B. Anguita

Effect of the apoptosis rate observed in oocytes and cumulus cells on embryo development in prepubertal goats.

Oocyte quality is the main factor that determines blastocyst yield; any factor that could affect it, such as apoptosis, could impair subsequent embryonic development. Our aim was to investigate the incidence of apoptosis in prepubertal goat oocytes and cumulus cells, assessed by Annexin-V staining and TUNEL assay, and their effect on embryo development. Oocyte-cumulus complexes (COCs) from slaughtered females were collected and classified depending on COC morphology as: Healthy (H) and Early Atretic (EA). Each one of these groups was classified depending on oocyte diameter: A: 110-125microm, B: 125-135microm and C: >135microm. The COCs were IVM for 27h, IVF with fresh semen and IVC for 8 days after insemination. Apoptosis analyses were performed before and after maturation. Annexin-positive oocytes decreased with diameter in the EA class (immature oocytes: A: 42.6%; B: 30.3%; C: 21%; IVM-oocytes: A: 17.5%; B: 4.8%; C: 0%), while TUNEL assay showed a decrease of apoptosis in the largest oocytes before and after IVM only in Healthy oocytes (immature oocytes: A: 51.5%; B: 43.3%; C: 12.1%; IVM-oocytes: A: 31.7%; B: 12%; C: 0%). Blastocyst rate increased with increasing oocyte diameter, and it was higher in H than in EA oocytes (Healthy; A: 0%; B: 5.3%; C: 14.4%; Early atretic: A: 0.3%; B: 4.1%; C: 5.1%). Oocyte diameter and COC morphology had no effect on the percentage of apoptosis in blastocyst cells. In conclusion, oocyte developmental competence in prepubertal goats is influenced by oocyte diameter and COC morphology.

124 TAXOL™ COULD PROMOTE EMBRYO DEVELOPMENT OF BOVINE OOCYTES VITRIFIED BY OPS

Stabilizing the cytoskeleton system during vitrification could be beneficial for improving post-thawed survival and subsequent development of vitrified oocytes. Taxol™, paclitaxel, is a microtubule stabilizer that has been found to improve development competence of vitrified mouse and human oocytes. The objective of this work was to study the effect of a Taxol pretreatment before OPS vitrification on the post-thaw cow and calf oocyte development. Oocytes were aspirated from slaughterhouse-derived ovaries and matured in TCM-199. Oocytes were randomly assigned to one of 3 experimental groups: (1) control oocytes matured in vitro for 24 h, (2) oocytes matured for 22 h and vitrified by the OPS method (Vajta et al. 1998 Mol. Reprod. Dev. 51, 53–58), and (3) oocytes matured for 22 h and vitrified by OPS method with 1 µM Taxol. OPS and Taxol–OPS oocytes were transferred back into the maturation dishes and matured for 2 additional h before being subjected to fertilization. Fertilization was performed using frozen–thawed Percoll-selected sperm. At 22 h after insemination, presumptive zygotes were pipetted and then cultured in drops of 25 µL SOF medium and 5% fetal calf serum under paraffin oil at 38.5°C in 5% CO2, 5% O2, 90% N2, and maximum humidity. The Taxol–OPS group provided a significantly higher cleavage rate than the OPS group in cows (41.9% and 34.0%, respectively) or in calves (33.7% and 23.5%, respectively). However, cleavage rate in the experimental groups was significantly lower than in the control group (78.3% and 69.7% for cow and calf control groups, respectively). Blastocyst yield was also higher for the Taxol–OPS group (3.2%) than the OPS group (0%) in cow oocytes. There was no blastocyst development when calf oocytes were vitrified with or without Taxol pretreatment. As expected, cow and calf vitrification groups triggered a significantly lower blastocyst yield when compared with their control (26.7% and 14.9% for cow and calf control groups, respectively). In conclusion, this study showed that supplementation of 1 µM Taxol could promote embryo development after thawing. Further research is indicated to clarify the function of Taxol and its optimal concentration in order to improve the rate of embryo development. Table 1. Effect of Taxol pretreatment on development of cow and calf oocytes vitrified by OPS