59 PRODUCTION OF HANDMADE CLONED GOAT EMBRYOS WITH TWO TYPES OF DONOR CELLS AND CULTURED IN THREE MEDIA

Abstract

The study was carried out to see the developmental efficiency of handmade cloned goat embryos with 3 different media: RVCL (Research Vitro Cleave, Cook, Brisbane, Australia), EDM (Embryo Development Media) and modified SOF (mSOF) and 2 types of donor cells: fetal fibroblast and adult fibroblast. Oocytes were isolated from abattoir goat ovaries, matured in maturation medium, and incubated in 5% CO₂ in air at 38.5°C for 24 h. Then, the oocytes were made cumulus free by treatment with hyaluronidase (0.5 mg mL^-1) and zona free by pronase (2 mg mL^-1). Protrusion cone formation in oocytes was found 95 to 100% in T20 (TCM-199 + 20% FBS). The zona-free oocytes were bisected with an ultra-sharp micro blade on the basis of visible protrusion cones on the surface of oocytes using T20 medium containing 2.5 μg mL^-1 cytochalasin-B. Fetal and adult fibroblast cells were used from confluent monolayer at passage 5 after trypsinizing in 0.25% trypsin-EDTA. One somatic cell was attached with one enucleated demioocyte by phytohemagglutinin and further fused with another enucleated demioocyte through electric pulse with a combination of alternating current (4 V) and direct current (2.10 kV cm^-1 for 5 μs with a single pulse) in fusion medium (0.3 M mannitol, 0.1 mM MgCl₂, 0.05 mM CaCl₂, and 3 mg mL^-1 BSA). Then, triplets were chemically activated with 5 μM Ca ionophore for 5 min and 2 mM 6-DMAP for 4 h and cultured in the 3 media. Cleavage and morulae formation were observed at Day 7 from 183 triplets with fetal fibroblasts as donor cells in media RVCL (78.60 ± 2.23, 38.97 ± 2.1), mSOF (72.62 ± 1.89, 33.81 ± 1.9), and EDM (73.96 ± 1.66, 26.20 ± 2.04), respectively. Simultaneously, cleavage and morulae formation were observed at Day 7 from 203 triplets with adult fibroblasts as donor cells in media RVCL (73.97 ± 3.57, 33.14 ± 2.68), mSOF (76.22 ± 4.36, 26.15 ± 0.99), and EDM (65.97 ± 3.11, 20.78 ± 2.77), respectively. Among the 3 media, morulae formation was significantly higher in RVCL. Hence, in the subsequent experiment, RVCL medium was used exclusively in culture for 172 triplets. Cleavage and morulae formation at Day 7 was not significantly different (P < 0.05) in 2 types of donor cells; fetal fibroblasts (77.46 ± 3.65, 38.70 ± 2.66) and adult fibroblasts (75.74 ± 3.04, 33.77 ± 1.43), respectively. The data were analyzed using SYSTAT 7.0 (Systat, SPSS Inc., Chicago, IL, USA) after arcsine transformation, one-way ANOVA followed by Fisher’s LSD test. PCR analysis was performed with highly polymorphic 286-bp fragment of MHC-II DRB gene of cloned embryo and its donor cell. Similar bands were observed in both the cloned embryos and fibroblast cells in agar gel electrophoresis. In conclusion, development of handmade cloned embryos was higher in RVCL medium compared with the other two media tested, and efficiency of morulae formation was similar in both types of donor cells. Further study is required to optimize blastocyst production.