Factors Affecting The Efficiency Of Sex Determination By PCR Of Bovine Embryos

Determining the sex of bovine preimplantation embryos by using the method of polymerase chain reaction (PCR) is a routine technology in dairy industry. Given the greater sensitivity of PCR, the most significant problem is that of inaccurate results. In this study, the factors which could affect the amplify results of PCR reaction were discussed. The results are as follows:1. There were significantly differences on the rate of no reaction between biopsied embryos with zona pellucida and without, respectively 4.58% and 1.87% (P<0.05). The result showed that zona pellucida had effect on PCR reaction. No differences were found in accuracy of female calf delivery which resulted from the transfer of sexed embryos (P>0.05, 95.65% vs 95.56%). So the samples with or without zona pellucida had no obvious effect on the accuracy of sex determination by PCR.2. The quality of embryos had an obvious effect on the accuracy of female calf delivery. With the degrade of embryo quality, the accuracy was increased. The accuracy of female calf delivery between grade 1 and grade 2 were significantly different (P<0.01, 94.85% vs 98.36%).3. Cell sampling by biopsy had an obvious effect on PCR reaction. The no reaction rate of the group with less than five cells was higher than that with more than or equal to five cells (P<0.05, 4.38% vs 2.12%). Furthmore, cell sampling influenced the accuracy of female calf delivery. The group of biopsied with fewer than five cells had a significantly lower accuracy than that of biopsied with more than or equal to five cells (P<0.01, 88.10% vs 98.47%).4. Viability of biopsied cells had no effect on PCR reaction, but had an effect on accuracy of female calf delivery. No differences were found in no reaction rate between group of viable cells and disperse or dead cells, respectively 3.19% and 2.19% (P>0.05). There were significant differences on the accuracy of female calf delivery between viable cells and disperse or dead cells (P<0.05, 93.39% vs 86.57%).5. Embryos were classified into 4 stages (morula, early blastocyst, blastocyst, and expanded blastocyst) and 3 quality grades(1-excellent,2-good,3-fair) . The sexing of 1258embryos by PCR ananlysis resulted in identification of 625 female and 633 male embryos. The sex ratio of sexed embryos was not difFerent from the 1:1 ratio (P>0.05,l:1.012).