| Glutamine has the ability to protect pig sperm at low temperature,especially by improving sperm motility after thawing.As an osmotic cryoprotectant,glycerin can protect sperm from freezing damage caused by low temperature.It is widely used in cryopreservation of sperm in various animals.However,glycerin also has an adverse effect and has a certain toxicity to sperm.In this test,different concentrations of glutamine(Gln)were used to replace part of glycerin as a cryoprotectant for pig semen.After being stored in liquid nitrogen for 4 weeks,it was thawed,and the sperm motility,plasma membrane integrity and acrosome of the frozen-thawed Songliao black pig sperm Integrity,mitochondrial membrane potential,membrane lipid disorder,protamine deficiency and in vitro fertilization and other technical links have been studied,so the experiment is divided into experiment 1:3%glycerol control group(no Gln added),experiment 2:2%glycerol(No Gln added)treatment group,experiment three:2%glycerol+Gln(20 mM,40 mM,80 mM,100 mM)treatment group.It aims to explore the effect of replacing part of glycerol with glutamine(Gln)on frozen-thaw pig sperm,and provide technical support for cold storage and commercial production of pig semen.The test results are as follows:1.Compared with 3%glycerol(44.28 ± 2.01,47.94 ±2.13,46.16±2.85,49.18 ±1.63,46.11 ±3.35)control group,2%glycerol treatment group(31.43±2.19,43.71 ±2.27,32.21±1.82)33.18±1.83,32.47±1.61)sperm motility,plasma membrane integrity,acrosome integrity,viability and mitochondrial membrane potential were all significantly decreased(P<0.05),2%glycerol+80 mM Gln treatment group(42.94±2.99,50.99 ± 2.19,48.25±1.24)sperm motility,plasma membrane integrity and acrosome integrity were significantly improved(P<0.05),2%glycerol+100 mM Gln treatment group(52.98 ±2.63,53.82 ± 2.84,52.08 ± 2.26,55.12 ± 3.08,54.72 ± 1.77)sperm motility,plasma membrane integrity,acrosome integrity,viability and mitochondrial membrane potential were all significantly increased(P<0.05);2.Compared with the 3%glycerol control group(8.83 ± 0.15),the 2%glycerol treatment group(8.30±0.20)had a significant decrease in the undisturbed level of cell membrane lipids of live sperm(P<0.05).After replacing part of glycerol with glutamine The lipid level of living sperm cell membrane was not disturbed significantly(P<0.05),and the difference was most significant when glutamine was 100 mM(11.83±0.20);3.Compared with the 3%glycerol control group(19.07 ± 1.87),the 2%glycerol treatment group(22.3±1.78)significantly increased the level of sperm protamine deficiency(P<0.05),and the 2%glycerol+ 100 mM Gln treatment group(11.80±1.77)The lack of sperm and protamine decreased significantly(P<0.05);4.Compared with the 3%glycerol control group(1.11±0.33),the level of sperm tyrosine phosphorylation between the 2%glycerol treatment group(0.93 ±0.10)and the 2%glycerol+100 mM Gln treatment group(1.11±0.34)No significant difference(P>0.05);5.Compared with the 3%glycerol control group(56.24 ± 5.51),the 2%glycerol treatment group(40.58±8.34)significantly reduced the sperm in vitro fertilization and cleavage rate(P<0.05),and the 2%glycerol+100 mM Gln treatment group(77.83±5.57)The cleavage rate of sperm in vitro fertilization increased significantly(P<0.05).The above results indicate that the replacement of part of glycerol with glutamine as a cryoprotectant for pig sperm can significantly improve sperm motility,plasma membrane,acrosome,mitochondrial membrane potential,viability and membrane lipid disorder,especially when glutamine is 100 mM At the same time,100 mM glutamine instead of part of glycerol as a cryoprotectant for pig sperm can also significantly improve the protamine deficiency level of sperm and the cleavage rate of in vitro fertilization.This study optimized the pig semen cryoprotectant system,which will provide commercial value for cold storage of pig semen. |
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