316 DEVELOPMENTAL COMPETENCE OF OOCYTES SELECTED BY THE BRILLIANT CRESYL BLUE STAINING IN PREPUBERTAL AND ADULT CATTLE

Abstract

Transvaginal ultrasound guided ovum pickup (OPU) can be carried out repeatedly not only in adult cows but also in prepubertal calves. Moreover, recently the brilliant cresyl blue (BCB) staining has been used successfully to select oocytes homologous stained with BCB as an indirect measure of oocyte growth for in vitro fertilization (IVF) in several farm animals. The aim of this study was to evaluate the effectiveness of BCB staining on the selection of developmentally competent oocytes, collected from cows and calves by OPU, before in vitro maturation by assessing the embryonic development to the blastocyst stage after IVM/IVF. OPU was performed once weekly for a period of 7 weeks on Japanese Black prepubertal calves (9 months old, n = 4) and adult cows (n = 4) without gonadotropin stimulation. Calf and cow oocytes with homogeneous cytoplasm were exposed to 26 µM BCB for 90 min and classified according to their cytoplasmic coloration: blue coloration (BCB+) or colorless (BCB-). Classified oocytes were then matured for 20 h in TCM-199 supplemented with 5% calf serum (CS). Matured oocytes were inseminated with Percoll-separated spermatozoa (3 × 10⁶ mL) for 6 h in BO solution supplemented with 5 mM hypotaurine and 2 U mL^-1 heparin. Presumptive zygotes were cultured in CR1aa supplemented with 5% CS for 8 days. Data were analyzed by Student's t-test. The mean (± SEM) percentage of oocytes classified as BCB+ in calves was significantly lower than that in cows (34.4 ± 2.9% and 69.2 ± 2.1%, respectively; P < 0.01). In cows, BCB+ oocytes showed significantly higher cleavage and blastocyst formation percentages (72.5% and 42.4%, respectively) than those of BCB− oocytes (47.0% and 13.0%, respectively). In contrast, in calves there were no significant differences in cleavage and blastocyst formation percentages between BCB+ oocytes (56.9% and 25.3%, respectively) and BCB− oocytes (65.4% and 22.4%, respectively). The mean (± SEM) numbers of usable oocytes and blastocysts obtained per calf (19.0 ± 1.5 and 4.5 ± 0.6, respectively) were similar to those obtained per cow (16.4 ± 1.1 and 5.2 ± 0.6, respectively). No significant difference was observed in the numbers between calves and cows. These results indicate that the selection of developmentally competent oocytes before IVM/IVF, using the BCB staining, was effective for cow oocytes but not for calf oocytes, and that blastocysts could be produced by OPU-IVF of oocytes from 9-month-old prepubertal calves at an efficiency equivalent to that achieved from adult cows.