In vitro characterisation of fresh and frozen sex-sorted bull spermatozoa

peer-reviewedThis study sought to compare the in vitro characteristics of fresh and frozen non-sorted (NS) and sex-sorted (SS) bull spermatozoa. Experiment 1: Holstein–Friesian ejaculates (n = 10 bulls) were split across four treatments and processed: (1) NS fresh at 3 × 106 spermatozoa, (2) X-SS frozen at 2 × 106 spermatozoa, (3) X-SS fresh at 2 × 106 spermatozoa and (4) X-SS fresh at 1 × 106 spermatozoa. NS frozen controls of 20 × 106 spermatozoa per straw were sourced from previously frozen ejaculates (n = 3 bulls). Experiment 2: Aberdeen Angus ejaculates (n = 4 bulls) were split across four treatments and processed as: (1) NS fresh 3 × 106 spermatozoa, (2) Y-SS fresh at 1 × 106 spermatozoa, (3) Y-SS fresh at 2 × 106 spermatozoa and (4) X-SS fresh at 2 × 106 spermatozoa. Controls were sourced as per Experiment 1. In vitro assessments for progressive linear motility, acrosomal status and oxidative stress were carried out on Days 1, 2 and 3 after sorting (Day 0 = day of sorting. In both experiments SS fresh treatments had higher levels of agglutination in comparison to the NS fresh (P < 0.001), NS frozen treatments had the greatest PLM (P < 0.05) and NS spermatozoa exhibited higher levels of superoxide anion production compared with SS spermatozoa (P < 0.05). Experiment 1 found both fresh and frozen SS treatments had higher levels of viable acrosome-intact spermatozoa compared with the NS frozen treatments (P < 0.01).ACCEPTEDpeer-reviewe

Epididymis Response Partly Compensates for Spermatozoa Oxidative Defects in snGPx4 and GPx5 Double Mutant Mice

We report here that spermatozoa of mice lacking both the sperm nucleaus glutathione peroxidase 4 (snGPx4) and the epididymal glutathione peroxidase 5 (GPx5) activities display sperm nucleus structural abnormalities including delayed and defective nuclear compaction, nuclear instability and DNA damage. We show that to counteract the GPx activity losses, the epididymis of the double KO animals mounted an antioxydant response resulting in a strong increase in the global H2O2-scavenger activity especially in the cauda epididymis. Quantitative RT-PCR data show that together with the up-regulation of epididymal scavengers (of the thioredoxin/peroxiredoxin system as well as glutathione-S-transferases) the epididymis of double mutant animals increased the expression of several disulfide isomerases in an attempt to recover normal disulfide-bridging activity. Despite these compensatory mechanisms cauda-stored spermatozoa of double mutant animals show high levels of DNA oxidation, increased fragmentation and greater susceptibility to nuclear decondensation. Nevertheless, the enzymatic epididymal salvage response is sufficient to maintain full fertility of double KO males whatever their age, crossed with young WT female mice

Probes and Techniques for Sperm Evaluation by Flow Cytometry

P. 67-78Flow cytometry has become an important technique in sperm evaluation and is increasingly used both for routine assessment and for research in veterinary science. We have revised the literature, describing fluorescent probes that have been used for analysing spermatozoa by flow cytometry, regarding: viability, acrosomal status, capacitation, mitochondrial status, apoptotic markers, oxidative stress markers, DNA damage, sperm counting and sperm sizing. Details and problems of some techniques are reviewed, with special attention to the occurrence of non‐sperm particles in the samples (‘debris’). New and promising aspects of flow cytometry, such as sperm sorting using viability markers as selection criteria, are highlighted. The relationship between flow cytometry analyses and fertility and their future improvements are considered.S

Anandamide Capacitates Bull Spermatozoa through CB1 and TRPV1 Activation

Anandamide (AEA), a major endocannabinoid, binds to cannabinoid and vanilloid receptors (CB1, CB2 and TRPV1) and affects many reproductive functions. Nanomolar levels of anandamide are found in reproductive fluids including mid-cycle oviductal fluid. Previously, we found that R(+)-methanandamide, an anandamide analogue, induces sperm releasing from bovine oviductal epithelium and the CB1 antagonist, SR141716A, reversed this effect. Since sperm detachment may be due to surface remodeling brought about by capacitation, the aim of this paper was to investigate whether anandamide at physiological concentrations could act as a capacitating agent in bull spermatozoa. We demonstrated that at nanomolar concentrations R(+)-methanandamide or anandamide induced bull sperm capacitation, whereas SR141716A and capsazepine (a TRPV1 antagonist) inhibited this induction. Previous studies indicate that mammalian spermatozoa possess the enzymatic machinery to produce and degrade their own AEA via the actions of the AEA-synthesizing phospholipase D and the fatty acid amide hydrolase (FAAH) respectively. Our results indicated that, URB597, a potent inhibitor of the FAAH, produced effects on bovine sperm capacitation similar to those elicited by exogenous AEA suggesting that this process is normally regulated by an endogenous tone. We also investigated whether anandamide is involved in bovine heparin-capacitated spermatozoa, since heparin is a known capacitating agent of bovine sperm. When the spermatozoa were incubated in the presence of R(+)-methanandamide and heparin, the percentage of capacitated spermatozoa was similar to that in the presence of R(+)-methanandamide alone. The pre-incubation with CB1 or TRPV1 antagonists inhibited heparin-induced sperm capacitation; moreover the activity of FAAH was 30% lower in heparin-capacitated spermatozoa as compared to control conditions. This suggests that heparin may increase endogenous anandamide levels. Our findings indicate that anandamide induces sperm capacitation through the activation of CB1 and TRPV1 receptors and could be involved in the same molecular pathway as heparin in bovines

L’analyse automatisée du sperme, l’andrologie 2.0

International audienceThere are mainly two automatic spermiology technologies, CASA (Computer-Assisted Semen Analysis) and SQA (Sperm Quality Analyser). Automatons save time and provide standardized and reproducible evaluation of sperm concentration and motility. However, they remain to be perfected for morphology analysis. To date, they allow rapid screening of altered versus normozoospermic sperm, but cannot replace a trained technician and the manual analysis. In parallel, semen analysis systems on smartphones or using kits are now freely available. They are inexpensive and feasible at home and allow an initial estimate of presence of spermatozoa before medical care. Artificial intelligence systems with autonomous learning "machinelearning" are also being developed to improve the characterization of sperm, including the choice of gamete to be injected in ICSI (Intra Cytoplasmic Sperm Injection).Il existe deux technologies principales d’automates de spermiologie, les CASA et les SQA. Ces automates permettent un gain de temps et l’évaluation standardisée et reproductible de la concentration et de la mobilité des spermatozoïdes. Cependant, ils restent à être perfectionnés pour l'analyse de la morphologie. A ce jour, s’ils autorisent un dépistage rapide des spermes, ils ne peuvent pas remplacer un technicien formé et une analyse manuelle. En parallèle, des systèmes d'analyse de sperme sur smartphones ou utilisant des kits sont désormais disponibles en accès libre. Ils sont peu coûteux, réalisables chez soi et donnent une première estimation de la présence de spermatozoïdes avant une prise en charge médicale. Des systèmes d'intelligence artificielle avec apprentissage autonome («machine-learning») sont également en cours de développement et permettent d'améliorer la caractérisation des spermatozoïdes, y compris dans le choix du gamète à injecter en ICSI

Computer-assisted semen analysis

International audienceComputer-assisted semen analysis (CASA) systems are equipment that use different methodologies. When a laboratory wishes to acquire these systems, it is important to know how they perform. CASA has been “relegated” to the “advanced semen examinations” section in the latest version of the World Health Organization (WHO) lab manual because there is a lack of literature to demonstrate its full utility in routine infertility diagnosis. However, some systems perform very well in assessing sperm motility and sperm count. In the context of assisted reproductive techniques (ARTs), these systems save time and can provide a standardized assessment of the characteristics of semen prepared for ART. CASA systems are evolving steadily, artificial (AI) systems are being developed, and smartphone-based sperm analysis systems are now available for home analysis. It is clear that andrology and ART are rapidly changing fields, whereas AI and automation are only just beginning. But in a few years’ time, there is no doubt that these CASA systems should improve further and enable what the WHO manual has been calling for since 2010: greater reproducibility and standardization

Impact du stress oxidant sur la qualité nucléaire spermatique

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Impact of slow freezing on sperm nuclear quality and telomere length

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Détection de la maladie résiduelle du sarcome d’Ewing dans le tissu ovarien et testiculaire par une méthode sensible et spécifique

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