Application Study Of Soybean Lecithin And Proanthocyanidins In The Goat Sperm Freezing

With the development of modern animal husbandry,the market demand and quality requirements of frozen semen in artificial insemination is increasing.Egg yolk is the most common ingredient which was used in mammalian semen extender to preserve spermatozoa during the freezing-thawing process.However,it is hard to porduce a standard extender because of the wide variability of EY composition and possible pathogenic microbial contamination.In order to explore the freezing dilluents with stable composition and non-contamination,the soybean lecithin（SL）was used to substitute egg yolk during the goat semen freezing.Firstly,this study aimed to select the optimum concentration of soybean lecithin.Secondly,the synergistic effects of soybean lecithin and egg yolk were investigated.Finally,the antioxidant protection effect of proanthocyanidins（PC）during goat semen freezing was studed with the addition of different concentrations of proanthocyanidins.The results were as follows:1. The aim of this study was to evaluate the effect of soybean lecithin on the replacement of egg yolk in the cryopreservation of goat semen.This study used 20%egg yolk（V/V）as control group,added 0.5%,1%,2%,3%soybean lecithin（m/V）to the basic dilution of goat as treatment groups.After freezing–thawing,the samples were evaluated for sperm quality parameters,including sperm viability,motility,plasma membrane integrity,acrosome integrity and mitochondrial activity.Antioxidant indexes were also detected by relevant detection kits,such as superoxide dismutase（SOD）,reactive oxygen species（ROS）and malondialdehyde（MDA）.The results showed that there were no significant differences in the sperm viability,motility,plasma membrane integrity,acrosome integrity and mitochondrial activity after thawing between 20%egg yolk group and 2%soybean lecithin group（P>0.05）.However,they were much higher than those of 0.5%,1%and 3%soybean lecithin groups（P<0.05）.2%soybean lecithin group got lowest ROS and MDA contents,and the highest SOD activity,which were much lower or higher than those of 0.5%,1%and 3%soybean lecithin groups（P<0.05）.There was no significant differences between 20%egg yolk group and 2%soybean lecithin group in antioxidant indexes（P>0.05）.After laparoscopic insemination,the egg yolk group and soybean lecithin group obtained 81.82%and 78.75%pregnancy rates after 45 d B-ultrasonic examination and 72.73%and 78.75%farrowing rate.It was concluded that 2%soybean lecithin can effectively alternate egg yolk as cryoprotectant during goat semen cryopreservation.2. The semen samples were pooled and diluted in 20%egg yolk-based extender or 2%soy lecithin-based extender（control group）,while diluted in 10%EY+2%SL,20%EY+1%SL,20%EY+2%SL（treatment group）.After thawing,The results showed that,the sperm viability,motility,plasma membrane integrity,acrosome integrity and mitochondrial activity were have no significant between 20%egg yolk and10%EY+2%SL group（P>0.05）,and they were higher than the groups of20%EY+1%SL and 20%EY+2%SL（P<0.05）.The ROS and MDA values of the10%EY+2%SL group were the lowest in treatment groups and the SOD values were the highest,while there was no significant differences between the 20%EY and 2%SL control groups（P>0.05）.However,the antioxidant ability of 10%EY+2%SL group was much higher than those of 20%EY+1%SL and 20%EY+2%SL groups（P<0.05）.Thus,it is concluded that SL can effectively replace or partially replace EY as cryoprotectant during goat semen cryopreservation.The addition of different concentrations of SL in20%EY increased the oxidative damage of goat frozen sperm.3. PC were added to the 2%SL based extender to the final concentration of 0μg·m L^-1,10μg·m L^-1,20μg·m L^-1,40μg·m L^-1,60μg·m L^-1.After freezing–thawing,sperm viability,motility,plasma membrane integrity,acrosome integrity,mitochondrial activity,antioxidant index and apoptotic state were detected.The results showed that the sperm viability,motility,acrosome integrity,mitochondrial activity and plasma membrane integrity after thawing（58.49%,53.45%,55.37%,55.16%and 50.46%,respectively）were the highest,which were significantly higher than those of EY and SL0 groups（P<0.05）.The SOD value(224.87 U·m L^-1)and GSH-PX level(129.6 U·L^-1)of SL3 group were the highest,and the Pan caspase apoptotic rate（54.33%）and TUNEL apoptotic rate（41.36%）of SL3 group were the lowest,which were significantly different with EY and SL0 groups（P<0.05）.When the concentration of PC increased to 60μg·m L-1,the quality indexes of thawed semen were significantly decreased,and the toxicity of PC was found in the goat semen freezing.In conclusion,40μg·m L^-1 PC addition in the goat semen freezing could reduce sperm oxidative damage,decrease apoptotic level and improve sperm quality.