Study On Sex Determination Of Bovine Early Embryo By PCR And LAMP

In modern livestock reproduction, people want to control the sex ratio of cattle offspring according their desire, increase breeding speed and the use of excellent quality cow,so the technology of sex control has important role in livestock. With the development of agriculture, and especially the rapid development of embryo engineering in flock and herds, the sex control has become more and more important in the farm animal industry. The objective of this study is to establish the sex control methods including PCR and LAMP which can be applied in the field. By using these methods, the economic benefit of farmer can be increased, and the cattle breeding in Xinjiang and countrywide can be rapidly improved. This study has been done as follows:1. By designing specific primer, changing Mg²⁺, annealed temperature and cycle condition, we have established the optimal reaction condition of PCR , which can be applied for the prodution.The optimal system of double PCR is a reaction mixture consisting of 2.5μl lOxBuffer, 2.5mM Mg²⁺, 0.2mM dNTP, 2units of Taq DNA polymerase, 0.4μM of sex primer, 0.15μM of common primer (sex and common primer can be added in same reaction respectively)in 25μl final volume. The stage (33 cycles) are predenetured at 94℃ for 5min, denetured at 94 ℃ for 30s, annealed at 55℃ for 30s, expanded 72℃ 30s for 32 cycles, then 72℃ for 5min.The optimal first system of nest PCR is a reaction mixture consisting of 1μl 10×Buffer, 2.5mM Mg²⁺, 0.2mM dNTP, lunits of Taq DNA polymerase, 0.1μM of each primer, in10μl final volume. The stage (20 cycles) are predenetured at 94℃ for 4min, denetured at 94℃ for 30s, annealed at 63℃ for 30s, expanded 72℃30s and then 19 cycles at 72℃ for 2min; The optimal second system of nest PCR is a reaction mixture consisting of 2//1 10xBuffer,2.5mM Mg²⁺,0.2mM dNTP,2units of Taq DNA polymerase,0.251μM of each primer, in 20μl final volume. The stage (30 cycles) are predenetured at 94℃ for 5min, denetured at 94℃ for 30s, annealed at 63℃ for 30s, expanded 72℃30s and for 30 cycles, then 72℃ for 5min.From the determing of 46 embryos in the field, the result was that the method of PCR can be applied.2. LAMP (Loop-mediated isothermal amplification) is a new genic amplification technology. Merits of this kit is rapid, accurate, sensitive and convenient. From the determing of different samples , 126 normal embryos and 42 sex embryos in the field ,we have proved the method of LAMP can be applied for the bovine sex determination.3. The result of same sample using PCR was consistent with LAMP, that was to say, only if we control the lab condition, we can select the one of methods (PCR and LAMP) according to the pratical condition.