Studies on in vitro production of equine embryos by Intracytoplasmic Sperm Injection (ICSI) using non-sorted, or sex-sorted, frozen/thawed stallion sperm: Effects on post-thaw sperm quality, cleavage and blastocyst rates, and characterization of cellular events during the first 24 hours post-fertilization via confocal microscopy.

Intracytoplasmic sperm injection (ICSI) using frozen/thawed (F/T) stallion sperm is a common procedure in the equine breeding industry. Historically, sex-sorted (SS) F/T stallion sperm has yielded lower cleavage (<30 %) and blastocyst rates (<5 %) after ICSI when compared to non-sorted (NS) F/T sperm. Recently, a new technology for sperm sex-sorting (Genesis III) has been validated by a commercial company. In Experiment 1, the post-thaw quality between NS-F/T and SS-F/T stallion sperm produced with this technology was compared. The post-thaw sperm motility was higher in NS-F/T (41 %) than in SS-F/T (12 %) sperm (P < 0.05), while sperm viability was similar between groups (54 % vs. 57 %, respectively). Conversely, the percentage of sperm with DNA with an increased susceptibility to denaturation, as determined by the Sperm Chromatin Structure Assay (SCSA), was higher in the SS-F/T group (80 %) than in the NS-F/T group (5 %; P < 0.05). In Experiment 2, higher cleavage (NS-F/T: 113/160 [71 %] vs. SS-F/T: 79/164 [48 %]) and blastocyst rates per injected oocytes (NS-F/T: 42/160 [26 %] vs. SS-F/T: 25/164 [15 %]) were observed in NS-F/T than in SS-F/T sperm (P < 0.05). When comparing the blastocyst rate per cleaved embryos (NS-F/T: 42/133 [37 %] vs. SS-F/T: 23/79 [32 %]) differences were not observed between groups. Furthermore, the percentage of blastocysts developing at days 7 (33 vs. 26 %), 8 (19 vs. 35 %), 9 (38 vs. 22 %), or 10 (10 vs. 17 %) after ICSI, and the number of blastocysts/ICSI cycle was similar between groups (NS-F/T: 1.4 vs. SS-F/T 0.7 blastocysts/cycle). In Experiment 3, the sperm DNA decondensation, sperm aster formation, and pronuclei configuration during the first 24 h post-ICSI of in vitro-matured equine oocytes fertilized with NS-F/T or SS-F/T sperm was studied using laser confocal microscopy. Overall, 50-80 % of the oocytes fertilized with either NS-F/T or SS-F/T sperm yielded similar pronuclear formation within the first 8 h post-ICSI; pronuclear apposition or metaphase plate formation within 12-18 h post-ICSI; and had undergone the first mitotic division by 24 h post-ICSI. This study provides comparisons regarding post-thaw sperm quality, efficiency of blastocyst production by ICSI, and the characterization of cellular events occurring during the first 24 h post-ICSI, using either NS-F/T or SS-F/T sperm.