42 Comparison of three permeating cryoprotectant mixtures for equine immature oocyte vitrification

Equine oocyte vitrification would benefit the growing intracytoplasmic sperm injection (ICSI) programs, but further optimisation of the protocol is necessary to reach clinical efficiency. The present study aimed to identify the optimum permeating cryoprotectant (CPA) mixture for the vitrification of immature equine oocytes. Slaughterhouse-derived cumulus-oocyte complexes (COCs) were partially denuded by pipetting until ± four cumulus layers remained. Then, COCs were vitrified using three different permeating CPA mixtures in a 50:50 ratio: ethylene glycol-dimethyl sulfoxide (DMSO) (ED), propylene glycol–DMSO (PD), and propylene glycol-ethylene glycol (PE)). For vitrification, oocytes were kept in base solution (BS; medium 199 with Hanks’ salts and 0.4% (w/v) bovine serum albumin). Four to six oocytes were equilibrated for 25 s in equilibration solution (BS with 20% (v/v) of the CPA mix), transferred to vitrification solution (BS with 40% (v/v) of the CPA mix and 0.5 M galactose) and loaded onto a cryo-device (Ortiz-Escribano et al. 2018 Equine Vet J. 50, 391-397), then plunged into liquid nitrogen within 40 s. For warming, COCs were transferred for 5 min into 4 mL of warming medium (WM) containing BS supplemented with 0.3 or 0.5 M galactose, resulting in six groups: ED-0.5 (n = 110), ED-0.3 (n = 85), PD-0.5 (n = 115), PD-0.3 (n = 79), PE-0.5 (n = 107), and PE-0.3 (n = 90). Warmed COCs were washed in BS. All procedures were performed at 39°C. Once warmed, COCs were matured in 500 µL of M199 with Earles’ salts and 10% fetal bovine serum (FBS) at 38.5°C in 5% CO₂ for ±30 h. Oocytes with an extruded polar body were injected by piezo-assisted ICSI and then cultured in 20-µL droplets of Dulbecco’s modified Egale medium-F12 with 10% FBS under paraffin oil for 7 to 10 days at 38.2°C in 5% O₂, 5% CO₂, and 90% N₂. A control group (n = 210) with non-vitrified oocytes was included in every replicate (4). The effects of the CPA mixture, galactose concentration in the WM, and their interaction on maturation, cleavage, and blastocyst rates were fitted in generalised mixed-effects models using the replicate as random. The maturation rate was significantly higher in the control (58.6 ± 3.4%) than in PD (44.3 ± 3.6%; P = 0.02) and PE (42.6 ± 3.5%; P = 0.007), but ED reached a similar maturation rate (48.7 ± 3.6%; P = 0.1). The cleavage rate in the PE (65.5 ± 5.3%) was similar to that in the control (77.1 ± 4.3%; P = 0.3), whereas cleavage in the control was greater than in PD (51.1 ± 5.5%; P = 0.002) and ED (53.5 ± 5.3%; P = 0.005). Blastocyst rates were lower for all vitrified groups (ED = 6.2 ± 2.6%, PD = 3.5 ± 2.0%, and PE = 9.4 ± 3.3%) compared with the control (34.2 ± 5.1%; P < 0.01). Although not statistically significant (P > 0.4), PE-0.3 exhibited the highest blastocyst rate (15.1 ± 5.9%) compared with the other groups (2.9 ± 2.8 to 7.9 ± 4.5%). The galactose concentration in WM did not affect maturation, cleavage, or blastocyst rates (P > 0.1). In conclusion, the PE mixture presented the highest cleavage rate compared with the other CPA combinations, and PE-0.3 gave rise to the highest blastocyst rate. As such, PE provides a valuable option for equine immature oocyte vitrification and for future research towards an optimised protocol.