Study On Sexing Bovine Preimplantation Embryo With Polymerase Chain Reaction

Sexing of pre-implantation embryos was studied by PCR method. To make thetechnique applied in commercial production, the original method was optimized. Theresults are as follows:Four pairs of bovine Y-chromosome-specific PCR primers(B90,B12,B34,B56)and three bovine autosome specific primers(A34,A56,A78) were designed. Inaddition, one pair of bovine Y-chromosome-specific PCR primers(B78) and one pairof bovine autosome specific primers(A12) were quoted. Two primer combinationswere selected based on the results of amplifying the bovine genomic DNA andfibroblasts with all these primers. The two primer combinations are A12/B34 andA12/B78.Multi-PCR system was established based on combination A12/B34 and theconditions were optimized by varying the following parameters individually and incombination: the concentration of primers, the concentration of dNTP, theconcentration of Mg2+, the content of Taq DNA polymerase, the annealingtemperature and the number of cycles in the program. The results indicated that theconcentration of primers, the annealing temperature and the number of cycles havemore effection on the efficiency of multi-PCR , but the concentration of dNTP, theconcentration of Mg2+ and the content of Taq DNA polymerase have fewer. finally, asteady and high efficiency multi-PCR system was established.In this experiment, two-temperature multi-PCR was firstly used to sexing thepre-implantation embryos. The denature time and the annealing time are both 1second and there is no prolongation step in the program. The total time of PCR wasreduced to about 37 minutes. The reaction program consisted of initial denaturing at95℃for 4 minutes, followed by 30 cycles of 94℃for 1 second and 56℃for 1second, and after circulations 72℃for 3 minutes.27 embryos were sexed with improved multi-PCR.14 and 13 of them were judgedas female and male, respectively. Finally, all were transferred to recipient cattles,ofwhich 14 were diagnosed as pregnant.Now 6 recipient cattles gave birth to 3 femaleand 3 male calves,all with the predicted sex.