different between the two abstinence periods. Despite the decrease in semen volume, sperm concentration and total sperm number after short abstinence periods, all mean values of the conventional semen parameters remained above the lower reference limits as reported by the WHO.

Conclusion: The data from this most comprehensive study of its kind challenges the generally accepted guidelines of the prolonged abstinence periods since the results show that 4 h of sexual abstinence yielded significantly better sperm samples from a functional point of view. Although this study was performed on normozoospermic men, future studies with infertile men might yield similar findings that could lead to employing short abstinence as a strategy to improve the outcome of ART and fertility preservation.

https://doi.org/10.1016/j.mefs.2017.07.005

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Marriage: WHO5 analysis and sperm function2017-07-26T15:51:12+02:00

Not just the marriage of Figaro: but the marriage of WHO/ESHRE semen analysis criteria with sperm functionality

Gerhard van der Horst, Stefan S du Plessis

Department of Medical Bioscience, University of the Western Cape, Belville, South Africa; Division of Medical Physiology, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa

Abstract:

The authors present a critical review of the WHO5 (2010) manual of semen analysis and what it should be used for: The analysis of sperm quality and not analysis to predict fertility outcome per se. We show the strengths and shortcoming of WHO5 and then ask for a better “marriage” among these parameters and the outcome of sperm functionality and fertilization/live birth outcome. For many decades the basis of the WHO manual for semen analysis has not changed and we emphasize that sperm functionality testing has not really been considered/performed in the routine andrology laboratory. There is a need to first develop more objective and quantitative methodology such as computer-aided sperm analysis, to analyse sperm quality and sperm functionality that relates in many instances to fertilization/live birth outcome: 1) sperm cervical mucous penetration using computer aided sperm analysis (CASA), 2) endpoint of capacitation, hyperactivation as measured accurately by CASA, 3) acrosome reaction quantitatively, 4) chromatin maturity and DNA fragmentation quantitatively, 4) where possible oocyte binding tests (hemizona), 5) relationships of vitality and hypo-osmotic swelling test using modern technology 6) measurement of oxidative stress, 7) analysis of semen using proteomics (proteins that are importantly functionally expressed in seminal plasma) as well as 8) metabolomics representing a systematic study of the unique metabolic fingerprints (chemical) that specific cellular processes leave behind and inform us about function/dysfunction, 9) patient profile (obesity, smoking, age, stress, female cryptic choice, environment and many other patient characteristics) as important determinants in fertility outcome. We believe we can intelligently in the end construct a matrix which combine all these factors and others in the future that inform us about potential fertility outcome. But then realize WHO5/ESHRE current guidelines are not particularly informative in the above context.

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Improved sperm kinematics in semen samples collected after 2 h versus 4-7 days of ejaculation abstinence2017-05-24T17:39:14+02:00

Improved sperm kinematics in semen samples collected after 2 h versus 4-7 days of ejaculation abstinence

Alipour H, Van Der Horst G, Christiansen OB, Dardmeh F, Jørgensen N, Nielsen HI, Hnida C

Biomedicine Group, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark; Department of Medical Bioscience, University of the Western Cape, Bellville, South Africa; Department of Obstetrics and Gynecology, Aalborg University Hospital, Aalborg, Denmark; Department of Clinical Medicine, Aalborg University, Aalborg, Denmark; University Department of Growth and Reproduction and International Center for Research and Research Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC), Rigshospitalet, Copenhagen, Denmark; Fertility Unit, Department of Obstetrics and Gynecology, Aalborg University Hospital, Aalborg, Denmark

Abstract

STUDY QUESTION:
Does a short abstinence period of only 2 h yield spermatozoa with better motility characteristics than samples collected after 4-7 days?

SUMMARY ANSWER:
Despite lower semen volume, sperm concentration, total sperm counts and total motile counts, higher percentages of motile spermatozoa with higher velocity and progressiveness were detected in samples obtained after 2 h.

WHAT IS KNOWN ALREADY:
Most studies that have assessed the effect of abstinence periods on sperm motility parameters in men with a sperm concentration below 15 million/ml have detected a higher percentage of motile spermatozoa in samples obtained after short abstinence periods. Studies of men with sperm concentrations above 15 million/ml have reported significantly decreased motile sperm counts after 24 h of abstinence compared with longer abstinence periods.

STUDY DESIGN, SIZE, DURATION:
This study had a controlled repeated-measures design based on semen samples from 43 male partners, in couples attending for IVF treatment, who had a sperm concentration above 15 million/ml. Data were collected between June 2014 and December 2015 in the Fertility Unit of Aalborg University Hospital (Aalborg, Denmark).

PARTICIPANTS/MATERIALS, SETTING, METHODS:
Participants provided a semen sample after 4-7 days of abstinence followed by another sample after only 2 h. For both ejaculates, sperm concentration, total sperm counts, motility groups and detailed kinematic parameters were assessed and compared by using the Sperm Class Analyzer (SCA) computer-aided sperm analysis system before and after density gradient selection. The laboratory’s local manual method (Makler chamber) was used for comparison.

MAIN RESULTS AND THE ROLE OF CHANCE:
The second raw ejaculate demonstrated lower semen volume (P < 0.0001), sperm concentration (P = 0.003) and sperm counts in all motility sub-groups (P < 0.001) but higher percentages of spermatozoa with higher velocity (P < 0.01), progressiveness (P < 0.001) and hyperactivation (P < 0.001), compared with the first raw ejaculate.

LIMITATIONS, REASONS FOR CAUTION:
The first ejaculate in this study was also used for the IVF/ICSI treatments and therefore only patients with a semen volume ≥2 ml and concentration ≥15 million/ml were included. Further validation in large prospective randomized controlled trials, more purposely directed at normozoospermic males with partners having problems conceiving when there appears to be no female factor, is needed to confirm the potential advantage of using a second semen sample in improving fertilization and pregnancy rates in assisted reproduction.

WIDER IMPLICATIONS OF THE FINDINGS:
Despite the significantly lower semen volume, sperm concentration and total sperm counts in all motility sub-groups, the significantly higher percentage of spermatozoa with better motility characteristics (velocity, progressiveness and hyperactivation) in the second ejaculate, may provide and allow for a simpler and more effective selection of higher quality spermatozoa. This could prove to be an advantage for ART procedures such as intracytoplasmic sperm injection where a large number of spermatozoa is not needed. It can also be speculated that pooling two consecutive ejaculates obtained after 4-7 days and after 2 h, could be an advantage for intrauterine insemination where a large number of motile spermatozoa are needed.

STUDY FUNDING/COMPETING INTEREST(S):
This study was supported by internal grants from the Department of Health Science and Technology, Faculty of Medicine, Aalborg University (Aalborg, Denmark). The SCA® was provided by a grant from ‘Ferring Pharmaceuticals’ to Aalborg University Hospital (H.I.N). G.V.D.H. is an external senior scientific consultant to Microptic S/L (Barcelona, Spain). H.A. has provided scientific input and presentations for Microptic S/L (Barcelona, Spain) on several occasions. All other authors declare no conflict of interest.

doi: 10.1093/humrep/dex101

Sperm structure and sperm motility of the African and Rockhopper penguins2017-05-24T16:17:51+02:00

Sperm structure and sperm motility of the African and Rockhopper penguins with special reference to multiple axonemes of the flagellum

Patrick Siyambulela Mafundaa; Liana Maree; Antoinette Kotze; Gerhard van der Horst

Department of Medical Bioscience, University of the Western Cape, Bellville, South Africa; Department of Research and Scientific Services, National Zoological Gardens of South Africa, Pretoria, South Africa; Genetics Department, University of the Free State, Bloemfontein, South Africa

Abstract: This study evaluated the semen of two penguin species from separate genera with reference to unique features in sperm structure using light microscopy and transmission electron microscopy. Ejaculates from African penguin (n = 51) and Rockhopper penguin (n = 9) contained on average more than 60% motile spermatozoa and a sperm concentration of 3274 × 106/ml and 1423 × 106/ml, respectively. The percentage progressive motility was similar for the two species as well as all the kinematics parameters. The sperm morphology of these two penguin species is almost identical and largely resembles that of non-passerine birds in terms of the filiform head, small acrosome and mid-piece containing 13 spherical mitochondria, arranged around the proximal and distal centrioles in a single helix. Apart from a shorter mid-piece, penguin sperm morphometrics were similar to other non-passerine birds. The ultrastructure of the sperm principal piece revealed the typical 9 + 2 microtubular arrangement without any outer dense fibres. An unusual feature in both African and Rockhopper penguin spermatozoa was the occurrence of multiple axonemes contained in one plasmalemma in 4% of spermatozoa. These double, triple and quadruple axonemal arrangements have not been described previously albeit multiple tails were reported in other bird species. It is unclear whether such a unique structural feature will be of any advantage for sperm motility and might rather be a result of the absence of sperm competition. Multiple axonemes found in penguin flagella could be an apomorphism that distinguish them from other bird spermatozoa.

https://doi.org/10.1016/j.theriogenology.2017.05.009

Reproductive toxicity after levetiracetam administration in male rats: Evidence for role of hormonal status and oxidative stress2022-05-04T15:57:07+02:00

Reproductive toxicity after levetiracetam administration in male rats: Evidence for role of hormonal status and oxidative stress

Merve Baysal; Sinem Ilgin; Gozde Kilic; Volkan Kilic; Seyda Ucarcan; Ozlem Atli

Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Anadolu University, Eskisehir, Turkey; Department of Biology, Faculty of Science, Anadolu University, Eskisehir, Turkey.

Abstract: Levetiracetam (LEV) is an antiepileptic drug commonly used in the treatment of epilepsy because of its excellent safety profile in all age groups. It is remarkable that there are no studies evaluating the toxic effects of this drug on the male reproductive system, as it is commonly used in male patients of reproductive age. From this point of view, our aim was to evaluate the possible toxic effects of LEV on the male reproductive system. Therefore, LEV was administered to male rats orally at 50, 150, and 300 mg/kg for 70 consecutive days. At the end of this period, alterations to body and organ weights were calculated, and sperm concentration, motility, and morphology were investigated by a computer-assisted sperm analysis system. Sperm DNA damage was determined by comet assay and histopathological examination of the testes was carried out. Serum testosterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels were measured by ELISAs to determine the effects of hormonal status, while glutathione, superoxide dismutase, catalase, and malondialdehyde levels in the testes were measured by colorimetric assay kits to determine the role of oxidative status in potential toxicity. According to the results, sperm quality was decreased by LEV treatment in a dose-dependent manner. LEV induced significant DNA damage in the 150 and 300 mg/kg LEV-administered groups. Histopathology of the testes showed that LEV resulted in testicular injury in the 300 mg/kg LEV-administered group. Serum testosterone, FSH, and LH levels were significantly decreased in the 300 mg/kg LEV-administered group. Glutathione, superoxide dismutase, and catalase levels were significantly decreased in all experimental groups while malondialdehyde levels were significantly increased in 150 and 300 mg/kg LEV-administered groups. According to these results, it was determined that LEV administration decreased sperm quality and it was alleged that hormonal alteration and oxidative stress are potential contributors to reproductive toxicity.

https://doi.org/10.1371/journal.pone.0175990

Sperm Chromatin Dispersion Test before Sperm Preparation Is Predictive2016-11-23T15:27:16+01:00

Sperm Chromatin Dispersion Test before Sperm Preparation Is Predictive of Clinical Pregnancy in Cases of Unexplained Infertility Treated with Intrauterine Insemination and Induction with Clomiphene Citrate

Frank W. R. C. Vandekerckhove, Ilse De Croo, Jan Gerris, Etienne Vanden Abbeel and Petra De Sutter

Centre for Reproductive Medicine, University Hospital, Ghent, Belgium.

Background/aims: A large proportion of men with normal sperm results as analyzed using conventional techniques have fragmented DNA in their spermatozoa. We performed a prospective study to examine the incidence of DNA fragmentation in sperm in cases of couples with previously unexplained infertility and treated with intrauterine insemination. We evaluated whether there was any predictive value of DNA fragmentation for pregnancy outcome in such couples.

Methods: The percentage of DNA fragmentation and all classical variables to evaluate sperm before and after sperm treatment were determined. We studied the probable association between these results and pregnancy outcome in terms of clinical and ongoing pregnancy rate per started first cycle. We also assessed the optimal threshold level to diagnose DNA fragmentation in our center.

Results: When using threshold levels of 20, 25, and 30%, the occurrence of DNA fragmentation was 42.9, 33.3, and 28.6%, respectively. Receiver operating characteristic (ROC) analysis of all cases revealed an area under the curve of 80% to predict the clinical pregnancy rate per cycle from testing the sperm motility (a + b) before treatment. We failed to generate an ROC curve to estimate pregnancy outcome from the amount of DNA fragmentation before treatment. However, when selecting only those men with a pretreatment DNA fragmentation of at least 20%, the pretreatment result was statistically different between couples who achieved a clinical pregnancy and those who did not.

Conclusion: DNA fragmentation is often diagnosed in couples with unexplained infertility. Each center should evaluate the type of test it uses to detect DNA fragmentation in sperm and determine its own threshold values.

doi: 10.3389/fmed.2016.00063

Standardization of pretreatment and staining technique to perform automatic human sperm morphology with ASMA2016-07-01T15:21:46+02:00

Standardization of pretreatment and staining technique to perform automatic human sperm morphology with ASMA (assisted sperm morphometry analyses)

Clara Ramírez, José Ramón Alonso, Pedro Jiménez, Rocío Reyes, Jordi Ramis, Josep Maria Gris, Carlos Aulesa

Unidad de Andrología, Laboratorios Clínicos, Hospital Universitario Vall d’Hebron, Barcelona, Spain; Servicio de Reproducción Asistida, Hospital Universitario Vall d’Hebron, Barcelona, Spain

Abstract

Objective: Standardization ofpretreatment and staining technique to realizemorphological semen evaluation with computer-assisted sperm morphometry analysis system, with semen system analyzer SCA 5.4 (Sperm Class Analyzer, Microptic).

Material and methods: Morphological analysis was performed with semen system analyzer SCA 5.4 (Microptic SL, Barcelona, Spain). The staining method was a modification of Hemacolor kit (Merck). Between 60 and 125 semen samples were chosen randomly from our laboratory.

Results: Pretreatment of semen samples was a centrifugation for 5 minutes at 300 g, seminal plasma was rejected and the pellet was homogenized with .2 mL of the seminal plasma itself. Not change in sperm morphology have been found with this pretreatment. Staining was performed with Hemacolor kit (Merck) but with some modifications. Fixer has been buffered with phosphate buffer pH 7.2 at 10%, time recommended by the manufacturer has been reduced. Fixation for 5 seconds, 30 seconds with Eosin staining and 2 seconds with staining Azur. Finally it was washed for 30 seconds with pH 7.2 phosphate buffer, as indicated by the manufacturer. After the pretreatment and staining we have got reference values for our methodology.

Conclusions: An automation methodology to perform sperm morphology reduces coefficient variations of determination thereby we can increase its technical reliability and remove the subjectivity of the manual analysis. This standardization can be the first step to study the diagnostic value of advanced morphology in infertility and urological diseases.

DOI: 10.1016/j.androl.2016.05.002

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Unraveling the sperm bauplan: relations between sperm head morphology and sperm function in rodents2016-12-30T09:48:19+01:00

Unraveling the sperm bauplan: relations between sperm head morphology and aperm function in rodents

María Varea-Sánchez; Maximiliano Tourmente; Markus Bastir; Eduardo R.S. Roldan

Department of Biodiversity and Evolutionary Biology, Museo Nacional de Ciencias Naturales (CSIC), Madrid, Spain; Department of Paleobiology, Museo Nacional de Ciencias Naturales (CSIC), Madrid, Spain.

Rodents have spermatozoa with features not seen in other species. Sperm heads in many rodent species bear one or more apical extensions, known as «hook(s)». The process by which hooks have evolved, together with their adaptive significance, are still controversial issues. In order to improve our understanding of the biological meaning of these sperm head adaptations, we analyzed hook curvature angles, hook length and overall hook shape in muroid rodents by using geometric morphometrics. We also searched for relationships between hook design and measures of inter-male competition to assess if postcopulatory sexual selection is an important selective force driving changes in this sperm structure. Finally, we sought possible links between aspects of sperm hook design and sperm velocity as a measure of sperm performance. Results showed that one hook curvature angle is under strong selective pressure. Similarly, hook length appears to be strongly selected by sexual selection with this selective force also exhibiting a stabilizing role reducing inter-male variation in this trait. The adaptive significance of changes in hook structure was underlied by the finding that there is a strong and significant covariation
between hook dimensions and shape and between hook design and sperm swimming velocity.
Overall, this study strongly suggests that postcopulatory sexual selection has an important effect on the design of the sperm head that, in turn, is important for enhancing sperm velocity, a function crucial to reaching the vicinity of the female gamete, and winning fertilizations under competitive situations.

doi: 10.1095/biolreprod.115.138008

Ameliorative potentials of quercetin against cotinine-induced toxic effects on human spermatozoa2016-12-30T09:48:19+01:00

Ameliorative potentials of quercetin against cotinine-induced toxic effects on human spermatozoa

Dale Goss; Ibukun P. Oyeyipo; Bongekile T. Skosana; Bashir M. Ayad; Stefan S. du Plessis

Division of Medical Physiology. Faculty of Medicine and Health Sciences. Stellenbosch University. South Africa; Department of Physiology. College of Health Sciences. Osun State University. Osogbo. Osun State. Nigeria.

OBJECTIVES: Cotinine, the principal metabolite of nicotine found in smokers’ seminal plasma, has been shown to adversely affect sperm functionality while quercetin, a flavonoid with diverse properties is associated with several in vivo and in vitro health benefits. The aim of this study was to investigate the potential benefits of quercetin supplementation against damage caused by the by-products of tobacco smoke in human sperm cells.
METHODS: Washed human spermatozoa from 10 normozoospermic donors were treated with nutrient medium (control), quercetin (30 mmol/L) and cotinine (190 mg/mL, 300 ng/mL) with or without quercetin for 60 and 180 min incubation periods. Computer-aided sperm analysis was used to assess sperm motility while acrosomereacted cells were identified under a fluorescent microscope using fluorescein isothiocyanate-labelled Pisum Sativum Agglutinin as a probe, viability was assessed by
means of a dye exclusion staining technique (eosin/nigrosin) and oxidative stress by flow cytometry using dihydroethidium as a probe. Values were expressed as mean ± S.E.M. as compared by ANOVA.
RESULTS: Higher cotinine concentrations reduced the number of viable cells after 60 and 180 min of exposure while viability of cells was increased in the cotinine aliquots supplemented with quercetin after 180 min of exposure when compared with cotinine only treated group.
Conclusion: This study indicates that the ameliorating ability of quercetin on cotinineinduced decline in sperm function is associated with increased number of viable cells.

doi:10.1016/j.apjr.2016.03.005

Thyroxine is useful to improve sperm motility2016-03-01T00:00:59+01:00

Thyroxine is useful to improve sperm motility

Gabriela Ruth Mendeluk. Ph.D.; Mónica Rosales M.D.

Laboratory of Male Fertility. Hospital de Clínicas “José de San Martín”. Faculty of Pharmacy and Biochemistry. University of Buenos Aires. Buenos Aires. Argentina; Laboratory of Endocrinology. Hospital de Clínicas “José de San Martín”. Faculty of Pharmacy and Biochemistry. University of Buenos Aires. Buenos Aires. Argentina.

BACKGROUND: The aim of this study was to evaluate the non-genomic action of thyroxine on sperm kinetic and its probable use to improve sperm recovery after an enrichment method like “swim-up” in comparison to the available one pentoxifylline.
MATERIAL AND METHODS: A total of 50 patients consulting for infertility were recruited. Conventional sperm assay were performed according to WHO criteria- 2010. A CASA system was employed to assess kinetic parameters and concentration. The number of motile sperm recovered after the preparation technique was calculated.
RESULT(S): Addition of T4 (0.002ug/ml) to semen samples increased hypermotility at 20 min(control: 14.18 ± 5.1% Vs. 17.66 ± 8.88 % ; p<0.03; data expressed as mean ± SD )and remained unchanged after 40 min. Significant differences were found in the motile sperm recovered after “swim-up” (control: 8.93 x 106 ± 9.52 x 106 Vs. 17.20 x 106 ± 21.16 x106; p<0.03), achieving all the tested samples the desired threshold value for artificial insemination outcome, while addition of pentoxifylline increased the number of recovered sperm after swim-up in 60% of the studied cases. No synergism between treatments could be proved.
CONCLUSION(S): We are proposing a new and physiologic tool to improve artificial insemination. The discussion opens our minds to think in unknown pathways involved in sperm capacitation and gives innovative arguments to understand infertility.

Computer Aided Sperm Analysis, a useful tool to evaluate patient’s response to varicocelectomy2016-03-01T00:00:28+01:00

Computer Aided Sperm Analysis, a useful tool to evaluate patient’s response to varicocelectomy

Julia I Ariagno, Gabriela R Mendeluk, María J Furlan, M Sardi, P Chenlo, Susana M Curi, Mercedes N Pugliese, Herberto E Repetto, Mariano Cohen

Departamento de Bioquímica Clínica, Laboratorio de Fertilidad Masculina, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires (CP1120), Argentina; Departamento de Urologia, Hospital de Clinicas “José de San Martín”, Universidad de Buenos Aires, Buenos Aires (CP 1120), Argentina

Pre-operative and post-operative sperm parameter values from infertile men with varicocele were analysed by computer-aided sperm analysis (CASA) to assess if sperm characteristics improved after varicocelectomy. Semen samples of men with proven fertility (n = 38) and men with varicocele-related infertility (n = 61) were also analysed. Conventional semen analysis was performed according to WHO (2010) criteria and a CASA system was employed to assess kinetic parameters and sperm concentration. Seminal parameters values in the fertile group were very far above from those of the patients, either before or after surgery. No significant improvement in the percentage normal sperm morphology (p=0.10), sperm concentration (p = 0.52), total sperm count (p = 0.76), subjective motility (%) (p = 0.97) nor kinematics (p = 0.30) was observed after varicocelectomy when all groups were compared. Neither was significant improvement found in percentage normal sperm morphology (p = 0.91), sperm concentration (p = 0.10), total sperm count (p = 0.89) or percentage motility (p = 0.77) after varicocelectomy in paired comparisons of pre-operative and post-operative data. Analysis of paired samples revealed that the total sperm count (0.01) and most sperm kinetic parameters: curvilinear velocity (p = 0.002), straight-line velocity (p = 0.0004), average path velocity (p = 0.0005), linearity (p = 0.02), and wobble (p = 0.006) improved after surgery. CASA offers the potential for accurate quantitative assessment of each patient’s response to varicocelectomy.

Asian Journal of Andrology.
Manuscript ID: AJA-5138

Evaluation of sperm motility using two cryoprotectants2016-12-30T09:48:19+01:00

Evaluation of sperm motility using two cryoprotectants

Sandra Abalde Graña; Mª Esther Rendal Vázquez; Mariana García García; Mª Jesús López Piñón; Marcos Carballal Rodríguez; Catarina Barbero Cancelo; Rosario Olvido Fernández Mallo; Inma Míguez Torre; Teresa Bermúdez González; Sonia Pértega Díaz; Jacinto Sánchez Ibáñez

Unidad de Criobiología-Banco de Tejidos. CHUAC. A Coruña; Unidad de Fecundación in vitro. CHUAC. A Coruña; Unidad de Estadística. CHUAC. A Coruña

Infertility is a problem that affects a lot of couples. One of its causes is a decreased semen quality due to, for example, gonadotoxic treatments. The cryopreservation of human semen is the technique that allows sperm preserving and storing without losing their fertilizing capacity; being a fundamental tool in assisted reproduction. The aim of this study was to optimize the cryopreservation technique. To this end, a study carried out on samples of patients under study by fertility problems, in which two cryoprotectant media (SpermCryo™ All-round and CryoSperm™) and the execution or non-execution of an immersion of the samples in liquid nitrogen (before storage) were compared; and the effect of the time between ejaculation and the processing on the quality of the sample. Variations were studied with an automatic analyzer by performing pre- and post-thaw sperm motility tests.
The results show no difference between the two cryoprotectants media, but seems to have a tendency to obtain better postthaw mobility with either depending on sample characteristics. Moreover, the liquid nitrogen bath had no apparent effects on post-thaw results. However, we must highlight the importance of time in the processing of semen samples once liquefied, to avoid decreased sperm quality.
To improve post-thaw outcomes the key lies in the necessity to adjust the freezing protocol to the sample characteristics and a correct implementation of the protocol cryopreservation (selection and addition of cryoprotectant media…); favoring the management of infertility and the success of assisted reproduction techniques.

Rev. Iberoam. Fert Rep Hum, 2016

Computer Assisted Semen Analysis, an overcoming tool2016-12-30T09:48:19+01:00

Computer Assisted Semen Analysis, an overcoming tool

Ariagno Julia I, Chenlo Patricia H, Mendeluk Gabriela R

Laboratorio de Fertilidad Masculina. Hospital de Clínicas “José de San Martín. Facultad de Farmacia Bioquímica. U.B.A., Argentina

Sperm motility is a key parameter while assessing semen quality. It provides information about sperm energy state, a requisite needed to achieve the active movement that enables the gamete to reach the oviduct site were fertilization takes place. The standard method to assess motility is subjective; it estimates speeds thus having a great inter-laboratory variation. In fact its use is against to the current tendency oriented to objectify all the biochemical studies.
Development of Assisted Computer Semen Analysis (CASA) has not only helped to increase the assessment accuracy and reliability, but has also provided much more information than that obtained by the conventional subjective method. However, certain factors (eg the operator, optics, software configuration, etc.) can affect the results obtained by the use of this technology. In accordance with current regulations we validated the CASA-SCA modules for mobility and concentration, before its use in the andrology clinic for evaluation of patient´s samples. Accuracy, method detection limit and reportable range were determined; a study on method comparison was done and the reference values were verified.
We want to highlight that CASA does not supplant the work of the expert who necessarily has to edit the images and who finally has to validate the clinical report. They can not predict semen sample «fertility». However, properly validated they provide detailed and accurate data useful for ejaculate characterization, exceeding the information that has so far been given by the subjective method.

SAEGRE

Comparative evaluation of Nabi and Beltsville extenders for cryopreservation of rooster semen2016-12-30T09:48:19+01:00

Comparative evaluation of Nabi and Beltsville extenders for cryopreservation of rooster semen

Nabi MM, Kohram H, Zhandi M, Mehrabani-Yeganeh H, Sharideh H, Zare-Shahaneh A, Esmaili V

Department of Animal Science, Faculty College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran; Department of Animal Science, Faculty College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran; Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahid Chamran University, Ahvaz, Iran; Department of Animal Science, Faculty College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran; Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran

Two experiments were conducted to evaluate the new rooster semen freezing extender which is containing a low level of glycerol and soybean lecithin as an alternative protective agent in the extender. The aim of the first experiment was to evaluate a new extender for freeze-thawing rooster semen known as «Nabi» extender compared to Beltsville. Second experiment was also performed to determine whether the Nabi extender has negative reactions on fertilization after artificial insemination (AI) or no. In the first experiment, post-thaw motion parameters, mitochondrial function and sperm apoptosis were analyzed using Sperm Class Analyzer (SCA), rhodamine-123 and Annexin-V, respectively for frozen-thawed semen in Nabi and Beltsville extender. Results showed that total motility, progressive motility, velocity parameters (VCL, VSL, VAP, LIN and STR) and live spermatozoa with active mitochondria were significantly higher in Nabi compare to Beltsville extender (P < 0.01). Also, the percentages of post-thawed live and early apoptotic spermatozoa were significantly higher in Nabi compared to Beltsville extender (14.46 ± 0.95 vs. 19.27 ± 0.95 and 14.83 ± 4.51 vs. 39.27 ± 4.51, respectively). For apoptotic spermatozoa, the percentages of post-thawed late apoptotic spermatozoa were significantly lower in Nabi (29.66 ± 3.11) compared to Beltsville extender (69.07 ± 3.11), but the type of extender had no effect on the percentages of post-thawed necrotic spermatozoa. In the second experiment, 20 broiler breeder hens (Ross 308) were inseminated with thawed semen using the new freezing diluents or fresh semen for determination of fertility rate. Fertility rate with thawed semen (with Nabi extender) was lower compared to fresh semen (by approximately 8% points). It can be concluded that Nabi extender would improve post-thawed rooster sperm in vitro quality compared to Beltsville extender. The fertility rates of insemination in hens with freeze-thaw sperm were comparable with fresh sperm.

doi: 10.1016/j.cryobiol.2015.11.005

Effect of inbreeding depression on bull sperm quality and field fertility2017-03-23T16:48:31+01:00

Effect of inbreeding depression on bull sperm quality and field fertility

Jesús Dorado; Rosa Morales Cid; Antonio Molina; Manuel Hidalgo; Julia Ariza; Miguel Moreno-Millán and Sebastián Demyda-Peyrás

Department of Animal Medicine and Surgery, University of Cordoba, Cordoba, Spain; Department of Genetics, University of Cordoba, Cordoba, Spain; Department of Cell Biology, Physiology and Immunology, University of Cordoba, Cordoba, Spain; Instituto de Genetica Veterinaria (IGEVET), CCT La Plata, CONICET – Facultad de Ciencias Veterinarias – Universidad de La Plata, Buenos Aires, Argentina.

Abstract: The present study investigated the effect of inbreeding depression on sperm quality using automated and objective methods and subsequent effects on beef bull field fertility. Individual inbreeding coefficient (F) values and field fertility data were determined using a dataset of AI bulls belonging to the Spanish Retinta Breeders Association (Asociación Nacional de Criadores de Ganado Vacuno Selecto de Raza Retinta (ANCRE)). Animals were clustered in two groups according to the F values as follows: (1) a high inbreeding group (HI; F ≥ 13.5%, mean 16.3); and (2) a non-inbreeding group (NI; F = 0%). In total, 17 different assessments were performed in both experimental groups, including evaluation of sperm morphology, acrosomal and DNA status, sperm plasma membrane integrity and function (hypo-osmotic swelling test), 10 kinetic parameters and the structure of sperm subpopulations. Sperm morphology, acrosomal and DNA status and osmotic tolerance were similar in both groups. Three velocity parameters (curvilinear velocity, straight line velocity and average path velocity) and the amplitude of lateral head displacement were higher in HI (P < 0.05). Cluster analysis of kinematic parameters revealed three different sperm subpopulations (sP1, sP2 and sP3), with the proportion of the sP1 population (highly active but non-progressive spermatozoa) being significantly (P < 0.05) higher in the HI group. Field fertility was assessed using two calving record datasets. In a smaller database including only bulls evaluated in the present study, there was a significant increase in the calving interval of cows sired with HI bulls. Conversely, in an extended genetic analysis of the ANCRE database, inbreeding only explained a small part of the variation in calving interval, and the results of regression analysis were not significant among bulls. The findings of the present study suggest that high inbreeding levels have a moderate effect on bull semen quality, with an increased percentage of highly active but non-progressive spermatozoa, but only when F values reached a certain threshold. This motility pattern could explain, in part, the higher calving interval produced by inbred bulls under field conditions.

http://dx.doi.org/10.1071/RD15324

Comparison of two histologic stains in the evaluation of sperm head morphometric measurements in frozen-thawed bull semen2016-12-30T09:48:19+01:00

Comparison of two histologic stains in the evaluation of sperm head morphometric measurements in frozen-thawed bull semen

A. Quintero-Moreno, M. L. Ramirez, H. Nava-Trujillo, M. Hidalgo

Laboratorio de Andrología, Unidad de Investigación en Producción Animal (UNIPA). Universidad del Zulia. Facultad de Ciencias Veterinarias. Apdo. 15252, Maracaibo 4005-A. Venezuela; Animal Reproduction Group, Faculty of Veterinary Science, University of Cordoba, 14071 Cordoba, Spain

This study was designed to compare the performance of the kit Hemacolor (HC) in two protocols (A, B) and Toluidine blue stain (TB) for staining the bull sperm head in samples of frozen-thawed semen. Automated Sperm Morphology Analysis (ASMA) was performed to determine the sperm measurements: head size (length, width, area and perimeter). TB was found to be the best procedure for staining the frozen-thawed bull sperm (p<0.0001). The use of this method rendered the highest number of cells correctly analyzed (88.29%) and the lowest coefficient of variation on the image processing (4.54) and morphometric measurements. TB provided good colour intensity and optimum contrast of the sperm head with the surrounding background that allows efficient boundary detection and reduces the number of stained foreign particles. The staining methods affected significantly the sperm head dimensions (p<0.0001) with increased values from HC (protocol A) than HC (protocol B) and TB, respectively (HC>TB). HC provide more intense grey-level values, resulting in enlarged cells, which influence the size morphometric parameters. Based on these findings, we recommend TB for its accurate and reproducible morphometric results.

Acta Microscopica Vol. 24, No. 2, 2015, pp. 103-110

Effect of different media additives on capacitation of frozen-thawed ram spermatozoa as a potential replacement for estrous sheep serum2016-12-30T09:48:19+01:00

Effect of different media additives on capacitation of frozen-thawed ram spermatozoa as a potential replacement for estrous sheep serum

García-Álvarez O, Maroto-Morales A, Jiménez-Rabadán P, Ramón M, Del Olmo E, Iniesta-Cuerda M, Anel-López L, Fernández-Santos MR, Garde JJ, Soler AJ

SaBio IREC (CSIC-UCLM-JCCM), Campus Universitario, Albacete, Spain; Regional Center of Animal Selection and Reproduction (CERSYRA) JCCM, Valdepeñas, Spain; SaBio IREC (CSIC-UCLM-JCCM), Campus Universitario, Albacete, Spain

Capacitation is a key process through which spermatozoa acquire their fertilizing ability. This event is required for the successful application of assisted reproductive technologies such as IVF. The aim of the present study was to investigate the effect of using a synthetic oviductal fluid medium supplemented with either heparin-hypotaurine alone, in combination with progesterone (P4), 17β-estradiol (E2), or BSA, or just β-cyclodextrin, in replacement for estrous sheep serum (ESS) for ram sperm capacitation. After incubation in the corresponding media for 15 (time 0) or 60 minutes, sperm function was evaluated by computerized sperm motility analysis and flow cytometry (plasma membrane status and fluidity). Treatments rendering the best results in regards to sperm function parameters related to capacitation were used for an IVF test. Herein, neither heparin-hypotaurine (alone), or in combination with P4, or E2, nor β-cyclodextrin induced capacitation-related changes in frozen-thawed ram spermatozoa. Only the medium supplemented with heparin-hypotaurine-BSA was able to induce changes compatible with in vitro capacitation relating to sperm motility pattern and plasma membrane fluidity, comparable to those in ESS-containing medium. Both media yielded sperm parameter values that differed (P < 0.05) from those obtained in the rest of the media tested. However, after the IVF trial, BSA was unable to support cleavage rates (21.80%) comparable to those obtained with ESS (52.60%; P < 0.05). We conclude that heparin-hypotaurine, P4, E2, β-cyclodextrin, or BSA is not suitable for replacing ESS in capacitation and fertilization media for ram spermatozoa.

doi: 10.1016/j.theriogenology.2015.05.032

Mass-specific metabolic rate influences sperm performance through energy production in mammals2016-12-30T09:48:19+01:00

Mass-specific metabolic rate influences sperm performance through energy production in mammals

Tourmente M, Roldan ER

Reproductive Ecology and Biology Group, Museo Nacional de Ciencias Naturales (CSIC), Madrid, Spain

Mass-specific metabolic rate, the rate at which organisms consume energy per gram of body weight, is negatively associated with body size in metazoans. As a consequence, small species have higher cellular metabolic rates and are able to process resources at a faster rate than large species. Since mass-specific metabolic rate has been shown to constrain evolution of sperm traits, and most of the metabolic activity of sperm cells relates to ATP production for sperm motility, we hypothesized that mass-specific metabolic rate could influence sperm energetic metabolism at the cellular level if sperm cells maintain the metabolic rate of organisms that generate them. We compared data on sperm straight-line velocity, mass-specific metabolic rate, and sperm ATP content from 40 mammalian species and found that the mass-specific metabolic rate positively influences sperm swimming velocity by (a) an indirect effect of sperm as the result of an increased sperm length, and (b) a direct effect independent of sperm length. In addition, our analyses show that species with higher mass-specific metabolic rate have higher ATP content per sperm and higher concentration of ATP per μm of sperm length, which are positively associated with sperm velocity. In conclusion, our results suggest that species with high mass-specific metabolic rate have been able to evolve both long and fast sperm. Moreover, independently of its effect on the production of larger sperm, the mass-specific metabolic rate is able to influence sperm velocity by increasing sperm ATP content in mammals.

doi: 10.1371/journal.pone.0138185

Nutrition and Reproductive Health: Sperm versus Erythrocyte Lipidomic Profile and -3 Intake2016-12-30T09:48:19+01:00

Nutrition and Reproductive Health: Sperm versus Erythrocyte Lipidomic Profile and -3 Intake

Gabriela Ruth Mendeluk, Mariano Isaac Cohen, Carla Ferreri, and Chryssostomos Chatgilialoglu

Laboratory of Male Fertility, Hospital de Cl´ınicas “Jos´e de SanMart´ın”, INFIBIOC, Faculty of Pharmacy and Biochemistry, University of Buenos Aires, 5950-800 Buenos Aires, Argentina; Urology Division, Hospital de Cl´ınicas “Jos´e de SanMart´ın”, University of Buenos Aires, 5950-800 Buenos Aires, Argentina; Consiglio Nazionale delle Ricerche (CNR), Istituto per la Sintesi Organica e la Fotoreattivit`a (ISOF), 40129 Bologna, Italy; Institute of Nanoscience and Nanotechnology, National Center of Scientific Research “Demokritos”, Agia Paraskevi, 15310 Athens, Greece

Fatty acid analyses of sperm and erythrocyte cell membrane phospholipids in idiopathic infertile patients evidenced that erythrocyte contents of EPA, DHA, omega-6–omega-3 ratio and arachidonic acid provide a mathematical correspondence for the prediction of EPA level in sperm cells. The erythrocyte lipidomic profile of patients was significantly altered, with signatures of typical Western pattern dietary habits and no fish intake. A supplementation with nutritional levels of EPA and DHA and antioxidants was then performed for 3 months, with the follow-up of both erythrocyte and sperm cell membranes composition as well as conventional sperm parameters. Some significant changes were found in the lipidomic membrane profile of erythrocyte but not in sperm cells, which correspondently did not show significant parameter ameliorations. This is the first report indicating that membrane lipids of different tissues do not equally metabolize the fatty acid elements upon supplementation. Molecular diagnostic tools are necessary to understand the cell metabolic turnover and monitor the success of nutraceuticals for personalized treatments.

doi: 10.1155/2015/670526

Effect of environmental pH on sperm kinematic characteristics2020-01-07T10:05:04+01:00

Effect of environmental pH on sperm kinematic characteristics

H. Alipour, F. Dardmeh, M.C. Dissing, H.I. Nielsen

Laboratory of Reproductive biomedicine, Biomedicine Group, Department of Health Science and Technology, Faculty of Medicine, Aalborg University, Aalborg, Denmark; ORIGIO A/S, Maaloev, Denmark

Semen preparation medium have an important role in assisted reproduction techniques and their composition influences sperm binding and motility.
Some studies have assessed the influence of pH on sperm kinetics. However, no study to date has assessed the effect of environmental pH on subtle differences in the details of the sperm movement (kinematics) of human sperm provided by computerized sperm analysis systems. This study was designed to assess the effect of two different media pH levels on kinematic parameters of the human sperm.
Samples were prepared using the 40%/80% Pureception (Sage, USA) density gradient and resuspended in customized sperm culture media with pH levels of 7.9 and 8.3 (Origio, Denmark). Kinematic parameters of the sperm in both groups were analyzed using the Sperm Class Analyzer (Microptic S.L., Spain) at 0, 6 and 24 hours following the addition of media.
Results of this study illustrated a general insignificant decrease in the ratio of progressively motile and hyperactive sperm after 6 and 24 hours. However a significant difference between the test groups was observed in the curvilinear, straight line and Mean path velocity and Straightness after 6 and 24 hours. Linearity and Wobble showed significant difference after 24 hours.
This study demonstrated a difference in the sperm motion pattern and velocity in different environmental pH levels. Based on these findings, further investigations are required to elucidate knowledge about possible effect of marginal pH changes, affecting the sperm motion characteristics at different stages in the dynamic environment of the female reproductive tract.

Dietary probiotic supplement positively affects sperm motility in an obese model2016-12-30T09:48:19+01:00

Dietary probiotic supplement positively affects sperm motility in an obese model

F. Dardmeh, H. Alipour, P. Gazerani, G. van der Horst, B. Kjærgaard, E. Brandsborg, H.I. Nielsen

Biomedicine Group, Department of Health Science and Technology,, Faculty of Medicine, Aalborg University, Aalborg, Denmark; SMI®, Department of Health Science and Technology, Faculty of Medicine, Aalborg University, Aalborg, Denmark; Department of Medical Bioscience, University of the Western Cape, Bellville, South Africa; Department of Clinical Medicine, The Faculty of Medicine, Aalborg University Hospital, Aalborg, Denmark; Bifodan A/S, Hundested, Denmark

Obesity in adult men in recent years has inconsistently been associated with low semen quality and sub-fecundity. Probiotics have gained high interest as alternatives to pharmacological compounds. However, their possible effect on male fertility has been less investigated. This study aimed at assessing the use of L.Rhamnusus on obese male fertility characteristics. We proposed that this probiotic can not only reduce the weight but in parallel would enhance sperm motility in obese male mice.
Diet-induced obese C57BL/6NTac mice were randomly assigned to 2 groups and treated with a single daily dose (1x109CFU) of L.Rhamnusus (test group) or physiological saline (control group) for 4 weeks. Sperm motility and kinematics were assessed by the Sperm Class Analyzer (SCA).
The control group maintained a raising trend in weight gain leading to a significant difference on week 5 continuing to week 8 whereas the DIO mice in the test group did not gain significant weight after the start of probiotic test. The test group showed a significantly higher progressive motility compared to the control group after 4 weeks of receiving the probiotic treatment.
L.Rhamnusus supplementation demonstrated a higher percentage of progressive sperm, suggesting a possible increase in pregnancy. The effect mechanism of L.Rhamnusus could be either through direct influence on sperm motility or indirectly due to the promotion of weight loss. The latter hypothesis is due to the fact that weight-loss leads to scrotal temperature decrease and hormonal balance affecting sperm kinetics and kinematics during maturation in the epididymis. These hypotheses require further investigation.

Effect of short abstinence time on sperm motility parameters2016-12-30T09:48:19+01:00

Effect of short abstinence time on sperm motility parameters

H. Alipour, F. Dardmeh, G. Van Der Horst, G. Manoharan, A. Askeland, H.I. Nielsen

Laboratory of Reproductive Biomedicine, Biomedicine group, Dept. of Health Science and Technology, Aalborg University, Aalborg, Denmark; Department of Medical Biosciences, University of the Western Cape, Cape town, South Africa

The importance of establishing an optimal period of sexual abstinence has been an area of focus due to its influence on sperm quality. The ”WHO laboratory manual for the examination and processing of human semen” suggests an ejaculatory abstinence of 2-7 days before sperm collection. Several previous studies have evaluated the effect of abstinence period on sperm motility and presented controversial results, making the WHO recommendation debatable. However, no study to date has assessed the effect of a short (2 hours) abstinence period on sperm quality. The present study compared the sperm motility (as a biomarker of sperm quality) of samples collected after 2-7 days of abstinence with a consecutive ejaculation after 2 hours. The neat samples were analysed using the Sperm Class Analyzer (Microptic S.L., Spain) computer assisted sperm analysing system. The results displayed a significant increase in the ratio of progressively motile spermatozoa and a significant decrease in the percentage of immotile sperm after 2-hours of abstinence compared to a 2-7 day abstinence period.
Based on the results of this study, decreasing the abstinence time to as low as 2 hours could provide a better chance for the treatment of patients with male factor infertility. Further investigation is still required in order to compare the effect of abstinence time on additional sperm quality parameters.

Influence of two commercially available lubricants on sperm motility and kinematics2016-12-30T09:48:19+01:00

Influence of two commercially available lubricants on sperm motility and kinematics

H.I. Nielsen, F. Dardmeh, K. Kannik, A.B.K. Jeppesen, L. Søresen, M.B. Hjelm, H. Alipour

Department of Health Science and Technology, Biomedicine Group, Faculty of Medicine, Aalborg University, Aalborg, Denmark

The use of lubricants is occasionally requested during intercourse and semen collection for assisted reproduction techniques. This might pose a problem because several lubricants have been reported to decrease sperm functional parameters, especially in regards to sperm motility. This study aimed to examine the influence of 2 commercially available personal lubricants, a water-based (Apotekets Glid) and a silicone-based (Klick Silk Glide), on human sperm motility. Semen samples were divided into three equal parts and added to the two lubricant test groups (Sperm Preparation Medium + 10 % lubricant) and a control group containing only medium. A detailed analysis of motility and different kinematic parameters was performed using the Sperm Class Analyzer® computer-aided sperm analysis system at 0, 0.5, 1, and 3 hours on the control and test groups.
The silicone-based lubricant demonstrated no significant difference compared to the control group. The water-based lubricant, however, had significantly lower fast progressive motility, total progressive motility, curvilinear velocity, straight line velocity, average path velocity, lateral head displacement, beat cross frequency, and hyperactivation compared to the control.
The results of this study indicate that the Klick Silk Glide silicone-based lubricant is less detrimental for sperm in terms of motility and kinematic parameters which have been related to pregnancy rates. Therefore, it can be suggested that when trying to conceive, both naturally and artificially, the Klick Silk Glide lubricant could have lower adverse effects on the sperm motility parameters compared to the water-based Apotekets Glid lubricant.

Differences in ATP generation via glycolysis and oxidative phosphorylation and relationships with sperm motility in mouse species2016-12-30T09:48:19+01:00

Differences in ATP Generation Via Glycolysis and Oxidative Phosphorylation and Relationships with Sperm Motility in Mouse Species

Tourmente M, Villar-Moya P, Rial E, Roldan ER

Reproductive Ecology and Biology Group, Museo Nacional de Ciencias Naturales (Consejo Superior de Investigaciones Científicas), 28006 Madrid; Mitochondrial Bioenergetics Research Group, Centro de Investigaciones Biológicas (Consejo Superior de Investigaciones Científicas), 28040 Madrid, Spain; Reproductive Ecology and Biology Group, Museo Nacional de Ciencias Naturales (Consejo Superior de Investigaciones Científicas), 28006 Madrid

Mouse sperm produce enough ATP to sustain motility by anaerobic glycolysis and respiration. However, previous studies indicated that an active glycolytic pathway is required to achieve normal sperm function and identified glycolysis as the main source of ATP to fuel the motility of mouse sperm. All the available evidence has been gathered from the studies performed using the laboratory mouse. However, comparative studies of closely related mouse species have revealed a wide range of variation in sperm motility and ATP production and that the laboratory mouse has comparatively low values in these traits. In this study, we compared the relative reliance on the usage of glycolysis or oxidative phosphorylation as ATP sources for sperm motility between mouse species that exhibit significantly different sperm performance parameters. We found that the sperm of species with higher oxygen consumption/lactate excretion rate ratios were able to produce higher amounts of ATP, achieving higher swimming velocities. Additionally, we show that the species with higher respiration/glycolysis ratios have a higher degree of dependence upon active oxidative phosphorylation. Moreover, we characterize for the first time two mouse species in which sperm depend on functional oxidative phosphorylation to achieve normal performance. Finally, we discuss that sexual selection could promote adaptations in sperm energetic metabolism tending to increase the usage of a more efficient pathway for the generation of ATP (and faster sperm).ç

doi: 10.1074/jbc.M115.664813

No evidence of trade-offs in the evolution of sperm numbers and sperm size in mammals2015-08-06T00:00:14+02:00

No evidence of trade-offs in the evolution of sperm numbers and sperm size in mammals

M. Tourmente, J. Delbarco Trillo, E.R.S. Roldan

Reproductive Ecology and Biology Group, Museo Nacional de Ciencias Naturales (CSIC), Madrid, Spain

Post-copulatory sexual selection, in the form sperm competition, has influenced the evolution of several male reproductive traits. However, theory predicts that sperm competition would lead to trade-offs between numbers and size of spermatozoa because increased costs per cell would result in a reduction of sperm number if both traits share the same energetic budget. Theoretical models have proposed that, in large animals, increased sperm size would have minimal fitness advantage compared with increased sperm numbers. Thus, sperm numbers would evolve more rapidly than sperm size under sperm competition pressure. We tested in mammals whether sperm competition maximizes sperm numbers and size, and whether there is a trade-off between these traits. Our results showed that sperm competition maximizes sperm numbers in eutherian and metatherian mammals. There was no evidence of a trade-off between sperm numbers and sperm size in any of the two mammalian clades as we did not observe any significant relationship between sperm numbers and sperm size once the effect of sperm competition was taken into account. Maximization of both numbers and size in mammals may occur because each trait is crucial at different stages in sperm’s life; for example size-determined sperm velocity is a key determinant of fertilization success. In addition, numbers and size may also be influenced by diverse energetic budgets required at different stages of sperm formation.

doi: 10.1111/jeb.12698

Sperm head phenotype and male fertility in ram semen2016-12-30T09:48:20+01:00

Sperm head phenotype and male fertility in ram semen

Maroto-Morales A, Ramón M, García-Álvarez O, Montoro V, Soler AJ, Fernández-Santos MR, Roldan ER, Pérez-Guzmán MD, Garde JJ

SaBio IREC (UCLM-CSIC-JCCM), Campus Universitario, Albacete, Spain; Regional Center of Animal Selection and Reproduction (CERSYRA) JCCM, Valdepeñas, Spain; Reproductive Ecology and Biology Group, Museo Nacional de Ciencias Naturales (CSIC), Madrid, Spain; SaBio IREC (UCLM-CSIC-JCCM), Campus Universitario, Albacete, Spain

Although there is ample evidence for the effects of sperm head shape on sperm function, its impact on fertility has not been explored in detail at the intraspecific level in mammals. Here, we assess the relationship between sperm head shape and male fertility in a large-scale study in Manchega sheep (Ovis aries), which have not undergone any selection for fertility. Semen was collected from 83 mature rams, and before insemination, head shapes were measured for five parameters: area, perimeter, length, width, and p2a (perimeter2/2×π×area) using a computer-assisted sperm morphometric analysis. In addition, a cluster analysis using sperm head length and p2a factor was performed to determine sperm subpopulations (SPs) structure. Our results show the existence of four sperm SPs, which present different sperm head phenotype: SP1 (large and round), SP2 (short and elongated), SP3 (shortest and round), and SP4 (large and the most elongated). No relationships were found between males’ fertility rates and average values of sperm head dimensions. However, differences in fertility rates between rams were strongly associated to the proportion of spermatozoa in an ejaculate SP with short and elongated heads (P < 0.001). These findings show how the heterogeneity in sperm head shape of the ejaculate has an effect on reproductive success, and highlight the important role of modulation of the ejaculate at the intraspecific level.

doi: 10.1016/j.theriogenology.2015.07.038

Comparison of different counting chambers using a computer-assisted semen analyzer2016-12-30T09:48:20+01:00

Comparison of different counting chambers using a computer-assisted semen analyzer

Nanni Peng, Xiangli Zou, and Lingwei Li

Reproductive Medical Center, People’s Hospital of Luohu District, Shenzhen, Guangdong, China

A routine computer-assisted sperm analysis is an important diagnostic test in the andrology laboratory. To evaluate the accuracy and precision of the  different types of counting chambers for human semen analysis in combination with a computer-assisted semen analyzer (CASA), a quality-control study that  compared human sperm analysis results obtained using different counting chambers (Makler chamber, disposable 8-cell GoldCyto chamber, disposable  4-cell Leja chamber, a plain glass slide, and a tissue culture dish cover with a 2424mm2 coverslip) in conjunction with the CASA systems was performed.  Significantly higher counts of sperm concentration were obtained from the reusable Makler chamber than from the other counting chambers. Sperm motility  from drop loaded counting chambers was significantly higher than that of capillary-loaded chambers. A plain glass slide and a tissue culture dish cover used  with a coverslip showed rather better performance in semen assessment. Disposable chambers are suitable for routine semen analysis with CASA in a  diagnostic andrology setting. With the proper workflow and quality control, a plain glass slide and the tissue culture dish cover are acceptable alternatives for  routine counting chambers with CASA as necessary. The type of counting chamber should be specified in test reports.

doi: 10.3109/19396368.2015.1063175

Performance of rodent spermatozoa over time is enhanced by increased ATP concentrations: The role of sperm competition2016-12-30T09:48:22+01:00

Performance of rodent spermatozoa over time is enhanced by increased ATP concentrations: The role of sperm competition

Maximiliano Tourmente, Pilar Villar-Moya, María Varea-Sánchez, Juan J. Luque-Larena, Eduardo Rial and Eduardo R. S. Roldan

Reproductive Ecology and Biology Group, Museo Nacional de Ciencias Naturales – Consejo Superior de Investigaciones Científicas, Madrid, Spain; Departamento de Ciencias Agroforestales, Universidad de Valladolid, Palencia, Spain; Department of Cellular and Molecular Medicine, Centro de Investigaciones Biológicas – Consejo Superior de Investigaciones Científicas, Madrid, Spain

Sperm viability, acrosome integrity, motility, and swimming velocity are determinants of male fertility and exhibit an extreme degree of variation among closely related species. Many of these sperm parameters are associated with sperm ATP content, which has led to predictions of tradeoffs between ATP content and sperm motility and velocity. Selective pressures imposed by sperm competition have been proposed as evolutionary causes of this pattern of diversity in sperm traits. Here, we examine variation in sperm viability, acrosome integrity, motility, swimming velocity and ATP content over time, among 18 species of closely related muroid rodents, to address the following questions: (a) Do sperm from closely related species vary in ATP content after a period of incubation? (b) Are these differences in ATP levels related to differences in other sperm traits? (c) Are differences in ATP content and sperm performance over time explained by the levels of sperm competition in these species? Our results revealed a high degree of interspecific variability in changes in sperm ATP content, acrosome integrity, sperm motility and swimming velocity over time. Additionally, species with high sperm competition levels were able to maintain higher levels of sperm motility and faster sperm swimming velocity when they were incubated under conditions that support sperm survival. Furthermore, we show that the maintenance of such levels of sperm performance is correlated with the ability of sperm to sustain high concentrations of intracellular ATP over time. Thus, sperm competition may have an important role maximizing sperm metabolism and performance and, ultimately, the fertilizing capacity of spermatozoa.

doi: 10.1095/biolreprod.114.127621

Freezability of Andalusian donkey (Equus asinus) spermatozoa: effect of extenders and permeating cryoprotectants2015-07-01T00:00:05+02:00

Freezability of Andalusian donkey (Equus asinus) spermatozoa: effect of extenders and permeating cryoprotectants

D. Acha, M. Hidalgo, I. Ortiz, M. J. Gálvez, J. J. Carrasco, V. Gómez-Arrones and J. Dorado

Veterinary Reproduction Group, Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, University of Cordoba, Campus de Rabanales (Edif. Hospital Clínico Veterinario), Ctra. Madrid-Cádiz, km 396, 14071 Córdoba, Spain; Equine Reproduction Center, Centro de Selección y Reproducción Animal, (CENSYRA-Extremadura Government), Camino Santa Engracia, S/N (Estación Pecuaria), 06007 Badajoz, Spain

The aim of this study was to compare the effect of two semen extenders and four permeating cryoprotectants on post-thaw sperm quality of Andalusian donkeys. First, 32 ejaculates were pooled, split and frozen in either Gent B or INRA 96 with egg yolk and glycerol. Second, 12 pooled semen samples were simultaneously frozen in Gent B (glycerol) or Gent A containing ethylene glycol (EG; 1 or 1.5%) or dimethyl sulfoxide (DMSO; 1.5 or 2%). Finally, nine pooled samples were simultaneously cryopreserved in Gent A containing 1% EG (as control), dimethylformamide (DMFA; 1 or 2.5%) or a combination of 1% EG and 1.5% DMFA. Gent B yielded a higher (P < 0.01) post-thaw sperm motility than modified INRA96. EG 1% increased the sperm membrane integrity (P < 0.001), whereas DMSO affected sperm motility and membrane integrity (P < 0.001). DMFA 2.5% yielded higher (P < 0.001) values for sperm motility and membrane integrity. We concluded that Gent B improves in vitro post-thaw sperm quality of donkey spermatozoa, but the replacement of glycerol with 1% EG or 2.5% DMFA increased sperm protection against cryodamage. The use of DMSO for freezing donkey semen was unsuccessful and a toxic effect is suspected. These extenders should be included in the pre-freeze test for each donkey.

doi: 10.1071/RD14449

The future of computer-aided sperm analysis2022-05-04T15:56:57+02:00

The future of computer-aided sperm analysis

Sharon T Mortimer, Gerhard Van der Horst, David Mortimer

Oozoa Biomedical Inc, West Vancouver, BC, Canada; University of the Western Cape, Bellville, South Africa

Computer-aided sperm analysis (CASA) technology was developed in the late 1980s for analyzing sperm movement characteristics or kinematics and has been highly successful in enabling this field of research. CASA has also been used with great success for measuring semen characteristics such as sperm concentration and proportions of progressive motility in many animal species, including wide application in domesticated animal production laboratories and reproductive toxicology. However, attempts to use CASA for human clinical semen analysis have largely met with poor success due to the inherent difficulties presented by many human semen samples caused by sperm clumping and heavy background debris that, until now, have precluded accurate digital image analysis. The authors review the improved capabilities of two modern CASA platforms (Hamilton Thorne CASA-II and Microptic SCA6) and consider their current and future applications with particular reference to directing our focus towards using this technology to assess functional rather than simple descriptive characteristics of spermatozoa. Specific requirements for validating CASA technology as a semi-automated system for human semen analysis are also provided, with particular reference to the accuracy and uncertainty of measurement expected of a robust medical laboratory test for implementation in clinical laboratories operating according to modern accreditation standards.

doi: 10.4103/1008-682X.154312

Differences in preservation of canine chilled semen using simple sperm washing, single-layer centrifugation and modified swim-up preparation techniques2016-12-30T09:48:22+01:00

Differences in preservation of canine chilled semen using simple sperm washing, single-layer centrifugation and modified swim-up preparation techniques

J. Dorado, M. J. Gálvez, S. Demyda-Peyrás, I. Ortiz, J. M. Morrell, F. Crespo, J. Gósalvez and M. Hidalgo

Animal Reproduction Group, Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, University of Cordoba, Campus de Rabanales (Edif. Hospital Clínico Veterinario), Ctra. Madrid-Cádiz, km 396, 14071 Córdoba, Spain; Department of Clinical Sciences, Division of Reproduction, Swedish University of Agricultural Sciences, Ullsväg 14C, Box 7054, SE-75007 Uppsala, Sweden; Veterinary Services of the Spanish Army (FESCCR-Ministry of Defense), C/Arsenio Guitiérrez Palacios s/n, 05005 Avila, Spain; Department of Biology, Genetics Unit, Autonomous University of Madrid, 20849 Madrid, Spain

This study compared the efficacy of simple sperm washing (SW), single-layer centrifugation (SLC) and modified swim-up (SU) techniques in the preparation of dog spermatozoa for cooling. Eighteen ejaculates, collected from three dogs (six per dog), were pooled (three ejaculates per pool) and divided into three aliquots: (1) one aliquot was washed and cooled at 5°C for 72 h, considered as control (SW-control), (2) the second aliquot was selected by SLC through Androcoll-C and subsequently cooled in the same way as the SW-control samples (SLC-AC) and (3) the last aliquot was selected by a modified SU method with Androcoll-C and cooled as mentioned above (SU-AC). Assessment of sperm motility, sperm morphology, sperm membrane integrity and acrosome integrity were performed on aliquots of fresh semen and chilled–rewarmed samples. Sperm membrane integrity and progressive motility were significantly (P < 0.05) improved by SU-AC compared with SW-control. Morphological sperm abnormalities decreased significantly (P < 0.001) in SLC-AC samples compared with SW-control samples. These sperm variables did not differ between SLC-AC and SU-AC methods (P > 0.05). The recovery rates were not significantly (P > 0.05) different between SW-control, SLC-AC and SU-AC samples. Our results confirm that SU-AC may be a successful method for the preparation of dog spermatozoa for cooling.

doi: 10.1071/RD15071

An Efficient Technique to Detect Sperm Reactive Oxygen Species: The CellRox Deep Red® Fluorescent Probe2016-12-30T09:48:22+01:00

An Efficient Technique to Detect Sperm Reactive Oxygen Species: The CellRox Deep Red® Fluorescent Probe

Maíra Bianchi Rodrigues Alves, André Furugen Cesar de Andrade, Rubens Paes de Arruda, Leonardo Batissaco, Shirley Andrea Florez-Rodriguez, Renata Lançoni, Bruna Marcele Martins de Oliveira, Mariana Andrade Torres, Gisele Mouro Ravagnani, Tamie Guibu de Almeida, Vinícius Silva Vellone and Eneiva Carla Carvalho Celeghini

Laboratory of Research in Pathology of Reproduction, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Sao Paulo (USP), Pirassununga, SP, Brazil; Laboratory of Andrology and Embryo Technology, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Sao Paulo (USP), Pirassununga, SP, Brazil; Laboratory of Semen Biotechnology and Andrology, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Sao Paulo (USP), Pirassununga, SP, Brazil

Heat stress and testicular traumas increase sperm Reactive Oxygen Species (ROS) resulting in Oxidative Stress (OS) and injury of sperm. The fluorescent probe CellROX Deep Red® is not already used to detect ROS in spermatozoa. On this way, this study aims to evaluate its efficiency in detecting ROS in ram sperm. For that, it was used ejaculates from rams performing the in vitro induction of sperm OS in T0, T50 and T100. T0 was semen sample that was not submitted to OS induction, T50 was 50% induced to OS induction and T100 was entire submitted to OS induction. The production of ROS was detected by CellROX Deep Red®. Data polynomial regression analysis was performed and the result of determination coefficient was 0.728. Besides, sixteen rams were submitted to scrotal insulation during 72 hours performing the in vivo induction of OS sperm. Analyses of sperm motility, morphology, DNA fragmentation and ROS (detected by CellROX Deep Red®) were done two times before the scrotal insulation and two times afterwards. Data analysis of variance was performed from the periods (before x after) and it is observed that after scrotal insulation the total and progressive motility decrease (p<0.05) and total and major sperm defects and sperm DNA fragmentation increase (p<0.05). Moreover, after insulation, it was observed increase (p<0.05) in sperm showing moderate and intense OS. Thus, it is possible to conclude that CellROX® fluorescent probe is able to detect ROS production in ram sperm by in vitro and in vivo induction being a new technique to detect sperm OS.

doi: 10.4172/2168-9652.1000157

DNA integrity of canine spermatozoa during chill storage assessed by the sperm chromatin dispersion test using bright-field or fluorescence microscopy2015-03-13T00:00:53+01:00

DNA integrity of canine spermatozoa during chill storage assessed by the sperm chromatin dispersion test using bright-field or fluorescence microscopy

M. Hidalgoa, M. Urbanoa, I. Ortiza, S. Demyda-Peyrasb, M.R. Murabitoa, M.J. Gálveza, J. Doradoa

Veterinary Reproduction Group, Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, University of Córdoba, Córdoba, Spain; MERAGEM Research Group, Department of Genetics, Faculty of Veterinary Medicine, University of Cordoba, Cordoba, Spain

The objective of this study was to evaluate the effect of chill storage on canine sperm DNA fragmentation assessed by the sperm chromatin dispersion test using bright-field microscopy with Wright solution (sDF-B) or fluorescence microscopy with propidium iodide (sDF-F). The relationship and agreement between the results obtained with both staining methods were analyzed. The values of DNA fragmentation indexes (sDF-F and sDF-B) were compared at each time of chill storage (0, 24, 48, 72, and 96 hours). Additionally, the sperm DNA fragmentation rate (slope) was compared between the methods during chill storage. Good agreement and no significant differences between values obtained with both staining procedures were observed. Finally, the effect of chill storage for up to 96 hours was assessed on sperm motility parameters and DNA fragmentation indexes. Significant differences were found after 48 hours of chill storage, obtaining greater values of fragmented DNA. Progressive sperm motility was lower just after 96 hours of chill storage, and no effect was found in total sperm motility. In conclusion, the Sperm-Halomax kit, developed for canine semen and based on the sperm chromatin dispersion test, can be used accurately under bright-field or fluorescence microscopy to assess the sperm DNA integrity of canine semen during chill storage. The sperm DNA fragmentation index increased after 48 hours of chill storage, thereby detecting sperm damage earlier than other routine sperm parameters, such as sperm motility.

doi: 10.1016/j.theriogenology.2015.03.030

 

Effect of different extenders and storage periods on motility and fertility of ram Sperm2016-09-16T10:06:47+02:00

Effect of different extenders and storage periods on motility and fertility of ram Sperm

Rossen Georgiev Stefanov; Georgi Anev; Desislava Vasileva Abadjieva

Institute of Biology and Immunology of Reproduction-BAS, bul. Tsarigradsko Shose 73, p.c. 1113, Sofia, Bulgaria; Agricultural Institute, BG – Turgovishte, Bulgaria.

The aim of this study was to test the effect of extenders containing different sugar in their composition on ram sperm motility and pregnancy rate of ewe’s following artificial insemination. Semen were collected from ten North-east Bulgarian fine-fleece breed and tested for quality. Semen was diluted with different extenders, with di- and trisaccharides. A series of experiments were repeated in triplicate. Total motility was determined by using Sperm Analysis (SCA, Microptic, Spain). A total of 200North-east Bulgarian fine-fleece breed mature ewes were used for cervical insemination with a sperm dose at the concentrationof 100 x 106 spermatozoa. Pregnancies were diagnosed 60 days after AI by – a real-time ultrasonic scan device (Alloka SSD500). In conclusion, our experiments demonstrated that higher sperm motility after storage at 4°C for 24 hours and 48 hourshas a ram spermatozoa diluted with extender 1, with combination of disaccharides (sucrose and lactose) and trisaccharides (rafinosa). This semen extender (number 1) can be used for successful insemination of ewes and to enhance pregnancy rate after artificial insemination.

doi: 10.14432/j.macvetrev.2014.12.036

Descriptive analysis of sperm head morphometry in Iberian ibex (Capra pyrenaica): Optimum sampling procedure and staining methods using Sperm-Class Analyzer®2016-12-30T09:48:22+01:00

Descriptive analysis of sperm head morphometry in Iberian ibex (Capra pyrenaica): Optimum sampling procedure and staining methods using Sperm-Class Analyzer®

M.C. Esteso, E. Rodríguez, A. Toledano-Díaz, C. Castaño, J. Pradiee, A. López-Sebastián, J. Santiago-Moreno

Departamento de Reproducción Animal, SGIT-INIA, Avda. Puerta de Hierro km 5.9, 28040 Madrid, Spain; Departamento de Reproducción Animal, SGIT-INIA, Avda. Puerta de Hierro km 5.9, 28040 Madrid, Spain; Departamento de Reproducción Animal, SGIT-INIA, Avda. Puerta de Hierro km 5.9, 28040 Madrid, Spain; Cnpq, Brazil

Sperm morphology has been identified as one characteristic which can be useful in prediction of fertility in a species. The development of computer automated sperm morphometry analysis allows for objective analysis of sperm head dimensions. The aim of the current study was to develop an optimum sampling procedure to characterize the Iberian ibex (Capra pyrenaica) sperm head morphometrically. Fresh semen from 11 males was collected using transrectal ultrasonic-guided massage of accessory sex glands and electroejaculation and prepared on slides for morphometric analysis to evaluate technical variation and standardize automated sperm morphometry analysis procedures by Sperm-Class Analyzer®. Three staining methods (Diff-Quik®, Hemacolor®, Spermblue®), number of sperm cells necessary to sample and repeatability of the staining technique were assessed. There were significant differences in size of sperm head depending on stain used. Hemacolor® was stain most suitable for sperm head morphometry evaluation (length = 8.42 μm; width = 4.21 μm; area = 29.37 μm2; perimeter = 21.93 μm; elongation = 0.33; elipticity = 2.01; regularity = 0.95; rugosity = 0.77). Morphometric values obtained from samples of 50, 100, 150, 175 and 200 sperm heads were compared. The most efficient method of analyzing sperm morphometry was to evaluate 100 sperm cells at 60× objective magnification. Thus, this study has allowed for description of optimal sample processing to determine morphometric parameters of sperm heads (size and shape) in Iberian ibex by Sperm-Class Analyzer® and provides a basis for future studies on the relationship with freezability and fertility in this species.

doi: 10.1016/j.anireprosci.2015.01.014

Sperm-egg penetration assay assessment of the contraceptive effects of glycerol and egg yolk in rooster sperm diluents2016-12-30T09:48:22+01:00

Sperm-egg penetration assay assessment of the contraceptive effects of glycerol and egg yolk in rooster sperm diluents

Abouelezz FM, Castaño C, Toledano-Díaz A, Esteso MC, López-Sebastián A, Campo JL, Santiago-Moreno J

Department of Animal Reproduction, INIA, Madrid, Spain; Department of Poultry Production, Faculty of Agriculture, Assiut University, Asyut, Egypt; Department of Animal Reproduction, INIA, Madrid, Spain; Department of Animal Breeding, INIA, Madrid, Spain; Department of Animal Reproduction, INIA, Madrid, Spain

Glycerol (GLY) and egg yolk (EY) are good cryoprotectants of avian and mammalian sperm, but in birds, they strongly inhibit the eventual fertilization of ova. Using the sperm penetration (SP-holes) assay and fertility trials, the present study investigates (1) the possible mechanism by which this contraceptive effect occurs in chickens and (2) the maximum concentrations of GLY and EY tolerated by fresh rooster sperm. Seventy Black-Barred Andaluza hens (five per treatment) were inseminated four times (twice per week) with 0.1 mL of fresh semen from roosters of the same breed diluted 1:1 (v:v) with Lake and Ravie medium containing different concentrations of GLY or EY. No adverse effects on acrosome integrity, sperm motility, or viability were seen with any concentration of GLY or EY. The number of SP-holes on perivitelline layer samples taken from above the germinal disc became progressively lower at GLY concentrations of 1.5% or greater (P > 0.05). No holes caused by sperms were seen in unfertilized eggs. The corresponding fertility results showed similar reductions when the GLY concentration was 1.5% or greater. No changes in the number of SP-holes were seen with increasing EY concentrations (0%-7.5%), nor were any differences in fertility observed, except for a reduction when 15% EY was used. The results therefore reveal that GLY affects the transit of sperms through the oviduct in their attempt to reach the infundibulum area, limiting their access to the ovum perivitelline layer. Egg yolk had no such effect, nor did it influence acrosome reaction capacity; its mechanism of contraceptive action therefore remains unknown. The maximum GLY and EY concentrations tolerated by the rooster sperm were 0.75% and 7.5%, respectively.

doi: 10.1016/j.theriogenology.2015.02.002

Insemination of bocachico fish (Prochilodus magdalenae) with fresh or cryopreserved semen: effect of spermatozoa/oocyte ratio2016-12-30T09:48:22+01:00

Insemination of bocachico fish (Prochilodus magdalenae) with fresh or cryopreserved semen: effect of spermatozoa/oocyte ratio

Víctor J Atencio García, MSc; José A Espinosa, MSc; José G Martínez, MSc; Sandra C Pardo Carrasco, PhD

Centro de Investigación Piscícola, Facultad de Medicina Veterinaria y Zootecnia, Universidad de Córdoba; Laboratory of Animal Genetics and Evolution – LEGAL, Institute of Biological Sciences, Federal University of Amazonas, Manaus, Brasil; Facultad de Ciencias Agrarias, Departamento de Producción Animal, BIOGEM, Universidad Nacional de Colombia Sede Medellín, Colombia

Background: cryopreservation is an important biotechnological tool in the conservation of biodiversity, particularly for endangered species. Objective: to evaluate six different spermatozoa/oocyte ratios using fresh and cryopreserved semen in bocachico fish (Prochilodus magdalenae). Methods: fresh semen was collected and its quality determined to verify cryopreservation feasibility. The semen was put in 5 mL straws and mixed with a solution (5.5% glucose, 12% egg yolk, and 10% dimethyl sulfoxide –DMSO-) in a 1:4 dilution (semen:solution). The semen was frozen in nitrogen vapor dry shipper for 30 min, rapidly transferred to storage thermos, and submerged directly into liquid nitrogen (LN; -196 °C). Straws were thawed at 60 ºC for 45 seconds. Motility, velocity, and sperm progressivity of fresh and cryopreserved semen were assessed using Sperm Class Analyzer (SCA®) software. Each proportion of spermatozoa/oocyte was assessed with 2 g of eggs (1,630 ± 87 eggs/g) to evaluate fertility (F), hatching (H), and larval survival (LS) rates. Results: the best reproductive performance for fresh semen was obtained inseminating with 160,000 spermatozoa/oocyte (F = 75.0%, H = 67.7%, LS = 32.7%). Similarly, the best reproductive performance for cryopreserved semen was achieved with 320,000 spermatozoa/oocyte (F = 70.0%, H = 48.6%, LS = 19.5%). Conclusion: it is possible to achieve adequate reproductive performance in bocachico fish using cryopreserved sperm (10% DMSO, 5.5% glucose, and 12% egg yolk) at twice the spermatozoa/oocyte ratio used with fresh semen.

doi: 10.17533/udea.rccp.v28n4a07

Testicular degeneration affected plasma, acrosomal and mitochondrial membrane integrity, and DNA fragmentation in ram spermatozoa2016-12-30T09:48:22+01:00

Testicular degeneration affected plasma, acrosomal and mitochondrial membrane integrity, and DNA fragmentation in ram spermatozoa

Bianchi Rodrigues Alves M, Furugen Cesar de Andrade A, Paes de Arruda R, Batissaco L, Lançoni R, Andrade Torres M, Mouro Ravagnani G, Florez-Rodriguez SA, Marcelle Martins de Oliveira B, Vellone V, Carla Carvalho Celeghini E.

Center of Biotechnology in Animal Reproduction, Department of Animal Reproduction, Faculty of Veterinary and Animal Science, Pirassununga, SP, Brazil.

Testicular degeneration, an important cause of male infertility, adversely affects sperm motility and morphology. However, few studies describe effects on integrity of plasma and acrosomal membranes, mitochondrial membrane potential, and DNA fragmentation; therefore, they were evaluated in the present study. Testicular degeneration was induced in 17 White Dorper rams (scrotal insulation for 72h). Semen was collected (artificial vagina) twice before insulation and twice thereafter (15-day intervals between post-insulation collections). Sperm motility and morphology were analysed by SCA software (Sperm Class Analyser(®), MICROPTIC(®), Barcelona, Spain) and differential interference contrast microscopy (DIC, model 80i, Nikon, Tokyo, Japan), respectively. Membrane integrity and potential were assessed with fluorescent probes: Hoescht 33342, propidium iodide, FITC-PSA, and JC-1 (Celeghini et al. 2010 Arq. Bras. Med. Vet. Zootec. 62, 536-543) and imaged with fluorescence microscopy (Nikon Model 80i, Nikon, Tokyo, Japan). Fragmentation of DNA was evaluated with a Halomax(®) kit (Halotech(®) DNA, Madrid, Spain). Data were analysed with Statview software (Stat View 1998, SAS Institute Inc., Cary, NC, USA). Data obtained from the periods (before×after insulation) were evaluated by analysis of variance (ANOVA) and means were compared using Tukey’s test. Total motility (before: 87.53±1.21%; after: 46.53±4.46%) and progressive motility (before: 58.64±2.00%; after: 31.33±3.82%) were reduced (P<0.01) by scrotal insulation, as were sperm major defects (before: 10.64±1.65%; after: 54.30±3.67%) and total defects (before: 20.50±2.40%; after: 63.85±3.41%; P<0.0001). Sperm with intact plasma and acrosomal membranes and high mitochondrial potential (PIAIH) decreased (P<0.0001) after insulation. In that regard, 53.19±2.20 and 28.48±3.48% of sperm were classified as PIAIH before v. after insulation, respectively. Furthermore, plasma membrane integrity, acrosome membrane integrity, and high mitochondrial potential were assessed independently. The quantity of plasma membrane integrity cells (before: 62.01±2.07%; after: 33.92±3.94%), acrosome membrane integrity cells (before: 57.17±2.30%; after: 31.47±3.77%), and high mitochondrial potential cells (before: 85.72±1.42%; after: 57.28±3.12%) were also reduced (P<0.0001) after insulation. Likewise, DNA integrity decreased (P=0.002) from 98.87±0.26% before insulation to 91.88±2.6% afterward. In conclusion, sperm plasma and acrosomal membrane integrity, mitochondrial membrane potential, and DNA fragmentation were adversely affected by testicular degeneration in rams induced by scrotal insulation.

doi: 10.1071/RDv27n1Ab265

The utility of nanowater for ram semen cryopreservation2016-12-30T09:48:22+01:00

The utility of nanowater for ram semen cryopreservation

Murawski M, Schwarz T, Grygier J, Patkowski K, Oszczęda Z, Jelkin I, Kosiek A, Gruszecki TM, Szymanowska A, Skrzypek T, Zieba DA, Bartlewski PM.

Department of Animal Biotechnology, University of Agriculture in Cracow, 30-274 Kraków, Poland. Institute of Animal Sciences, University of Agriculture in Cracow, 31-120 Kraków, Poland. Department of Animal Biotechnology, University of Agriculture in Cracow, 30-274 Kraków, Poland joanna.grygier7@gmail.com. Department of Biology and Animal Breeding, University of Life Sciences in Lublin, 20-950 Lublin, Poland. Nantes Nanotechnology Systems, 59-700 Bolesławiec, Poland. Center for Interdisciplinary Research, Catholic University of Lublin, 20-705 Lublin, Poland. Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada N1G 2W1.

Nanowater (NW; water declusterized in the low-temperature plasma reactor) has specific physicochemical properties that could increase semen viability after freezing and hence fertility after artificial insemination (AI) procedures. The main goal of this study was to evaluate ram semen quality after freezing in the media containing NW. Ejaculates from 10 rams were divided into two equal parts, diluted in a commercially available semen extender (Triladyl®; MiniTüb GmbH, Tiefenbach, Germany) prepared with deionized water (DW) or NW, and then frozen in liquid nitrogen. Semen samples were examined for sperm motility and morphology using the sperm class analyzer system and light microscopy. Cryo-scanning electron microscopy (cryo-SEM) was employed to determine the size of extracellular water crystals in frozen semen samples. Survival time at room temperature, aspartate aminotransferase (AspAT) and alkaline phosphatase (ALP) concentrations post-thawing as well as conception/lambing rates after laparoscopic intrauterine AI of 120 ewes were also determined. There were no significant differences between DW and NW groups in sperm progressive motility (26.4 ± 12.2 and 30.8 ± 12.4%) or survival time (266.6 ± 61.3 and 270.9 ± 76.7 min) after thawing and no differences in the percentages of spermatozoa with various morphological defects before or after freezing. There were, however, differences (P < 0.05) in AspAT (DW: 187.1 ± 160.4 vs. NW: 152.7 ± 118.3 U/l) and ALP concentrations (DW: 2198.3 ± 1810.5 vs. NW: 1612.1 ± 1144.8 U/l) in semen samples post-thawing. Extracellular water crystals were larger (P < 0.05) in ejaculates frozen in NW-containing media. Ultrasonographic examinations on day 40 post-AI revealed higher (P < 0.05) conception rates in ewes inseminated with NW (78.3%) compared with DW semen (58.3%), and the percentages of ewes that carried lambs to term were 73.3% and 45.0% in NW and DW groups, respectively (P < 0.01). In summary, the use of a semen extender prepared with NW was associated with a substantial improvement in the fertilizing ability of frozen-thawed ram semen and lamb productivity of inseminated ewes.

doi: 10.1177/1535370214557219

Soybean lecithin-based extender preserves spermatozoa membrane integrity and fertilizing potential during goat semen cryopreservation2016-12-30T09:48:22+01:00

Soybean lecithin-based extender preserves spermatozoa membrane integrity and fertilizing potential during goat semen cryopreservation

Chelucci S, Pasciu V, Succu S, Addis D, Leoni GG, Manca ME, Naitana S, Berlinguer F

Department of Veterinary Medicine, University of Sassari, Sassari, Italy; Department of Veterinary Medicine, University of Sassari, Sassari, Italy; Centro di Competenza Biodiversità Animale, Sassari, Italy; Department of Veterinary Medicine, University of Sassari, Sassari, Italy; Centro di Competenza Biodiversità Animale, Sassari, Italy

Soybean lecithin may represent a suitable alternative to egg yolk for semen cryopreservation in livestock species. However, additional studies are needed to elucidate its effects on spermatozoa functional properties. Semen collected from five Sarda bucks was cryopreserved in Tris-based extender and glycerol (4% v:v) with different supplementations. In a preliminary experiment, different soybean lecithin concentrations were tested (1%-6% wt/vol) and results in terms of viability, percentages of progressive motile and rapid spermatozoa, and DNA integrity after thawing showed that the most effective concentration was 1%. In the second experiment, semen was frozen in a Tris-based extender with no supplementation (EXT), with 1% lecithin (EXT LC), and 20% egg yolk (EXT EY). The effectiveness of these extenders was also compared with a commercial extender. The EXT EY led to the highest viability and motility parameters after freezing and thawing (P < 0.0001). No significant differences were observed in intracellular ATP concentrations. Additional molecular features revealed that sperm functionality was affected in EXT EY, as demonstrated by lower DNA and acrosome integrity (P < 0.05), and higher lipid peroxidation compared with spermatozoa cryopreserved in EXT LC (P < 0.0001). Results obtained in the heterologous in vitro fertilization test showed that EXT LC better preserved spermatozoa functionality, as demonstrated by the higher fertilization rates compared with the other media (66.2 ± 4.5% for EXT LC vs. 32.7 ± 4.5%, 38.7 ± 4.5%, 39.6 ± 5.2% for EXT, EXT EY, and commercial extender; P < 0.01). The present study demonstrated that lecithin can be considered as a suitable alternative to egg yolk in goat semen cryopreservation, because it ensures higher fertilization rates and a better protection from membrane damage by cold shock.

doi: 10.1016/j.theriogenology.2014.12.012

Use of hypometabolic TRIS extenders and high cooling rate refrigeration for cryopreservation of stallion sperm: Presence and sensitivity of 5′ AMP-activated protein kinase (AMPK)2016-12-30T09:48:22+01:00

Use of hypometabolic TRIS extenders and high cooling rate refrigeration for cryopreservation of stallion sperm: Presence and sensitivity of 5′ AMP-activated protein kinase (AMPK)

Alex Córdova, Pablo Strobel, Andrés Vallejo, Pamela Valenzuela, Omar Ulloa, Rafael A. Burgos, Bruno Menarim, Joan Enric Rodríguez-Gil, Marcelo Ratto, Alfredo Ramírez-Reveco

Instituto de Ciencia Animal, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Chile; Instituto de Ciencias Clínicas, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Chile; Instituto de Farmacología y Morfofisiología, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Chile; Haras Militar Pupunahue, DGFER-Ejército de Chile, Chile; Unitat Reproducció Animal, Universitat Autònoma de Barcelona, Spain; Ross University School of Veterinary Medicine, Basseterre, West Indies

This study evaluated the effect of the use of hypometabolic TRIS extenders in the presence or the absence of AMPK activators as well as the utilization of high cooling rates in the refrigeration step on the freezability of stallion sperm. Twelve ejaculates were cryopreserved using Botucrio® as a control extender and a basic TRIS extender (HM-0) separately supplemented with 10 mM metformin, 2 mM 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), 2 mM Adenosine monophosphate (AMP), 40 μM compound C AMPK inhibitor or 2 mM AMP + 40 μM compound C. Our results showed that the utilization of a hypometabolic TRIS extender supplemented or not with AMP or metformin significantly improves stallion sperm freezability when compared with a commercial extender. Additionally, high cooling rates do not affect stallion sperm quality after cooling and post-thawing. Finally, stallion spermatozoa present several putative AMPK sperm isoforms that do not seem to respond to classical activators, but do respond to the Compound C inhibitor.

doi:10.1016/j.cryobiol.2014.10.008

Sperm motility evaluation following long-term storage (5 years) of cryopreserved sea bream (Sparus aurata L., 1758) semen2014-11-01T16:29:35+01:00

Sperm motility evaluation following long-term storage (5 years) of cryopreserved sea bream (Sparus aurata L., 1758) semen

A. Fabbrocini, R. D’Adamo, S. Pelosi, L. F. J. Oliveira, F. Del Prete, F. Silvestri, V. Vitiello, G. Sansone

Consiglio Nazionale delle Ricerche, Istituto di Scienze Marine, Lesina, Italy; Instituto Oceanografico – USP Praca do Oceanografico, Sao Paulo, Brazil; The Capes Foundation – Ministry of Education of Brazil, Brasilia, Brazil; Centro Interdipartimentale di Ricerche per la Gestione delle Risorse Idrobiologiche e per l’Acquacoltura, Universita degli Studi di Napoli Federico II, Portici, Italy; Dipartimento di Biologia, Universita degli Studi di Napoli Federico II, Napoli, Italy

The aim of this study was to evaluate, by computer-assisted analysis, the motility parameters of cryopreserved sea bream spermatozoa after prolonged storage (up to 5 years) in liquid nitrogen in comparison to the performances of fresh semen and of semen thawed 1 month after freezing (cryopreservation medium: 1% NaCl containing 5% DMSO; freezing rate: 10°C min 1; stored in liquid nitrogen). Semen samples were thawed 1 month and 5 years after cryopreservation. Sperm motility was analyzed by means of the Sperm Class Analyzer (SCA, Microptic, Barcelona, Spain). The percentages of motile sperm and rapid sperm curvilinear velocity >100 lm s 1 only), and the curvilinear, straight-line and average path velocities (lm s 1) were evaluated. The percentages of total motile and rapid sperm, as well the relative velocity levels, were slightly lower in thawed semen than in fresh semen (for example, 85 vs 95% for total motile sperm; 70 vs 80% for rapid sperm; 200 vs 300 lm s 1 for VCLt; 250 vs 350 lm s 1 for VCLr). Data of all trials did not differ in relation to storage time. It can therefore be concluded that long-term storage of large amounts of cryopreserved semen was homogeneous, providing high
quality sea bream semen for use in fertilization trials in both aquaculture and laboratory research.

doi: 10.1111/jai.12726

Presence and function of dopamine transporter (DAT) in stallion sperm: dopamine modulates sperm motility and acrosomal integrity2016-12-30T09:48:22+01:00

Presence and function of dopamine transporter (DAT) in stallion sperm: dopamine modulates sperm motility and acrosomal integrity

Urra JA, Villaroel-Espíndola F, Covarrubias AA, Rodríguez-Gil JE, Ramírez-Reveco A, Concha II.

Escuela de Graduados, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Valdivia, Chile; Instituto de Bioquímica y Microbiología, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile; Instituto de Ciencia Animal, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Valdivia, Chile; Instituto de Bioquímica y Microbiología, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile; Unitat de Reproducció Animal, Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain; Instituto de Ciencia Animal, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Valdivia, Chile

Dopamine is a catecholamine with multiple physiological functions, playing a key role in nervous system; however its participation in reproductive processes and sperm physiology is controversial. High dopamine concentrations have been reported in different portions of the feminine and masculine reproductive tract, although the role fulfilled by this catecholamine in reproductive physiology is as yet unknown. We have previously shown that dopamine type 2 receptor is functional in boar sperm, suggesting that dopamine acts as a physiological modulator of sperm viability, capacitation and motility. In the present study, using immunodetection methods, we revealed the presence of several proteins important for the dopamine uptake and signalling in mammalian sperm, specifically monoamine transporters as dopamine (DAT), serotonin (SERT) and norepinephrine (NET) transporters in equine sperm. We also demonstrated for the first time in equine sperm a functional dopamine transporter using 4-

[4-(Dimethylamino)styryl]-N-methylpyridinium iodide (ASP(+)), as substrate. In addition, we also showed that dopamine (1 mM) treatment in vitro, does not affect sperm viability but decreases total and progressive sperm motility. This effect is reversed by blocking the dopamine transporter with the selective inhibitor vanoxerine (GBR12909) and non-selective inhibitors of dopamine reuptake such as nomifensine and bupropion. The effect of dopamine in sperm physiology was evaluated and we demonstrated that acrosome integrity and thyrosine phosphorylation in equine sperm is significantly reduced at high concentrations of this catecholamine. In summary, our results revealed the presence of monoamine transporter DAT, NET and SERT in equine sperm, and that the dopamine uptake by DAT can regulate sperm function, specifically acrosomal integrity and sperm motility.

doi: 10.1371/journal.pone.0112834

Sperm flagellum volume determines freezability in red deer spermatozoa2016-12-30T09:48:22+01:00

Sperm flagellum volume determines freezability in red deer spermatozoa

Ros-Santaella JL, Domínguez-Rebolledo AE, Garde JJ

SaBio, Instituto de Investigación en Recursos Cinegéticos (IREC), CSIC-UCLM-JCCM, Campus Universitario, Albacete, Spain; Department of Animal Science and Food Processing, Faculty of Tropical AgriSciences, Czech University of Life Sciences, Prague, Czech Republic; Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP), Mocochá, Yucatán, México; SaBio, Instituto de Investigación en Recursos Cinegéticos (IREC), CSIC-UCLM-JCCM, Campus Universitario, Albacete, Spain

The factors affecting the inter-individual differences in sperm freezability is a major line of research in spermatology. Poor sperm freezability is mainly characterised by a low sperm velocity, which in turn is associated with low fertility rates in most animal species. Studies concerning the implications of sperm morphometry on freezability are quite limited, and most of them are based on sperm head size regardless of the structural parts of the flagellum, which provides sperm motility. Here, for the first time, we determined the volumes of the flagellum structures in fresh epididymal red deer spermatozoa using a stereological method under phase contrast microscopy. Sperm samples from thirty-three stags were frozen and classified as good freezers (GF) or bad freezers (BF) at two hours post-thawing using three sperm kinetic parameters which are strongly correlated with fertility in this species. Fourteen stags were clearly identified as GF, whereas nineteen were BF. No significant difference in sperm head size between the two groups was found. On the contrary, the GF exhibited a lower principal piece volume than the BF (6.13 µm3 vs 6.61 µm3, respectively, p = 0.006). The volume of the flagellum structures showed a strong negative relationship with post-thawing sperm velocity. For instance, the volume of the sperm principal piece was negatively correlated with sperm velocity at two hours post-thawing (r = -0.60; p<0.001). Our results clearly show that a higher volume of the sperm principal piece results in poor freezability, and highlights the key role of flagellum size in sperm cryopreservation success.

doi: 10.1371/journal.pone.0112382

Cryopreservation of rainbow trout Oncorhynchus mykiss spermatozoa: effects of extender supplemented with different antioxidants on sperm motility, velocity and fertility2015-05-08T09:24:01+02:00

Cryopreservation of rainbow trout Oncorhynchus mykiss spermatozoa: effects of extender supplemented with different antioxidants on sperm motility, velocity and fertility

Kutluyer F, Kayim M, Öğretmen F, Büyükleblebici S, Tuncer PB

Tunceli University, Fisheries Faculty, 62000 Tunceli, Turkey; Tunceli University, Fisheries Faculty, 62000 Tunceli, Turkey; Muğla Sıtkı Koçman University, Faculty of Fisheries, 48000 Muğla, Turkey; Aksaray University, Faculty of Veterinary Medicine, Department of Reproduction and Artificial Insemination, Aksaray, Turkey; Aksaray Vocational School, Aksaray University, Aksaray, Turkey

In present study, it was examined whether addition of different antioxidants to the cryopreservation extenders had an effect on semen post-thaw fertility and motility in rainbow trout (Oncorhynchus mykiss) and also it was investigated the sperm characteristics post-thaw sperm characteristics and fertility. The collected semen was pooled to minimize individual variation. Each pooled ejaculate was split into 12 equal aliquots and diluted with base extenders supplemented with the antioxidants, and a base extender with no additives (control). The pooled semen samples diluted at the ratio of 1:10 by the extenders were subjected to cryopreservation. Antioxidants were separately added to the extenders (one per experimental group): catalase (250 U/l), superoxide dismutase (250 U/l), peroxidase (250 U/l), oxidized glutathione (1.5 mmol/l), reduced glutathione (1.5 mmol/l), L-methionine (1.5 mmol/l), uric acid (0.25 mmol/l), L-ascorbic acid (0.5 mmol/l), α-tocopherol (2.0 mmol/l), β-carotene (0.5 mmol/l) and carnitine (0.5 mmol/l). After dilution the semen was aspirated into 0.25 ml straws, the straws were placed on the tray, frozen for 10 min, and plunged into liquid nitrogen. Our results indicated that the post-thaw motility rate increased in extenders supplemented with uric acid, L-methionine, SOD, L-carnitine, α-tocopherol and L-reduced glutathione (p<0.05). The motility duration of frozen thawed semen increased in extenders supplemented with uric acid, L-methionine, SOD, α-tocopherol and L-reduced glutathione (p<0.05). Fertilization rate and hatching rate of frozen-thawed semen was not affected by the tested antioxidants. Consequently, the tested antioxidants affected the motility parameters and cryopreservation extenders could be supplement with antioxidants. This study suggested usage of antioxidants in the cryopreservation of rainbow trout.

doi: 10.1016/j.cryobiol.2014.10.005

Fragmentation of sperm DNA using the TUNEL method2016-12-30T09:48:22+01:00

Fragmentation of sperm DNA using the TUNEL method

P.H. Chenlo, S.M. Curi, M.N. Pugliese, J.I. Ariagno, M. Sardi-Segovia, M.J. Furlan, H.E. Repetto, E. Zeitler, M. Cohen, G.R. Mendeluk

Departamento de Bioquímica Clínica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Hospital de Clínicas ‘‘José de San Martín’’, Buenos Aires, Argentina

Objectives: To establish the validity of the TUNEL assay in determining sperm DNA fragmentation, the relationship between the degree of fragmentation and the seminal parameters and the sample needed to conduct the test.
Material and methods: We used semen samples from healthy fertile men (n = 33), patients who consulted for infertility with a prescription for the TUNEL assay (n = 77) and patients with intracytoplasmic sperm injection failure (n = 20), analyzed according to the 2010 WHO. The TUNEL/propidium iodide test was performed by flow cytometry, on baseline and post-swim-up samples.
Results: The cutoff value for the TUNEL assay (ROC curves) was 26%, with a sensitivity and specificity of 85% and 89%, respectively. The pre-swim-up and post-swim-up medians of the results from the TUNEL assay showed no significant differences (17.0% vs. 12.9%, respectively). However, 39.1% of the samples showed a difference greater than 15 in absolute value between the results of the baseline and post-swim-up TUNEL assays. The linear correlation study of the morphology, mobility and vitality using the post-swim-up TUNEL assay showed a greater correlation than preselection, with significant results (r: −0.394, p < .0001; r: −0.461, p < .0001; r: −0.526, p < .0001).
Conclusions: The TUNEL assay is a valid test for clinical use. DNA fragmentation is a factor independent from traditional semen tests. We found a greater susceptibility to damage generated in the laboratory procedures in the samples with lower quality. The sample of choice for evaluating DNA fragmentation will depend on whether the clinician is treating a natural or assisted fertilization.

Actas Urol Esp. 2014;38(9):608-612

Photobiomodulation with light-emitting diodes improves sperm motility in men with asthenozoospermia2016-12-30T09:48:22+01:00

Photobiomodulation with light-emitting diodes improves sperm motility in men with asthenozoospermia

Ban Frangez H1, Frangez I, Verdenik I, Jansa V, Virant Klun I.

Reproductive Unit, Department of Obstetrics and Gynecology, University Medical Centre Ljubljana, Slajmerjeva 3, 1000, Ljubljana, Slovenia

Sperm motility is an important parameter of male fertility and depends on energy consumption. Photobiomodulation with light-emitting diode (LED) is known to stimulate respiratory chain in mitochondria of different mammalian cells. The aim of this research was to evaluate the effect of photobiomodulation with LED on sperm motility in infertile men with impaired sperm motility-asthenozoospermia. Thirty consecutive men with asthenozoospermia and normal sperm count who visited the infertility clinic of University Medial Centre Ljubljana between September 2011 and February 2012 were included in the study. Semen sample of each man was divided into five parts: one served as a non-treated (native) control and four parts were irradiated with LED of different wavelengths: (1) 850 nm, (2) 625, 660 and 850 nm, (3) 470 nm and (4) 625, 660 and 470 nm. The percentage of motile sperm and kinematic parameters were measured using a Sperm Class Analyser system following the WHO recommendations. In the non-treated semen samples, the average ratio of rapidly progressive sperms was 12 % and of immotile sperm 73 %. Treating with LED significantly increased the proportion of rapidly progressive sperm (mean differences were as follows: 2.83 (1.39-4.28), 3.33 (1.61-5.05), 4.50 (3.00-5.99) and 3.83 (2.31-5.36) for groups 1-4, respectively) and significantly decreased the ratio of immotile sperm (the mean differences and 95 % CI were as follows: 3.50 (1.30-5.70), 4.33 (2.15-6.51), 5.83 (3.81-7.86) and 5.50 (2.98-8.02) for groups 1-4, respectively). All differences were highly statistically significant. This finding confirmed that photobiomodulation using LED improved the sperm motility in asthenozoospermia regardless of the wavelength.

doi: 10.1007/s10103-014-1653-x

Effects of red palm oil and rooibos on sperm motility parameters in streptozotocin-induced diabetic rats2022-05-04T15:56:41+02:00

Effects of red palm oil and rooibos on sperm motility parameters in streptozotocin-induced diabetic rats

Ayeleso AO, Oguntibeju OO, Aboua YG, Brooks NL

Department of Bio-medical Sciences, Faculty of Health and Wellness Sciences, Cape Peninsula University of Technology, Bellville South Africa; Department of Wellness Sciences, Faculty of Health and Wellness Sciences, Cape Peninsula University of Technology, Cape Town, South Africa

BACKGROUND: Diabetes mellitus characterized by hyperglycaemia could affect sperm quality as a result of increased oxidative stress. This study was performed to investigate the effects of red palm oil (RPO), aqueous rooibos tea extracts (RTE) as well as their combination (RPO + RTE) on sperm motility parameters in streptozotocin-induced diabetic rats.
MATERIALS AND METHODS: Diabetes was induced by a single administration of streptozotocin (50 mg/kg) and the rats were treated with red palm oil (2 ml/day) and / or aqueous rooibos tea extract (2%) for 7 weeks. Sperm motility parameters were measured using Computer Assisted Sperm Analyzer (CASA).
RESULTS: Hyperglycaemia negatively affected the sperm progressive motility significantly at p<0.05. There was a significant decrease (p<0.05) in sperm linearity (LIN) in the diabetic group when compared with the normal control group. RPO supplemented diabetic rats exhibited increased progressive sperm motility, sperm linearity (LIN) and wobble (WOB). Significant decreases (p<0.05) in straight line velocity (VSL) and average path velocity (VAP) of the sperms were observed in all the diabetic groups when compared to the control group. Significant (p<0.05) elevated levels of WOB and LIN were observed following RTE treatment and co-administration with RPO respectively.
CONCLUSION: The present study suggests that red palm oil and / or rooibos administration exhibited no adverse effects on sperm motility parameters but rather showed some beneficial effects.
KEYWORDS: Diabetes mellitus; Rats; Red palm oil; Rooibos; Sperm; Streptozotocin

doi: 10.4314/ajtcam.v11i5.2

Sperm characterization and identification of sperm sub-populations in ejaculates from pampas deer (Ozotoceros bezoarticus)2016-12-30T09:48:22+01:00

Sperm characterization and identification of sperm sub-populations in ejaculates from pampas deer (Ozotoceros bezoarticus)

Beracochea F, Gil J, Sestelo A, Garde JJ, Santiago-Moreno J, Fumagalli F, Ungerfeld R

Departamento de Fisiología, Universidad de la República, Montevideo, Uruguay; Departamento de Reproducción, Facultad de Veterinaria, Universidad de la República, Montevideo, Uruguay; Jardín Zoológico de la Ciudad de Buenos Aires – Fundación Bioandina Argentina, Argentina; SaBio IREC (CSIC-UCLM-JCCM), Campus Universitario sn, Albacete, Spain; Departamento de Reproducción Animal, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Madrid, Spain; Departamento de Fisiología, Universidad de la República, Montevideo, Uruguay

Pampas deer (Ozotoceros bezoarticus) is a native endangered species. Knowledge of the basic spermiogram characteristics and the morphometric descriptors is necessary to effectively develop sperm cryopreservation. In other species, sperm sub-population is related to sperm cryo-resistance. The objective was to provide a general description of the sperm, including sperm head morphometric descriptors, its repeatability, and the existence of sperm sub-populations. Sperm were obtained from adult males by electroejaculation during the breeding season. The motility score was 3.4 ± 0.2 (mean ± SEM) and progressive motility was 59.4 ± 3.7%. Ejaculated volume was 413.9 ± 51.0 μl, the total number of sperm ejaculated was 321.2 ± 55.4 × 10(6). Also, 63.3 ± 3.1% of the sperm were morphologically abnormal and 23.7 ± 2.3% had acrosome damage. The sperm head length was 7.6 ± 0.01 μm, width 4.4 ± 0.01 μm, area 28.1 ± 0.07 μm(2) and the perimeter was 21.9 ± 0.04 μm. There was a positive relationship among morphometric descriptors and the motility score, overall motility and progressive motility. Also length (P=0.011), width (P=0.003), area (P=0.006) and perimeter (P=0.009) of sperm head obtained in two different collections were positively related. Overall, the low concentration, volume, overall quality and abnormal morphology, and wide variation of these variables may be a limitation for the development of sperm cryopreserved banks. There were three sperm sub-populations with different morphometric characteristics. The morphometric descriptors are maintained similarly among different collections.

doi: 10.1016/j.anireprosci.2014.07.013

Sperm selection by Capripure(®) density-gradient centrifugation versus the dextran swim-up procedure in wild mountain ruminants2016-12-30T09:48:23+01:00

Sperm selection by Capripure(®) density-gradient centrifugation versus the dextran swim-up procedure in wild mountain ruminants

Santiago-Moreno J, Esteso MC, Castaño C, Toledano-Díaz A, Rodríguez E, López-Sebastián A

Departamento de Reproducción Animal, INIA, 28040 Madrid, Spain; Departamento de Reproducción Animal, INIA, 28040 Madrid, Spain

This study compares the effectiveness of two methods of sperm selection – Capripure(®) density-gradient centrifugation (DGC) and dextran swim-up (DSU) – in semen samples from Iberian ibex (Capra pyrenaica) and European mouflon (Ovis musimon). During the increasing photoperiod, Capripure(®) DGC improved the percentage of sperm with progressive motility (P<0.05) in ibexes, and selected 60.6% of the initial total number of spermatozoa contained in the ejaculate samples. In mouflon, Capripure(®) DGC selection was unaffected by photoperiod, had no influence on any sperm variable, and selected 47.8% of the initial total number of mouflon spermatozoa in ejaculate samples. Photoperiod had no influence on the effectiveness of DSU in either ibexes or mouflons. In the ibexes, DSU reduced (P<0.05) the percentage of sperm cells with morphological abnormalities, but only selected 11.3% of the initial total number of spermatozoa in ejaculate samples. In the mouflons, DSU had no significant influence on any sperm variable, and selected 27.8% of the initial total number. Capripure(®) DGC improved ibex and mouflon sperm motility (P<0.05) following 30min and 2h of post-centrifugation, stress-inducing incubation, respectively. In both ibexes and mouflon, sperm cells showing non-progressive motility were found after only 20 h of post-centrifugation incubation following Capripure(®) DGC selection. In conclusion, Capripure(®) DGC would seem a useful method for selecting the best spermatozoa from both ibex and mouflon ejaculates.

doi: 10.1016/j.anireprosci.2014.07.003

Effects of glucose concentration on the sperm motility of the catfish Sorubim cuspicaudus2016-12-30T09:48:23+01:00

Effects of glucose concentration on the sperm motility of the catfish Sorubim cuspicaudus

Víctor J. Atencio, María Dorado, Emilio Navarro, Francisco Pérez, Tatiana Roathan, Maricela Arias, José A. Espinosa

University of Cordoba

The aim of the study was to evaluate the effect of glucose concentration (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10%) in the activation of sperm motility of the catfish Sorubim cuspicaudus. Semen was obtained by hormonal induction, with salmonid GnRH analogue (10 mg/Kg body weight) of males undergoing spermiation (n = 13). The osmolarity of the extender and the seminal plasma (273.28±7.2 mOsm/Kg) was measured with an osmometer (Osmomette III, Precision Systems, USA). The semen and extender were mixed in proportion 1:3. The total motility and activation time were evaluated in the glucose solution and post-dilution, activated
with 75 μL of distilled water in a Makler chamber with the assistance of software Sperm Class Analyzer (SCA, Microptic, Spain). The results are shown in table 1. The motility activation was recorded when semen was mixed with solution of glucose lower of 5%; but from glucose solutions with 6% or more, was not recorded motility activation, which was later acquired by adding distilled water and was registred decreased of motility when the osmolarity was increased in the extender. Glucose solution to 6% can be used as extender to the cryopreservation of semen of the catfish Sorubim cuspicaudus.

World Aquaculture Society: World Aquaculture 2014

Antioxidant effect of rosemary (Rosmarinus officinalis L.) extract in soybean lecithin-based semen extender following freeze-thawing process of ram sperm2016-12-30T09:48:23+01:00

Antioxidant effect of rosemary (Rosmarinus officinalis L.) extract in soybean lecithin-based semen extender following freeze-thawing process of ram sperm

Motlagh MK, Sharafi M, Zhandi M, Mohammadi-Sangcheshmeh A, Shakeri M, Soleimani M, Zeinoaldini S

Department of Animal Science, Faculty of Agriculture and Natural Resources, Arak University, Arak 38156-8-8349, Iran; Department of Poultry Sciences, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran; Department of Animal Science, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran; Department of Animal and Poultry Science, College of Aburaihan, University of Tehran, Pakdasht, Tehran, Iran; Department of Transgenic Animal Science, Stem Cell Technology Research Center, Tehran, Iran; Department of Animal Science, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran; Department of Transgenic Animal Science, Stem Cell Technology Research Center, Tehran, Iran; Department of Hematology, Faculty of Medical Science, Tarbiat Modares University, Tehran, Iran

The aim of current study was to evaluate effect of rosemary aqueous extract on post-thawed ram sperm quality in a soybean lecithin-based (SL) extender. Ram semen samples were obtained, extended with SL extender and supplemented with 0% (SL-R0), 2% (SL-R2), 4% (SL-R4), 6% (SL-R6), and 8% (SL-R8) rosemary aqueous extract. Following equilibration, the straws were frozen, and then plunged into the liquid nitrogen. After thawing, sperm motility and velocity parameters, plasma membrane functionality, viability, acrosomal and capacitation status were evaluated. Membrane lipid peroxidation was also analyzed through the malondialdehyde (MDA) concentration. Our results showed that SL-R4 and SL-R6 groups resulted in higher (p < 0.05) percentages of total motility, progressive motility, and plasma membrane functionality, as compared with other groups. Highest (p < 0.05) viable and lowest (p < 0.05) dead spermatozoa were observed in SL-R6 group compared to the other groups. The acrosomal and capacitation status were not affected (p > 0.05) by different levels of rosemary aqueous extract. Lower (p < 0.05) MDA concentration has been observed in SL-R4 and SL-R6 groups. The results of this study demonstrate that supplementation of SL extender with rosemary aqueous extract influences post-thawed ram sperm quality in a dose dependent manner.

doi: 10.1016/j.cryobiol.2014.07.007

Melatonin-mediated effects on killifish reproductive axis2016-12-30T09:48:23+01:00

Melatonin-mediated effects on killifish reproductive axis

Lombardo F, Gioacchini G, Fabbrocini A, Candelma M, D’Adamo R, Giorgini E, Carnevali O.

Dipartimento di Scienze della Vita e dell’Ambiente, Università Politecnica delle Marche, Via Brecce Bianche, Ancona, Italy; Consiglio Nazionale delle Ricerche, Istituto di Scienze Marine, UOS Lesina, FG, Italy; Dipartimento di Scienze della Vita e dell’Ambiente, Università Politecnica delle Marche, Via Brecce Bianche, Ancona, Italy

The aim of this study was to investigate the melatonin-mediated effects upon the neuroendocrine axis of the brackish killifish (Fundulus heteroclitus), a suitable experimental model to study reproductive events. The ability of melatonin to enhance reproductive capacity (fecundity, embryo survival and hatching rate) inducing the transcriptional activity of gonadotropin releasing hormone (gnrh), luteinizing hormone receptor (lhr) and melatonin receptor (mtnr) was investigated in adult females. Moreover, the melatonin-mediated enhancement of killifish sperm motility and velocity was found consistent with higher fecundity of melatonin-exposed fishes. As a further extent, Fourier Transform Infrared (FT-IR) microspectroscopy evidenced a reduction of lipid unsaturation level on isolated spermatozoa from treated males. Moreover, the reduction of mtnr gene expression during embryo development and lower biometric parameters documented in the larvae from melatonin-exposed parents suggest that melatonin acts as a hormonal mediator able to transfer the environmental signal to oocytes and then to embryos as inheritance of adaptive environmental changes. These results support the positive role of melatonin on killifish reproduction and its role as a maternal factor on embryo and larval development.

doi: 10.1016/j.cbpa.2014.02.008

Obesity is associated with increased seminal insulin and leptin alongside reduced fertility parameters in a controlled male cohort2015-05-08T09:28:37+02:00

Obesity is associated with increased seminal insulin and leptin alongside reduced fertility parameters in a controlled male cohort

Leisegang K, Bouic PJ, Menkveld R, Henkel RR

Department of Medical Biosciences, University of the Western Cape, Private Bag X17, Bellville 7535, South Africa

BACKGROUND: Obesity appears to be associated with male reproductive dysfunction and infertility, although this has been inconsistent and inconclusive. Insulin and leptin are known mediators and modulators of the hypothalamus-pituitary-testes axis, contributing to the regulation of male reproductive potential and overall wellbeing. These hormones are also present in semen influencing sperm functions. Although abdominal obesity is closely associated with insulin resistance (hyperinsulinaemia), hyperleptinaemia and glucose dysfunction, changes in seminal plasma concentrations of insulin, leptin and glucose in obese males has not previously been investigated.
METHODS: This small case controlled study assessed serum and seminal concentrations of insulin, leptin and glucose in obese (BMI > =30; n = 23) and non-obese (BMI < 30; n = 19) males. Following a detailed medical history and examination, participants meeting the inclusion criteria were entered for data analysis. Body parameters such as BMI, waist and hip circumference and the waist hip ratio were measured. Serum and semen samples were collected and assayed for insulin, leptin and glucose. Semen samples also underwent a standard semen analysis, with sperm mitochondrial membrane potential (MMP) and DNA fragmentation (DF).
RESULTS: Obesity was associated with increased serum and seminal insulin and leptin, with no significant difference in seminal glucose. Serum and seminal concentrations of insulin and leptin were positively correlated. Furthermore, obesity was associated with decreased sperm concentration, sperm vitality and increased MMP and DF, with a non-significant impact on motility and morphology.
CONCLUSIONS: Hyperinsulinaemia and hyperleptinaemia are associated with increased seminal insulin and leptin concentrations, which may negatively impact male reproductive function in obesity. Insulin was also found to be highly concentrated in the seminal plasma of both groups. This data will contribute to the contradictive information available in the literature on the impact of obesity and male reproduction.

doi: 10.1186/1477-7827-12-34

Structural evolution of CatSper1 in rodents is influenced by sperm competition, with effects on sperm swimming velocity2015-05-08T09:29:04+02:00

Structural evolution of CatSper1 in rodents is influenced by sperm competition, with effects on sperm swimming velocity

Vicens A, Tourmente M, Roldan ER

Reproductive Ecology and Biology Group, Museo Nacional de Ciencias Naturales (CSIC), c/Jose Gutierrez Abascal 2, 28006 Madrid, Spain

BACKGROUND:
Competition between spermatozoa from rival males for success in fertilization (i.e., sperm competition) is an important selective force driving the evolution of male reproductive traits and promoting positive selection in genes related to reproductive function. Positive selection has been identified in reproductive proteins showing rapid divergence at nucleotide level. Other mutations, such as insertions and deletions (indels), also occur in protein-coding sequences. These structural changes, which exist in reproductive genes and result in length variation in coded proteins, could also be subjected to positive selection and be under the influence of sperm competition. Catsper1 is one such reproductive gene coding for a germ-line specific voltage-gated calcium channel essential for sperm motility and fertilization. Positive selection appears to promote fixation of indels in the N-terminal region of CatSper1 in mammalian species. However, it is not known which selective forces underlie these changes and their implications for sperm function.
RESULTS:
We tested if length variation in the N-terminal region of CatSper1 is influenced by sperm competition intensity in a group of closely related rodent species of the subfamily Murinae. Our results revealed a negative correlation between sequence length of CatSper1 and relative testes mass, a very good proxy of sperm competition levels. Since CatSper1 is important for sperm flagellar motility, we examined if length variation in the N-terminus of CatSper1 is linked to changes in sperm swimming velocity. We found a negative correlation between CatSper1 length and several sperm velocity parameters.
CONCLUSIONS:
Altogether, our results suggest that sperm competition selects for a shortening of the intracellular region of CatSper1 which, in turn, enhances sperm swimming velocity, an essential and adaptive trait for fertilization success.

doi: 10.1186/1471-2148-14-106

Diagnostic value of fine needle aspiration cytology in testicular disorders of red deer (Cervus elaphus): a case report2015-05-08T10:16:44+02:00

Diagnostic value of fine needle aspiration cytology in testicular disorders of red deer (Cervus elaphus): a case report

Pintus E, Ros-Santaella JL, Garde JJ

Department of Pathology and Veterinary Clinic, University of Sassari, Via Vienna 2, 07100 Sassari, Italy

We used fine needle aspiration cytology (FNAC) to diagnose Sertoli cell-only pattern and hypospermatogenesis in an Iberian red deer (Cervus elaphus hispanicus). Cytologic diagnosis was confirmed by histology and epididymal sperm analysis. We conclude that FNAC can be an important diagnostic tool in testicular diseases of wildlife.

doi: 10.7589/2013-11-304

Increasing precision and reducing variation in sperm assessments using the Sperm Class Analyzer®2016-12-30T09:48:23+01:00

Increasing precision and reducing variation in sperm assessments using the Sperm Class Analyzer®

Rothman C, Sims CA, Shamonki J, Schiewe MC

California Cryobank (CCB), QC/R&D Lab, Los Angeles, CA 90025, USA.

Background
CCB endeavors to optimize precision of sperm count and motility calculations by reducing potential procedural sources of variation. The adaptation of real-time video imaging software has made Computer Assisted Semen Analysis (CASA) more versatile and practical for the end users. In fact, this technology allows for remote capturing of video images (i.e., satellite labs) to be processed at one central location, thus eliminating inter-technician/facility variation.
Objective
This prospective validation study aims to compare sperm concentration and motility determinations by manual (Makler chamber) or automated (Sperm Class Analyzer {SCA}, Microptics) methods.  Two different technicians (one per method) were used to eliminate technical bias.
Methods
50 specimens were acquired from men being evaluated as potential sperm donors.  Consent was obtained from all participants. Semen analysis was performed on each sample in concurrence with an IRB-approved, FDA-supported validation trial of the SCA unit. The standard manual evaluation of each sample consisted of duplicate or triplicate counting of 20 grids per analysis on a Maker chamber. For the SCA analysis we loaded Leja SC 20 disposable slides with 5 μl of sample per chamber and placed it on a standard phase contrast microscope. Using the SCA Motility program, at least 5 separate fields were captured using a 10X objective under green-filtered low light. Centralized regions from the opening to the base of the chamber were documented. The highest and lowest sperm concentration outliers were eliminated.
Results
The SCA-CASA system assessed an average of 848 sperm/analysis, while Makler chambers evaluated 83 sperm/analysis. The mean concentration of specimens analyzed with the Makler was 10.9% lower than those analyzed with the SCA (64±3.6 vs 71.8±2.3, x106/ml). The manual method produced greater dispersion in motility estimates; total motility estimates were lower (48±5.4% vs 61±3.8%; 22% variation) and progressive motility estimates higher (32±4% vs 21±2%; 55% variation).
Discussion
The reproducibility of sperm count and motility calculation will always be subject to a degree of variation inherent to this biological product. Eliminating procedural variation and optimizing technician precision are important QC laboratory goals. A centralized SCA unit has the potential to provide superior evaluation and video documentation of specimens. Additional verification studies are needed to resolve why differences in motility exceed 20% differences in motility when compared to manual estimates.
Support
Microptic, Barcelona, Spain; ASPIRE IRB, LaMesa, CA.

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Cryopreservation of canine semen after cold storage in a Neopor box: effect of extender, centrifugation and storage time2016-12-30T09:48:23+01:00

Cryopreservation of canine semen after cold storage in a Neopor box: effect of extender, centrifugation and storage time

Hidalgo M, Portero JM, Demyda-Peyrás S, Ortiz I, Dorado J

Animal Reproduction Group, Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, University of Cordoba, Córdoba 14071, Spain; MERAGEM Research Group, Department of Genetics, Faculty of Veterinary Medicine, University of Cordoba, Cordoba 14071, Spain

The aim of this work was to assess the combined effect of sperm centrifugation, semen extender and storage time before freezing on post-thaw sperm quality and freezability on chilled stored canine semen in a Neopor box. Sperm parameters evaluated were total and progressive sperm motility by Computer-Assisted Sperm Analysis (CASA) and sperm viability and acrosome integrity using a triple fluorescent stain. Sperm quality and freezability indexes were also studied. First, the effect of centrifugation and two commercial extenders from Minitübe (Biladyl A and CaniPRO Freeze A) was evaluated in chilled semen after 24 and 45 hours of cold storage. No significant differences were observed between treatments in almost all the sperm parameters assessed. Secondly, chilled semen was frozen after 24 and 45 hours of cold storage in a Neopor box. The best results were obtained when semen was centrifuged, chilled with CaniPRO Freeze A and then frozen after 24 hours of cold storage, showing no differences in both post-thaw sperm quality and freezability in comparison with semen immediately frozen after collection. In conclusion, dog semen centrifuged after collection and extended with CaniPRO Freeze can be frozen after 24 hours of cold storage in a Neopor box, obtaining similar results to semen immediately frozen after collection.

doi: 10.1136/vr.102010

Role of zinc trafficking in male fertility: from germ to sperm2015-05-08T10:18:29+02:00

Role of zinc trafficking in male fertility: from germ to sperm

Foresta C, Garolla A, Cosci I, Menegazzo M, Ferigo M, Gandin V, De Toni L

Department of Medicine and Centre for Human Reproduction Pathology, University of Padova, Padova 35128, Italy

STUDY QUESTION: What are the dynamics of zinc (Zn) trafficking in sperm, at the testicular, epididymal and ejaculate levels?
SUMMARY ANSWER: Zn transporters are peculiarly expressed in the cells of the germ line and Zn uptake is maximal at the post-epididymal phase, where Zn is involved in the regulation of sperm functions.
WHAT IS KNOWN ALREADY: Zn is known to influence several phases of sperm life, from germ cell development to spermiation. Zn trafficking across the membrane is allowed by specific families of transporters known as the ZnTs, which are involved in effluent release, and the Zips, which mediate uptake.
STUDY DESIGN, SIZE, DURATION: We enrolled 10 normozoospermic healthy participants in an infertility survey programme, as well as 5 patients affected by testicular germ cell cancer, and 18 patients presenting with obstructive azoospermia, without mutations of the CFTR gene, and undergoing assisted reproductive technologies.
PARTICIPANTS/MATERIALS, SETTING, METHODS: The research study was performed at our University Clinic. Semen samples, or biopsies or fine needle aspirates from the testis or epididymis, were obtained from each of the participants. Protein expression of main members of the ZnT and Zip families of Zn transporters was examined in human testis and epididymis samples by immunofluorescence. Quantification of sperm Zn content was performed by flow cytometry, atomic absorption spectrometry (AA) and autometallography.
MAIN RESULTS AND THE ROLE OF CHANCE: Intratubular cells of the germ line displayed a high redundancy of Zip family members involved in Zn uptake, while ZnT transporters were more represented in epididymis. Testicular and epididymal spermatozoa contained less Zn than ejaculated spermatozoa (2.56 ± 0.51 and 12.58 ± 3.16 versus 40.48 ± 12.71 ng Zn/10(6)cells, respectively). Gain of hypermotility and acrosomal reaction were significantly linked to the loss of Zn content in ejaculated spermatozoa.
LIMITATIONS, REASONS FOR CAUTION: This was an ancillary study performed on a small cohort of normozoospermic subjects. Although these results clarify the Zn trafficking during different phases of sperm life, no conclusive information can be drawn about the fertilizing potential of sperm, and the overall pregnancy outcomes, after Zn supplementation.
WIDER IMPLICATIONS OF THE FINDINGS: Our data disclose the dynamics of Zn trafficking during over the sperm lifespan.
STUDY FUNDING/COMPETING INTEREST(S): No external funding was sought or obtained for this study. No conflict of interest is declared.

doi: 10.1093/humrep/deu075

Effect of Cissampelos capensis rhizome extract on human spermatozoa in vitro2015-05-08T10:19:07+02:00

Effect of Cissampelos capensis rhizome extract on human spermatozoa in vitro

Shalaweh SM1, Erasmus N, Weitz F, Henkel RR.

Department of Medical Biosciences, University of the Western Cape, Bellville, South Africa.

Cissampelos capensis is commonly known by the Afrikaans name ‘dawidjies’ or ‘dawidjieswortel’. C. capensis is the most important and best-known medicinal plant of the family Menispermaceae used by the Khoisan and other rural people in the western regions of South Africa. Among numerous other ailments, it is traditionally taken to treat male fertility problems. Yet, no studies have investigated the effects of this plant or its extracts on human spermatozoa. The aim of study was to investigate the effects of C. capensis extracts on sperm function. A total of 77 semen samples were collected. Spermatozoa were washed with HTF-BSA medium and incubated with different concentrations of C. capensis (0, 0.05, 0.5, 5, 50, 200 μg ml-1 ) for 1 h at 37 °C. Sperm motility, vitality, acrosome reaction, reactive oxygen species (ROS), capacitation, Annexin V binding, DNA fragmentation and mitochondrial membrane potential (Δψm ) were determined. While viability, Annexin V positivity and Δψm were not affected, the percentages of ROS-positive, TUNEL-positive, capacitated and hyperactivated spermatozoa increased significantly and dose-dependently. It is concluded that the alkaloids present in the extract of C. capansis rhizomes triggered sperm intrinsic superoxide production leading to sperm capacitation and DNA fragmentation.

doi: 10.1111/and.12264

Evaluation of ethylene glycol as a cryoprotectant in the sperm cryopreservation of trans-andean shovelnose catfish (sorubim cuspicaudus, pimelodidae)2016-12-30T09:48:25+01:00

Evaluation of ethylene glycol as a cryoprotectant in the sperm cryopreservation of trans-andean shovelnose catfish (sorubim cuspicaudus, pimelodidae)

VÍCTOR J. ATENCIO-GARCÍA, M.Sc.; MARÍA DORADO, M.Sc., Biol; EMILIO NAVARRO, Acuicultor; FRANCISCO PÉREZ, Acuicultor;BRINER HERRERA, Acuicultor; JORGE MOVILLA, Acuicultor; JOSÉ A. ESPINOSA-ARAUJO, M.Sc.

Universidad de Córdoba, Carrera 6 # 76-103. Montería, Colombia;Autoridad Nacional de Acuicultura y Pesca – A UNAP. Repelón, Colombia

The catfish Sorubim cuspicaudus cryopreservation semen was evaluated using three levels (5, 10, 15%) of ethylene glycol (ETG). Males (n = 13) undergoing spermiation and in final maturation females (n = 6) were induced with 0.4 ml Ovaprim®/Kg, after 12 and 14 post-induction the semen was collected in 2 ml Eppendorf vials. The different cryoprotectants solutions were prepared with glucose 6% (w/v) skimmed milk powder 5% (w/v) and distilled water. The semen was diluted in ratio 1:3 (semen:extender), packed in macrotubes of 2.5 ml and frozen in liquid nitrogen (NL) vapor for 30 minutes, then the macrotubes were stored in cryogenic tanks submerged directly in NL. The sperm were thawed in serological bath to 35 °C for 90 seconds. The total motility, total progressivity and velocities in fresh and thawed semen were analyzed with the Sperm Class Analyzer software (SCA Microptic SL, Spain). Fertility and hatching rates were assessed with 1.0-1.5 g of oocytes in experimental up flow incubators 2 L, a completely randomized design was used. The hatching rate of fresh semen was 51.8 ± 21.0%, with no significant differences with semen cryopreserved with ETG 5% (38.6±13.9%) (p&gt;0.05), while ETG 15% (9.6±2.9%), recorded the lower hatching rate (p&lt;0.05). The results suggest that the cryoprotectant solution composed of ETG 5%, glucose 6% and powdered milk 5% is a viable alternative for semen cryopreservation of the catfish Sorubim cuspicaudus.

doi: 10.15446/abc.v19n2.41288

Vérification de performances d’un système CASA Sperm Class Analyzer SCA2016-12-30T09:48:25+01:00

Vérification de performances d’un système CASA Sperm Class Analyzer SCA

Nassima Tarhouchi, Julie Le Bachelet, Daniel Przyrowski, Olivier Moreau.

Laboratoire Bioatlantique AMP diagnostique

Le Sperm Class Analyzer (SCA® MICROPTIC) est un système de gestion et d’analyse des principaux paramètres du sperme humain. Le logiciel comprend différents modules: numération, mobilité, cytologie… Le laboratoire s’est intéressé au programme « motility » afin d’évaluer la numération et la mobilité des spermatozoïdes.
La vérification des méthodes est une confirmation par des preuves tangibles que les exigences, spécifiées par le laboratoire, ont été satisfaites et sont adaptées à l’utilisation prévue. Ce processus permet de vérifier les performances et de garantir la fiabilité des résultats de mesure obtenus dans notre laboratoire.
Conformément à la norme NF EN ISO 15189 : 2012, toutes performances analytiques doivent être préalablement vérifiées. C’est pourquoi le programme « motility » du SCA, a été vérifié en se basant sur des critères d’acceptation définis par le laboratoire, en ayant prit comme référence le guide de l’OMS : « WHO laboratory manual for the examination and processing of human semen, fifth edition ».

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Human semen cryopreservation: a sperm DNA fragmentation study with alkaline and neutral Comet assay2016-12-30T09:48:25+01:00

Human semen cryopreservation: a sperm DNA fragmentation study with alkaline and neutral Comet assay

J. Ribas-Maynou, A. Fernandez-Encinas, A. Garcıa-Peiro, E. Prada, C. Abad, M. J. Amengual, J. Navarro and J. Benet

Departament de Biologia Cellular, Fisiologia i Immunologia, Universitat Autonoma de Barcelona, Bellaterra, Catalunya, Spain, Centro de Infertilidad Masculina y Analisis de Barcelona (CIMAB), Edifici Eureka, PBM5, Parc de Recerca de la UAB (PRUAB), Bellaterra, Spain, Servei de Ginecologia, Hospital Universitari Mutua de Terrassa, Terrassa, Spain, Servei d’Urologia, Corporacio Sanitaria Parc Taulı, Institut Universitari Parc Taulı – UAB, Sabadell, Spain, and UDIAT, Centre Diagnostic, Corporacio Sanitaria Parc Taulı, Institut Universitari Parc Taulı – UAB, Sabadell, Spain

Sperm cryopreservation is widely used for both research and reproduction purposes, but its effect on sperm DNA damage remains controversial. Sperm DNA fragmentation (SDF) has become an important biomarker to assess male infertility. In particular, the differentiation between single- and double-stranded DNA fragmentation (ssSDF and dsSDF) has clinical implications for male infertility where ssSDF is associated with reduced fertility, whereas dsSDF is associated with increased risk of miscarriage. In this study, semen samples from 30 human males have been analysed in both fresh and cryopreserved using the alkaline and neutral Comet assays.
Results show an increase of about 10% of ssSDF, assessed by the alkaline Comet assay, regardless of the male fertility status. Neutral Comet analysis of dsSDF does not show any statistical increase when comparing fresh and cryopreserved samples in any of the patient groups. Results support previous reports that oxidative stress is the major effector in DNA damage during sample cryopreservation, as, on one hand, ssSDF has previously been related to oxidative damage and, on the other hand, we have not found any effect on dsSDF. Therefore, there might be a slight risk of decreased fertility after using a freezed sample, but no evidence for increased miscarriage risk from cryopreserved spermatozoa should be expected.

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Validation of the Sperm Class Analyser CASA system for sperm counting in a busy diagnostic semen analysis laboratory2015-05-08T10:20:39+02:00

Validation of the Sperm Class Analyser CASA system for sperm counting in a busy diagnostic semen analysis laboratory

Dearing CG, Kilburn S, Lindsay KS.

Andrology Laboratory, Hammersmith Hospital, Imperial College NHS Trust , London , UK

Sperm counts have been linked to several fertility outcomes making them an essential parameter of semen analysis. It has become increasingly recognised that Computer-Assisted Semen Analysis (CASA) provides improved precision over manual methods but that systems are seldom validated robustly for use. The objective of this study was to gather the evidence to validate or reject the Sperm Class Analyser (SCA) as a tool for routine sperm counting in a busy laboratory setting. The criteria examined were comparison with the Improved Neubauer and Leja 20-μm chambers, within and between field precision, sperm concentration linearity from a stock diluted in semen and media, accuracy against internal and external quality material, assessment of uneven flow effects and a receiver operating characteristic (ROC) analysis to predict fertility in comparison with the Neubauer method. This work demonstrates that SCA CASA technology is not a standalone ‘black box’, but rather a tool for well-trained staff that allows rapid, high-number sperm counting providing errors are identified and corrected. The system will produce accurate, linear, precise results, with less analytical variance than manual methods that correlate well against the Improved Neubauer chamber. The system provides superior predictive potential for diagnosing fertility problems.

PMID: 24359435
Effect of freezing extender composition and male line on semen traits and reproductive performance in rabbits2015-05-08T10:20:55+02:00

Effect of freezing extender composition and male line on semen traits and reproductive performance in rabbits

Viudes-de-Castro MP, Lavara R, Safaa HM, Marco-Jiménez F, Mehaisen GM, Vicente JS

Centro de Investigación y Tecnologia Animal-Instituto Valenciano de Investigaciones Agrarias Agrarias (CITA-IVIA), Polígono La Esperanza n° 100, 12400, Segorbe, Castellón, Spain; Departamento de Ciencia Animal: Laboratorio de Biotecnologia de la Reproducción, Instituto de Ciencia y Tecnologia Animal, Universidad Politécnica de Valencia, 46071, Valencia, Spain; Animal Production Department, Faculty of Agriculture, Cairo University, 12613, Giza, Egypt

This study was conducted to elucidate the effect of different freezing extenders on two lines selected for hyperprolificacy and longevity (H and LP, respectively). In extender A, dimethyl sulphoxide (Me2SO) and sucrose were used as cryoprotectants. In extenders B and C, the sucrose was replaced by 20% egg yolk, and in extender C the Me2SO was substituted by acetamide. Semen was packaged in 0.25 ml plastic straws and cooled at 5°C for 45 min, and then was frozen in liquid nitrogen vapour for 10 min before being plunged into the liquid nitrogen. Thawing was carried out by immersing the straws in a water bath at 50°C for 10 s. Frozen-thawed semen characteristics and reproductive parameters were affected by freezing. Extender C showed significantly lower post-thawing quality traits than any of the three extenders. Acrosome integrity was significantly improved when Me2SO was used as cryoprotectant. Sucrose replacement by 20% egg yolk had no effect on acrosome integrity but provided significantly lower sperm motility and viability. Freezing extender affected fertility rate, total born, number of implantation sites and gestational losses, obtaining better results when extender A was used. The acrosomal integrity after frozen-thawed process showed a significant correlation with fertility at 12th day and also at birth, indicating that an increase in acrosomal integrity leads to an increase in both fertilities (12th day and at birth). A positive correlation between motility of semen and implantation sites was found. The post-thawing quality traits of semen were not affected by the genetic line, although LP line showed higher total born and lower foetal and gestational losses. The findings of this study suggest that freezing extender composition has a significant effect on the success of rabbit sperm for preservation, and when Me2SO was used as permeable cryoprotectant sucrose provided better protection compared with egg yolk and improved reproductive traits, and, on the other hand, the male genotypes used in the present study had no effect on frozen-thawed sperm parameters but negatively affected some of the reproductive parameters.

doi: 10.1017/S1751731114000135

Effect of breeding season on the kinematic parameters and morphology of ram’ sperm from synthetic population bulgarian milk sheep breed2016-12-30T09:48:25+01:00

Effect of breeding season on the kinematic parameters and morphology of ram’ sperm from synthetic population bulgarian milk sheep breed

D. Abadjieva; M. Chervenkov; R. Stefanov; N. Metodiev; E. Kistanova; D. kacheva; and E. Raycheva

Bulgarian Academy of Sciences, Institute of Biology and Immunology of Reproduction, BG – 1113 Sofia, Bulgaria; Institute of Animal Science, BG – 2232 Kostinbrod, Bulgaria

The aim of this study was the investigation of the breeding season effect on the kinematic and main spermatological parameters of the rams from Synthetic Population Bulgarian Milk sheep breed (SPBM), new Bulgarian breed certifiated in 2005. The experiment was carried out with seven rams. Two consecutive ejaculates from each ram were obtained by artificial vagina before and during the breeding campaign (n=28). Overall sperm motility and the individual kinematic parameters of motile spermatozoa were assessed by the computer-aided sperm analysis system Sperm Class Analyzer (SCA). The sperm morphology was estimated after sperm blue stain and calculated as a percent of abnormal cells among 100 sperm cells from several filds on the slide.

It was found that the ejaculates obtained from SPBM rams during the breeding season had better features of sperm motion kinetics. The values of the velocity parameters (P< 0.05), motility (P< 0.05), and percentages of spermatozoa with rapid (P< 0.01) and medium (P< 0.001) speed were higher than those from the ejaculates collected before the breeding season. Minor and not signifiant changes in the kinematic parameters of motile spermatozoa in consecutive ejaculates were observed. No signifiant differences were established in morphological status of spermatozoa in nonbreeding and breeding season. It seems that the better sperm motility kinematic parameters during the breeding season ensure the higher sperm fertility and success on the future insemination.

Bulgarian Journal of Agricultural Science, 20 (No 4) 2014, 967-972

Verification of reference values for semen analysis according to WHO 2010 in Buenos Aires2016-12-30T09:48:25+01:00

Verification of reference values for semen analysis according to WHO 2010 in Buenos Aires

Susana Mercedes Curi, Patricia Haydee Chenlo, Mercedes Norma Pugliese, Julia Irene Ariagno, Herberto Ernesto Repetto, José Vazquez, Melba Sardi Segovia, Gabriela Ruth Mendeluk

Laboratorio de Fertilidad Masculina, Departamento de Bioquímica Clínica, INFIBIOC, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Córdoba 2351 (1120), Buenos Aires, Argentina.

WHO 2010 provided reference values for semen analysis; however clinical laboratories should verify them in healthy fertile males. In order to be able to transfer this reference interval to our unit, it was proposed to examine the available data in the Buenos Aires population. The range of data obtained in this population was also provided for the following: total number of progressively motile and morphologically normal spermatozoa in the ejaculate, interkinematic parameter values and functional tests, in an attempt to establish standardization in these parameters. The process of reference value verification was carried out according to C28-A2 CLSI (Clinical and Laboratory Standards Institute) guidelines; to that aim, twenty men born in Argentina who reside in Buenos Aires province or city were recruited to participate. They were of proven fertility in the last 12 months. Individuals with pathologies by andrological consultation were excluded. Samples were processed according to 2010 WHO. In Buenos Aires City, WHO reference values were confirmed whereas semen volume was not confirmed. The range of values for total progressive and morphologically normal spermatozoa as well as kinetic parameter values, enrichment by swim up and functional test results were established for the male fertile population studied in an attempt to contribute to the construction of future reference values.

Acta Bioquím Clín Latinoam 2014; 48 (4): 429-35

Assessment of cryopreserved sperm motility from colombian creole stallions by Sperm Class Analyzer2013-12-01T09:09:19+01:00

Assessment of cryopreserved sperm motility from colombian creole stallions by Sperm Class Analyzer

Giovanni Restrepo, Daniel Ocampo, Andrés Velásquez

Department of Agrarian Sciences, Politécnico Colombiano Jaime Isaza Cadavid, Colombia.

Reproduction plays a key role in the genetic improvement in horses. Equine semen cryopreservation is a complementary technique to reproductive biotechnology processes. However, only 40% of the horses produce semen that can be cryopreserved, with a high variability in fertility. The reliability of the methods of semen evaluation is important to determine their fertility potential, but the conventional analysis by microscopic evaluation has not been very effective in this regard. Computer-assisted semen analysis (CASA) provides an objective measurement of sperm motility. This study aimed to compare the evaluation of equine sperm motility through conventional and SCA® (CASA) methods. Semen of three
horses of the Colombian creole breed (three ejaculates each) was frozen in extender (4% egg yolk, 5% dimethylformamide). Thawed sperm motility was conventionally determined by phase contrast microscopy and by SCA ® software. For statistical analysis was fitted a generalized linear model (GLM) and was determined the association between variables using correlation and regression analysis. It was found an average of total motility of 54.3% ± 10.1 % and 61.8% ± 13.9% for conventional and SCA® evaluation, respectively. No significant difference was observed between analysis methods. We conclude that equine semen analysis by conventional method and SCA® system is comparable to analyze the motility of criopreserved equine semen.

Rev. U.D.CA Act. & Div. Cient. 16(2):445-450

Effect of single layer centrifugation using Androcoll-E-Large on the sperm quality parameters of cooled-stored donkey semen doses2016-12-30T09:48:25+01:00

Effect of single layer centrifugation using Androcoll-E-Large on the sperm quality parameters of cooled-stored donkey semen doses

Ortiz I, Dorado J, Ramírez L, Morrell JM, Acha D, Urbano M, Gálvez MJ, Carrasco JJ, Gómez-Arrones V, Calero-Carretero R, Hidalgo M

Animal Reproduction Group, Department of Animal Medicine and Surgery, University of Cordoba, 14071 Cordoba, Spain; Department of Clinical Sciences, Swedish University of Agricultural Sciences (SLU), Box 7054, SE-75007, Uppsala, Sweden; Equine Center for Assisted Reproduction Services, CENSYRA-Extremadura Government, 06007 Badajoz, Spain

The aim of this study was to determine the effect of single layer centrifugation (SLC) using Androcoll-E-Large on donkey sperm quality parameters after 24 h of cool-storage. Ejaculates were collected from Andalusian donkeys and then cooled at 5°C. SLC was carried out after 24 h of cool-storage using Androcoll-E-Large. In the first experiment, all sperm parameters assessed (total and progressive sperm motility, viability, sperm morphology and sperm kinematics VCL, VSL, VAP, LIN, STR, WOB, ALH and BCF) were statistically compared between semen samples processed or not with Androcoll-E-Large. Significant differences (P<0.05) were found between SLC-selected and unselected semen samples for all parameters assessed, obtaining better results after SLC. In the second experiment, semen samples were classified in two groups according to their sperm progressive motility (PM) before SLC. Then, the increments obtained in semen quality parameters after SLC were compared between groups. No significant differences were found between groups, indicating that SLC improved the sperm quality parameters of entire set of semen samples processed with independence to their original PM. In conclusion, SLC with Androcoll-E-Large can be used in donkeys, increasing the sperm quality of cooled-stored donkey semen doses after 24 h of cool storage.

doi: 10.1017/S1751731113002097

Effect of cryopreservation and single layer centrifugation on canine sperm DNA fragmentation assessed by the sperm chromatin dispersion test2015-05-08T10:21:29+02:00

Effect of cryopreservation and single layer centrifugation on canine sperm DNA fragmentation assessed by the sperm chromatin dispersion test

Urbano M, Dorado J, Ortiz I, Morrell JM, Demyda-Peyrás S, Gálvez MJ, Alcaraz L, Ramírez L, Hidalgo M

Animal Reproduction Group, Department of Animal Medicine and Surgery, University of Cordoba, Cordoba, Spain

The aims of this study were: 1) to assess the effect of freezing and thawing on dog sperm DNA fragmentation index (sDFI) using the sperm chromatin dispersion test (SCDt); and 2) to determine whether or not the sperm selection by single layer centrifugation (SLC) using Androcoll-C improves sperm DNA longevity in SLC-selected frozen-thawed dog semen samples. Semen samples were collected from 4 dogs using digital manipulation. After collection, ejaculates were pooled and cryopreserved following a standard protocol. Sperm motility and morphology were assessed before freezing and after thawing as a control for the cryopreservation method used. In experiment 1, sDFI was analyzed immediately before freezing and after thawing (baseline values), showing no significant differences between fresh and frozen-thawed semen samples. In experiment 2, frozen-thawed semen samples were processed or not by SLC using Androcoll-C and longevity of DNA were assessed in terms of sDFI after 24h of in vitro incubation at physiological temperature (38°C). The results showed low values of sDFI in SLC-selected semen in comparison to unselected samples. In conclusion, no effect of cryopreservation was observed on baseline values of dog sperm DNA fragmentation. Additionally, SLC-selection using Androcoll-C improved longevity of frozen-thawed sperm DNA assessed by the SCDt.

doi: 10.1016/j.anireprosci.2013.10.005

In vivo effects of Aspalathus linearis (rooibos) on male rat reproductive functions2022-05-04T15:56:31+02:00

In vivo effects of Aspalathus linearis (rooibos) on male rat reproductive functions

Opuwari CS1, Monsees TK.

Department of Medical Biosciences, University of the Western Cape, Bellville, South Africa

Aspalathus linearis (rooibos tea) may improve sperm function owing to its antioxidant properties. To test this hypothesis, male rats were given 2% or 5% rooibos tea for 52 days. No significant alterations were observed in body and reproductive organs weight, serum antioxidant capacity and testosterone level. Seminiferous tubules displayed complete spermatogenesis. However, a significant (P < 0.05) decrease in tubule diameter and germinal epithelial height was observed. Epithelial height of caput epididymides showed a significant increase. Unfermented rooibos significantly enhanced sperm concentration, viability and motility. Fermented rooibos also significantly improved sperm vitality (P < 0.01), but caused a significant increase in spontaneous acrosome reaction (P < 0.05), whereas unfermented did not. Creatinine was significantly enhanced in all treated rats, consistent with significant higher kidney weights. Rooibos significantly reduced alanine transaminase level, while 2% fermented rooibos significantly decreased aspartate transaminase level (P < 0.01). In conclusion, treatment with rooibos improved sperm concentration, viability and motility, which might be attributed to its high level of antioxidants. However, prolonged exposure of rooibos might result in subtle structural changes in the male reproductive system and may induce acrosome reaction, which can impair fertility. Intake of large amounts of rooibos may also harm liver and kidney function.

doi: 10.1111/and.12158

Sperm DNA fragmentation assay by sperm chromatin dispersion (SCD)2017-05-25T13:09:18+02:00

Sperm DNA fragmentation assay by sperm chromatin dispersion (SCD): correlation between DNA fragmentation and outcome of intracytoplasmic sperm injection

T. Sivanarayana; Ch. Ravi Krishna; G. Jaya Prakash; K. M. Krishna; K. Madan; G. Sudhakar; G. A. Rama Raju

Gynecology Division, Krishna IVF Clinic, Visakhapatnam, India; Embryology Research Group, Krishna IVF Clinic, Visakhapatnam, India; Genetics Department, Krishna IVF Clinic, Visakhapatnam, India; Department of Human Genetics, Andhra University, Visakhapatnam, India

Abstract

Purpose: The aim of the present study was to investigate the relationship between sperm DNA fragmentation index (sDFI) and outcome of intracytoplasmic sperm injection (ICSI).

Methods: All the patients were divided into two groups based on sperm DNA fragmentation analysis by the sperm chromatin dispersion (SCD) method. A total of 237 patients were in the DNA fragmentation normal group (sDFI ≤ 30 %), and 140 patients were in the DNA fragmentation abnormal group (sDFI ≥ 30 %). The relationship of sDFI with the outcome of ICSI was analyzed.

Results: A significant difference in semen parameters was observed between the DNA fragmentation normal and abnormal groups [count, motility and morphology (p < 0.05)]. However, no significant difference was seen between the number of oocytes retrieved and fertilization rates between the two groups, whereas the number of embryos progressed to day 3 and the blastocyst formation rate in the remaining embryos after transfer were significantly more in the DNA fragmentation normal group (p < 0.05). A significant negative correlation was noted between DFI values of more than 30 % and number of pregnancies and deliveries (p < 0.05). A higher DFI was also associated with increased abortion rates.

Conclusions: In the present study, sperm with DNA fragmentation showed a negative correlation with semen parameters. Further, sperm with damaged DNA have potential adverse effects on embryo progression, clinical pregnancy rate, and ongoing pregnancies.

doi: 10.1007/s12522-013-0168-7

Gamete quality of streaked prochilod Prochilodus lineatus (Characiformes) after GnRHa and dopamine antagonist treatment2015-05-08T10:22:29+02:00

Gamete quality of streaked prochilod Prochilodus lineatus (Characiformes) after GnRHa and dopamine antagonist treatment

Viveiros AT, Gonçalves AC, Di Chiacchio IM, Nascimento AF, Romagosa E, Leal MC

Department of Animal Science (DZO),Federal University of Lavras,(UFLA),PO Box 3037,Lavras,MG,37200-000,Brazil; Department of Animal Science,Federal University of Lavras (UFLA),P.O. Box 3037,Lavras,Minas Gerais,37200-000,Brazil; Institute of Fisheries,APTA,SAA,SP,Brazil

The efficiency of Ovaprim™ salmon gonadotropin-releasing hormone agonist (GnRHa) and dopamine antagonist on the induction of spawning and spermiation in Prochilodus lineatus in comparison with the commonly used method using pituitary extract (PE) was evaluated. Females received PE at 0.5 + 5.0 mg/kg and Ovaprim™ at 0.05 + 0.45 ml/kg or at 0.125 + 0.375 ml/kg. All males received a first dose of PE at 0.4 mg/kg and then PE at 4.0 mg/kg or Ovaprim™ at 0.25 ml/kg. Oocyte, egg, larvae and sperm quality were evaluated. All females spawned and oocyte, egg and larvae quality were similar between Ovaprim™-treated (both doses) and PE-treated females. Data from females were pooled and the mean values were: 242 g ova weight, 15% ova index, 1209 oocytes/g ova, 284,539 oocytes/female, 183 oocytes/g body weight, 1.18 mm oocyte diameter, 49% fertilization rate, 43% hatching rate and 89% normal larvae. Sperm quality was similar between Ovaprim™-treated and PE-treated males. Data from males were pooled and the mean values of semen were: volume of 3.0 ml, 14.9 × 109 sperm/ml, osmolality of 283 mOsm/kg, pH of 7.4, 71% motile sperm, 217 μm/s curvilinear velocity, 102 μm/s straight-line velocity and 189 μm/s average path velocity. Ovaprim™ treatment can be used for commercial reproduction of P. lineatus, without any loss of gamete quality in comparison with PE treatment.

doi: 10.1017/S0967199413000440

Dietary fish oil can change sperm parameters and fatty acid profiles of ram sperm during oil consumption period and after removal of oil source2015-05-08T10:22:50+02:00

Dietary fish oil can change sperm parameters and fatty acid profiles of ram sperm during oil consumption period and after removal of oil source

Alizadeh A, Esmaeili V, Shahverdi A, Rashidi L

Department of Animal Science, Saveh Branch, Islamic Azad University, Saveh, Iran; Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran; Institute of Standard and Industrial Research of Iran (ISIRI), Karaj, Iran

OBJECTIVE: The effects of dietary fish oil on semen quality and sperm fatty acid profiles during consumption of n-3 fatty acids as well as the persistency of fatty acids in ram’s sperm after removing dietary oil from the diet were investigated.
MATERIALS AND METHODS: In this experimental study, we randomly assigned 9 Zandi rams to two groups (isoenergetic and isonitrogenous diets): control (CTR; n=5) and fish oil (FO; n=4) for 70 days with a constant level of vitamin E in both groups. Semen was collected at the first week and at the last week of the feeding period (phase 1). After the feeding period, all rams were fed a conventional diet and semen samples were collected one and two months after removal of FO (phase 2). The sperm parameters and fatty acid profiles were measured by computer assisted semen analyzer (CASA) and gas chromatography (GC), respectively. The completely randomized design was used and data were analyzed with SPSS version 16.
RESULTS: Dietary FO had significant positive effects on all sperm quality and quantity parameters compared with the CTR during the feeding period (p<0.05). The positive effects of FO on sperm concentration and total sperm output were observed at one and two months after removal of FO (p<0.05), whereas other sperm parameters were unaffected. Before feeding, C14 (myristic acid), C16 (palmitic acid), C18 (stearic acid), C18:1 (oleic acid) and C22:6 (docosahexaenoic acid: DHA) were the primary sperm FA. FO in the diet increased sperm DHA, C14:0 and C18:0 during the feeding period (p<0.05).
CONCLUSION: The present study showed not only manipulation of ram sperm fatty acid profiles by dietary FO and sperm parameters during the feeding period, but also the persistency of unique effects of dietary omega-3 fatty acids up to two months following its removal from the diet. Also, we recommend that sperm fatty acid profiles should be comprehensively analyzed and monitored.

doi: 24611147

A multi-regional study on new approaches to investigate the quality of human sperm – including DNA fragmentation, proteomics and metabolomics2016-12-30T09:48:25+01:00

A multi-regional study on new approaches to investigate the quality of human sperm – including DNA fragmentation, proteomics and metabolomics

Hiva Alipour, Ingolf Nielsen, Fereshteh Dardmeh, Gerhard Van der Horst

Aalborg University, Aalborg University Hospital, University of the Western Cape

Background: Preliminary data has also shown that there is less fragmented sperm in 2nd and 3rd ejaculates compared to first ones which could be a major factor in determining the pregnancy outcome. Assessing this factor objectively and relating it to other parameters in sperm quality in this study could result in new prediction criteria for the pregnancy outcome. Materials and Methods: As one of the goals, this study will focus on analyzing the seminal fluid from the semen sample using NMR and Mass Spectrometry in research for a correlation between the results of these methods and the DNA fragmentation, Kinetic parameters and finally implantation rates and pregnancy outcome. Results: As another goal in this study we plan to assess whether the second consecutive sample obtained within 12 hours of the first sample has a lower DNA fragmentation rate which will be very important in lowering the number of failed implantations and repeated abortions. Conclusion: Collecting and analyzing the samples from different locations (different countries and continents) using the SCA (Sperm Class Analyzer, Microptic, Spain) which has eliminated the inter technician variation and provides quantitative data and a means to assess samples at the exact same scale, will provide a comparison of the average sperm statistics and also the quality of consecutive sperm samples in these different locations. This would specially prove to be of utmost importance in determining and endorsing a global scale for the computer assisted sperm analysis results which seem to be taking over the old visual (subjective) analysis of sperm among the laboratories and clinics worldwide.

Conference: 14th Royan Congress on Reproductive Biomedicine, At Tehran, Iran, Volume: 7, Suppl. 1

Sperm DNA Fragmentation Assays Correlate with Sperm Abnormal Morphology and the Pregnancy Outcome2013-05-07T10:56:28+02:00

Sperm DNA Fragmentation Assays Correlate with Sperm Abnormal Morphology and the Pregnancy Outcome

Adriana Fortunato, Rita Leo, Sofia Casale, Giuseppina Nacchia, Francesca Liguori, Elisabetta Tost

Animal Physiology and Evolution laboratory Stazione Zoologica Anton Dohrn, Naples, Italy; DF center, Piazza Municipio 4, 80138- Naples, Italy

Abstract: 

Purpose: A retrospective study of 89 hypo-fertile male patients attending for in vitro fertilization was undertaken in order to evaluate possible correlations among sperm DNA damage, sperm analysis parameters and pregnancies.

Methods: Sperm parameters (concentration, normal morphology and multiple anomalies) were evaluated according to the World Health Organization guidelines. DNA damages weresimultaneously evaluated on each sperm sample by i) sperm chromatin decondensation test; ii) sperm DNA fragmentation evaluated by Sperm Chromatin Dispersion and Halo sperm Kit; iii) sperm DNA fragmentation evaluated by the Terminal Uridine Nick-End Labelling procedure.

Results: Statistical analysis was performed by using analysis of variance and least squares regression. The sperm chromatin dispersion and fragmentation assays showed a statistically significant positive correlation (P=0.0017) in all the samples confirming the good efficacy of either of the two tests in detecting sperm DNA damage. Both the two test negatively correlated with normal sperm morphology (P=0.008), however only by using Halo sperm test we obtained a significant correlation with multiple sperm pathological morphologies (P=0.029) and an inverse correlation with pregnancies outcome (P=0.013). No correlation was detected among DNA damages, sperm concentration and chromatin decondensation.

Conclusions: These data suggest a similar efficacy of sperm chromatin dispersion and Terminal Uridine Nick-End Labelling in detecting sperm DNA integrity. Due to the higher sensitivity of Halo test, its prognostic role in the diagnosis of fertilizing ability of a semen sample and possible pregnancy rate is discussed.

See full article here

Journal of Fertilization – DOI: 10.4172/2375-4508.1000101
Received April 09, 2013; Accepted May 04, 2013; Published May 07, 2013

Cryopreservation of collared peccary (Pecari tajacu) semen using different freezing curves, straw sizes, and thawing rates2015-05-08T10:24:24+02:00

Cryopreservation of collared peccary (Pecari tajacu) semen using different freezing curves, straw sizes, and thawing rates

Silva MA, Peixoto GC, Castelo TS, Lima GL, Silva AM, Oliveira MF, Silva AR

Laboratory of Animal Germplasm Conservation-LCGA, Universidade Federal Rural do Semi-Árido-UFERSA, Mossoró, RN, Brazil

The objective of this study was to verify the effect of different freezing curves, straw sizes, and thawing rates on the cryopreservation of collared peccary semen. Twelve ejaculates were obtained from captive adult males by electroejaculation, and evaluated for sperm motility, kinetic rating, viability, morphology, and functional membrane integrity. The ejaculates were diluted in a coconut water extender (ACP-116c) with egg yolk and glycerol, packaged into 0.25 mL or 0.50 mL plastic straws and cryopreserved in liquid nitrogen following a slow (-10 °C/min) or a fast (-40 °C/min) freezing curve. After one week, samples were thawed at 37 °C/1 min or 70 °C/8s and evaluated as reported for fresh semen, and also for kinematic parameters (computerized analysis). A significant decrease in sperm motility and kinetic rating was observed after glycerol addition at 5 °C and also after thawing for all the treatments (P<0.05). Regarding post-thaw semen variables, no differences were verified between freezing curves when the same straw size and thawing rate were taken as reference (P>0.05). In general, values for sperm characteristics found after thawing at 37 °C were better preserved than at 70 °C (P<0.05), both in the use of 0.25 mL or 0.50 mL straws, which were similar for semen packaging (P>0.05). The evaluation of the kinematic parameters of sperm motility confirmed these results at values varying from 20% to 30% motile sperm for the samples tha wed at 37 °C, and values fewer than 12% motile sperm for samples thawed at 70 °C (P<0.05). In conclusion, we recommend the use of a fast freezing curve that reduces the time spent on the cryopreservation of collared peccary semen, which could be packaged both in 0.25 mL or 0.50 mL straws, but the thawing should be conducted at 37 °C/1 min.

doi: 10.1016/j.cryobiol.2013.04.009

Dynamics of sperm subpopulations based on motility and plasma membrane status in thawed ram spermatozoa incubated under conditions that support in vitro capacitation and fertilisation2015-05-08T10:24:49+02:00

Dynamics of sperm subpopulations based on motility and plasma membrane status in thawed ram spermatozoa incubated under conditions that support in vitro capacitation and fertilisation

García-Álvarez O, Maroto-Morales A, Ramón M, del Olmo E, Jiménez-Rabadán P, Fernández-Santos MR, Anel-López L, Garde JJ, Soler AJ

SaBio IREC (Instituto de Investigacion en Recursos Cinegeticos), Campus Universitario s.n. 02071 Albacete, Spain; CERSYRA (Centro Regional de Seleccion y Reproduccion Animal) JCCM, Avda. Del Vino 6, 13300 Valdepeñas, Spain

The present study evaluated modifications occurring in thawed ram spermatozoa during incubation in different media that supported in vitro capacitation and fertilisation, and examines how these changes relate to IVF. Thawed sperm samples were incubated under capacitating (Cap) and non-capacitating (non-Cap) conditions for 0, 1 and 2h and used in an IVF test. During incubation, changes related to membrane status and the motility pattern of spermatozoa were assessed, the latter being used to characterise sperm subpopulations. A significantly greater increase (P≤0.05) in the percentage of spermatozoa with higher membrane fluidity was observed in samples incubated with Cap medium from the beginning of incubation. In addition, changes over time in the distribution of the motile subpopulation were particularly evident when spermatozoa were incubated with Cap medium, with a noted increase in spermatozoa classified as ‘hyperactivated like’, with major changes occurring after 1h incubation. Both characteristics (i.e. membrane fluidity and the percentage of the hyperactivated-like subpopulation) were significantly related with in vitro fertility, and only sperm samples incubated with the Cap medium were capable of fertilising oocytes. These results support the idea that changes in sperm membrane fluidity and motility pattern (i.e. an increase in hyperactivated spermatozoa) are needed for fertilisation to take place.

doi: 10.1071/RD13034

Photoperiod and melatonin treatments for controlling sperm parameters, testicular and accessory sex glands size in male Iberian ibex: A model for captive mountain ruminants2016-12-30T09:48:25+01:00

Photoperiod and melatonin treatments for controlling sperm parameters, testicular and accessory sex glands size in male Iberian ibex: A model for captive mountain ruminants

Santiago-Moreno J, Toledano-Díaz A, Castaño C, Coloma MA, Esteso MC, Prieto MT, Delgadillo JA, López-Sebastián A

Departamento de Reproducción Animal, INIA, Avda. Puerta de Hierro km 5.9, 28040 Madrid, Spain

This study examines whether photoperiod and/or melatonin treatments can improve sperm variables outside the breeding season in the Iberian ibex-a model species for wild mountain ruminants-thus helping in the collection of high quality sperm beyond the normal breeding season for depositing in genetic resource banks. Adult Iberian ibex males (n=17) were divided into four treatment groups: (1) controls under the natural photoperiod (control group; n=4), (2) treatment with melatonin implants on December 22nd, February 22nd and April 22nd (group WS-M; n=5), (3) treatment with short photoperiod cycles, i.e., 2 months of long days followed by melatonin implants (to emulate 2 months of short days) throughout the year (group PHPld+M; n=4), and (4) treatment with melatonin implants on June 22nd and August 22nd (group SS-M; n=4). The interaction treatment x season had a strong influence on testis size (P<0.05), the size of the seminal vesicles (P<0.001), the percentage of abnormal sperms (P<0.05), and percentage non-progressive (P<0.05) and progressive (P<0.001) sperm motility. In groups WS-M and PHPld+M, the normal springtime physiological reductions in testis size, non-progressive sperm motility and acrosome integrity were prevented. The values for the studied sperm variables were, however, reduced in the natural breeding season at the end of the experimental period in group PHPld+M, although not in group WS-M. The pattern of melatonin administration in group SS-M conferred no advantages on reproductive functionality. These results suggest that lengthening the short day period after the winter solstice (the WS-M treatment) extends reproductive activity in this species, allowing good quality sperm to be recovered for conservation purposes during the non-breeding season.

doi: 10.1016/j.anireprosci.2013.04.006

Sperm cell population dynamics in ram semen during the cryopreservation process2015-05-08T10:25:17+02:00

Sperm cell population dynamics in ram semen during the cryopreservation process

Ramón M, Pérez-Guzmán MD, Jiménez-Rabadán P, Esteso MC, García-Álvarez O, Maroto-Morales A, Anel-López L, Soler AJ, Fernández-Santos MR, Garde JJ

CERSYRA, Junta de Comunidades de Castilla-La Mancha, Valdepeñas, Spain

BACKGROUND:
Sperm cryopreservation has become an indispensable tool in biology. Initially, studies were aimed towards the development of efficient freezing protocols in different species that would allow for an efficient storage of semen samples for long periods of time, ensuring its viability. Nowadays, it is widely known that an important individual component exists in the cryoresistance of semen, and efforts are aimed at identifying those sperm characteristics that may allow us to predict this cryoresistance. This knowledge would lead, ultimately, to the design of optimized freezing protocols for the sperm characteristics of each male.
METHODOLOGY/PRINCIPAL FINDINGS:
We have evaluated the changes that occur in the sperm head dimensions throughout the cryopreservation process. We have found three different patterns of response, each of one related to a different sperm quality at thawing. We have been able to characterize males based on these patterns. For each male, its pattern remained constant among different ejaculates. This latter would imply that males always respond in the same way to freezing, giving even more importance to this sperm feature.
CONCLUSIONS/SIGNIFICANCE:
Changes in the sperm head during cryopreservation process have resulted useful to identify the ability of semen of males for freezing. We suggest that analyses of these response patterns would represent an important tool to characterize the cryoresistance of males when implemented within breeding programs. We also propose follow-up experiments to examine the outcomes of the use of different freezing protocols depending on the pattern of response of males.

doi: 10.1371/journal.pone.0059189

An essential role for insulin and IGF1 receptors in regulating sertoli cell proliferation, testis size, and FSH action in mice2016-12-30T09:48:25+01:00

An essential role for insulin and IGF1 receptors in regulating sertoli cell proliferation, testis size, and FSH action in mice

Pitetti JL, Calvel P, Zimmermann C, Conne B, Papaioannou MD, Aubry F, Cederroth CR, Urner F, Fumel B, Crausaz M, Docquier M, Herrera PL, Pralong F, Germond M, Guillou F, Jégou B, Nef S

Department of Genetic Medicine and Development, National Center of Competence in Research, Frontiers in Genetics, University of Geneva, 1211 Geneva 4, Switzerland

Testis size and sperm production are directly correlated to the total number of adult Sertoli cells (SCs). Although the establishment of an adequate number of SCs is crucial for future male fertility, the identification and characterization of the factors regulating SC survival, proliferation, and maturation remain incomplete. To investigate whether the IGF system is required for germ cell (GC) and SC development and function, we inactivated the insulin receptor (Insr), the IGF1 receptor (Igf1r), or both receptors specifically in the GC lineage or in SCs. Whereas ablation of insulin/IGF signaling appears dispensable for GCs and spermatogenesis, adult testes of mice lacking both Insr and Igf1r in SCs (SC-Insr;Igf1r) displayed a 75% reduction in testis size and daily sperm production as a result of a reduced proliferation rate of immature SCs during the late fetal and early neonatal testicular period. In addition, in vivo analyses revealed that FSH requires the insulin/IGF signaling pathway to mediate its proliferative effects on immature SCs. Collectively, these results emphasize the essential role played by growth factors of the insulin family in regulating the final number of SCs, testis size, and daily sperm output. They also indicate that the insulin/IGF signaling pathway is required for FSH-mediated SC proliferation.

doi: 10.1210/me.2012-1258

Fertility of cryopreserved ovine semen is determined by sperm velocity2015-05-08T10:26:10+02:00

Fertility of cryopreserved ovine semen is determined by sperm velocity

Del Olmo E, Bisbal A, Maroto-Morales A, García-Alvarez O, Ramon M, Jimenez-Rabadan P, Martínez-Pastor F, Soler AJ, Garde JJ, Fernandez-Santos MR.

SaBio IREC (CSIC – UCLM – JCCM), Campus Universitario s. n. 02071 Albacete, Spain; Regional Center of Animal Selection and Reproduction (CERSYRA) JCCM, 13300 Valdepeñas, Spain; ITRA-ULE, INDEGSAL, University of León, León, Spain; SaBio IREC (CSIC – UCLM – JCCM), Campus Universitario s. n. 02071 Albacete, Spain

The present study aims to examine the predictive value of some sperm parameters on male fertility. Semen samples from six Manchega rams were collected and cryopreserved. Sperm quality was assessed after thawing and after 2h of incubation, either in the freezing extender (37°C) or after dilution in Synthetic Oviductal Fluid (SOF) (38°C, 5% CO2), attempting to mimic the physiological conditions of the female reproductive tract. The following sperm parameters were evaluated: motility and kinetic parameters by computer-assisted semen analyzer (CASA), and sperm viability (propidium iodide), mitochondrial membrane potential (JC-1), apoptotic-like membrane changes (YO-PRO-1), acrosomal status (PNA-FITC), and intracellular calcium (fluo-3) by flow cytometry. Results showed no significant differences between incubation media neither after thawing nor after incubation. There were no significant correlations between fertility and sperm parameters assessed by flow cytometry. However, after incubation in the freezing extender, sperm samples from males with poor fertility yielded less linearity and velocity (P<0.05) as indicated by motility parameters analyzed by CASA. These results indicate that kinematic sperm motility parameters evaluation by CASA might be useful to identify samples with poor fertility.

doi:10.1016/j.anireprosci.2013.02.007

A transgenic minipig model of Huntington’s Disease2016-12-30T09:48:25+01:00

A transgenic minipig model of Huntington’s Disease

Baxa M, Hruska-Plochan M, Juhas S, Vodicka P, Pavlok A, Juhasova J, Miyanohara A, Nejime T, Klima J, Macakova M, Marsala S, Weiss A, Kubickova S, Musilova P, Vrtel R, Sontag EM, Thompson LM, Schier J, Hansikova H, Howland DS, Cattaneo E, DiFiglia M, Marsala M, Motlik J

Laboratory of Cell Regeneration and Plasticity, Institute of Animal Physiology and Genetics, v.v.i., AS CR, Libechov, Czech Republic Faculty of Science, Department of Cell Biology, Charles University in Prague, Prague, Czech Republic; Laboratory of Cell Regeneration and Plasticity, Institute of Animal Physiology and Genetics, v.v.i., AS CR, Libechov, Czech Republic Faculty of Science, Department of Cell Biology, Charles University in Prague, Prague, Czech Republic Neurodegeneration Laboratory, Department of Anesthesiology, University of California, San Diego, La Jolla, CA, USA Sanford Consortium for Regenerative Medicine, San Diego, La Jolla, CA, USA; Laboratory of Cell Regeneration and Plasticity, Institute of Animal Physiology and Genetics, v.v.i., AS CR, Libechov, Czech Republic; Laboratory of Cell Regeneration and Plasticity, Institute of Animal Physiology and Genetics, v.v.i., AS CR, Libechov, Czech Republic Department of Neurology, Massachusetts General Hospital, Boston, MA, USA; Vector Development Laboratory, Human Gene Therapy Program, Department of Pediatrics, University of California, San Diego, La Jolla, CA, USA; Neurodegeneration Laboratory, Department of Anesthesiology, University of California, San Diego, La Jolla, CA, USA; Neurodegeneration Laboratory, Department of Anesthesiology, University of California, San Diego, La Jolla, CA, USA Sanford Consortium for Regenerative Medicine, San Diego, La Jolla, CA, USA; Novartis Institutes for Biomedical Research, Neuroscience Discovery, Basel, Switzerland IRBM Promidis, Pomezia, Italy; Department of Genetics and Reproduction, Veterinary Research Institute, Brno, Czech Republic; Department of Clinical Genetics and Fetal Medicine, Palacky University, University Hospital Olomouc, Olomouc, Czech Republic; Department of Biological Chemistry University of California, Irvine, CA, USA Department of Psychiatry and Human Behavior, University of California, Irvine, CA, USA; Department of Biological Chemistry University of California, Irvine, CA, USA Department of Psychiatry and Human Behavior, University of California, Irvine, CA, USA Department of Neurobiology and Behavior University of California, Irvine, CA, USA; Institute of Information Theory and Automation v.v.i., AS CR, Prague, Czech Republic; Laboratory for Study of Mitochondrial Disorders, First Faculty of Medicine, Department of Pediatrics and Adolescent Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic; CHDI Foundation, Princeton, NY, USA; Department of Pharmacological Sciences and Centre for Stem Cell Research, Università degli Studi di Milano, Milan, Italy; Department of Neurology, Massachusetts General Hospital, Boston, MA, USA; Neurodegeneration Laboratory, Department of Anesthesiology, University of California, San Diego, La Jolla, CA, USA Sanford Consortium for Regenerative Medicine, San Diego, La Jolla, CA, USA Institute of Neurobiology, Slovak Academy of Sciences, Kosice, Slovak Republic

BACKGROUND: Some promising treatments for Huntington’s disease (HD) may require pre-clinical testing in large animals. Minipig is a suitable species because of its large gyrencephalic brain and long lifespan.
OBJECTIVE: To generate HD transgenic (TgHD) minipigs encoding huntingtin (HTT)1-548 under the control of human HTT promoter.
METHODS: Transgenesis was achieved by lentiviral infection of porcine embryos. PCR assessment of gene transfer, observations of behavior, and postmortem biochemical and immunohistochemical studies were conducted.
RESULTS: One copy of the human HTT transgene encoding 124 glutamines integrated into chromosome 1 q24-q25 and successful germ line transmission occurred through successive generations (F0, F1, F2 and F3 generations). No developmental or gross motor deficits were noted up to 40 months of age. Mutant HTT mRNA and protein fragment were detected in brain and peripheral tissues. No aggregate formation in brain up to 16 months was seen by AGERA and filter retardation or by immunostaining. DARPP32 labeling in WT and TgHD minipig neostriatum was patchy. Analysis of 16 month old sibling pairs showed reduced intensity of DARPP32 immunoreactivity in neostriatal TgHD neurons compared to those of WT. Compared to WT, TgHD boars by one year had reduced fertility and fewer spermatozoa per ejaculate. In vitro analysis revealed a significant decline in the number of WT minipig oocytes penetrated by TgHD spermatozoa.
CONCLUSIONS: The findings demonstrate successful establishment of a transgenic model of HD in minipig that should be valuable for testing long term safety of HD therapeutics. The emergence of HD-like phenotypes in the TgHD minipigs will require more study.

doi: 10.3233/JHD-130001

Postcopulatory sexual selection increases atp content in rodent spermatozoa2015-05-08T10:43:54+02:00

Postcopulatory sexual selection increases atp content in rodent spermatozoa

Maximiliano Tourmente, Melissah Rowe, M. Mar González-Barroso, Eduardo Rial, Montserrat Gomendio, and Eduardo R. S. Roldan

Reproductive Ecology and Biology Group, Museo Nacional de Ciencias Naturales (CSIC) 28006, Madrid, Spain; Natural History Museum, University of Oslo, NO-0318, Oslo, Norway; Department of Cellular and Molecular Medicine, Centro de Investigaciones Biológicas (CSIC) 28040, Madrid, Spain

Sperm competition often leads to increase in sperm numbers and sperm quality, and its effects on sperm function are now beginning to emerge. Rapid swimming speeds are crucial for mammalian spermatozoa, because they need to overcome physical barriers in the female tract, reach the ovum, and generate force to penetrate its vestments. Faster velocities associate with high sperm competition levels in many taxa and may be due to increases in sperm dimensions, but they may also relate to higher adenosine triphosphate (ATP) content. We examined if variation in sperm ATP levels relates to both sperm competition and sperm swimming speed in rodents. We found that sperm competition associates with variations in sperm ATP content and sperm-size adjusted ATP concentrations, which suggests proportionally higher ATP content in response to sperm competition. Moreover, both measures were associated with sperm swimming velocities. Our findings thus support the idea that sperm competition may select for higher ATP content leading to faster sperm swimming velocity.

doi: 10.1111/evo.12079

Determination of the effectiveness of an automatic quantification system SCA (Sperm Class Analyzer) used for the estimation and quality assessment of phytoplankton in intensive culture2013-01-23T00:00:13+01:00

Determination of the effectiveness of an automatic quantification system SCA (Sperm Class Analyzer) used for the estimation and quality assessment of phytoplankton in intensive culture

B. Álvarez-Blázquez, MJ. Lago, C. Gómez, J. Iglesias

Instituto Español de Oceanografía (IEO). Subida a Radio Faro 50, 36390 Vigo, Pontevedra, Spain

The marine microalgae culture system has evolved since the beginning of aquaculture, where large volumes were produced to attain an adequate biomass, extensive culture, intensive continuous cultures and finally the semi continuous systems that are presently used, using photo bioreactors, which optimize the use of light and nutrients to attain optimal microalgae production in much lower volumes and shorter times, reducing space and time required, as well as costs related to material and hand labor.
The objective of this experiment was to validate an automatic sperm count normally used in the veterinary industry (SCA), for its use in microalgae count. Results indicate that the SCA is valid for this use. Furthermore, SCA can be of great utility and accuracy, when configured for the analysis of quality parameters in the intensive microalgae culture.

XIV Congreso Nacinoal de Acuicultura. Gijón Spain

Quantification and identification of sperm subpopulations using computeraided sperm analysis and species-specific cut-off values for swimming speed2015-05-08T10:44:56+02:00

Quantification and identification of sperm subpopulations using computeraided sperm analysis and species-specific cut-off values for swimming speed

L Maree, G van der Horst

Department of Medical Bioscience , University of the Western Cape, Private Bag X17, Bellville, 7535 , South Africa

Motility is an essential characteristic of all flagellated spermatozoa and assessment of this parameter is one criterion for most semen or sperm evaluations. Computeraided sperm analysis (CASA) can be used to measure sperm motility more objectively and accurately than manual methods, provided that analysis techniques are standardized. Previous studies have shown that evaluation of sperm subpopulations is more important than analyzing the total motile sperm population alone. We developed a quantitative method to determine cut-off values for swimming speed to identify three sperm subpopulations. We used the Sperm Class Analyzer® (SCA) CASA system to assess the total percentage of motile spermatozoa in a sperm preparation as well as the percentages of rapid, medium and slow swimming spermatozoa for six mammalian species. Curvilinear velocity (VCL) cut-off values were adjusted manually for each species to include 80% rapid, 15% medium and 5% slow swimming spermatozoa. Our results indicate that the same VCL intervals cannot be used for all species to classify spermatozoa according to swimming speed. After VCL intervals were adjusted for each species, three unique sperm subpopulations could be identified. The effects of medical treatments on sperm motility become apparent in changes in the distribution of spermatozoa among the three swimming speed classes.

doi: 10.3109/10520295.2012.757366

Study of human semen: implementation of an objective method2016-12-30T09:48:25+01:00

Study of human semen: implementation of an objective method

Patricia Haydee Chenlo, Julia Irene Ariagno, Mercedes Norma Pugliese, Herberto Ernesto Repetto, Lydia Melba Sardi Segovia, Gabriela Ruth Mendeluk, Susana Mercedes Curi

Departamento de Bioquímica Clínica. Facultad de Farmacia y Bioquímica-UBA, INFIBIOQ. Hospital de Clínicas “José de San Martín”. Bs. As, Argentina. Hospital de Clínicas “José de San Martín” – Av. Córdoba 2351 (1120) CABA. Argentina

The aim of this study was to standardize and validate a CASA system, SCA (Sperm Class Analyzer) for the parameters of sperm concentration and motility according to international standards. The type of speed for the classification of degrees and the proper depth of the camera were standardized. For the validation, accuracy, detection limit, measuring range and method comparison study were determined. High linear correlation was found in the classification of motion using curvilinear velocity or average speed (r=0.99, p<0.001), establishing the use of the first to obtain results comparable with other objectives. Likewise, the use of cameras 10 microns in depth was also standardized since better images are obtained for the analysis without affecting mobility. Average imprecision was 13.29%, 14.26% and 18.75% for counting, progressive and mobile grades respectively. High correlation was found with the manual method, both in concentration (r=0.97, p<0.0001), progressive mobiles (r=0.84, (p<0.001) and mobile grade a (r=0.82, p<0.0001). Linearity (r=0.99, p<0.001) was proved between 0.98 and 125 x 106 x 106 sperm/mL. It is concluded that the proposed method meets the requirements for use in the clinic, being image editing by a qualified operator indispensable.

Acta Bioquím Clín Latinoam 2013; 47 (1): 61-69

Sperm form and function in the absence of sperm competition2015-05-08T10:46:18+02:00

Sperm form and function in the absence of sperm competition

Gerhard van der Horst and Liana Maree

Department of Medical Bioscience, University of the Western Cape, Bellville, South Africa

SUMMARY
Sperm competition is a post-copulatory, sexual selection force that, together with phylogeny and fertilization mode, has been regarded as one of the main factors explaining the diversity in sperm size across species. This universal sperm selection mechanism favors traits that enhance a male’s fertilizing ability and paternity success. Surprisingly, however, sperm characteristics and semen quality in monogamous species, with low risk of sperm competition, have barely received any attention. In this review, we consider sperm competition and monogamy as two ends of the selective spectrum, and discuss its effect on spermstructure and function. We address the issue of a lack of sperm competition by comparing sperm traits of essentially monogamous speciestheir largely degenerative sperm features and high degree of polymorphisms could be norms for monogamous species. Further, the level of sperm competition in humans is discussed by comparing its mating strategy, relative testis size, and sperm traits to other primate species. In terms of sperm concentration, sperm swimming speed, and sperm morphology, humans seem to be closer aligned to the low-risk sperm competition situation in gorillas than to promiscuous chimpanzees.

doi: 10.1002/mrd.22277

Vitamin E (Trolox) addition to Tris-egg yolk extender preserves ram spermatozoon structure and kinematics after cryopreservation2016-12-30T09:48:26+01:00

Vitamin E (Trolox) addition to Tris-egg yolk extender preserves ram spermatozoon structure and kinematics after cryopreservation

Silva SV, Soares AT, Batista AM, Almeida FC, Nunes JF, Peixoto CA, Guerra MM

Andrology Laboratory (ANDROLAB), Veterinary Medicine Department – UFRPE, Dom Manuel de Medeiros Street, s/n. Dois Irmãos, Recife, PE, CEP: 52171-900, Brazil

Several studies reveal that vitamin E acts as a cellular stabilizer of unsaturated lipids against oxidative deterioration, thus maintaining structural and functional integrity at the subcellular level. The objective of this study was to evaluate Vitamin E (Trolox) addition to freezing extender for ram spermatozoa. Semen samples were diluted in Tris-yolk egg medium without antioxidant (control group) and with Trolox in different concentrations (30, 60 and 120μM). After thawing (37°C/30s), samples were subjected to analysis for plasma membrane integrity (PMi), acrosome integrity (Aci), mitochondrial membrane potential (MMP), sperm kinematics, and ultrastructural integrity. The Trolox 60 and 120μM groups showed higher percentages of iPMs (P<0.05) when compared to the control group. Differences were observed among groups in sperm kinematic indicators (progressive motility, linearity, straightness, oscillation index, straight-line velocity and average path velocity), with higher values (P<0.05) for the Trolox 60 and 120μM groups. On ultrastructural assessment, Trolox addition at the three concentrations preserved spermatozoon head plasma membranes, while for the spermatozoon tail, plasma membrane preservation at 60μM was higher (P<0.05) than the other groups. The Trolox 60 and 120μM groups presented more mitochondrial ultrastructural preservation than the other groups (P<0.05). These results indicate that Trolox addition to Tris-egg yolk at 60 and 120μM provides greater structural integrity (plasma membrane and mitochondria) and kinematics for ram spermatozoa after cryopreservation.

doi: 10.1016/j.anireprosci.2012.12.002

An improved experimental model for understanding the impact of sperm DNA fragmentation on human pregnancy following ICSI2015-05-08T10:47:20+02:00

An improved experimental model for understanding the impact of sperm DNA fragmentation on human pregnancy following ICSI

Nuñez-Calonge R, Caballero P, López-Fernández C, Guijarro JA, Fernández JL, Johnston S, Gosálvez J

Clínica Tambre, Madrid, Spain

Using donor oocytes of proven fertility, the effect of sperm DNA fragmentation (SDF) and motility on reproductive success was examined in 70 couples undergoing ICSI. Both SDF and sperm motility were assessed at the time of sperm injection and using the same sperm sample that was processed for ICSI. While there was no difference in the fertilization rate, cleavage rate, embryo quality, or sperm motility between pregnant and nonpregnant couples, the SDF of nonpregnant couples (SDF = 23.9%) was higher than that of pregnant couples (SDF = 17.0%; U Mann-Whitney 347; P = .002). Using a combination of the sensitivity and specificity measures from the production of ROC (receiver-operating characteristic) curves and the Youden index, we determined a threshold SDF value for our data set of 17% for predicting pregnancy (77.8% sensitivity and 71.1% specificity). Our results suggest that proven donor oocytes in combination with SDF assessment at the time of sperm injection represent a useful experimental model for reducing the confounding influences of sperm DNA repair by the oocyte and iatrogenic sperm damage.

doi: 10.1177/1933719112459238

Cryopreservation of aoudad (Ammotragus lervia sahariensis) sperm obtained by transrectal ultrasound-guided massage of the accessory sex glands and electroejaculation2016-12-30T09:48:26+01:00

Cryopreservation of aoudad (Ammotragus lervia sahariensis) sperm obtained by transrectal ultrasound-guided massage of the accessory sex glands and electroejaculation

Santiago-Moreno J, Castaño C, Toledano-Díaz A, Esteso MC, López-Sebastián A, Guerra R, Ruiz MJ, Mendoza N, Luna C, Cebrián-Pérez JA, Hildebrandt TB

Departamento de Reproducción Animal, INIA, Madrid, Spain

This study examines (1) the effectiveness of transrectal, ultrasound-guided massage of the accessory sex glands (TUMASG) combined with electroejaculation for obtaining aoudad (Ammotragus lervia sahariensis) sperm samples for cryopreservation, and (2) the effectiveness of a Tris-citric acid-glucose-based medium (TCG; usually used for freezing ibex sperm) and a TES-Tris-glucose-based medium (TTG; typically used in the cryopreservation of mouflon sperm) as sperm extenders. After TUMASG, just one to three electrical pulses were required for ejaculation to occur in five of the six animals studied; one ejaculated after TUMASG alone. Transrectal, ultrasound-guided massage of the accessory sex glands would therefore appear to be useful in obtaining sperm samples from this species, requiring few subsequent electrical electroejaculation stimuli and sometimes none at all. After thawing, the membrane integrity (assessed by nigrosin-eosin staining) of sperm extended with TTG was greater than that of sperm extended with TCG (P < 0.05). The total percentage of sperm showing an intact acrosome, as assessed by fluorescein isothiocyanate-conjugated peanut (Arachis hypogea) agglutinin, was also higher in the TTG-extended sperm (P < 0.05), and the percentage of dead sperm with a damaged acrosome was lower (P < 0.05). No differences were seen between TCG and TTG in terms of apoptotic manifestations (DNA damage, caspase activity, mitochondrial membrane potential, and plasmalemma stability). Therefore, TTG appears to be a better extender than TCG for cryopreserving aoudad sperm.

doi: 10.1016/j.theriogenology.2012.10.011

HSL-knockout mouse testis exhibits class B scavenger receptor upregulation and disrupted lipid raft microdomains2016-12-30T09:48:26+01:00

HSL-knockout mouse testis exhibits class B scavenger receptor upregulation and disrupted lipid raft microdomains

Casado ME, Huerta L, Ortiz AI, Pérez-Crespo M, Gutiérrez-Adán A, Kraemer FB, Lasunción MÁ, Busto R, Martín-Hidalgo A

Servicio de Bioquímica-Investigación, Hospital Universitario Ramón y Cajal, Instituto Ramón y Cajal de Investigación Sanitaria, Madrid, Spain

There is a tight relationship between fertility and changes in cholesterol metabolism during spermatogenesis. In the testis, class B scavenger receptors (SR-B) SR-BI, SR-BII, and LIMP II mediate the selective uptake of cholesterol esters from HDL, which are hydrolyzed to unesterified cholesterol by hormone-sensitive lipase (HSL). HSL is critical because HSL knockout (KO) male mice are sterile. The aim of the present work was to determine the effects of the lack of HSL in testis on the expression of SR-B, lipid raft composition, and related cell signaling pathways. HSL-KO mouse testis presented altered spermatogenesis associated with decreased sperm counts, sperm motility, and infertility. In wild-type (WT) testis, HSL is expressed in elongated spermatids; SR-BI, in Leydig cells and spermatids; SR-BII, in spermatocytes and spermatids but not in Leydig cells; and LIMP II, in Sertoli and Leydig cells. HSL knockout male mice have increased expression of class B scavenger receptors, disrupted caveolin-1 localization in lipid raft plasma membrane microdomains, and activated phospho-ERK, phospho-AKT, and phospho-SRC in the testis, suggesting that class B scavenger receptors are involved in cholesterol ester uptake for steroidogenesis and spermatogenesis in the testis.

doi: 10.1194/jlr.M028076

Effect of different thawing rates on post-thaw viability, kinematic parameters and chromatin structure of buffalo (Bubalus bubalis) spermatozoa2016-12-30T09:48:26+01:00

Effect of different thawing rates on post-thaw viability, kinematic parameters and chromatin structure of buffalo (Bubalus bubalis) spermatozoa

Rastegarnia A, Shahverdi A, Rezaei Topraggaleh T, Ebrahimi B, Shafipour V

Department of Clinical Science, Faculty of Veterinary Medicine, Urmia Branch, Islamic Azad University, Urmia, Iran

OBJECTIVE: The aim of the present study was to evaluate three thawing rates on the post thaw motility, viability and chromatin structure of buffalo semen frozen in 0.5-ml straws.
MATERIALS AND METHODS: In this experimental study semen was collected with artificial vagina (42℃) from four buffalo bulls.Split pooled ejaculates (n=4) were extended at 37℃ with a Bioxcell® extender. Semen was cooled to 4℃ within 2 hours, equilibrated at 4℃ for 4 hours, then filled in 0.5 ml French straws, and frozen in programmable cell freezer before plunging into liquid nitrogen. Straws were thawed at water bath temperatures of 37, 50 or 70℃ for 30, 15 and 6 seconds, respectively. Semen was incubated at 37℃ for 2 hours and evaluated for post thaw motility, viability, acrosomal and DNA integrity of spermatozoa. Analysis of variance (ANOVA) was used for comparisons of means. When the ANOVA test showed statistical differences, the mean of the treatments were compared using Duncan’s multiple range tests.
RESULTS: The initial postthaw motility (0 hour) averaged 62.7 ± 7.2%, 73.1 ± 9.77%, and 74.9 ± 8.58% for the three thaw rates, respectively. Kinematic parameters such as average path velocity, linearity and beat/cross frequency in the thaw rate of 70℃ for 6 seconds were superior to other rates studied (p<0.05). After 2 hours of incubation, proportions of progressive motility and Kinematic parameters decreased in all groups (p>0.05). A positive correlation was detected between sperm motility and thawing rate after two hours incubation times. The percentage of viable spermatozoa and spermatozoa with an intact acrosome and plasma membrane integrity were not different between the groups of samples thawed at different temperatures (p>0.05). The percentage of spermatozoa with chromatin dispersion forthe thaw rate of 70℃ for 6 seconds was significantly higher than for the to other rates studied (p< 0.05). In contrast with motility and viability, the DNA integrity of post thaw spermatozoa remained unaffected during 2 hours incubation.
CONCLUSION: The post thaw motility and kinematic parameters of buffalo spermatozoa were significantly improved immediately after thawing by increasing the thawing rate from 37℃ in 30 seconds to70℃ in 6 seconds. However, this relative advantage had disappeared after incubation in a water bath at 37℃ for two hours.A thaw rate of 70℃ for 6 seconds was associated with higher chromatin dispersion than the other thaw rates studied. Sperm thawing over at 50 degrees could be safely used to improve motility recovery after sperm cryopreservation in buffalo bulls.

CELL JOURNAL(Yakhteh), Vol 14, No 4, Winter 2013

Effect of freezing and thawing rates on sperm motility in Bocachico Prochilodus magdalenae (Pisces, Characiformes)2015-05-08T10:48:52+02:00

Effect of freezing and thawing rates on sperm motility in Bocachico Prochilodus magdalenae (Pisces, Characiformes)

José G. Martínez, M.Sc, Sandra Pardo C, Ph.D

Universidade Federal do Amazonas, Instituto de Ciências Biológicas, Laboratório de Evolução e Genética Animal -LEGAL, Manaus, Brasil. Universidad Nacional de Colombia sede Medellín, Facultad de Ciencias Agrarias, Departamento de Producción Animal, BIOGEM, Medellín, Colombia

Objective. To determine the freezing and thawing rates necessary to maintain sperm viability during cryopreservation of Bocachico semen. Materials and methods. Four interactional treatments were implemented between two freezing (rapid and slow) and two thawing (rapid and slow) curves, in a 2×2 factorial as follows: rapid freezing-rapid thawing, rapid freezing-slow thawing, slow freezing-rapid thawing, and slow freezing-slow thawing. After thawing by Sperm Class Analyzer (SCA) curvilinear velocity (VCL) and straight-line (VSL) (µm sec-1) were analyzed; total, rapid, medium, and slow motility, were compared among treatments. Results. The rapid freezing-slow thawing treatment was lethal for all variables of velocity and motility, causing a significant (p<0.01) post-thaw inmotility of 100%. The slow freezing-rapid thawing interaction had a significantly higher effect than the other treatments (p<0.05), particularly on variables such as rapid motility (10.1 ± 1.1%), medium motility (30.16 ± 4.1%), and curvilinear velocity (51.5 ± 4.75 µm sec.-1) also decreased the percentage of sperm with slow motility (41.7 ± 4.45%). Independently of the applied thawing rate, the freezing rate generated the main significant effect on total motility. Conclusions. It is possible to conclude that the interaction effect between freezing and thawing rates is nil (except for slow motility) during cryopreservation process. However, the independent effects of these factors (main effects) on remaining motility variables are positively significant and decisive to the maintenance of these features, especially the freeze factor (when it is slow). This becomes the first successful report of sperm cryopreservation from Bocachico Prochilodus magdalenae in the world and may be used in conservation programs for this endangered species.

Rev.MVZ Cordoba vol.18 no.1 Córdoba Jan./Apr. 2013

Cryopreserved semen in ecotoxicological bioassays: sensitivity and reliability of cryopreserved Sparus aurata spermatozoa2015-05-08T10:49:17+02:00

Cryopreserved semen in ecotoxicological bioassays: sensitivity and reliability of cryopreserved Sparus aurata spermatozoa

Fabbrocini A, D’Adamo R, Del Prete F, Langellotti AL, Rinna F, Silvestri F, Sorrenti G, Vitiello V, Sansone G

Consiglio Nazionale delle Ricerche, Istituto di Scienze Marine, via Pola, 4, 71010 Lesina, Foggia, Italy

The aim of this study was to evaluate the feasibility of using cryopreserved S. aurata semen in spermiotoxicity tests. Cryopreservation is a biotechnology that can provide viable gametes and embryos on demand, rather than only in the spawning season, thus overcoming a limitation that has hindered the use of some species in ecotoxicological bioassays. Firstly, the sperm motility pattern of cryopreserved semen was evaluated after thawing by means of both visual and computer-assisted analyses. Motility parameters in the cryopreserved semen did not change significantly in the first hour after thawing, meaning that they were maintained for long enough to enable their use in spermiotoxicity tests. In the second phase of the research, bioassays were performed, using cadmium as the reference toxicant, in order to evaluate the sensitivity of cryopreserved S. aurata semen to ecotoxicological contamination. The sensitivity of the sperm motility parameters used as endpoints (motility percentages and velocities) proved to be comparable to what has been recorded for the fresh semen of other aquatic species (LOECs from 0.02 to 0.03 mg L(-1)). The test showed good reliability and was found to be rapid and easy to perform, requiring only a small volume of the sample. Moreover, cryopreserved semen is easy to store and transfer and makes it possible to perform bioassays in different sites or at different times with the same batch of semen. The proposed bioassay is therefore a promising starting point for the development of toxicity tests that are increasingly tailored to the needs of ecotoxicology and environmental quality evaluation strategies.

doi: 10.1016/j.ecoenv.2012.07.024

Pregnancy in the Caspian miniature horse using frozen semen cryopreserved with the EquiPRO CryoGuard freeze medium and customized freezing protocols2016-12-30T09:48:26+01:00

Pregnancy in the Caspian miniature horse using frozen semen cryopreserved with the EquiPRO CryoGuard freeze medium and customized freezing protocols

Hiva Alipour DVM, Mina Sharbatoghli MSc, Poopak Eftekhari Yazdi PhD, Abdolhossein Shahverdi PhD, Mohamad Taghi Daneshzadeh DVM, Masoud Afshani DVM, Seied Jalal Mirian DVM, Hormoz Hamidi DVM, Ahmad Reza Mohammadi DVM, Mojtaba Rezazadeh Valojerdi PhD

Department of Embryology, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran; Agricultural and Resource Research Center, Jihad-e-Agriculture, Tehran, Iran

The primary goal of this project was to establish a protocol to freeze the sperm of the Caspian miniature horse in an attempt to start an intensive artificial insemination program to effectively increase the population of this breed, which has been listed as “Critical Rare Breed” by the American Livestock Breed Conservancy and is in danger of extinction. Commercially available equine freezing medium (EquiPRO CyoGuard Complete egg-yolk extender) was used for the initial setup of two different freeze protocols: slow and fast. The fast-freeze protocol had slightly better postthaw results and was used for a fertility demonstration. Five mares of proven fertility, aged 3 to 12 years, were used in the fertility trials, two of which resulted in pregnancy. This is the first report of pregnancy in the Caspian miniature horse using frozen semen, and the results seem to be a promising start to an extensive program to help this endangered breed, although further research on freezing protocols and conditions for this process are necessary to further improve the survival of semen and pregnancy rate.

doi:10.1016/j.jevs.2012.07.005

Characterisation of sperm cell motility rate of Southern African indigenous cockerel semen following analysis by Sperm Class Analyser2012-07-01T12:21:18+02:00

Characterisation of sperm cell motility rate of Southern African indigenous cockerel semen following analysis by sperm class analyser

M.B Makhafola, D.O Umesiobi, M.L Mphaphathi, M.B Masenya, T.L Nedambale

Agricultural Research Council, Animal Production Institute, Germplasm Conservation and Reproductive Biotechnologies, Private Bag X 2, Irene, 0062; Central University of Technology, Free State School of Agriculture and Environmental Science, Private Bag X 20539 Bloemfontein 9300; Tshwane University of Technology, Department of Animal Sciences, Private Bag X680, Pretoria 0001, South Africa

Abstract: The Sperm Class Analyser (SCA) system (CASA) is advantageous and provides precise additional information that cannot be attained through the subjective manual sperm cell assessment methods. The aim of the study was to characterise Naked Neck (NN), Ovambo (OV) and Potchefstroom Koekoek (PK) cockerels semen macroscopically and microscopically. A total of 198 ejaculates were collected from 33 cockerels; Ovambo (n=11), Potchefstroom Koekoek (n=11) and Naked Neck (n=11) by means of the abdominal massage technique. Semen was analysed macroscopically (colour, semen volume and semen pH) and microscopically (sperm concentration, morphology, sperm motility and velocity rate). The sperm motility rate was evaluated with the aid of the Sperm Class Analyser (SCA) system. The sperm morphology was performed by the staining sperm cells with nigrosin-eosin breed significantly P < 0.05 affected the body weight of Ovambo (2.5±0.4kg), Naked Neck (2.0±0.3kg) and Potchefstroom Koekoek (2.3±0.3kg) cockerels. The semen volume of Naked Neck (0.5±0.2ml) breed was significantly higher, when compared to Ovambo (0.4±0.2ml), but similar to Potchefstroom Koekoek (0.3±0.2ml) breed. The overall sperm motility rate of Ovambo (95.0±7.2%), Potchefstroom Koekoek (86.0±13.7%) Naked Neck (76.4±22.2%) group was significantly different. The normal sperm morphology in the Naked Neck (76.4±22.2%) was lower when compared to Potchefstroom Koekoek (86.0±13.7%) and Ovambo (95.0±7.2%) group of cockerel. a positive correlation existed between body weight and semen volume for Potchefstroom Koekoek (r=0.1477) cockerels only. Moreover, there was a positive correlation between the body weight and total sperm motility for Naked Neck (r=0.3848), Ovambo (r=0.4871) and Potchefstroom Koekoek (r=0.2230) cockerels. However, a negative correlation existed between body weight and semen volume for Naked Neck (r=-0.4502) and Ovambo (r=-0.1244). In conclusion, body weight positively affected semen volume and sperm motility rate but this was breed dependent. The Sperm of Potchefstroom Koekoek and Ovambo resulted in a better motility and morphology rate. The Sperm Class Analyser or CASA provided more precise, repeatable and objective information. Evaluation of the sperm motility rate of South African indigenous cockerels holds potential for future use in semen evaluation.

See full article here

Journal of Animal Science Advances
July 2012

Motile sperm subpopulations in frozen-thawed dog semen: changes after incubation in capacitating conditions and relationship with sperm survival after osmotic stress2016-12-30T09:48:26+01:00

Motile sperm subpopulations in frozen-thawed dog semen: changes after incubation in capacitating conditions and relationship with sperm survival after osmotic stress

Peña AI, Barrio M, Becerra JJ, Quintela LA, Herradón PG

Unit of Reproduction and Obstetrics, Department of Animal Pathology, Faculty of Veterinary Medicine, University of Santiago de Compostela, Lugo, Spain

In this study we investigated the changes that in vitro incubation under capacitating conditions could induce on the motile sperm subpopulations present in frozen-thawed dog semen samples. In addition, cryopreserved dog spermatozoa were exposed to CCM (canine capacitating medium) solutions of 300, 150, 100 and 75 mOsm and the proportions of live spermatozoa with swollen tails were recorded (HOST+). Finally, frozen-thawed dog semen samples were submitted to a second cycle of freezing and thawing and the overall sperm motility, as well as the motile sperm subpopulations structure, was determined. Cryopreserved dog semen samples were structured in four sperm subpopulations with different motility characteristics: Subpopulation (Sp) 1 contained moderately rapid and progressive spermatozoa (25.2 ± 8.5%), Sp 2 included poorly motile and non progressive sperm (15.3 ± 8.1%), Sp 3 was represented by moderately slow non progressive sperm (14.9 ± 5.9%), and Sp 4 contained the most rapid and progressive sperm (20.8 ± 14.7%). After 3h of incubation under capacitating conditions, percentages of spermatozoa assigned to Sp 2 (6.1 ± 3.4%) and 3 (4.9 ± 2.8%) significantly decreased, whereas those assigned to Sp 1 (17.0 ± 11.2%) and 4 (16.2 ± 12.8%) did not significantly change. Significant correlations were found between percentages of HOST+, for the 3 osmolarities tested, and percentages of spermatozoa included in Sp 1 and 4 after 3 h of incubation in capacitating conditions or in Sp 4 after double freezing and thawing. These results indicated that subpopulations with the most rapid and progressive sperm seemed to be highly resistant to in vitro incubation in capacitating conditions and to osmotic stress, suggesting they are likely to be the source of the fertilizing population.

doi: 10.1016/j.anireprosci.2012.06.016

The effect of temperature and pH on the motility and viability of ostrich sperm2015-05-08T10:50:09+02:00

 The effect of temperature and pH on the motility and viability of ostrich sperm

Bonato M, Cornwallis CK, Malecki IA, Rybnik-Trzaskowska PK, Cloete SW

Department of Animal Sciences, University of Stellenbosch, Matieland, South Africa

As the chemical environment of semen diluents can have a profound effect on sperm quality, we examined the effect of temperature and pH on the motility and viability of sperm in the ostrich. Semen was collected from four males, each male being replicated three times. Ejaculates were diluted and incubated for 10 min at 20°C and 40°C in four different buffers, temperature adjusted at pH 6, 7, 8 and 9 respectively. Average path velocity (VAP), curvilinear velocity (VCL), straight-line velocity (VSL), linearity (LIN), beat cross frequency (BCF) and amplitude of lateral displacement (ALH) were then recorded for each sample using CASA. The viability of sperm was assessed using nigrosin-eosin staining. Sperm incubated at 40°C had higher motility parameters, except for ALH. At 40°C, VAP, VSL and LIN increased with pH while VCL, BCF and ALH were higher for lower pHs. The viability of sperm was not affected by temperature but decreased at pH values>7. A pH in the neutral range appeared to yield higher quality sperm after in vitro storage at 20°C. However, the effect of different pH levels and temperatures on sperm longevity needs to be investigated further to develop viable ostrich specific diluents.

doi: 10.1016/j.anireprosci.2012.06.008

DNA fragmentation and membrane damage of bocachico Prochilodus magdalenae (Ostariophysi: Prochilodontidae) sperm following cryopreservation with dimethylsulfoxide and glucose2015-05-08T10:51:00+02:00

DNA fragmentation and membrane damage of bocachico Prochilodus magdalenae (Ostariophysi: Prochilodontidae) sperm following cryopreservation with dimethylsulfoxide and glucose

José Gregorio Martínez; Víctor Atencio García; Sandra Pardo Carrasco

Universidad Nacional de Colombia, Facultad de Ciencias Agropecuarias, Departamento de Producción Animal, Grupo de Investigación Biodiversidad y Genética Molecular (BIOGEM). Calle 59A No. 63-020, Código Postal 4-72 Medellín, Colombia; Universidad de Córdoba, Facultad de Medicina Veterinaria y Zootecnia, Departamento de Ciencias Acuícolas, Centro de Investigación Piscícola, Carrera 6 No. 76-103, Montería, Colombia

The endangered bocachico Prochilodus magdalenae is a native freshwater fish of Colombia, the most captured species locally and one of the most important species for ex-situ conservation (germplasm banks). The aim of this study was to examine the effect of three concentrations of Dimethylsulfoxide (DMSO) (5%, 10%, 15%) and three of glucose (305, 333, 361 mM) in the extender on spermatic DNA fragmentation (F-DNA) (by Halomax®, Chromatin dispersion) and membrane damage (D-Me) (by eosin-nigrosin staining). After assessment of sperm quality by computer analysis of motility, one part of semen from males was diluted separately with three parts of extender and filled into 0.5 ml straws. Freezing was carried out in liquid nitrogen vapor dry shipper for 30 minutes and thawed at 60ºC for 8 seconds in a water bath and evaluated for the percentage of cells found with F-DNA and D-Me. The results demonstrated that cryopreservation causes greater F-DNA (13.62 ± 1.6% to 28.91 ± 3.25) and D-Me (24.27 ± 1.1% to 58.33 ± 2.81%) when compared with pre-freezing semen (PFS) (6.71 ± 1.54% and 2.34 ± 0.5%, respectively for F-DNA and D-Me). A significant interaction was found between DMSO and glucose concentration in this experiment. Use of extender: 10% DMSO + 305 mM glucose + 12% chicken egg yolk and, 10% DMSO + 333 mM glucose + 12% chicken egg yolk, allow for lower F-DNA and D-Me during cryopreservation of bocachico semen. A high correlation between F-DNA and D-Me was found (r = 0.771).

doi: 10.1590/S1679-62252012005000018

Comparison of slow freezing and vitrification methods for Venda cockerel’s spermatozoa2015-05-08T10:51:28+02:00

Comparison of slow freezing and vitrification methods for Venda cockerel’s spermatozoa

Masindi L. Mphaphathi, Dibungi Luseba, Ben Sutherland, Tshimangadzo L. Nedambale

Agricultural Research Council, Animal Production Institute, Germplasm Conservation & Reproductive Biotechnologies, Irene, RSA; Tshwane University of Technology, Faculty of Science, Department of Animal Sciences, Pretoria, RSA; University of the Free State, Department of Animal, Wildlife and Grassland Sciences, Bloemfontein, RSA

An improvement in avian semen cryopreservation is essential and has the potential to improve the cryo-gene banking efficiency. This study compared two cryopreservation methods (slow freezing and vitrification) and the effect of different thawing/warming temperatures (5℃, 25℃ and 41℃) on Venda cockerel’s spermatozoa. Semen samples from Venda cockerels were diluted with modified Kobidil+ extender supplemented with 8% dimethyl sulfoxide. Semen from each ejaculate was stained with nigrosin/eosin for viability examination. The cryopreserved samples were either slow cooled in 0.25 mL straw or vitrified in a solid surface vitrification (SSV) device. Semen straw or cryovial was stored in liquid nitrogen container. The straw or cryovial with sperm was thawed or warmed at 5?C, 25?C and 41℃ and analysed by a Computer-Aided Sperm Analysis (CASA). There was a significant difference in live/normal sperm between the semen donors. Cockerels spermatozoa cryopreserved by slow freezing (43%) and thawed at 5?C had a significantly higher survival and motility rate compared to vitrification (2.5%) method. In conclusion, there was higher rate of live/normal morphology sperm. Cryopreservation process reduces sperm motility and velocity rate regardless of cryoprevervation method and thawing or warming temperatures. However, slow freezing was a better method to maintain motility of spermatozoa following cryopreservation.

DOI: 10.4236/ojas.2012.23028

Factors influencing the success of an artificial insemination program in Florida goats2015-05-08T10:51:44+02:00

Factors influencing the success of an artificial insemination program in Florida goats

F. A. Arrebola, B. Pardo, M. Sanchez, M. D. Lopez and C. C. Perez-Marin

IFAPA Centro de Hinojosa del Duque, Junta de Andalucía, 14270 Hinojosa del Duque, Cordoba, Spain; Department of Animal Production, Faculty of Veterinary Medicine, University of Cordoba, Campus de Rabanales, 14014 Cordoba, Spain; ACRIFLOR, Campus de Rabanales, 14014 Cordoba, Spain; Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, University of Cordoba, Campus de Rabanales, 14014 Cordoba, Spain

An artificial insemination (AI) program using cooled semen was evaluated over a 7-year period in Florida goats. The effect of the following variables on  pregnancy rates was analysed: production system, year and season of AI, synchronisation treatment, bucks, AI technicians, semen deposition site, days in milk  at AI, milk yield and parity. Animals were reared under field conditions on commercial farms in southern Spain. Semen was collected from nine bucks and  cooled at 4°C until use. A total of 3,941 goats were synchronised using intravaginal progesterone sponges and inseminated 46.0 ± 0.5 h. after sponge removal.  Pregnancy was diagnosed by ultrasonography 42-46 days after AI, obtaining an average pregnancy rate of 48.7%. Logistic regression showed that production  system, AI year and season, bucks and semen deposition site had a significant effect (p < 0.05) on pregnancy rate, while the other variables analysed were  removed from the model. The final statistical model accounted for 59.7% of the cases analysed, suggesting that other factors not studied here may influence  pregnancy rates in Florida goats.

doi: 10.5424/sjar/2012102-223-11

Sperm cryopreservation of freshwater fish bocachico (Prochilodus magdalenae) in DMSO and glucose and its effects on fertilization and hatching efficiency2015-05-08T10:52:02+02:00

Sperm cryopreservation of freshwater fish bocachico (Prochilodus magdalenae) in DMSO and glucose and its effects on fertilization and hatching efficiency

J.G. Martínez, A.M. Tarazona-Morales, S.C. Pardo-Carrasco

Universidad Nacional de Colombia – Medellín – Facultad de Ciencias Agropecuarias, Departamento de Producción Animal, Grupo de Investigación en Biodiversidad y Genética Molecular BIOGEM, código postal 472, Colombia

Internal cryoprotectants (dimethylsulfoxide – DMSO), as well as external ones (glucose) have been of great importance for sperm cryopreservation in freshwater fish. The aim of this study was to evaluate both the fertilization and hatching rates of eggs fertilized with bocachico (Prochilodus magdalenae) spermatozoa cryopreserved in different combinations of DMSO and glucose. Nine treatments were evaluated by a combination of three concentrations of DMSO: 5% (701 mM), 10% (1402 mM), 15% (v/v; 2103 mM) and three concentrations of glucose: 5.5% (305 mM), 6% (333 mM), 6.5% (w/v; 361 mM). Semen from males obtained by abdominal stripping 6 h after hormonal induction with carp pituitary extract was submitted to each treatment. The semen was frozen in 0.5 ml straws in a nitrogen vapor dry shipper for 30 min and then in liquid nitrogen (-196°C). Five days later they were placed in water with a temperature of 60°C for 8 sec and analyzed. A high total motility (71.0 ± 7.0%) was observed when DMSO concentration was 10% and glucose was 6%, and a high linearity displacement (62.8 ± 6.3%) was observed when DMSO concentration was 5% and glucose was 5.5%. In conclusion, we found that for the purposes of cryopreservation of bocachico spermatozoa, the combinations of 10% DMSO + 5.5 or 6% glucose and 5% DMSO + 5.5 or 6% glucose produced the best results in terms of fertilization and hatching rates. This becomes the first report to successfully demonstrate the fertilizing capacity and larvae obtaining capabilities of cryopreserved bocachico semen.

Anim. Reprod., v.9, n.1, p.00-00, Jan./Mar. 2012

Improved semen collection method for wild felids: Urethral catheterization yields high sperm quality in African lions (Panthera leo)2015-05-08T10:52:53+02:00

Improved semen collection method for wild felids: Urethral catheterization yields high sperm quality in African lions (Panthera leo)

I. Lueders, I. Luther, G. Scheepers, G. van der Horst

Geolifes-Animal Fertility and Reproductive Research, Frohmestr 7, 22 457 Hamburg, Germany

For wild and domestic felids, electroejaculation (EE) is the most common semen collection method. However, the equipment is expensive, there is a risk of urine contamination and animals usually show strong muscular contraction despite general anesthesia. Accordingly, we tested the feasibility of a different approach using urethral catheterization (UC) in seven African lions, previously described for domestic cats only. After general anesthesia with the 2-agonist medetomidine (which also stimulates semen release into the urethra) and ketamine, a transrectal ultrasound was performed to locate the prostate. A commercial dog urinary catheter (2.6 or 3.3 mm in diameter) was advanced approximately 30 cm into the urethra to allow semen collection into the lumen of the catheter by capillary forces. After retraction, sperm volumes between of 422.86  296.07 l yielded motility of 88.83  13.27% (mean  SD) with a mean sperm concentration of 1.94  109/ml. Here we describe a simple, field friendly and effective method to attain highly concentrated semen samples with excellent motility in lions and potentially other wild felid species as an alternative to electroejaculation.

Theriogenology 78 (2012) 696–701

Taking advantage of the use of supervised learning methods for characterization of sperm population structure related with freezability in the Iberian red deer2016-12-30T09:48:26+01:00

Taking advantage of the use of supervised learning methods for characterization of sperm population structure related with freezability in the Iberian red deer

MANUEL RAMÓN, FELIPE MARTÍNEZ-PASTOR, OLGA GARCÍA-ÁLVAREZ, ALEJANDRO MAROTO-MORALES, ANA JOSEFA SOLER, PILAR JIMÉNEZ-RABADÁN, MARIA ROCÍO FERNÁNDEZ-SANTOS, RODOLFO BERNABÉU, JOSÉ JULIÁN GARDE

Biology of Reproduction Group, National Wildlife Research Institute (IREC) UCLM-CSIC-JCCM, 02071, Albacete, Spain; Department of Medicine and Animal Surgery, Faculty of Veterinary Medicine, University of Murcia, 30071, Murcia, Spain; cITRA-ULE, INDEGSAL, University of León, 24071, León, Spain; Molecular Biology (Cell Biology), University of León, 24071, León, Spain; CERSYRA, Castilla-La Mancha, 13300, Valdepeñas, Spain; Escuela Técnica Superior de Ingenieros Agrónomos, University of Castilla-La Mancha, 02071, Albacete, Spain

Using Iberian red deer as a model, this study presents a supervised learning method, the Support Vector Machines (SVM), to characterize sperm population structure related with freezability. Male freezability was assessed by evaluating motility, membrane status and mitochondrial membrane potential of sperm after a freezing-thawing procedure. The SVM model was generated using sperm motility information captured by computer-assisted sperm analysis (CASA) from thawed semen, belonging to six stags with marked differences on their freezability. A total of 1369 sperm tracks were recorded for seven kinematic parameters and assigned to four motility patterns based on them: weak motile, progressive, transitional and hyperactivated-like. Then, these data were split in two sets: the training set, used to train the SVM model, and the testing set, used to examine how the SVM method and three other unsupervised methods, a non-hierarchical, a hierarchical and a multistep clustering procedures, performed the sperm classification into subpopulations. The SVM was revealed as the most accurate method in the characterization of sperm subpopulations, showing all the sperm subpopulations obtained in this way high significant correlations with those sperm parameters used to characterize freezability of males. Given its superiority, the SVM method was used to characterize the sperm motile subpopulations in Iberian red deer. Sperm motile data from frozen–thawed semen belonging to 25 stags were recorded and loaded into the SVM model. The sperm population structure revealed that those males showing poor freezability were characterized by high percentages of sperm with a weak motility pattern. In opposite, males showing good freezability were characterized by higher percentages of sperm with a progressive and hyperactivated-like motility pattern and lower percentages of sperm with a weak motile pattern. We also identified a sperm subpopulation with a transitional motility pattern. This subpopulation increased as the freezability of males improved, and may be used as indicative of overall sperm motility.

doi:10.1095/biolreprod.113.112110

Effect of post-mortem time on post-thaw characteristics of Spanish ibex (Capra pyrenaica) spermatozoa2016-12-30T09:48:26+01:00

Effect of post-mortem time on post-thaw characteristics of Spanish ibex (Capra pyrenaica) spermatozoa

M.R. Fernández-Santosa, A.J. Solera, M. Ramóna, J.L. Ros-Santaellaa, A. Maroto-Moralesa, O. García-Álvarezc, A. Bisbala, J.J. Gardea, M.A. Colomad, J. Santiago-Morenod

Biology of Reproduction Group (IREC), UCLM-CSIC-JCCM, Albacete, Spain; Regional Development Institute (IDR), UCLM, Albacete, Spain; CERSYRA (JCCM), Valdepeñas, Spain; Department of Animal Reproduction, INIA, Madrid, Spain

Viable epididymal sperm can be obtained in the Spanish ibex during 24 h after death, but it has been observed a significant effect of the post-mortem time on fertility success, so only goats inseminated with semen recovery during the first 8 h became pregnant. The aim of this study was to determine the effect of post-mortem time on epididymal semen samples from of Spanish ibex. For this purpose, sperm samples from 36 males were collected at different post-mortem times, from 2 to 24 h, and cryopreserved. Thawed samples were incubated for 2 h at 37 °C without dilution or after dilution in a modified Tyrode medium, in order to study the sperm resistance to dilution. Moreover, flow cytometry was used to assess the sperm viability (PI), phospolipid disorder of the plasma membrane (M540), mitochondrial membrane potential (Mitotracker Deep Red), indirect apoptosis markers (YOPRO-1) and sperm chromatin stability (SCSA®). Sperm motility was evaluated by computer-assisted sperm analysis (CASA). Our results have shown that post-mortem time caused a reduction in mitochondrial membrane potential. In this regard, the loss of energy could be responsible for the loss of maintenance of the membrane with a consequent increase in permeability leading to a decrease in sperm viability and motility, losing linearity and speed. Moreover, the loss of maintenance of the membrane influence the extent to which sperm will survive the cryopreservation process, as it shows the results obtained from the dilution–incubation resistance test. Finally, one important finding of this study is the demonstration of no effect of post-mortem time on post-thaw DNA integrity, giving us the possibility of using sperm samples from valuable males, even if it was not possible to process during the first 8 h.

doi:10.1016/j.anireprosci.2011.09.011

Objective assessment of the cryoprotective effects of dimethylformamide for freezing goat semen2016-12-30T09:48:26+01:00

Objective assessment of the cryoprotective effects of dimethylformamide for freezing goat semen

Bezerra FS, Castelo TS, Alves HM, Oliveira IR, Lima GL, Peixoto GC, Bezerra AC, Silva AR

Laboratory of Animal Germplasm Conservation, Universidade Federal Rural do Semi-Árido, Rio Grande do Norte, Brazil

The aim of this work was to assess the cryoprotective effects of dimethylformamide (DMF) for freezing goat semen, using an objective analysis by computer-assisted sperm analysis (CASA). Twenty-one ejaculates (seven per animal) were collected from three stud bucks with the aid of an artificial vagina and immediately evaluated for gross and microscopic characteristics. The semen was diluted in two steps with a Tris-egg yolk extender containing 6% glycerol or 6% DMF, frozen in 0.50-mL straws, and stored in liquid nitrogen. Samples were accessed for sperm morphology, sperm membrane structural and functional integrity, and by CASA, immediately after thawing. There were differences (P<0.05) between glycerol and DMF with regard to subjective progressive motility (23.9±2.2% vs. 16.6±2.0%), objective progressive motility (3.5±0.4% vs. 1.8±0.3%), linearity (53.9±1.6% vs. 48.1±1.4%) and amplitude of lateral head (2.3±0.1 vs. 2.9±0.1 mm), which confirmed the efficiency of glycerol. In conclusion, dimethylformamide could be used as an alternative cryoprotectant for goat semen freezing. However it was showed that no benefits were derived by using dimethylformamide to replace glycerol at an equal 6% concentration.

doi: 10.1016/j.cryobiol.2011.09.136

Influence of season on the freezability of free-range poultry semen2016-12-30T09:48:26+01:00

Influence of season on the freezability of free-range poultry semen

Santiago-Moreno J, Castaño C, Toledano-Díaz A, Coloma MA, López-Sebastián A, Prieto MT, Campo JL.

Dpto. Reproducción Animal, INIA, Madrid, Spain

The purpose of the present study was to examine the seasonal variation in freezing damage in free-range rooster sperm. Over a period of 1 year, heterospermic semen samples were collected weekly by massage from the roosters of 14 Spanish chicken breeds, all housed under natural photoperiod and climatic conditions. All samples were frozen in straws using DMA as a cryoprotectant, placing them first in nitrogen vapour and then plunging them into liquid nitrogen. No seasonal effects on fresh sperm quality were found. Neither did season affect the percentage of viable frozen-thawed spermatozoa nor the percentage with an intact acrosome. However, the collection season influenced (p < 0.05) most frozen-thawed sperm motility values. The percentage of immotile frozen-thawed spermatozoa was lower (p < 0.05) in spring-collected sperm than in summer- or autumn-collected samples. The percentage of spermatozoa showing progressive motility was higher in spring-collected sperm compared with winter-, summer- or autumn-collected samples (p < 0.05). The curvilinear velocity (VCL), straight-line velocity (VSL) and average path velocity (VAP) values of spring-collected sperm were also higher (p < 0.05). In conclusion, spring would appear to be the best season for collecting and freezing the semen of free-range Mediterranean chicken breeds.

doi: 10.1111/j.1439-0531.2011.01921.x

Comparative study on semen characteristics of Kolbroek and Large White boars following computer aided sperm analysis® (CASA)2015-05-08T10:54:25+02:00

Comparative study on semen characteristics of Kolbroek and Large White boars following computer aided sperm analysis® (CASA)

Matshidiso Bailekae Masenya, M. L. Mphaphathi, M. H. Mapeka, P. H. Munyai, M. B. Makhafola, F. V. Ramukhithi, P. P. Malusi, D. O. Umesiobi2 and T. L. Nedambale

Agricultural Research Council, Animal Production Institute, Germplasm Conservation & Reproductive Biotechnologies, Private Bag X2, Irene, 0062, South Africa; Department of Agriculture, Central University of Technology, Private Bag X20539, Bloemfontein, 9300, South Africa; Tshwane University of Technology, Department of Animal Sciences, Private Bag X680, Pretoria 0001, South Africa; University of the Free State, Department of Animal, Wildlife and Grassland Sciences, Private Bag X 339, Bloemfontein, South Africa

Consistent estimates of boar fertility potential from objective semen evaluation could be a valuable tool for boar selection. The objective of this study was to evaluate semen characteristics of Kolbroek and Large White boars following computer aided sperm analysis® (CASA). Eight ejaculates were collected separately from individual Kolbroek (n = 4) and Large White (n = 4) boars using the gloved-hand technique. Following semen collection, semen was evaluated for macroscopic and microscopic characteristics. Analysis of variance (ANOVA) was used to test the differences between the breeds (P<0.05). The bodyweight of Kolbroek (154.7 ± 8.5) was significantly lower compared to Large White (189.9 ± 7.7) boar. There was also a positive correlation between bodyweight and semen volume of both Kolbroek (r = 0.2197) and Large White (r = 0.2577) boar. However, no significant differences were observed in Kolbroek and Large White boar semen volume (140 and 170 ml), sperm concentration (0.727 and 0.761 × 109 sperm cell/ml), pH (7.0 and 7.0), total motility (95 and 91%) and morphology (84 and 82%). In conclusion, the bodyweight of Kolbroek and Large White boar was positively correlated with ejaculated semen volume. Sperm characteristics of both Kolbroek and Large White boar were similar. Sperm class analyser® provided a precise and more objective information of sperm motility characteristics.

doi: 10.5897/AJB11.2010

Influence of sampling factors on canine sperm motility parameters measured by the Sperm Class Analyzer2016-12-30T09:48:26+01:00

Influence of sampling factors on canine sperm motility parameters measured by the Sperm Class Analyzer

Dorado J, Rijsselaere T, Muñoz-Serrano A, Hidalgo M

Animal Reproduction Group, Department of Medicine and Animal Surgery, Faculty of Veterinary Medicine, University of Cordoba, Cordoba, Spain

The aim of the present study was to investigate the effect of different technical settings and semen processing on sperm motility parameters measured by the Sperm Class Analyzer (SCA). Semen was collected from 3 dogs, pooled, and diluted in phosphate buffered saline and subsequently assessed by the SCA for the different sperm motility characteristics. The data were statistically analyzed by ANOVA and the repeatability was assessed by coefficient of variation (CV). After a principal component analysis, the reliability was determined with intra-class correlation coefficient (ICC). In experiment 1, the CV’s were below 10% for all evaluated parameters. Significant differences (P < 0.05) were found between the different sperm concentrations (25, 50, and 75 x 10(6) spermatozoa/ml) in all of the motion parameters assessed, yielding the highest ICC (0.81) at 25 x 10(6) spermatozoa/ml. No significant differences (P > 0.05) in SCA read-outs were found between the number of microscopic fields captured (1, 2, 3, 4, or 5 fields), yielding the highest ICC (0.83) when 3 fields were captured. No significant differences (P > 0.05) in motility parameters were found between the number of cells analyzed in each field (20, 50, and 100 spermatozoa) with the exception of beat cross frequency. Reliability of the SCA was good (ICC = 0.71 to 0.90) for all motility measurements when 20 (ICC = 0.89) or 50 (ICC = 0.77) cells were captured in each field, but only just acceptable (ICC = 0.51 to 0.70) when 100 cells were counted (ICC = 0.67). The frame settings significantly (P < 0.05) influenced most of the measured motility characteristics. Scanning 60 frames at a frame rate of 30 Hz improved the reliability of the results (ICC = 0.92). In conclusion, we suggest that the measurements with the SCA are ideally performed at a sperm concentration of 25 x 10(6) spermatozoa/ml, counting at least 100 cells in three microscopic fields. We also propose that the SCA should analyze 60 frames at 30 Hz to yield consistent results of a set of measurements or a measuring instrument thus obtaining reliable motility results.

doi: 10.3109/19396368.2011.627081

Response of thawed epididymal red deer spermatozoa to increasing concentrations of hydrogen peroxide, and importance of individual male variability2015-05-08T10:54:53+02:00

Response of thawed epididymal red deer spermatozoa to increasing concentrations of hydrogen peroxide, and importance of individual male variability

Domínguez-Rebolledo AE, Martínez-Pastor F, Bisbal AF, Ros-Santaella JL, García-Álvarez O, Maroto-Morales A, Soler AJ, Garde JJ, Fernández-Santos MR

Reproductive Biology Group, National Wildlife Research Institute (IREC), UCLM-CSIC-JCCM, Albacete, Spain

Oxidative stress represents a challenge during sperm manipulation. We have tested the effect of increasing hydrogen peroxide (H(2)O(2)) levels on red deer spermatozoa after cryopreservation, and the role of male-to-male variation in that response. In a first experiment, eight thawed samples were submitted to 0, 25, 50, 100 and 200 μm H(2)O(2) for 2 h at 37 °C. Intracellular reactive oxygen species (H(2)DCFDA-CM) increased with H(2)O(2) concentration, but we only detected a decrease in sperm function (motility by CASA and chromatin damage by sperm chromatin structure assay) with 200 μm. Lipoperoxidation assessed by the thiobarbituric acid reactive substance (TBARS) method increased slightly with 50 μm H(2)O(2) and above. In a second experiment, samples from seven males were submitted to 0 and 200 μm H(2)O(2) for 2 h, triplicating the experiment within each male. Males differed at thawing and regarding their response to incubation and H(2)O(2) presence. We found that the kinematic parameters reflected male-to-male variability, whereas the response of the different males was similar for lipid peroxidation and viability. A multiparametric analysis showed that males grouped differently if samples were assessed after thawing, after incubation without H(2)O(2) or after incubation with H(2)O(2) . Red deer spermatozoa are relatively resilient to H(2)O(2) after thawing, but it seems to be a great male-to-male variability regarding the response to oxidative stress. The acknowledgement of this individual variability might improve the development of optimized sperm work protocols.

doi: 10.1111/j.1439-0531.2010.01677.x

Undiluted or extended storage of ram epididymal spermatozoa as alternatives to refrigerating the whole epididymes2016-12-30T09:48:26+01:00

Undiluted or extended storage of ram epididymal spermatozoa as alternatives to refrigerating the whole epididymes

Tamayo-Canul J, Alvarez M, López-Urueña E, Nicolas M, Martinez-Pastor F, Anel E, Anel L, de Paz P

ITRA-ULE, INDEGSAL, University of León, León, Spain

The effect of storage procedure at 5°C on the quality of ram spermatozoa from the cauda epididymis was analyzed. Two strategies were tested at 0, 24, 48 and 72h post-mortem: (1) spermatozoa held in the epididymal fluid and stored either in the cauda epididymis (In-EPID) or in vitro (Ex-EPID), (2) epididymal spermatozoa extended in three media at 320, 370 and 420 mOsm/kg (D320, D370, D420). Analyzed parameters were: osmolality, pH, motility, acrosomal status and viability. In experiment 1, osmolality of the In-EPID samples, but not in Ex-EPID, increased with post-mortem time. Motility of In-EPID spermatozoa in samples, after 24h post-mortem, was higher compared to the Ex-EPID samples, although differences decreased at 48 and 72h. In experiment 2, total (TM) and progressive motility (PM) were not significantly affected by storage time for D320 and In-EPID samples. However, the motility of D370 and D420 samples significantly decreased with time. TM and PM of D320 were significantly higher than D370 and D420 at 72h. At 24h, sperm viability was higher for In-EPID (80.7±3.4%) than for the extended samples (44.8±2.9%, 37.7±3.9% and 48.6±6.0% for D320, D370 and D420, respectively), which also decreased faster with time. At 24h, the percentage of damaged acrosomes was low and similar for the four methods of storage, but damaged acrosomes increased with time for D320 and D370. Storing the spermatozoa in the epididymis is a good strategy for maintaining sperm quality in ram, at least for 48h. The D320 extender preserve motility of epididymal spermatozoa but does not protect the status of the acrosome.

doi: 10.1016/j.anireprosci.2011.04.011

Effects of reduced glutathione and catalase on the kinematics and membrane functionality of sperm during liquid storage of ram semen2016-12-30T09:48:26+01:00

Effects of reduced glutathione and catalase on the kinematics and membrane functionality of sperm during liquid storage of ram semen

D.R. Câmara, M.M.C. Mello-Pinto, L.C. Pinto, O.O. Brasil, J.F. Nunes, M.M.P. Guerra,

Department of Veterinary Medicine, Federal University of Alagoas, Fazenda São Luiz, s/n, Zona Rural de Viçosa, Viçosa-AL, Brazil; Northeast Biotechnology Network, Brazil; Laboratory of Sheep and Goat Semen Technology, Ceará State University, Av. Paranjana, 1700 – Campus do Itaperi, Fortaleza-CE, Brazil; Laboratory of Andrology, Department of Veterinary Medicine, Federal Rural University of Pernambuco, R. Dom Manoel de Medeiros, s/n, Dois Irmãos, Recife-PE, CEP 52171-900, Brazil

The objective of this study was to evaluate the effects of reduced glutathione (GSH) and catalase (CAT) supplementation on the kinematics and membrane functionality of sperm during the liquid storage of ram semen, cooled at 5 °C, for up to 24 h. Semen samples from four rams were pooled, diluted with Tris-egg yolk extender without antioxidants (control) or supplemented with either CAT (100, 200, and 400 U/mL) or GSH (100, 200, and 400 mM) at a final concentration of 50 × 106 sperm/mL. Sperm kinematics, which was analyzed by computer-assisted sperm analysis (CASA), and membrane functionality, which was analyzed using the hypo-osmotic swelling test (HOST), were determined after the addition of the semen samples at different processing times (fresh/diluted, 1.5, 6, 12, and 24 h, at 5 °C). No significant differences were recorded in the kinematics or membrane functionality between treatments at different times. The supplementation of diluents with 100 and 200 U/mL of CAT prevented the harmful effects of cooling on total sperm motility. No significant differences were observed in progressive sperm motility throughout processing, regardless of the treatment and time of evaluation. Supplementation with 400 mM GSH resulted in an earlier reduction (P < 0.05) of total sperm motility, a decrease in rapid sperm rate and a reduction in curvilinear velocity during incubation, at 5 °C. The cooling induced a reduction (P < 0.05) in the percentage of sperm with a functional plasma membrane (HOST), especially after 1.5 h of incubation. Based on the results of the present study, the addition of CAT (100 and 200 U/mL) reduced the deleterious effects of cooling on total motility in ram sperm maintained at 5 °C for 24 h, although it did not affect the functionality of the sperm membranes. However, the addition of 400 mM GSH caused negative effects on the velocity parameters of the sperm.

doi:10.1016/j.smallrumres.2011.05.010

Dimethylformamide as a cryoprotectant for canine semen diluted and frozen in ACP-106C2015-05-08T10:56:22+02:00

Dimethylformamide as a cryoprotectant for canine semen diluted and frozen in ACP-106C

Mota Filho AC, Teles CH, Jucá RP, Cardoso JF, Uchoa DC, Campello CC, Silva AR, Silva LD

Carnivore Reproduction Laboratory, Veterinary College, State University of Ceará, Fortaleza, CE, Brazil

The objective was to assess the effect of adding various concentrations of dimethylformamide on characteristics of canine semen diluted in powdered coconut water (ACP-106C; ACP Biotecnologia, Fortaleza, CE, Brazil) and frozen at -196°C. Fifteen ejaculates were collected by manual stimulation from five adult Boxer dogs. The sperm-rich fraction was diluted in ACP-106C (ACP Biotecnologia) containing 10% egg yolk and divided into four aliquots. The cryoprotectants used for each aliquot were 6% glycerol (control group; CG) or 2%, 4%, or 6% dimethylformamide (DF2, DF4, and DF6, respectively). After thawing, total motility (mean ± SEM) for CG (58.4 ± 24.6) was higher (P < 0.05) than that of the other groups (2% dimethylformamide, 24.4 ± 12.3; 4% dimethylformamide, 26.5 ± 16.1; and 6% dimethylformamide, 21.7 ± 17.9). Furthermore, there was a greater percentage of fast, average, and slow moving sperm (assessed with computer-aided semen analysis; CASA) in CG in comparison with the other three groups. Therefore, based on concentrations tested in this study, dimethylformamide, together with ACP-106C (ACP Biotecnologia) and 10% egg yolk as a diluent, yielded unsatisfactory in vitro results for freezing canine semen.

doi: 10.1016/j.theriogenology.2011.05.010

Quality of frozen-thawed semen in brown bear is not affected by timing of glycerol addition2015-05-08T10:56:36+02:00

Quality of frozen-thawed semen in brown bear is not affected by timing of glycerol addition

Alvarez-Rodríguez M, Alvarez M, Gomes-Alves S, Borragan S, Martinez-Pastor F, de Paz P, Anel L.

University of León, 24071 León, Spain.

We have tested several freezing protocols for brown bear semen, modifying the time when glycerol was added (before and after cooling to 5 °C). No differences were found among protocols, indicating a good tolerance of brown bear semen to glycerol. This finding indicates that freezing protocols for brown bear semen could be modified to fit practical solutions which would facilitate preparation of the seminal samples in the field with the addition of glycerol at ambient temperature.

doi: 10.1016/j.theriogenology.2010.12.009

Effect of relaxin on human sperm functions2015-05-08T10:57:04+02:00

Effect of relaxin on human sperm functions

Ferlin A, Menegazzo M, Gianesello L, Selice R, Foresta C

University of Padova, Department of Histology, Microbiology and Medical Biotechnologies, Section of Clinical Pathology and Center for Male Gamete Cryopreservation, Padova, Italy

Relaxin is a circulating hormone with functions in pregnancy, parturition, and other aspects of female reproduction. It is also secreted from the prostate gland into the seminal fluid; however, the role of relaxin in male reproduction is debated. Studies conducted in the past have suggested possible actions on human spermatozoa, but the data were contrasting. Here, we show that the relaxin receptor RXFP1 (Relaxin Family Peptide Receptor 1) is expressed in human spermatozoa, and it mainly localizes in the astrodome. In vitro studies on human sperm demonstrated that this hormone attenuates the natural decline in sperm motility and maintains higher mitochondrial activity and lower apoptosis level. Furthermore, relaxin induced an increase in sperm hyperactivation, intracellular calcium and cAMP, and acrosome reaction. These effects were abolished by the use of the specific anti-RXFP1 antibody. Relaxin concentrations were low in the blood (x ± SD, 0.16 ± 0.03 nM) and very high in the seminal plasma (x ± SD, 10.3 ± 4.0 nM), confirming its secretion mainly by the prostate. Taken together, these data demonstrate that relaxin influences positively many sperm functions linked to fertilizing ability, and it preserves sperm functionality, with possible practical value in assisted reproduction techniques.

doi: 10.2164/jandrol.110.012625

Characterization of the kisspeptin system in human spermatozoa2016-12-30T09:48:26+01:00

Characterization of the kisspeptin system in human spermatozoa

Pinto FM, Cejudo-Román A, Ravina CG, Fernández-Sánchez M, Martín-Lozano D, Illanes M, Tena-Sempere M, Candenas ML

Instituto de Investigaciones Químicas, CSIC-Universidad de Sevilla, Spain

Kisspeptin, the product of the KISS1 gene, plays an essential role in the regulation of spermatogenesis acting primarily at the hypothalamic level of the gonadotropic axis. However, the presence of kisspeptin and its canonical receptor, KISS1R, in spermatozoa has not been explored nor the direct effects of kisspeptin on sperm function have been studied so far. In the present study, we analysed the expression of kisspeptin and its receptor in sperm cells by western blot and immunocytochemistry assays and evaluated the effects of exposure to kisspeptin on sperm intracellular Ca(2+) concentration,

[Ca(2+)]i, sperm motility, sperm hyperactivation and the acrosome reaction. Changes in [Ca(2+)]i were monitored using Fura-2, sperm kinematic parameters were measured using computer-assisted sperm analysis (CASA), and the acrosome reaction was measured using fluorescein isothiocyanate-coupled Pisum sativum agglutinin lectin (FITC-PSA method). We found that kisspeptin and its receptor are present in sperm cells, where both are mainly localized in the sperm head, around the neck and in the flagellum midpiece. Exposure to kisspeptin caused a slow, progressive increase in [Ca(2+)]i, which reached a plateau about 3-6 min after kisspeptin exposure. In addition, kisspeptin modulated sperm progressive motility causing a biphasic (stimulatory and inhibitory) response and also induced transient sperm hyperactivation. The effects of kisspeptin on sperm motility and hyperactivation were inhibited by the antagonist of KISS1R, peptide 234. Kisspeptin did not induce the acrosome reaction in human spermatozoa. These data show for the first time that kisspeptin and its receptor are present in human spermatozoa and modulate key parameters of sperm function. This may represent an additional mechanism for their crucial function in the control of male fertility.

doi: 10.1111/j.1365-2605.2011.01177.x

Centrifugation on PureSperm® density-gradient improved quality of spermatozoa from frozen-thawed dog semen2015-05-08T10:57:32+02:00

Centrifugation on PureSperm® density-gradient improved quality of spermatozoa from frozen-thawed dog semen

Dorado J, Alcaráz L, Duarte N, Portero JM, Acha D, Demyda S, Muñoz-Serrano A, Hidalgo M.

Department of Medicine and Animal Surgery, University of Cordoba, Córdoba, Spain.

The main objective of this study was to investigate if centrifugation through PureSperm(®) density-gradient can improve the post-thaw semen quality of dog semen. Semen from 5 dogs was collected and cryopreserved following a standard protocol. After thawing, semen samples were selected by centrifugation on PureSperm(®). Assessments of sperm motility (assessed by computerized-assisted semen analysis), morphology (Diff-Quick staining) and viability (triple fluorescent stain of Propidium iodine/isothiocyanate-labeled peanut (Arachis hypogaea) agglutinin/Rhodamine 123), were performed on aliquots of fresh semen, unselected samples and selected preparations. Cryopreservation had a significant (P < 0.001) effect on all studied semen parameters. PureSperm(®) centrifugation yielded sperm suspensions with improved motility and viability (P < 0.001). The washing step significantly reduced (P < 0.001) all of the kinematics parameters evaluated as well as reduced the proportion of viable spermatozoa with intact acrosomes (P < 0.05). We concluded that PureSperm(®) centrifugation is a successful method for improving the quality of frozen-thawed dog spermatozoa. However, washing after density-gradient centrifugation dramatically reduces the post-thaw semen quality, indicating that the inclusion of such a washing step is unnecessary.

doi:10.1016/j.theriogenology.2011.02.026

Time trends, environmental factors and genetic basis of semen traits collected in Holstein bulls under commercial conditions2016-12-30T09:48:29+01:00

Time trends, environmental factors and genetic basis of semen traits collected in Holstein bulls under commercial conditions

Karoui S, Díaz C, Serrano M, Cue R, Celorrio I, Carabaño MJ

Animal Breeding Department, National Institute for Agriculture and Food Research and Technology, Ctra. de La Coruña Km 7.5, 28040 Madrid, Spain

The fact that results of artificial insemination (AI) are declining in highly selected dairy cattle populations has added a renewed interest to the evaluation of male fertility. Data from 42,348 ejaculates collected from 1990 to 2007 on 502 Holstein bulls were analysed in a Bayesian framework to provide estimates of the evolution of semen traits routinely collected in AI centres throughout the last decades of intense selection for production traits and estimate genetic parameters. The traits under consideration were volume (VOL), concentration (CONC), number of spermatozoa per ejaculate (NESPZ), mass motility score (MM), individual motility (IM), and post-thawing motility (PTM). The environmental factors studied were year-season and week of collection, which account for changes in environmental and technical conditions along time, age at collection, ejaculate order, time from previous collection (TPC) and time between collection and freezing (TCF) (only for PTM). Bull’s inbreeding coefficient (Fi), bull’s permanent environmental and additive genetic effects were also considered. The use of reduced models was evaluated using the Bayes factor. For all the systematic effects tested, strong or very strong evidence in favour of including the effect in the model was obtained, except for Fi for motility traits and TCF for PTM. No systematic time trends for environment or bull effects were observed, except for PTM, which showed an increasing environmental trend, associated with improvements in freezing-thawing protocols. Heritability estimates were moderate (0.16-0.22), except for IM, which presented a low value (0.07). Genetic correlations among motilities and between motilities and CONC were large and positive

[0.38-0.87], VOL showed a negative correlation with CONC (-0.13) but with ample HPD 95%. The magnitude of heritabilities would allow an efficient selection if required and grants the use of these traits as indicators of the sperm viability component of bulls breeding soundness.

doi: 10.1016/j.anireprosci.2011.02.008

Simple and economic colloidal centrifugation protocols may be incorporated into the clinical equine sperm processing procedure2015-05-08T10:58:17+02:00

Simple and economic colloidal centrifugation protocols may be incorporated into the clinical equine sperm processing procedure

Gutiérrez-Cepeda L, Fernández A, Crespo F, Gosálvez J, Serres C

Animal Medicine and Surgery Department, Veterinary Faculty, UCM, Avda. Puerta de Hierro s/n, Ciudad Universitaria, 28040 Madrid, Spain; Centro Militar de Cría Caballar, (FESCCR-Ministerio de Defensa), C/Arsenio Gutiérrez Palacios s/n, 05005 Ávila, Spain; Biology Department, Genetic Unity, UAM, C/Darwin 2, Ciudad Universitaria de Cantoblanco, 28049 Madrid, Spain

For many years in human assisted-reproduction procedures there have been special protocols to prepare and improve sperm quality. Colloidal centrifugation (CC) is a useful technique that has been proved to enhance semen quality by selection of the best spermatozoa for different species. Its use is recommended to improve fertility of subfertile stallions but current CC protocols are clinically complicated in the equine sperm processing technique due to economic and technical difficulties. The aim of this study was to determine the optimal processing procedures to adapt the use of a CC product (EquiPure™) in the equine reproduction industry. A total of nineteen ejaculates were collected from 10 Purebred Spanish Horses (P.R.E horses) using a Missouri artificial vagina. Gel-free semen aliquots were analyzed prior to treatment (control). Semen was subjected to one of six CC protocols with EquiPure™ and centrifuged samples were statistically evaluated by ANOVA and Duncan tests (p<0.05) for sperm quality and recovery rate. We obtained higher values by colloidal centrifugation in LIN, STR and BCF variables and DNA fragmentation index trended to be lower in most of the CC protocols. The studied protocols were shown to be as efficient in improving equine sperm quality as the current commercial EquiPure™, with the added advantage of being much more economical and simple to use. According to these results it seems to be possible to incorporate single layer and or high colloidal centrifugation volume protocols what would make them simple, economic and clinically viable for the equine sperm processing procedure.

doi: 10.1016/j.anireprosci.2011.02.001

Effects of antioxidants and duration of pre-freezing equilibration on frozen-thawed ram semen2015-05-08T10:58:29+02:00

Effects of antioxidants and duration of pre-freezing equilibration on frozen-thawed ram semen

Câmara DR, Silva SV, Almeida FC, Nunes JF, Guerra MM

Department of Veterinary Medicine, Federal University of Alagoas, Viçosa-AL. Brazil

The objective was to evaluate the effects of various antioxidants and duration of pre-freezing equilibration on cryopreservation of ram semen. Semen samples from four rams were pooled, diluted with Tris-egg yolk extender without antioxidants (control), or supplemented with reduced glutathione (GSH: 0.5, 1.0, and 2.0 mM), superoxide dismutase (SOD: 5, 10, and 20 U/mL), or catalase (CAT: 5, 10, and 20 U/mL), and cryopreserved, immediately after thermal equilibrium was reached at 5 °C (0 h), or 12 or 24 h after equilibration. Total antioxidant capacity was determined in the in natura extenders and after addition of semen samples for various durations of processing (fresh/dilute, throughout refrigeration, and post-thaw). Plasma membrane (PI-CFDA), acrosome integrity (FITC-PNA), and mitochondrial membrane potential (JC-1) were determined in fresh/diluted, refrigerated, and post-thaw samples. Post-thaw sperm motility was assessed with a computerized analysis system (CASA). There were no significant differences in acrosome damage or mitochondrial membrane potential after refrigeration and freeze-thaw, regardless of antioxidant addition. Sperm plasma membrane integrity was worse (P < 0.05) with cryopreservation immediately after equilibration (average 20.1 ± 8.3; mean ± SD) than after 12 h of equilibration (average 42.5 ± 10.9); however, the addition of SOD and CAT (10 and 20 U/mL) resulted in no significant difference between post-equilibration intervals of 0 and 12 h. Total antioxidant activity was not different (P > 0.05) among treatments after sperm addition or throughout the refrigeration and post-thaw. In conclusion, adding GSH, SOD or CAT did not increase the total antioxidant capacity of semen, nor did it enhance the quality of the post-thaw sperm. However, maintenance of ram semen at 5 °C for 12 h prior to cryopreservation reduced membrane damage of frozen-thawed sperm.

doi: 10.1016/j.theriogenology.2011.02.013

Study for the diagnostic evaluation of the sperm DNA fragmentation index in a patient population of the fertility unit2011-01-01T17:09:33+01:00

Study for the diagnostic evaluation of the sperm DNA fragmentation index in a patient population of the fertility unit

Albert Blanco, Jordi Ramis, Carlos Aulesa, José Mª Gris

Laboratorio de Andrología, Unidad de Laboratorios Clínicos Vall d’Hebron, Barcelona. Servicio de Esterilidad Hospital Maternal Vall d’Hebron.

Summary:

Introduction: At present, parameters obtained via the common seminogram fail to provide complete information on the fertilizing potential of semen, as it is observed that between 10 to 15% of sterile males show a normal seminogram.

Objective: This study has verified the clinical utility of determination of the sperm DNA Fragmentation Index (DFI) as a diagnosis aid to male subfertility.

Design: 60 samples were processed from patients at the Fertility Unit, of which 42 patients showed proven infertility due to their clinical history, the remaining 18 were fertile males. The corresponding seminogram and FI were carried out using Halosperm® (Halotech D, Madrid) commercial equipment for the preparation of samples and a Sperm Class Analyzer (SCA) type Computer Assister Semen Assay® (CASA) for reading the sperm fragmentation.

Results: The FI sensitivity in diagnosis of subfertility was 54.8% and the specificity 66.7%, with a final diagnostic efficiency of 58.3%. The seminogram showed a sensitivity of 8.7% and a specificity of 50%, with diagnostic efficiency of 78.9%. The areas under the curve (AUC) are calculated from the ROC curves; it is shown that the FI presents a AUC=0.601 and the seminogram a AUC=0.679, which indicates that the seminogram has a higher diagnostic value.

Conclusions: Further to the data obtained, we reach the conclusion that the FI test does not appear to show a higher diagnostic value than the classic parameters of the seminogram. Likewise, we have observed that a pathological result in the FI can be a factor to consider for those couples who still have doubts about exploring other assisted reproductive techniques or Artificial Insemination by donor (AID).

Full article here

Revista Iberoamericana de Fertlidad y Reproducción Humana
Vol. 28 nº1 Enero-Febrero-Marzo 2011

Sperm structure and motility in the eusocial naked mole-rat, Heterocephalus glaber: a case of degenerative orthogenesis in the absence of sperm competition?2022-05-04T15:56:23+02:00

Sperm structure and motility in the eusocial naked mole-rat, Heterocephalus glaber: a case of degenerative orthogenesis in the absence of sperm competition?

Gerhard van der Horst, Liana Maree1, Sanet H Kotzé and M Justin O’Riain

Department of Medical Bioscience, University of the Western Cape, Bellville, South Africa; Division of Anatomy and Histology, Department of Biomedical Sciences, Stellenbosch University, Parow, South Africa; Department of Zoology, University of Cape Town, Cape Town, South Africa

Background: We have studied sperm structure and motility in a eusocial rodent where reproduction is typically restricted to a single male and behaviourally dominant queen. Males rarely compete for access to the queen during her estrus cycle, suggesting little or no role for sperm competition.
Results: Our results revealed an atypical mammalian sperm structure with spermatozoa from breeding, subordinate and disperser males being degenerate and almost completely lacking a “mammalian phylogenetic stamp”. Sperm structure is characterized by extreme polymorphism with most spermatozoa classified as abnormal. Sperm head shapes include round, oval, elongated, lobed, asymmetrical and amorphous. At the ultrastructural level, the sperm head contains condensed to granular chromatin with large open spaces between the chromatin. Nuclear chromatin seems disorganized since chromatin condensation is irregular and extremely inconsistent. The acrosome forms a cap (ca 35%) over the anterior part of the head. A well defined nuclear fossa and neck with five minor sets of banded protein structures are present. The midpiece is poorly organized and contains only 5 to 7 round to oval mitochondria. The flagellar pattern is 9+9+2. A distinct degenerative feature of the tail principal piece is the absence of the fibrous sheath. Only 7% motile spermatozoa were observed which had exceptionally slow swimming speeds.
Conclusion: In this species, sperm form has simplified and degenerated in many aspects and represents a specialised form of degenerative orthogenesis at the cellular level.

doi:10.1186/1471-2148-11-351

Modification of spermatozoa quality in mature small ruminants2015-05-08T10:59:45+02:00

Modification of spermatozoa quality in mature small ruminants

Martin GB, de St Jorre TJ, Al Mohsen FA, Malecki IA

UWA Institute of Agriculture M082, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia

This review is based largely, but not entirely, on the assumption that gamete quality is directly linked to sperm output and thus testicular mass, an approach made necessary by the absence of a large body of data on factors that affect gamete quality in ruminants. On the other hand, there is a change in the efficiency of sperm production per gram of testicular tissue when the testis is growing or shrinking, a clear indicator of changes in the rates of cell loss during the process of spermatogenesis, probably through apoptosis. We therefore postulate that the spermatozoa that do survive when the testis is shrinking are of a lower quality than those that are produced when the testis is growing and the rate of sperm survival is increasing. In adult small ruminants in particular, testicular mass and sperm production are highly labile and can be manipulated by management of photoperiod (melatonin), nutrition, genetics and behaviour (‘mating pressure’). Importantly, these factors do not act independently of each other – rather, the outcomes in terms of sperm production are dictated by interactions. It therefore seems likely that spermatozoa quality will be affected by these same factors, but definitive answers await detailed studies.

doi: 10.1071/RD11902

Noninsulin-dependent diabetes mellitus: effects on sperm morphological and functional characteristics, nuclear DNA integrity and outcome of assisted reproductive technique2016-12-30T09:48:29+01:00

Noninsulin-dependent diabetes mellitus: effects on sperm morphological and functional characteristics, nuclear DNA integrity and outcome of assisted reproductive technique

G. A. Rama Raju, G. Jaya Prakash, K. Murali Krishna, K. Madan, T. Siva Narayana & C. H. Ravi Krishna

Gynecology Division, Krishna IVF Clinic, Visakhapatnam, India; Embryology Research Group, Krishna IVF Clinic, Visakhapatnam, India; Genetic Department, Krishna IVF Clinic, Visakhapatnam, India

The aim of the study was to compare the semen characteristics and nuclear DNA fragmentation in spermatozoa of diabetic and nondiabetic men undergoing assisted reproduction and correlate them with pregnancy outcome. Semen characteristics and nuclear DNA fragmentation were analysed using computeraided semen analysis system and sperm chromatin dispersion assay (SCD), respectively. Spermatozoa from diabetic patients showed significantly lower progressive (Type A) motility (14.64 ± 9.60 versus 17.99 ± 11.51, P < 0.02) and increased nuclear DNA fragmentation (37.05 ± 12.68 versus 21.03 ± 10.13, P < 0.001). Furthermore, a positive correlation was observed in diabetic patients in terms of blastocyst formation rate (38.13% versus 55.46%, P < 0.001), pregnancy rate (28.57% versus 46.34%, P < 0.001) and miscarriage rate (50.0% versus 24.56%, P < 0.001). The higher percentage of sperm DNA damage because of oxidative stress seen in diabetic patients may be responsible for the poor embryonic development and pregnancy outcome in these individuals.

doi: 10.1111/j.1439-0272.2011.01213.x

CASA derived human sperm abnormalities: correlation with chromatin packing and DNA fragmentation2015-05-08T11:00:38+02:00

CASA derived human sperm abnormalities: correlation with chromatin packing and DNA fragmentation

T. Sivanarayana, Ch. Ravi Krishna, G. Jaya Prakash, K. Murali Krishna, K. Madan, B. Sireesha Rani, G. Sudhakar, G. A. Rama Raju

Gynecology Division, Krishna IVF Clinic, Visakhapatnam, India; Embryology Research Group, Krishna IVF Clinic, Visakhapatnam, India; Genetics Department, Krishna IVF Clinic, Visakhapatnam, India; Department of Human Genetics, Andhra University, Visakhapatnam, India

Purpose The present study was undertaken to evaluate the effects of morphokinetic abnormalities of human spermatozoa on chromatin packing and DNA integrity and possible beneficial effects of sperm selection in ICSI.
Methods Semen samples from 1002 patients were analysed for morphology and motility using CASA. Protamine status and DNA fragmentation were analysed by chromomycin A3 staining and sperm chromatin dispersion assay respectively.
Results Sperms with elongated, thin, round, pyri, amorphous, micro and macro forms were significantly higher in teratozoospermic and oligoasthenoteratozoospermic groups. Significant difference in chromatin packing and DNA fragmentation index was observed in these abnormal groups compared with normal. Similarly significant correlation was also seen between abnormal motility parameters and DNA fragmentation index in asthenozoospermic group compared with normal.
Conclusions Specific abnormal morphological forms have higher incidence of chromatin packing abnormalities and DNA fragmentation. Using these sperms in ICSI might have an impact on fertilization, embryo development and abortion rates. These can be selectively avoided during ICSI procedure to improve ART outcome.

doi: 10.1007/s10815-012-9885-9

Reduced glutathione content in human sperm is decreased after cryopreservation: Effect of the addition of reduced glutathione to the freezing and thawing extenders2016-12-30T09:48:29+01:00

Reduced glutathione content in human sperm is decreased after cryopreservation: Effect of the addition of reduced glutathione to the freezing and thawing extenders

Gadea J, Molla M, Selles E, Marco MA, Garcia-Vazquez FA, Gardon JC

Dept. Physiology, University of Murcia, Murcia 30100, Spain

In this study, total glutathione content was determined in human spermatozoa before and after cryopreservation. Total GSH in fresh semen was 4.47±0.46nmol/10(8) cells. Following semen cryopreservation, GSH decreased to 1.62±0.13nmol/10(8) cells, a 64% reduction (p<0.01). This decrease in GSH content was associated with a decrease in sperm progressive motility (68% of reduction, p<0.01). Addition of 1mM GSH to the freezing extender increased the percentage of total motility and sperm viability. It also modified the motility pattern measured by CASA with changes in the straight-line and average path velocities and wobble of the curvilinear trajectory. Addition of GSH to the freezing media reduced spermatozoa ROS levels and increased the level of sulfhydryl groups on membrane proteins. Nevertheless, no effect of GSH addition on lipid membrane disorder or chromatin condensation was detected. Addition of 1 or 5mM GSH to the thawing media increased the percentage of motile and progressively motile spermatozoa, but no effect on viability was detected. In conclusion, the antioxidant defensive capacity of the GSH is severely altered by the freeze-thawing process. The addition of GSH to the freezing and thawing extender could be of partial and limited benefit in improving the function of frozen human spermatozoa.

doi: 10.1016/j.cryobiol.2010.12.001

Melatonin protects ram spermatozoa from cryopreservation injuries in a dose-dependent manner2016-12-30T09:48:29+01:00

Melatonin protects ram spermatozoa from cryopreservation injuries in a dose-dependent manner

Succu S, Berlinguer F, Pasciu V, Satta V, Leoni GG, Naitana S

Department of Animal Biology, University of Sassari, Sassari, Italy

Cryopreservation harms spermatozoa at different levels and thus impairs their fertilizing ability. The role of melatonin in protecting spermatozoa from different kind injuries has been widely reported. Thus, this study tested whether the addition of melatonin to ram semen freezing extender could exert a protective effect and ameliorate postthawing sperm function. Melatonin was added to recommended ram extender to yield five different final concentrations: 0.001, 0.01, 0.1, 1, and 10 mm. A control group without melatonin supplementation was included. Spermatozoa viability, motility parameters, and intracellular ATP concentrations were evaluated both before and after cryopreservation, while DNA integrity and in vitro fertilizing ability were evaluated only after thawing. Obtained results showed that the concentration of 1 mm melatonin led to higher viability rates, higher percentages of total motile and progressive motile spermatozoa, higher percentages of spermatozoa with average rapid and medium velocity, higher intracellular ATP concentrations, and higher DNA integrity among semen frozen in control and melatonin-supplemented extenders (P<0.05). In addition, results obtained after the IVF test showed that at 1 mm concentration, melatonin led to a faster first embryonic division and to higher total cleavage rates compared to the other experimental groups (P<0.05). No difference in embryo output was observed among the six experimental groups. In conclusion, the addition of melatonin to ram semen freezing extender protected spermatozoa during cryopreservation in a dose-dependent manner. These results are likely to be mediated by its well-known antioxidant properties, even if a direct action of the indolamine cannot be ruled out.

doi: 10.1111/j.1600-079X.2010.00843.x

Effect of exogenous DNA on bovine sperm functionality using the sperm mediated gene transfer (SMGT) technique2015-05-08T11:09:23+02:00

Effect of exogenous DNA on bovine sperm functionality using the sperm mediated gene transfer (SMGT) technique

Canovas S, Gutierrez-Adan A, Gadea J

Department of Physiology, Veterinary Faculty, University of Murcia, Murcia, Spain

Sperm mediated gene transfer (SMGT) could provide the opportunity to carry out transgenesis on a mass scale using spermatozoa as vectors for exogenous DNA. However, the efficiency of sperm-mediated DNA transfer is still questionable, and the mode of transmission to the egg has not yet been well understood. Our aim was to investigate the capacity of bovine spermatozoa to carry exogenous DNA and its relationship to sperm functionality. We studied these parameters using flow cytometry to measure viability (necrosis and apoptosis) and capacitation status, computer-assisted semen analysis (CASA) to measure motility parameters and in vitro fertilization (IVF) to assess fertilizing capacity. Furthermore, we studied the effect of capacitation status on interaction with exogenous DNA, and the role of heparin supplementation in this process. Bull spermatozoa showed a high capacity to bind DNA quickly and reached a maximum after 30 min, with approximately half of the DNA-bound spermatozoa being viable. Incubation with exogenous DNA induced a decrease in sperm viability and motility and increased the proportion of apoptotic cells, but did not affect the cleavage rate in IVF assay. Heparin increased high-lipid disorder and the number of sperm with DNA bound (viable and dead). In conclusion, this study shows that live spermatozoa can bind exogenous DNA with a slight negative effect in some parameters of sperm function that in our opinion, would not drastically compromise fertility.

doi: 10.1002/mrd.21205

Evaluation of sperm quality at a national level: Logistic, recruitment, analytical and statistical problems encountered in switzerland2015-05-08T11:09:37+02:00

Evaluation of sperm quality at a national level: Logistic, recruitment, analytical and statistical problems encountered in switzerland

Josefina Vargas, Roumen Parapanov, Marc Van Den Bergh, Eric Stettler, Pierre Crettaz, Alfred Senn, Marc Germond

Fondation Faber, Lausanne, Switzerland, Kantonspital Baden Ag, Fertilitätslabor, Switzerland, Swiss Army Medical Services, Switzerland, Federal Office of Public Health, Switzerland

Aim:
For decades, numerous studies have reported a decline in sperm quality among industrialised countries. Explanations for this decline point toward environmental factors acting on animals and humans. More recently, the detrimental role of endocrine disruptors during foetal development of the testicles has also been hypothesised. In order to test whether these effects are operating in Switzerland, a survey study among Swiss young men was initiated in 2005 and will cover within the next two years the entire country. The aim of this report is to present the differences in sperm quality observed so far in various geographic regions of the Switzerland.
Method:
One month before recruitment, all conscripts are informed about the study. When interested, the volunteers send a consent form and two questionnaires to the Swiss army physician in charge of supervising the study and warranting anonymity. Four recruiting centres (Lausanne, Rüti, Windisch, Monte Ceneri) are actively involved in the sample collection phase. At the end of their recruiting camp, volunteers are invited in a nearby laboratory for biological investigations. Sperm samples are analysed according to WHO recommendations. Sperm concentration, motility and morphology are measured using a computerised system (CASA, SCA Microptic, Spain).
Results:
Results are grouped according to a geographic stratification of Switzerland into (1) Plateau west and central, (2) Jura, (3) Alps, (4) Plateau north and east. Medians for sperm concentration were significantly lower in regions 3 and 4 compared to the two other regions, whereas sperm motility was less affected.
Conclusion:
The geographic differences observed in sperm quality throughout Switzerland are currently further investigated by enlarging the cohort and identifying possible explanations for such differences. A human biomonitoring is underway in order to identify the presence of various endocrine disruptors in serum and urine samples collected from the volunteers.

doi: 10.1016/S1472-6483(10)62403-0

Impact of spontaneous smoking cessation on sperm quality: case report2010-05-10T00:00:45+02:00

Impact of spontaneous smoking cessation on sperm quality: case report

E. Prentki Santos, S. López-Costa, P. Chenlo, M. N. Pugliese, S. Curi, J. Ariagno, H. Repetto, M. Sardi, L. Palaoro & G. Mendeluk

Laboratory of Andrology and Male Fertility, Clinical Hospital, INFIBIOC, University of Buenos Aires, Argentina; Urology Division, Tobacco Cessation Program, Ramos Mejía Hospital, Government of the City of Buenos Aires, Argentina

We evaluated sperm quality after a 3-month smoking cessation programme by sperm analysis, objective sperm motility analysis, protein tyrosine phosphorylation in capacitating conditions and DNA fragmentation (TUNEL). Sperm analysis after smoking cessation revealed a distinctive improvement in sperm concentration, fast spermatozoa (‡35 lm/s), sperm vitality, percentage of spermatozoa recuperated after an enrichment technique and protein tyrosine phosphorylation. However, no changes were observed in the number of germinal cells in the ejaculate, sperm morphology and sperm DNA fragmentation. It is concluded that physicians should strongly advise their patients to quit smoking before undergoing medical treatment or assisted reproduction techniques to achieve pregnancy.

doi: 10.1111/j.1439-0272.2010.01089.x

Computerized sperm motility analysis in toxicity bioassays: a new approach to pore water quality assessment2018-04-17T12:05:51+02:00

Computerized sperm motility analysis in toxicity bioassays: a new approach to pore water quality assessment

Fabbrocini A, Di Stasio M, D’Adamo R

Consiglio Nazionale delle Ricerche-Istituto di Scienze Marine, UOS Lesina, via Pola 4, 71010 Lesina (FG), Italy

The aim of this study was to test the sensitivity of computerized sperm motility analysis in the sea urchin Paracentrotus lividus as the endpoint in toxicity bioassays. The tested matrices were pore water samples collected in an agriculture-impacted Mediterranean lagoon, Lake Varano (Italy). Two standardized bioassays were also conducted as controls, the P. lividus spermiotoxicity test and the Vibrio fischeri (Microtox®) test. VCL (curvilinear velocity), VSL (straight line velocity), VAP (average path velocity), and the percentage of rapid spermatozoa recorded by the Sperm Class Analyzer® system showed high sensitivity and discrimination ability, to a degree comparable with the larval development endpoint of the spermiotoxicity test. The test evaluated in this study requires small volumes of matrices, involves minimal sample manipulation, and can easily be extended to many other bioindicator species. It may therefore be considered a promising «quick response tool» following hazardous events that may adversely affect an aquatic ecosystem.

doi: 10.1016/j.ecoenv.2010.05.003

The HSP90AA1 sperm content and the prediction of the boar ejaculate freezability2016-12-30T09:48:29+01:00

The HSP90AA1 sperm content and the prediction of the boar ejaculate freezability

Casas I, Sancho S, Ballester J, Briz M, Pinart E, Bussalleu E, Yeste M, Fàbrega A, Rodríguez-Gil JE, Bonet S.

Biotechnology of Animal and Human Reproduction (TechnoSperm), Department of Biology, Institute of Food and Agricultural Technology (INTEA), University of Girona, Campus Montilivi, s/n, 17071 Girona, Spain

In a previous study we reported that the immunolabelling of GLUT3, HSP90AA1, and Cu/ZnSOD proteins on boar sperm did not show differences between good and poor freezability ejaculates, in terms of a qualitative analysis based on location and reactivity of these proteins at 17 degrees C and at 240 min post-thaw. Since predicting the ejaculate freezability is considerably important in sperm cryopreservation procedures, the objective of the present study was to quantify the expression of these three proteins in good and poor freezability ejaculates. For this purpose, 10 ejaculates from 9 Piétrain boars were cryopreserved and their sperm quality assessed in the three main steps of the freezing process (17 degrees C, 5 degrees C, and 240 min post-thaw). After this assessment, the 10 ejaculates were clustered for freezability on the basis of their sperm progressive motility and membrane integrity at 240 min post-thaw. From the whole ejaculates, only four good and four poor freezability ejaculates displaying the most divergent values were selected for a western blot assay using sperm samples coming from the three mentioned freezing steps. Protein levels through densitometry were significantly different between good and poor freezability ejaculates for Cu/ZnSOD at 240 min post-thaw (P <or= 0.01) and for HSP90AA1 at 17 degrees C and 5 degrees C (P <or= 0.05). This last finding claims the introduction of tests based on molecular markers in spermatozoa to accurately predict the freezability of ejaculates in order to promote the use of frozen semen on artificial insemination programmes.

doi: 10.1016/j.theriogenology.2010.04.021

Development of extender based on soybean lecithin for its application in liquid ram semen2016-12-30T09:48:29+01:00

Development of extender based on soybean lecithin for its application in liquid ram semen

de Paz P, Esteso MC, Alvarez M, Mata M, Chamorro CA, Anel L

Cell Biology, University of León, 24071, León, Spain

The soybean lecithin is used as a phospholipids source for the commercial extenders available for freezing bull semen which allows replacing the traditional membrane protective of animal origin (egg yolk). These extenders have been tested for freezing semen in various livestock species but specific adjustments cannot be made due to trade protection. The aim of the present study was to develop a soybean-based extender analyzing the optimal conditions of preparation, handling, and storage in order to optimize its use in liquid ram semen. Its effect on the quality of liquid ram semen was also studied. Different TES-Tris-Fructose-based extenders were prepared using two soybean types (S20 and S95) differentiated by their lipid composition (complex or simple, respectively). These extenders were made up in two temperatures: 20 degrees C (PT20) or 37 degrees C (PT37); centrifuged and filtered at 20 degrees C and stored at 15 degrees C or 5 degrees C (ST15 and ST05) for several periods (from 6 hours to 7 days). Three different concentrations of soybean (0.5%, 2%, and 3.5%) were evaluated for each extender. The amount and nature of phospholipids present in the extender were evaluated by high performance liquid chromatography (HPLC) method according to the different parameters applied in their preparation. In general, the highest quantity of phospholipids is observed in S20 extender. Centrifugation-filtration process during the extender preparation reduces by 50% the quantity of phospholipids in medium for different experiments. The quantity of phospholipids was not affected significantly by preparation temperature in S20 extender. Storage temperature affects the phospholipids present in the extender (S20 and S95) with minimum values for the storage at 5 degrees C. As for the storage time, both extenders (S20 and S95) showed a stable quantity of phospholipids in the course of the time, for 2 days at 15 degrees C and for 7 days at 5 degrees C. The extender obtained with a higher concentration of soybean (3.5%) showed a higher content of phospholipids under different conditions tested. Finally, sperm motility and viability in new extenders were analyzed. We observed that the sperm quality is not affected by storage temperature for S20 extender. Sperm motility was higher in S20-2% extender and control (UL). Our results suggest that a soybean lecithin extender obtained from S20 soybean at 20 degrees C, centrifuged and filtered, preserve the sperm motility and viability at 15 degrees C and 5 degrees C as an egg-yolk extender.

doi: 10.1016/j.theriogenology.2010.03.022

Motility and fertility of the subtropical freshwater fish streaked prochilod (Prochilodus lineatus) sperm cryopreserved in powdered coconut water2016-12-30T09:48:29+01:00

Motility and fertility of the subtropical freshwater fish streaked prochilod (Prochilodus lineatus) sperm cryopreserved in powdered coconut water

Viveiros AT, Nascimento AF, Orfão LH, Isaú ZA

Department of Animal Sciences, Federal University of Lavras, UFLA, P.O. Box 3037, Lavras, MG, 37200-000, Brazil

Streaked prochilod (Prochilodus lineatus) is a freshwater fish inhabiting many South American rivers. The objective was to determine the effectiveness of coconut water (ACP), combined with methylglycol, as a freezing medium for streaked prochilod sperm. A secondary objective was to compare a computer-assisted sperm analyzer (CASA) system versus subjective microscropic examination as a means of assessing sperm motility. As a control, glucose and methylglycol was used, according to our previous study. Sperm diluted in each medium was loaded into 0.5 mL straws, frozen in liquid nitrogen vapor (in a dry shipper), and stored in liquid nitrogen (-196 degrees C). Half of the samples were evaluated for sperm motility, both subjectively and with CASA; the remainder were evaluated for fertility. There was no difference (P > 0.05) between subjective or CASA assessment of post-thaw sperm motility. Although sperm motility was higher in sperm cryopreserved in ACP (85%) than in glucose (75%), cryopreservation in either extender yielded similar fertilization rates (46-48%) and sperm velocities. There were positive correlations (r = 0.56-0.8) between all sperm velocities and fertilization rate. In conclusion, streaked prochilod sperm cryopreserved in glucose or ACP and methylglycol was fertile, and thus could be used for research or commercial settings. Furthermore, although the CASA system provided objective data regarding sperm motility, in the present study, subjective evaluation of sperm motility was practical and a good indication of sperm quality; it could readily be done by well-trained personnel under field or laboratory conditions.

doi: 10.1016/j.theriogenology.2010.03.018

Morphometric dimensions of the human sperm head depend on the staining method used2015-05-08T11:11:41+02:00

Morphometric dimensions of the human sperm head depend on the staining method used

L. Maree, S.S. du Plessis, R. Menkveld and G. van der Horst

Department of Medical Biosciences, University of the Western Cape, Private Bag X17, Bellville 7530, South Africa. Division of Medical Physiology; Department of Biomedical Sciences, Stellenbosch University, PO Box 19063, Tygerberg 7505, South Africa; Andrology Laboratory, Department of Obstetrics and Gynaecology, Tygerberg Academic Hospital and Stellenbosch University, Tygerberg 7505, South Africa

BACKGROUND: Assessment of sperm morphology (including morphometry) is extensively used to determine one of the qualities of a semen sample and depends on the differential staining of spermatozoa. A staining technique should cause as little change to sperm dimensions and form as possible in order to reliably evaluate the morphometric features of the sperm. Various staining techniques have been employed, but only a few have been recommended by the World Health Organization and are amenable to automated sperm morphometry analysis. Our study was aimed at comparing the effect of three staining techniques [Papanicolaou (PAP), Rapidiff® (RD) and SpermBlue® (SB)] on human sperm head dimensions and to compare these with the head dimensions in fresh semen.
METHODS: Smears made from human semen samples (n = 24) were stained according to the three staining techniques and sperm head morphometry was assessed with the Sperm Class Analyzer. Head dimensions of fresh spermatozoa were measured with a digital calliper on a computer screen. The minimum number of spermatozoa to be analyzed to represent the sperm population and the degree of inter-laboratory variation were determined. Electron micrographs from the same semen samples were used to determine the actual acrosome coverage of the spermatozoa in the semen (n = 7) in order to verify the results of the automatic analyses.
RESULTS: The osmolality of human semen differs from that of the RD and PAP fixatives and stains, but is more similar to the SB fixative and stain. At least 100 spermatozoa should be analyzed to include a representative sample of the sperm population. RD caused sperm heads to swell, PAP caused them to shrink and SB had no significant effect on sperm head dimensions when compared with spermatozoa in fresh semen. Very little inter-laboratory variations were found. The percentage acrosome coverage was significantly different between the three staining techniques, as well as between the RD and PAP stains and the manual measurements obtained using the electron micrographs.
CONCLUSIONS: Different staining techniques change the morphometric dimensions of the human sperm head, probably due to the fact that either the fixatives or stains are not iso-osmotic in relation to human semen. Since these changes in sperm head dimensions are not uniform, care should be taken when selecting a staining technique. Ideally, stained spermatozoa should have dimensions as close to spermatozoa in fresh semen as possible, as was found with the SB staining method, resulting in accurate evaluations of sperm head morphometry.

doi: 10.1093/humrep/deq075

Variation of semen parameters in healthy medical students due to exam stress2015-05-08T11:22:39+02:00

Variation of semen parameters in healthy medical students due to exam stress

Fanuel Lampiao

College of Medicine, Blantyre, Malawi

Aim
This study was aimed at investigating semen parameters that vary most in samples of healthy donors undergoing stressful examination period.
Methods
Samples were left to liquefy in an incubator at 37°C, 5% CO2 for 30 minutes before volume was measured. Concentration and motility parameters were measured by means of computer assisted semen analysis (CASA) using Sperm Class Analyzer® (Microptic S.L, Madrid, Spain).
Results
Sperm concentration was significantly decreased in samples donated close to the exam period as well as samples donated during the exam period when compared to samples donated at the beginning of the semester.
Conclusion
Stress levels of donors might prove to be clinically relevant and important when designing experiment protocols.

Malawi Med J. 2009 Dec; 21(4): 166–167.

SpermBlue®: A new universal stain for human and animal sperm which is also amenable to automated sperm morphology analysis2016-12-30T09:48:29+01:00

SpermBlue®: A new universal stain for human and animal sperm which is also amenable to automated sperm morphology analysis

G van der Horst; L. Maree

Department of Medical Biosciences, University of the Western Cape, Private Bag X17, Bellville, South Africa

Our study was aimed at exploring a simple procedure to stain differentially the acrosome, head, midpiece, and flagellum of human and animal sperm. A further prerequisite was that sperm morphology of the stained samples could be analyzed using automated sperm morphology analysis (ASMA). We developed a new staining process using SpermBlue® fixative and SpermBlue® stain, which are iso-osmotic in relation to semen. The entire fixation and staining processes requires only 25 min. Three main steps are required. First, a routine sperm smear is made by either using semen or sperm in a diluting medium. The smear is allowed to air dry at room temperature. Second, the smear is fixed for 10 min by either placing the slide with the dried smear in a staining tray containing SpermBlue® fixative or by adding 1 ml SpermBlue® fixative to the slide. Third, the fixed smear is stained for 15 min by either immersing the slide in a staining tray containing SpermBlue® stain or adding four drops of SpermBlue® stain to the fixed smear. The stained slide is dipped gently in distilled water followed by air drying and mounting in DPX® or an equivalent medium. The method is simple and suitable for field conditions. Sperm of human, three monkey species, horse, boar, bull, ram, mouse, rat, domestic chicken, fish, and invertebrate species were stained successfully using the SpermBlue® staining process. SpermBlue® stains human and animal sperm different hues or intensities of blue. It is possible to distinguish clearly the acrosome, sperm head, midpiece, principal piece of the tail, and even the short end piece. The Sperm Class Analyzer® ASMA system was used successfully to quantify sperm head and midpiece measurements automatically at either 600 times or 1000 times magnification for most of the species studied.

Biotechnic and Histochemistry, Volume 84, Issue 6 December 2009 , pages 299 – 308

Semen molecular and cellular features: these parameters can reliably predict subsequent ART outcome in a goat model2016-12-30T09:48:29+01:00

Semen molecular and cellular features: these parameters can reliably predict subsequent ART outcome in a goat model

Berlinguer F, Madeddu M, Pasciu V, Succu S, Spezzigu A, Satta V, Mereu P, Leoni GG, Naitana S

Department of Animal Biology, University of Sassari, Via Vienna 2, 07100 Sassari, Italy

Currently, the assessment of sperm function in a raw or processed semen sample is not able to reliably predict sperm ability to withstand freezing and thawing procedures and in vivo fertility and/or assisted reproductive biotechnologies (ART) outcome. The aim of the present study was to investigate which parameters among a battery of analyses could predict subsequent spermatozoa in vitro fertilization ability and hence blastocyst output in a goat model. Ejaculates were obtained by artificial vagina from 3 adult goats (Capra hircus) aged 2 years (A, B and C). In order to assess the predictive value of viability, computer assisted sperm analyzer (CASA) motility parameters and ATP intracellular concentration before and after thawing and of DNA integrity after thawing on subsequent embryo output after an in vitro fertility test, a logistic regression analysis was used. Individual differences in semen parameters were evident for semen viability after thawing and DNA integrity. Results of IVF test showed that spermatozoa collected from A and B lead to higher cleavage rates (0 < 0.01) and blastocysts output (p < 0.05) compared with C. Logistic regression analysis model explained a deviance of 72% (p < 0.0001), directly related with the mean percentage of rapid spermatozoa in fresh semen (p < 0.01), semen viability after thawing (p < 0.01), and with two of the three comet parameters considered, i.e tail DNA percentage and comet length (p < 0.0001). DNA integrity alone had a high predictive value on IVF outcome with frozen/thawed semen (deviance explained: 57%). The model proposed here represents one of the many possible ways to explain differences found in embryo output following IVF with different semen donors and may represent a useful tool to select the most suitable donors for semen cryopreservation.

doi: 10.1186/1477-7827-7-125

Effect of eggyolk on the kinematics and acrosome membrane integrity of cooled-rewarmed canine spermatozoa2016-12-30T09:48:29+01:00

Effect of eggyolk on the kinematics and acrosome membrane integrity of cooled-rewarmed canine spermatozoa

J. Dorado, M. J. Galvez, M. R. Murabito, S. Demyda, L. J. De Luca, M. Moreno, and M. Hidalgo

AAnimal Reproduction Group, University of Cordoba, Spain; BDairy Production Department, University of Buenos Aires, Argentina; CDepartment of Genetics, University of Cordoba, Spain; DMAEC-AECID grant holder

Tris-egg yolk-based diluents provide adequate cryoprotection for the sperm of most species. This study was conducted to compare the ability of Tris-glucose extender containing 2 different concentrations of egg yolk to maintain sperm motility and acrosome integrity of canine spermatozoa during 72 h of preservation. For this purpose, a total of 20 ejaculates from 4 clinically healthy dogs (2 Spanish Greyhound, 1 German Pointer, and 1 Crossbreed) were collected by digital manipulation. The sperm-rich fraction of each ejaculate was divided into 2 aliquots. Then, they were diluted in Tris-based extender and centrifuged at 700g for 8 min. Sperm pellets were resuspended in eitherTris buffer added to 20% (EY20) or 10% centrifuged egg yolk (EY10) and cooled to 5◦C over 72 h. The effects of these extenders on motility and acrosome integrity were assessed objectively using a computer-aided semen analyzer (Sperm Class Analyzer, Microptic SL, Spain) and Spermac® staining, respectively. Each cooled-rewarmed semen sample was evaluated after 24, 48, and 72 h of preservation. Sperm motion parameters shown by computer-assisted semen analysis (CASA) are progressively motile (PMS) and motile spermatozoa (MS), curvilinear velocity (CLV), average path velocity (APV), progressive speed (SLV), and lateral head displacement (LHD). Datawere statistically analysed byANOVA. Dependent variables expressed as percentageswere arsine-transformed before analysis. Differences between mean values were evaluated by the Duncan method. Data were presented as mean±SEM. Differences were considered significant when P <0.05. Analyses were performed using the statistical package SPSS 12.0.A total of 98 172 motile sperm trajectories were analyzed by CASA: 52 259 in EY20 and 45 913 in EY10.After 24, 48, and 72 h of preservation,MSand PMSwere statistically higher (P <0.01) in EY20. No significant differences were found for LHD using either extender over a 72-h period. No significant differences were observed for CLV using either extender during the first 2 days. At Day 3, CLV data were significantly higher (P <0.01) in EY20. Similarly, from Day 2, APV was significantly higher (P <0.001) in EY20. After 24 h of preservation, SLV was statistically higher (P <0.001) in EY10, whereas the opposite tendency was found at Day 3. No significant differences were observed for SLV using either extender after 48 h of preservation. During the first 2 days, acrosome integrity was statistically higher (P <0.001) in EY20. At hour 72, higher acrosome integrity (P <0.001) was observed in EY10. In conclusion, we have observed that the EY20 extender provided higher motility after 72 h of chilled preservation; however, the acrosome membrane integrity was better preserved in EY10.

Reproduction, Fertility and Development

Characterization of ram (Ovis aries) sperm head morphometry using the Sperm-Class Analyzer2016-12-30T09:48:29+01:00

Characterization of ram (Ovis aries) sperm head morphometry using the Sperm-Class Analyzer

Maroto-Morales A, Ramón M, García-Alvarez O, Soler AJ, Esteso MC, Martínez-Pastor F, Pérez-Guzmán MD, Garde JJ

Biology of Reproduction Group, National Wildlife Research Institute (IREC), UCLM-CSIC-JCCM, Albacete, Spain

Sperm morphology has been identified as a characteristic that can be useful in the prediction of fertilizing capacity. The aim of the current study was to characterize ram sperm heads morphometrically as a basis for future studies on the relationship between sperm quality and male fertility. For this purpose, ejaculates from 241 mature rams (Ovis aries) belonging to 36 different dairy herds were used to evaluate sperm head morphometry by means of the Sperm-Class Analyzer. Sperm samples, collected by artificial vagina, were diluted in phosphate buffered saline (PBS) for the analysis. A microscope slide was prepared from single-diluted fresh sperm samples. Slides were air-dried and stained with Hemacolor. A minimum of 115 sperm heads were analyzed from each male. Each sperm head was measured for four primary parameters (area, perimeter, length, width), and four derived parameters of head shape were obtained. Significant differences in sperm head morphometry were found between rams (CV for morphometric parameters ranging from 0.9 to 10.1), and there were marked differences in the sperm morphometric composition of the ejaculates. For all parameters, within-animal CVs were greater than between-animal CVs. Within-animal CVs ranged from 4.2 to 10.6, showing the high degree of sperm polymorphism present in the sheep ejaculate. Significant differences in sperm head morphometry were found between rams belonging to the different herds (i.e., origin). An important part of the variability observed on morphometric parameters was due to the male itself, with an explained variance ranging from 3.6% for regularity to 34.0% for p2a (perimeter(2)/

[4xpixarea]). The explained variance by the herd of origin of the males ranged from 0.6% for regularity to 10.8% for area. Our results suggest that a genetic component might be responsible for the observed sperm head morphometry differences between herds.

doi: 10.1016/j.theriogenology.2009.10.003

Objective assessment of goat sperm head size by computer-assisted sperm morphometry analysis (ASMA)2015-05-08T11:24:13+02:00

Objective assessment of goat sperm head size by computer-assisted sperm morphometry analysis (ASMA)

M. Hidalgo, J. Dorado

Animal Reproduction, Department of Medicine and Animal Surgery, University of Cordoba, Spain

The aim of the present study was to identify different objective categories of sperm head size evaluation, using both morphometric and statistical analyses. For this purpose, semen samples (n = 16) were collected from 4 Florida male goats and assessed using a computer-assisted sperm morphometric analysis (ASMA) to obtain sperm head size measurements. The sperm heads were grouped into categories according to 25th and 75th percentiles (the lower and upper quartile, respectively) of their area values. Thereafter, a discriminant analysis was implemented on all the sperm morphometric parameters assessed, to obtain a classification matrix for sperm head size. Sperm heads by this method were classified into 3 categories depending on their size (small, medium and large), with a globally correct assignment of 95.5%. Moreover, significant differences (p < 0.001) were recorded between individual animals for all the sperm head morphometric parameters assessed. In conclusion, by using both statistical and morphometric analyses, it was possible to recognize the three categories of sperm head size. It is expected that research could be useful in defining the relationship between sperm head size measurements and actual fertility data.

doi:10.1016/j.smallrumres.2009.10.006

Out-of-season sperm cryopreserved in different media of the Amazonian freshwater fish pirapitinga (Piaractus brachypomus)2016-12-30T09:48:29+01:00

Out-of-season sperm cryopreserved in different media of the Amazonian freshwater fish pirapitinga (Piaractus brachypomus)

Nascimento AF, Maria AN, Pessoa NO, Carvalho MA, Viveiros AT

Dept Veterinary Medicine, Federal University of Lavras, UFLA, P.O. box 3037, Lavras, MG, 37200-000, Brazil

The pirapitinga (Piaractus brachypomus) is a freshwater fish that inhabits the Amazon and Orinoco River basins. The use of cryopreserved sperm has been considered to facilitate procedures during the artificial reproduction. The aim of the present study was to develop a freezing protocol for pirapitinga sperm collected outside the spawning season. Sperm samples were diluted in four freezing media prepared by a combination of two extenders (glucose and BTS-Beltsville Thawing Solution) and two cryoprotectant agents (DMSO and methylglycol) loaded into 0.5-mL straws, frozen in a nitrogen-vapor shipping dewar (dry-shipper) and stored in liquid nitrogen at -196 degrees C. Post-thaw sperm motility was evaluated both subjectively using a light microscope and by a computer-assisted sperm analyzer (CASA). Curvilinear, average path and straight-line velocities were also determined. There were no differences (P>0.05) in post-thaw sperm motility between evaluations performed subjectively and using the CASA. Sperm samples cryopreserved in glucose-methylglycol yielded the greatest post-thaw sperm motility (81%) and fastest sperm velocities when compared to the samples frozen in the other three media (P<0.05). Out-of-season sperm cryopreserved in glucose and methylglycol under the conditions described above is of high quality and can therefore be used to facilitate artificial reproduction procedures, as only females will need handling for hormonal induction and gamete collection during the spawning season. Although the CASA system provides precise data on sperm motility, the subjective evaluation is practical and can be conducted by well-trained personnel at commercial fish farms as an acceptable evaluation of sperm quality.

doi: 10.1016/j.anireprosci.2009.07.002

Effects of H2O2 exposure on human sperm motility parameters, reactive oxygen species levels and nitric oxide levels2015-05-08T11:24:41+02:00

Effects of H2O2 exposure on human sperm motility parameters, reactive oxygen species levels and nitric oxide levels

S. S. du Plessis, D. A. McAllister, A. Luu, J. Savia, A. Agarwal& F. Lampiao

Divison of Medical Physiology, University of Stellenbosch, Tygerberg, South Africa;Center for Reproductive Medicine, Glickman Urological and Kidney Institute, Cleveland Clinic, Cleveland, OH, USA

Research has revealed that reactive oxygen species (ROS) negatively affect sperm function, both in vivo and in vitro. Sperm preparation techniques for assisted reproductive technologies (ART) are potential causes for additional ROS production. This study aimed to correlate the concentration of exogenous H(2)O(2) with sperm motility parameters and intracellular ROS and nitric oxide (NO) levels to reiterate the importance of minimising ROS levels in ART. Human spermatozoa from 10 donors were incubated and exposed to different exogenous H(2)O(2) concentrations (0, 2.5, 7.5 and 15 mum). Subsequently, motility was determined using computer-aided semen analysis, while ROS (2,7-dichlorofluorescin diacetate) and NO (diaminofluorescein-2/diacetate) were analysed using fluorescence-activated cell sorting. Results showed that H(2)O(2) did affect the sperm parameters. Exogenous H(2)O(2) was detrimental to motility and resulted in a significant increase in overall ROS and NO levels. A significant increase in static cells was seen as well. It is important to elucidate the mechanisms between intracellular ROS levels with sperm motility parameters. While this experiment demonstrated a need to reduce exogenous ROS levels during ART, it did not illustrate the cause and effect relationship of intracellular ROS and NO levels with sperm motility. Further research needs to be conducted to define a pathological level of ROS.

doi: 10.1111/j.1439-0272.2009.00980.x

Effects of dilution and centrifugation on the survival of spermatozoa and the structure of motile sperm cell subpopulations in refrigerated Catalonian donkey semen2015-05-08T11:24:54+02:00

Effects of dilution and centrifugation on the survival of spermatozoa and the structure of motile sperm cell subpopulations in refrigerated Catalonian donkey semen

J. Miró, E. Taberner, M. Rivera, A. Peña, A. Medrano, T. Rigau, A. Peñalba

Department of Animal Medicine and Surgery, School of Veterinary Medicine, Autonomous University of Barcelona, E-08193 Bellaterra, Spain

The aim of this work was to study the effects of dilution and centrifugation (i.e., two methods of reducing the influence of the seminal plasma) on the survival of spermatozoa and the structure of motile sperm cell subpopulations in refrigerated Catalonian donkey (Equus asinus) semen. Fifty ejaculates from nine Catalonian jackasses were collected. Gel-free semen was diluted 1:1, 1:5 or 1:10 with Kenney extender. Another sample of semen was diluted 1:5, centrifuged, and then resuspended with Kenney extender until a final dilution of 25  106 sperm/ml was achieved (C). After 24 h, 48 h or 72 h of refrigerated storage at 5 8C, aliquots of these semen samples were incubated at 37 8C for 5 min. The percentage of viable sperm was determined by staining with eosin-nigrosin. The motility characteristics of the spermatozoa were examined using the CASA system (Microptic, Barcelona, Spain). At 24 h, more surviving spermatozoa were seen in the more diluted and in the centrifuged semen samples (1:1 48.71%; 1:5 56.58%, 1:10 62.65%; C 72.40%). These differences were maintained at 48 h (1:1 34.31%, 1:5 40.56%, 1:10 48.52%, C 66.30%). After 72 h, only the C samples showed a survival rate of above 25%. The four known donkey motile sperm subpopulations were maintained by refrigeration. However, the percentage of motile sperms in each subpopulation changed with dilution. Only the centrifuged samples, and only at 24 h, showed exactly the same motile sperm subpopulation proportions as recorded for fresh sperm. However, the 1:10 dilutions at 24 and 48 h, and the centrifuged semen at 48 h, showed few variations compared to fresh sperm. These results show that the elimination of seminal plasma increases the survival of spermatozoa and the maintenance of motility patterns. The initial sperm concentration had a significant (P < 0.05) influence on centrifugation efficacy, but did not influence the number of spermatozoa damaged by centrifugation. In contrast, the percentage of live spermatozoa in the fresh semen significantly influenced the number of spermatozoa damaged by centrifugation, but not centrifugation efficacy.

doi:10.1016/j.theriogenology.2009.06.012

Functional polymorphism in H2BFWT-5’UTR is associated with susceptibility to male infertility2015-05-08T11:25:19+02:00

Functional polymorphism in H2BFWT-5’UTR is associated with susceptibility to male infertility

Lee J, Park HS, Kim HH, Yun YJ, Lee DR, Lee S

Department of Pharmacology, CHA Stem Cell Institute, School of Medicine, CHA University, Bundang-Gu, Seongnam-Si, Kyunggi-Do, Korea

H2B histone family, member W, testis-specific (H2BFWT) gene encodes a testis-specific histone that becomes incorporated into sperm chromatin. A male infertility-associated single nucleotide polymorphism (-9C > T) within the 5′ untranslated region (5’UTR) of the H2BFWT gene was identified by direct sequencing. Statistical association studies showed the polymorphism significantly associated with male infertility (n = 442, P = 0.0157), especially in non-azoospermia (n = 262, P = 0.018). Furthermore, this polymorphism is also associated with sperm parameters, especially sperm count (n = 164, P = 0.0127) and vitality (n = 164, P = 0.0076). We investigated how the genetic variant at 5’UTR confers susceptibility to non-azoospermia. Western blotting of His-tag H2BFWT revealed a difference at the translational level between -9T and the wild-type -9C in the absence of change at the transcriptional level. Reporter assays showed that this reducing translational change originated from an upstream open reading frame (uORF) generated by the -9C to -9T change. Finally, in vivo H2BFWT expression in sperm was significantly dependent on the -9C > T genotype from non-azoospermia (P = 0.0061). Therefore, this polymorphism could affect the translational efficiency of a quantitatively important histone protein by the uORF. Our data implicate H2BFWT as a susceptibility factor for male infertility, possibly with other genetic and environmental factors.

doi: 10.1111/j.1582-4934.2009.00830.x

Effects of cryopreservation on the motile sperm subpopulations in semen from Asturiana de los Valles bulls2016-12-30T09:48:29+01:00

Effects of cryopreservation on the motile sperm subpopulations in semen from Asturiana de los Valles bulls

Muiño R, Peña AI, Rodríguez A, Tamargo C, Hidalgo CO

Unidad de Reproducción y Obstetricia, Departamento de Patología Animal, Facultad de Veterinaria, Universidad de Santiago de Compostela (USC), Lugo, Spain

The aim of this study was to identify different motile sperm subpopulations in ejaculates from an autochthonous bull breed (Bos taurus) and to determine possible modifications in these subpopulations resulting from cryopreservation. Ejaculates were collected and cryopreserved following a conventional protocol. The overall sperm motility and the kinematic parameters of individual spermatozoa were evaluated in fresh ejaculates, after 4h at 5 degrees C, and at 0 and 2h postthaw. A multivariate clustering procedure separated 23,585 motile spermatozoa into four subpopulations: Subpopulation 1 showed medium velocity (VCL: 99.4+/-17.8 microm/sec) and high progressiveness (LIN: 65.1+/-14.0%); Subpopulation 2 included spermatozoa with high velocity (VCL: 148.7+/-25.6 microm/sec) but a nonprogressive trajectory (LIN: 33.1+/-10.5%); Subpopulation 3 represented slowly motile (VCL: 58.3+/-24.3 microm/sec) and nonprogressive sperm (LIN: 39.6+/-18.3%); and Subpopulation 4 included very rapid (VCL: 152.8+/-25.7 microm/sec) and highly progressive sperm (LIN: 70.9+/-13.7%). Subpopulation 4 was present in the greatest quantity in fresh ejaculates (36%), but after cooling, it significantly decreased (21%) concomitantly with an increase (P<0.001) in Subpopulation 2 (from 21% in fresh to 34% in postcooled semen). After freezing and thawing, the overall sperm motility was reduced, mainly due to Subpopulation 2 decreasing from 34% after cooling to 14% after thawing. Differences among bulls in the frequency distribution of spermatozoa within subpopulations were evidenced after thawing by different proportions of spermatozoa in Subpopulations 2 and 4. The current results indicate that a structure of four sperm subpopulations may be a common characteristic of bovine ejaculates and that the cooling phase of cryopreservation seems to be the determinant of postthaw semen quality.

doi: 10.1016/j.theriogenology.2009.06.009

Effects of season and feeding level on reproductive activity and semen quality in Payoya buck goats2015-05-08T11:25:42+02:00

Effects of season and feeding level on reproductive activity and semen quality in Payoya buck goats

Zarazaga LA, Guzmán JL, Domínguez C, Pérez MC, Prieto R

Departamento de Ciencias Agroforestales, Universidad de Huelva, Carretera de Palos de la Frontera s/n, 21819 Palos de la Frontera, Huelva, Spain

The aim of this study was to determine if there is a seasonal pattern of reproductive activity in male Payoya goats and if this seasonality can be modulated by a higher level of nutrition. For a period of 16 months, 10 adult bucks were divided into two experimental groups that differed in their feeding level. The high nutrition group (H, n=5) received 1.6 times their maintenance food requirements. The control nutrition group (C, n=5) received a diet that supported 1.1 times their maintenance requirements. Body weight and testosterone concentrations were determined weekly, and testicular weight was determined every 2 weeks. Sexual behaviour and semen characteristics were determined monthly. Feeding level did not affect the onset or the end of the reproductive activity as measured by testosterone concentrations, with high testosterone concentrations between July and November. Ejaculation latency was positively influenced by feeding level: 43.2+/-2.2s vs. 61.6+/-3.2s for H and C group, respectively (P<0.001). The percentage of males that ejaculated or that were sexually active was higher in the H group (P<0.01). No differences between feeding levels were observed in the different semen characteristics studied. However, major differences between months were observed for all studied variables. These results lead us to conclude that Payoya bucks exhibit large seasonal variation in their reproductive activity. Higher feeding level allowed a better sexual behaviour in bucks in late spring, when male effect is used on the local livestock to breed females.

doi: 10.1016/j.theriogenology.2009.01.007

Clinical implementation of the Halosperm® test kit in combination with SCA®2015-05-08T11:26:21+02:00

Clinical implementation of the Halosperm® test kit in combination with SCA®

Kellie Williams, PhD and J. Kevin Thibodeaux, PhD, HCLD

Tulsa Fertility Center, 115 East 15th St., Tulsa OK 74119

Fertility Magazine, Volume 12, pages 66-68

Evaluation of automated system Sperm Class Analyzer (SCA) for semen analysis2016-12-30T09:48:29+01:00

Evaluation of automated system Sperm Class Analyzer (SCA) for semen analysis

Carlos Aulesa, M. Cabrera, R. Alonso, M. Benítez y M. Martínez

Unidad de Seminología, Laboratorios Clínicos, Ciutat Sanitària Vall d’Hebron, Barcelona, España

Objective
Evaluation of the efficiency of the New Sperm Class Analyzer® (SCA v.3.2.0) in sperm automated analysis for the calculation of the motility, concentration and morphology parameters using the latest CASA (Computer-Assisted Sperm Analysis) technology in software for spermiogram analysis.
Materials and methods
Analysis of 150 semen samples from the clinical areas of Fertility and Urology, 42 samples were analyzed with the SCA concentration module and 65 samples were analyzed with the SCA motility module using a disposable 10 micron deep Leja Chamber in both. At the same time the samples were analyzed by the manual method using the Neubauer Improved chamber for counting and slides with 22×22mm cover slip heated to 37°C with an average of 200 spermatozoa for motility, in order to test the precision, linearity and accuracy of SCA system compared with the manual method. The morphology analysis was evaluated using 50 pre-stained slides and evaluating 200 cells both manually by an ESHRE qualified technician and the SCA method and then calculating the correlation coefficient.
Results
The counting with SCA system compared with the manual method has shown a within-day imprecision of 10.13% with a range between 3.2% and 17.1% depending on the count level; correlation with the manual method was r=0.98 (P=0.001). The linearity of the SCA was shown to be linear between 0.5 million and 190 million sperm/ml. The motility module showed a higher within-day imprecision in counting WHO ¿type b¿ and ¿type c¿ spermatozoa (21.81%) than the ¿type a¿ and ¿type d¿, with an average value of 10.32%, also depending on the count rate. The reliability of the motility parameter was evaluated up to a of 120 million sperm/ml. The morphology module, with a customised configuration of parameters, had a significant positive correlation (r=0.899; P=0.0001) compared with the manual method.
Conclusions
The comparison of the concentration and motility of spermatozoa between the manual method and the SCA modules using 10 microns deep Leja chambers was positively evaluated. A series of premises are established for using the automated morphology performed on the Sperm Class Analyzer. The clinical usefulness of some of the new kinetic and morphometric parameters of sperm provided by the semen analyser is also established.

Laboratorio Clinico. 2009;02:8-16.

Study of three consecutive electroejaculations in brown bear (Ursus Arctos)2016-12-30T09:48:30+01:00

Study of three consecutive electroejaculations in brown bear (Ursus Arctos)

L. Anel, S. Borragan, M. Alvarez, F. Martinez-Pastor, M. Mata-Campuzano, S. Gomes-Alves, E. Anel, P. de Paz

University of León, León, Spain; Cabárceno Nature Park, Cantabria, Spain; IREC (CSIC-UCLM-JCCM), Albacete, Castilla-La Mancha, Spain

The preservation of threatened species, such as the cantabric brown bear, requires the establishment of genetic resource banks. In these species it is important to increase the efficiency of the electroejaculation techniques so as to collect as many gametes as possible from each collection, and to decrease risks of anesthesia and immobilization. Our objective was to study several characteristics of brown bear semen quality obtained in three consecutive electroejaculations (a, b, and c) in the same anesthetic session. Ejaculates were collected from 11 adult males living in the Cabárceno Nature Park during the breeding season (May–July). Animals were anesthetized by administration of tiletamine + zolazepan (Zoletil 100®) and ketamine (Imalgene 1000®) before being subjected to electroejaculation (6 to 10 V; 250 to 300 mA). From each ejaculate we assessed motility (CASA: SCA, Microptic, Barcelona, Spain), osmolality (mOsm k–1), and viability (VIAB: % viable spermatozoa (spz), SYBR-14 and propidium iodide) and mitochondrial membrane potential (MIT: % spz, JC-1) by flow cytometry. Our results show that total spz (×106 spz) varies widely among individuals depending on the number of electroejaculation. In five males we observe a decreasing pattern (a: 454.50; b: 341.7; c: 138.0); in other two males it is observed an increasing pattern (a: 24.9; b: 70.3; c: 334.3) while in the remaining four males we see a varied pattern, with the sperm production peak in the second electroejaculation (a: 53.4; b: 270.6; c: 107.5). Motility parameters do not show differences among the three electroejaculations, showing a reduction of the progressive motility in the males with increasing pattern with respect to the other patterns. Also, spermatozoa physiology indicators show a relation with the sperm production patterns. For viability (%) it is shown a rising tendency in the increasing pattern (a: 64.0, b: 80.0, c: 79.5) and a reduction tendency in the decreasing pattern (a: 68.7, b: 61.0, c: 58.7). The same is observed in the case of mitochondrial membrane potential (%) (increasing pattern

[a: 77.0, b: 89.0, c: 87.0]; decreasing pattern [a: 80.0, b: 76.3, c: 55.7]). Ejaculates of the varied pattern show irregular data for these parameters. On the other hand, osmolality changes depending on the number of electroejaculation (increasing pattern [a: 324.0, b: 289.0, c: 298.3]; decreasing pattern [a: 333.0, b: 297.0, c: 283.0]; varied pattern [a: 264.0, b: 294.6, c: 318.54]) which would determine a change in the spz microenvironment that regulates their physiological activity. Although the high individual variability observed does not lead to solid conclusions, our results indicate that consecutive electroejaculations can be useful for increasing the technique yield in brown bear.

Reproduction, Fertility and Development 21(1) 175–175

Effects of cryopreservation on head morphometry and its relation with chromatin status in brown bear (Ursus arctos) spermatozoa2016-12-30T09:48:30+01:00

Effects of cryopreservation on head morphometry and its relation with chromatin status in brown bear (Ursus arctos) spermatozoa

Alvarez M, García-Macías V, Martínez-Pastor F, Martínez F, Borragán S, Mata M, Garde J, Anel L, De Paz P

Animal Reproduction and Obstetrics, University of León, 24071 León, Spain

The Cantabrian brown bear (Ursus arctos) is a highly endangered species in Spain and basic studies are necessary in order to bank its germplasm. Sperm heads are mainly made up of chromatin, thus their shape depends partly on chromatin structure. Thawed semen from 10 bears was used to analyze chromatin status by sperm chromatin structure assay (SCSA) and head morphometry by the computer-assisted sperm morphology assessment (CASMA) system. Morphometry was analyzed before and after freezing-thawing in order to evaluate the effects of cryopreservation on sperm heads. Each spermatozoon was measured for four primary parameters (length, L; width, W; area, A; perimeter, P) and derived parameters (ellipticity: L/W, circularity: 4piA/ P2, elongation: (L-W)/(L+W), regularity: piLW/ 4A). All the derived parameters significantly differed between bears. Likewise, cryopreservation affected head morphometry by reducing its size. Clustering based on morphometric parameters separated three subpopulations, one of them being significantly more influenced by the cryopreservation process. We obtained high correlations between head morphometry and SCSA parameters: standard deviation of DNA fragmentation index (SD-DFI) was correlated with perimeter and area (r=0.75 and r=0.62, respectively) and DFIm and DFIt (moderate and total DNA fragmentation index) were correlated with perimeter (r=0.65 and r=0.67, respectively). Nevertheless, classification of males according to SCSA or head morphometry did not completely agree so the two assays might explain male variability differently. We conclude that cryopreservation affected morphometry at least in a subset of spermatozoa. These results might improve future application of sperm banking techniques in this species.

doi: 10.1016/j.theriogenology.2008.06.097

The in vitro effect of benzo[a]pyrene on human sperm hyperactivation and acrosome reaction2015-05-08T11:27:28+02:00

The in vitro effect of benzo[a]pyrene on human sperm hyperactivation and acrosome reaction

Dyutiman Mukhopadhyay, M.S., Parag Nandi, M.S., Alex C. Varghese, Ph.D., Rohit Gutgutia, M.D., Samir Banerjee, Ph.D., Asok K. Bhattacharyya, Ph.D., D.Sc.

Department of Zoology, University of Calcutta; Institute of Reproductive Health and Toxicology; Department of Environmental Science, University of Calcutta; IVF Division, Advanced Medicare and Research Institue Ltd.; Department of Biochemistry, University of Calcutta, Calbutta, India

Objective
To evaluate the in vitro effect of benzo[a]pyrene on sperm hyperactivation and acrosome status in normozoospermic semen samples of nonsmokers analyzed by computer-assisted semen analysis (CASA).
Design
Experimental in vitro study.
Setting
Andrology laboratory.
Patient(s)
Thirteen proven fertile, normozoospermic, and nonsmoking men.
Intervention(s)
Spermatozoa were washed free of seminal plasma and were treated with different concentrations of benzo[a]pyrene and compared with controls treated with medium alone. The benzo[a]pyrene concentrations were: 100, 50, 25, and 12.5 µg/mL.
Main Outcome Measure(s)
Effect of varying concentrations of benzo[a]pyrene on sperm hyperactivation and acrosomal reaction.
Result(s)
A statistically significant increase in sperm hyperactivation was observed in presence of benzo[a]pyrene at concentrations of =50 µg/mL. The result of the acrosome halo test showed that concentrations of benzo[a]pyrene =25 µg/mL statistically significantly decreased the percentage of halo formation, indicating an inappropriate (false) acrosome reaction.
Conclusion(s)
Benzo[a]pyrene statistically significantly affected sperm functional competence as evidenced by increased hyperactivation as well as premature acrosomal reaction.

Presented at the 64th annual meeting of American Society of Reproductive Medicine (ASRM), November 8-12, 2008, San Francisco, California.
(Gutgutia R, et al. In vitro analysis of the effect of benzo[a]pyrene on sperm hyperactivation [abstract]. Fertil Steril 2008;90:S185.)

Testis size, sperm characteristics and testosterone concentrations in four species of shrews (Mammalia, Soricidae)2016-12-30T09:48:30+01:00

Testis size, sperm characteristics and testosterone concentrations in four species of shrews (Mammalia, Soricidae)

Parapanov RN, Nusslé S, Crausaz M, Senn A, Hausser J, Vogel P

Department of Ecology and Evolution, University of Lausanne, CH-1015 Lausanne, Switzerland

The aim of this study was to establish and compare the sperm characteristics in four shrew species in the context of the sperm competition hypothesis. As expected, the large relative testis size in promiscuous species was associated with a high number of cauda epididymal spermatozoa and a high concentration of circulating testosterone. In addition, in Sorex and Neomys, species with high intensity of sperm competition, the spermatozoa stored in cauda epididymis were characterized by high percentage of progressive motility whereas in Crocidura and Suncus, the cauda epididymal spermatozoa were motile but with very low percentage of progressive motility. This capability is achieved only following the passage through the vas gland, a specialized region for sperm storage located along the vas deferens in these shrew species. The hypothesis that sperm competition is positively correlated with spermatozoa length could not be confirmed. In Crocidura and Suncus, the total sperm length is increased by the large sperm head due to a big acrosome. This trait, specific to the subfamily Crocidurinae, may results from a selective pressure independent of the context of sperm competition, related to a specific, but as yet unclear role, for the acrosome during the fertilization.

doi: 10.1016/j.anireprosci.2008.09.013

The effect of low-level laser irradiation on dog spermatozoa motility is dependent on laser output power2015-05-08T11:28:03+02:00

The effect of low-level laser irradiation on dog spermatozoa motility is dependent on laser output power

Corral-Baqués MI, Rivera MM, Rigau T, Rodríguez-Gil JE, Rigau J

Post-Degree Laser Medical Study, Rovira i Virgili University, Reus, Spain; Animal Reproduction Unit, Faculty of Veterinary Medicine, Autonomous University of Barcelona, Bellaterra, Spain

Biological tissues respond to low-level laser irradiation and so do dog spermatozoa. Among the main parameters to be considered when a biological tissue is irradiated is the output power. We have studied the effects on sperm motility of 655 nm continuous wave diode laser irradiation at different output powers with 3.34 J (5.97 J/cm(2)). The second fraction of fresh dog sperm was divided into five groups: control, and four to be irradiated with an average output power of 6.8 mW, 15.4 mW, 33.1 mW and 49.7 mW, respectively. At 0 min and 45 min after irradiation, pictures were taken and a computer aided sperm analysis (CASA) performed to analyse different motility parameters. The results showed that different output powers affected dog semen motility parameters differently. The highest output power showed the most intense effects. Significant changes in the structure of the motile sperm subpopulation were linked to the different output powers used.

doi: 10.1007/s10103-008-0606-7

Male gamete survival at stake: causes and solutions2015-05-08T11:28:17+02:00

Male gamete survival at stake: causes and solutions

Alex C Varghese, Stefan S du Plessis, Ashok Agarwal

Reproductive Research Centre, Glickman Urological and Kidney Institute, Cleveland Clinic, Cleveland, Ohio, USA; Division of Medical Physiology, University of Stellenbosch, Tygerberg, South Africa; Correspondence: Andrology Laboratory and the Reproductive Research Centre, Glickman Urological and Kidney Institute and Department of Obstetrics-Gynecology, Cleveland Clinic, 9500 Euclid Avenue, Desk A19.1, Cleveland, Ohio 44195, USA

Over the years, the development of assisted reproductive technology to bypass male factor infertility has improved drastically. Considered one of the most perplexing disorders in the reproductive field, male factor infertility is prevalent and may be on the rise. Unfortunately, its aetiology remains elusive. One of the main reasons lies in the complex machinery and structure of the hydrodynamic sperm cell. Its polyunsaturated fatty acid cell membrane, the protamines in its genetic material and the absence of antioxidants in its cytoplasm ensure that the spermatozoon is highly susceptible to environmental effects. The spermatozoon’s genesis, storage, and transport through the male reproductive tract are also susceptible, genetically and pathologically, to environmental effects. This review aims to include all the possible causes of disruption to this unique cell and their probable solutions, in the hope of clearing up the ambiguity that surrounds male factor infertility.

doi: 10.1016/S1472-6483(10)60416-6

First Evaluation of Human Sperm Quality in Various Geographic Regions of Switzerland2017-06-13T13:02:40+02:00

First Evaluation of Human Sperm Quality in Various Geographic Regions of Switzerland

Michel Crausaz, Josefina Vargas, Roumen Parapanov, Yves Chollet, Marc Wisard, Eric Stettler, Alfred Senn, and Marc Germond

Service de chirurgie thoracique et vasculaire, University Hospital of Lausanne; Fondation FABER, Lausanne, Switzerland; Centre for Medically Assisted Procreation (CPMA)

Abstract: A decline in human sperm quality and quantity has been reported in numerous Western countries. This observation was also accompanied by an increase in urogenital malformations. The need for epidemiological studies dealing with unbiased populations in order to understand the causes of these observations is obvious. In Switzerland, the large majority of young men are asked to attend a military camp to be drafted into the army. A few weeks before this camp, conscripts were contacted and invited to participate in a large national study on semen quality. The participation was totally voluntary and anonymous. From September 2005 to June 2007, 770 volunteers filled out a questionnaire, underwent a clinical examination and provided sperm, blood and urine samples. Using self-rated health assessments, the observed cohort could be considered as healthy and no testicular cancer was found. Moreover, the testicular volumes, measured using Prader’s orchidometry and ultrasonography, were comparable to those already published for young male populations. The median sperm concentration was 47 × 106/ml, which is close to the concentration reported in Denmark, known to have the highest incidence of testicular cancer in Europe. Statistically significant differences were observed between regions with a lower sperm concentration for men residing in the Alps (43 × 106/ml) and in the Zürich area (36 × 106/ml) compared to men from West Plateau (54 × 106/ml) and from the Jura (54 × 106/ml). Such a regional discrepancy could be related to environmental factors, including endocrine disruptors. In order to confirm such regional differences more volunteers from the already studied regions should be studied and other parts of the country should be investigated. The rather low sperm concentration of Swiss young volunteers should be considered as a national health issue and investigated further.

doi:10.2533/chimia.2008.395

Semen evaluation of two selected lines of rabbit bucks2015-05-08T11:30:12+02:00

Semen evaluation of two selected lines of rabbit bucks

Safaa H.M., Vicente J.S., Lavara R., Viudes de Castro M.P.

Animal Production Department, Faculty of Agriculture, Cairo University, 12613 GiZa. Egypt; Departamento de Ciencia Animal, Universidad Politécnica de Valencia. Camino de Vera s/n. 46022 valencia. Spain; Centro de Investigación y Tecnología Animal. Apdo 187. Polígono La Esperanza, nº 100. 12400 SeGoRBe. Spain

Twenty rabbit bucks of 9 months of age were used to evaluate semen quality of two lines of New Zealand rabbit bucks selected for litter size at weaning (A line) and growth rate from weaning to slaughter (R line). The morphological semen characteristics indicated that the A line spermatozoa had greater acrosome integrity (+3.6 percentage units; P<0.01) and smaller sperm head size (for example, ¿1.46 ¿m2 for sperm head area) than in the R line. Seminal functional traits were also significantly higher for the A line (+13.4 percentage units for viability, +10.6 percentage units for hypo-osmotic swelling test (HOST) and +3.3 g/L for seminal plasma protein. However, no differences were detected between lines for motility parameters and seminal plasma protein electrophoretic profiles. Both lines had the same twelve bands with the following molecular weights to the nearest 1 kD: 124, 117, 99, 86, 75, 62, 40, 32, 21, 19, 10 and 6 kD. A relationship (r=0.308 for A line and 0.359 for R line; P<0.01) was found between the integrity of the plasmatic membrane (viability rate) and tail membrane (HOST) of the spermatozoa in the A line, but not in the R line, which had greater sperm head size. There was also a significant positive correlation coefficient between sperm concentration and either viability or some kinetic traits (r=0.567 and 0.575 for VCL, r=0.584 and 0.561 for VSL and r=0.588 and 0.588 for VAP, for A and R lines, respectively; P<0.001). We concluded that the A line seems to have better semen characteristics than the R line. We also found an interesting correlation among the seminal morphological, functional and kinetic traits, which could possibly be used to facilitate semen evaluation.

doi: 10.4995/wrs.2008.622

Human sperm in the third dimension2016-12-30T09:48:30+01:00

Human sperm in the third dimension

Claire Garrett, Joselito Chua, KG Tan, Eduard Sanchez and HW Gordon Baker

University of Melbourne Department of Obstetrics and Gynaecology, Royal Women’s Hospital, Parkville, Australia, University of Melbourne Department of Computer Science and Software Engineering, Parkville, Australia, Microptics SL, Barcelona, Spain

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Seminal diagnostic2015-05-08T11:31:15+02:00

Seminal diagnostic

A. Sebastianelli, L. Caponecchia

Unit of Andrology and Physiopathology of Reproduction, Centre for Sterility in Couples and Cryopreservation of Gametes, «S. Maria Goretti» Hospital, ASL of Latina, Italy

Journal of Andrological Sciences 2008;15(suppl 1):18-23

Morphological and functional parameters of endangered Bermeya goat breed semen2016-12-30T09:48:30+01:00

Morphological and functional parameters of endangered Bermeya goat breed semen

C. O. Hidalgo, A. Rodríguez, C. Díez, D. Martín, M. Carbajo, A. Martínez, J. de la Fuente, A. T. Palasz, J. M. Benito and C. Tamargo

Servicio Regional de Investigación y Desarrollo Agroalimentatio, Villaviciosa, Asturias, Spain

The Bermeya goats are an endangered autochthonous breed distributed in the north of Spain. To ensure their genetic diversity and long-term survival, morphological and functional parameters of the semen must be known in order to preserve the current genetic stock in a germplasm bank. The aim of this work was to establish basic characteristics and post-thaw survival of Bermeya goat’s semen obtained by electro-ejaculation, that is not well described in the literature. The semen was collected by electro-ejaculation from 7 bucks, 1 to 7 years old, twice per week, for 9 weeks (n = 83). Fresh semen was evaluated for volume (V), concentration (C), motility, morphology, functional integrity of the sperm (spz) membranes (hypoosmotic swelling test; HOST), and acrosome integrity rate (NAR). Individual and progressive sperm motility were analyzed by means of a computer-assisted sperm analysis system (CASA: SCA 2002®, Microptic, Barcelona, Spain) immediately after dilution with the extender at 37°C, and after cooling to 4°C; five fields per sample (diluted to 204 × 106 spz mL–1) were evaluated under a phase contrast microscope (100×). The NAR and morphological abnormalities of sperm head, midpiece, tail, and cytoplasmic droplets were determined by counting 100 spz under 1000×. For freezing, ejaculates with at least 80% motile spz were diluted at 32°C with Krebs-Ringer solution containing 20% egg yolk and 14% glycerol to a final concentration of 400 × 106 spz mL–1, cooled to 4°C for 90 min, aspirated into 0.25-mL plastic straws (IMV®, L’Aigle, France), frozen at 7 cm above liquid nitrogen (LN2) phase for 10 min, and then plunged into the LN2. Straws were thawed in a water bath at 39°C for 30 s for post-thaw survival analysis. Data were analyzed by the GLM and FREQ procedures (SAS; SAS Institute, Inc., Cary, NC, USA) and expressed as means ± standard error. Fresh semen characteristics were: V = 1.7 ± 0.1 mL; C = 2619 × 106 ± 153 spz mL–1; total and progressive motility were 89.0 ± 2.1% and 66.9 ± 2.1%, respectively. Percentages of head abnormalities were 4.8 ± 0.5; midpiece: 3.8 ± 0.7; tail: 4.7 ± 1.0; cytoplasmic droplets: 8.3 ± 0.7; intact acrosome: 91.8 ± 0.6; and membrane integrity: 49.2 ± 2.1. At 4°C, the % of total motile spz was 62.6 ± 1.6, and the post-thaw survival rate was 46.3 ± 1.5. There were only individual differences (P < 0.001) between bucks on sperm concentration, head abnormalities, and cytoplasmic droplets. In conclusion, our results indicate that semen quality is related to each individual animal and that electro-ejaculation allows collection of semen of satisfactory quality to use as fresh and for cryopreservation. However, the validity of our results for possible future sperm banking of endangered Bermeya goats semen must be confirmed by field trials.

doi: 10.1071/RDv20n1Ab14

Effect of different thawing rates on post-thaw sperm viability, kinematic parameters and motile sperm subpopulations structure of bull semen2016-12-30T09:48:30+01:00

Effect of different thawing rates on post-thaw sperm viability, kinematic parameters and motile sperm subpopulations structure of bull semen

Muiño R, Rivera MM, Rigau T, Rodriguez-Gil JE, Peña AI

Department of Animal Pathology, Faculty of Veterinary Medicine of Lugo, Unit of Reproduction and Obstetrics, University of Santiago de Compostela, 27002 Lugo, Spain

The aim of the present study was to evaluate three thawing rates for bull semen frozen in 0.25-ml straws: placing the straws in a water bath at 37 degrees C for 40s, at 50 degrees C for 15s or at 70 degrees C for 5s. In a first experiment, the three thawing rates were compared in relation to post-thaw sperm motility, determined subjectively, and sperm plasma and acrosomal membrane integrity, examined by flow cytometry, after 0 and 5h of incubation at 37 degrees C. In a second experiment, the three thawing rates were evaluated based on post-thaw sperm motility, determined using a CASA system, after 0 and 2h of incubation at 37 degrees C. In addition, for the motile spermatozoa, the individual motility descriptors were analysed using a multivariate clustering procedure to test the presence of separate sperm subpopulations with specific motility characteristics in the thawed bull semen samples. Finally, it was investigated if the thawing rate had any influence on the relative frequency distribution of spermatozoa within the different subpopulations. In terms of overall post-thaw motility or plasma and acrosomal sperm membrane integrity there were no significant differences between the three thawing methods evaluated. The statistical analysis clustered all the motile spermatozoa into four separate subpopulations with defined patterns of movement: (1) moderately slow and progressive sperm (27%); (2) «hyperactivated-like» sperm (15.4%); (3) poorly motile non-progressive sperm (34.3%); (4) fast and progressive sperm (23.3%). The thawing rate had no significant influence on the frequency distribution of spermatozoa within the four subpopulations, but there was a significant effect (P<0.05) of the interaction between thawing rate and incubation time. Higher proportions of spermatozoa with fast and progressive movement were observed after 2h of post-thaw incubation when the thawing was at the faster rates (35 degrees C/40s: 8.3%, 50 degrees C/15s: 18.1% and 70 degrees C/5s: 16.5%). Whether this subtle difference might affect to the in vivo fertility of the thawed bovine semen is not known.

doi: 10.1016/j.anireprosci.2007.11.028

Current and future perspectives on intracytoplasmic sperm injection: a critical commentary2016-12-30T09:48:30+01:00

Current and future perspectives on intracytoplasmic sperm injection: a critical commentary

Alex C Varghese, Eric Goldberg, Ashok Agarwal

Molecular Research and Investigation Centre, Kolkata, India; DeCode Life Foundation, Kolkata, India; Department of Biochemistry, University of Calcutta, Kolkata, India; Reproductive Research Center, Glickman Urological and Kidney Institute and Department of Obstetrics–Gynecology, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, Ohio 44195, USA

Intracytoplasmic sperm injection (ICSI) is an increasingly popular means of treating infertility in couples who wish to conceive. However, there are many potential complications that can be faced by the clinician while performing ICSI. These complications and other related issues are discussed, with an emphasis on understanding how these issues are being resolved, or how they can be resolved in the future. Matters of sperm selection and injection are discussed, as well as the effect of ICSI on fertilization, embryonic growth and development, and the health of ICSI-conceived children. These aspects are viewed from various perspectives, including genetic, mechanistic, developmental and clinical. Since new studies on ICSI are published regularly, it is important that the established protocol is revised often, and that the role of ICSI in infertility therapy is continually re-evaluated.

doi: 10.1016/S1472-6483(10)60540-8