A Preliminary Study On Embryonic Chimeras Between Buffalo And Cattle

This study was undertaken to systematically examine factors affecting the chimeric efficiency of buffalo and bovine embryos, so as to establish a preliminary procedure for making interspecific chimeras between cattle and buffalo.Bovine inner cell mass (ICM) was isolated from blastocysts by either mechanical method or immunosurgery, and then injected into buffalo blastocysts that ICM was removed to reconstruct a chimeric embryo. In addition, chimeric embryos were also reconstructed by aggregating blastomeres from early bovine and buffalo embryos isolated by either enzymatic or mechanical method in a droplet of culture medium containing 100fig/ml phytohemagglutinin (PHA) . Significantly more ICM were isolated from bovine blastocysts by immunosurgery when the anti-cattle rabbit serum was inactivated at 63.5 C in comparison with him inactivated at 56癈 (100% vs. 0%. P<0.01) . Supplementing isolating medium with 6% FCS resulted in a significant decrease in the ICM isolating efficiency (0% vs. 97.6%, P<0.01) . More chimeric buffalo blastocysts hatched when their ICM were replaced with bovine ICM isolated by immunosurgery (80% ) compared with the ICM isolated by microsurgery (43.8%, p<0.05) although the survival rate of chimeric blastocysts did not differ each other (91.4% vs. 87.5%, p>0.05) .Aggregating buffalo morulae with bovine embryos at either 8-cell or blastula stage did not differ significantly in their aggregation rate (85.7% vs. 77.8%, p>0.05) and proportion developing to blastocysts (72.2% vs. 42%; p>0.05). There were also no significant difference in aggregation rate (92.3% vs. 86.7%, p>0.05) and blastocyst development (58.3% vs. 46.2%; p>0.05 ) between the chimeric embryos derived from 8-cell embryonic blastomeres isolated by 0.25% Pronase and microsurgery.In conclusions, (1) Interspecific chimeric embryos (cattle and buffalo) reconstructed by either blastomere aggregation or ICM exchanging can develop to the blastocyst and hatched blastocyst stage; (2) Anti-cattle rabbit serum for isolating cattle ICM should be inactivated at 63.5 C for 30 min and the isolation needs to be performed in serum-free medium; (3) Immunosurgery is superior to microsurgery in isolating ICM for chimeric embryo reconstruction; (4) The aggregation rate and embryonic development of chimeric embryos are not influenced by the stages of embryos to be aggregated;(5) Methods for isolation of blastomeres (enzymatic treament and microsurgery) do not affect their aggregation rate and embryonic development.