Optimization of a Bovine Intracytoplasmic Sperm Injection System Using Sex-Sorted Sperm: Screening with Slaughterhouse-Derived Oocytes and Validation with Ovum Pick-Up-Derived Oocytes

Sex-sorted sperm permit the preferential production of offspring of a specified sex, but flow sorting and cryopreservation can compromise sperm function and reduce the efficiency of conventional in vitro fertilization (IVF). We sequentially evaluated oocyte maturation duration, sperm processing, post-injection incubation medium, and nine activation regimens using slaughterhouse-derived oocytes, and then evaluated the best-performing combination under the tested sequential conditions using ovum pick-up (OPU)-derived oocytes. In conventional IVF, the presumptive normal fertilization rate, based on the presence of two pronuclei (2PN), was 2.45% with X-sorted sperm and 46.13% with conventional sperm. The final selected combination, consisting of 22 h maturation, Brackett–Oliphant (BO)-based swim-up, oocyte maturation medium (OM) during post-injection incubation, and treatment with 7% ethanol for 5 min [ETH (5)], resulted in a presumptive normal fertilization (2PN) rate of 52.92%, a blastocyst rate of 10.46%, and an ICM/TE ratio of 0.82. In the validation using OPU-derived oocytes, the blastocyst formation rate after ssICSI was 13.46% when calculated relative to injected oocytes and 16.84% when calculated relative to surviving oocytes. These rates differed significantly from the blastocyst formation rate after ssIVF (7.88%), whereas cleavage rates did not differ significantly between groups. ETH (5) provided the most favorable in vitro balance of presumptive 2PN formation, blastocyst development, and ICM/TE cell allocation among the tested regimens. Because the factors were optimized sequentially, their interactions were not estimated, which represents a limitation of this study. Furthermore, implantation, pregnancy, and calving rates require validation in independent in vivo embryo-transfer experiments.