Effects of egg yolk extender on fertility of cooled donkey semen

Abstract

Artificial insemination (AI) with cooled semen is largely used worldwide in horses with acceptable fertility rates and low costs. However, fertility data for cooled donkey semen is scarce. For cooled sperm preservation, donkey semen needs an adequate environment to protect the sperm from cold shock. A variety of extenders have been proposed for cooling, storage and transport of donkey semen in the last years but there are not enough fertility data. Only two previous studies reported a 26 to 40% per cycle pregnancy rate in jennies using cooled donkey semen. Effective protocols are essential to increase fertility and reproductive efficiency in commercial programs. The aim of this study was to determine per cycle pregnancy rate in jennies inseminated with cooled donkey semen using chicken egg yolk-based extender. Semen samples from one native Argentinian donkey stallion of proven fertility were diluted in Equiplus extender (Minitube, Tiefenbach, Germany) at 50 × 106 sperm/ml. One sample was used for AI with diluted fresh semen (G1), and the other two samples were cooled diluted in Equiplus (G2) and Equiplus with the addition of 1% chicken egg yolk (G3) in accordance with a previous report (Alonso. et.al. Brazilian Journal of Veterinary Research and Animal Science. 2021; 58: 40). Samples were cooled in Equitainer (Hamilton Biovet, MA, USA) for 24h. A total of eight adult and fertile nativeArgentinian jennies were randomly assigned to three groups and inseminated using a cross-over design with fresh semen (G1), cooled semen (G2) or cooled semen with chicken egg yolk (G3). Ovulation was induced using 1500 IU EV of hCG (Human Chorionic Gonadotropin, Ovusyn, Zoetis) and insemination 24h post-induction with 500 × 106 progressive motile sperm calculated after cooling. Before AI, semen parameters such as volume, concentration, progressive motility, HOS test, and sperm vitality were assessed and at 24 hours after cooling no significant difference between semen extended in Equiplus or semen extended in Equiplus with egg yolk was detected. AI was performed with the technique described by Bragulat. et.al. (Journal of Equine Veterinary Science 113 2022; 13, 40). The interval between AI and ovulation was 12 to 24h and pregnancy diagnosis was performed by ultrasound 14 days post-ovulation. For statistical analysis, RStudio (RStudio, Inc., Boston, MA URL) (R Core Team, 2018) and the Q-Cochran test were used. The results show differences in pregnancy rates between G1 (75%; 6/8) and G2 (0%; 0/8) (p<0.05) but no differences were detected (p=0.5) between G1 (75%; 6/8) and G3 (50%; 4/8). In conclusion, based on our preliminary data, refrigerated donkey semen with egg yolk-based extender can be used for AI with similar pregnancy rates as fresh semen in jennies.