235 DEVELOPMENT OF IN VIVO-MATURED OOCYTES COLLECTED FROM JAPANESE BLACK CATTLE STIMULATED WITH DIFFERENT DURATIONS OF FOLLICULAR GROWTH

Abstract

We demonstrated that in vivo-matured oocytes (mOC) collected by ovum-pick up (OPU) from cows after stimulation of follicular growth (FG) are suitable for producing good quality blastocysts (BL). However, it is not known whether duration of FG affects developmental competence of mOC. The purpose of this study was to examine development of mOC after stimulation with different duration of FG. Japanese black donor cows (n = 4 per each group), were treated with a CIDR at Day 0. Follicle of diameter >8 mm were removed on Day 5. A total 20 AU of FSH was administrated to cows twice daily with decreasing doses from the evening of Day 6 to the morning of Day 10. In the conventional group (48PG), a administration of PGF_2α (0.75 mg of cloprostenol), CIDR withdrawal, and administration of GnRH (0.2 mg of fertirelin acetate) were performed on the evening of Day 8, morning of Day 9, and morning of Day 10, respectively. In the experimental group (72PG), administration of PGF2a, CIDR withdrawal, and administration of GnRH were performed on the evening of Day 9, the morning of Day 10, and the morning of Day 11, respectively. The mOC were collected from follicles >5 mm by OPU at 25 to 26 h following GnRH administration. Collected mOC were inseminated with 3 × 10⁶ sperm mL^–1 in BO solution on 30 h after GnRH. After 6 h of IVF, presumptive zygotes were cultured for 168 h in 5% CS + CR1aa, using a micro-well culture dish (Dai-Nippon-Print) and time-lapse cinematography (CCM-1.4MZS; Astec) for individual embryo observation. The kinetics of early embryo was analysis by CCM-1.4 software. To assess the quality of BL, prognostic factors were used as follows: (1) less than 27 hpi (hours post-insemination) at the first cleavage (1st CD), (2) 2 blastomeres at the end of 1st CD, and (3) absence of multiple fragments at the end of the 1st CD (Sugimura et al. 2012 PLoS ONE 7, e36627; Imai et al. 2014 Reprod. Fertil. Dev. 26, 182). Data were analysed by Student's t-test or chi-square test. The number of mOC were 12.5 ± 4.7 and 10.3 ± 2.7 (means ± s.e.) oocytes per session in 48PG and 72PG. There was no significant difference in cleavage rate or BL formation rate (97.5 ± 1.5 v. 98.2 ± 1.8%, 66.3 ± 8.2 v. 66.8 ± 3.5%, respectively). The time for 1st CD was shorter in 48PG (26.1 ± 0.3 v. 27.8 ± 0.4; P < 0.01), and the rate of 1st CD less than 27 hpi was superior in 48PG compared with 72PG (74.3 v. 42.9%; P < 0.05). However, the rate of 2 blastomeres and absence of multiple fragments were not different between 48PG and 72PG. The number of BL tended to decrease in 72PG compared with 48PG (28.6 v. 48.6%; P = 0.087). These results indicate that duration of FG did not affect the rate of cleavage and BL formation. However, extension of duration of FG might reduce the quality of BL.