Production of Female Bovine Embryos with Set-Sorted Sperm by Intracytoplasmic Sperm Injection: Efficency and in vitro Developmental Competence

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The production of embryos with a preselected sex sperm is important in the livestock industry. This study examined the efficiency of producing female embryos by intracytoplasmic sperm injection (ICSI) with flow cytometry sorted (ssICSI) and unsorted (usICSI) bovine sperm, as well as their developmental competence in vitro. For comparison, bovine embryos were also produced by in vitro fertilization (IVF) with sorted (ssIVF) and unsorted (usIVF) bovine sperm. The semen used in this study was from a bull selected for its high fertility and blastocyst developmental competence among four bulls compared. We first examined and compared pronuclear (PN) formation and cleavage rates of the produced embryos among the treatment groups. Our results showed that PN formation rates (judged by 2PN) and cleavage rates in ssIVF group (23.1% and 43.6%) were lower than those in the usIVF (71.1% and 71.6%), usICSI (73.1% and 92.8%) and ssICSI (75% and 79.1%) groups, respectively (P < 0.05). Moreover, the blastocyst formation rate in ssIVF group was lower than those in the usIVF, usICSI, and ssICSI groups (2.7% vs. 30.2%, 28.7% and 24.7%, P < 0.05). Importantly, we found that the blastocyst formation rate in the ssICSI group was similar to that in the usISCI group, indicating that ICSI can rescue the damages introduced to sperm by flow cytometry-mediated sex-sorting. Of note, we achieved a blastocyst formation rate in the ssICSI group to be comparable to the usIVF group. We then examined embryo quality by counting the number of both normal and apoptotic cells in blastocysts. It was found that, despite the fact that blastocyst formation rate in the ssIVF group was significantly lower than those in the usIVF, usICSI and ssICSI groups, there was no difference in total and apoptosis cell numbers among these groups (P > 0.05). Finally, karyotyping analysis showed that the proportion of female embryos in the ssICSI and ssIVF groups was 100%, while was 58.8% and 57.8% in the usIVF and usICSI groups, respectively. In conclusion, ICSI with flow cytometry sorted bovine sperm provides an alternative approach to produce embryos with predetermined sex.