Nonelectrophoretic And Electrophoretic Bovine Embryo PCR Sexing --Research And Comparision

In order to develop a more efficient and applicable method of embryo sexing, 8 pairs of primers for sex chromosome DNA sequences were chosen and synthesized basing on some reported DNA sequences on XY chromosome. Blood samples of Jin-nan cattle and Hostem cattle were labeled to extract genome DNA, concentration of which was gauged. Both electrophoretic and nonelectrophoretic PCR sexing was used in the research and nonelectrophoretic PCR sexing both had reached an accuracy of 100% through a series of PCR condition system ameliorating.FSH was injected to cause superovulation of Jin-nan female cattle, followed by insemination procedure; then on day 7, embryos were flushed and collected for examining the applicability of nonelectrophoretic method in embryo sexing. After being graded, embryos were put in pipes for routine freezing in cryoprotectant with ethylene glycol. Pipes with embryos were stored in liquid nitrogen until been used. After thawing, embryos were sampled by needle and then there was a DNA releasing procedure right before PCR. Each embryo had 2-4 samples; one was amplified by SE first. In case of successful amplification, other samples were all amplified by DYZ-1; otherwise another sample was amplified until a successful amplification occurred. The result of DYZ-1 was compared with result of SE for the same embryo to confirm if it was right. Nonelectrophoretic PCR sexing seems more sensitive to low DNA concentration than SE, meanwhile it takes about 1 hour less than SE. It reached an efficiency of 90% even when the template concentration was only 1-2 copies.This study successfully applied nonelectrophoretic PCR to bovine embryo sexing for the very first time in our country, it also reduced a traditional step in embryo sexing. Furthermore, compared with abroad research, it further shortened the time for sexing, therefore made PCR sexing in field more applicable. In 5 pairs of microsatellite polymorphic DNA primers used in this research, 2 pairs being tested sex specific showed potential usability for bovine embryo sexing.