Sperm Motility and Lactate production at different sperm concentrations

Lactate production is associated with total spermatozoa concentration. It negatively affects preservation of total and progressive motility, showing an effect of by-products of anaerobic metabolism on long-term storage. Moreover, our data show that non-progressive motile spermatozoa are highly associated to lactate concentration, and thus, anaerobic glycolysis. More studies are required to determine relative contributions of aerobiosis and anaerobiosis to spermatozoa motility under different storage conditions.Peer reviewe

Glucose use and lactate production by equine fresh semen in human and equine extender

This study shows that this human semen extender doesn’t support equine semen preservation. Sperm cells’ glucose consumption and lactate production seem to be negligible, as these parameters were not affected by sperm concentrations in our study. Our results suggest that spermatozoa are able to cleave complex carbohydrates as glucose concentration in INRA96 increased over time

Sperm Quality Assessment in Stallions: How to Choose Relevant Assays to Answer Clinical Questions

peer reviewedStallion sperm analysis is indicated for infertility diagnosis, pre-sale expertise, production of fresh or frozen doses, and frozen straw quality control. Various collection methods are described, and numerous assays can be performed on semen. Determining an approach for each of these cases is challenging. This review aims to discuss how to obtain relevant clinical results, answering stallion owners’ concerns. Semen can be collected with an artificial vagina on a phantom or a mare, by elec- tro-ejaculation under anesthesia, or after pharmacological induction. The collection method influ- ences the semen volume and concentration, while the total sperm number depends on the testicular production and collection frequency. In the seminal plasma, acidity, pro-oxidant activity, and some enzymes have repercussions for the semen quality and its conservation. Moreover, non-sperm cells of seminal plasma may impact semen conservation. Motility analysis remains a core parameter, as it is associated with fresh or frozen dose fertility. Computer-assisted motility analyzers have im- proved repeatability, but the reproducibility between laboratories depends on the settings that are used. Morphology analysis showing spermatozoa defects is useful to understand production and maturation abnormalities. Staining of the spermatozoa is used to evaluate viability, but recent ad- vances in flow cytometry and in fluorochromes enable an evaluation of multiple intracellular pa- rameters. Spermatozoa protein expression already has clinical applications, for example, as a fertil- ity and freezing ability predictor. At present, stallion semen analysis ranges from macroscopic eval- uation to assessing spermatozoa proteins. However, clinically, all these data may not be relevant, and the lack of standardization may complicate their interpretation

Uterine fluid contains factors inhibiting myeloperoxidase enzymatic activity in mares

peer reviewe

The secretions of oviduct epithelial cells increase the equine in vitro fertilization rate: are osteopontin, atrial natriuretic peptide A and oviductin involved?

ABSTRACT: BACKGROUND: Oviduct epithelial cells (OEC) co-culture promotes in vitro fertilization (IVF) in human, bovine and porcine species, but no data are available from equine species. Yet, despite numerous attempts, equine IVF rates remain low. Our first aim was to verify a beneficial effect of the OEC on equine IVF. In mammals, oviductal proteins have been shown to interact with gametes and play a role in fertilization. Thus, our second aim was to identify the proteins involved in fertilization in the horse. Methods & results In the first experiment, we co-incubated fresh equine spermatozoa treated with calcium ionophore and in vitro matured equine oocytes with or without porcine OEC. We showed that the presence of OEC increases the IVF rates. In the subsequent experiments, we co-incubated equine gametes with OEC and we showed that the IVF rates were not significantly different between 1) gametes co-incubated with equine vs porcine OEC, 2) intact cumulus-oocyte complexes vs denuded oocytes, 3) OEC previously stimulated with human Chorionic Gonadotropin, Luteinizing Hormone and/or oestradiol vs non stimulated OEC, 4) in vivo vs in vitro matured oocytes. In order to identify the proteins responsible for the positive effect of OEC, we first searched for the presence of the genes encoding oviductin, osteopontin and atrial natriuretic peptide A (ANP A) in the equine genome. We showed that the genes coding for osteopontin and ANP A are present. But the one for oviductin either has become a pseudogene during evolution of horse genome or has been not well annotated in horse genome sequence. We then showed that osteopontin and ANP A proteins are present in the equine oviduct using a surface plasmon resonance biosensor, and we analyzed their expression during oestrus cycle by Western blot. Finally, we co-incubated equine gametes with or without purified osteopontin or synthesized ANP A. No significant effect of osteopontin or ANP A was observed, though osteopontin slightly increased the IVF rates. CONCLUSION: Our study shows a beneficial effect of homologous and heterologous oviduct cells on equine IVF rates, though the rates remain low. Furthers studies are necessary to identify the proteins involved. We showed that the surface plasmon resonance technique is efficient and powerful to analyze molecular interactions during fertilization

Validation of Calcein Violet as a New Marker of Semen Membrane Integrity in Domestic Animals

peer reviewedMany fluorochromes routinely used in semen quality analysis emit in the green and red channels, limiting their possible combination for multiple parameter analysis. The use of fluorophores emitting in different light channels broadens the possibilities of combination to expand the range of simultaneously evaluated criteria. This is of great interest in cases of small ejaculated volumes, such as those naturally occurring in roosters, small dog breeds and drones (Apis mellifera). The purpose of this experiment is to establish Calcein Violet (CaV), a blue fluorochrome, as a marker of viability and acrosomal integrity in domestic animals in order to free the red and green channels. SYBR®14/Propidium Iodide (PI) was used as reference dye, heat-treated samples as negative controls, serial staining combination for validation and epifluorescence microscopy for observation. Dead spermatozoa marked in red with PI showed no blue fluorescence either from the head or the tail. Live spermatozoa showed a decreasing blue emission from head to tail when single stained with CaV. Unreacted acrosomes showed intense blue fluorescence irrespective of plasma membrane integrity. This needs to be further confirmed for species with small and difficult to observe heads. Establishment of CaV as a marker of membrane integrity by fluorescence microscopy is a decisive first step towards further technical development and use with flow cytometry

Le Cortisol et la DHEA comme marqueurs de la placentite chez la jument gestante : une étude préliminaire

peer reviewedPlacentitis in mares is a subclinical cause of abortion, high- lighting the importance of early detection for successful treatment. Sexual steroids could serve as biomarkers, but cross-reactivity and a limited detection range of immunoassays may hinder ac- curate measures. This study evaluated some steroids as poten- tial markers of placentitis using liquid chromatography coupled to mass spectrometry (LC-MS), known to improve detection and quantification of steroids with good selectivity, specificity, and allowing multiplexing capabilities (Conley et al., Reproduction. 2019;158:197-208; Ledeck et al., Theriogenology. 2022;189:86- 91). From the 7th month of pregnancy, mares were transrectally scanned to measure the combined thickness of the uterus and placenta (CTUP), and blood samples were collected at a fixed time of day. Ten healthy mares (HM) and nine mares diagnosed with non-experimentally-induced placentitis (PM) between 8 and 10 months were enrolled, based on ultrasonographic placentitis signs: heterogeneous echogenicity and/or thickened CTUP. Post- partum allantochorion examination confirmed the diagnosis. Sub- sequently, PM were further excluded from the study as they re- ceived treatments for placentitis. Serum concentrations of pro- gesterone, 17α-hydroxyprogesterone (17αOHP), dehydroepiandros- terone (DHEA), and cortisol were assayed using LC-MS. Non- normally distributed data were presented as medians, and groups were compared at the same month of pregnancy using the Mann- Whitney test. There were no significant differences in 17αOHP and progesterone concentrations between groups at any month. DHEA concentrations were significantly higher (p=0.0297) at 8 months in PM (3.552μg/L) compared to HM (2.240μg/L). A sim- ilar trend was observed at 9 months, with DHEA concentra- tions of 2.126μg/L and 1.445μg/L in PM and HM, respectively (p=0.0604). In contrast, HM had significantly higher (p=0.022) cortisol concentrations at 7 months (71.75μg/L) compared to PM (38.62μg/L). This difference tended (p=0.0529) to be observed at 8 months with cortisol concentrations of 52.25μg/L (HM) and 37.28μg/L (PM). The DHEA/cortisol ratio was significantly higher in PM than in HM at 9 months (p=0.013) and tended to be in- creased at 7 and 8 months (respectively, p=0.0659 and p=0.0529). Placentitis has been shown to increase pregnenolone production (Ousey et al., Theriogenology. 2005;63:1844-1856). In this prelim- inary study, PM were observed to metabolize pregnenolone into DHEA rather than cortisol, without changes in 17αOHP concen- trations. Reduced cortisol concentrations at 7 months could be an early but non-specific biomarker of placentitis. Conversely, elevated DHEA Ievels only appear together with ultrasonographic signs at 8 months but could become a more specific biomarker, which dif- fers from previous findings in mares with experimentally-induced placentitis (Canisso et al., Equine Veterinary Journal. 2017;49:244- 249). These changes result in a higher DHEA/cortisol ratio in PM at 9 months. However, further research should explore mod- ifications of steroids’ pathways in PM to confirm the value of DHEA and cortisol for early diagnosis of placentitis in mares.Study of equine placental sexual steroids’ metabolism using Mass Spectrometry (LC-MS/MS) and in vitro model to develop early diagnosis methods of placental pathologie

Immunohistochemical expression of Myeloperoxidase in the equine endometrium

editorial reviewe

Cas clinique: Fibrome Ovarien chez une jument - Considérations hormonales

peer reviewedThis report shows that, as in humans, AMH as well as steroids productions are low in case of ovarian fibroma, thus preserving normal cyclicity