Cryopreservation Of Giant Panda Semen In Qinling Mountains

At present,the low conception rate of artificial insemination of giant pandas makes the reproduction of captive giant pandas difficult.Timely research on the quality of giant panda semen and its cryopreservation technology development are of great significance to maintain the population size of giant pandas in the Qinling Mountains,and establish a germplasm resource base of the Qinling subspecies,and finally protect the endangered plight of giant pandas.Oxidative damage to sperm during freezing is a major factor affecting the quality of frozen sperm,meanwhile,the addition of antioxidants to sperm cryopreservation solution has become an important method to improve the quality of frozen semen of domestic animals.In this experiment,we used ELISA and other techniques to explore the correlation between seminal plasma indexes and semen quality of giant pandas in Qinling Mountain based on the previous detection of seminal plasma indexes,and analyzed the effects of lycium barbarum polysaccharide(LBP),laminarin(LJP),and resveratrol(RSV)on the conventional quality and mitochondrial activity of giant panda frozen semen and screened the optimal dose of antioxidant,we also observed the ultrastructural changes of giant panda spermatozoa after freezing and thawing,and provided experimental data for the establishment of ultra-low temperature cryopreservation technology for giant panda semen in Qinling Mountain and the improvement of conception rate of artificial insemination.The experimental results are as follows:1.Semeon was collected from four giant pandas aged from 12 to 17 years,the mean sperm viability,plasma membrane integrity and acrosome integrity of fresh sperm were 82.75±5.74%,81.04±4.35% and 47.10±8.11%,respectively.the sperm viability,plasma membrane integrity and acrosome integrity of Panda B and Panda C were significantly higher than those of Panda A and Panda D.The seminal plasma index analysis showed a positive correlation between the contents of acid phosphatase and zinc in seminal plasma and the results of sperm viability,plasma membrane integrity and acrosome integrity,the increase of ROS content in seminal plasma of Panda D was significant and correlated negatively with semen quality.2.The effect the effects of ultra-low temperature freezing on giant panda spermatozoa were performed using a scanning and transmission electron microscopy by making SEM sections,the result showed that the damage was mainly caused to the head and neck of spermatozoa after ultra-low temperature cryopreservation.3.Three antioxidants were added to the semen before freezing and tested by ELISA and other techniques.The results showed that the 50 μM and 100 μM RSV and2.0 mg/m L LBP addition groups significantly increased the sperm quality and antioxidant capacity of frozen sperm after thawing,and the 100 μM RSV addition group significantly down-regulated the content of ROS;1.0 mg/m L LBP,4.0 mg/m L LBP and the combined addition group could significantly increase the antioxidant capacity of sperm,and the combined addition group significantly up-regulated the sperm viability;2.0 mg/m L and 4.0 mg/m L LBP significantly up-regulated the activity of sperm acrosome protease.RSV,LJP and LBP significantly improved sperm quality by increasing antioxidant enzyme activity and inhibited ROS production during freezing.Among them,50 μM RSV and 2.0 mg/m L LBP had the best effect on sperm protection during cryoprotection.In conclusion,the results of this study showed that ultra-low temperature freezing caused some damage to the sperm structure of giant panda,and the addition of 2.0 mg/m L LBP or the addition of 50 μM RSV to the cryoprotectant could reduce the oxidative damage during the cryopreservation of giant panda semen and improve the sperm quality of giant panda semen after cryopreservation.