| Animal sex manipulationtechnology is a biotechnology that changes the sex ratio of offspring through human intervention and obtains offspring that meet human expectations.In cattle production,X-and Y-bearing sperm sorting technology combined with artificial insemination is one of the most widely used technologies in sex manipulation.Researchers have been exploring methods for accurate,efficient,and rapid separation of sperm.Among them,the separation of bovine sperm by flow cytometry is the most mature and widely used technologies in X-and Y-bearing sperm separation.As for sperm separation technology,both the establishment and optimization of the method cannot be separated from the efficient and rapid evaluation system as the technical guarantee.So far,the identification and analysis techniques for the purity of sperm separation in my country mainly include: flow cytometry reanalysis,FISH,etc.But these methods have some limitations in a way.In view of the practical requirements for rapid,accurate and efficient laboratory methods for testing the purity of X and Y sperm separation,this study is based on qPCR and uses the relationship between the Ct value in the test results and the copy number of the starting template to establish a rapid test.The method for separating the purity of frozen bovine semen is designed to ensure the accuracy of detection,improve detection efficiency,and better promote the promotion and application of bovine sex-controlled semen in production practice.This research is divided into three parts,the main research content and results are as follows:1)qPCR technology establishes a method to quickly detect the separation purity of frozen bovine semen:In this experiment,recombinant plasmids containing specific fragments of cattle X and Y sperm KP677336.1 and SRY genes were constructed separately and used as reference templates for sex-related DNA quantification.Then mix the recombinant plasmid standards of different known concentrations in proportion,and perform qPCR detection.And construct a standard curve based on the relationship between the Ct value and the copy number of the substrate.Finally,specific primers are used to perform qPCR detection on the sample to be tested,and the obtained Ct value is compared with the standard curve,and the concentration ratio of X sperm and Y sperm in the sample to be tested is calculated.The results show that this experiment has established a method to quickly detect the separation purity of bovine frozen semen using qPCR technology,which can effectively calculate the separation purity of the sample to be tested.2)Flow cytometry reanalysis method to compare and verify the detection results of qPCR: In this study,frozen bovine semen was resuscitated by ultrasound,and the tail was quasi-free,dyed with Hoechst 33342 fluorescent dye,and the sample to be tested was re-analyzed by flow cytometry,and the analysis results were compared with the results of of semen separation purityby qPCR detection.The results showed that the results of the two detection methods were not significantly different,which further verified the accuracy of the fluorescence quantitative PCR method in detecting the purity of the frozen bovine semen.3)Comparison of fluorescence in situ hybridization to verify the detection results of qPCR method: In this study,a Y sperm specific probe with a length of 30 bp was designed and synthesized,and the purity of the samples to be tested was evaluated by fluorescence in situ hybridization technology.It is proposed to compare the analysis results with the results of qPCR method to detect the purity of semen analysis.The results show that the synthesized Y sperm specific probe has a weak fluorescence signal and no specificity,and it is impossible to realize the identification of different sperm.Therefore,the FISH method using this probe in this study cannot be used to verify the test results of the qPCR method.In summary,this experiment successfully established a qPCR-based evaluation method for the rapid evaluation of the purity of bovine sex-controlled semen,which was further verified by flow reanalysis.The method is characterized by fast speed,low cost,and does not require large-scale equipment.It is expected to be used in frozen semen production enterprises and testing units. |
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