Study On Bovine Embryo Sexing By PCR And LAMP Methods

Embryo sexing is one of the important parts of embryo engneering techniques, which has played an important role in livestock breeding, reproduction and prevention of genetic diseases. The purpose of this study was to establish a rapid,reliable and convenient method for the bovine embryo sexing. For this aim, the main results were as follows:1． PCR system of bovine embryo sexing was established by using bovine Y chromosome–specific sequence primers. The BOV97M and bovine 1.715 satellite DNA sequences were selected as the primers of male and bovine-specific DNA, respectively. In consecutive and multiplex PCR, the first 11 cycles were done with male specific primers, then followed by additional 25 cycles with bovine-specific primers(control primers). PCR amplification of BOV97M resulted in a 141bp band and of Bovine satellite 1.715 resulted in a 216 bp band. The consecutive and multiplex PCR system was optimized by using DNA sample extracted from male and female bovine whole blood. The accuracy and sensitivity were 100%(66/66), 10pg bovine genomic DNA (about 3 cells). Adding control primers during the cycling process, results in wasting time, inconvenience and easy contamination, therefore, it is unsuitable for this PCR amplification system to be used as bovine embryo sexing kit.2． The PCR amplification system of bovine embryo sexing was established by using a pair of primers based on bovine amelogenin sequences. This PCR system can amplify a single band of 467bp from female cattle sample, two bands of 467bp and 341bp from male cattle's. This PCR system was optimized under a variety of conditions, including Mg2+ concentration, annealing temperature, DNA polymerase concentration, primer concentration, denaturant, cycles and final volumes. The optimal system is a reaction mixture consisting of 50mM KCl, 10 mM Tris-HCl ( pH 8.8 ), 0.1% Triton-X 100, 2.0mM MgCl2, 0.2mM of dNTP, 3 units of Taq DNA polymerase, 20pM of each primer, in 50μl final volume. The first stage (20 cycles) was annealed at 53℃ and then 30 cycles at 54℃. Accuracy and sensitivity were 100%(60/60), 10pg bovine genomic DNA, respectively. The advantages of this amplification system included using one pair of primers, accuracy, sensitivity and rapidity. So it was suitable for the system to be used as bovine embryo sexing kit.3． The Japanese LAMP(Loop-mediated isothermal amplification) method bovine embryo sexing kit was evaluated under lab and field conditions. The accuracy and sensitivity were 100%(60/60), 1pg genomic DNA, and coincidence of the transfer sex determined embryo was 3/3. Merits of this kit were rapid, accurate, sensitive and convenient. On the other hand, it easily led to fault result (female misidentified as male) when the DNA template .国家"863"计划"奶牛和肉牛胚胎工程高效繁育技术"项目基金资助concentration were above 500ng. It was difficult to eliminate non-specific amplified bands. Above all, the LAMP kit had advantages over PCR kit under field conditions.4． we had done some exporation research to esteblish LAMP method bovine embryo sexing kit. Eight bovine embryo sexing LAMP primers were designed to establish LAMP sexing system. It was found that the LAMP reaction only resulted in amplified a few products without betaine, and DMSO inhibited LAMP reaction. Meanwhile, the design of primers and optimization of the LAMP method system were studied.