Sperm vitrification supports in vitro blastocyst development after ICSI in horses

In the last decade, the introduction of advanced assisted reproductive techniques has significantly impacted the horse breeding industry. Specifically, in vitro embryo production by intracytoplasmic sperm injection (ICSI) has gained relevance and begun to be used to conserve wild equids. Kinetic sperm vitrification permits cell cryopreservation without permeating cryoprotective agents, facilitating the international commercialization and transportation of samples. This study aimed to assess embryo morpho-kinetic dynamics up to the blastocyst stage of ICSI embryos produced by injection of frozen (F) or vitrified (V) spermatozoa into horse eggs. Semen collection, as well as sperm vitrification and analysis were done as described by Consuegra et al. (Animal Reproduction Sciences. 2019; 206:69-77). Computer-assisted sperm analysis (Sperm Class Analyzer® CASA) and flow cytometry with double stain PNA-FITC (peanut agglutinin-fluorescein isothiocyanate) and PI (propidium iodide) were performed to assess acrosomal integrity and sperm viability respectively. Frozen and vitrified sperm cells from a single ejaculate of a horse and a donkey were used. Oocyte collection, in vitro maturation, ICSI, time-lapse imaging andembryo culture were performed as reported by Bragulat et. al. (Theriogenology 2023; 195:199-208). Sperm analysis before cryopreservation showed: total motility (TMOT) 98.34%; progressive sperm motility (PMOT) 78.60%; intact-membrane sperm (IMS) 86.00%, and normal form (NF) 79.37%. After thawing, frozen sperm (F) analysis showed: TMOT 84.36%; PMOT 61.28%; IMS 77.50% and NF 86.21% and for vitrified sperm (V): TMOT 80.64%; PMOT 61.28%, IMS 81.00% and NF 68.97%. A total of 89 matured oocytes were injected. No significant differences were found in cleavage rates (F: 35/43, 81.39%; V: 35/46, 76.08%) or blastocyst rates at day 8 (F: 3/43, 6.97%; V: 8/46, 17.39%). Preliminary data from time-lapse imaging showed similar embryo morpho-kinetic dynamics between groups. Average hours after ICSI for time to cleavage were (mean ± SEM): F, n = 4, 27.00 ± 1.41; V, n = 3, 27.67 ± 1.20. Second cell division: F, n = 3, 38.33 ± 6.64; V, n = 3, 32.33 ± 3.38. Morula stage: F, n = 1, 85; V, n = 1, 120. Blastocyst stage: F, n = 1, 150; V, n = 1, 180. Our results demonstrate for the first time that vitrified sperm cells can be used successfully to produce blastocysts by ICSI without compromising developmental competence in horses.