66 IN VITRO DEVELOPMENT OF YAK (POEPHAGUS MUTUS) CLONED EMBRYOS BY INTERSPECIES SOMATIC NUCLEAR TRANSFER
L. Su A , F.L. Du B C , L.Y. Sung B , S. Yang A , B.S. Jeong B , S. Casinghino B , T.L. Nedambale B , J. Xu C , C.X. Tian B , X. Yang B and W. Ji AA Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China
B Department of Animal Science, Center for Regenerative Biology, University of Connecticut, Storrs, CT 06269, USA
C Evergen Biotechnologies Inc, Storrs, CT 06269, USA. Email: fdu@canr.uconn.edu
Reproduction, Fertility and Development 17(2) 183-183 https://doi.org/10.1071/RDv17n2Ab66
Submitted: 1 August 2004 Accepted: 1 October 2004 Published: 1 January 2005
Abstract
Interspecies nuclear transfer (NT) is an important tool for preservation of endangered animal species. This study was carried out to clone Yak (Poephagus mutus) embryos by using Yak skin fibroblasts and bovine (Bos taurus) recipient cytoplasts, and to compare the efficiency of YAK interspecies NT (bovine cytoplast-Yak donor cell) and bovine somatic NT (bovine cytoplast-bovine donor cell). Recipient oocytes were extracted from antral follicles of bovine ovaries, and subsequently cultured in maturation medium for 18–20 h in 5% CO2 and 95% humidified air at 39°C. Cumulus cells were removed from the oocytes by vortexing also facilitated further enucleation. Yak skin fibroblast cells were prepared from cultured ear explants of an adult 5-year-old female. Fibroblasts were cultured at passage 6–9 in 10% FBS DMEM at 37°C in 5% CO2 humidified air. The donor cell at a diameter of 19–20 μm was inserted into the perivitelline space of an enucleated oocyte. A bovine female cell line at similar passage number was used for bovine somatic NT as control. Somatic cell-cytoplast pairs were then fused by applying two direct current pulses at 2.0 kV/cm for a duration of 6–10 μs/pulse. Fused embryos were activated in 10 μg/mL cycloheximide and 2.5 μg/mL cytochalasin D in M199 plus 7.5% FBS for 5 h. Reconstructed Yak embryos were cultured in CR1aa plus 6 mg/mL BSA for 2 days (initiation of activation = Day 0) at 39°C, 5% CO2, 5% O2, and 90% N2, and then in 7.5% FBS CR1aa medium for 5 successive days on bovine cumulus monolayers. Expanding and hatching blastocysts on Day 7 were recorded and cryopreserved for further embryo transfer trials. The percentage of cleavage and the development to morulae and blastocysts were statistically analyzed using a General Linear Model (GLM, Univariate, SPSS 9.0, SPSS Inc, Chicago, IL, USA). As indicated in Table 1, the results demonstrated that the efficiencies of fusion rate as well as developmental potential in vitro were significantly higher in the bovine somatic NT group compared to those of the Yak interspecies NT group. However, the morphology and cell number per embryo of interspecies Yak cloned embryos were indistinguishable from those of bovine NT embryos. Our data suggest that bovine oocytes possess the capability of reprogramming/reactivation of the genome from differentiated somatic Yak nuclei.
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