60 Evaluation of the production of cloned embryos by interspecies nuclear transfer (bovine-sheep)

The main objective of this study was to evaluate the behaviour of interspecies somatic cell nuclear transfer (iSCNT) between cattle and sheep. First, primary somatic cell cultures were generated from a Simbrah cow owned by UANL, selected on basis of its conformation and outstanding productive characteristics. From this specimen, located near de city of Linares, Mexico, three skin samples were taken and transported for 13 ± 2 h, to Mexico City in tubes containing Delbucco’s Modified Eagle Medium supplemented with 10% fetal bovine serum. The samples were processed by 2 different methods: enzymatic disintegration with collagenase type I and II (0.02%), obtaining averages of 2 655 000 ± 1629 cells in 3 mL of recovered culture medium, and by mechanical disintegration (manual fragmentation of the sample into smaller portions), obtaining averages of 2 936 000 ± 1713 cells in 3 mL of recovered culture medium. The time needed to achieve results with the enzymatic method was 9.3 ± 5.1 days, and 10.6 ± 8.6 days for the mechanical method (P > 0.05, Student’s t-test). Subsequently, oocytes were aspirated from sheep ovaries, obtained from a slaughterhouse. Out of 169 ovaries, 232 oocytes were obtained, and those surrounded by a minimum of four layers of cumulus cells were subjected to in vitro maturation. From these, 90 matured oocytes were retrieved, representing 39% of the total sample. Groups of two enucleated oocytes (using the handmade cloning technique), were used as cytoplasts and a somatic cell obtained from enzymatic disintegration of the Simbrah cow was used as a karyoplast. The two cytoplasts and nuclear donor cell (triplets) were fused by a single pulse of 100 V/mm DC for 9 μseg, resulting in 44 (97%) reconstructed embryos. Simultaneously, ovine parthenogenetic embryos were generated and used as controls. The results obtained for cleavage (55% cloned (33/60), and 65% parthenogenetic (36/55)) and early embryo development showed a higher numerical difference for iSCNT, compared to parthenogenic embryos that reached the morula stage (46% (15/33) vs 16% (6/36), respectively, P < 0.05, Student’s t-test), but not at the blastocyst stage (0% (0/33) vs 54% (19/36), respectively, P < 0.05, Student’s t-test). Our findings demonstrate the successful production of interspecies morula-stage embryos through handmade cloning, utilising bovine cultured cells and ovine in vitro-matured oocytes.