131 BOVINE OOCYTE VITRIFICATION IS IMPROVED BY INCREASED PYRUVATE CONCENTRATION AND THE INCLUSION OF HYALURONAN IN THE VITRIFICATION MEDIUM

Abstract

Bovine oocytes have limited cryo-tolerance and are susceptible to damage partially caused by intracellular ice formation and reactive oxygen species. The ability to routinely cryopreserve bovine oocytes has remained elusive, with high rates of degeneration upon warming or fertilization failure. Hyaluronan has been shown to increase bovine blastocyst survival after vitrification, and the addition of an antioxidant improves mouse embryo survival. The aim of this study was, therefore, to determine if supplementation of the base medium with hyaluronan and increasing the pyruvate concentration would improve the success of bovine oocyte vitrification. Bovine oocytes were recovered by aspiration of abattoir ovaries. Oocytes were matured for 21 h in a defined system using G-MAT supplemented with recombinant albumin (2.5 mg mL^-1) and epidermal growth factor (EGF; 100 ng mL^-1). All oocytes were partially denuded using hyaluronidase and divided into 3 groups: Control (non-vitrified, n = 789), G-MOPS^TM (Vitrolife Sweden AB, Kungsbacka, Sweden; n = 697), and G-MOPS supplemented with 0.125 mg mL^-1 hyaluronan with a 1.5-fold pyruvate increase (designated G-VIT; n = 697). Oocytes were soaked in either G-MOPS or G-VIT for 45 min prior to being vitrified. G-MOPS or G-VIT was then used as the base medium for all vitrification and warming steps. The vitrification procedure was carried out at 37°C using the Cryoloop. Oocytes were incubated in 8% dimethyl sulfoxide (DMSO) and ethylene glycol (EG) for 1 min and 16% DMSO and EG (with 0.65 M sucrose and 10 mg mL^-1 Ficol, MW 400 000) for under 30 s, before loading onto the Cryoloop and submerging into liquid nitrogen. Warming was performed using 0.25 M sucrose for 1 min, 0.125 M sucrose for 5 min, and no sucrose for at least 10 min. All oocytes (within 1 h of warming) were then fertilized for 18 h and their survival assessed before being washed and moved to G1.3 (with 8 mg mL^-1 BSA). Embryos were again washed, cleavage rates assessed, and embryos were moved to G2.3 (with BSA) on the morning of Day 4 post-fertilization. Blastocyst formation was assessed at 144 h. Differences between treatments were assessed by Fisher's exact test. Survival, cleavage, and blastocyst development in the G-VIT and G-MOPS groups were 76.9 vs. 58.2% (P < 0.001), 48.5 vs. 41.6% (P < 0.05), and 15.4 vs. 4.1% (P < 0.001), respectively. The cleavage and blastocyst development rates of control oocytes were higher than for both vitrification groups (61.7 and 26.7%, respectively; P < 0.001). Survival and development of vitrified oocytes improved with the addition of hyaluronan and increased pyruvate concentration. Ongoing research into the role of macromolecules and antioxidants should further increase the success of oocyte cryopreservation.

This study was supported by a grant from Vitrolife.