70 VITRIFICATION OF IMMATURE OVINE OOCYTES WITH CRYOLOOP: EFFECT OF CYTOCHALASIN B PRETREATMENT ON SUBSEQUENT DEVELOPMENT

Abstract

Oocyte cryopreservation is still a challenge in most mammalian species because of their extreme sensitivity to chilling injuries. Relaxation of the cytoskeleton during vitrification may improve post-thaw survival and subsequent development; however, a previous study in immature [germinal vesicle (GV) stage] oocytes from prepubertal lambs reported that pretreatment with cytochalasin B (CB) did not improve maturation (Silvestre MA et al. 2006 Anim. Reprod. Sci. 93, 176–182). We previously reported that GV oocytes from mature ewes can be vitrified using a cryoloop with high survival, maturation, and subsequent in vitro fertilization (Moawad AR and Campbell KHS 2008 Reprod. Fertil. Dev. 20, 122). The aim of this study was to evaluate the effects of CB pretreatment prior to vitrification of GV oocytes (from mature ewes) on subsequent development. Cumulus–oocyte complexes obtained at slaughter were randomly divided into 2 groups and incubated with or without 7.5 μg mL^–1 CB for 60 min before vitrification. Oocytes from each group were vitrified or used as toxicity and controls. For vitrification, oocytes were equilibrated in 10% ethylene glycol (EG) and 0.25 m trehalose (T) in HEPES/TCM-199 plus 10% fetal bovine serum (BM) for 3 min. Oocytes were then exposed to vitrification solution (20% EG and 20% DMSO in BM), loaded into the cryoloop within 1 min, and immersed in liquid nitrogen. Oocytes were warmed by exposure to (1) 10% EG and 1 m T in BM, (2) 0.5 m T in BM, and (3) BM for 3 min in each solution at 39°C. Oocytes were then matured, fertilized, and cultured in vitro as previously described. The frequency of cleavage 24 and 48 h post-insemination (pi), development to morula (5 days pi), blastocyst (7 days pi), and total cell numbers of blastocyst-stage embryos were evaluated. Cleavage was significantly lower (P < 0.001) in vitrified and CB-vitrified groups at both 24 h pi (15.2 v. 14.7%) and 48 h pi (27.3 v. 23.5%) than in other groups. Development to morula stage was significantly lower (P < 0.001) in vitrified and CB-vitrified oocytes (12.1 v. 17.7%) than in toxicity, CB-control, and control groups (43.1, 42.6, and 52.8%, respectively); however, no significant difference (P > 0.05) was observed between CB-vitrified and CB-toxicity groups. There was a significant decrease (P < 0.01) in development to blastocyst in CB-vitrified (2.9%) compared with CB-control (22.9%) and control (24.5%), but this did not differ significantly (P > 0.05) from toxicity and CB-toxicity groups (9.8 v. 11.4%). No blastocysts developed from the vitrified group. Hatched blastocysts were observed only in CB-control and control groups (8.2 v. 5.7%). Total cell numbers were significantly greater (P < 0.05) in control blastocysts than in toxicity, CB-vitrified, and CB-control (143 v. 87.25, 73, and 82, respectively). However, this did not differ significantly from the CB-toxicity group (115). These results support our previous data and suggest that pretreatment of GV-stage ovine oocytes with CB prior to vitrification has a positive effect on subsequent development.