In contrast to man and many other mammalian species, conventional in vitro fertilization (IVF) with horse gametes is not reliably successful. The apparent inability of stallion spermatozoa to penetrate the zona pellucida in vitro is most likely due to incomplete activation of spermatozoa (capacitation) because of inadequate capacitating or fertilizing media. In vivo, the oviduct and its secretions provide a microenvironment that does reliably support and regulate interaction between the gametes. This review focuses on equine sperm–oviduct interaction. Equine sperm–oviduct binding appears to be more complex than the presumed species-specific calcium-dependent lectin binding phenomenon; unfortunately, the nature of the interaction is not understood. Various capacitation-related events are induced to regulate sperm release from the oviduct epithelium and most data suggest that exposure to oviduct secretions triggers sperm capacitation in vivo. However, only limited information is available about equine oviduct secreted factors, and few have been identified. Another aspect of equine oviduct physiology relevant to capacitation is acid–base balance. In vitro, it has been demonstrated that stallion spermatozoa show tail-associated protein tyrosine phosphorylation after binding to oviduct epithelial cells containing alkaline secretory granules. In response to alkaline follicular fluid preparations (pH 7.9), stallion spermatozoa also show tail-associated protein tyrosine phosphorylation, hyperactivated motility and (limited) release from oviduct epithelial binding. However, these ‘capacitating conditions’ are not able to induce the acrosome reaction and fertilization. In conclusion, developing a defined capacitating medium to support successful equine IVF will depend on identifying as yet uncharacterized capacitation triggers present in the oviduct.

Extracellular vesicles (EVs) play a possible role in cell–cell communication and are found in various body fluids and cell conditioned culture media. The aim of this study was to isolate and characterize EVs in culture medium conditioned by bovine embryos in group and to verify if these EVs are functionally active. Initially, ultracentrifuged bovine serum albumin (BSA) containing medium was selected as suitable EV-free embryo culture medium. Next, EVs were isolated from embryo conditioned culture medium by OptiPrep^TM density gradient ultracentrifugation. Isolated EVs were characterized by nanoparticle tracking analysis, western blotting, transmission, and immunoelectron microscopy. Bovine embryo-derived EVs were sizing between 25–230 nm with an average concentration of 236.5 ± 1.27 × 10⁸ particles/mL. Moreover, PKH67 EV pre-labeling showed that embryo-secreted EVs were uptaken by zona-intact bovine embryos. Since BSA did not appear to be a contaminating EV source in culture medium, EV functionality was tested in BSA containing medium. Individual embryo culture in BSA medium enriched with EVs derived from conditioned embryo culture medium showed significantly higher blastocyst rates at day 7 and 8 together with a significantly lower apoptotic cell ratio. In conclusion, our study shows that EVs play an important role in inter embryo communication during bovine embryo culture in group.

In contrast to various other mammalian species, conventional in vitro fertilization (IVF) with horse gametes is not reliably successful. In particular, stallion spermatozoa fail to penetrate the zona pellucida, most likely due to incomplete activation of stallion spermatozoa (capacitation) under in vitro conditions. In other mammalian species, specific capacitation triggers have been described; unfortunately, none of these is able to induce full capacitation in stallion spermatozoa. Nevertheless, knowledge of capacitation pathways and their molecular triggers might improve our understanding of capacitation-related events observed in stallion sperm. When sperm cells are exposed to appropriate capacitation triggers, several molecular and biochemical changes should be induced in the sperm plasma membrane and cytoplasm. At the level of the sperm plasma membrane, (1) an increase in membrane fluidity (2) cholesterol depletion and (3) lipid raft aggregation should occur consecutively; the cytoplasmic changes consist of protein tyrosine phosphorylation and elevated pH, cAMP, and Ca²⁺ concentrations. These capacitation-related events enable the switch from progressive to hyperactivated motility of the sperm cells, and the induction of the acrosome reaction. These final capacitation triggers are indispensable for sperm cells to migrate through the viscous oviductal environment, penetrate the cumulus cells and zona pellucida and, finally, fuse with the oolemma. This review will focus on molecular aspects of sperm capacitation and known triggers in various mammalian species. Similarities and differences with the horse will be highlighted to improve our understanding of equine sperm capacitation / fertilizing events.

Induction of hyperactivated motility is considered essential for triggering the release of oviduct-bound mammalian spermatozoa in preparation for fertilization. In this study, oviduct-bound stallion spermatozoa were exposed for 2 h to: i) pre-ovulatory and ii) post-ovulatory oviductal fluid; iii) 100% and iv) 10% follicular fluid (FF); v) cumulus cells, vi) mature equine oocytes, vii) capacitating and viii) non-capacitating medium. None of these triggered sperm release or hyperactivated motility. Interestingly, native FF was detrimental to sperm viability, an effect that was negated by heat inactivation, charcoal treatment and 30 kDa filtration alone or in combination. Moreover, sperm suspensions exposed to treated FF at pH 7.9 but not pH 7.4 showed Ca²⁺-dependent hypermotility. Fluo-4 AM staining of sperm showed elevated cytoplasmic Ca²⁺ in hyperactivated stallion spermatozoa exposed to treated FF at pH 7.9 compared to a modest response in defined capacitating conditions at pH 7.9 and no response in treated FF at pH 7.4. Moreover, 1 h incubation in alkaline, treated FF induced protein tyrosine phosphorylation in 20% of spermatozoa. None of the conditions tested induced widespread release of sperm pre-bound to oviduct epithelium. However, the hyperactivating conditions did induce release of 70–120 spermatozoa per oviduct explant, of which 48% showed protein tyrosine phosphorylation and all were acrosome-intact, but capable of acrosomal exocytosis in response to calcium ionophore. We conclude that, in the presence of elevated pH and extracellular Ca²⁺, a heat-resistant, hydrophilic, <30 kDa component of FF can trigger protein tyrosine phosphorylation, elevated cytoplasmic Ca²⁺ and hyperactivated motility in stallion sperm, but infrequent release of sperm pre-bound to oviduct epithelium.

In many species, sperm binding to oviduct epithelium is believed to be an essential step in generating a highly fertile capacitated sperm population primed for fertilization. In several mammalian species, this interaction is based on carbohydrate-lectin recognition. D-galactose has previously been characterized as a key-molecule that facilitates sperm-oviduct binding in the horse. We used oviduct explant and oviduct apical plasma membrane (APM) assays to investigate the effects of various carbohydrates, glycosaminoglycans, lectins, S-S reductants and the capacitating factors albumin, Ca²⁺ and HCO₃^- on sperm-oviduct binding in the horse. Carbohydrate-specific lectin staining indicated that N-acetylgalactosamine, N-acetylneuraminic acid (sialic acid), and D-mannose or D-glucose were the most abundant carbohydrates on equine oviduct epithelia whereas D-galactose moieties were not detected. However, in a competitive binding assay, sperm-oviduct binding density was not influenced by any tested carbohydrates, glycosaminoglycans, lectins or D-penicillamine, nor did the glycosaminoglycans induce sperm tail-associated protein tyrosine phosphorylation. Furthermore, N-glycosidase F (PNGase) pretreatment of oviduct explants and APM did not alter sperm-oviduct binding density. By contrast, a combination of the sperm capacitating factors albumin and HCO₃^- severely reduced (>10 fold) equine sperm-oviduct binding density by inducing rapid head-to-head agglutination, both of which events were independent of Ca²⁺ and an elevated pH (7.9). Conversely, neither albumin, HCO₃^- or any other capacitating factor could induce release of oviduct-bound sperm. In conclusion, a combination of albumin and HCO₃^- markedly induced sperm head-to-head agglutination which physically prevented stallion sperm to bind to oviduct epithelium.

Background: The success of in vitro embryo production (IVEP) is influenced by donor mare and stallion. Objectives: To determine whether donor mare and stallion influence the pregnancy rate after transfer of in vitro produced (IVP) blastocysts and to identify factors influencing the likelihood of obtaining one or more pregnancies from a single IVEP session. Study Design: Retrospective clinical study. Methods: The influence of the following variables on pregnancy outcome was assessed in 2292 transfers of IVP blastocysts: speed of in vitro embryo development, embryo grade after thawing, age of the donor mare, blastocyst rate, age and breed of the recipient mare, year of embryo production, month and year of embryo transfer. Finally, the variability in pregnancy rate between individual mares and stallions, and the effect of mare‐stallion combination was investigated. Results: Overall, 85% of successful IVEP sessions resulted in at least one pregnancy. When a single blastocyst was produced, 78% of sessions yielded a pregnancy; this increased to 91% and 98% when two or at least three blastocysts were produced, respectively. Slow in vitro embryo development, poor embryo grade after thawing, advanced donor mare age, and performing embryo transfer late in the season had a negative impact on the pregnancy rate. The variation in pregnancy rates between most mares and stallions was low (~20%). For some mares, the likelihood of pregnancy varied by as much as 50% depending on the stallion used. Main Limitations: Due to the limited sample size, variation in pregnancy rates between donors was assessed descriptively. Conclusions: IVEP is a very efficient way to obtain pregnancies. The likelihood of pregnancy after transfer of an IVP embryo has improved over time, and individual mare and stallion identity seems to have a low impact. However, for some underperforming mares, results differ markedly depending on the stallion used for intracytoplasmic sperm injection.