种公猪精液蛋白组高通量iTRAQ检测及其繁殖性能调控机制研究

The improvement of boar reproductive performance is one of the core problem in animal husbandry. The sperm fertilization ability, which is regulated by semen proteins, is one of the important indicators reflecting boar reproductive ability. Semen proteomics is a hot spot in current research of animal reproduction, and the relationships and mechanism between boar semen proteins and sperm fertilization ability or boar breeding performance are eagerly need to be elucidated. This project intends to take the selected boars of high breeding and low breeding by a scientific method, and the top boars selected by whole genome selection, as the main research objects from a large boar group based on a criterion of qualified conventional semen indexes. Semen differentially expressed proteins were detected with a high flux screening iTRAQ technology, and then a series of new reproductive performance related proteins will be got by using biometric and bioinformatics methods to analyze the correlation between the proteins and boar breeding performance. Then the expression and distribution of the objective proteins (such as GPxs) were detected in boar reproductive organs, sperm and oocyte. And the effect of objective proteins in the process of fertilization was studied by using artificial insemination, in vitro fertilization and related technologies, so as to explore and elucidate the effect and mechanism of objective protein actions with the sperm fertilization ability. This project can provide protein markers for accurate evaluation of semen fertilization capacity, improving the efficiency of artificial insemination and boar reproduction; also it can provide important reference basis for boar breeding; At the same time, it can also provide reference for the research and treatment of infertility in human reproduction.

提高种公猪繁殖能力是畜牧业发展的核心问题之一，精子受精能力是公猪繁殖性能的重要指标，受到精液蛋白的影响和调控。精液蛋白组学是当前动物繁殖领域的研究热点，精液蛋白与精子受精能力和公猪繁殖性能的关系与作用机制亟待阐释。本项目拟将以科学方法获得的高繁殖力和低繁殖力的合格种公猪、以及全基因组选择的顶级公猪作为主要研究对象，采用高通量iTRAQ蛋白检测技术筛选精液差异表达蛋白，并用生物统计和生物信息学方法分析精液蛋白与公猪繁殖性能的相关性，得到一批新的繁殖性能相关目标蛋白；再通过检测目标蛋白（如GPxs等）在公猪生殖器官、精子和卵子的表达分布，利用体外受精、人工授精等技术研究目标蛋白在受精过程的作用，探索并阐释目标蛋白对精子受精能力的影响及作用机制。本项目研究可为准确评定精液受精能力提供蛋白标记、提高人工授精和种公猪繁殖效率；还可为公猪育种提供重要参考；同时也为人类生殖不孕不育的研究和治疗提供参考。

提高种公猪繁殖能力是当前养猪业亟需解决的重要科学问题。精液蛋白可作为潜在的动物繁殖性能的生物标记，在受精过程中具有重要作用。而猪精液蛋白含量与精子受精能力及公猪繁殖能力之间的关系与调控作用尚有待于研究和阐释。本项目通过对公猪繁殖成绩大数据统计分析，建立了杜洛克、长白和大白种公猪繁殖能力评价的数学模型。利用iTRAQ技术筛选出一批不同繁殖性能种公猪的精液差异蛋白，并进行了相关生物信息学分析，为种公猪繁殖性能的理论研究和选育生物标记的研发提供了重要参考依据。成功进行了猪源OPN重组蛋白的体外生产和单克隆抗体的制备，研究发现精子OPN相对含量与窝产活仔数呈显著正相关，OPN蛋白在各级生精细胞、精子、卵子和卵丘细胞中均有表达，在精子上主要定位于精子头的赤道部，能够影响精子顶体反应、精子cAMP含量和精子体外受精能力，参与ECM-受体相互作用和PI3K-Akt信号通路。研究了猪精液CRISP2/3蛋白相对含量与大白种公猪繁殖性能的相关性，发现精子蛋白CRISP2相对含量与参配母猪的窝均产仔数、窝均活仔数和窝均健仔数呈显著正相关，精子蛋白CRISP3相对含量与参配母猪的分娩率、繁殖效率呈显著正相关，精清蛋白CRISP3相对含量与参配母猪的窝均产仔数、窝均活仔数和繁殖效率呈显著正相关；并研究了CRISP2/3蛋白的组织表达和细胞定位，研究发现精子CRISP2影响精子的体外受精能力。研究了GPx6蛋白在公猪生殖系统的时空表达和组织细胞定位，发现GPx6具有显著降低精子cAMP含量、抑制精子顶体反应、降低体外受精卵裂率的作用，但对精子ROS水平没有显著影响；KEGG通路分析表明GPx6主要参与花生四烯酸代谢和谷胱甘肽代谢。本项目研究为种公猪繁殖理论研究和养猪生产中高繁殖能力种公猪的选育提供了重要参考依据。