Studies On The Methods Of PCR Sexing Pre-implantation Embryos And Associated Techniques

The splitting and embryo cells sampling techniques of mouse and bovine pre-implantation embryos, the cryo-preservation of split-sampled mouse and bovine embryos , and the sexing of pre-implantation bovine embryos by PCR were studied systematicly. Genomic DNA of six farm animal species such as cattle, yak, goat, sheep, mouse and pig were amplificated by PCR with bovine Y-chromosome specific primers in this study, and the conservative sequences in different species were cloned and sequenced. The results were as follows:1. Simple technique of mouse and cattle embryos splitting and embryo cells(5-10cells) sampling under stereo-microscope with glass needle or metal razor blade. was established. Development rates of split-sampled mouse and bovine embryos culture in vitro for 48 hours were 76.7%(46/60) and 80%(16/20), respectively. The pregnant rate of recipients of split -sampled bovine fresh and frozen-thawed embryos after transfer were 47.1% and 42.9%, respectively.2. The total development rates of split-sampled mouse embryos in vitro after frozen-thawed by traditional freezing method in 10% Glycerol, 10% ethylene glycol, 10% glycerol + 10% dextran + 0.1M sucrose and 10% ethylene glycol+ 10% dextran + 0.1M sucrose freezing solution were 56.9%(259/455), 81.8% (317/), 84.8% (540/738) and 59.5% (308/518) , respectively. Short time (0-4hours ) in vitro culture of splitted angd sampled mouse embryos before freezing did not significantly effect on their development rate after frozen-thawed..3. Quick-freezing technique of split-sampled mouse and bovine embryos was established. The total development rate of quick-freezing split-sampled mouse embryos culture 48 hours in vitro was 73.3% (33/45) ,. The rate of pregnant recipients of frozen-thawed split-sampled bovine embryos, frozen in 25% Glycerol + 0.25M sucrose +20%serum PBS, was 57.1%(4/7) .4. Five pairs of bovine male-specific PCR primers were designed based on the sequences of bovine Y-specific repeat sequence, testis-specific protein gene from Y-chromosome (TSPY) and sex determining region Y-protein gene (SRY) from GenBank. Meanwhile, based on skeletal alpha action precursor gene and Bovine 1.715 satellite DNA, 4 pairs of bovine-specific PCR primers were designed as internal control primers. After PCR...