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

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

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

Comparison of combined thickness of the uterus and the placenta between 2 breeds of mares and their potential relationship with estrogens during normal pregnancy.

peer reviewe

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

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

Development and validation of a liquid chromatography coupled to mass spectrometer (LC-MS) method for the simultaneous quantification of estrone-3-sulfate, progesterone, estrone and estradiol in serum of mares and American bisons

peer reviewedSteroid concentrations in serum are fluctuating during pregnancy of many mammal species. The current knowledge about endocrinology of gestation is mainly based on immunoassays. However, the lack of specificity of these assays hampers the reliability of the results. In the present work, we developed and validated a methodology associating liquid chromatography (LC) and mass spectrometry (MS) to simultaneously quantify, with high specificity and accuracy, estrone-3-sulfate (E3S), progesterone (PRO), estrone (E1) and estradiol (E2) in serum of two different mammal species. The sample preparation procedure is based on a simple protein precipitation and a derivatization with dansyl chloride. After the chromatographical separation, compounds were analyzed with a triple-quadrupole mass spectrometer operating in multiple reaction monitoring. Mare and American bison serum samples were analyzed with the validated method and results were compared with concentrations measured with commercial radioimmunoassay (RIA), enzyme linked immunosorbent assay (ELISA) and chemiluminescent microparticle immunoassay (CMIA). Following these criterions: relative standard deviation <15% and relative bias <15%, lower limits of quantification of 0.5 ng/mL (E3S), 0.1 ng/mL (PRO) and 2 pg/mL (E1 and E2) were achieved. Most of the comparison between immunoassays and LC-MS showed poor correlation and proportional differences. Our LC-MS method is able to simultaneously quantify several steroid hormones with high specificity, accuracy and sensitivity in serum of two different mammal species. Our method constitutes a useful and performant tool for veterinary clinicians and LC-MS should thus be used to update and refine the current knowledge about the endocrinology of pregnancy in mammals

A case of true hermaphrodism in a horse

peer reviewedTrue hermaphroditism in horses, is a complex and poorly under- stood disorder of sexual development characterized by the presence of both male and female gonads as separate organs or in a combined structure called ovotestis. A 3-year-old Spanish nullipa- rous mare with a history of stallion behaviour and an abnormal reproductive exam was presented at the Equine Clinic of the University of Li ege. Externally, a small vulva with an enlarged clitoris was observed. Transrectal ultrasonography showed a thin (<1 cm) uterus, leading to a small heterogeneous structure in the normal place of the left ovary. No structure could be identified in the presumed location of the right ovary. Speculum exam was compatible with a vaginal agenesis, as the cervix opened craniad the urethral meatus. Oestradiol, progesterone and testosterone concen- trations were compatible with a stallion’s endocrinology. Gonads were laparoscopically found in the usual location of the ovaries in both flanks and excised. Histopathology of both gonads showed atrophic testicular tissue with hyperplastic Leydig cells. The left gonad also contained ovarian tissue with some scarce primordial follicles. Clitoral enlargement is the first symptom most commonly identified in animals with true hermaphroditism and can be explained by the production of testosterone by the gonads, however, the aetiology of the vaginal agenesis is unknown. True hermaph- roditism in horses is generally related to 64XX syndrome with or without SRY gene translocation (impending karyotyping analysis). After 5 days the mare was discharged from the clinic, and in absence of testosterone the stallion behaviour disappeared in a couple of months