Optimization of in vitro fertilization outcomes with bovine x-sorted semen

The increasing economic pressure in dairy farming has elevated the demand for female calves due to their superior utility in milk production. Assisted reproductive technologies, especially in vitro fertilization (IVF) combined with X-sorted semen, offer a pathway to selectively produce female offspring. However, X-sorted semen has reduced fertilization potential compared to unsorted semen, primarily due to the mechanical and chemical stresses introduced during the sorting and cryopreservation processes. These stresses result in lower spermatozoa motility, compromised membrane integrity, and premature capacitation, contributing to suboptimal blastocyst rates. This study aimed to optimize bovine IVF outcomes using commercially available X-sorted semen by refining processing protocols and co-incubation conditions to improve spermatozoa motility, fertilization rates, and embryo development. Bovine cumulus-oocyte complexes (COCs) were aspirated from ovaries of Holstein-Friesian cows collected at the abattoir, matured in vitro, and fertilized in vitro using commercially available X-sorted semen from a single Holstein-Friesian bull, processed through modified Percoll® (Merck Life Science S.r.l., Milano, Italy) gradients (G1: 90%-45%, 4 mL; G2: 90%-45%, 0.8 mL; and G3: 78.7%-67.5%, 0.8 mL). Spermatozoa concentration and motility were analyzed. Post-IVF embryo development was assessed after co-incubation of gametes for either 10 or 18 hours. Presumptive zygotes (n=33-35/group) were fixed for pronuclei (PN) assessment or cultured in vitro for 8 days to evaluate blastocyst formation and morphology. Data were analyzed using Fisher’s exact test or T-test, with P < 0.05 considered statistically significant. G2 was excluded due to poor performance, while G3 yielded significantly higher sperm motility (P = 0.0462) and improved fertilization rates (27/31, 87% 2PN) compared to G1 (9/34, 26% 2PN; P < 0.0001). Despite high PN formation with G3, blastocyst rates were low. However, shortening the gamete co-incubation time to 10 hours significantly enhanced blastocyst yield (G3: 56/225, 25% vs G1: 19/124, 15%). In summary, we observed that increasing gradient density and reducing the co-incubation time of gametes positively impacted X-sorted IVF, enhancing both sperm motility and embryo production rates. While studies indicate that different bulls may require specific conditions for optimal fertilization and embryo yield, small adjustments to semen processing, tailored to the characteristics of sorted sperm, can significantly improve IVP efficiency. Funded by: MUR PRIN2017, No. 20172N2WL3_002; Piano di Sostegno alla Ricerca UNIMI: Linea 2 – Azione A.