141 SUPERIORITY OF FEMALE EMBRYO PRODUCTION SYSTEM BY IN VIVO-MATURED OOCYTE AND X-SORTED SPERM IN BROWN SWISS COWS

Abstract

Embryo transfer using a female embryo is an effective tool for offspring production on dairy industry; however, embryo production by embryo recovery (ER) using X-sorted semen is not sufficient because non-fertilized oocytes are recovered frequently. In Holstein cows, we developed a system for high blastocyst production that was performed by IVF using X-sorted sperm and in vivo-matured oocytes obtained by ovum pickup (OPU) after superstimulation. The purpose of this study was to adjust this system to Brown Swiss cows, comparing between ER and embryo production from oocytes derived from OPU with or without superstimulation. In the ER group, cows (n = 10) received a CIDR (Day 0) and 2 mg of oestradiol-benzoate on Day 1. A total of 30 Armour Units of FSH were injected into cows twice a day, with decreasing doses from the evening of Day 5 to the morning of Day 9. On the evening of Day 7 or 8, 0.75 mg of prostaglandin F_2α (cloprostenol) was injected. The CIDR was removed on Day 8 or 9 and 0.2 mg of gonadotropin-releasing hormone (GnRH; fertirelin acetate) were injected on Day 9 or 10. At oestrus, AI was carried out twice, 12 h apart, with a total of 4 straws of X-sorted semen per cow. In the OPU group, cows (n = 7) were subjected to OPU without any pretreatment, collected immature oocytes were in vivo matured for 20 to 22 h, followed by IVF using X-sorted sperm for 6 h; then, presumptive zygotes were in vitro cultured (IVC) for 9 days. In the in vivo-matured oocyte group (matured group), a CIDR was inserted (Day 0) in cows (n = 4), all follicles larger than 8 mm were removed on Day 5. Administration of FSH, prostaglandin F_2α, and GnRH, as well as withdrawal of CIDR, were performed as in the ER group. In vivo-matured oocytes were collected from follicles larger than 5 mm by OPU at 25 to 26 h following GnRH injection; collected oocytes with expanded cumulus cells were fertilized with X-sorted sperm 30 h after GnRH. After 6 h of IVF, presumptive zygotes were transferred to in vitro culture, as in the OPU group. Data were compared among 3 groups; the ER group was analysed for number of CL, collected embryos, and normal embryos, against the number of aspirated follicles, collected oocytes used for IVF, and formed blastocysts in the OPU and matured groups, respectively, by Tukey-kramer test after ANOVA. There were no differences between the number of CL in the ER group and the number of follicles in the OPU and matured groups (16.4 ± 5.3 v. 31.6 ± 22.7 v. 18.5 ± 4.7, mean ± s.d., respectively). Also the number of collected embryos in the ER group and number of oocytes for IVF in the OPU and matured groups (12.8 ± 7.6 v. 14.9 ± 11.8 v. 17.8 ± 7.7, respectively) was similar. However, the number of blastocysts in the matured group (13.0 ± 5.9; P < 0.01) was higher than that in the OPU group (3.0 ± 2.2) and in the ER group (2.8 ± 3.7). For female embryo production in Brown Swiss cows using X-sorted sperm, the system of IVF with in vivo matured oocytes obtained by OPU is more effective than ER or OPU without pretreatment.