Effect of meiotic stage and warming medium equilibration atmosphere on equine oocyte vitrification

DANIEL ANGEL; HELOISA CANESIN; J BROM-DE-LUNA; KATHERINE HINRICHS; SERGIO MORADO; GABRIEL DALVIT; DIANA GOMEZ; POSADA, NATALIA; RODRIGO URREGO; CATALINA VELEZ

Cambridge

Simposio; XIIth International Symposium on Equine Reproduction; 2018

The objectives of this study were to evaluate a commercial human oocyte vitrification method (Cryotech®) for vitrification of immature and in vitro-matured equine oocytes, with and without cumulus cells, and to evaluate the effect of warming medium equilibration atmosphere on oocyte developmental competence. Oocyte-cumulus complexes were obtained from slaughterhouse-derived ovaries. The COCs (n = 551) were held overnight at room temperature (RT), pipetted to a standard ~4 layers of cumulus cells, then divided into immature (Imm) or in vitro matured (Mat) groups. Imm oocytes were immediately vitrified (ImmCV). Mat oocytes were subjected to in vitro maturation culture, then, without evaluation for meiotic stage, either vitrified (MatCV) or denuded of cumulus and then vitrified (MatDV). Vitrification and warming were performed as specified by the manufacturer with minor modifications. Two methods were used to equilibrate the warming solutions: air or 5% CO2 in air, generating ImmCVCO2, ImmCVAir, MatCVCO2, MatCVAir, MatDVCO2, and MatDVAir groups. After warming, Imm oocytes were in vitro matured for 30 h and Mat oocytes were cultured for 2 h. Oocytes were denuded and those with a polar body were fertilized by intracytoplasmic sperm injection (ICSI), and cultured as previously described (Canesin et al, Cryobiology, 2018, Jan 3). To control for ICSI procedures, oocytes recovered by transvaginal ultrasound-guided follicle aspiration (not vitrified) were subjected to maturation, ICSI and in vitro embryo culture concurrently with a warmed replicate at least once per week. Cleavage was evaluated at Day 2; blastocyst formation was evaluated from Days 7-10. Differences between groups were evaluated by Chi-square. Recovery rate (oocytes recovered/oocytes vitrified) was lower in ImmCV (67%) than in MatCV (84%; P < 0.001) or MatDV (79%; P < 0.05). Survival rate was similar among groups (93 to 98%). There was no difference in maturation rate among ImmCV, and Control oocytes (39 vs-50%; P = 0.083), nor between equilibration atmospheres within ImmCV (40% for both). Equilibration of warming medium in CO2 decreased cleavage rate (48%) compared to that for Controls (78%; P < 0.01); Air-warmed oocytes were intermediate (65%; P > 0.05). Within Air warming, presence of cumulus on Mat oocytes did not affect cleavage rate (63% for MatCV and 71% for MatDv; P > 0.1). Blastocyst rate for all vitrified groups were significantly lower than that for control (0-7% vs 23%; P < 0.001). In conclusion, the vitrification method used was associated with high survival and maturation rate, but developmental competence was low for both Imm and Mat oocytes. Equilibration of warming medium in 5% CO2 decreased cleavage rates after ICSI. Further studies are needed to increase developmental competence of vitrified/warmed equine oocytes.