In vitro fertilization has remained elusive in the horse, as evidenced by low sperm penetration rates when IVF has been attempted with in vivo- or in vitro-matured oocytes. It is likely that the low sperm penetration rates observed in IVF studies are due to the inability to appropriately capacitate or hyperactivate, or both, stallion sperm in the laboratory. The acquisition of hyperactivated sperm motility has been observed within the oviducts of mammals at the time of fertilization and is required for zona pellucida penetration in conjunction with the acrosome reaction (AR). Although the zona pellucida is considered the prime physiological inducer of AR, previous studies have shown a low incidence of AR in zona pellucida-bound stallion spermatozoa after 1 h of in vitro binding. This low incidence suggests that, besides the zona pellucida glycoproteins, another major factor might be responsible for AR. Protein-bound progesterone, present in equine follicular fluid (FF), has been demonstrated to induce AR in stallion spermatozoa. In this context, the aims of this study were (1) to hyperactivate stallion sperm in FF and (2) to verify whether this hyperactivation supports equine IVF. Pooled FF, aspirated from the preovulatory follicles of oestrous mares, was used and its progesterone concentration was determined by immune enzymatic assay. Spermatozoa from fertile stallions selected by a swim-up procedure were pre-incubated for 6 h in capacitating medium (modifed Whittens's medium (WM) supplemented with 25 mM NaHCO3 and 7 mg mL–1 of BSA) and then incubated for 6 h at 37°C in either FF or capacitating WM. Sperm motility was assayed by computer-assisted semen analysis, rates of AR were assessed by fluorescein isothiocyanate-PNA staining and rate of apoptosis was assessed by an annexin V test. For IVF, spermatozoa were incubated at 10 × 106 sperm mL–1 in capacitating WM for 6 h and then diluted to 1 × 106 sperm mL–1 in capacitating WM with or without 10% of FF. Five mature mare oocytes were transferred into droplets (100 μL) of the sperm suspensions covered with mineral oil and then incubated for 18 h at 38.5°C in 5% CO2 in humidified air. After that, oocytes were transferred to an embryo culture medium (DMEM/F-12) for an additional 3 days. Data were analysed by ANOVA. Treatment of sperm with FF resulted in a significant (P ≤ 0.05) decrease of 3 motility variables indicative of hyperactivation: straight line velocity, straightness and linearity. The highest rate of AR (29.44%) and a lower rate of apoptosis (16.93%) were obtained after 4 h of incubation in follicular fluid. By coupling capacitating conditions with the induction of hyperactivation using follicular fluid, we have obtained reproducible percentages of 8-cell-stage embryos (18.56%) in our IVF experiments. Conversely, sperm incubated in capacitating conditions but not treated with FF did not fertilize (0%). It is concluded that mare FF does not impair sperm viability, stimulates equine sperm hyperactivation in vitro, induces the AR and supports equine IVF.
Evaluation of Zona Pellucida Function for Sperm Penetration During In Vitro Fertilization in Pigs
