Sperm vitrification in horses and donkeys

Sperm vitrification is an alternative freezing method, which includes high cooling rates and non-permeable cryoprotectants agents. The first attempt in equids was using the spheres technique by directly dropping small volumes of the sperm into liquid nitrogen. Later, vitrification was developed using 0.25 mL straws with outer covers, which resulted in similar progressive motility when compared to conventional freezing in donkeys (44.3 ± 15.0 % vs. 44.7 ± 18.2 %) or even higher in horses (48.2 ± 2.3 % vs. 37.3 ± 2.2 %). Subsequently, the vitrification of larger volumes of sperm in 0.5 mL straws was evaluated, but the results showed poor sperm quality after different warming procedures. Finally, fertility was assessed in vitro and in vivo. In horses, the sperm fertilizing capacity was assessed utilizing heterologous in vitro fertilization (IVF) and vitrified sperm showed the ability to penetrate cattle oocytes, leading to pronuclear formation and hybrid embryo cleavage. In donkeys, fertility trials were conducted in vivo on a small group of jennies, showing no statistically significant difference in pregnancy rates between artificial insemination (AI) with vitrified semen (22%) or frozen semen (10%); however, the uterine inflammatory response after AI with vitrified semen solved faster than frozen semen. In conclusion, sperm vitrification has been optimized in horses and donkeys using 0.25 mL straws with outer covers and can be considered as an alternative to conventional freezing.