138 comparison of kinetics and patterns of the first cleavage of in vivo and in vitro-matured holstein oocytes after in vitro fertilization with x-sorted sperm

Previously, it was reported that a high rate of good quality blastocysts were produced by IVF of in vivo-matured oocytes, obtained by ovum pick up (OPU) after superstimulation in Holstein cows, using X-sorted sperm (Matoba et al. 2012 Reprod. Domest. Anim. 47, 515). In this system, an early first cleavage within 28 h after IVF was found to be a potent marker for the selection of embryos with high developmental competence (Matoba et al. 2013 Reprod. Fertil. Dev. 25, 266). However, we have limited knowledge on the timing and normality of embryonic cleavages in in vitro-matured oocytes after IVF. The purpose of the present study was to compare the kinetics and patterns of the first cleavage of in vivo- and in vitro-matured bovine oocytes after IVF with X-sorted sperm. In vivo-matured oocytes (Group A) were collected by OPU from non-lactating Holstein cows just before ovulation after superstimulation. Immature oocytes were either collected by OPU without hormonal treatment or by aspiration of ovaries at the local abattoir and matured in vitro (Group B or C). All the oocytes were inseminated with 5 × 106 sperm mL–1 of X-sorted sperm, except half of oocytes in Group C inseminated by non-sorted sperm (Group D) and cultured in CR1aa supplemented with 5% calf serum and 0.25 mg mL–1 of linoleic acid albumin at 38.5°C in 5% CO2, 5% O2, and 90% N2 for 216 h. Embryo kinetics were observed individually using a microwell culture dish and time-lapse cinematography (Sugimura et al. 2010 Biol. Reprod. 83, 970–978). Photographs of each embryo were taken in every 15 min during the IVC period and analysed by time-lapse cinematography software. Cleavage pattern was categorized as normal (2 even blastomeres without fragment or protrusion) or abnormal (2 uneven blastomeres, with fragment or protrusion and those dividing into 3–5 blastomeres) at the first cleavage. Data were analysed by ANOVA, chi-squared, or discriminant function. A total of 268 cleaved embryos were used. The blastocyst rate in Group A was higher than in Groups B and C (61.3 v. 40.0 and 25.0%, respectively; P < 0.05). The timing of first cleavage was longer in Group A compared with Groups C and D (28.3 ± 3.8 v. 27.6 ± 3.8 and 26.7 ± 1.9 h, respectively) and in Group B (28.1 ± 4.0 h) compared with in Group D (P < 0.05). Higher rates of normal cleavage were observed in Groups A, B, and D than in Group C (53.5, 44.4, and 54.8 v. 16.7%, respectively; P < 0.01). The frequency of blastocysts derived from the early (28.3 h) and normal pattern cleaving oocytes were greater in Groups A and B than in Group C (29.0 and 20.0 v. 8.3%, respectively; P < 0.05) and similar in Group D (22.6%). Our results reveal that IVF embryos produced from in vivo-matured oocytes with sex-sorted sperm had superior normality than those produced from in vitro-matured oocytes and similar normality to embryos inseminated with non-sorted sperm. Supported by the Research and Development projects for application in promoting new policy of agriculture, forestry and fisheries (22016) and by JSPS and HAS under the Japan-Hungary Research Cooperative Program.