猪精液液态保存过程中精子葡萄糖代谢的研究

Liquid semen storage is of great importance for the application of pig artificial insemination. The technique for liquid storage of pig semen is hindered, however, by problems such as complicated extender composition, limited duration and unstable outcome. Although it is accepted that energy supply and prevention of oxidative stress are the two major issues for extended liquid semen storage, research are few on sperm glucose metabolism under such conditions. Furthermore, the existence of the PPP pathway and the need for the Krebs cycle in sperm of different species has yet to be confirmed. Studies on sperm glucose metabolism during liquid storage of pig semen are thus important not only for understanding sperm energy metabolism but also for improving the technique of liquid semen storage. The objective of this project is to study sperm glucose metabolism during liquid semen storage. To this end, pig semen will be preserved at different temperatures in the basic PBS extender that does not contain any energy substrates but supplemented with radio labeled or non-labeled glucose, intermediate metabolites of glucose and/or inhibitors of glucolysis, PPP or mitochondria electron transport. At different times of preservation, sperm motility, viability and other parameters of aging or apoptosis, and glucose metabolites will be evaluated by using techniques such as Sperm Class Analyzer (SCA), flow cytometry and liquid scintillation spectrometry to determine the pathways of sperm glucose metabolization. Data obtained will also be used to formulate an optimal system that provides the best energy and antioxidant supplies for extended liquid storage of pig semen.

精液液态保存对猪人工授精推广至关重要，但目前还存在着稀释液成分复杂，保存时间短，效果不稳定等问题。有效提供能量和防止氧化应激是延长精液液态保存时间的两大问题。然而，目前关于精子体外长时间培养的糖代谢研究很少，不同物种精子PPP途径是否存在，三羧酸循环是否必需，尚无结论。因此，研究猪精液液态保存过程中的精子葡萄糖代谢无论对于丰富精子能量代谢理论，还是对于延长精液保存时间、提高人工授精效率，都具有重要理论和实践意义。本项目拟采用不含任何能量物质的PBS为基础稀释液，通过添加放射标记或不标记的葡萄糖和糖代谢中间产物以及糖酵解、PPP途径和电子传递的抑制剂，不同温度下液态保存猪精液不同时间用精子分析仪检测精子活力和老化凋亡等分子指标，用液闪仪检测糖代谢产物，研究猪精液液态保存过程中精子对葡萄糖的利用和代谢途径，并在此基础上研制出改进精子能量代谢和还原能力的精液保存体系，延长猪精液液态保存时间。

有效提供能量和抑制氧化应激损伤是延长猪精液液态保存时间的两大问题。葡萄糖代谢方式的研究对调控猪精子能量代谢，提高还原能力，延长精液保存时间具有重要意义。本研究采用不含任何能量物质的PBS为基础稀释液，通过添加葡萄糖和糖代谢中间产物以及糖酵解、PPP途径和电子传递的抑制剂，在不同温度下液态保存猪精液于不同时间检测精子活力、活率、顶体完整率等指标，研究猪精液液态保存过程中精子对葡萄糖的利用和代谢途径，并在此基础上研制出改进精子能量代谢精液保存体系，延长猪精液液态保存时间。结果发现：（1）较低温度如15℃有利于猪精液液态保存（2）稀释液中添加适当浓度的葡萄糖（3mM）有利于猪精子的存活，添加过量的葡萄糖对猪精子的存活有害。（3）液态保存过程中，猪精子葡萄糖代谢主要通过糖酵解途径，有氧氧化途径也参与精子活力的维持。（4）葡萄糖代谢的PPP途径，除了提供能量外，还为猪精子提供抗氧化能力，但在猪精液液态保存过程中似乎不依赖于的PPP途径。