El sistema CASA SCA® es una herramienta útil en investigación, y ha sido ampliamente utilizado en muchos estudios de diferentes especies. A continuación algunas de las publicaciones científicas y artículos publicados por usuarios de SCA®:
Can the Sperm Class Analyser (SCA) CASA-Mot system for human sperm motility analysis reduce imprecision and operator subjectivity and improve semen analysis?
Chey Dearing, Channa Jayasena and Kevin Lindsay
School of Health & Sport Science and School of Nursing, Eastern Institute of Technology, Taradale Campus, Hawkes Bay, New Zealand; Andrology Laboratory, Hammersmith Hospital, Imperial College NHS Trust, London, UK; Andrology Laboratory, Hammersmith Hospital, Imperial College NHS Trust, London, UK
Abstract: Semen analysis (SA) is considered mandatory for suspected male infertility although its clinical value has recently become questionable. Sperm motility is an essential parameter for SA, but is limited by high measurement uncertainty, which includes operator subjectivity. Computer-assisted sperm analysis (CASA) can reduce measurement uncertainty compared with manual SA. The objective of this study was to determine whether the Sperm Class Analyser (SCA) CASA-Mot system could reduce specific components of sperm motility measurement uncertainty compared with the World Health Organization (WHO) manual method in a single laboratory undertaking routine diagnostic SA. The study examined: (i) operator subjectivity; (ii) precision, (iii) accuracy against internal and external quality standards; and (iv) a pilot sub-study examining the potential to predict an IVF fertilisation rate. Compared with the manual WHO method of SA on 4000 semen samples, SCA reduces but does not completely eliminate operator subjectivity. Study SCA and CASA-Mot are useful tools for well-trained staff that allow rapid, high-number sperm motility categorization with less analytical variance than the manual equivalent. Our initial data suggest that SCA motility may have superior predictive potential compared with the WHO manual method for predicating IVF fertilization.
Human Fertility – https://doi.org/10.1080/14647273.2019.1610581
Received 13 Dec 2018, Accepted 21 Mar 2019, Published online: 06 May 2019
Humanin levels in human seminal plasma and spermatozoa are related to sperm quality
M. Rao, Z. Wu, Y. Wen, R. Wang, S. Zhao, L. Tang
Department of Reproduction and Genetics, The First Affiliated Hospital of Kunming Medical University, Kunming, China
Background: Humanin has reportedly been expressed in testis and spermatozoa, but no study has yet reported its presence in human seminal plasma (SP).
Objective: The aim of this study was to investigate the presence of humanin in human SP and to determine the correlation between humanin levels in SP/spermatozoa and sperm quality.
Materials and methods: Semen samples for SP/sperm humanin level measurement were collected from 164 patients who attended our andrology clinic for fertility evaluation. The localization of humanin in spermatozoa was evaluated using an immunofluorescence method, and SP/sperm humanin levels were measured with ELISA. Correlations between SP/sperm humanin levels and sperm parameters were analyzed.
Results: Humanin was expressed in the midpiece of the spermatozoa. Humanin concentrations in the SP ranged from 24.4 to 285.1 pg/mL, with a median of 89.7 pg/mL. The SP humanin concentrations in patients with normospermia were significantly higher than those in patients with oligospermia (p < 0.001), asthenospermia (p = 0.002), and oligoasthenospermia (p < 0.001). Spearman analysis showed a positive and significant correlation between SP humanin concentration and sperm concentration (r = 0.75, p < 0.001), and progressive sperm motility (r = 0.29, p < 0.001). Sperm humanin level was significantly and positively associated with progressive sperm motility (r = 0.70, p < 0.001). In addition, a significantly higher level of humanin was found in swim‐up spermatozoa than in non‐swim‐up spermatozoa (p = 0.03).
Conclusions: Seminal plasma and sperm humanin levels were significantly and positively correlated with sperm quality, especially sperm motility. Further studies of the origin of SP humanin and its role in spermatogenesis should be conducted.
Andrology – https://doi.org/10.1111/andr.12614
Received: 27-Aug-2018 / Revised: 26-Feb-20 19 / Accepted: 28-Feb-2019 / First published: 28 March 2019
Improved sperm motility after 4 h of ejaculatory abstinence: role of accessory sex gland secretions
Dale Goss, Bashir Ayad, Gerhard van der Horst, Bongekile Skosana and Stefan S. du Plessis
Division of Medical Physiology, Stellenbosch University, Private Bag X1, Matieland, 7602, Stellenbosch, South Africa
Abstract: Various studies have sought to determine the typical v. optimal abstinence period after which semen samples should be collected, with many contradictory results reported. Several factors influence the semen microenvironment, and thus sperm parameters. In this study we focused on the secretions of the prostate, seminal vesicles and the epididymis. Semen samples were obtained from healthy normozoospermic males (n = 16) after 4-day and 4-h periods of ejaculatory abstinence, and standard semen analysis was performed using computer-aided sperm analysis, whereas seminal plasma citric acid, neutral α-glucosidase and fructose concentrations were measured using assay kits. There were significant decreases in total sperm count (P < 0.001), sperm concentration (P < 0.05) and semen volume (P < 0.05) after 4 h compared with 4 days ejaculatory abstinence. Furthermore, increases were observed in total sperm motility (P < 0.05) and sperm progressive motility (P < 0.01) after a 4-h abstinence period, accompanied by significant reductions in citric acid (P < 0.05), α-glucosidase (P < 0.01) and fructose (P < 0.01) concentrations. In addition, due to the decreased number of spermatozoa, these concentrations translated to a significant decrease in fructose (P < 0.05) per spermatozoon, indicating an intrinsic mechanism capitalising on alternative sources of energy for increased metabolic function and subsequent sperm motility.
Reproduction, Fertility and Development – https://doi.org/10.1071/RD18135
Submitted: 11 April 2018 Accepted: 3 January 2019 Published online: 26 March 2019
The effect of the staining technique on morphological and morphometric parameters of boar sperm
Magdalena Czubaszek, Katarzyna Andraszek, Dorota Banaszewska, Renata Walczak-Jędrzejowska
Department of Animal Genetics and Horse Breeding, Siedlce University of Natural Sciences and Humanities, Siedlce, Poland; Department of Breeding Methods and Poultry Breeding, Siedlce University of Natural Sciences and Humanities, Siedlce, Poland; Department of Andrology and Repoductive Endocrinology, Medical University of Łodz, Łodz, Poland
Abstract: Sperm morphology and morphometry are important parameters in predicting fertility. Sperm are considered to be normal if the shape and size of the head, midpiece and tail fall within the classification for a given species. It is important to select the appropriate technique for staining the semen of a given species, because, as many authors have pointed out, some methods work well for one species but are not suitable for analysing another. The aim of the study was to assess the morphometric parameters of boar sperm following the use of different staining techniques and to verify the hypothesis that the staining technique affects the morphometric parameters of sperm. The staining method was found to significantly affect the dimensions of the boar sperm head. The semen stained by the SpermBlue technique had the closest morphometric sperm head parameters to those of the unstained sperm, so this technique, rather than the routinely used eosin and gentian complex, should be the leading technique in the evaluation of boar sperm morphometry. Silver nitrate staining reveals the structure of the sperm in the most detail; this method can be considered universal, and can be used independently or to supplement routine diagnostics. As the staining technique should interfere as little as possible with the structure of the sperm, while revealing its morphology in as much detail as possible, it is crucial to establish the natural dimensions of the unstained sperm head before determining the optimal technique and its reference values. The recommended or most commonly-used techniques are not always the best options for the staining and analysis of sperm of a given species.
Full article here
PLOS ONE – https://doi.org/10.1371/journal.pone.0214243
Published: March 25, 2019
155 Whole genome association analysis suggests an influence of inbreeding on bull sperm morphometry
F. Azcona, M. Sole, J. Dorado, P. Ross, E. Terán, S.E. Demyda-Peyrás
Instituto de Genética Veterinaria, La Plata, Buenos Aires, Argentina; Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata, La Plata, Buenos Aires, Argentina; Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden; Departamento de Medicina y Cirugía Animal, Universidad de Córdoba, Córdoba, España; University of California, Davis, Davis, California, USA
Abstract: Inbreeding depression, the genetic condition caused by mating related individuals, is particularly important in several cattle breeds with limited effective populations. This condition is often associated with decreases in performance, including fertility. Furthermore, sperm head morphometry was associated with fertility in several species. To our knowledge, the influence of inbreeding on sperm morphometry has not been widely reported in cattle. In this study, a Sperm Class Analyzer (SCA™, Microptic S.L., Barcelona, Spain) was used to characterise 7 sperm morphometry parameters in 59 Retinta bulls, including sperm head length, width, perimeter, ellipticity, elongation, regularity and rugosity. Two replicates of at least 100 sperm heads, from 2 frozen semen samples, were assessed per individual (n=200 sperm per bull). Additionally, all individuals were genotyped with the Axiom Bos1 high density SNP Array (Affymetrix, Santa Clara, CA, USA). The molecular-based inbreeding factor (FROH; mean 12.5%, range 1.75 to 33.0) had very low correlations with all sperm morphometry parameters. On average, sperm heads from bulls with higher FROH had a smaller area, but variability was high. Correlations between inbreeding and sperm shape were low and negative, length (r=−0.1449; P<0.01), width (r=−0.2494; P<0.01), and rugosity (r=−0.1086; P>0.01) being the most informative. Whole-genome association study (GWAS) analysis, performed using molecular inbreeding as co-factor, revealed 8 SNP, located on chromosomes BTA2, BTA5, BTA7, and BTA11, significantly associated with sperm head regularity and rugosity. Genomic analysis revealed that genes SLF1 and TMEFF2 are located close to enriched SNP. Gene SLF1 (SMC5-SMC6 complex localization factor 1) is associated with the regulation of protein complex and cytoskeleton assembly, whereas TMEFF2 (transmembrane protein with EGF like and 2 follistatin like domains 2) is associated with integral components of cell membrane and cell spreading and development. Therefore, we inferred that SLF1 and TMEFF2 may be involved in variations of sperm head shape. In this preliminary study, there was evidence of a potential influence of inbreeding on sperm morphometry in a beef cattle breed. However, additional studies, ideally including more individuals and additional breeds, are necessary to determine the validity of this potential association.
Quantitative assessment of heavy metal effects on sperm function using computer-aided sperm analysis and cytotoxicity assays
Hardneck F, Israel G, Pool E, Maree L.
Department of Medical Bioscience, University of the Western Cape, Bellville, South Africa
Abstract: One known environmental risk factor impacting on human reproduction is heavy metal pollution. Although some metals (e.g., Cu, Se and Zn) have protective effects on the male reproductive system in low doses, heavy metals can accumulate to toxic levels and result in poor semen quality and decreased sperm function. We investigated the effect of CuSO4 and CdCl2 (10, 50, 100 and 250 µg/ml or 500 µg/ml) on human sperm motility and vitality by using computer-aided sperm analysis (CASA) and two cytotoxicity assays (WST-1 and XTT). Several sperm motility parameters were significantly reduced after 5 hr of exposure to the highest concentrations of CuSO4 (250 µg/ml) and CdCl2 (500 µg/ml). The WST-1 assay also revealed significantly lower absorbance values for 50, 100 and 250 µg/ml CuSO4 and for 500 µg/ml CdCl2 ; however, no significant effect was seen with XTT. The calculated average IC50 value was 50.31± 4.34 µg/ml for CuSO4 and 392.32 ±76.79 µg/ml for CdCl2 . The effects of these metals were confirmed with MgCl2 , a positive control. This study provides threshold concentrations for the harmful effect of CuSO4 and CdCl2 on human spermatozoa and recommends the use of WST-1 as vitality assay in future in vitro studies.
Lysine acetylation modulates mouse sperm capacitation
Carla Ritagliati, Guillermina M. Luque, Cintia Stival, Carolina Baro Graf, Mariano G. Buffone & Dario Krapf
Laboratory of Cell Signal Transduction Networks, Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET-UNR, Rosario, 2000, Argentina; Laboratory of Cellular and Molecular Reproductive Biology, Instituto de Biología y Medicina Experimental (IBYME), CONICET, Buenos Aires, C1428ADN, Argentina
Abstract: Mammalian sperm are unable to fertilize the egg immediately after ejaculation. To gain fertilization competence, they need to undergo a series of modifications inside the female reproductive tract, known as capacitation. Capacitation involves several molecular events such as phosphorylation cascades, hyperpolarization of the plasma membrane and intracellular Ca2+ changes, which prepare the sperm to develop two essential features for fertilization competence: hyperactivation and acrosome reaction. Since sperm cells lack new protein biosynthesis, post-translational modification of existing proteins plays a crucial role to obtain full functionality. Here, we show the presence of acetylated proteins in murine sperm, which increase during capacitation. Pharmacological hyperacetylation of lysine residues in non-capacitated sperm induces activation of PKA, hyperpolarization of the sperm plasma membrane, CatSper opening and Ca2+ influx, all capacitation-associated molecular events. Furthermore, hyperacetylation of non-capacitated sperm promotes hyperactivation and prepares the sperm to undergo acrosome reaction. Together, these results indicate that acetylation could be involved in the acquisition of fertilization competence of mammalian sperm.
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CRISPR-Cas9-mediated mutation revealed BSPH2 protein is dispensable for male fertility
Eskandari-Shahraki M, Prud’homme B, Manjunath P
Maisonneuve-Rosemont Hospital Research Centre, Montreal, Canada; Departments of Pharmacology and Physiology, Faculty of Medicine, University of Montreal, Montreal, Canada; Department of Medicine, Faculty of Medicine, University of Montreal, Montreal, Canada.
Abstract: Members of the Binder of SPerm (BSP) superfamily have been identified in both human and mouse epididymis. These proteins are known to bind sperm membrane and promote sperm capacitation. Studies suggest that BSPH2 might play a different role in sperm functions from its counterparts; however, the role of BSPH2 remains mainly unexplored. To investigate whether the absence of one member of the BSP family could affect fertility, mice lacking Bsph2 expression were generated using clustered regularly interspaced short palindromic repeats (CRISPR) associated 9 (Cas9) technology. Knockout (KO) male mice were mated with wild-type (WT) females, and the number and weight of the pups were determined. Sperm motility in WT and KO was assessed using sperm class analyzer (SCA). Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used for protein identification. Fertility analysis of null Bsph2 mice did not reveal any phenotype. No differences were noticed on average litter size or average pup weight. Normal testis weight and morphology were observed in Bsph2+/- and Bsph2-/- compared to the WT. Quantitative polymerase chain reaction analyses revealed that Bsph1 messenger RNA expression was increased in mutant mice, whereas LC-MS/MS analysis displayed no increase in protein expression level. Taken together, we show the existence of redundant function for murine BSPH2 and the lack of BSPH2 itself does not lead to sterility.
Sperm motility assessment using computer assisted semen analysis (CASA): a comparison of standard microscope slides and coverslips and the 20 µm MicroCell™
Callum Robinson, Peter Roberts, Phillip Matson
School of Medical and Health Sciences, Edith Cowan University, Joondalup, Western Australia 6027, Australia; Fertility North, Joondalup, Western Australia 6027, Australia
Abstract: Computer assisted semen analysis (CASA) uses instrumentation that makes precise measurements of sperm motility, but the values obtained can be influenced by a number of technical aspects. Motility of human sperm was measured using a Sperm Class Analyzer (Microptic S.L., Barcelona, Spain), and the effect of using different counting chamber/slide configurations was investigated. Results for 20μm MicroCell slides (Vitrolife Sweden AB, Göteborg, Sweden) were compared with microscope slides and 22mmx22mm coverslips loaded with either 5µl (CV.5µl) or 10µl (CV.10µl) semen. Operator-correction of readings for all slide configurations resulted in a significantly lower number of sperm assessed due to the elimination of non-sperm bodies. Following operator-correction, the MicroCell chamber and CV.10µl slide gave similar readings for both progressive motility and immotility for up to 5 minutes, whereas the CV.5µl had a progressive increase in immotile sperm. The interval to analysis was therefore standardised at 2 minutes prior to the measurement of kinetic parameters, and the MicroCell values were significantly different to the CV.10µl for curvilinear velocity (VCL), average path velocity (VAP) and straight line velocity (VSL), and the CV.5µl for VAP. It is concluded that the same configuration be used within the same study, and that care should be taken when comparing different studies that have used different slide/chamber configurations.
J Reprod Stem Cell Biotechnol 7:1-7
Bottlenose dolphin (Tursiops truncatus) sperm revisited: Motility, morphology and ultrastructure of fresh sperm of consecutive ejaculates
Gerhard van der Horst, Katarina Medger, Daniela Steckler, Ilse Luther, Paul Bartels
University of the Western Cape, Private Bag X17, Bellville 7535, South Africa; National Zoological Garden, South African National Biodiversity Institute, PO Box 754, Pretoria 0001, South Africa; Department of Zoology and Entomology, University of Pretoria, Hatfield/Pretoria 0028, South Africa; Section of Reproduction, Faculty of Veterinary Science, University of Pretoria, Onderstepoort, Pretoria 0110, South Africa; GEOsperm, Brits 0250, South Africa; Department of Nature Conservation, Tshwane University of Technology, Pretoria-West 0001, South Africa.
Abstract: Computer aided sperm analysis systems allow detailed examination of sperm motility and morphology variables, which are important for the understanding of the spermatology of a species and the development of assisted reproductive techniques. Cetacean biology is too complex to study in the wild and data from captive individuals provide an important alternative for the conservation of these charismatic animals. The present study evaluates ejaculate and sperm characteristics, including sperm motility, kinematic variables and quantitative sperm morphology and ultrastructure, of consecutive ejaculates from Atlantic bottlenose dolphins (Tursiops truncatus). Sperm concentrations and total and progressive motilities were greater in the second than the first ejaculate, with all ejaculates being of very high quality (6.9–1127 × 106/ml sperm concentration, 75% to 91% total motility and 89% to 96% normal sperm). Most sperm in an ejaculate (≥84%) were highly (VCL>150 μm/s) and progressively motile with very few abnormal sperm. The sperm have small heads, a short but very bulky midpiece and a long tail. Detailed sperm morphometrics using CASA indicated there were similarities from one ejaculate to the next. The large mitochondria with extensive cristae mitochondriales are tightly packed in the midpiece resulting in a large midpiece volume. All the semen and sperm characteristics indicate high quality sperm and support the assumption that a multimale mating system is present in T. truncatus.
CASA in invertebrates
Gerhard van der Horst, Monique Bennett and John D. D. Bishop
Department of Medical Biosciences, University of the Western Cape, Robert Sobukwe Road, Bellville, 7535, South Africa; Marine Biological Association of the United Kingdom, Citadel Hill Laboratory, Plymouth PL1 2PB, UK
Abstract: Sperm movement has been described in several phyla of invertebrates. Yet, sperm motility has only been quantified using computer-aided sperm analysis (CASA-Mot) in externally fertilising species (broadcast spawners) of two phyla, molluscs and echinoderms. In the present study we quantified in detail the nature of the sperm tracks, percentage motility groupings and detailed kinematics of rapid-, medium- and slow-swimming spermatozoa in the oyster Crassostrea gigas and four species never previously studied by CASA-Mot, namely the molluscs Choromytilus meridionalis, Donax serra and Haliotis midae and the echinoderm Parechinus angulosus. A feature common to all these species are the helical tracks, the diameter of which seems to be species specific. Using CASA-Mot, the behaviour of spermatozoa was also studied over time and in the presence of egg water and Ca2þ modulators such as caffeine and procaine hydrochloride. For the first time, we show that hyperactivation can be induced in all species in the presence of egg water (sea water that was mixed with mature eggs and then centrifuged) and/or caffeine, and these hyperactivated sperm tracks were characterised using CASA-Mot. We relate the different patterns of sperm motility and behaviour to reproductive strategies such as broadcast spawning and spermcasting, and briefly review studies using CASA-Mot on other invertebrates.
Current perspectives of CASA applications in diverse mammalian spermatozoa
Gerhard van der Horst, Liana Maree and Stefan S. du Plessis
Department of Medical Bioscience, University of the Western Cape, Private Bag X17, Bellville, 7535, South Africa; Division of Medical Physiology, Faculty of Medicine and Health Sciences, Stellenbosch University, PO Box 19063, Tygerberg, 7505, South Africa
Abstract: Since the advent of computer-aided sperm analysis (CASA) some four decades ago, advances in computer technology and software algorithms have helped establish it as a research and diagnostic instrument for the analysis of spermatozoa. Despite mammalian spermatozoa being the most diverse cell type known, CASA is a great tool that has the capacity to provide rapid, reliable and objective quantitative assessment of sperm quality. This paper provides contemporary research findings illustrating the scientific and commercial applications of CASA and its ability to evaluate diverse mammalian spermatozoa (human, primates, rodents, domestic mammals, wildlife species) at both structural and functional levels. The potential of CASA to quantitatively measure essential aspects related to sperm subpopulations, hyperactivation, morphology and morphometry is also demonstrated. Furthermore, applications of CASA are provided for improved mammalian sperm quality assessment, evaluation of sperm functionality and the effect of different chemical substances or pathologies on sperm fertilising ability. It is clear that CASA has evolved significantly and is currently superior to many manual techniques in the research and clinical setting.
The effects of gelatin supplementation prior to cooling on ram semen quality and fertility
Anderson Marques Pinto Bandeira; José Eduardo Matos; Alexandre Nízio Maria; Paulo César Falanghe Carneiro; Phillip H. Purdy; Hymerson Costa Azevedo
Universidade Federal de Sergipe, São Cristóvão, Brazil; Embrapa Tabuleiros Costeiros, Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA), Aracaju, SE, Brazil; Agricultural Research Service (ARS), United States Department of Agriculture (USDA), Fort Collins, United States of America
Abstract: The physical and chemical characteristics of gelatin have been used to justify its inclusion in extenders to preserve the sperm quality and improve results of cervical artificial insemination with cooled semen. The objective of this study was to evaluate the effects of gelatin supplementation in cooling extender on the quality and fertility of ram semen stored at 5°C. Semen samples (n = 24) of Santa Inês rams (n = 6) were diluted in Glycine-Yolk-Milk extender without (control) or with 1.5% of gelatin. The samples were loaded into 0.25 mL straws, cooled to 5°C and stored vertically for 48 and 72 hours. Sample quality was evaluated using straw homogeneity tests based on pH, osmolality and the proportion of spermatozoa (PS) in both upper and lower segments of straws (US and LS), analyses of sperm motility, plasma and acrosomal membrane integrity, and by fertility after artificial insemination. Differences between the US and LS of straws were found for pH and PS (%). They were significant only in the control group at both times: pH – 5.96 vs. 5.71 at 48 h and 6.13 vs. 5.89 at 72 h; PS – 21.66 vs. 78.34 at 48 h and 20.87 vs. 79.13 at 72 h. Storage in gelatin had very little, to no effect on the sperm kinetics or on the sperm membrane integrity evaluations. The addition of gelatin to the extender did not affect the pregnancy rate which ranged from 4.4 to 26.1%. We conclude that gelatin is effective in maintaining the physical and chemical homogeneity of the semen samples. Further research is needed in order to optimize the use of gelatin supplementation and elucidate any potential benefits.
Animal Reproduction – DOI: 10.21451/1984-3143-AR2017-0021
A novel flush technique to simulate natural dispersal of spermatozoa in the female reproductive tract and expedite motility assessment of fresh ejaculated Merino (Ovis aries) sperm
N. H. Boshoff, H. Lambrechts, L. Maree, S. W. P. Cloete, G. van der Horst
Department of Animal Sciences, Stellenbosch University, P/Bag X1, Matieland, 7602, South Africa; Department of Medical Biosciences, University of the Western Cape, P/Bag X17, Bellville, 7535, South Africa; Directorate Animal Sciences: Elsenburg, P/Bag X1, Elsenburg, 7607, South Africa
Abstract: Motility is an important criterion in the assessment and quantification of the quality of ejaculated and epididymal sperm samples. Ovine sperm spermatozoa are particularly susceptible to cryodamage, and shortening the interval from collection to cryostorage may potentially minimize the negative effects of cryopreservation, thereby improving the post-thaw viability of ram spermatozoa. The use of the swim-up technique (SUT) in quantifying spermatozoa motility is well documented, especially for the isolation of highly motile spermatozoa for assisted reproductive purposes. However, this technique is time consuming and involves a swim-up period of 10 minutes before the motility of a sample is recorded by using computerassisted sperm analysis (CASA) software such as the Sperm Class Analyser® (SCA®). The novel flush technique (FT) allows for capturing of sperm motility tracks via the SCA® system shortly after semen collection, that is, within a minute. This study compared fresh ejaculated sperm motility traits by using the SUT and FT. Motility evaluations were performed using 45 semen samples obtained from 15 adult Merino rams (Ovis aries) at weekly intervals. Motility recordings were captured at 100 frames per second, using a calibrated 20 µm deep Leja slide. The percentage total motile spermatozoa of samples subjected to the FT was 89.2%, which was significantly higher than that recorded by the SUT (83.9%). The results also indicated that the wobble (WOB) parameter showed significantly higher values when using the FT, and parameters curvilinear velocity (VCL) and amplitude of the lateral head displacement (ALH) indicated significantly higher values when using the SUT. Establishing the ideal spermatozoa concentration and analysis of sperm subpopulation motility characteristics would assist in the optimization of the FT, and its use in CASA motility analysis of ovine spermatozoa. Standardization of CASA analysis of ovine semen samples, which would enable the selection of quality spermatozoa samples for use in field insemination (fresh samples) or in vitro fertilization programs, and laparoscopic AI cryopreserved samples warrants further investigation.
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Clinical application of the standardisation of computer-assisted sperm morphometry analysis with ASMA (assisted sperm morphometry analysis) using a semen system analyser SCA 5.4 (Sperm Class Analyzer, Microptic)
Clara Ramírez, José Ramón Alonso, Pedro Jiménez, Jordi Ramis, Josep María Gris, Carlos Aulesa
Unidad de Andrología, Laboratorios Clínicos, Hospital Universitario Vall d’Hebron, Barcelona, Spain; Servicio de Reproducción Asistida, Hospital Universitario Vall d’Hebron, Barcelona, Spain
Introduction: Use is made of computer-assisted sperm morphometry analysis (ASMA) in order to analyse sub-populations as a function of several pathologies.
Material and methods: A total of 703 patients were classified into 7 pathology groups, and their sperm sub-populations were analysed. Pathology groups were classified into infertility, male factor stress (anxiety, insomnia and/or depression), diabetes mellitus type 1 or 2, varicocele, urogenital infections, oncological patients, and other endocrine diseases. Morphological analysis was performed using the semen analyser system SCA 5.4 (Sperm Class Analyzer) (Microptic S.L., Barcelona, Spain). Pre-treatment and staining method used has been previously described by this group.
Results: Automatic human sperm morphology showed that the study of sperm sub-populations has a clinical usefulness in the diagnosis of varicocele. Higher values of 30% elongated sperms had a sensitivity of 37% and a specificity of 79% (1.54 odds ratio with a 95% confidence interval from 1.05 to 2.26). Furthermore, groups of infertility, stress, and diabetes showed new sperm sub-populations which had not been described previously, so they could become a new diagnostic tool.
Discussion: The coefficients of variation of sperm morphology were decreased using automation, enabling the study sperm subpopulations in different pathologies. There is a diagnostic usefulness for varicocele. There could be a prediction to choose the best assisted reproductive technique for infertility patients. However, for the rest of groups new sperm subpopulations could be identified that could make it a diagnostic tool, but requires a higher casuistry to conclude the study.
Revisiting the relationship between the ejaculatory abstinence period and semen characteristics
Bashir M Ayad, Gerhard Van der Horst, Stefan S Du Plessis
Division of Medical Physiology, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa.
Variation in the ejaculatory abstinence period suggested by different guidance bodies have resulted in a growing concern among researchers and clinicians over what the precise period of ejaculatory abstinence ought to be for an optimal semen sample. Several studies have thus been undertaken to examine the association between the length of sexual abstinence and semen characteristics. Not all studies, however, have arrived at the same conclusions. This study aims to review all existing literature published during the past few decades pertaining to the influence of ejaculatory abstinence on semen quality. For the purpose of this systematic review, all data related to sexual abstinence duration and seminal parameters were re-analysed to homogenize the current data. Thorough PubMed, MEDLINE and Google Scholar, a literature search was conducted using the keywords “sexual abstinence”, “ejaculatory abstinence”, “semen”, “spermatozoa”, “semen analysis”, “sperm parameters”, “motility”, “reactive oxygen species (ROS)” and “DNA fragmentation”. After carefully reviewing all the literature, 30 relevant papers, both written in English and published between January 1979 and December 2016, were included in this review. The weight of the evidence suggests that the decline in semen volume and sperm concentration with shorter abstinence periods is accompanied by a substantial improvement in sperm motility characteristics, especially progressive motility and velocity. Nevertheless, available data are insufficient to support definitive conclusions regarding the influence of the ejaculatory abstinence period on advanced semen parameters (ROS, DNA fragmentation and seminal plasma antioxidant capacity) and pregnancy rates. In conclusion, taking all data into account, shortening of the abstinence period may be beneficial to sperm quality. Furthermore, we recommend that the current guidelines regarding the prescribed abstinence period should be revisited.
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Cut-off values for normal sperm morphology and toxicology for automated analysis of rat sperm morphology and morphometry
G van der Horst, B Skosana, A Legendre, P Oyeyipo & SS du Plessis
Department of Medical Bioscience, University of the Western Cape, Bellville; Division of Medical Physiology, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa; Laboratory of Experimental Toxicology, Fontenay aux Roses Cedex, France
We used automated sperm morphology analysis to investigate rat sperm morphometry and morphology in Sprague-Dawley and Wistar rats in three research centers to develop normal baseline values for sperm morphometry and to quantify the percentage of morphologically normal sperm in healthy rats. The participating centers were IRSN in Paris, France (Sprague-Dawley rats), University of the Western Cape, South Africa (Wistar rats) and Stellenbosch University (Wistar rats), South Africa. All three centers used identical sperm isolation techniques from the cauda epididymis, the same staining protocols, identical computer-aided sperm morphometry analysis (CASMA) software and microscopes with similar optics. With CASMA, fully automated analysis of the different parts of stained sperm, e.g., head, acrosome, mid-piece, can be performed, many sperm morphometric features can be measured accurately and eventually normal sperm morphology can be defined. We found that it is possible to distinguish sperm morphometric characteristics of Sprague-Dawley and Wistar rats. We also developed cut-off values for evaluating the percentage normal sperm in these two rat strains using the automatic analysis mode. Normal sperm morphology varied between 67 and 74% by contrast with previous findings of > 90%.
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Effects of chronic musculoskeletal pain on fertility potential in lean and overweight male patients
Fereshteh Dardmeh, Hiva Alipour, Hans Ingolf Nielsen, Sten Rasmussen and Parisa Gazerani
Department of Health Science and Technology, Biomedicine Group, The Faculty of Medicine, Aalborg University, Aalborg, Denmark; SMI®, Department of Health Science and Technology, The Faculty of Medicine, Aalborg University, Aalborg, Denmark; Department of Clinical Medicine, The Faculty of Medicine, Aalborg University Hospital, Aalborg, Denmark
Abstract: Both chronic pain and obesity are known to affect reproductive hormone profiles in male patients. However, the effect of these conditions, alone or in combination, on male fertility potential has received less attention. 20 chronic musculoskeletal pain patients and 20 healthy controls were divided into lean and overweight subgroups according to their BMI. Current level of chronic pain (visual analogue scale) and pressure pain thresholds (PPTs) in 16 predefined sites, classically described and tested as painful points on the lower body, were measured. Levels of reproductive hormone and lipid profiles were assessed by ELISA. Sperm concentration and motility parameters were analyzed using a computer-aided sperm analysis system. Sperm concentration, progressive motility, and percentage of hyperactivated sperm were generally lower in the chronic pain patients in both lean and overweight groups. The overweight control and the lean chronic pain groups demonstrated a significantly lower percentage of progressively motile sperm compared with the lean control group, suggesting that musculoskeletal chronic pain may have a negative influence on sperm quality in lean patients. However, due to the potential great negative influence of obesity on the sperm parameters, it is difficult to propose if musculoskeletal chronic pain also influenced sperm quality in overweight patients. Further research in chronic pain patients is required to test this hypothesis.
Pain Research and Management – https://doi.org/10.1155/2017/4628627
Received 30 August 2017; Revised 3 November 2017; Accepted 13 November 2017; Published 24 December 2017
Diabetes mellitus- induction: Effect of different streptozotocin doses on male reproductive parameters
Temidayo S. Omolaoye, Bongekile T. Skosana, Stefan S. du Plessis
Division of Medical Physiology, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa; Division of Medical Physiology, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa
Abstract: Diabetes mellitus (DM) is reported to be involved in male reproductive impairment, and its impact is evident in the increased prevalence of infertility. Various studies have reported that a single parenteral injection of <40 mg/kg Streptozotocin (STZ) is ineffective in ablating pancreatic β-cells and creating a rat model to investigate the effect of DM on the male reproductive system. This study therefore aims to validate these claims. Adult male Wistar rats received either a single intraperitoneal injection of STZ (30 mg/kg or 60 mg/kg) or saline (0.9%, Control). Diabetes was confirmed after 72 h if plasma glucose levels were ≥14 mmol/L. Body weight, glucose level, fluid and food intake were measured weekly. Animals were sacrificed after 8 weeks of treatment by an overdose of sodium pentobarbital (160 mg/kg body weight). The testis and epididymis were harvested and weighed prior to preparation for histological evaluation. Epididymal sperm morphology was analysed using computer aided sperm analysis (CASA). STZ60 animals presented with significantly lower body weights compared to both control and STZ30 groups. Animals in both STZ30 and STZ60 groups showed decreased normal sperm morphology compared to control. Histological evaluation of the testes showed a decrease in the number of spermatozoa in the seminiferous tubules of animals in the STZ30 and STZ60 groups compared to control. A complete absence of spermiogenesis was observed in the seminiferous tubules of STZ60 animals. These findings prove that an STZ concentration of 30 mg/kg, which is much lower than the reported 40 mg/kg, has adverse effects on the male reproductive system via its diabetogenic effect and can therefore be used to study the impact of DM on male fertility.
Grooves surrounding the micropyle decrease the inseminating dose in fish
Matheus Pereira-Santos, Eduardo Shimoda, André Furugen Cesar de Andrade, Luciano Andrade Silva, Takafumi Fujimoto, José Augusto Senhorini, George Shigueki Yasui and Laura Satiko Okada Nakaghi
Aquaculture Center, São Paulo State University, Jaboticabal-SP, Brazil; Department of Pharmacy, Cândido Mendes University, Campos dos Goytacazes, Brazil; Department of Veterinary, University of São Paulo, Pirassununga, Brazil; Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Japan; and National Center for Research and Conservation of Continental Fish, Chico Mendes Institute of Biodiversity Conservation, Pirassununga, Brazil
Abstract: In fish with external fertilization, sperm must reach the oocyte through the micropyle to enter the cytoplasm. Fertilization success is then influenced by characteristics of oocytes or sperm. In this study, we evaluated oocyte morphology and sperm motility parameters and their effects on the inseminating dose in a teleost fish Astyanax altiparanae . Interestingly, we found one of the lowest yet described inseminating doses in teleosts (2390 spermatozoa oocyte ⁻¹ ml ⁻¹ ). Such a fertilization efficacy may be explained by the long duration of sperm motility (>75 s), the small oocyte diameter (695.119 µm), large micropyle diameter (7.57 µm), and the presence of grooves on the oocyte surface that guides spermatozoon to the fertilization area. Additionally, we have described for the first time a structure that combines grooves on the chorion surface and a ridge in the micropylar area.
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Lactobacillus rhamnosus PB01 (DSM 14870) supplementation affects markers of sperm kinematic parameters in a diet-induced obesity mice model
Fereshteh Dardmeh, Hiva Alipour, Parisa Gazerani, Gerhard van der Horst, Erik Brandsborg, Hans Ingolf Nielsen
Biomedicine Group, Department of Health Science and Technology, Faculty of Medicine, Aalborg University, Aalborg, Denmark; SMI®, Department of Health Science and Technology, Faculty of Medicine, Aalborg University, Aalborg, Denmark; Department of Medical Bioscience, University of the Western Cape, Bellville, South Africa; Bifodan A/S, Hundested, Denmark
Probiotics have been proposed as alternatives to pharmacological products in several medical conditions including the modulation of obesity, which is frequently associated with poor semen quality. However, effects of probiotics on male fertility have been less investigated. This study assessed the effect of Lactobacillus rhamnosus PB01 (DSM-14870) on sperm kinematic parameters in Normal-weight (NW) and diet-induced obese (DIO) models. NW and DIO C57BL/6NTac mice were divided into two subgroups with or without a single daily dose (1x109CFU) of L. rhamnosus for four weeks. Sperm motility and kinematics together with blood lipid profiles and reproductive hormone levels were assessed using the sperm class analyzer system. Probiotic supplementation increased serum testosterone, LH and FSH levels in both NW and DIO groups resulting in significantly (P<0.05) higher velocity (VSL, VCL and VAP) and percentages of progressively motile sperm and significantly lower percentages of immotile sperm. Other kinematic parameters (Lin, STR, ALH and BCF) were also increased in both probiotic supplemented DIO and NW groups at the 10% level of significance. Probiotic supplemented DIO mice demonstrated significantly higher percentages of progressively motile sperm versus DIO controls. This study demonstrated the potential of L. rhamnosus PB01 as a regulatory agent with positive effects on weight loss and reproductive-hormones, significantly improving sperm motility and kinematic parameters in male DIO models.
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Association between obesity and sperm quality
G. A. Ramaraju, S. Teppala, K. Prathigudupu, M. Kalagara, S. Thota, M. Kota, R. Cheemakurthi
Center for Assisted Reproduction, Krishna IVF Clinic, Visakhapatnam, India
There is awareness of likelihood of abnormal spermatozoa in obese men; however, results from previous studies are inconclusive. Advances in computer-aided sperm analysis (CASA) enable precise evaluation of sperm quality and include assessment of several parameters. We studied a retrospective cohort of 1285 men with CASA data from our infertility clinic during 2016. Obesity (BMI ≥30) was associated with lower (mean ± SE) volume (−0.28 ± 0.12, p-value = .04), sperm count (48.36 ± 16.51, p-value = .002), concentration (−15.83 ± 5.40, p-value = .01), progressive motility (−4.45 ± 1.92, p-value = .001), total motility (−5.50 ± 2.12, p-value = .002), average curve velocity (μm/s) (−2.09 ± 0.85, p-value = .001), average path velocity (μm/s) (−1.59 ± 0.75, p-value = .006), and higher per cent head defects (0.92 ± 0.81, p-value = .02), thin heads (1.12 ± 0.39, p-value = .007) and pyriform heads (1.36 ± 0.65, p-value = .02). Obese men were also more likely to have (odds ratio, 95% CI) oligospermia (1.67, 1.15–2.41, p-value = .007) and asthenospermia (1.82, 1.20–2.77, p-value = .005). This is the first report of abnormal sperm parameters in obese men based on CASA. Clinicians may need to factor in paternal obesity prior to assisted reproduction.
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Effects of GnRH vaccination in wild and captive African Elephant bulls (Loxodonta africana) on reproductive organs and semen quality
Imke Lueders, Debbie Young, Liana Maree, Gerhard van der Horst, Ilse Luther, Stephan Botha, Brendan Tindall, Geoffrey Fosgate, André Ganswindt, Henk J. Bertschinger
GEOlifes-Animal Fertility and Reproductive Research, Hamburg, Germany; Endocrine Research Laboratory, Department of Anatomy and Physiology, Faculty of Veterinary Science, University of Pretoria, Onderstepoort, South Africa; African Elephant Research Unit, Plettenberg Bay, South Africa; Department of Medical Biosciences, University of the Western Cape, Bellville, South Africa; Department of Research and Scientific Services, National Zoological Gardens of South Africa, Pretoria, South Africa; African Lion Safari, Cambridge, ON, Canada, 7 Robberg Veterinary Clinic, 56 Longships, Plettenberg Bay, South Africa; Department of Production Animal Studies, Faculty of Veterinary Science, University of Pretoria, Onderstepoort, South Africa
Objectives: Although the African elephant (Loxodonta africana) is classified as endangered by the International Union for Conservation of Nature (IUCN), in some isolated habitats in southern Africa, contraception is of major interest due to local overpopulation. GnRH vaccination has been promoted as a non-invasive contraceptive measure for population management of overabundant wildlife. We tested the efficacy of this treatment for fertility control in elephant bulls.
Methods: In total, 17 male African elephants that were treated with a GnRH vaccine were examined in two groups. In the prospective study group 1 (n = 11 bulls, ages: 8–36 years), semen quality, the testes, seminal vesicles, ampullae and prostate, which were all measured by means of transrectal ultrasound, and faecal androgen metabolite concentrations were monitored over a three-year period. Each bull in the prospective study received 5 ml of Improvac® (1000 μg GnRH conjugate) intramuscularly after the first examination, followed by a booster six weeks later and thereafter every 5–7 months. In a retrospective study group (group 2, n = 6, ages: 19–33 years), one examination was performed on bulls which had been treated with GnRH vaccine for 5–11 years.
Results: In all bulls of group 1, testicular and accessory sex gland sizes decreased significantly after the third vaccination. In six males examined prior to vaccination and again after more than five vaccinations, the testis size was reduced by 57.5%. Mean testicular height and length decreased from 13.3 ± 2.6 cm x 15.2 ± 2.8 cm at the beginning to 7.6 ± 2.1 cm x 10.2 ± 1.8 cm at the end of the study. Post pubertal bulls (>9 years, n = 6) examined prior to vaccination produced ejaculates with viable spermatozoa (volume: 8–175 ml, sperm concentration: 410-4000×106/ml, total motility: 0–90%), while after 5–8 injections, only 50% of these bulls produced ejaculates with a small number of immotile spermatozoa. The ejaculates of group 2 bulls (vaccinated >8 times) were devoid of spermatozoa. Faecal androgen metabolite concentrations measured in captive males decreased significantly after the fourth vaccination. None of the males entered musth during the treatment period.
Conclusions: Our results showed a marked decrease in semen quality, testicle and secondary sex gland sizes following repeated GnRH vaccinations. After 2–4 years of continuous treatment every 5–7 months, the effects were similar to surgical castration.
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Short abstinence: A potential strategy for the improvement of sperm quality
Bashir M. Ayad, Gerhard Van der Horst, Stefan S. du Plessis
Division of Medical Physiology, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa
Objective: To determine the effect of short (4 h) and long (4 days) abstinence periods on sperm quality based on functional and biochemical parameters in a population of normozoospermic men.
Methods: Two semen samples were collected in succession from potentially fertile, normozoospermic men (n = 100) after an abstinence period of 4 days and 4 h respectively. The mean values of semen volume, pH, viscosity, sperm concentration, percentage of total and progressively motile sperm, sperm kinematics/velocity, normal morphology, acrosome status, DNA fragmentation, intracellular superoxide (O2−•) levels and seminal antioxidant status were compared between the two abstinence duration groups.
Results: A significant increase in total and progressive motility and velocity parameter values were observed after short abstinence compared with long abstinence periods. Sperm DNA fragmentation and intracellular O2−• levels were not significantly different between the two abstinence periods. Despite the decrease in semen volume, sperm concentration and total sperm number after short abstinence periods, all mean values of the conventional semen parameters remained above the lower reference limits as reported by the WHO.
Conclusion: The data from this most comprehensive study of its kind challenges the generally accepted guidelines of the prolonged abstinence periods since the results show that 4 h of sexual abstinence yielded significantly better sperm samples from a functional point of view. Although this study was performed on normozoospermic men, future studies with infertile men might yield similar findings that could lead to employing short abstinence as a strategy to improve the outcome of ART and fertility preservation.
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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
The authors present a critical review of the WHO5 (2010) manual of semen analysis and what it should be used for: The analysis of sperm quality and not analysis to predict fertility outcome per se. We show the strengths and shortcoming of WHO5 and then ask for a better “marriage” among these parameters and the outcome of sperm functionality and fertilization/live birth outcome. For many decades the basis of the WHO manual for semen analysis has not changed and we emphasize that sperm functionality testing has not really been considered/performed in the routine andrology laboratory. There is a need to first develop more objective and quantitative methodology such as computer-aided sperm analysis, to analyse sperm quality and sperm functionality that relates in many instances to fertilization/live birth outcome: 1) sperm cervical mucous penetration using computer aided sperm analysis (CASA), 2) endpoint of capacitation, hyperactivation as measured accurately by CASA, 3) acrosome reaction quantitatively, 4) chromatin maturity and DNA fragmentation quantitatively, 4) where possible oocyte binding tests (hemizona), 5) relationships of vitality and hypo-osmotic swelling test using modern technology 6) measurement of oxidative stress, 7) analysis of semen using proteomics (proteins that are importantly functionally expressed in seminal plasma) as well as 8) metabolomics representing a systematic study of the unique metabolic fingerprints (chemical) that specific cellular processes leave behind and inform us about function/dysfunction, 9) patient profile (obesity, smoking, age, stress, female cryptic choice, environment and many other patient characteristics) as important determinants in fertility outcome. We believe we can intelligently in the end construct a matrix which combine all these factors and others in the future that inform us about potential fertility outcome. But then realize WHO5/ESHRE current guidelines are not particularly informative in the above context.
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Improved sperm kinematics in semen samples collected after 2 h versus 4-7 days of ejaculation 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
Does a short abstinence period of only 2 h yield spermatozoa with better motility characteristics than samples collected after 4-7 days?
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.
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.
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.
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.
Standardization of pretreatment and staining technique to perform automatic human sperm morphology with ASMA (assisted sperm morphometry analyses)
Clara Ramírez, José Ramón Alonso, Pedro Jiménez, Rocío Reyes, Jordi Ramis, Josep Maria Gris, Carlos Aulesa
Unidad de Andrología, Laboratorios Clínicos, Hospital Universitario Vall d’Hebron, Barcelona, Spain; Servicio de Reproducción Asistida, Hospital Universitario Vall d’Hebron, Barcelona, Spain
Objective: Standardization ofpretreatment and staining technique to realizemorphological semen evaluation with computer-assisted sperm morphometry analysis system, with semen system analyzer SCA 5.4 (Sperm Class Analyzer, Microptic).
Material and methods: Morphological analysis was performed with semen system analyzer SCA 5.4 (Microptic SL, Barcelona, Spain). The staining method was a modification of Hemacolor kit (Merck). Between 60 and 125 semen samples were chosen randomly from our laboratory.
Results: Pretreatment of semen samples was a centrifugation for 5 minutes at 300 g, seminal plasma was rejected and the pellet was homogenized with .2 mL of the seminal plasma itself. Not change in sperm morphology have been found with this pretreatment. Staining was performed with Hemacolor kit (Merck) but with some modifications. Fixer has been buffered with phosphate buffer pH 7.2 at 10%, time recommended by the manufacturer has been reduced. Fixation for 5 seconds, 30 seconds with Eosin staining and 2 seconds with staining Azur. Finally it was washed for 30 seconds with pH 7.2 phosphate buffer, as indicated by the manufacturer. After the pretreatment and staining we have got reference values for our methodology.
Conclusions: An automation methodology to perform sperm morphology reduces coefficient variations of determination thereby we can increase its technical reliability and remove the subjectivity of the manual analysis. This standardization can be the first step to study the diagnostic value of advanced morphology in infertility and urological diseases.
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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.
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.
Thyroxine is useful to improve sperm motility
Gabriela Ruth Mendeluk. Ph.D.; Mónica Rosales M.D.
Laboratory of Male Fertility. Hospital de Clínicas “José de San Martín”. Faculty of Pharmacy and Biochemistry. University of Buenos Aires. Buenos Aires. Argentina; Laboratory of Endocrinology. Hospital de Clínicas “José de San Martín”. Faculty of Pharmacy and Biochemistry. University of Buenos Aires. Buenos Aires. Argentina.
BACKGROUND: The aim of this study was to evaluate the non-genomic action of thyroxine on sperm kinetic and its probable use to improve sperm recovery after an enrichment method like “swim-up” in comparison to the available one pentoxifylline.
MATERIAL AND METHODS: A total of 50 patients consulting for infertility were recruited. Conventional sperm assay were performed according to WHO criteria- 2010. A CASA system was employed to assess kinetic parameters and concentration. The number of motile sperm recovered after the preparation technique was calculated.
RESULT(S): Addition of T4 (0.002ug/ml) to semen samples increased hypermotility at 20 min(control: 14.18 ± 5.1% Vs. 17.66 ± 8.88 % ; p<0.03; data expressed as mean ± SD )and remained unchanged after 40 min. Significant differences were found in the motile sperm recovered after “swim-up” (control: 8.93 x 106 ± 9.52 x 106 Vs. 17.20 x 106 ± 21.16 x106; p<0.03), achieving all the tested samples the desired threshold value for artificial insemination outcome, while addition of pentoxifylline increased the number of recovered sperm after swim-up in 60% of the studied cases. No synergism between treatments could be proved.
CONCLUSION(S): We are proposing a new and physiologic tool to improve artificial insemination. The discussion opens our minds to think in unknown pathways involved in sperm capacitation and gives innovative arguments to understand infertility.
Computer Aided Sperm Analysis, a useful tool to evaluate patient’s response to varicocelectomy
Julia I Ariagno, Gabriela R Mendeluk, María J Furlan, M Sardi, P Chenlo, Susana M Curi, Mercedes N Pugliese, Herberto E Repetto, Mariano Cohen
Departamento de Bioquímica Clínica, Laboratorio de Fertilidad Masculina, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires (CP1120), Argentina; Departamento de Urologia, Hospital de Clinicas “José de San Martín”, Universidad de Buenos Aires, Buenos Aires (CP 1120), Argentina
Pre-operative and post-operative sperm parameter values from infertile men with varicocele were analysed by computer-aided sperm analysis (CASA) to assess if sperm characteristics improved after varicocelectomy. Semen samples of men with proven fertility (n = 38) and men with varicocele-related infertility (n = 61) were also analysed. Conventional semen analysis was performed according to WHO (2010) criteria and a CASA system was employed to assess kinetic parameters and sperm concentration. Seminal parameters values in the fertile group were very far above from those of the patients, either before or after surgery. No significant improvement in the percentage normal sperm morphology (p=0.10), sperm concentration (p = 0.52), total sperm count (p = 0.76), subjective motility (%) (p = 0.97) nor kinematics (p = 0.30) was observed after varicocelectomy when all groups were compared. Neither was significant improvement found in percentage normal sperm morphology (p = 0.91), sperm concentration (p = 0.10), total sperm count (p = 0.89) or percentage motility (p = 0.77) after varicocelectomy in paired comparisons of pre-operative and post-operative data. Analysis of paired samples revealed that the total sperm count (0.01) and most sperm kinetic parameters: curvilinear velocity (p = 0.002), straight-line velocity (p = 0.0004), average path velocity (p = 0.0005), linearity (p = 0.02), and wobble (p = 0.006) improved after surgery. CASA offers the potential for accurate quantitative assessment of each patient’s response to varicocelectomy.
Asian Journal of Andrology.
Manuscript ID: AJA-5138
Evaluation of sperm motility using two cryoprotectants
Sandra Abalde Graña; Mª Esther Rendal Vázquez; Mariana García García; Mª Jesús López Piñón; Marcos Carballal Rodríguez; Catarina Barbero Cancelo; Rosario Olvido Fernández Mallo; Inma Míguez Torre; Teresa Bermúdez González; Sonia Pértega Díaz; Jacinto Sánchez Ibáñez
Unidad de Criobiología-Banco de Tejidos. CHUAC. A Coruña; Unidad de Fecundación in vitro. CHUAC. A Coruña; Unidad de Estadística. CHUAC. A Coruña
Infertility is a problem that affects a lot of couples. One of its causes is a decreased semen quality due to, for example, gonadotoxic treatments. The cryopreservation of human semen is the technique that allows sperm preserving and storing without losing their fertilizing capacity; being a fundamental tool in assisted reproduction. The aim of this study was to optimize the cryopreservation technique. To this end, a study carried out on samples of patients under study by fertility problems, in which two cryoprotectant media (SpermCryo™ All-round and CryoSperm™) and the execution or non-execution of an immersion of the samples in liquid nitrogen (before storage) were compared; and the effect of the time between ejaculation and the processing on the quality of the sample. Variations were studied with an automatic analyzer by performing pre- and post-thaw sperm motility tests.
The results show no difference between the two cryoprotectants media, but seems to have a tendency to obtain better postthaw mobility with either depending on sample characteristics. Moreover, the liquid nitrogen bath had no apparent effects on post-thaw results. However, we must highlight the importance of time in the processing of semen samples once liquefied, to avoid decreased sperm quality.
To improve post-thaw outcomes the key lies in the necessity to adjust the freezing protocol to the sample characteristics and a correct implementation of the protocol cryopreservation (selection and addition of cryoprotectant media…); favoring the management of infertility and the success of assisted reproduction techniques.
Rev. Iberoam. Fert Rep Hum, 2016
Computer Assisted Semen Analysis, an overcoming 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.
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.
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.
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
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.
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.
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.
Effect of environmental pH on sperm kinematic characteristics
H. Alipour, F. Dardmeh, M.C. Dissing, H.I. Nielsen
Laboratory of Reproductive biomedicine, Biomedicine Group, Department of Health Science and Technology, Faculty of Medicine, Aalborg University, Aalborg, Denmark; ORIGIO A/S, Maaloev, Denmark
Semen preparation medium have an important role in assisted reproduction techniques and their composition influences sperm binding and motility.
Some studies have assessed the influence of pH on sperm kinetics. However, no study to date has assessed the effect of environmental pH on subtle differences in the details of the sperm movement (kinematics) of human sperm provided by computerized sperm analysis systems. This study was designed to assess the effect of two different media pH levels on kinematic parameters of the human sperm.
Samples were prepared using the 40%/80% Pureception (Sage, USA) density gradient and resuspended in customized sperm culture media with pH levels of 7.9 and 8.3 (Origio, Denmark). Kinematic parameters of the sperm in both groups were analyzed using the Sperm Class Analyzer (Microptic S.L., Spain) at 0, 6 and 24 hours following the addition of media.
Results of this study illustrated a general insignificant decrease in the ratio of progressively motile and hyperactive sperm after 6 and 24 hours. However a significant difference between the test groups was observed in the curvilinear, straight line and Mean path velocity and Straightness after 6 and 24 hours. Linearity and Wobble showed significant difference after 24 hours.
This study demonstrated a difference in the sperm motion pattern and velocity in different environmental pH levels. Based on these findings, further investigations are required to elucidate knowledge about possible effect of marginal pH changes, affecting the sperm motion characteristics at different stages in the dynamic environment of the female reproductive tract.
Dietary probiotic supplement positively affects sperm motility in an obese 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
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
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
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
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.
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.
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.
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 f