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Thesis

Effect Of Donor Cell Type On The In Vitro Development Of Buffalo Somatic Cell Nuclear Transfer Embryos

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TNV_PT_01004.pdf(1.04 MB)

(Ph.D. Thesis)

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2005
Veerapandian, C | Ramadass, P | Subramanian, A | Balasubramanian, S
Tamil Nadu Veterinary and Animal Sciences University
Ph.D

Somatic Cell Nuclear Transfer (SCNT) is an important technology which has immense potential application in animal production and pharmaceuticals. Research works on SCNT in Bubalus bubalis were limited and with the objective of optimizing various aspects of SCNT in buffaloes, experiments were carried out on maturation of oocytes, donor cell type, fusion, activation and culture of NT embryos. A total of 2,982 oocytes retrieved by slicing method from a total of 662 buffalo ovaries collected from the Chennai slaughter house were utilized for the study. A recovery rate of 4.5 oocytes and a yield of 3.43 good quality oocytes (A,B and C grades) per ovary were recorded. The supplementation of cysteamine (100 μM) and Epidermal Growth Factor (10 ng/ml) to the maturation media, TCM 199 with FBS, FSH, LH and Estradiol, significantly (P<0.01) improved the maturation rate (mean V SE) based on extrusion of the first polar body, when compared to the control (77.59 V 1.08 vs. 63.87V2.00 per cent).In the experiment designed to standardize the electrofusion protocol, two DC pulses of 1.4 - 1.6 kV/cm for 35 μsec, for fetal fibroblasts (FF) and adult skin fibroblasts (AF) and similar pulses for 25 μsec, for cumulus (C) were found to give the best fusion and development rates. The NT embryos activated at 24 h post maturation (hpm) had better developmental rates than those activated at 30 hpm. The fusion at 24 hpm and activation at 28-30 hpm gave better fusion and development rates compared to fusion and immediate activation at 24 hpm. In another experiment, in which reconstructs were made with different donor cell types and their fusion and development rates (per cent, mean V SE) were compared, the contemporary cumulus (FC, 41.00 V 1.80) cells had significantly (P<0.05) lower fusion rates compared to FF (52.6 V 2.98) and AF (46.9 V 1.80). The cleavage rate was the highest in FF (42.27 V 3.10) followed by FC (39.3 V 3.87) and AF (35.07 V 2.93). However the reconstructs with FC showed the maximum development rates to 4,  8 and  16 cell stages (25.47 V 3.97, 11.68 V 2.38 and 2.62 V 1.05) followed by FF (24.84 V 2.84, 7.97 V 1.66 and 2.17 V 0.72) and AF (15.51 V 2.36, 2.53 V 1.06 and 0.66 V 0.46, respectively). The sex of the FF had no influence on the development rates and 2-5 cell passages in the preparation of karyoplasts were found to give better results compared to 6-12 passages. The mean cleavage and progression to further development stages were found to be the best in G 1 /G 2 , followed by TCM 199 and the least in SOFaa medium. In G 1 /G 2 alone 12.19 V 5.66 and 1.75 V 1.49 per cent (mean V SE) developed to morula and blastocyst stages respectively. The transfer of a total of 16 embryos to six healthy recipients on fourth or fifth day of estrous cycle did not result in any conception. In conclusion, the results of the present study showed that FF, AF and C cells could be successfully reprogrammed and developed to pre- implantation embryos and FC cells would be the most ideal karyoplast for SCNT in water buffaloes. An electrofusion regimen of two DC pulses of 1.4 - 1.6 kV/cm for 35 μsec and fusion at 24 hpm followed by activation at 28-30 hpm were suitable for buffalo SCNT. The development of NT embryos was best supported by G 1 /G 2 followed by TCM 199 media.

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