96 Comparative study of the morphokinetic parameters of embryos produced in vitro with sex-sorted vs unsorted bovine sperm

The protocols used for flow cytometric sex-sorting of sperm have been substantially improved in the last decade, but the use of sex-sorted bull sperm can result in variable blastocyst rates after IVF. Thus, we aimed to perform a comparative study of the morphokinetics of embryos that were in vitro produced with either cryopreserved sex-sorted (SS) or unsorted (control, CON) bovine sperm cells. For this, single ejaculates were collected from three Holstein-Friesian and one Jersey bull, and split in two equal aliquots: the SS aliquot was subjected to flow cytometric sorting for X-chromosome sperm (90% purity; 4 × 10⁶ sperm/0.25-mL straw), and the CON aliquot was processed using standard methods for conventional semen (15 × 10⁶ sperm/0.25-mL straw). Both SS and CON straws were cryopreserved and stored in liquid nitrogen until use. Cumulus–oocyte complexes from slaughterhouse ovaries were used for IVF with SS or CON sperm. In total, 136 presumptive zygotes were cultured in microwell dishes and imaged every 10 min until Day 9 using a time-lapse monitoring system. The times when key developmental stages occurred were recorded: time of the first (t1) and second (t2) cleavage; the last cleavage before entering the lag phase (t3); cleavage resumption after lag phase (tRCl); the onset of blastocyst expansion (tSB); and the time of blastocyst hatching (tHB). The cleavage rate and blastocyst rate (both expressed as % of initial number of oocytes placed in culture), and the hatched blastocyst rate (expressed as % of blastocysts) were compared between treatments using the Mann–Whitney U test. Treatment differences for embryo transitions between states were explored with multistate Cox proportional hazards regression models. There was no significant difference (P > 0.05 in all cases) between CON and SS regarding cleavage (88.9% ± 4.2% vs 82.8% ± 8.0%), blastocyst (41.7% ± 19.8% vs 51.6% ± 8.0%), and hatching (74.2% ± 26.5% vs 61.1% ± 8.0%) rates. The oocytes fertilized with SS sperm had 35.1% less likelihood of reaching t1 (P = 0.046) and needed more time to reach state t1 compared with oocytes fertilized with CON semen (31.05 vs 29.25 h). Having completed the t1 state, there were no differences between treatments in the ability to achieve subsequent developmental stages. In conclusion, our results indicated that oocytes fertilized with SS sperm had compromised ability to complete the first cleavage, but thereafter developmental potential was not affected.