Giant Panda Semen Collection And Cryopreservation

Giant panda (Ailuropoda melanoleuca) is endangered wild animal, the population is declining, because their natural reproductive capacity is low, the artificial insemination rate of fresh semen is about 33%, and that of frozen semen is lower. Now, artifical insemination is the most widely used and effective assisted reproductive technology, the artificial insemination of frozen semen has become the important means of livestock breed improvement, is equally important to rare wild animal, such as giant panda, but their genetic resources are very scarce, so the related research is relatively laggard.Semen cryopreservation is the important component of artificial insemination, the quality of frozen sperm directly influences the pregnancy rate of artificial insemination. Sperm suffers oxidative damage during frozen-thawed process, it is a important factor of hurting frozen sperm. ROS destroies the structural and functional integrity of sperm, leads to the quality losses of frozen semen. It has have some research results that adding antioxidants to the semen of livestock during cryopreservation process, however, the application of antioxidants to giant panda semen is seldomly reported. This experiment gathers giant panda semen, detects frozen-thawed sperm motility, viability, plasma membrane integrity and acrosome integrity, filtrates the best cryopreservation method and the most suited thawing medium; On this basis, vitamins and antioxidases are respectively added to the thawing medium of giant panda semen, then by detecting sperm quality and the content of MDA, it is found that antioxidants and their optimum combination which can effectively protect frozen sperm. The results are as follows:1. The motility, viability, plasma membrane integrity and acrosome integrity of frozen-thawed sperm using 0.25ml straw are higher than that using frozen particles, though are not significant between them, the straw has these advantages, including standard dose, clear mark, being difficultly contaminated and easy thawing, so straw cryopreservation method should be used; As compared to M199, Ham's F-10 can distinctly improve plasma membrane integrity and acrosome integrity of frozen sperm, so Ham's F-10 is suitable as the thawing medium of giant panda semen.2.2 mmol/ml VC, 0.2 mg/ml VE or 0.50 mg/ml VB12 is respectively added to the thawing medium of giant panda semen, may improves effectively frozen-thawed sperm quality and declines the content of MDA. The sperm acrosome integrity in 1.0mg/ml VE group is lower than that in control group, the content of MDA is highest in group adding 4 mmol/ml VC or 1.0mg/ml VE, reveals that their high dose promote lipid peroxidation; Protective effect of frozen sperm in group adding of VC, VE and VB12 simultaneity is better than that of Vc and VE simultaneity.3. SOD, CAT and GSH alone adds to thawing medium may protect frozen sperm, especially the concentration with 50U/ml of SOD,200U/ml of CAT and 5mg/ml GSH; Combined addition of SOD and CAT is superior to that of SOD and GSH in the protective effect of giant panda frozen sperm.In a word, it is better that freezing giant panda semen using 0.25ml straw and thawing semen using Ham's F-10; respectively adding 2 mmol/ml VC 0.2 mg/ml VE,0.50 mg/ml VB12 to thawing medium or that in combination; alone adding SOD, CAT and GSH with the concentration:50U/ml, 200U/ml,5mg/ml or adding of SOD and CAT simultaneity, can improve frozen semen quality and antioxidative damage ability of frozen sperm. This experiment can provide a theoretical reference for the development of giant panda semen cryopreservation and application of antioxidants in semen cryopreservation.