Production of Water Buffalo Clone Embryos Using Ear Skin Fibroblasts as Donor Cell in Nuclear Transfer.

The present study investigated the possibility of producing water buffalo clone embryos using ear skin fibroblasts from ear skin tissues tested for viability postcollection and cryopreservation. Water buffalo ear skin samples were collected and maintained in 4ºC at different time points: 12, 24, 48, 72, 96, 120 and 168 hr post-collection. After storage at specified time, tissue samples were processed for primary culture. Fibroblasts obtained from ear skin tissues were cryopreserved using different freezing procedures and were then used as donor nuclei for nuclear transfer. The maximum time lapse when ear skin fibroblast proliferation did not decrease significantly was at 120-hr post-collection (hpc). The post-thaw viability of the fibroblasts cryopreserved with the alternative rapid freezing procedures was comparable with that of the control group. The fibroblasts survived the alternative cryopreservation procedure and reached cell confluency at sub-culture. When used further for nuclear transfer, no significant differences were observed from the control group in terms of rates of cleavage and development to blastocyst, and cell numbers of the blastocyst formed. The present findings indicate that ear skin fibroblasts derived from tissues stored up to 120 hpc can be used as donor nuclei without compromising the developmental competence of the reconstructed embryos. [ABSTRACT FROM AUTHOR]