Articles

Articles 2017-01-04T09:00:18+00:00

SCA® CASA system is a useful tool for research, and it has been widely used in many species studies. Find some of the scientific papers and articles published by our SCA® users:

Marriage: WHO5 analysis and sperm function 2017-07-20T16:35:59+00:00

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.

Erratum: Please note that in Table 2 Normal morphology and Vitality must be switched around. Normal morphology 3 – 4 and Vitality 55 – 63

Full article here

Improved sperm kinematics in semen samples collected after 2 h versus 4-7 days of ejaculation abstinence 2017-05-24T17:39:14+00:00

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 penguins 2017-05-24T16:17:51+00:00

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 stress 2017-05-10T16:45:07+00:00

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 Predictive 2016-12-14T15:29:35+00:00

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

Unraveling the sperm bauplan: relations between sperm head morphology and sperm function in rodents 2016-12-30T09:48:19+00:00

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 spermatozoa 2016-12-30T09:48:19+00:00

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 motility 2016-03-30T16:39:04+00:00

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.

Evaluation of sperm motility using two cryoprotectants 2016-12-30T09:48:19+00:00

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 Aided Sperm Analysis, a useful tool to evaluate patient’s response to varicocelectomy 2016-03-30T16:17:08+00:00

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

Computer Assisted Semen Analysis, an overcoming tool 2016-12-30T09:48:19+00:00

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 semen 2016-12-30T09:48:19+00:00

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 fertility 2017-03-23T16:48:30+00:00

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 semen 2016-12-30T09:48:19+00:00

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 serum 2016-12-30T09:48:19+00:00

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 mammals 2016-12-30T09:48:19+00:00

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 Intake 2016-12-30T09:48:19+00:00

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 characteristics 2016-12-30T09:48:19+00:00

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.

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Dietary probiotic supplement positively affects sperm motility in an obese model 2016-12-30T09:48:19+00:00

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 parameters 2016-12-30T09:48:19+00:00

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 kinematics 2016-12-30T09:48:19+00:00

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 species 2016-12-30T09:48:19+00:00

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 mammals 2015-09-21T16:46:21+00:00

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 semen 2016-12-30T09:48:20+00:00

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 analyzer 2016-12-30T09:48:21+00:00

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 competition 2016-12-30T09:48:22+00:00

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 cryoprotectants 2015-07-07T15:54:59+00:00

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 analysis 2016-12-30T09:48:22+00:00

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 techniques 2016-12-30T09:48:22+00:00

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 Probe 2016-12-30T09:48:22+00:00

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 microscopy 2015-07-07T16:00:02+00:00

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 Sperm 2016-09-16T10:06:46+00:00

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+00:00

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 diluents 2016-12-30T09:48:22+00:00

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

Example use of low-cost system for capturing the kinetic parameters of sperm cells in Atlantic Salmon (Salmo salar) 2016-12-30T09:48:22+00:00

Example use of low-cost system for capturing the kinetic parameters of sperm cells in Atlantic Salmon (Salmo salar)

Jorge Parodi, Alfredo Ramírez-Reveco, Guillermo Guerra

Laboratorio de Fisiología de la Reproducción, Núcleo de Producción Alimentaria, Escuela de Medicina, Veterinaria, Facultad de Recursos Naturales, Universidad Católica de Temuco, Temuco, Chile; Laboratorio de Criobiología y Análisis de Funcionalidad Espermática, Instituto de Ciencia Animal, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Valdivia, Chile

A spermiogramme is an analysis performed to assess the quality of semen. Motility parameters are primarily obtained by subjectively observing samples or by using complex systems, such as computer-assisted sperm analysis (CASA). Here, we describe an easy and low-cost analysis system for obtaining quantifiable kinetic observations using Salmo salar semen model. In this work, we observed and captured video images of both fresh and stored Atlantic salmon semen to describe the possibility of analysis using the ImageJ CASA plug-in application for the kinetic parameters obtained from the videos. The semen is exposed to “powermilt”, a commercial activating solution, and the curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP) values are described. When the samples were activated after having been stored, differences were detected in sperm quality, using the low-cost plug-in application. However, this system was not able to detect small variations in the same recorded sample, suggesting limits in sample observation. The results indicate that it is possible to quantify the kinetic parameters of semen samples using a low-cost video system and free software.

doi: 10.4236/abb.2015.62007

Insemination of bocachico fish (Prochilodus magdalenae) with fresh or cryopreserved semen: effect of spermatozoa/oocyte ratio 2016-12-30T09:48:22+00:00

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 spermatozoa 2016-12-30T09:48:22+00:00

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 cryopreservation 2016-12-30T09:48:22+00:00

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 cryopreservation 2016-12-30T09:48:22+00:00

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+00:00

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) semen 2016-08-29T16:31:02+00:00

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 integrity 2016-12-30T09:48:22+00:00

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 spermatozoa 2016-12-30T09:48:22+00:00

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 fertility 2015-05-08T09:24:00+00:00

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 method 2016-12-30T09:48:22+00:00

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 asthenozoospermia 2016-12-30T09:48:22+00:00

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 rats 2016-12-30T09:48:22+00:00

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+00:00

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 ruminants 2016-12-30T09:48:23+00:00

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 cuspicaudus 2016-12-30T09:48:23+00:00

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 sperm 2016-12-30T09:48:23+00:00

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 axis 2016-12-30T09:48:23+00:00

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 cohort 2015-05-08T09:28:36+00:00

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 velocity 2015-05-08T09:29:03+00:00

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 report 2015-05-08T10:16:42+00:00

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+00:00

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 time 2016-12-30T09:48:23+00:00

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 sperm 2015-05-08T10:18:28+00:00

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 vitro 2015-05-08T10:19:06+00:00

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) 2016-12-30T09:48:23+00:00

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 SCA 2016-12-30T09:48:25+00:00

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 assay 2016-12-30T09:48:25+00:00

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 laboratory 2015-05-08T10:20:38+00:00

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 rabbits 2015-05-08T10:20:55+00:00

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 breed 2016-12-30T09:48:25+00:00

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 Aires 2016-12-30T09:48:25+00:00

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 Analyzer 2016-08-30T09:13:52+00:00

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 doses 2016-12-30T09:48:25+00:00

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 test 2015-05-08T10:21:28+00:00

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 functions 2015-05-08T10:22:11+00:00

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) 2017-05-25T13:09:17+00:00

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 treatment 2015-05-08T10:22:28+00:00

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 source 2015-05-08T10:22:49+00:00

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 metabolomics 2016-12-30T09:48:25+00:00

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

Cryopreservation of collared peccary (Pecari tajacu) semen using different freezing curves, straw sizes, and thawing rates 2015-05-08T10:24:24+00:00

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 fertilisation 2015-05-08T10:24:48+00:00

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 ruminants 2016-12-30T09:48:25+00:00

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 process 2015-05-08T10:25:17+00:00

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 mice 2016-12-30T09:48:25+00:00

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 velocity 2015-05-08T10:26:10+00:00

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 Disease 2016-12-30T09:48:25+00:00

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 spermatozoa 2015-05-08T10:43:53+00:00

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 culture 2015-05-29T08:44:41+00:00

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 speed 2015-05-08T10:44:56+00:00

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 method 2016-12-30T09:48:26+00:00

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 competition 2015-05-08T10:46:18+00:00

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 cryopreservation 2016-12-30T09:48:26+00:00

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 ICSI 2015-05-08T10:47:20+00:00

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 electroejaculation 2016-12-30T09:48:26+00:00

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 microdomains 2016-12-30T09:48:26+00:00

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) spermatozoa 2016-12-30T09:48:26+00:00

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) 2015-05-08T10:48:51+00:00

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 spermatozoa 2015-05-08T10:49:17+00:00

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 protocols 2016-12-30T09:48:26+00:00

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

Motile sperm subpopulations in frozen-thawed dog semen: changes after incubation in capacitating conditions and relationship with sperm survival after osmotic stress 2016-12-30T09:48:26+00:00

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 sperm 2015-05-08T10:50:08+00:00

 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 glucose 2015-05-08T10:50:59+00:00

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 spermatozoa 2015-05-08T10:51:28+00:00

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 goats 2015-05-08T10:51:43+00:00

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 efficiency 2015-05-08T10:52:01+00:00

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+00:00

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 deer 2016-12-30T09:48:26+00:00

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) spermatozoa 2016-12-30T09:48:26+00:00

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 semen 2016-12-30T09:48:26+00:00

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 semen 2016-12-30T09:48:26+00:00

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) 2015-05-08T10:54:24+00:00

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 Analyzer 2016-12-30T09:48:26+00:00

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 variability 2015-05-08T10:54:52+00:00

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 epididymes 2016-12-30T09:48:26+00:00

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 semen 2016-12-30T09:48:26+00:00

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-106C 2015-05-08T10:56:22+00:00

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 addition 2015-05-08T10:56:35+00:00

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 functions 2015-05-08T10:57:04+00:00

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 spermatozoa 2016-12-30T09:48:26+00:00

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 semen 2015-05-08T10:57:31+00:00

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 conditions 2016-12-30T09:48:27+00:00

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 procedure 2015-05-08T10:58:16+00:00

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 semen 2015-05-08T10:58:29+00:00

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

Sperm structure and motility in the eusocial naked mole-rat, Heterocephalus glaber: a case of degenerative orthogenesis in the absence of sperm competition? 2015-05-08T10:59:29+00:00

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 ruminants 2015-05-08T10:59:44+00:00

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 technique 2016-12-30T09:48:29+00:00

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 fragmentation 2015-05-08T11:00:38+00:00

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 extenders 2016-12-30T09:48:29+00:00

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 manner 2016-12-30T09:48:29+00:00

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) technique 2015-05-08T11:09:23+00:00

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 switzerland 2015-05-08T11:09:36+00:00

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 report 2016-03-30T16:53:40+00:00

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 assessment 2015-05-08T11:09:48+00:00

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 freezability 2016-12-30T09:48:29+00:00

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 semen 2016-12-30T09:48:29+00:00

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 water 2016-12-30T09:48:29+00:00

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 used 2015-05-08T11:11:40+00:00

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 stress 2015-05-08T11:22:38+00:00

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 analysis 2016-12-30T09:48:29+00:00

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 model 2016-12-30T09:48:29+00:00

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 spermatozoa 2016-12-30T09:48:29+00:00

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 Analyzer 2016-12-30T09:48:29+00:00

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) 2015-05-08T11:24:12+00:00

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+00:00

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 levels 2015-05-08T11:24:40+00:00

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 semen 2015-05-08T11:24:54+00:00

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 infertility 2015-05-08T11:25:18+00:00

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 bulls 2016-12-30T09:48:29+00:00

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 goats 2015-05-08T11:25:41+00:00

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+00:00

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 analysis 2016-12-30T09:48:29+00:00

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+00:00

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) spermatozoa 2016-12-30T09:48:30+00:00

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 reaction 2015-05-08T11:27:28+00:00

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+00:00

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 power 2015-05-08T11:28:03+00:00

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 solutions 2015-05-08T11:28:17+00:00

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 Switzerland 2017-06-13T13:02:39+00:00

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 bucks 2015-05-08T11:30:11+00:00

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 dimension 2016-12-30T09:48:30+00:00

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 diagnostic 2015-05-08T11:31:15+00:00

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 semen 2016-12-30T09:48:30+00:00

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 semen 2016-12-30T09:48:30+00:00

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 commentary 2016-12-30T09:48:30+00:00

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

Effects of cryopreservation on bull spermatozoa distribution in morphometrically distinct subpopulations 2016-12-30T09:48:30+00:00

Effects of cryopreservation on bull spermatozoa distribution in morphometrically distinct subpopulations

Rubio-Guillén J, González D, Garde JJ, Esteso MC, Fernández-Santos MR, Rodríguez-Gíl JE, Madrid-Bury N, Quintero-Moreno A.

Unidad de Investigación en Producción Animal (UNIPA), Facultad de Ciencias Veterinarias, La Universidad del Zulia (LUZ), Maracaibo, Venezuela.

Assisted sperm morphometry analysis (ASMA) was used in this study to determine the effects of cryopreservation on bull spermatozoa distribution in morphometrically distinct subpopulations. Ejaculates were collected from five bulls and were divided. One portion was diluted at 30 degrees C in a skim milk-egg yolk medium, containing glycerol. A microscope slide was prepared from single extended sperm samples prior to freezing. The remainder of each sample was frozen in nitrogen vapours. After thawing, sperm smears were prepared as described above. All slides were air dried and stained with Hemacolor. The sperm-head dimensions for a minimum of 200 sperm heads were analysed from each sample by means of the Sperm-Class Analyser (SCA), and the mean measurements recorded. Our results showed that applying the ASMA technology and multivariate cluster analyses, it was possible to determine that three separate subpopulations of spermatozoa with different morphometric characteristics coexist in bull ejaculates (large, average and small spermatozoa). The mean values of each sperm head dimension among the three subpopulations of spermatozoa were significantly different (p < 0.001). Besides, there were significant (p < 0.001) differences in the distribution of these three sperm subpopulations between fresh and thawed samples. Thus, the percentage of representation of the subpopulation that includes those spermatozoa whose dimensions are the biggest, decreased from 52.06% in extended fresh samples to 15.51% in the thawed ones. Contrarily, the percent of representation of the subpopulation containing the smallest spermatozoa, increased from 8.70% in extended fresh samples to 34.04% in the thawed ones. In conclusion, the present study confirms the heterogeneity of sperm head dimensions in bull semen, heterogeneity that vary through the cryopreservation procedure.

Reprod Domest Anim.
PMID: 17635770

The relationship between sperm morphology and chromatin integrity in the koala (Phascolarctos cinereus) as assessed by the Sperm Chromatin Dispersion test (SCDt) 2016-12-30T09:48:30+00:00

The relationship between sperm morphology and chromatin integrity in the koala (Phascolarctos cinereus) as assessed by the Sperm Chromatin Dispersion test (SCDt)

Johnston SD, López-Fernández C, Gosálbez A, Zee Y, Holt WV, Allen C, Gosálvez J

School of Animal Studies, The University of Queensland, Gatton 4343, Australia

Koala (Phascolarctos cinereus) sperm nuclei show a tendency to swell after cryopreservation, but it is uncertain whether this phenomenon is associated with DNA fragmentation. In this study, we validated a modified version of the sperm chromatin dispersion test (SCDt) for use with koala spermatozoa, which is the first use of the test for a marsupial. Cryopreserved spermatozoa (multiple straws) from a single koala were used to explore the relationship between sperm morphology, viability, chromatin dispersion, and DNA fragmentation. A SCDt prototype kit (Sperm Halomax) was specifically developed for koala spermatozoa with the use of a lysing solution that did not contain dithiothreitol. DNA fragmentation of lysed and nonlysed spermatozoa was examined in microgel slides and validated by means of in situ nick translation (ISNT). The SCDt was then applied to the analysis of extended and frozen-thawed semen samples of 3 different koalas. Spermatozoa were classified into 3 distinct koala sperm morphotypes (KSMs) after the SCDt: 1) KSM-1, rod-shaped cells with no halo of DNA; 2) KSM-2, rounded nuclei with various degrees of halo formation about a dense chromatin core; and 3) KSM-3, rod-shaped or rounded nuclei consisting of an inner chromatin core but with large dispersed halos of stellar chromatin. Although ISNT after the SCDt did not label KSM-1, both KSM-2 and KSM-3 stained positively for DNA fragmentation. ISNT was not able to differentiate between KSM-2 and KSM-3. Although application of the SCDt to the spermatozoa of another 3 koalas showed no difference in the percentage of the 3 sperm morphotypes found between extended and frozen-thawed semen, thawed spermatozoa incubated at 35 degrees C for 2 hours showed an increase in the incidence of KSM-3 and a corresponding decrease in KSM-2. We propose that KSM-1 and KSM-2 represent nuclei that show either no, or only limited, sperm DNA fragmentation, respectively. It is likely that the halos formed around KSM-2 are from DNA that is damaged as part of the normal processing of the spermatozoa and is a consequence of the lack of cysteine residues and associated stabilizing disulfide bonds in marsupial sperm DNA. “True” sperm DNA damage is most likely associated with KSM-3, which shows a massive dispersion of chromatin similar to that described in other species. A model of koala sperm chromatin structure is proposed to explain the behavior of the sperm nuclei after the SCDt. Further studies are required to determine whether DNA damage found in KSM-2 is indicative of single-stranded DNA breakage associated with an inherent lack of cysteine residues in marsupial sperm chromatin. Conversely, it will also be important to establish whether KSM-3 is caused by an increased incidence of double-stranded DNA breakage and whether this abnormality is correlated with impaired fertility as it is in other species.

doi: 10.2164/jandrol.107.003350

Expression of enkephalin-degrading enzymes in human semen and implications for sperm motility 2015-05-08T11:33:01+00:00

Expression of enkephalin-degrading enzymes in human semen and implications for sperm motility

Subirán N, Agirregoitia E, Valdivia A, Ochoa C, Casis L, Irazusta J

Department of Physiology, Faculty of Medicine and Dentistry, University of the Basque Country, Leioa, Spain

OBJECTIVE: To investigate the presence of two enkephalin-degrading enzymes (aminopeptidase N [APN] and neutral endopeptidase 24.11 [NEP]) in different fractions of human semen, their distribution in sperm cells, and their effect on sperm motility.
DESIGN: We performed expression assays for APN and NEP by real-time polymerase chain reaction, Western blot, and immunofluorescence techniques in sperm cells and performed motility analysis after incubation of semen samples with enzyme inhibitors and the opioid receptor antagonist naloxone.
SETTING: Assisted reproduction unit and academic research laboratory.
PATIENT(S): Semen from 50 normozoospermic healthy human donors.
INTERVENTION(S): Spermatozoa isolated from semen on a discontinuous Percoll gradient (40%-80%), followed by swim-up, were used for all techniques except for sperm motility analysis, for which fresh semen was used.
MAIN OUTCOME MEASURE(S): Immunoblotting blots, indirect immunofluorescence antibody assays, cycle threshold values for real-time polymerase chain reaction, and percentage of motile sperm.
RESULT(S): We found APN in the equatorial segment of the upper post-acrosomal region of the sperm head, in the neck and along the tail of spermatozoa, in prostasomes, and in seminal fluid, whereas NEP was present in a very restricted area of a few sperm cells and in prostasomes. Messenger RNA of both enzymes was detected in spermatozoa. The inhibition of enkephalin-degrading enzymes attenuated the time-dependent decrease of sperm motility; this effect was reversed by naloxone.
CONCLUSION(S): Enkephalin-degrading enzymes are present in human semen and may be involved in the control of sperm motility, mainly by the regulation of endogenous opioid peptides.

doi:10.1016/j.fertnstert.2007.06.056

Effects of cryopreservation on semen quality and the expression of sperm membrane hexose transporters in the spermatozoa of Iberian pigs 2016-12-30T09:48:30+00:00

Effects of cryopreservation on semen quality and the expression of sperm membrane hexose transporters in the spermatozoa of Iberian pigs

Sancho S, Casas I, Ekwall H, Saravia F, Rodriguez-Martinez H, Rodriguez-Gil JE, Flores E, Pinart E, Briz M, Garcia-Gil N, Bassols J, Pruneda A, Bussalleu E, Yeste M, Bonet S

Biotechnology of Porcine Reproduction, University of Girona, Campus de Montilivi, s/n, 17071 Girona, Spain

This study evaluated the effects of cooling, freezing and thawing on the plasma membrane integrity, kinetics and expression of two sugar transporters glucose transporter-3 and -5 (GLUT-3 and GLUT-5) in spermatozoa from Iberian boars. Semen samples were collected twice weekly from eight young, fertile Iberian boars of the ‘Entrepelado’ and ‘Lampiño’ breeds. The samples were suspended in a commercial extender and refrigerated to 17 degrees C for transport to the laboratory (step A), where they were further extended with a lactose-egg yolk-based extender and chilled to 5 degrees C (step B) prior to freezing in the presence of glycerol (3%). Spermatozoa were assessed for plasma membrane integrity and sperm motility at each of the steps, including post-thaw (step C). Aliquots were also prepared for immunocytochemical localisation of the sugar transporters (fixed and thin smears for transmission and scanning electron microscopy levels respectively) and for SDS-PAGE electrophoresis and subsequent western blotting, using the same antibodies (rabbit anti-GLUT-3 and anti-GLUT-5 polyclonal antibodies). The results showed lower percentages of progressively motile spermatozoa at step C in both breeds, while the percentage of live spermatozoa was significantly lower only in the ‘Entrepelado’ breed. The results obtained from electron microscopy clearly showed that Iberian boar spermatozoa expressed the hexose transporters, GLUT-3 and GLUT-5. The pattern of expression, in terms of location and concentration, was characteristic in each case but, in the case of isoform GLUT-5, it remained constant during the different steps of freezing-thawing protocol. These results indicate that cryopreservation affects the status of sperm cells of Iberian boars by altering the distribution of some membrane receptors and decreasing the percentage values of parameters linked to sperm quality.

doi: 10.1530/REP-07-0118

Scrotal heat stress effects on sperm viability, sperm DNA integrity, and the offspring sex ratio in mice 2016-12-30T09:48:30+00:00

Scrotal heat stress effects on sperm viability, sperm DNA integrity, and the offspring sex ratio in mice

Pérez-Crespo M, Pintado B, Gutiérrez-Adán A

Dpto. de Reproducción Animal y Conservación de Recursos Zoogenéticos, INIA, Madrid, Spain

Evidence exists to suggest detrimental effects of heat stress on male fertility. This study was designed to assess the effects of scrotal heat stress on mature and developing sperm in a mouse model. After receiving shock heat treatment (42 degrees C for 30 min), mature spermatozoa were recovered from the epididymis hours (6) or Days (7, 14, 21, 28, 60) later, to determine the variables: number of spermatozoa, sperm viability, motility and progressive motility, sperm DNA integrity as established by the TUNEL method, embryo implantation rate, and sex ratio of the fetuses conceived using the heat-exposed spermatozoa. Our results indicate that transient mild heat treatment does not affect in the same way the different types of male germ cells. Spermatocytes present within the testis at the time of heat stress resulted into a lower concentration of spermatozoa with reduced viability and low motility. Even though, DNA integrity of spermatozoa resulting from spermatocytes was also compromised by heat stress, the higher degree of DNA damage was found among spermatozoa resulting from spermatids present within the testis at the time of heat stress. At last, heat shock effect on spermatozoa present in the epididymis at the time of thermal stress resulted into a sex ratio distortion. These findings point to a higher sensitivity of spermatocytes to heat exposure and also suggest a different response of X and Y chromosome-bearing spermatozoa to heat stress that warrants further investigation.

doi: 10.1002/mrd.20759

Supplementation of the dilution medium after thawing with reduced glutathione improves function and the in vitro fertilizing ability of frozen-thawed bull spermatozoa 2015-05-08T11:33:37+00:00

Supplementation of the dilution medium after thawing with reduced glutathione improves function and the in vitro fertilizing ability of frozen-thawed bull spermatozoa

Gadea J, Gumbao D, Cánovas S, García-Vázquez FA, Grullón LA, Gardón JC

Department of Physiology, School of Veterinary, University of Murcia, Murcia, Spain

In this study, we evaluated the effects of glutathione (l-gamma-glutamyl-l-cysteinylglycine; GSH) supplementation of the thawing extender on bull semen parameters to compensate for the decrease in GSH content observed during sperm freezing. To address these questions fully, we used a set of functional sperm tests. These included tests of sperm motility assayed by computer-assisted semen analysis, membrane lipid packing disorder, spontaneous acrosome reaction, free radical production [reactive oxygen species (ROS) generation], sperm chromatin condensation, DNA fragmentation by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling and acridine orange staining measured by flow cytometry. Finally, the in vitro penetrability of in vitro matured oocytes and the in vitro production of embryos were evaluated. The main findings emerging from this study were that addition of GSH to the thawing medium resulted in: (i) a higher number of non-capacitated viable spermatozoa; (ii) a reduction in ROS generation; (iii) lower chromatin condensation; (iv) lower DNA fragmentation; (v) higher oocyte penetration rate in vitro and (vi) higher in vitro embryo production compared with control group. Nevertheless, GSH had no significant effect on motion parameters or the occurrence of the spontaneous acrosome reaction. Addition of GSH to the thawing extender could be of significant benefit in improving the function and fertilizing capacity of frozen bull spermatozoa.

doi: 10.1111/j.1365-2605.2007.00756.x

Effectiveness of butylated hydroxytoluene as egg yolk substitute for cryopreservation of boar spermatozoa 2015-05-08T11:34:03+00:00

Effectiveness of butylated hydroxytoluene as egg yolk substitute for cryopreservation of boar spermatozoa

L. Rodriguez-Vilar, M. Hernandez, C. Lopez-Sanchez, J. M. Vazquez, E. A. Martinez and J. Roca

University of Murcia

Butylated hydroxytoluene (BHT) has proven to be efficient as a supplement for cryopreservation boar spermatozoa (Roca et al. 2004 J. Androl. 25, 397–405). Moreover, it has been successfully used as an egg yolk substitute to cryopreserve goat spermatozoa (Khalifa and El-Saidy 2006 Anim. Reprod. Sci. 93, 303–315). The objective of this study was to evaluate the effectiveness of BHT as an egg yolk substitute for freezing boar spermatozoa. Nine sperm-rich ejaculate fractions were collected from 3 boars (3 ejaculates per boar) using the gloved-hand method. After centrifugation (2400g for 3 min), the sperm pellet of each ejaculate was split into 5 aliquots. The aliquots were diluted (to a final concentration of 1 × 109 sperm/mL) in a Tris-citric-glucose extender with 3% glycerol and supplemented with 20% egg yolk (positive control, PC aliquot) or BHT at the final concentrations of 0 (negative control, NC aliquot), 0.2, 0.4, and 0.8 mM. Diluted semen samples were dispensed into 0.5-mL straws, and frozen in a programmable cell freezer at 20°C min. Thawing was carried out in a water bath at 70°C for 8 s. Post-thaw sperm survival was assessed according to total sperm motility (TSM, %) using a CASA system (SCA®; Microptic, Barcelona, Spain), and plasma membrane integrity (PMI, %) and acrosome membrane integrity (AMI, %) using a flow cytometric procedure (SYBR-14/propidioum iodide/FITC-phycoerythrin), at 30 and 150 min post-thawing in diluted Beltsville thawing solution with spermatozoa held in a waterbath at 37°C (3 straws per ejaculate). Data were analyzed using a ANOVA mixed model including the main effects of aliquot, boar, post-thaw assessment time, and their interactions, with ejaculate and straw as random effects. All main effects had significant influence (P ≤ 0.01) in all post-thaw sperm assessments. However, no interactions (P ≥ 0.05) among main effects were shown. Data were combined for the 2 post-thaw assessment times. The best (P ≤ 0.05) post-thaw sperm quality (mean ± SEM) was achieved in PC aliquots (47.11 ± 3.10, 58.98 ± 2.78, and 51.35 ± 3.42 for TSM, PMI, and AMI, respectively). In NC aliquots, the percentage of TSM, PMI, and AMI were always below 1% (P ≤ 0.05). BHT has a beneficial (P ≤ 0.05) effect on post-thaw sperm assessments, and no differences (P ≥ 0.05) among concentrations were shown. The mean post-thaw sperm quality in the BHT aliquots was 8.50 ± 0.80, 20.29 ± 0.53, and 16.03 ± 0.55 for TSM, PMI, and AMI, respectively. On the basis of these data, we can conclude that BHT has a protective effect for boar spermatozoa during the cryopreservation process. However, BHT alone is insufficient to replace the protective effect of egg yolk.

doi: 10.1071/RDv19n1Ab130

Sperm characterization of Asturcon ponies after collection and equilibration/refrigeration before freezing 2016-12-30T09:48:33+00:00

Sperm characterization of Asturcon ponies after collection and equilibration/refrigeration before freezing

C. Tamargo, C. Díez, J. De La Fuente, M. Carbajo, J. M. Benito and C. O. Hidalgo

Servicio Regional de Investigación y Desarrollo Agroalimentario; University of Castilla-La Mancha;

The need to conserve farm animal biodiversity is accepted by many countries through the ratification of the convention of biological diversity, and sperm quality is known to be an important criterion in the evaluation of breeding soundness. The aim of this work was to characterize the semen of a local breed of ponies ‘Asturcon’ (maintained free over the mountains all year around) before its incorporation into a germplasm bank. Semen was obtained from six stallions (6–17 years of age) using an artificial vagina, 3 days/week, during 12 weeks. Immediately after collection, gel-free semen was evaluated for volume, sperm concentration, and motility. Semen motility was again evaluated after equilibration/refrigeration. For evaluation of individual (IM) and progressive motility (PM) rates, semen was diluted (20 × 106 spermatozoa/mL) and analyzed with a CASA System (SCA; Microptic S.L., Barcelona, Spain). Five fields per sample were evaluated (minimum 500 spermatozoa/sample) under a phase contrast microscope (100×). Semen samples were subjected to a hypo-osmotic swelling test (HOS) test to detect the presence of swollen tails in a 100 mM citrate–fructose solution. Percentages of altered acrosomes and morphological abnormalities were determined by counting 100 spermatozoa (1000×). Then, semen was diluted and centrifuged for 10 min at 600g. After the supernatant was discarded, the pellet was re-suspended in freezing medium (skim milk extender containing 2% egg yolk and 2.5% glycerol) to a final concentration of 100 × 106 spermatozoa/mL, and equilibrated/cooled (60 min) to 4°C. Statistical analysis was carried out by means of the GLM and CORR procedures and Duncan test for means (SAS Institute, Inc., Cary, NC, USA). A significant effect between males (P < 0.05) on semen quality, such as volume of the ejaculate, sperm concentration, and morphological abnormalities, were detected among stallions. On the other hand, positive and significant correlations were found between the sperm motility immediately after collection and after equilbration/refrigeration (r = 0.73; P < 0.05); moreover, sperm motilities (both fresh and refrigerated) correlated with the results of the HOS test (r = 0.56; P < 0.001, and r = 0.27, P < 0.05, respectively). These preliminary results confirm that the sperm of the Asturcon ponies breed can be collected and will survive the equilibration/refrigeration procedures. Conservation and development of local breeds is important because they represent a unique source of genes for improving health and performance traits of industrial breeds. However, complementary studies on the ability of the stallion sperm to survive freezing/thawing procedures in rates higher than 30% are needed to ensure that genetic banks are correctly created.

doi: 10.1071/RDv19n1Ab20

Effect of spermatozoa head morphometric dimensions on freezability in brown bear (Ursus Arctos) 2016-12-30T09:48:33+00:00

Effect of spermatozoa head morphometric dimensions on freezability in brown bear (Ursus Arctos)

L. Anel, V. Garcia-Macias, F. Martinez-Pastor, M. Alvarez, S. Borragan, J. Bernardo, S. Alves, P. Paz

Universidad de León, León, Castille and León, Spain

Having recently observed that survival of red deer spermatozoa after cryopreservation seemed to reflect the size of sperm heads, we hypothesized that cryoresistance of brown bear spermatozoa might also be dependent on head size, since in a preliminary study we had also observed significant differences in sperm head sizes among male brown bears (median values for area 22.2 µm2, perimeter 18.2, length 6.1 and width 4.4 µm). In the present report, we analyzed the post-thaw survival of spermatozoa of 6 brown bears that were assigned to 2 groups (3 bears/group) based on sperm size: Group A with large-size sperm heads; Group B with small-size heads. Ejaculates were obtained by electroejaculation of adult brown bears (semi-free ranging in Cabarceno Park, Cantabria, Spain) under general anesthesia (7 mg kg-1 tiletamine + zolazepan and 2 mg kg-1 ketamine). Semen was diluted (Tes-Tris-fructose, 8% glycerol, 20% egg yolk, EDTA, and Equex paste), loaded in 0.25-mL straws, and frozen in a biofreezer at 20°C min-1 to -100°C. After storage in liquid nitrogen, samples were thawed in water at 65°C for 6 s and survival was measured. Sperm motility (TM: total, and PM: progressive; %) was assessed microscopically, and sperm viability, acrosome integrity (PI/PNA-FITC), and mitochondrial status (JC-1) were assayed for fresh and thawed sperm by flow cytometry. Recovery rates (RR: thawed/fresh × 100) were calculated for all parameters. For measurement of head size, fresh sperm samples were fixed in glutaraldehyde and slides were air-dried for 2 h. The samples were then stained with Diff-Quik® staining at 37°C. The area (Ar), perimeter (P), length (L), and width (W) of the heads of >100 spermatozoa per slide were measured (Sperm Class Analyzer®; Microptic S.L., Barcelona, Spain). Data were analyzed with the SAS ver8 system, and the Wilcoxon test was applied. The respective morphometric dimensions of the 2 groups were practically identical (Ar = 22; P = 18; L = 6; W = 4). The post-thaw recovery rates of spermatozoa from Group A were: TM: 60.1 ± 29.3; PM: 54.8 ± 36.0; viability: 99.4 ± 8.0; acrosomes: 96.2 ± 3.1; mitochondria: 70.9 ± 15.5. The recovery rates for Group B were: TM: 78.7 ± 13.8; PM: 69.0 ± 18.8; viability: 93.8 ± 5.2; acrosomes: 98.2 ± 9.8; mitochondria: 72.5 ± 22.5. Because of the high variability of recovery rates between males within each group, there were no statistical differences between the 2 groups. The absence of differences can be explained by the small number of males examined and the high variability between them. More studies are necessary to determine whether large sperm cells of brown bears are more susceptible to damage during cryopreservation.

doi: 10.1071/RDv19n1Ab242

Male fertility and sex ratio at birth in red deer 2015-05-08T11:35:04+00:00

Male fertility and sex ratio at birth in red deer

Montserrat Gomendio, Aurelio F. Malo, Ana J. Soler, Maria R. Fernández-Santos, Milagros C. Esteso, Andrés J. García, Eduardo R. S. Roldan, Julian Garde

Reproductive Ecology and Biology Group, Department of Evolutionary Ecology, Museo Nacional de Ciencias Naturales [Consejo Superior de Investigaciones Científicas (CSIC)], 28006-Madrid, Spain

Efforts to test sex ratio theory have focused mostly on females. However, when males possess traits that could enhance the reproductive success of sons, males would also benefit from the manipulation of the offspring sex ratio. We tested the prediction that more-fertile red deer males produce more sons. Our findings reveal that male fertility is positively related to the proportion of male offspring. We also show that there is a positive correlation between the percentage of morphologically normal spermatozoa (a main determinant of male fertility) and the proportion of male offspring. Thus, males may contribute significantly to biases in sex ratio at birth among mammals, creating the potential for conflicts of interest between males and females.

1 December 2006, Science 314, 1445 (2006). 10.1126/science.1133064.

Functional significance of the sperm head morphometric size and shape for determining freezability in Iberian red deer (Cervus elaphus hispanicus) epididymal sperm samples 2016-12-30T09:48:33+00:00

Functional significance of the sperm head morphometric size and shape for determining freezability in Iberian red deer (Cervus elaphus hispanicus) epididymal sperm samples

Esteso MC, Soler AJ, Fernandez-Santos MR, Quintero-Moreno AA, Garde JJ

Biology of Reproduction Group, Department of Game Resources (IDR), Castilla-La Mancha University (UCLM), Albacete, Spain

In the present study, computer-automated sperm head morphometry of epididymal samples was used to determine if sperm head area and shape are useful measurements for separating “good” and “bad” Iberian red deer freezers. A microscope slide was prepared from single diluted sperm fresh samples collected from 38 mature stags. Slides were air-dried and stained with Hemacolor. The sperm head area and shape (length/width) for a minimum of 145 sperm heads were determined for each male by means of the Sperm-Class Analyser. The remainder of each sample was frozen. After thawing, sperm cryosurvival was judged in vitro by microscopic assessments of individual sperm motility and of plasma membrane and acrosome integrities. All sperm parameters evaluated at thawing were placed in a statistical database and a multivariate cluster analysis performed. Mean sperm parameters of the 2 clusters generated (“bad” and “good” freezers) were compared by ANOVA. Our results show that sperm quality at thawing for all sperm parameters evaluated was significantly higher (P < .01) for “good” freezers than for the “bad” ones (sperm motility index: 67.4+/-2.0 vs 57.1+/-2.8; NAR: 67.1+/-2.5 vs 54.5+/-3.5; viability: 68.8+/-2.0 vs 60.1+/-2.8; HOST: 71.3+/-2.2 vs 63.1+/-3.1). Additionally, differences (P < .01) in epididymal sperm head area and shape were found between “good” and “bad” freezers before freezing, with the smallest overall sperm head dimensions found in the “good” freezers group (area: 32.04 mum(2) vs 34.42 mum(2)). Thus, the lower the sperm head area in the fresh samples, the greater the sperm cryoresistance. Our results show that the 2 groups of males also differ in sperm head shape in fresh samples (good: 1.96 vs poor: 1.72; P < .01). It is possible that sperm head area and shape influence total sperm volume, thus causing differences in heat exchange as well as in movements of water, ions, and cryoprotectants and, in turn, on sperm freezability.

J Androl. 2006 Sep-Oct;27(5):662-70. Epub 2006 May 25.

Role of sialic acid in bovine sperm-zona pellucida binding 2015-05-08T11:36:08+00:00

Role of sialic acid in bovine sperm-zona pellucida binding

Velásquez JG, Canovas S, Barajas P, Marcos J, Jiménez-Movilla M, Gallego RG, Ballesta J, Avilés M, Coy P

Department of Physiology, Veterinary Faculty, University of Murcia, Murcia, Spain

Sperm binding activity has been detected in zona pellucida (ZP) glycoproteins and it is generally accepted that this activity resides in the carbohydrate moieties. In the present study we aim to identify some of the specific carbohydrate molecules involved in the bovine sperm-ZP interaction. We performed sperm binding competition assays, in vitro fecundation (IVF) in combination with different lectins, antibodies and neuraminidase digestion, and chemical and cytochemical analysis of the bovine ZP. Both MAA lectin recognising alpha-2,3-linked sialic acid and neuraminidase from Salmonella typhimurium with catalytic activity for alpha-2,3-linked sialic acid, demonstrated a high inhibitory effect on the sperm-ZP binding and oocyte penetration. These results suggest that bovine sperm-ZP binding is mediated by alpha-2,3-linked sialic acid. Experiments with trisaccharides (sialyllactose, 3′-sialyllactosamine and 6′-sialyllactosamine) and glycoproteins (fetuin and asialofetuin) corroborated this and suggest that at least the sequence Neu5Ac(alpha2-3)Gal(beta1-4)GlcNAc is involved in the sperm-ZP interaction. Moreover, these results indicate the presence of a sperm plasma membrane specific protein for the sialic acid. Chemical analysis revealed that bovine ZP glycoproteins contain mainly Neu5Ac (84.5%) and Neu5GC (15.5%). These two types of sialic acid residues are probably linked to Galbeta1,4GlcNAc and GalNAc by alpha-2,3- and alpha-2,6-linkages, respectively, as demonstrated by lectin cytochemical analysis. The use of a neuraminidase inhibitor resulted in an increased number of spermatozoa bound to the ZP and penetrating the oocyte. From this last result we hypothesize that a neuraminidase from cortical granules would probably participate in the block to polyspermy by removing sialic acid from the ZP.

doi: 10.1002/mrd.20619

The effect of cryopreservation on sperm head morphometry in Florida male goat related to sperm freezability 2016-12-30T09:48:33+00:00

The effect of cryopreservation on sperm head morphometry in Florida male goat related to sperm freezability

Hidalgo M, Rodríguez I, Dorado JM

Animal Reproduction Group, Department of Animal Medicine and Surgery, University of Cordoba, Campus Rabanales, Hospital Clínico, Ctra. Madrid-Cádiz, Km. 396, 14014 Cordoba, Spain

The Sperm Class Analyzer was used to investigate the effect of freeze-thawing procedure on Florida buck sperm head morphometry, and to relate possible changes in sperm head dimensions to cryopreservation success. Semen samples (n=76) were frozen with tris and milk-based extenders and thawed. Sperm quality samples (motility, morphology, acrosome), and sperm head morphometric values (length, width, area, perimeter, ellipticity) were compared between fresh and frozen-thawed samples. Sperm freezability was judged according to the sperm quality parameters assessed. Fertility data was obtained after artificial insemination with cryopreserved semen. Cryopreservation success was different between freezing methods. Sperm head dimensions were significantly (p<0.05) smaller in cryopreserved tris and milk spermatozoa respectively than in those of the fresh samples. The sperm head morphometric parameters that had changed after cryopreservation were lower in suitable semen samples after thawing and with successful pregnancies after artificial insemination. These data suggest that changes in sperm head morphometry might reflect spermatozoa injury occurred during cryopreservation.

doi:10.1016/j.anireprosci.2006.07.003

Immunolocalization and possible functional role of PSP-I/PSP-II heterodimer in highly extended boar spermatozoa 2015-05-08T11:36:35+00:00

Immunolocalization and possible functional role of PSP-I/PSP-II heterodimer in highly extended boar spermatozoa

Caballero I, Vázquez JM, García EM, Roca J, Martínez EA, Calvete JJ, Sanz L, Ekwall H, Rodríguez-Martínez H

Departamento de Medicina y Cirugía Animal, Facultad de Veterinaria, Universidad de Murcia, Murcia, Spain

PSP-I/PSP-II heterodimer is a major protein of boar seminal plasma which is able to preserve, in vitro, the viability, motility, and mitochondrial activity of highly extended boar spermatozoa for at least 5 hours. However, little is known about the binding pattern of the heterodimer to the sperm plasma membrane and its eventual relation with the maintenance of the sperm functionality. The present study investigated the effect of exposing highly extended boar spermatozoa (1 million/mL) to 1.5 mg/mL of PSP-I/PSP-II for 0.5, 5, and 10 hours at 38 degrees C on sperm characteristics and the changes in PSP-I/PSP-II localization as a result of both the addition of PSP-I/PSP-II to the extender and the incubation time. Exposure of the spermatozoa to PSP-I/PSP-II preserved sperm viability, motility, and mitochondrial activity when compared to nonexposed spermatozoa. This protective effect lasted for 10 hours (P < .05). After immunolabeling of highly extended semen with rabbit monospecific polyclonal antibody against PSP-I/PSP-II, the percentage of immunopositive spermatozoa declines over time from 71% (0.5 hours) to 49% (10 hours). However, more than 80% of spermatozoa remained labeled during the 10-hour incubation period if PSP-I/PSP-II was added. Scanning electron microscopy revealed 4 different binding patterns. The heterodimer was mainly localized to the acrosomal area, being redistributed to the postacrosomal area or lost during in vitro incubation. In conclusion, the protective effect of the heterodimer appears to be related to its adhesion to the acrosomal area, and the loss of this protective effect coincides with a stepwise redistribution of PSP-I/PSP-II during incubation.

doi: 10.2164/jandrol.106.000539

The effects of cryopreservation on the morphometric dimensions of Iberian red deer (Cervus elaphus hispanicus) epididymal sperm heads 2016-12-30T09:48:33+00:00

The effects of cryopreservation on the morphometric dimensions of Iberian red deer (Cervus elaphus hispanicus) epididymal sperm heads

Esteso MC, Fernandez-Santos MR, Soler AJ, Montoro V, Quintero-Moreno A, Garde JJ

Grupo de Biología de la Reproducción, Instituto de Investigación en Recursos Cinegéticos, IREC, (CSIC, UCLM, JCCM), Campus Universitario, Albacete, Spain

Computer-automated sperm-head morphometry was used in this study to determine the effects of cryopreservation on red deer sperm-head morphometry. Epididymal sperm samples were collected from 40 mature stags and were divided. One portion was diluted at room temperature in a Tris-citrate egg yolk medium, containing 6% glycerol. A microscope slide was prepared from single extended sperm samples prior to freezing. The remainder of each sample was frozen in nitrogen vapours. After thawing, sperm smears were prepared as described above. All slides were air dried and stained with Hemacolor. The sperm-head dimensions for length, width, area, perimeter and shape factor (length/width), for a minimum of 135 spermatozoa were determined for each slide by means of the Sperm-Class Analyser (SCA). Firstly, our results show that cryopreservation substantially reduced (p < 0.001) sperm motility and plasma membrane and acrosome integrities. In addition, sperm heads were significantly smaller in cryopreserved spermatozoa than in the companion extended samples for area (32.05 microm2 vs 32.56 microm2; p < 0.05), length (8.46 microm vs 8.53 microm; p < 0.0001) and shape factor (1.833 vs 1.849; p < 0.0001) for all stags. These differences were found within 29 of 40 stags (75%) for at least three of the morphometric parameters. The individual variability (CV) of sperm head measurements from extended samples was negatively correlated (p < 0.005) with the per cent of change in sperm head measurements after cryopreservation for area (r = -0.465), width (r = -0.483) and perimeter (r = -0.375). Thus, the lower the sperm head variability in the extended samples, the greater the sperm change as a consequence of the cryopreservation. These results suggest that the variability (heterogeneity) in sperm head dimensions of individual stags may be a good indicator of sperm freezability.

Reprod Domest Anim. 2006 Jun;41(3):241-6.

Sperm design and sperm function 2015-05-08T11:37:30+00:00

Sperm design and sperm function

Malo AF, Gomendio M, Garde J, Lang-Lenton B, Soler AJ, Roldan ER

Museo Nacional de Ciencias Naturales (CSIC), Reproductive Ecology and Biology Group, José Gutierrez Abascal 2, 28006 Madrid, Spain

Spermatozoa vary enormously in their form and dimensions, both between and within species, yet how this variation translates into fertilizing efficiency is not known. Sperm swimming velocity is a key determinant of male fertilization success, but previous efforts to identity which sperm phenotypic traits are associated with swimming velocity have been unsuccessful. Here, we examine the relationship between the size of several sperm components and sperm swimming velocity in natural populations of red deer (Cervus elaphus hispanicus) where selective pressures to enhance male reproductive success are expected to be strong. Our results show that there is little within-male and considerable between-male variation in sperm dimensions. Spermatozoa with longer midpieces swim more slowly, a finding which does not support the hypothesis that the size of the midpiece determines the amount of energy which is translated into swimming speed. In contrast, spermatozoa with elongated heads, and those in which the relative length of the rest of the flagellum is longer, swim faster. Thus, the hydrodynamic shape of the head and the forces generated by the relative size of the rest of the flagellum seem to be the key determinants of sperm swimming velocity.

doi:10.1098/rsbl.2006.0449

Influence of staining and sampling procedures on goat sperm morphometry using the Sperm Class Analyzer 2016-12-30T09:48:33+00:00

Influence of staining and sampling procedures on goat sperm morphometry using the Sperm Class Analyzer

Hidalgo M, Rodríguez I, Dorado J.

Reproduction and Obstetrics Unit, Faculty of Veterinary Science, University of Cordoba, 14071 Cordoba, Spain

Computer-assisted sperm morphometry analysis (ASMA) has improved the assessment of sperm morphology, but the results depend on the use of adequate sampling and staining procedures of spermatozoa from individual species. In this study, the Sperm Class Analyzer ASMA system was used for the morphometric analysis of goat sperm heads. Semen samples, obtained from four bucks, were used to evaluate the influence of three staining procedures (Diff-Quik, Hemacolor and Harris’ Haematoxylin) on the accuracy of image processing and sperm morphometry, the effect of the number of cells analysed and the repeatability of the method. These experiments were performed to obtain objective, accurate and reliable sperm morphometric measurements of goat spermatozoa. Diff-Quik and Harris’ Haematoxylin were significantly (p < 0.05) more accurate than Hemacolor. However, Diff-Quik obtained the highest proportion of correctly analysed sperm heads (86.06%) and the lowest coefficients of variation on the image processing and morphometric measurements. The staining methods affected significantly the sperm dimensions ( p < 0.001) with increased values from Diff-Quik than Hemacolor and Harris’ Haematoxylin, respectively (Diff-Quik > Hemacolor > Harris’ Haematoxylin). No differences in morphometric parameters were found when 100, 150, 175 or 200 spermatozoa were analysed. The repeatability of results obtained was very high since no differences were found when measuring the same sperm on multiple attempts. In conclusion, to obtain objective, accurate and repeatable sperm morphometric measurements by the Sperm Class Analyzer system in goats, the analysis of 100 spermatozoa from slides which have been previously stained with Diff-Quik is recommended.

Theriogenology. 2006 Feb.

Sperm DNA fragmentation in a random sample of the Spanish boar livestock 2015-05-08T11:38:29+00:00

Sperm DNA fragmentation in a random sample of the Spanish boar livestock

C. López-Fernández, B. Pérez-Llano, P. García-Casado, R. Sala, A. Gosálbez, F. Arroyo, J.L. Fernández, J. Gosálvez

Unidad de Genética, Departamento de Biología, Universidad Autónoma de Madrid, C/Darwin No. 2, 28049 Madrid, Spain

A collection of 180 chilled boar semen samples, randomly chosen from stocks currently used for routine characterization of standard seminal quality, were studied for DNA fragmentation status using the sperm chromatin dispersion test and the DNA fragmentation index (DFI: percent of abnormal cell versus normal cells for DNA fragmentation) was determined. Values for sperm motility, acrosome status, coiled tails and abnormal head morphology, including presence and position of cytoplasmic droplets were also obtained. The DFI in the whole sample presented a wide range of variation with values oscillating between practically 0% and 47.95% and do not fit to a normal distribution. The most frequent classes (83.3%) presented a DFI lower than a 5%. Significant correlations between sperm DNA fragmentation and sperm motility, acrosome status, frequency of distal droplets, coiled tails and abnormal head morphology, were not observed. However, the presence of proximal cytoplasmic droplets showed a significant correlation with the level of DNA fragmentation observed in the ejaculated spermatozoa.

Anim. Reprod. Sci. (2006), doi:10.1016/j.anireprosci.2006.11.015

Analysis of the principal components within the morphometric parameters of stallion spermatozoa 2016-12-30T09:48:33+00:00

Analysis of the principal components within the morphometric parameters of stallion spermatozoa

M Hidalgo, I Rodríguez and J Dorado

Animal Reproduction Unit, University of Córdoba, Spain

Computer-assisted sperm morphometry systems usually analyse a large number of objective variables, which can be used for classification techniques using  statistical modelling procedures. The aim of this study was to reduce the morphometric parameters of stallion spermatozoa to principal components, which  explain as much as possible of the information included in the original variables. Eajaculates were collected by artificial vagina from 8 adult stallions of the  Arabian, Thoroughbred, Anglo-Arabian and Spanish-Thorougbred breeds. Morphometric slides were stained with Harris’ haematoxylin and at least 200  spermatozoa were analysed using the Sperm Class Analyser (Microptic, Spain). The Principal components (PC) matrix was calculated statistically over the  morphometry data obtained. Morphometric parameters were classified in 4 PC. The PC1 was correlated with the sperm head size parameters

[length(0.87),  width(0.79) area(0.96) perimeter(0.94)], PC2 with the head shape [ellipticity(0.99), rugosity()0.96), elongation(0.99)], PC3 with the midpiece size [width (0.93)  rea(0.92)] and PC4 with the midpiece insertion parameters [distance(0.98), angle(0.99)]. The reduction of the data to PC permitted grouping sets of  highly correlated variables, which could explain 90.5% of the total variance. It is concluded that morphometric parameters of stallion spermatozoa were reduced  to four PC for head size and shape, and midpiece size and insertion respectively, which retained a higher percentage of the information obtained with the original variables (Supported by PTR1995-0826-OP).

Reprod Dom Anim 41, 311–377 (2006)

Variations in buck semen motility assessed by CASA system: Correlations with environmental temperature 2016-12-30T09:48:33+00:00

Variations in buck semen motility assessed by CASA system: Correlations with environmental temperature

J Dorado, M Hidalgo and I Rodríguez

Animal Reproduction Unit, University of Cordoba, Spain

Several factor affect goat semen production and sperm quality and knowledge of these factors is very important to improve goat reproductive efficiency. This  study determined the influence of environmental temperature (T) upon buck semen motility using Sperm Class Analyser (SCA, Microptic). Animals were  located in southwest of Iberian Peninsula (3753¢N-446¢W; altitude 110 m). Temperature measurements (minimum, Tmin; mean, Tm; maxim, Tmax and difference in temperature, Td as Tmax-Tmin) were recorded daily at local meteorological centre. Semen data from two fertile adult Florida bucks collected  during 12 months were evaluated. In total, 110 ejaculates were diluted in DPBS to obtained 50 · 106 spermatozoa/ ml, incubated for 5 min. at 37C and  analysed to determine percentage of motile spermatozoa (MS, %) and rapidly progressive (RPMS, %), curvilinear velocity (CLV, lm/sec), average path  velocity (APV, lm/ sec), progressive speed (SLV, lm/sec), linearity (LIN); straightness (STR), wobble (WOB), beat cross frequency (BCF, Hz) and lateral head displacement (LHD,  lm). Bilateral correlation was established between those parameters. There was significant correlation in semen motility kept at different T. Tmin, Tmax and Tm  only showed correlation with MS (r = 0.21 for Tmin and r = 0.22 for Tmax and Tm). Although, Td had a negative correlation with LIN (r = (0.22), STR (r =  (0.19) and WOB (r = (0.22) and positive correlation with BCF (r = 0.20) and LHD (r = 0.21). We concluded that motion quality measurements (LIN and STR)  were significantly lower when Td was higher, whilst motility and velocity parameters were not affected.

Reprod Dom Anim 41, 311–377 (2006)

Estimation of seasonal effect on sperm motility in fresh semen from bulls 2016-12-30T09:48:33+00:00

Estimation of seasonal effect on sperm motility in fresh semen from bulls

C Tamargo, M Carbajo, C Díez, S de la Varga and CO Hidalgo

SERIDA, Gijón, Spain, Facultad de Veterinaria, León, Spain

Seasonal changes can affect sperm motility, which is essential for fertilization. Computer-assisted semen analysis (CASA) provides more accurately values than those obtained with traditional semen analysis methods. The aim of our experiment was to investigate the possible seasonal effect on sperm motility,  assessed by a subjective measurement system complemented with a CASA system. Ejaculates (n = 373) from seven Asturiana de los Valles adult bulls were weekly collected by means of artificial vagina for one year. Immediately after collection, mass motility (MM) was evaluated and qualified from 1 (bad) to 3 (very good) by observing wave motion under a phase contrast microscope (10x). Individual (IM) and progressive motility (PM) rates were assessed by a  Sperm Class Analyzer (SCA 2002 Microptic, Spain). Semen was diluted (20 · 106 spermatozoa/ml) and loaded in warmed slides. Five fields per sample were evaluated (minimum 500 spermatozoa/sample) under a phase contrast microscope (10x). Statistical analysis was carried out by means of the GLM procedure and Duncan test for means. Values for PM were maximal during spring (72.54 ± 1.28 a,b) and winter months (69.05 ± 1.55 a), and decreased in summer (64.66 ± 1.36 c) and autumn (67.80 ± 1.18 b,c) (a,b,c: p < 0.05). Individual motility highly correlated with PM (r = +0.06, p < 0.001). We conclude that the new computer-assisted semen analysis is a useful tool for analyzing seasonal changes affecting sperm motility in our local Asturiana de los Valles breed (Performed in collaboration with ASEAVA).

Reprod Dom Anim 41, 311–377 (2006)

Morphometric and chromatin assessment in brown bear (Ursus Arctos) ejaculated spermatozoa 2016-12-30T09:48:33+00:00

Morphometric and chromatin assessment in brown bear (Ursus Arctos) ejaculated spermatozoa

V García-Macías, F Martínez-Pastor, P Paz, M Álvarez, S Borragan, F Martínez, E Anel and L Anel

Animal Reproduction, Cell Biology and Anatomy, University of León, Cabarceno Park, Cantabria, Spain

The Brown bear is a highly endangered species in Spain. The study of its spermatology will allow us to acquire the knowledge for establish a germplasm bank.  Sperm heads consist mainly of chromatin, thus their shape should be related to chromatin status. Potential problems in chromatin may result in subtle  changes in sperm head shape that could be detectable with computer-assisted morphology assessment (CASMA). Our objectives are to describe sperm  morphology results in brown bear ejaculates and to connect these parameters with those derived from sperm chromatin structure assay (SCSA). Four bears were electroejaculated under general anaesthesia. From each ejaculated we applied SCSA (Evenson, 2002; J Androl; 23:25–43) and morphometry  assessment (Diff-Quik and Sperm Class Analyser; Microptic S.L.). For SCSA we obtained the mean and standard deviation of the DNA fragmentation index  (SD-DFI). CASMA results included the area, perimeter, length, width and elipticity of heads. We analysed data using ANOVA (male as factor) and Spearman  correlations between morphometry and SCSA. The CASMA system detected subtle morphometric differences among spermatozoa from different bears (21.75 ± 2.14; 21.76 ±  2.32; 20.53 ± 2.40; 22.24 ± 1.69 area media ± IQ range for each bear). SD-DFI (6.9, 11.5, 5.5 and 12.1 for each bear) and the area rendered good correlations (r =  1; p < 0.01). Thus, SCSA and sperm head morphometry may be powerful tools to improve semen examination in the brown bear (Financed by CICYT

[CGL  2004-0278/BOS] and CANTUR).

Reprod Dom Anim 41, 311–377 (2006)

Expression and localization of δ, κ and µ opioid receptors in human spermatozoa and implications for sperm motility 2016-12-30T09:48:33+00:00

Expression and localization of δ, κ and µ opioid receptors in human spermatozoa and implications for sperm motility

Ekaitz Agirregoitia, Asier Valdivia, Arkaitz Carracedo, Luis Casis, Javier Gil, Nerea Subiran, Carmen Ochoa and Jon Irazusta

Department of Physiology, Faculty of Medicine and Dentistry, University of the Basque Country, PO Box 699, Bilbao, 48080 Bizkaia, Spain

CONTEXT: Endogenous opioid peptides signal through δ, κ and µ-opioid receptors. Some of these peptides such as endorphins and enkephalins are present in the male reproductive tract, but the presence of the corresponding receptors in human sperm cells has not yet been reported.
OBJECTIVE: To study the expression and localization of δ, κ and µ-opioid receptors on human spermatozoa and the implication in sperm motility.
METHODS: The expression of receptors was studied by RT-PCR, Western blot and immunofluorescence techniques. We evaluated the effects of activation of each opioid receptor by specific agonist and antagonist.
RESULTS: Human spermatozoa express δ, κ and µ-opioid receptors. These receptors were located in different parts of the head, in the middle region and in the tail of the sperm. Progressive motility of spermatozoa, an important parameter to evaluate male fertility, was found to be significantly reduced after incubation with the µ-receptor agonist morphine, whereas this effect was antagonized in the presence of the corresponding antagonist, naloxone. The δ receptor antagonist naltrindole significantly reduced progressive motility immediately after its addition. However, the δ receptor agonist DPDPE had no significant effect. Finally, neither the κ receptor agonist U50488 nor its antagonist nor-binaltorphimine significantly affected the progressive motility of human spermatozoa.
CONCLUSION: We report for first time the presence of functional δ-, κ- and µ-opioid receptors in human sperm membranes. These findings are indicative of a role for the opioid system in the regulation of sperm physiology.

doi: 10.1210/jc.2006-0599

Morphometric characterization of epididymal and ejaculated spermatozoa from brown bear (Ursus Arctos) 2016-12-30T09:48:33+00:00

Morphometric characterization of epididymal and ejaculated spermatozoa from brown bear (Ursus Arctos)

V. Garcia-Macias, F. Martinez-Pastor, M. Alvarez, P. Paz, S. Borragan, M. Celada, E. Anel and L. Anel

Reproduction and Obstetrics, University of León, León, Spain; Cell Biology, University of León, León, Spain; Cabarceno Park, Cantabria, Spain

Sperm morphology is an useful characteristic for estimating potential fertility. Currently, we are obtaining baseline information on various aspects of reproduction in the brown bear (Ursus arctos) with the intention of using the knowledge to establish a germplasm bank for the species. In the present report, we describe the results obtained using assisted sperm morphology analysis (ASMA, Sperm Class Analyzer®; Microptic S.L, Barcelona, Spain) to analyze the morphological differences in epidydimal (caput, corpus, and cauda) and ejaculated brown bear spermatozoa. A post-mortem epididymal sperm sample was
obtained from an adult brown bear after accidental death. The epididymides were excised, washed, and dissected into the three major segments; caput, corpus and cauda. Then multiple incisions were made in the tissue to allow migration of spermatozoa into the surrounding medium. Semen was collected by electroejaculation from five adult brown bears living in a semi-free ranging environment in the Cabarceno Park (Cantabria, Spain). Anesthesia was induced using tiletamine + zolazepan (Zoletil 100®; Virbac, Carras, France; 7 mg/kg), and ketamine (Imalgene 1000®; Rhone Merieux, Lyon, France; 2 mg/kg). The electroejaculation unit (PT Electronics®; Boring, Oregon) was connected to a 3-lateral electrode transrectal probe (26 mm in diameter, 320 mm in length). Ejaculation occurred at 6–10 V/250–300 mA. For head morphometry assessment, sperm samples were fixed in glutaraldehyde and slides were smeared and air-dried for 2 h. The samples were then stained with Diff-Quik® staining (37°C; 10 min in the red component and 15 min in the blue component). The area, perimeter, length and width, and ellipticity (length/width) of heads were measured from at least 100 spermatozoa/slide. As shown in Table 1, values obtained for each measure were similar in both epididymal and ejaculated spermatozoa. These results provide normal morphometry values for brown bear spermatozoa, a  potentially useful characteristic for predicting fertility.

Reproduction, Fertility and Development 18(2) 217–218

Estimation of sperm quality in fresh and frozen–thawed semen from Asturiana de los Valles bulls 2016-12-30T09:48:33+00:00

Estimation of sperm quality in fresh and frozen–thawed semen from Asturiana de los Valles bulls

C. Tamargo, M. Carbajo, C. Diez, D. Martin and C. O. Hidalgo

Servicio Regional de Investigación y Desarrollo Agroalimentario

Artificial insemination and semen cryopreservation have significantly improved the breeding potential of male animals. However, current freezing techniques commonly result in reduced semen quality (Januskauskas et al. 1999 Theriogenology 52, 641-658), and surviving cells are affected post-thaw either structurally or functionally (Nagy et al. 2004 Anim. Reprod. Sci. 80, 225-235). In this work we analyze the impact of cryopreservation on Asturiana de los Valles bull sperm. Ejaculates (n = 373) from seven adult bulls were weekly collected by means of artificial vagina. Immediately after collection, routine parameters including volume (V), mass motility (MM), and concentration (C) of sperm cells were evaluated. Then the semen was extended with a commercial extender, loaded into 0.25-mL plastic straws at a concentration of 23 × 106 per straw, frozen and stored for further analysis. Four straws per ejaculate were thawed, pooled and analyzed for motion characteristics by means of a CASA system (Sperm Class Analyzer, SCA 2002® Microptic S. L., Barcelona, Spain) added to an optical phase-contrast microscope with heatable (37°C) stage. Immediately after thawing, we analyzed the % of motile spermatozoa (MS) and the % of progressively motile spermatozoa (PMS); then samples were incubated for 3 h at 37°C and MS and PMS were measured again (MS3 and PMS3, respectively). Functional integrity of the plasmallema was evaluated by the hypoosmotic swelling test (HOST) together with the % of typical tail coiling/swelling (percentage of HOST-positive spermatozoa, HOST-PS). The % of viable spermatozoa (VS)

[membrane integrity was evaluated by fluorescence microscopy with a dual staining system (propidium iodide (PI) and 6-carboxyfluorescein diacetate (CFDA)]. Sperm showing partial or complete red fluorescence (PI staining) were considered nonviable, whereas sperm showing complete green fluorescence were considered viable. Altered acrosomes (AA) and morphological abnormalities were also determined. The % of morphological abnormalities was classified according to their location in head (HA), midpiece (MA), and tail (TA). Proximal and distal cytoplasmic droplets were counted as separate abnormalities (CD). Data were analyzed by the MEANS procedure of SAS (SAS Institute, Inc., Cary, NC, USA). A significant (P < 0.05) decrease in the sperm motility was observed after freezing/thawing (MS: 80.20 ± 0.75 vs. 47.36 ± 1.04, and PMS: 68.73 ± 0.73 vs. 42.14 ± 0.96 for fresh and frozen-thawed semen, respectively). Also, the frozen-thawed sperm showed increased % of HA, MA, AA, HOST-PS, and VS (P < 0.05). These morphological abnormalities could contribute to decreasing sperm motility. The new computer and video technologies provide useful information about sperm quality and can be used in the daily routine of processing semen.

doi: 10.1071/RDv18n2Ab111

Description of genitalia and sperm recovered postmortem from a pygmy sperm whale, Kogia Breviceps 2016-12-30T09:48:34+00:00

Description of genitalia and sperm recovered postmortem from a pygmy sperm whale, Kogia Breviceps

F. Martinez-Pastor, V. Garcia-Macias, J. Garcia, M. Alvarez, E. Anel, P. Herraez, P. de Paz and L. Anel

Animal Reproduction, University of León, León, Spain; Cell Biology, University of León, León, Spain

A pygmy sperm whale (Kogia breviceps; adult male; 350 kg) was stranded and died on a beach near Cabo Bustos (Asturias, North of Spain) on March 12th, 2005. Finding specimens of this species is a rare event on Spanish shores, although this whale is not considered endangered. Postmortem examination was performed 24 h later. Genitalia (testicles and epididymides) were extracted. The postmortem report indicated that vas deferens and seminal glands seemed to contain an important amount of semen, which was not recovered. Refrigerated genitalia were send to our laboratory, arriving around 40 h postmortem. The refrigerated testicles were in poor physical condition upon arrival, indicating advanced tissue detoriation. The epididymides (very long) were not closely attached to the testicles, but were connected by a loose conjunctive membrane. We divided the epididymides into four regions that approximated the (1) caput, (2) mid-region, (3) corpus, and (4) cauda. Physical characteristics of the genitalia are described in Table 1. The left testicle was larger, and possibly more active, than the right one. A sperm sample was obtained from the cauda region after incising the tissue. Osmolality and pH of the sample were 428 mOsm/kg and 6.62, respectively (maybe due to tissue breakdown) and the sperm concentration was 1194 ! 106/mL. Spermatozoa were immotile, even after diluting in buffered medium; it is possible that postmortem damage occurred quickly. However, using flow cytometry we determined that 57% of cauda spermatozoa had intact plasma membranes and acrosomes (determined by staining with 37 mmol/mL propidium iodide and 1 !g/mL PNA-FITC; Sigma, Madrid, Spain). Examination by phase contrast microscopy (!600) showed many spermatozoa with abnormal heads and bent midpieces and flagella, even in the cauda (13% and 21%, respectively). Sperm head morphometry was studied using DiffQuick staining and an automated analysis system (SCA2000; Microptic, Barcelona, Spain). Mean sperm head size was 3.71 ± 0.19 ! 2.61 ± 0.12 !m in width and length, respectively. Computer analysis (AnalySiS-GmbH, Cologne, Germany) of phase contrast images revealed that the mean size of the sperm midpiece and flagellum were 3.44 ± 0.19 and 40.95 ± 2.02 !m, respectively. The information obtained after postmortem recovery of the testes and epididymis should be useful to future conservation efforts of the pygmy sperm whale and similar species. The rapid deterioration of the testicular tissue by 40 h postmortem was not expected since good quality sperm samples have been obtained at similar postmortem intervals in other species. Therefore, we recommend that postmortem sperm recovery should be accomplished as  rapidly as possible in this species.

doi:10.1071/RDv18n2Ab223

Epididymal sperm cryopreservation of one Somalia wild ass (Equus Africanus Somaliensis) using six different extenders 2016-12-30T09:48:34+00:00

Epididymal sperm cryopreservation of one Somalia wild ass (Equus Africanus Somaliensis) using six different extenders

M. Álvarez, F. Martínez-Pastor, V. García-Macías, S. Borragán, M. Celada, J. Bernardo, N. Gonzalez, S. Alves and L. Anel

Animal Reproduction, University of León, León, Spain; Cell Biology, University of León, León, Spain; Cabarceno Park, Santander, Cantabria, Spain

The Somalia wild ass (Equus africanus somaliensis) is a critically endangered taxon (IUCN 2004 red list) which could benefit from biological resource banking. In this work, we studied the effect of different extenders applied to the cryopreservation of epididymal sperm obtained from one male of this subspecies. This animal (13 years old; housed in Cabarceno Park, Cantabria, Spain) was castrated because of very aggressive behavior with other mature males. Genitalia were dissected and weighed (testicles: right, 166 g, and left, 179 g; cauda epididymis: right, 9.3 g, and left, 11.8 g). Sperm were flushed from the cauda epididymis, yielding 15 mL of sample. Sperm concentration was 15 ! 109 spermatozoa/mL, totaling 225 ! 109 (allowing 4500 doses at 50 ! 106 sperm/dose). Sperm motility (TM = % total motile; PM = % progressive; VAP = average path velocity) was assessed by CASA (Microptic, Barcelona, Spain). Viability (VIAB = % viable sperm) and acrosomal status (ACR = % viable spermatozoa with intact acrosomes) were assessed using propidium iodide (37 !mol/L) and PNA-FITC (1 ng/L) and flow cytometry. Chemicals were purchased from Sigma (Madrid, Spain). Part of the sample was divided into six aliquots and diluted 1:1 with different extenders: UL4: Tes-Tris-Fructose (TTF), 10% egg yolk (EG), and 4% glycerol (G); UL8: TTF, 20% EG, and 8% G; AND4: Andromed® (Minitüb, Tiefenbach, Germany) and 4% G; AND7: Andromed® and 7% G; GENT: Gent 1045; and INRA: INRA96 and 4% G. Andromed, Gent, and INRA are commercial extenders. Samples were cooled to 5°C (-0.2°C/min) and then diluted to 200 ! 106 sperm/mL. Samples were packed (0.5-mL straws) and frozen using a biofreezer (from 5°C to -15°C at -15°C/min, and from -15°C to -100°C at -25°C/min). Samples were thawed at 65°C for 6 s, and assessed as for pre-freezing (Table 1). Post-thawing motility recovery using AND7 was excellent. The highest viability recovery was achieved by UL4, although
that in AND7 was similar. The poor results of equine commercial extender Gent 1045 in this species are remarkable. Our results highlight the importance of species differences in the field of sperm cryopreservation. It is necessary to carry out continuous research for optimizing cryopreservation protocols in order to create germplasm banks for wild species.

doi:10.1071/RDv18n2Ab215

Addition of reduced glutathione to thawing medium improved the sperm motility and reduced ros generation in frozen ovine and caprine spermatozoa 2016-12-30T09:48:34+00:00

Addition of reduced glutathione to thawing medium improved the sperm motility and reduced ros generation in frozen ovine and caprine spermatozoa

J. C. Gardón, J. A. Rodriquez and J. Gadea

University of Murcia

The processes of cooling and freezing/thawing produce physical and chemical stress on the sperm membrane, and this stress is associated with oxidative stress and reactive oxygen species (ROS) generation that further reduce sperm viability and fertilizing ability. It is known that the process of freezing is associated with a significant reduction of the intracellular reduced glutathione (GSH) content. The aim of these experiments was to investigate the effects of addition of GSH to thawing extenders on motility parameters and ROS generation in frozen-thawed ovine and caprine spermatozoa. Frozen spermatozoa from eight rams (Ovis aries) and eight bucks (Capra hircus) (generously provided by Ovigen, Zamora, Spain) were thawed in a water bath at 37°C for 30 s and resuspended in sperm-TALP medium (Parrish et al. 1986 Theriogenology 25, 591-600) without (control) and with addition of 1 mM or 5 mM GSH. After 30 min of incubation at 37°C, sperm motility was evaluated using a computer-assisted sperm analysis (CASA) system (SCA, Microptic, Barcelona, Spain). The recorded parameters of motility were: % total, % progressive, curvilinear velocity, straight-line velocity, average path velocity, linearity of the curvilinear trajectory, straightness, amplitude of lateral head displacement, wobble of the curvilinear trajectory and beat cross frequency. Another set of sperm samples was incubated in the presence of (0.7 ¼M) 22,72-dichlorodihydrofluorescein diacetate (Gadea et al. 2005 J. Androl. 26, 396-404) to estimate production of ROS by flow cytometry. Data were analyzed by two-way ANOVA, considering the specific sperm treatment (GSH addition) and the males as the main variables. In ram frozen spermatozoa, all of the motility parameters were significantly improved when the medium was supplemented with GSH (P < 0.01) with even better results when 5 mM GSH was used. As an example, progresive motility increased from 31.16% (control) to 39.17 and 43.97%, respectively, for 1 and 5 mM GSH. Despite of the male effect detected (P < 0.01), all eight rams studied presented a similar pattern (interaction P > 0.05). The generation of ROS was significantly reduced when GSH was added (6.23a for control vs. 5.32b and 3.85c for 1 and 5 mM, respectively; P < 0.01). In buck frozen spermatozoa, % motility and progressive motility were significantly higher in GSH groups than in the control (P < 0.01), with no differences between 1 and 5 mM GSH. However, for the other motility parameters, the differences were not significant, which probably could be related to differences in the pattern shown by different animals (interaction of buck by treatment P < 0.05). ROS generation was significantly reduced when GSH was added (7.50a for control vs. 4.32b and 2.70b for 1 and 5 mM, respectively; P < 0.01). The addition of GSH to the thawing medium had a positive influence on the parameters studied in both species, increasing the motility patterns and reducing the ROS generation. In conclusion, we can assume that the addition of reduced glutathione to the thawing medium exerts a protective effect on spermatozoa functionality.

doi:10.1071/RDv18n2Ab94

Effect of collection rhythm on spermatozoa and droplet concentration of rabbit semen 2016-12-30T09:48:34+00:00

Effect of collection rhythm on spermatozoa and droplet concentration of rabbit semen

Castellini C., Lattaioli P., Cardinali R., Dal Bosco A.

Dpt. Biologia Vegetale, Biotecnologie Agroambientali e Zootecniche, Borgo 20 Giugno, 74 06100, PERUGIA, Italy

The aim of the paper was to analyse the effect of collection rhythms on spermatozoa and droplet concentration of rabbit semen. Thirty adult New Zealand White rabbit bucks were submitted to 3 collection rhythms: every day (D), every week (W), every 2 weeks (2W). The trial lasted 71 days and a total of 790 ejaculates were collected. Volume, concentration of spermatozoa, droplets and their dimensions were evaluated. Ejaculate volume and concentration of spermatozoa were the lowest (0.37±0.08 mL and 48.9±43.8×106 mL-1, respectively) when the samples were collected daily, whereas the ratio droplets/spermatozoa was the highest (6.4±3.3). The output of spermatozoa per week was the highest with W rhythm (173.4±105.3×106), followed by D (134.2±75.1×106) and by 2W (79.5±49.8×106 ) collection whereas the weekly output of droplets was the highest for D (810.2±615.7×106). In the following order of collection the spermatozoa concentration was similar in W and 2W while D showed a sharp decline of values after few collections. Semen droplets with respect to spermatozoa showed a more stable trend showing that droplets are able to respond better to high soliciting. Bucks submitted to an intensive rhythm were able to increase about 9-fold (respecting to W) the secretion of droplets by prostate glands. As a result, the ratio droplet/spermatozoa showed the highest values in daily collected semen (6.4±3.3). The repeatability of seminal traits showed that volume and spermatozoa concentration had the highest value followed by droplets, and ratio droplets/spermatozoa. In conclusion, it is possible to affirm that the collection rhythm, besides influencing the concentration of spermatozoa, also affects the rate of droplet production. A collection rhythm every 2 weeks was detrimental to both the spermatozoa and droplet output.

doi: 10.4995/wrs.2006.551

Kinematic changes during the cryopreservation of boar spermatozoa 2016-12-30T09:48:34+00:00

Kinematic changes during the cryopreservation of boar spermatozoa

Cremades T, Roca J, Rodriguez-Martinez H, Abaigar T, Vazquez JM, Martinez EA

Department of Medicine and Animal Surgery, Faculty of Veterinary Medicine, Campus de Espinardo, University of Murcia, E-30071 Murcia, Spain

The present study evaluates the effect that various steps of a conventional cycle of cryopreservation have on the patterns of movement exhibited by boar spermatozoa. Sperm-rich ejaculate fractions collected from 24 mature fertile boars (1 ejaculate per boar) were cryopreserved following a standard freeze-thaw procedure with 0.5-mL plastic straws. Overall sperm motility and the individual kinematic parameters of motile spermatozoa (assessed by the computer-aided sperm analysis system Sperm Class Analyzer

[SCA]) were recorded in 5 steps of the cryopreservation procedure. These steps were as follows: 1) at the time that the fresh semen was extended, 2) at 17 degrees C, after sperm concentration by centrifugation and re-extension of the pellet with lactose-egg yolk extender; 3) at 5 degrees C, after added freezing extender; 4) at the time that thawed semen was held in a water bath at 37 degrees C for 30 minutes; and 5) at the time that thawed semen was held in a water bath at 37 degrees C for 150 minutes. Data from individual motile spermatozoa, defined by 7 kinematic parameters (curvilinear velocity [VCL], straight-line velocity [VSL], average path velocity [VAP], linearity [LIN], straightness [STR], mean amplitude of lateral head displacement [ALH], and beat cross frequency [BCF]), were analyzed using a pattern analysis technique (PATN) to identify and quantify populations and subpopulations of motile sperm within the semen samples. After the first cluster analysis, 3 motile sperm populations (P) were identified (P1: progressive and/or vigorous cells [90.4%], P2: poorly progressive cells [8.3%], and P3: nonprogressive cells [1.3%]). These populations remained constant (P > .05) throughout the 5-step cryopreservation procedure. A second PATN was carried out within the P1 sperm population, which identified 3 sperm subpopulations (sP) (eg, sP1: cells with progressive and vigorous movement [58.7%], sP2: progressive cells only [24.6%], and sP3: vigorous cells only, hyperactive-like [16.7%]). Although the relative frequency of these 3 subpopulations varied among ejaculates (boars), there was no interaction with any cryopreservation step we examined. Whereas sP1 remained constant (P > .05), sP2 and sP3 varied significantly (P < .05) through the cryopreservation procedure, with the increase in sP3 after centrifugation at 17 degrees C and during cooling at 5 degrees C considered particularly relevant. In conclusion, the present study confirms the heterogeneity of sperm movement patterns in boar semen, patterns that vary through the cryopreservation procedure, especially after removal of the seminal plasma by centrifugation and subsequent extension at 17 degrees C and after the slow cooling at 5 degrees C, when obvious increases in hyperactivated movement appeared. The vast majority of spermatozoa, those exhibiting progressive and vigorous movement, remained constant during the cryopreservation procedure, although the proportion differed among boars.

J Androl. 2005 Sep-Oct;26(5):610-8.

Do parameters of seminal quality correlate with the results of on-farm inseminations in rabbits? 2015-05-08T11:45:37+00:00

Do parameters of seminal quality correlate with the results of on-farm inseminations in rabbits?

Lavara R, Moce E, Lavara F, Viudes de Castro MP, Vicente JS

Universidad Politecnica de Valencia, Departamento de Ciencia Animal, Laboratorio de Biotecnologia de la Reproduccion, Camino de Vera, 46071 Valencia, Spain

This study was conducted to determine if different sperm characteristics correlate with the in vivo fertility of rabbit sperm. A total of 2765 heterospermic inseminations were performed in commercial rabbitries using 50-pooled samples of fresh semen. Sperm motility and morphological evaluations were performed on each of the heterospermic pooled samples to asses the seminal quality, and the percentage of kindling rate (76.2%) and number of kits born alive (9.3) were recorded. Sperm motility parameters, assessed using a computer-assisted sperm analysis (CASA) system (Sperm Class Analyzer, Microptic, Barcelona, Spain), were: average path velocity, curvilinear velocity, straight-line velocity, linearity, amplitude of lateral head displacement, beat cross-frequency, wobble and percentage of total motile spermatozoa. Morphological analyses included the percentage of sperm with a normal apical ridge, the percentage of sperm with cytoplasmatic droplets and the percentage of abnormal sperm. Significant correlations were observed between kindling rate and the percentage of total motile cells (r=0.31; P<0.05), linearity index (r=-0.32; P<0.05) and the percentage of abnormal sperm in the sample (r=-0.32; P<0.05). Regression models including motility and the morphological parameters explained 45% of the variation in kindling rate. These results indicate that motility parameters, determined by CASA systems, in combination with sperm morphology analyses can provide some information about the fertilizing potential of rabbit sperm.

Theriogenology. 2005 Sep 15;64(5):1130-41.

Natural Mediterranean photoperiod does not affect the main parameters of boar-semen quality analysis 2015-05-08T11:46:16+00:00

Natural Mediterranean photoperiod does not affect the main parameters of boar-semen quality analysis

Rivera MM, Quintero-Moreno A, Barrera X, Palomo MJ, Rigau T, Rodriguez-Gil JE

Unit of Animal Reproduction, Department of Animal Medicine and Surgery, School of Veterinary Medicine, Autonomous University of Barcelona, E-08193 Bellaterra, Spain

Photoperiod is an important factor in the modulation of male reproduction in mammals. In boars, however, it is a controversial factor. The main aim of this work is to determine the precise effect of the natural, Mediterranean photoperiod on boar-semen quality. To do this, boars were housekept in strictly controlled temperature and humidity conditions, whereas light periods were also strictly adjusted to obtain a light-cycle in the farm. The work was performed over a period of one year, thus allowing for the determination of the putative yearly oscillations of boar-semen quality. Variations of the natural Mediterranean photoperiod do not induce substantial changes in overall semen-quality parameters like the percentages of viability, morphological abnormalities and total motility, the response to the osmotic resistance test and sperm motion characteristics. Only the motile-sperm subpopulation structure was significantly (P<0.05) changed depending on the variations of the natural photoperiod. Furthermore, the boar-semen ability for storage at 15-17 degrees C in a commercial extender was not modified by photoperiod changes. Our results indicate that the natural Mediterranean photoperiod does not induce strong changes in boar-semen characteristics, probably due to boar sperm having a strong capability of adaptation to the light variations of this photoperiod.

Theriogenology. 2005 Sep 1;64(4):934-46

Do different portions of the boar ejaculate vary in their ability to sustain cryopreservation? 2016-12-30T09:48:34+00:00

Do different portions of the boar ejaculate vary in their ability to sustain cryopreservation?

Pena FJ, Saravia F, Nunez-Martinez I, Johannisson A, Wallgren M, Rodriguez Martinez H.

Animal Reproduction and Obstetrics, Faculty of Veterinary Medicine, University of Extremadura, Avda de la Universidad s/n, 10071 Caceres, Spain; Section of Comparative Reproduction, Obstetrics and Udder Health, Department of Clinical Sciences, Swedish University of Agricultural Sciences (SLU), Box 7039, SE-750-07 Uppsala, Sweden

Previous studies have shown sperm quality post-cryopreservation differs depending on the fraction of the seminal plasma boar spermatozoa are fortuitously contained in. As such, spermatozoa contained in the first 10mL of the sperm-rich fraction (portion I) have better sustained handling procedures (extension, handling and freezing/thawing) than those contained in the ulterior part of a fractionated ejaculate (second portion of the sperm-rich fraction and the post-spermatic fraction, portion II). However, those studies were performed using pooled samples. In the present study, individual ejaculates were used. Split ejaculates (portions I and II) from five boars were frozen and thawed using a conventional freezing protocol, followed by computer-assisted motility and morphology analysis (CASA and ASMA, respectively), as well as an Annexin-V assay for spermatozoa from each boar and ejaculate portion. Significant differences between portions were observed in all ASMA-derived variables, except in one boar. Also significant differences were observed between boars and ejaculate portions in sperm quality post-thaw. We identified, however, boars showing best results of motility and sperm membrane integrity post-thaw in portion I, while in other boar the best results was observed in portion II. It is concluded that the identification of the ejaculate portion more suitable to sustain cryopreservation in each individual boar may be a readily applicable and easy technique to diminish variation in sperm freezability among boars.

Anim Reprod Sci. 2005 Aug 3.

Supplementation of the thawing media with reduced glutathione improves function and the in vitro fertilizing ability of boar spermatozoa after cryopreservation 2015-05-08T11:46:47+00:00

Supplementation of the thawing media with reduced glutathione improves function and the in vitro fertilizing ability of boar spermatozoa after cryopreservation

Gadea J, Gumbao D, Matás C, Romar R

Department of Physiology, School of Veterinary, University of Murcia, Spain

In this study, we evaluated the effects of glutathione (L-g-glutamyl-L-cysteinylglycine; GSH) supplementation of the thawing extender on semen parameters to compensate for the decrease in GSH content observed during sperm freezing. To fully address these questions, we used a set of functional sperm tests. These included tests of motility and motion parameters, changes in sulfhydryl group content in membrane proteins, capacitation status, measures of intra-cellular reactive oxygen species generation, sperm chromatin condensation, and in vitro penetration of immature oocytes. The main findings emerging from this study were that addition of GSH to the thawing media resulted in a lower number of capacitated viable spermatozoa, a decrease in the number of spermatozoa with changes in the sulfhydryl groups in membrane proteins, a reduction of the reactive oxygen species generation, a lower chromatin condensation, and a higher penetration ability of oocytes in vitro and a higher proportion of decondensated sperm heads. GSH appears to play an important role in sperm antioxidant defense strategy. Addition of GSH to the thawing extender could be of significant benefit in improving the function and fertilizing capacity of frozen boar spermatozoa.

doi: 10.2164/jandrol.05057

Identification of sperm morphometric subpopulations in two different portions of the boar ejaculate and its relation to postthaw quality 2015-05-08T11:47:14+00:00

Identification of sperm morphometric subpopulations in two different portions of the boar ejaculate and its relation to postthaw quality

Peña FJ, Saravia F, García-Herreros M, Núñez-martínez I, Tapia JA, Johannisson A, Wallgren M, Rodríguez-Martínez H

Section of Animal Reproduction and Obstetrics, Department of Herd Health and Medicine, Faculty of Veterinary Medicine, University of Extremadura, Cáceres, Spain

A statistical approach using sequentially principal component analysis (PCA), clustering, and discriminant analyses was developed to identify sperm morphometric subpopulations in well-defined portions of the fresh boar ejaculate. Semen was obtained as 2 portions (the first 10 mL of the sperm-rich fraction and the rest of the ejaculate, respectively) and frozen using a conventional protocol. Before freezing, an aliquot was used for computer-assisted sperm morphometry analysis (ASMA). Postthaw quality was evaluated using computer-assisted sperm analysis (CASA), and an annexin-V/PI assay evaluated sperm membranes. The PCA revealed that 3 variables represented more than 78% of the cumulative variance in sperm subpopulations. The clustering and discriminant analyses, based on 5780 individual spermatozoa, revealed the existence of 4 sperm subpopulations. The relative percentage of these subpopulations varied between boar and ejaculate portions. Linear regression models based on measured morphometric characteristics could account for up to 36% of the percentage of intact sperm membranes postthaw. The ASMA protocol used in our study was useful to detect subtle morphometric differences between spermatozoa, and the combination of this analysis with a multivariate statistical procedure gave new information on the biological characteristics of boar ejaculates that is not given by conventional sperm analysis.

doi: 10.2164/jandrol.05030

Sperm motility patterns and metabolism in Catalonian donkey semen 2016-12-30T09:48:34+00:00

Sperm motility patterns and metabolism in Catalonian donkey semen

Miro J, Lobo V, Quintero-Moreno A, Medrano A, Pena A, Rigau T

Unit of Reproduction, Department of Animal Medicine and Surgery, School of Veterinary Medicine, Autonomous University of Barcelona, E-08193 Bellaterra, Spain

The Sperm-Class Analyzer detected four subpopulations of spermatozoa with different motility characteristics in the ejaculate of the Catalonian donkey. Significant differences (P < 0.001) in the distribution of these subpopulations, as well as in total sperm number and percentage total motility, were seen in the diluted semen of four sampled donkeys. All the ejaculates evaluated showed excellent semen quality characteristics; the sperm they contained was more rapid than horse sperm. Principal components analysis showed sperm l-lactate production to be a good predictor of semen condition. This, plus the characteristics of the motility patterns of the different sperm subpopulations, provides an excellent overall indicator of semen quality.

Theriogenology. 2005 Apr 1;63(6):1706-16.

Post mortem time and season alter subpopulation characteristics of Iberian red deer epididymal sperm 2015-05-08T11:47:48+00:00

Post mortem time and season alter subpopulation characteristics of Iberian red deer epididymal sperm

Martinez-Pastor F, Diaz-Corujo AR, Anel E, Herraez P, Anel L, de Paz P

Cell Biology and Anatomy, University of León, 24071 León, Spain

We have studied the effect of post mortem time and season on sperm subpopulation pattern and characteristics. We used epididymal samples from free-ranging Iberian red deers harvested during the hunting season. We studied samples at different moments of the year (rut, transition period and post-rut), and at different times post mortem (up to 4 days). Sperm were extracted from the cauda epididymis and their motility was evaluated by means of a CASA system. A principal component and clustering analysis were carried out to identify subpopulations. Post mortem time caused a significant decrease in motility quality, and a general deterioration in subpopulation characteristics. We found three subpopulations the first day, and the one indicating good sperm quality decreased with post mortem time until it disappeared on the fourth day. This may indicate considerable impairment of the samples after 72 h post mortem, which could compromise their use in AI programs. With regard to season, subpopulation pattern and characteristics were better in the transition and post-rut periods. Moreover, we found one subpopulation formed by mature spermatozoa, which increased from rut to post-rut. This might be a negative fact, because samples collected after the rut may undergo hypermaturation, which possibly impairs fertility. Our results are of interest for the management of wildlife germplasm banks based on post mortem sperm recovery.

doi: 10.1016/j.theriogenology.2005.01.003

Cooling and freezing of boar spermatozoa: supplementation of the freezing media with reduced glutathione preserves sperm function 2015-05-08T11:48:02+00:00

Cooling and freezing of boar spermatozoa: supplementation of the freezing media with reduced glutathione preserves sperm function

Gadea J, García-Vazquez F, Matás C, Gardón JC, Cánovas S, Gumbao D.

Department of Physiology, Facultad de Veterinaria, Universidad de Murcia, 30100 Murcia, Spain

In this study, we evaluated the effects of glutathione (L-gamma-glutamyl-L-cysteinylglycine; GSH) supplementation of the freezing extender on semen parameters during the cooling (2 hours at 5 degrees C) and freezing phases of the cryopreservation process to compensate for the decrease in GSH content observed during sperm freezing. To fully address these questions, we incorporated a new set of functional sperm tests. These included tests of mitochondrial function, inducibility of the acrosome reaction, in vitro penetration (IVP) of oocytes, changes in sulfhydryl group content in membrane proteins, and capacitation status. The main findings emerging from this study were that the addition of GSH to the freezing media resulted in 1) an improvement in percent motility (%MOT) and motion parameters of thawed spermatozoa, as measured by both microscopic analysis and computer-assisted semen analysis (CASA); 2) a higher number of total viable spermatozoa; 3) a higher number of noncapacitated viable spermatozoa; and 4) a decrease in the number of spermatozoa with changes in the sulfhydryl groups in membrane proteins. This protective effect on sperm function was more pronounced with 1 mM of GSH than with 5 mM of GSH.

doi: 10.2164/jandrol.04155

Identification of sperm-head morphometric subpopulations in Iberian red deer epididymal sperm samples 2016-12-30T09:48:34+00:00

Identification of sperm-head morphometric subpopulations in Iberian red deer epididymal sperm samples

Esteso, M. C., Fernandez-Santos, M. R., Soler, A. J., Montoro, V., Martinez-Pastor, F., Garde, J. J

Grupo de Biología de la Reproducción, Instituto de Investigación en Recursos Cinegéticos, IREC, (CSIC, UCLM, JCCM), Campus Universitario, Albacete, Spain

Computer-assisted sperm morphometry analysis (CASMA) was used in this study to identify sperm morphometric subpopulations in Iberian red deer epididymal sperm samples. Epididymal sperm samples were collected from 37 mature stags and were divided. One portion was diluted in a Tris-citrate-egg yolk medium. A microscope slide was prepared from single extended sperm samples prior to freezing. The remainder of each sample was frozen in nitrogen vapours using a conventional protocol. After thawing, sperm smears were prepared as described for extended samples. All slides were air-dried and stained with Hemacolor. The sperm-head dimensions for a minimum of 145 sperm-heads were analyzed from each sample by means of the Sperm-Class Analyser, and the mean measurements recorded. Each sperm-head was measured for four primary sperm-head parameters, and five parameters of head shape. All sperm morphometric parameters evaluated were placed in a statistical database and a multivariate cluster analysis was performed. The clustering analyses, based on 10 867 individual spermatozoa, revealed the existence of three subpopulations (SP(1), SP(2), SP(3)) of spermatozoa with different morphometric characteristics (p < 0.001). The proportion of spermatozoa present in any of the three subpopulations remained constant (p > 0.05) through the cryopreservation process. Pre-freeze and post-thaw sperm quality was in vitro evaluated by microscopic assessments of individual sperm motility and of plasma membrane and acrosome integrities. In conclusion, our results show that applying the CASMA techniques and multivariate cluster analyses, it was possible to determine that three subtle subpopulations of spermatozoa with different morphometric characteristics coexist in red deer semen.

Reprod Domest Anim 44:206–211.

Characterization of ram (Ovis aries) sperm head morphometry using the Sperm-Class Analyzer 2016-12-30T09:48:34+00:00

Characterization of ram (Ovis aries) sperm head morphometry using the Sperm-Class Analyzer

Maroto-Morales, A., Ramón, M., García-Alvarez, O., Soler, A. J., Esteso, M. C., Martínez-Pastor, F., Pérez-Guzmán, M. D., Garde, J. J

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. Copyright 2010 Elsevier Inc. All rights reserved.

Theriogenology 73:437-48.

Sperm characteristics and in vitro fertilization ability of thawed spermatozoa from Black Manchega ram: electroejaculation and postmortem collection 2015-05-08T11:50:19+00:00

Sperm characteristics and in vitro fertilization ability of thawed spermatozoa from Black Manchega ram: electroejaculation and postmortem collection

García-Alvarez, O., Maroto-Morales, A., Martínez-Pastor, F., Garde, J. J., Ramón, M., Fernández-Santos, M. R., Esteso, M. C., Pérez-Guzmán, M. D., Soler, A. J.

Regional Center of Animal Selection and Reproduction (CERSYRA) JCCM, Valdepeñas, Spain

The aim of this study was to assess two models of sperm collection on the quality and fertility of thawed spermatozoa from Black Manchega rams, a threatened breed. Sperm samples were collected by electroejaculation and postmortem from each male. Samples were diluted with Biladyl and frozen. Motility (subjective and objective by means of computer-assisted semen analysis), membrane integrity, and acrosomal status (microscopy) were assessed on fresh and thawed semen; plasmalemma integrity, mitochondrial membrane potential, DNA integrity, and acrosomal status were evaluated by flow cytometry on thawed semen. Thawed spermatozoa were used in a heterologous in vitro fertilization test. After thawing, the proportion of live spermatozoa with intact membrane (YO-PRO-1-/PI-) was higher for postmortem samples (P<0.001), although the ratio of YO-PRO-1- spermatozoa within the PI- population was higher for ejaculated samples (P=0.007). Likewise, the proportion of live spermatozoa having high mitochondrial membrane potential (MitoTracker+) and intact acrosomes (PNA-) was higher for postmortem samples (P<0.001 and P<0.001, respectively). Considering only live spermatozoa, the ratio of MitoTracker+/PNA- cells was higher for electroejaculated samples (P=0.026 and P=0.003). Both electroejaculated and postmortem samples fertilized oocytes. Nevertheless, electroejaculated samples yielded a higher percentage of hybrid embryos (P=0.041). In conclusion, although postmortem spermatozoa had better sperm quality after thawing, electroejaculated spermatozoa showed higher ratios for sperm quality when only the live population was considered. Electroejaculated and postmortem samples might be used for germplasm banking of this threatened breed, but the fertility of postmortem spermatozoa might be lower.

Theriogenology 72:160-8.

DNA fragmentation assessment by flow cytometry and Sperm-Bos-Halomax (bright-field microscopy and fluorescence microscopy) in bull sperm 2015-05-08T11:51:00+00:00

DNA fragmentation assessment by flow cytometry and Sperm-Bos-Halomax (bright-field microscopy and fluorescence microscopy) in bull sperm

García-Macías, V., de Paz, P., Martinez-Pastor, F., Alvarez, M., Gomes-Alves, S., Bernardo, J., Anel, E., Anel, L.

Animal Reproduction and Obstetrics, University of León, 24071, León, Spain

The aim of this study was to find the relationship between fertility (as 90-day non-return rates) and DNA fragmentation assessed by two techniques [sperm chromatin structure assay (SCSA) and Sperm-Bos-Halomax (SBH)]. Furthermore, other quality parameters were achieved (motility, morphological abnormalities, cytoplasmic droplets, viability, capacitation and acrosomal and mitochondrial status) and their correlations with fertility were analysed. Bulls were divided into three fertility groups: high [non-return rate (NRR) > 80], medium (80 < NRR > 70) and low (70 < NRR > 40). The results of this study indicate that there is a good correlation between fertility and different parameters of sperm quality (SBH and SCSA parameters, % of spermatozoa with head, neck and total abnormalities, and % of spermatozoa with proximal cytoplasmic droplets) and differences between fertility groups were observed in some of them (SBH and SCSA parameters and % of spermatozoa with head, neck and total abnormalities). In this sense, SBH parameters rendered good correlations with fertility (r = -0.42 using bright light microscope and r = -0.47 with fluorescence). Also, standard deviation of DNA fragmentation index (SD-DFI) and DFIh (cells with High DNA fragmentation index) showed good correlations with fertility (r = -0.41 and r= -0.29). No correlations were observed between SCSA and SBH parameters. A multiple regression shows that four parameters (% of proximal cytoplasmic droplets, % of intact acrosomes in total population, SD-DFI and percentage of fragmented DNA detected by bright light microscope) present a good predictive value of the fertility of sperm samples (r2 = 0.34, p < 0.001).

Int J Androl 30:88-98.

Refrigerated storage of red deer epididymal spermatozoa in the epididymis, diluted and with vitamin C supplementation 2016-12-30T09:48:34+00:00

Refrigerated storage of red deer epididymal spermatozoa in the epididymis, diluted and with vitamin C supplementation

Fernandez-Santos, M. R., Dominguez-Rebolledo, A. E., Esteso, M. C., Garde, J. J., Martinez-Pastor, F.

Reproductive Biology Group, National Wildlife Research Institute (IREC), UCLM-CSIC-JCCM, and Game Research Institute (IDR), UCLM. Albacete, Spain

We have approached the problem of refrigerated storage of epididymal sperm samples from red deer by comparing three options: storing the genital (testicles within the scrotum), diluting the semen in extender or diluting the semen in extender supplemented with an anti-oxidant. Twenty-nine pairs of testes were collected. Spermatozoa from one of each of the pairs were immediately recovered, and diluted to 400 x 10(6) sperm/ml in Tris-citrate-fructose with 20% egg yolk. Control group was stored as such, and Anti-oxidant group was supplemented with 0.8 mm vitamin C. The remaining epididymides and the diluted samples were stored at 5 degrees C and spermatozoa were analysed at 0, 24, 96 and 192 h for: motility

[computer-assisted semen analysis (CASA)], acrosomal integrity, sperm viability (eosine/nigrosine staining), normal tails and chromatin status [sperm chromatin structure assay (SCSA)]. In general, seminal quality decreased with storage time. Vitamin C supported progressive motility better at 24 h (median 42% vs 23% Control and 15% epididymis), reduced the incidence of tail abnormalities and protected chromatin. Storing the semen in the epididymis slowed down motility loss, but slightly increased the occurrence of tail abnormalities and viability was lower at 192 h. However, regarding chromatin status, sperm stored in the epididymis was protected similarly to those diluted in the medium supplemented with vitamin C. Although the differences between the three groups were small, there were some advantages in supplementing the extender with vitamin C. Besides, refrigerating the epididymis may be a good option when immediate processing is not available.

Reprod Domest Anim 44:212–220.

Effect of basic factors of extender composition on post-thawing quality of brown bear electroejaculated spermatozoa 2016-12-30T09:48:34+00:00

Effect of basic factors of extender composition on post-thawing quality of brown bear electroejaculated spermatozoa

Anel, L., Gomes-Alves, S., Alvarez, M., Borragan, S., Anel, E., Nicolas, M., Martinez-Pastor, F., De Paz, P.

Animal Reproduction and Obstetrics, University of León, 24071 León, Spain

The improvement of freezing extenders is critical when defining sperm cryopreservation protocols for wild species, in order to create germplasm banks. The aim of this study was to evaluate the effect of additives (Equex Paste and EDTA) supplementation, egg-yolk (10 and 20%) and glycerol (4 and 8%) concentrations and extender osmolality (300 and 320 mOsm/kg) on the post-thawing quality of brown bear semen. Semen was obtained from 20 adult males by electroejaculation, and centrifugated individually (600 x g for 6 min). The pellets were diluted 1:1 in the corresponding extender TTF (TES-Tris-Fructose with the aforementioned variants) and cooled to 5 degrees C. Then, it was diluted down to 100 x 10(6) spz/mL, loaded in 0.25 mL straws and frozen at -20 degrees C/min. After thawing (in water at 65 degrees C for 6s), the semen samples were assessed for motility (CASA), viability (SYBR-14 with propidium iodide), acrosomal status (PNA-FITC with propidium iodide) and mitochondrial activity (JC-1). Extender supplementation with additives rendered significantly higher results for these sperm parameters. Comparing the two percentages of egg yolk, 20% egg yolk showed the highest motility results, percentages of viable spermatozoa and viable spermatozoa with intact acrosome. No differences were detected among samples frozen using 4 or 8% glycerol. For extender osmolality, 300 mOsm/kg showed higher values of VAP, VCL, VSL, and ALH than 320 mOsm/kg. Based on the best performance of sperm motility, viability and acrosome status, we conclude that the most suitable extender to cryopreserve brown bear spermatozoa was TTF adjusted to 300 mOsm/kg, supplemented with 20% egg yolk, 4-8% glycerol, and the additives 1% Equex paste and 2% EDTA.

Theriogenology accepted.

Antlers honestly advertise sperm production and quality 2015-05-08T11:52:25+00:00

Antlers honestly advertise sperm production and quality

Malo AF, Roldan ER, Garde J, Soler AJ, Gomendio M

Reproductive Ecology and Biology Group, Museo Nacional de Ciencias Naturales (CSIC), 28006 Madrid, Spain

We have applied a statistical protocol based on principal component analysis, clustering methods Evolutionary theory proposes that exaggerated male traits have evolved via sexual selection, either through female mate choice or male-male competition. While female preferences for ornamented males have been amply demonstrated in other taxa, among mammals sexual characters are commonly regarded as weapons whose main function is to enhance male competitiveness in agonistic encounters. One particularly controversial hypothesis to explain the function of male sexual characters proposes that they advertise male fertility. We test this hypothesis in red deer (Cervus elaphus), a species where sexual characters (antlers) reach an extreme degree of elaboration. We find that a global measure of relative antler size and complexity is associated with relative testes size and sperm velocity. Our results exclude the possibility that condition dependence, age or time of culling, drive these associations. Red deer antlers could signal male fertility to females, the ability to avoid sperm depletion throughout the reproductive season and/or the competitive ability of ejaculates. By contrast, male antlers could also signal to other males not only their competitive ability at the behavioural level (fighting ability) but also at the physiological level (sperm competition).

Proc R Soc Lond B Biol Sci. 2005 Jan 22;272(1559):149-57.

Comparison of two methods for obtaining spermatozoa from the cauda epididymis of Iberian red deer 2015-05-08T11:52:59+00:00

Comparison of two methods for obtaining spermatozoa from the cauda epididymis of Iberian red deer

Martinez-Pastor, F., Garcia-Macias, V., Alvarez, M., Chamorro, C., Herraez, P., de Paz, P., Anel, L.

Cell Biology and Anatomy, University of León, 24071 León, Spain

We have compared two methods for salvaging epididymal sperm from post-mortem samples from Iberian red deer. Of each pair of testicles (29 samples), one cauda epididymis was processed by means of cuts (sperm was immediately diluted with extender) and the other was detached from the corpus and flushed from the vas deferens with 1 mL of extender. Sperm was processed for cryopreservation, and analyzed just after recovery, pre-freezing and post-thawing. Total spermatozoa recovered, contamination (concentration of epididymal cells and red blood cells (RBCs)) and quality (motility by CASA, and acrosomal status, viability and mitochondrial status by flow cytometry) were used to compare both methods. The number of recovered spermatozoa was similar for both methods. Contamination was higher for the cuts method, but when considering the final dilution before freezing, only RBCs concentration was significantly higher. Motility was similar just after extraction, but higher for both pre-frozen and post-thawed flushed sperm. Pre-freezing acrosomal status (P < 0.05) and viability (P < 0.1) were better for flushing; however post-thawing results were similar for the two methods. A clustering analysis using CASA data showed that the subpopulation pattern of motile sperm was different depending on the method, being better for flushing. With regard to yield, lower contamination (especially RBCs) and, in general, better quality results, flushing seems to be a more recommendable method for post-mortem sperm recovery. The cuts method may be more practical on certain occasions, but care must be taken in order to achieve rapid extension of the sample and to avoid contamination in order to improve sample condition.

Theriogenology 65:471-85.

Washing increases the susceptibility to exogenous oxidative stress in red deer spermatozoa 2015-05-08T11:53:37+00:00

Washing increases the susceptibility to exogenous oxidative stress in red deer spermatozoa

Domínguez-Rebolledo, A. E., Fernández-Santos, M. R., García-Alvarez, O., Maroto-Morales, A., Garde, J. J., Martínez-Pastor, F.

National Wildlife Research Institute (IREC) (UCLM-CSIC-JCCM), 02071 Albacete, Spain

The effects of routine sperm work are often overlooked. We assessed the effect of washing cryopreserved epididymal spermatozoa from red deer (Cervus elaphus hispanicus, Helzheimer 1909). After thawing, epididymal samples (four stags) were diluted in TALP-HEPES. A split was left untouched, another was centrifuged (300 x g, 5 min) and resuspended, and a third was centrifuged and the supernatant substituted by fresh TALP-HEPES (washing). Each split was supplemented either with nothing, 1mM of the antioxidant Trolox, 100 microM of the oxidant Fe (with ascorbate), or both. The 3×4 treatments were incubated at 37 degrees C and assessed each hour up to 3h for motility (computer-aided sperm assessment) and viability/apoptosis plus mitochondrial status (YO-PRO-1, propidium iodide, Mitotracker Deep Red; flow cytometry). DNA damage at 4h was assessed using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay. Centrifugation alone affected neither sperm quality nor DNA, and the oxidant had no effect in control or centrifuged samples. Washed samples were not different than control, but oxidant decreased motility, mitochondrial status and viability, and altered the motility subpopulation pattern, being partially suppressed by Trolox. Spermatozoa with damaged DNA dramatically increased in the washed-oxidized sample (from 22.30+/-3.52% to 67.94+/-5.07%), but not when antioxidant was present. Although samples from different males behaved similarly, male-to-male variability was detected regarding susceptibility to oxidative damage after washing. We concluded that, although red deer thawed spermatozoa seemed resilient to centrifugation, the vulnerability to oxidative stress after washing makes it advisable to supplement manipulation media with antioxidants, especially taking into account male-to-male variability.

Theriogenology 72:1073-84.

Study on dynamic of sperm population in semen of stallion, boar and rabbit 2016-12-30T09:48:34+00:00

Study on dynamic of sperm population in semen of stallion, boar and rabbit

Quintero-Moreno A.

Unit of Reproduction, Department of Animal Medicine and Surgery, School of Veterinary Medicine, Autonomous University of Barcelona, E-08193 Bellaterra, Spain

The first aim of this study was to test the presence of separate sperm subpopulations with specific motility characteristics in stallion, boar and rabbit ejaculates by using a computer-assisted semen analysis system (CASA). Sperm motility descriptors were analyzed thorough the clustering of variables based on a covariance matrix. This matrix selects the descriptors of sperm motility that better explain the spermatozoon kinetics. Sperm subpopulations were obtained by disjointing cluster analysis where the observations are divided into clusters by which each observation belongs to one specific cluster. This test showed that three separate sperm subpopulations with different motility characteristics in boar, and four in stallion and rabbit, coexist in the ejaculates. There were significant (P<0.001) differences in the distribution of these subpopulations among individuals in all of the studied species, but no clear relationship between motile subpopulation structure and fertility was obtained. A second aim of the study was to test the possibility for a precise estimation of the fertilizing ability of mammalian ejaculate based upon the results of semen analysis. For this purpose, we tested the mathematical combination of several parameters of the boar and rabbit semen quality analysis as predictive “in vivo” fertility tools. The main mathematical relations utilized among parameters were logistic and linear regressions. In boar, two mathematical models obtained by logistic regression involving osmotic resistance test, hyperosmotic resistance test and viability of fresh samples, showed a significant (P<0.05) relationships between semen characteristics and conception rate. However, none of the obtained models produced a significant relation between semen characteristics and litter size. In rabbits, logistic and linear regression analysis rendered two mathematic, significant (P<0.05) models, with related some semen characteristic (sperm viability and abnormalities) with “in vivo” fertility and litter size. In stallion, the study of subpopulations in ejaculates which showed confirmed fertilizing capacity showed that these ejaculates had the majority of their motile spermatozoa included in a subpopulation with high progressiveness and low linear velocity. Moreover, all the ejaculates with proven fertility which have a total sperm count >20×109 spermatozoa/ejaculate showed all of their motile sperm included in this subpopulation. Our results support that the use of the values obtained in a standard boar, rabbit and stallion semen quality analysis to predict life fertilizing ability of a single ejaculate can reasonably be achieved by applying logistic and linear regression analyses to the parameters included in this analysis. Thus, our methodology can explain in a systematic manner mammal semen quality, relating it to conception rate and litter size.

Improving the effect of incubation and oxidative stress on thawed spermatozoa from red deer by using different antioxidant treatments 2015-05-08T11:54:39+00:00

Improving the effect of incubation and oxidative stress on thawed spermatozoa from red deer by using different antioxidant treatments

Domínguez-Rebolledo, A. E., Fernández-Santos, M. R., Bisbal, A., Ros-Santaella, J. L., Ramón, M., Carmona, M., Martínez-Pastor, F., Garde, J. J.

Biology of Reproduction Group, National Wildlife Research Institute (IREC) (UCLM-CSIC-JCCM), 02071 Albacete, Spain

Antioxidants could improve sperm media, extending the viability of spermatozoa and protecting their DNA. The protective ability of lipoic acid, melatonin, Trolox and crocin was tested on red deer spermatozoa incubated at 37 degrees C. Cryopreserved spermatozoa were thawed and incubated with 1 mM or 0.1 mM of each antioxidant, with or without oxidative stress (100 muM Fe(2+)). Motility (CASA), viability, mitochondrial membrane potential and acrosomal status were assessed. Lipoperoxidation (malondialdehyde production), intracellular reactive oxygen species (ROS) and DNA status (TUNEL) were checked at 4 h. Incubation alone increased ROS and decreased motility. Oxidative stress intensified these effects, increasing lipoperoxidation and DNA damage. Lipoic acid had little protective effect, whereas 1 mM melatonin showed limited protection. Trolox lowered ROS and lipoperoxidation both in oxidised and non-oxidised samples. In oxidised samples, Trolox prevented DNA and acrosomal damage, and ameliorated motility. Crocin at 1 mM showed similar results to Trolox, but noticeably stimulated motility and had no effect on lipoperoxidation. In a second experiment, a broader range of crocin and melatonin concentrations were tested, confirming the effects of crocin (positive effects noticeable at 0.5-0.75 mM), but showing an increase in lipoperoxidation at 2 mM. Melatonin was increasingly effective at 2.5 and 5 mM (ROS, lipoperoxidation and DNA status). Crocin seems a promising new antioxidant, but its particular effects on sperm physiology must be further studied, especially the consequences of motility stimulation and confirming its effect on lipoperoxidation. Melatonin might be useful at relatively high concentrations, compared to Trolox.

Reprod Fertil Dev 22:856-70.

Effects of long-term chilled storage of red deer epididymides on DNA integrity and motility of thawed spermatozoa 2016-12-30T09:48:37+00:00

Effects of long-term chilled storage of red deer epididymides on DNA integrity and motility of thawed spermatozoa

Fernández-Santos, M. R., Martínez-Pastor, F., Matias, D., Domínguez-Rebolledo, A. E., Esteso, M. C., Montoro, V., Garde, J. J.

Reproductive Biology Group, National Wildlife Research Institute (IREC), UCLM-CSIC-JCCM, and Institute of Regional Development (IDR), UCLM, Albacete, Spain

We have carried out a study on the influence of prolonged cold storage (5 degrees C) of Iberian red deer epididymides on post-thaw sperm motility and DNA integrity. Twenty-nine pairs of testes, with attached epididymides, were collected during November and December. Spermatozoa from one of each of the pairs were immediately recovered, evaluated and frozen (control). The remaining epididymides were cooled to 5 degrees C and stored for 24, 96 and 192 h (experimental groups), after which spermatozoa were collected and frozen. Samples were evaluated before freezing, after thawing, and after a 2-h period of incubation at 37 degrees C. Motility was evaluated by means of a CASA system and chromatin stability was assessed following the Sperm Chromatin Structure Assay (SCSA). Our results showed that, during the first 96 h, the motility (total and progressive) did not significantly decline when assessed after cryopreservation, although there was a significant decline when epididymides had been stored for 192 h at 5 degrees C (P<0.001). The present study demonstrates that motility and DNA status of thawed spermatozoa collected from refrigerated epididymes, at least 96 h post-mortem, were good enough to consider their eventual use. Most importantly, sperm DNA integrity after thawing was apparently not affected by storage time, even after 192 h.

Anim Reprod Sci 111:93-104.

Male fertility in natural populations of red deer is determined by sperm velocity and the proportion of normal spermatozoa 2016-12-30T09:48:37+00:00

Male fertility in natural populations of red deer is determined by sperm velocity and the proportion of normal spermatozoa

Malo AF, Garde JJ, Soler AJ, Garcia AJ, Gomendio M, Roldan ER

Reproductive Ecology and Biology Group, Museo Nacional de Ciencias Naturales (CSIC), 28006-Madrid, Spain

Male reproductive success is determined by the ability of males to gain sexual access to females, and by their ability to fertilize ova. Among polygynous mammals, males differ markedly in their reproductive success, and a great deal of effort has been made to understand how selective forces have shaped traits which enhance male competitiveness before and after copulation (i.e. sperm competition). However, the possibility that males may also differ in their fertility has been ignored, under the assumption that male infertility is rare in natural populations because selection against it is likely to be strong. In this study we examined which semen traits correlate with male fertility in natural populations of Iberian red deer. We found no trade-offs between semen traits. Our analyses revealed strong associations between (a) sperm production and sperm swimming velocity, (b) sperm motility and the proportion of morphologically normal spermatozoa, and (c) sperm viability and acrosome integrity. These last two variables had the lowest coefficients of variation suggesting that these traits have stabilized at high values and are unlikely to be related to fitness. In a fertility trial, our results show that there was a large degree of variation in male fertility, and that differences in fertility were mainly determined by sperm swimming velocity and also by the proportion of morphologically normal sperm. We conclude that male fertility does vary substantially in natural populations of Iberian red deer and that, when sperm numbers are equal, it is mainly determined by sperm swimming velocity and sperm morphology.

Biol Reprod 2004, Dec 1.

Use of chromatin stability assay, mitochondrial stain JC-1, and fluorometric assessment of plasma membrane to evaluate frozen-thawed ram semen 2016-12-30T09:48:37+00:00

Use of chromatin stability assay, mitochondrial stain JC-1, and fluorometric assessment of plasma membrane to evaluate frozen-thawed ram semen

Martinez-Pastor F, Johannisson A, Gil J, Kaabi M, Anel L, Paz P, Rodriguez-Martinez H.

Department of Anatomy and Histology, Swedish University of Agricultural Sciences (SLU), Box 7039, Uppsala SE-750-07, Sweden

Cryopreservation of semen imposes deleterious effects on spermatozoa, either killing a certain proportion of cells or causing subtle damages on sperm function in the surviving population, changes not easily revealed by conventional assays. We have tested three functional assessment techniques in frozen-thawed ram semen from six adult rams, cryopreserved following eight different protocols (four extenders, and glycerol being added at two temperatures). Semen samples were thawed and the following analyses were carried out: motility (CASA), membrane integrity (Hoescht 33258 and fluorometry), chromatin status (chromatin stability test and fluorescence-assisted cell sorting, FACS) and mitochondrial activity (JC-1 and FACS). Fluorometry outcome did not correlate with the other parameters and showed large variation, albeit discriminating among cryopreservation techniques (

[Formula: see text] ). Mitochondrial activity correlated, but with low values, with total and progressive motility. However, good sperm motility and high velocity values were associated to high mitochondrial membrane potential. The chromatin stability assay was also successfully carried out, and had a good relationship with male factor (%COMP [Formula: see text] and SD [Formula: see text] parameters). In conclusion, fluorometric assessment of membrane integrity albeit rendering poor results, merits improvement, being a low-cost and handy technique, especially for work in the field. On the other hand, both assessments of chromatin stability and mitochondrial status (JC-1 staining), combined with FACS, are reliable techniques that can be used for the functional assessment of frozen-thawed ram semen.

Anim Reprod Sci. 2004 Aug;84(1-2):121-33.

Boar sperm velocity and motility patterns under capacitating and non-capacitating incubation conditions 2015-05-08T11:56:22+00:00

Boar sperm velocity and motility patterns under capacitating and non-capacitating incubation conditions

García Herreros M, Aparicio IM, Núñez I, García-Marín LJ, Gil MC, Peña Vega FJ.

Department of Animal Health and Medicine (Reproduction and Obstetrics), Facultad de Veterinaria, University of Extremadura, Avd. de la Universidad s/n, 10071 Cáceres, Spain

This study compares the velocity and motility of boar sperm under capacitating and non-capacitating incubation conditions. Aliquots of pooled, washed boar sperm were incubated in either Tyrode’s complete medium (TCM; a capacitating medium), Ca2+-free TCM (TCM-Ca2+), or Ca2+ and NaHCO3-free TCM (Tyrode’s basal medium [TBM]; a non-capacitating medium). Motility patterns were determined every hour over a 3h period of incubation at 38 degrees C. Capacitation status was assessed by the chlortetracycline assay after 1 and 3h of incubation. Experiments were repeated five times. Compared to the TBM control, a significant increase was seen in the percentage of capacitated sperm after 1h of incubation in TCM: the kinematics of these sperm cells were favorably modified. However, the motility patterns of sperm cells incubated in TCM and TCM-Ca2+ were very similar. Under capacitating conditions (TCM), the coefficients of linearity (LIN) and straightness (STR) significantly increased over time (LIN values were significantly different after 3h of incubation, while STR values were significantly different after only 2 h). Significant correlations were seen between LIN and the percentage of cells showing the B pattern (r = 0.334, P < 0.05) and the number of acrosome reacted spermatozoa (r = 0.301, P < 0.05). This suggests that capacitated boar spermatozoa may have a species-specific motility pattern.

doi:10.1016/j.theriogenology.2004.05.003

Effects of centrifugation before freezing on boar sperm cryosurvival 2015-05-08T11:56:39+00:00

Effects of centrifugation before freezing on boar sperm cryosurvival

Carvajal G, Cuello C, Ruiz M, Vazquez JM, Martinez EA, Roca J

Department of Medicine and Animal Surgery, Faculty of Veterinary Medicine, Campus de Espinardo, University of Murcia, Murcia, Spain

Current protocols for boar sperm cryopreservation require the centrifugation of semen in order to separate sperm cells from the seminal plasma. This study evaluated the influence of different centrifugation regimes on both sperm recovery and yield (percentage of viable sperm with an intact acrosome relative to the initial sperm population) after centrifugation (experiment 1) as well as the influence of different centrifugation regimes on boar sperm cryosurvival (experiment 2). In both experiments, sperm-rich fractions from 3 boars were diluted, pooled, and cooled to 17 degrees C before centrifugation. In experiment 1, the g-forces tested were 400, 800, 1600, and 2400 x g for 3 or 5 minutes, using the standard regime (800 x g for 10 minutes) as a reference. Sperm recovery (Burker Chamber) and yield (triple fluorescent stain of PI/R123/FITC-PNA [DNA-specific fluorochrome propidium iodide/mitochondria-specific fluorochrome rhodamine-123/acrosome-specific fluorochrome fluorescein isothiocyanate-labeled peanut (Arachis hypogaea) agglutinin]) were calculated. The highest recovery and yield (P <.05) values were achieved using 2400 x g for 5 or 3 minutes and 1600 x g for 5 minutes, which showed no differences (P >.05) from the reference in terms of sperm yield. In experiment 2, cooled semen was centrifuged using 3 different regimes: C1 (2400 x g for 3 minutes), C2 (1600 x g for 5 minutes), and C3 (800 x g for 10 minutes). Pellets were diluted in lactose-egg yolk (LEY)-glycerol-Equex STM (1 x 10(9) cells/mL) and frozen in 0.5-mL straws. After thawing, sperm quality was assessed after 30 and 150 minutes of incubation (37 degrees C). Centrifugation regimes C1 and C2 showed significantly (P <.05) higher postthaw sperm motility (assessed with a computer-assisted semen analysis system), viability (evaluated as for experiment 1), and percentage of uncapacitated sperm (assessed with a chlortetracycline assay) than did C3. In addition, C1 had the highest (P <.05) oocyte penetrating ability (assessed with the homologous in vitro penetration test performed with immature oocytes). Malondialdehyde production, assessed with the thiobarbituric acid reactive species test, was unaffected (P >.05) by the centrifugation regime used. We conclude that high g-force (2400 x g) and short centrifugation time (3 minutes) do not affect sperm recovery and yield and that, moreover, they have a positive effect on the cryosurvival of boar sperm. Therefore, we recommend the use of short-term centrifugation with a relatively high g-force (2400 x g for 3 minutes) in boar sperm cryopreservation protocol.

J Androl. 2004 May-Jun;25(3):389-96.

Survival and fertility of boar spermatozoa after freeze-thawing in extender supplemented with butylated hydroxytoluene 2015-05-08T11:57:07+00:00

Survival and fertility of boar spermatozoa after freeze-thawing in extender supplemented with butylated hydroxytoluene

Roca J, Gil MA, Hernandez M, Parrilla I, Vazquez JM, Martinez EA

Department of Medicine and Animal Surgery, Faculty of Veterinary Medicine, University of Murcia, Murcia, Spain

This study evaluated the protective effect of butylated hydroxytoluene (BHT), a lipid-soluble antioxidant, against cryopreservation injuries to boar spermatozoa. In experiment 1, the lowest BHT concentrations able to reduce lipid peroxidation in boar spermatozoa were determined. Nine BHT concentrations (ranging from 0.025 to 3.2 mM) were evaluated, and the lowest (P <.05) production of malondialdehyde (MDA), as an indicator of lipid peroxidation, was obtained when BHT ranged from 0.2 to 1.6 mM. In experiment 2, sperm survivability was evaluated when BHT was added to a postthaw freezing extender by measuring the degree of sperm lipid peroxidation (using MDA production) and by measuring parameter such as motility, plasma membrane and acrosome integrity, and cell apoptosis. The ability of thawed spermatozoa to fertilize in vitro-matured oocytes and of embryos to develop to the blastocyst stage in vitro was also assessed. Pooled sperm-rich fractions collected from 3 mature Pietrain boars were frozen in 0.5-mL straws after dilution with lactose-egg yolk-glycerol-Orvus ES Paste extender supplemented with 0, 0.2, 0.4, 0.8, and 1.6 mM BHT. Postthaw sperm survival, evaluated 30 and 150 minutes after thawing, was higher in BHT-treated spermatozoa, being significant (P <.05) when the freezing extender was supplemented with 0.2, 0.4, and 0.8 mM BHT. The addition of BHT to the freezing extender resulted in a significant (P <.05) decrease in the MDA concentration in thawed spermatozoa, irrespective of the level of BHT used. BHT had no effect on oocyte cleavage rates, but the development to blastocyst was improved for embryos derived from spermatozoa frozen in extender supplemented with 0.4 mM BHT (16% vs 29% of blastocysts per total oocytes; P <.05). In conclusion, under the conditions tested in the present study, the addition of BHT to the freezing extender improved the overall efficiency of thawed boar spermatozoa.

J Androl. 2004 May-Jun;25(3):397-405.

Regression analyses and motile sperm subpopulation structure study as improving tools in boar semen quality analysis 2015-05-08T11:57:20+00:00

Regression analyses and motile sperm subpopulation structure study as improving tools in boar semen quality analysis

Quintero-Moreno A, Rigau T, Rodriguez-Gil JE

Unit of Animal Reproduction, Faculty of Veterinary Science, University of Zulia, Box 15252, Maracaibo 4005-A, Venezuela

A precise estimation of the fertilizing ability of a boar ejaculate would be very useful to improve pig assisted reproduction results. For this purpose, we tested the mathematical combination of several parameters of the boar semen quality analysis, including the computer-assisted semen motility analysis (CASA), as a predictive fertility tool. The utilized mathematical relations among parameters were logistic and linear regressions. Two mathematical models obtained by logistic regression involving Osmotic Resistance Test (ORT Test), Hyperosmotic Resistance Test (HRT Test) and viability of fresh samples, showed a significant (P<0.05) correlation between semen characteristics and conception rate. However, none of the obtained models produced a significant correlation model between semen characteristics and prolificacy. The CASA analyses show that three separate subpopulations of spermatozoa with different motility characteristics coexist in boar ejaculates. There were significant (P<0.001) differences in the distribution of these subpopulations among boars, but no clear relationship between motile subpopulation structure and fertility was obtained. Our results support the belief that the predictive use of the results obtained in a standard boar semen quality analysis can reasonably be achieved by applying logistic correlation analyses among several function parameters of boar semen quality analysis and in vivo conception rates obtained after artificial insemination (AI).

Theriogenology. 2004 Feb;61(4):673-90.

Sperm subpopulations in Iberian red deer epididymal sperm and their changes through the cryopreservation process 2015-05-08T11:57:32+00:00

Sperm subpopulations in Iberian red deer epididymal sperm and their changes through the cryopreservation process

Martinez-Pastor F, Garcia-Macias V, Alvarez M, Herraez P, Anel L, Paz P

Cell Biology and Anatomy and Animal Reproduction and Obstetrics, University of León, 24071, León, Spain

We have applied a statistical protocol based on principal component analysis, clustering methods, and discriminant analysis for the identification of sperm subpopulations in computer-assisted sperm analysis (CASA) data. Samples were obtained from the cauda epididymis of 11 Iberian red deer and cryopreserved following a standard protocol. Motility by CASA was analyzed just after sperm recovery, just before freezing, and after thawing, and eight motility descriptors for each individual spermatozoon were recorded. Sperm viability and acrosomal status were also assessed. Subpopulation analysis was performed in four sequential steps: principal component analysis using the eight motility descriptors; nonhierarchical clustering analysis (k-means) using the first two principal components; hierarchical clustering analysis (UPGMA); and selection of the final number of clusters. Three clusters were obtained for each motility analysis: slow and nonlinear; rapid and linear; and rapid, high ALH, nonlinear. We detected variations in the clusters between treatments (initial, prefreezing and postthawed). Indeed, motility increased and linearity decreased in the prefreezing analysis. A discriminant analysis isolated three descriptors that were used again in the same statistical analysis, giving four clusters that resembled the pattern found in the first classification. We also performed a clustering analysis of the males according to prefreezing/postthawed variation of total motility, viability, and acrosomal status. The proportion of the linear subpopulations in the prefreezing treatment, in both clustering analyses, correlated positively with postthawed viability recovery. Our results show that clustering analysis of CASA data gives useful and practical information that is not obtained by conventional sperm analysis.

Biol Reprod 2004, 10.1095/biolreprod.104.032730.

Zona pellucida binding ability and responsiveness to ionophore challenge of cryopreserved dog spermatozoa after different periods of capacitation in vitro 2015-05-08T11:57:46+00:00

Zona pellucida binding ability and responsiveness to ionophore challenge of cryopreserved dog spermatozoa after different periods of capacitation in vitro

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, 27002 Lugo, Spain

The present study was aimed to evaluate the functional status of cryopreserved dog spermatozoa after different periods (2, 8 and 24 h) of capacitation in vitro. Sperm motility, viability and binding capacity to the zona pellucida of canine oocytes derived from frozen-thawed ovaries were evaluated at each time point. Sperm viability was assessed by flow cytometry using the Ca(2+)-sensitive indicator Fluo 3 AM and PI, to simultaneously detect the proportion of live spermatozoa and the existence of live sperm subpopulations with different intracellular Ca(2+) content. In addition, the acrosome reaction frequency in ionophore-treated aliquots of spermatozoa incubated in capacitating (CCM) versus non-capacitating (NCM) medium, were evaluated by using eosin-nigrosin staining at the same time intervals. The number of spermatozoa bound to the zona pellucida decreased in about 50% (from 18.61 +/- 14.40 to 7.7 +/- 6.97) when sperm incubation was prolonged from 2 to 8h, however, sperm motility, viability and the subpopulation of live spermatozoa with higher intracellular Ca(2+) concentration decreased in lower extent (10-15%). In CCM-incubated samples, the rate of acrosomal exocytosis in response to ionophore challenge was high (>80%), independently of the evaluation period. NCM-incubated sperm were not affected by ionophore treatment, however, their intracellular Ca(2+) concentration was no different than that observed in CCM-incubated spermatozoa. It was concluded that, after being capacitated, motile and viable spermatozoa seem to lose their ability to bind to the zona pellucida, but this loss is not accompanied by a reduced response to ionophore challenge and it may not be related with changes in the intracellular Ca(2+) concentration of spermatozoa.

doi:10.1016/j.anireprosci.2003.11.007

Field and in vitro assay of three methods for freezing ram semen 2016-12-30T09:48:37+00:00

Field and in vitro assay of three methods for freezing ram semen

Anel L, de Paz P, Alvarez M, Chamorro CA, Boixo JC, Manso A, Gonzalez M, Kaabi M, Anel E

Animal Reproduction and Obstetrics, Leon University, Leon 24071, Spain

Glycerol has been the most widely used cryopreservation agent for spermatozoa and a wide range of factors affect its action on sperm viability and fertilizing capacity. We tested three methods for freezing ram semen packed in 0.25 ml straws (final cellular concentration: 100 x 10(6) spz/ml). Method M1: Two-thirds of the final volume of diluent was added as solution A (without glycerol) to the pure semen at 35 degrees C. The sample was cooled to 5 degrees C (-0.30 degrees C/min), one-third of final diluent volume was added as solution B (final concentration of glycerol 4%) and the sample was maintained at 5 degrees C for 2h. It was then frozen in a programmable biofreezer (-20 degrees C/min down to -100 degrees C). Method M2: The sample was diluted with a specific solution at 35 degrees C (final concentration of glycerol 3%), cooled to 5 degrees C (-0.20 degrees C/min) and left for 2h. After that, it was frozen in nitrogen vapours. Method M3: Semen was diluted 1:1 in a specific solution (concentration of glycerol 2%) and cooled to 5 degrees C (-0.25 degrees C/min). The sample was then diluted again in the same solution to the final cellular concentration (final concentration of glycerol 4%). It was left for 1h at 5 degrees C and then frozen in a programmable biofreezer (-20 degrees C/min down to -100 degrees C). Best total motility (TM) and progressive motility (PM) (75.8 and 55.18%) were obtained using Method M3. Methods M1 and M3 gave significantly higher values (P<0.05) for kinetic parameters: average path velocity (VAP) (81.3 and 85.2 microm/s), straight-line velocity (VSL) (72.8 and 77.3 microm/s) and linearity (LIN) (66.6 and 68.8%). Method M2 showed the lowest kinetic parameters of motility (VAP 74.4, VSL 67.3 and LIN 62.5) and the highest percentage of cells with damaged plasma membrane (53.8%). Method M1 gave the worst results in viability and acrosome status assessed using fluorescence probes (31.3%-dead cells with damaged acrosomes-versus 25.4% in M2 and 23.3% in M3). A field trial carried out on fertility showed a significantly higher percentage of pregnant or lambing ewes (P<0.05) with Method M3 (67.3% versus 51.1% for M1 and 58.8% for M2). We concluded that the use of a simple dilution medium (test-fructose-glycerol-egg yolk) with the addition of glycerol (to 2% at 35 degrees C and to 4% at 5 degrees C) in two steps together with a programmable biofreezer was a productive method for freezing ram semen.

Theriogenology. 2003 Oct 15;60(7):1293-308.

Effect of epididymis handling conditions on the quality of ram spermatozoa recovered post-mortem 2015-05-08T11:58:46+00:00

Effect of epididymis handling conditions on the quality of ram spermatozoa recovered post-mortem

Kaabi M, Paz P, Alvarez M, Anel E, Boixo JC, Rouissi H, Herraez P, Anel L

Animal Reproduction and Obstetrics, University of Leon, 24071 Leon, Spain

Post-mortem spermatozoa recovery is an important technique for obtaining germplasm reserves from genetically valuable animals or endangered species. However, there are many factors that influence the outcome of this technique. We have studied the effect of the interval between animal’s death and sperm recovery (0, 24 or 48 h) on the quality and freezability of ram spermatozoa from cauda epididymidis. Storage temperature of epididymis (room temperature or 5 degrees C) was also analysed. Spermatozoa were diluted with Tes-Tris-Fructose solution supplemented with egg yolk (10%) and glycerol (4%), and frozen using a programmable biofreezer (-20 degrees C/min). Pre-freeze and post-thaw sperm samples showed viable spermatozoa up to 48 h after the animal’s death, although their quality declined significantly as post-mortem storage time increased. Epididymis sperm stored at 5 degrees C showed better motility and a lower percentage of abnormal forms than epididymis stored at room temperature after 24 and 48 h. The fertilizing ability of cauda epididymis ram spermatozoa obtained at 0 and 24h after the animal’s death is similar to that of ejaculated spermatozoa. Therefore, a good protocol for post-mortem semen collection in rams when epididymal spermatozoa cannot be collected immediately, is to preserve the epididymis at 5 degrees C and process the samples in the first 24h after the animal’s death.

Theriogenology. 2003 Oct 15;60(7):1249-59.

Effects of thawing procedure on postthawed in vitro viability and in vivo fertility of red deer epididymal spermatozoa cryopreserved at -196 degrees C 2015-05-08T11:59:11+00:00

Effects of thawing procedure on postthawed in vitro viability and in vivo fertility of red deer epididymal spermatozoa cryopreserved at -196 degrees C

Soler AJ, Garcia AJ, Fernandez-Santos MR, Esteso MC, Garde JJ

Department of Agroforestry Science and Technology and Game Resources (IDR), Castilla-La Mancha University (UCLM), Albacete, Spain

In this study, we have determined the effects of individual factor and thawing procedure on in vitro viability and in vivo fertility of frozen-thawed red deer epididymal spermatozoa. The spermatozoa that were collected from 13 Iberian deer stags were diluted at room temperature in a Triladyl-20% egg yolk medium and frozen in nitrogen vapors. In the first experimental series, sperm samples were collected from 10 mature stags. For thawing, the frozen straws were subjected to 3 different procedures: I (37 degrees C for 20 seconds), II (60 degrees C for 8 seconds) and III (70 degrees C for 5 seconds). Sperm cryosurvival was judged in vitro by microscopic assessments of individual sperm motility (SM) and of plasma membrane and acrosome (NAR) integrities. Statistically significant variations were found (P <.05) between stags for most of the seminal parameters evaluated. The thawing procedure did not have an effect on the seminal characteristics evaluated after this process, except for SM (P <.05), with the best overall recovery rates after freezing and thawing found with the use of protocol I. Our results also show a differential resistance to return to isosmotic conditions of spermatozoa thawed using the different thawing protocols. In the second experimental series (insemination artificial trial), with spermatozoa from 3 stags, results of fertility were statistically higher (69.7% vs 42.4%, P =.014) when spermatozoa were thawed at 37 degrees C for 20 seconds than were warmed at 60 degrees C for 8 seconds. Therefore, thawing protocol I, which provides slow thawing rates, was the most beneficial for epididymal spermatozoa thawing of the cervid subspecies analyzed in this study. In summary, high in vitro survival and in vivo fertility of frozen-thawed deer epididymal spermatozoa were dependent on warming rates, but each stag exhibited its own sensitivity to cryopreservation.

J Androl. 2003 Sep-Oct;24(5):746-56.

Head dimensions of cryopreserved red deer spermatozoa are affected by thawing procedure 2016-12-30T09:48:37+00:00

Head dimensions of cryopreserved red deer spermatozoa are affected by thawing procedure

Esteso MC, Fernandez-Santos MR, Soler AJ, Garde JJ

Dpto. Ciencia y Tecnologia Agroforestal. Campus Universitario, 02071, Albacete, Spain

The objective of this study was to evaluate the effects of the thawing procedure on red deer spermatozoa distribution in morphologically distinct subpopulations after freezing and thawing. For this purpose, epididymal spermatozoa were thawed using two different thawing protocols (I = 37 degree celsius for 20 s vs. II = 70 degree celsius for 5 s). The spermatozoa, from 10 Iberian deer stags, were diluted at room temperature in a Triladyl-20 percent egg yolk medium and frozen in nitrogen vapor. Standard sperm freezability was judged by microscopic assessments of sperm motility. The thawing procedure had an effect on sperm motility percentage (P = 0.05), with the best overall recovery rates found with the use of protocol I (76.8 + or – 1.8 vs. 70.6 + or – 1.8). Moreover, the morphometric dimensions for a minimum of 200 sperm heads were analyzed from each sample by means of the Sperm-Class Analysez (SCA), and the mean measurements recorded. Deer sperm heads were significantly (P = 0.01) smaller when spermatozoa were thawed using protocol II than when using procedure I (area = 30.02 square micrometers vs. 30.32 square micrometers; width = 4.47 micrometers vs. 4.51 micrometers; length = 8.05 micrometers vs. 8.11 micrometers), but not for all stags. All sperm head measurements were placed in a statistical database and a multivariate cluster analysis performed. Mean measurements for all parameters of the major clusters for the two different thawing procedures were compared by ANOVA. The mean values for length, width, area, perimeter, shape factor and width/length in the major cluster of sperm head dimensions for thawing protocol I were significantly different from those for protocol II (P = 0.001). In addition, differences were found within all stags for whole morphometric parameters (P = 0.001), with the smallest overall sperm head dimensions found with the use of protocol II. Additionally, the rapid thawing protocol produced a dramatic loss of heterogeneity. Finally, our results showed that the greater the loss of heterogeneity, the greater the degree of sperm cryoinjury.

Cryo Letters. 2003 Jul-Aug;24(4):261-8.

Relationship between the characteristics of epididymal red deer spermatozoa and penetrability into zona-free hamster ova 2016-12-30T09:48:37+00:00

Relationship between the characteristics of epididymal red deer spermatozoa and penetrability into zona-free hamster ova

Soler AJ, Garde JJ

Department of Agroforestry Science and Technology and Game Resources (IDR), Castilla-La Mancha University, Albacete, Spain

A heterologous (zona-free hamster oocytes) in vitro fertilization (IVF) system was used to evaluate the relationship between sperm factors and penetration capacity of epididymal red deer spermatozoa. The sperm parameters evaluated in 36 sperm samples obtained postmortem from stags selectively shot during the rutting season were sperm motility, functional integrity of plasma membrane by means of the hypo-osmotic swelling test (HOST), and, simultaneously, viability and acrosomal status via a triple-stain technique. Zona-free hamster oocytes were used to evaluate the capacity of the different sperm assays to predict in vitro penetration. In order to increase the variability in sperm quality, we recovered samples from stags at different intervals between the death of the male and the collection of the genitalia. All measures of sperm quality declined progressively (P <.001) with increasing intervals between death and sample collection. In addition, many sperm parameters were related to penetration ability in vitro. Subsequently, sperm samples were rearranged in 2 categories according to the interval that had elapsed between death and the collection of the genitalia (group 1, short interval = 0-12 h; group 2, large interval = 18-40 hours). When samples were grouped, less correlation achieved significance, especially for group 1, than when samples were not divided. Also, correlation between the number of sperm per oocyte and sperm parameters for group 1, which had the highest values of sperm quality, failed to reach significance. It is concluded that the classical parameters accepted in assessing the viability of deer spermatozoa can be good predictors of the penetrating ability of the spermatozoa when satisfactory in vitro conditions are used for the development of the IVF system. Also, this study demonstrates that compatible heterologous gamete interaction allows thorough assessment of sperm function in a wild deer.

J Androl. 2003 May-Jun;24(3):393-400.

Identification of sperm subpopulations with specific motility characteristics in stallion ejaculates 2016-12-30T09:48:37+00:00

Identification of sperm subpopulations with specific motility characteristics in stallion ejaculates

Quintero-Moreno A, Miro J, Teresa Rigau A, Rodriguez-Gil JE

Unit of Reproduction, Department of Animal Medicine and Surgery, School of Veterinary Medicine, Autonomous University of Barcelona, E-08193 Bellaterra, Spain

The aim of this study was to test the presence of separate sperm subpopulations, with specific motility characteristics, in stallion ejaculates by using a computer-assisted semen motility analysis (CASA) system. Motility data were analyzed with a hierarchical clustering of variables based on a correlation or covariance matrix to select like parameters of sperm motility descriptors that better explain the kinetics of spermatozoa. The statistical analyses clustered the whole motile sperm population in both fresh and 24 h stored ejaculates into four separate groups. There were significant differences in the distribution of the four subpopulations (P < 0.001) as well as in the total sperm number and the percentage of total motility (P < 0.01) in fresh semen among the five stallions tested. Our results show that separate subpopulations of spermatozoa with different motility characteristics coexist in stallion ejaculates. These subpopulations were maintained, although with a less-progressive motion pattern, after 24 h of storage. The study of subpopulations in ejaculates that have confirmed fertilizing capacity showed that the majority of the motile spermatozoa in these ejaculates are included in a subpopulation with high progressive motility and low linearity, and the ejaculates with proven fertility that have a total sperm count > or = 20 x 10(9) spermatozoa/ejaculate show all of their motile sperm included in this subpopulation. Our results show that the use of the CASA system is a relatively simple approach to the study of sperm subpopulation patterns in equine ejaculates.

Theriogenology. 2003 May;59(9):1973-90.

Storage of red deer epididymides for four days at 5 degrees C: effects on sperm motility, viability, and morphological integrity 2015-05-08T12:00:32+00:00

Storage of red deer epididymides for four days at 5 degrees C: effects on sperm motility, viability, and morphological integrity

Soler AJ, Perez-Guzman MD, Garde JJ

Dpto de Ciencia y Tecnologia Agroforestal, ETSIA, UCLM, Albacete, Spain

The purpose of this study was to assess the sperm motility, the plasma membrane integrity and the morphology of red deer spermatozoa when maintained within epididymides stored for 4 days at 5 degrees C, and to evaluate whether such stored spermatozoa are able to withstand a refrigeration process. Thirty pairs of testes, with attached epididymides, were collected from 30 hunter-killed mature stags (Cervus elaphus hispanicus), and spermatozoa from each one of the pairs were immediately collected in Triladyl medium, evaluated and refrigerated (Control Group). The remaining testes and epididymides were gradually cooled to 5 degrees C and stored for 1, 2, 3, and 4 days (Experimental Groups), after which spermatozoa were processed as described previously for the control group. The effects on spermatozoa that had been stored within epididymides for various times were determined by assaying sperm motility index (SMI), plasma membrane integrity and sperm morphology (SM). In the same way, SMI and SM were assessed after spermatozoa refrigeration at 5 degrees C for 3 hours in different groups (SMI-R, SM-R). There was no significant decrease in plasma membrane integrity of spermatozoa recovered from epididymides stored at 5 degrees C for 4 days. Similarly, the percentage of morphologically normal spermatozoa remained unaffected during the first 3 days of storage. In contrast, during storage sperm motility evaluation revealed significantly (P<0.05) lower SMI values for samples from epididymides stored 2, 3, and 4 days (47.7+/-3.6, 45.5+/-4.4, 44.1+/-5.2) than that of the control group (57.6+/-1.6). Similar results were obtained after refrigeration of spermatozoa in Triladyl at 5 degrees C. These data suggest that it might be possible to recover functional spermatozoa from red deer epididymides stored at 5 degrees C during several days when epididymal spermatozoa cannot be collected and cryopreserved immediately.

J Exp Zoolog Part A Comp Exp Biol. 2003 Feb 1;295(2):188-99.

Effect of supranutritional level of dietary alpha-tocopheryl acetate and selenium on rabbit semen 2016-12-30T09:48:37+00:00

Effect of supranutritional level of dietary alpha-tocopheryl acetate and selenium on rabbit semen

Castellini C, Lattaioli P, Bosco AD, Beghelli D

Department of Animal Science, Borgo 20 Giugno, 74-06100 Perugia, Italy

This research examined the effects of dietary alpha-tocopheryl acetate (50 or 200 mg/kg diet) and selenium (Se, 0 or 0.5 ppm) supplementation on motion characteristics, oxidative stability and fertilizing ability of rabbit spermatozoa, fresh and stored for 24 h at 5 degrees C. The higher amount of dietary alpha-tocopheryl acetate increased the level of Vitamin E in the fresh semen (1.75 mmol/l versus 0.95 mmol/l) and its oxidative stability (thiobarbituric acid reactive substances-TBARS 12.44 nmol malondialdehyde/10(8) sperm versus 21.4 nmol malondialdehyde/10(8) sperm). Dietary Se increased gluthatione peroxidase activity (GPx) in erythrocytes (285 U/g Hb versus 207 U/g Hb), seminal plasma (270 U/l versus 190 U/l) and spermatozoa (1338 mU/10(9) sperm versus 1103 mU/10(9) sperm), whereas it did not show any effect on alpha-tocopherol level and TBARS. No synergy between Vitamin E and Se was shown. Storage for 24 h at 5 degrees C increased the TBARS level in all the experimental groups. Neither live and acrosome reacted spermatozoa, nor kinetic parameters, nor fertility rate were modified by dietary supplementation.

doi: 10.1016/S0093-691X(02)01083-X

Differential effects of glucose and fructose on hexose metabolism in dog spermatozoa 2016-12-30T09:48:37+00:00

Differential effects of glucose and fructose on hexose metabolism in dog spermatozoa

Rigau T, Rivera M, Palomo MJ, Fernández-Novell JM, Mogas T, Ballester J, Peña A, Otaegui PJ, Guinovart JJ, Rodríguez-Gil JE

Unit of Animal Reproduction, Department of Animal Medicine and Surgery, Autonomous University of Barcelona, E-08193 Bellaterra, Spain

Incubation of dog spermatozoa with 10 mmol l(-1) glucose or fructose rapidly increased the intracellular content of glucose 6-phosphate and fructose 6-phosphate, although the effect of fructose was greater. These effects were correlated with increases in ATP, ribose 5-phosphate and glycogen contents, and in the rates of formation of L-lactate and CO2. In all cases, except for ATP and glycogen, the effect of fructose was greater than that of glucose. The total hexokinase activity of the crude extracts of dog spermatozoa was more sensitive to fructose than to glucose at lower concentrations (0.1-3.0 mmol l(-1)). Both monosaccharides induced a fast and intense increase in the overall tyrosine phosphorylation of dog spermatozoa, although their specific induced-phosphorylation patterns differed slightly. Glut 3 and Glut 5 hexose transporters were the main hexose transporters in dog spermatozoa; however, other possible SGLT family-related hexose transporters were also localized. These data indicate that, at concentrations from 1 mmol l(-1) to 10 mmol l(-1), fructose has a stronger effect than glucose on hexose metabolism of dog spermatozoa. These differences appear to be related to variations in the sensitivity of hexokinase activity. Moreover, the differential hexose metabolism induced by the two sugars had distinct effects on the function of dog spermatozoa, as revealed by the diverse patterns of tyrosine phosphorylation.

doi: 10.1530/rep.0.1230579

Effects of glucose and fructose on motility patterns of dog spermatozoa from fresh ejaculates 2016-12-30T09:48:38+00:00

Effects of glucose and fructose on motility patterns of dog spermatozoa from fresh ejaculates

Rigau T, Farre M, Ballester J, Mogas T, Pena A, Rodriguez-Gil JE

Department of Animal Medicine and Surgery, School of Veterinary Medicine, Autonomous University of Barcelona, Bellaterra, Spain

This study was performed to gain insight about how fructose and glucose modulate dog spermatozoa motility in the absence of other motility-modulating factors. Incubation of dog spermatozoa from fresh ejaculates in a basal medium without sugars for 60 min at 37 degrees C induced a progressive decrease in the percentage of motile spermatozoa and in some mean motility parameters, such as mean velocity (VAP), linear coefficient (LIN) and dance (DNC), and an increase in the mean frequency of head displacement (BCF). This indicates a progressive loss of linearity and an increase in oscillatory movement. Addition of 10 mM fructose prevented these effects. Incubation in a basal medium with 10 mM glucose for 60 min at 37 degrees C provoked a fast and intense decrease of LIN and a slight increase of DNC, inducing a less linear and more oscillatory mean movement. Neither fructose nor glucose modified the percentage of motile spermatozoa. The response to both sugars was dose-dependent, with differences appearing at concentrations as low as 1 mM. An analysis of the spermatozoa subpopulation placed above the 95th percentile of the whole population and a factorial analysis of the data indicated that the changes in the mean values of the motility parameters were mainly due to a specific motile subpopulation that had a strong reaction to the two sugars. Our results indicate that fructose, at concentrations from 1 to 10 mM, induced a more linear and less oscillatory motility pattern than glucose. Moreover, from our results we suggest the presence of motile dog sperm subpopulations with an increased sensitivity to fructose and glucose.

Theriogenology. 2001 Sep 15;56(5):801-15.

Effect of seminal plasma on the characteristics and fertility of rabbit spermatozoa 2015-05-08T12:01:36+00:00

Effect of seminal plasma on the characteristics and fertility of rabbit spermatozoa

Castellini C, Lattaioli P, Moroni M, Minelli A

Dipardimento di Scienze Zootecniche, Universita di Perugia, Perugia, Italy

The effects of different dilutions of seminal plasma (SP) on the qualitative characteristics of rabbit spermatozoa and on their fertilising ability were analysed. Ejaculated semen was centrifuged twice and the sperm resuspended in media with decreasing ratios of SP/Tris: (1/2; 1/5; 1/10; 1/20; 1/30; 1/100) until the complete substitution was with SP. The control constituted sperm in undiluted SP. Samples were maintained at 37 degrees C and kinetic analysis done at fixed intervals (0-6h). Also the thiobarbituric reactive substances (TBA-RS) values were determined. Rabbit sperm suspended in Tris, or with extremely low content of SP, lost motility and viability within 1-3h, while sperm suspended in SP either undiluted or diluted up to 10-fold, showed similar motility during the 6h period (from 39 to 49%). Further dilutions of SP (1/20-1/30) had no effect during the initial 2h of storage but thereafter the decline of motility was more marked (after 6h: from 0 to 17%). Kinetic parameters followed the same trend and differences were particularly marked after storage: the highest values were in samples diluted up to 1/10; a sharp decline in motility characteristics was observed at higher dilutions. The addition of SP (1/2 v/v) to immotile sperm reactivated 35.5% of cells. However, SP did not significantly affect fertility rate or litter size possible involving an interaction with the female reproductive tract. SP reduced lipid oxidation (TBA-RS) of semen only after storage. A positive correlation between final TBA-RS and cell viability indicated that peroxidation was one of the cause of rabbit sperm deterioration during conservation.

doi:10.1016/S0378-4320(00)00181-0

Objective evaluation of the morphology of human epididymal sperm heads 2015-05-08T12:01:51+00:00

Objective evaluation of the morphology of human epididymal sperm heads

Soler C, Perez-Sanchez F, Schulze H, Bergmann M, Oberpenning F, Yeung C, Cooper TG

Department of Animal Biology, University of Valencia, Spain; Institute of Reproductive Medicine of the University, Munster, Germany

Spermatozoa were obtained from nine epididymal regions of six epididymides taken from five men undergoing castration for prostatic carcinoma (53-76 years) and from one man with testicular cancer (38 years). Spermatozoa were obtained by mincing tissue in phosphate-buffered saline, making air dried smears and staining with Hemacolor. The percentage of sperm heads categorised subjectively as normal (of uniform shape) or otherwise was calculated for each region. This confirmed that grossly swollen sperm heads (previously shown to be artefacts) were only present in proximal regions of the duct. A computer-aided sperm morphology analyser (Sperm Class Analyzer(R)) was used to provide objective measurements of sperm head area, perimeter, length and width of the uniform sperm heads and revealed that there was a gradual and statistically significant decline in sperm head size upon maturation, as occurs in other species. There was no significant difference between the morphometric parameters of spermatozoa obtained from the distal cauda epididymis and those obtained from the ejaculates of young normozoospermic patients.

Int J Androl. 2000 Apr;23(2):77-84.

Liquids storage (5 degrees C) of ram semen in different diluents 2016-12-30T09:48:38+00:00

Liquids storage (5 degrees C) of ram semen in different diluents

Lopez-Saez A, Ortiz N, Gallego L, Garde JJ

Dpto. Ciencia y Teconologia Agroforestal, ETSIA, Universidad de Castilla-La Mancha, Campus Universitario, Albacete, Spain

This study examined the effects of three diluents–skim milk (M), TesT (TE) and Tris-trehalose (TR)–on quality of ram semen stored at 5 degrees C over a long term. Percentage of motile spermatozoa (PM), motility score (MS), percentage of intact acrosomes (PIA), and percentage of swollen spermatozoa under hypoosmotic conditions (PS) were assessed after cooling and every 24 h up to day 16. There was a significant effect (p < .01) of diluent type in the conservation rates after cooling for PM. The TR diluent achieved greater values (94.7 +/- 1.36%) than TE (82.4 +/- 3.08%), while M showed an intermediate value (89.3 +/- 2.17%). In relation to time of storage, TR diluent showed the highest PM values up to day 12, but differed (p < .05) from TE medium only until day 4. The TR extender maintained a PM > 50% up to day 8, while TE and M were greater than 50% up to day 5. Skim milk maintained progressive movement of sperm cells for longer (MS = 3 until day 5) than the other diluents. There were no significant differences in PIA among the 3 media during the first 6 days of storage (except in day 2), and M showed the lowest value afterward. There was no significant extender effect on PS for the first 3 days, but after day 6 diluent M also showed the lowest values of this variable.

Arch Androl. 2000 Mar-Apr;44(2):155-64.

Post-mortem recovery and quality of the spermatozoa obtained from the cauda epididymis of Iberian red deer (Cervus elaphus hispanicus) 2015-05-08T12:02:32+00:00

Post-mortem recovery and quality of the spermatozoa obtained from the cauda epididymis of Iberian red deer (Cervus elaphus hispanicus)

Martínez F

Departamento de Biología Celular y Anatomía, Facultad de Ciencias Biológicas y Ambientales, Universidad de León

We have applied a statistical protocol based on principal component analysis, clustering methods and discriminant analysis for the identification of sperm subpopulations in CASA data. Samples were obtained from the cauda epididymis of 11 Iberian red deers, and cryopreserved following a standard protocol. Motility by CASA was analyzed just after sperm recovery, just before freezing and after thawing, and eight motility descriptors for each individual spermatozoon were recorded. Sperm viability and acrosomal status were also assessed. Subpopulation analysis was performed in four sequential steps: principal component analysis using the 8 motility descriptors; non-hierarchical clustering analysis (k-means) using the first two principal components; hierarchical clustering analysis (UPGMA); and selection of the final number of clusters. Three clusters were obtained for each motility analysis: slow and non-linear; rapid and linear; and rapid, high ALH, non-linear. We detected variations in the clusters between treatments (initial, pre-freezing and post-thawed). Indeed, motility increased and linearity decreased in the pre-freezing analysis. A discriminant analysis isolated three descriptors that were used again in the same statistical analysis, giving four clusters that resembled the pattern found in the first classification. We also performed a clustering analysis of the males according to pre-freezing/post-thawed variation of total motility, viability and acrosomal status. The proportion of the linear subpopulations in the pre-freezing treatment, in both clustering analyses, correlated positively with post-thawed viability recovery. Our results show that clustering analysis of CASA data gives useful and practical information that is not obtained by conventional sperm analysis.

Theriogenology.

Postmortem assessment of sperm characteristics of the red deer during the breeding season 2016-12-30T09:48:38+00:00

Postmortem assessment of sperm characteristics of the red deer during the breeding season

Garde JJ, Ortiz N, Garcia AJ, Gallego L, Landete-Castillejos T, Lopez A

ETSIA, Dpto. Ciencia y Tecnologia Agroforestal, Universidad de Castilla-La Mancha, Albacete, Spain

This study examined the effect of male age, time lapse between death of individual and collection of its sperm, breeding season (1993, 1995, or 1996), and testicle sampled (left or right) on the cell quality of spermatozoa obtained postmortem from the epididymis of red deer stags (Cervus elaphus hispanicus). A total of 142 sperm samples obtained from 71 free-ranging individuals shot during the breeding season were used to investigate these effects. The spermatozoa were obtained from the cauda epididymis of stags. Immediately after collection, an assessment was made of the proportion of motile spermatozoa (PM), normal morphology (PN), intact acrosomes (PIA), and the osmotic resistance degree of the plasmatic spermatozoa membrane as determined by the cell endosmosis test (E+). Gamete quality was influenced by both the age of the individuals and the lapse between death and collection of sperm (p < 0.001), whereas the year of collection and testicle sampled did not affect sperm quality. Sperm samples were classified in three groups: excellent, acceptable, or unacceptable, depending on the values achieved in the PM, PN, E+, and PIA variables. Acceptable samples had to achieve the following score: PM > 40%, PN > 40%, E+ > 40%, and PIA > 60%. Within this group, samples with a PM > 60% were classified as excellent. The percentage of samples classified as viable (strictly acceptable plus excellent) achieved 59.8% (85 out of 142). These results indicate that it is possible to obtain a remarkable percentage of viable sperm after the death of the deer. This finding might also be useful to obtain embryos of threatened species of wild ungulates.

Arch Androl. 1998 Nov-Dec;41(3):195-202.

Occurrence of prostasome-like membrane vesicles in equine seminal plasma 2016-12-30T09:48:38+00:00

Occurrence of prostasome-like membrane vesicles in equine seminal plasma

Minelli A, Moroni M, Martínez E, Mezzasoma I, Ronquist G

Dipartimento di Biologia Cellulare e Molecolare, Università di Perugia, Italy

Equine seminal plasma was shown to contain membrane vesicles that are similar to the well characterized prostasomes in human seminal plasma. Determination of nucleoside and nucleotide concentrations of these particles have shown that ATP, ADP and adenosine are the main components of the nucleotidic pool. 5′ nucleotidase, endopeptidase and dipeptidyl peptidase i.v. activities have been found on the surface of the particles. The interaction between these prostasome-like vesicles and spermatozoa was demonstrated by electron micrograph scans which revealed the steps of a fusion-like process leading to mixing of the membranes. In addition, endopeptidase activity, a marker enzyme of these seminal vesicles that is normally absent from equine spermatozoa, was shown to be acquired by these cells after interaction with the vesicles. The addition of these vesicles to equine spermatozoa resulted in the modification of adenylate catabolism. Therefore, a role in stabilizing the energy charge of the spermatozoa thus allowing longer viability is proposed for these organelles.

doi: 10.1530/jrf.0.1140237

Ureaplasma urealyticum reduces motility and induces membrane alterations in human spermatozoa 2015-05-08T12:05:30+00:00

Ureaplasma urealyticum reduces motility and induces membrane alterations in human spermatozoa

Nunez-Calonge R, Caballero P, Redondo C, Baquero F, Martinez-Ferrer M, Meseguer MA

Department of Gynecology, Ramon y Cajal Hospital, Institute Nacional of Health, Madrid, Spain

The in-vitro effects of several concentrations of Ureaplasma urealyticum on the motility, membrane integrity and morphology of washed spermatozoa from healthy donors were studied. A significant reduction in sperm motility and signs of membrane alteration, directly related to U.urealyticum concentration and contact time were observed. Scanning electron microscopy examination showed masses of U. urealyticum attached to the head and middle piece of some of deformed spermatozoa. It is suggested that U.urealyticum is involved in sperm changes leading to male infertility, particularly when there is heavy U. urealyticum colonization or specific infections with this microorganism.

Hum Reprod. 1998 Oct;13(1O):2756-61.

Effects of exposure to static magnetic fields on the morphology and morphometry of mouse epididymal sperm 2016-12-30T09:48:38+00:00

Effects of exposure to static magnetic fields on the morphology and morphometry of mouse epididymal sperm

Tablado L, Perez-Sanchez F, Nunez J, Nunez M, Soler C

Departament de Biologia Animal, Universitat de Valencia, Burjassot, Spain

Morphologic and morphometric sperm characteristics of mouse epididymal extracts from animals exposed to static magnetic fields were evaluated. For this purpose, animals were exposed for 35 days to a field of 0.7 T generated by a commercial permanent magnet for either 1 or 24 h per day. The values of morphometric parameters were obtained using the morphometric module of the Sperm Class Analyzer computerized image analysis system, and percentages of abnormalities were calculated. The size of sperm heads was unaffected by exposure to static magnetic fields. Lack of hook was a sperm head abnormality found significantly more frequently in animals exposed continually than in nonexposed animals, showing a possible alteration to the spermatogenic process after exposure to static magnetic fields. The percentage of sperm with coiled tails or of sperm with abnormal midpiece or tail was not altered by exposure.

Bioelectromagnetics. 1998;19(6):377-83.