Optimalisasi Produksi Embrio Domba Secara In Vitro: Penggunaan Medium CR1aa dan Pengaruh Status Reproduksi

Abstract

The aim of this study was to investigate the capacity of CR1aa as a simple medium for in vitro maturation, fertilization and culture of ovine embryo and to determine whether the ovarian reproductive status influenced the number of follicles, oocytes quality, nuclear maturation, fertilization and cleavage rates for ovine in vitro embryo production. In first experiment, oocytes were matured in Tissue Culture Medium (TCM)-199 as control and CR1aa as treatment. Both maturation medium were supplemented with 10% Fetal Bovine Serum (FBS), 10 IU/ml Follicle Stimulating Hormone (FSH), 10 IU/ml Luteinizing Hormone (LH), 1 ìg/ml Estradiol and 100 IU/ml penicillin-streptomycin. Matured oocytes were fertilized in BO or CR1aa medium, supplemented with 2.5 mM caffeine benzoate and 20 mg/ml heparin. Zygotes were cultured in TCM-199 or CR1aa medium, supplemented with 5% FBS, 5 mg/ml insulin, 100 IU/ml penicillin streptomycin. In second experiment, the pairs of ovary was classified into four groups: 1) ovaries with Corpus Luteum (CL) and Dominant Follicle (DF), 2) ovaries with CL, without DF, 3) ovaries with DF, without CL, 4) ovaries without both CL and DF. Results showed that in first experiment, the highest maturation rate was found in TCM -199 medium (73.27%) and significantly different (P<0.05) from CR1aa (52.88%). Fertilization rate in CR1aa medium (67.59%) was higher (P<0.05) than in BO medium (52.94%). Furthermore, there was no significant difference (P>0.05) between cleavage rate of ovine embryos in TCM-199 or CR1aa medium (39.45%; 43/109 vs 50.94%; 54/106). In second experiment, there was no significant different (P>0.05) in the number of follicles were found from all groups of ovaries. The number of oocytes with good quality from pairs of ovary with CL and DF was 7.94 ± 2.59, significantly different (P<0.05) with pairs of ovary with DF without CL (4.40 ± 1.50). The maturation rate of oocytes with good quality were 75.51% from pairs of ovary with CL and DF and significantly different (P<0.05) with pairs of ovary with DF without CL (42.86%). Fertilization and cleavage rates were similar (P>0.05) from all groups of ovaries. In conclusion, optimum result on ovine in vitro embryo production can be achieved from a combination of TCM -199 as maturation medium and CR1aa as fertilization and culture medium. Furthermore reproductive status of ovaries can influence in obtaining the number of good quality oocytes and maturation rate, but not in fertilization and cleavage rate.