Birth of twin kids following transfer of in-vitro produced goat embryos

Abstract

Post-implanted growth and development of embryo is deficient in many aspects as evidenced by the great difficulty in growing embryos, while maintaining viability as shown by development of early embryos in-vitro generally delayed or completely blocked at 8–16 cell stage embryo, and hardly small percentage of the in-vitro fertilized embryo develop to morula and blastocyst (Kharche et al. 2008b). Investigations on in-vitro embryo production and to assess the post transfer survival of in-vitro produced embryos in goats have their usefulness for both basic research and commercial application. Therefore, the present study was aimed to evaluate in-vitro embryo development potential and to asses post transfer survival rate of in-vitro produced embryos. Collection of ovaries: Ovaries of goats were obtained from slaughterhouse located at Agra. Ovarian tissues were transported to the laboratory in a thermos flask containing sterile and warm physiological normal saline solution (NSS) supplemented with antibiotics (100 µg/ml penicillin-G and 100 µg/ml streptomycin sulphate). Prior to oocytes retrieval, surplus tissues on the ovaries were removed and ovaries were washed several times in normal saline containing antibiotics to avoid any form of microbial contamination. Recovery of oocytes and in-vitro maturation (IVM): The oocytes were collected from each ovary in oocyte collection media (OCM: Dulbecco’s phosphate-buffered saline with 3 mg/ml BSA, 50µg/ml streptomycin and 60µg/ml penicillin) by follicle puncture method using 18-G needle. Isolated oocytes were washed 4-to-5 times in oocyte holding medium (OHM) containing (TCM-199 with Hepes modification, EGS 10%, sodium pyruvate 0.25 mM, gentamycin 50 µg/ml, glutamine 100 µg/ml, BSA 3 mg/ml) and graded. The oocytes were then washed 3-times with oocyte maturation medium (OMM) containing (TCM-199 supplemented with 10% FBS, sodium pyruvate 0.25 mM, glutamine 100 µg/ml, LH 10 µg/ ml, FSH 5µg/ml, BSA 3 mg/ml and gentamycin 50µg/ml) and were allowed to mature in 50µl droplets of IVM medium for 24 –27 h in a CO 2 incubator maintained at 38.5°C, 5% CO 2 and high humidity. In-vitro fertilization: After 27 h of in-vitro maturation oocytes were denuded by hyaluronidase enzyme and by passing repeatedly through fine pipette. The oocytes were washed 8–10 times in Fert TALP (TALP solution containing 10% oestrous goat serum, 8 mg/ml fatty acid free BSA and 100 µg / ml heparin) and put in 50 µl drop in culture petridish. Fertilization drops containing oocytes were inseminated with 25 to 50 µl of finally diluted semen so as to obtain a sperm concentration of 2–4 millions sperm /ml. The spermoocytes were co-incubated for 18 hr in a CO 2 incubator maintained at 38.5° C, 5% CO 2 and high humidity. In-vitro embryo development: Following 18 hr of sperm oocytes co-incubation, the oocytes were washed in KSOM medium containing 10% FBS and 3 mg/ml BSA and then cultured in 50 µl drop of embryo development medium. The zygotes were cultured under mineral oil in 5% CO 2 in air with high humidity at 38.5°C. After 48h of post insemination, oocytes were observed for cleavage and half of the culture medium was replenished with fresh medium. Embryos were morphologically evaluated under inverted phase contrast microscope to separate retarded embryos from normally developing embryos.