Capacitation of frozen-thawed semen improves ICSI-outcomes in horses

Abstract

The first report of a repeatable in vitro fertilization (IVF) protocol for fresh semen in horses in 2022 (Felix.et.al., Biology of Reproduction. 2022; 107(6): 1551-1564) paved the way for exploring capacitation applications in equine semen. The effect of equine sperm capacitation prior to intracytoplasmatic sperm injection (ICSI) on subsequent embryo development remains to be determined. In this study, 145 cumulus-oocyte complexes (COC's) were collected from slaughterhouse-derived ovaries and matured for 30-32 hours in TCM-199 with Earl's salts containing 10% (v/v) FBS and 10 µg/mL follicle stimulating hormone and 2 µg/mL luteinizing hormone (Stimufol^Ⓡ, Reprobiol, Ouffet, Belgium). After maturation, the COCs were denuded and the ones showing an extruded polar body were fertilized with piezo-drill ICSI. Half of the mature oocytes (N = 31) were injected with frozen-thawed (FT) semen that was first selected with a sperm separation device (VetCount^Ⓡ Harvester; MotilityCount ApS, Copenhagen, Denmark) and then capacitated in FERT-TALP with penicillamine, hypotaurine and epinephrine (Felix et al., 2022) for 10 hours. The remaining oocytes (N = 33) were subjected to control ICSI with FT-semen that was washed and centrifuged in G-MOPS (Vitrolife, Londerzeel, Belgium). The same batch of frozen-thawed semen was used for both techniques. All presumed zygotes were cultured individually in Global^Ⓡ medium (LGGG-050, LifeGlobal, Guilford, CT, USA) with 10% (v/v) FBS for 5 days, after which they were transferred to DMEM/F-12 containing 10% (v/v) FBS. Cleavage rates were assessed 36h after fertilization, and blastocyst formation was assessed between day 7 and 10 post-ICSI. Statistical analyses were performed using a Fisher's exact test. Cleavage and blastocyst rates were higher for ICSI with capacitated semen (29/31 (93.5%) and 20/31 (64.5%)) than for control ICSI (24/33 (72.7%) and 12/33 (36.4%), respectively) (P = 0.03 and P = 0.02). Capacitation of semen before ICSI could improve commercial ICSI results, but more research is needed to confirm these results and to assess suitability of different stallions for capacitation of FT semen.