70 Possibility for genomic evaluation using single blastomeres derived from 8-cell stage IVP embryos in Japanese Black cows

Pre-implantation genomic selection based on single nucleotide polymorphism (SNP) genotyping combined with in vitro embryo production (IVP) has significantly accelerated genetic improvement in cattle. The aims of this study were to examine the possibility for genomic evaluation from single blastomeres by blastomere separation of early IVP embryos in Japanese Black cows and to define the optimum oocyte collection method and embryo stage to maximize the percentage of blastocyst production from remaining blastomeres. Immature oocytes were collected either by the aspiration of follicles of ovaries obtained at an abattoir or by transvaginal ovum pickup (OPU) of Japanese Black cows. The oocytes were in vitro matured and fertilized (IVF). Presumptive zygotes were cultured individually in microwells. The 2- or 8-cell stage embryos were subjected to blastomere separation by pipetting after chemically remove the zona pellucida (2-cell abattoir group, n = 125; 2-cell OPU group, n = 35; 8-cell abattoir group, n = 121; and 8-cell OPU group, n = 185). When 2-cell stage embryos were used, both blastomeres were cultured individually to verify their ability to develop to a pair of blastocysts, then one blastocyst was subjected to SNP genotyping. When 8-cell stage embryos were used, a single blastomere was used for SNP genotyping and the remaining seven blastomeres were cultured together to verify their ability to develop to the blastocyst stage. Blastocysts and single blastomeres were subjected to DNA extraction and whole-genome amplification. Then, SNP genotyping was performed using SNP Chip (BovineLD-24 v4.0 kit, Illumina). The rate of blastocyst formation per number of the 2- or 8-cell stage embryo subjected to blastomere separation on Day 7 (Day 0 = IVF) was higher in the 8-cell OPU group than in the 8-cell abattoir group, 2-cell OPU group, and 2-cell abattoir group (82.1 ± 15.1%, 65.2 ± 13.0%, 65.2 ± 14.5%, 41.5 ± 17.4%, respectively; P < 0.05; one-way ANOVA). The call rate for an individual in blastocysts in the 2-cell abattoir group was greater than those in single blastomeres in the 8-cell OPU group (93.7% (max 99.6%) vs 81.9% (max 93.1%); P < 0.05; t-test). Furthermore, we predicted the genomic estimated breeding values (GEBV) of six carcass traits using the SNP genotypes. We obtained some SNP genotypes with call rates greater than 0.8 from a single blastomere and the accuracy of the GEBVs were all greater than 0.7. Our results revealed the possibility of genomic evaluation from single blastomeres derived from 8-cell stage OPU-IVP embryos while enabling 82% of blastocyst formation in Japanese Black cows.