The Presence of D-Penicillamine during the In Vitro Capacitation of Stallion Spermatozoa Prolongs Hyperactive-Like Motility and Allows for Sperm Selection by Thermotaxis

SIMPLE SUMMARY: Capacitation of stallion semen in vitro is still a suboptimal procedure. The main objective of this study was the use of thermotaxis as a novel method for sperm selection and determining the most adequate media for maintaining frozen/thawed horse sperm longevity in vitro. Our results show that the most common media (Whitten’s) used in this species is not the best for capacitating the semen in terms of hyperactive-like motility, and tyrosine phosphorylation being synthetic human tubal fluid supplemented with D-penicillamine is the most adequate in preserving these parameters during 180 min of incubation. Therefore, this media (with and without D-penicillamine) was chosen for performing thermotaxis. The selection conditions were a gradient of 3 °C of difference (35–38 °C) for 1 h. The results revealed that the selected fraction showed higher levels of tyrosine phosphorylation in the whole flagellum and lower levels of DNA fragmentation when compared to the unselected fraction (kept at 37 °C) when human tubal fluid with D-penicillamine was used. These results are promising for improving the in vitro embryo production rates in these species by improving the sperm selection methodology. ABSTRACT: Assisted reproductive technologies (ARTs) in the horse still yield suboptimal results in terms of pregnancy rates. One of the reasons for this is the lack of optimal conditions for the sperm capacitation in vitro. This study assesses the use of synthetic human tubal fluid (HTF) supplemented with D-penicillamine (HTF + PEN) for the in vitro capacitation of frozen/thawed stallion spermatozoa by examining capacitation-related events over 180 min of incubation. Besides these events, we explored the in vitro capacity of the spermatozoa to migrate by thermotaxis and give rise to a population of high-quality spermatozoa. We found that HTF induced higher levels of hyperactive-like motility and protein tyrosine phosphorylation (PTP) compared to the use of a medium commonly used in this species (Whitten’s). Also, HTF + PEN was able to maintain this hyperactive-like motility, otherwise lost in the absence of PEN, for 180 min, and also allowed for sperm selection by thermotaxis in vitro. Remarkably, the selected fraction was enriched in spermatozoa showing PTP along the whole flagellum and lower levels of DNA fragmentation when compared to the unselected fraction (38% ± 11% vs 4.4% ± 1.1% and 4.2% ± 0.4% vs 11% ± 2% respectively, t-test p < 0.003, n = 6). This procedure of in vitro capacitation of frozen/thawed stallion spermatozoa in HTF + PEN followed by in vitro sperm selection by thermotaxis represents a promising sperm preparation strategy for in vitro fertilization and intracytoplasmic sperm injection in this species.