Vitrification of pre-pubertal ovine cumulus-oocyte complexes: effect of cytochalasin B pre-treatment.

The aim of this study was to evaluate the effect of cytochalasin B (CCB) pre-treatment before vitrification on ability of immature oocytes from lamb ovaries to progress until metaphase II (MII) stage after vitrification/warming procedure. Cumulus-oocyte complexes (COCs) were obtained from ovaries of lambs, from 80 to 90 days old, collected from a local slaughterhouse. Before vitrification, COCs were randomly distributed in two experimental groups corresponding to the incubation with or without 7.5 microg/ml CCB for 30 min. In order to study cryoprotectant and CCB pre-treatment toxicity (toxicity test), oocytes were exposed to cryoprotectants, with or without CCB pre-treatment, but without plunging into N2 liquid. Vitrification solution was composed by 4.48 M EG plus 3.50 M DMSO supplemented with 0.25 M sucrose. Two-step addition was performed. After vitrification or toxicity test, COCs were matured in bicarbonate-buffered TCM 199 containing 10% foetal calf serum and 10 ng/ml epidermal growth factor. A sample of COCs was directly in vitro matured (control group). Rates of MII oocytes of toxicity groups both, with or without CCB pre-treatment were lower than control group (41.1-50.0 versus 79.9, respectively; P<0.05). After vitrification, a lower number of oocytes progressed to MII stage in comparison with non-vitrification groups (P<0.05). In vitrified groups both with or without CCB pre-treatment 8.0 and 12.7%, respectively, of immature oocytes reached MII stage by the end of in vitro maturation culture. No effect of CCB was observed, either in the toxicity or vitrified groups. In conclusion, no effect of CCB pre-treatment before vitrification was detected in this study with immature oocytes of pre-pubertal sheep. More studies are needed in order to increase ovine oocyte survival after vitrification.