| Cryopreservation techniques for goat semen are an important part of the goat breeding system,which can effectively preserve high-quality Germplasm Resources and accelerating the process of goat breeding efforts.However,goat sperm will be damaged by oxidative stress,cold stress and other adverse factors during freezing,resulting in low quality of goat sperm after thawing,which greatly limits the application of frozen goat semen.A large number of studies have shown that adding amino acid substances during semen cryopreservation can effectively improve the quality of frozen-thawed spermatozoa.Previously,our group found that glutamine,phenylalanine and leucine could be potential biomarkers of freeze resistance in Inner Mongolian cashmere goat semen by proteomicmetabolomic analysis of high-Freezability(HF)and low-Freezability(LF)semen.Therefore,the aim of this study was to investigate the effects of different concentrations of glutamine,phenylalanine and leucine on the semen quality of Inner Mongolian cashmere goats after frozen-thawed,and to select the best concentration by measuring sperm motility,structural integrity,antioxidant capacity and mitochondrial function.In order to provide theoretical basis and reference for the application of glutamine,phenylalanine and leucine in the cryopreservation of cashmere goat semen.The following results were obtained:1.Glutamine with different concentrations(2,4,6,8 mmol/L)was added to the cryopreservation dilution.The results showed that 4 mmol/L glutamine had a positive effect on the quality of cashmere goat spermatozoa after frozen-thawed.After frozen-thawed,sperm motility was 67.9 %,plasma membrane integrity was 54.5 %,acrosome integrity was79.2 %,and straight-line motility was 49.6 μm/s,average path speed 54.6 μm/s.Leucine with different concentrations(0.01,0.1,1,10 mmol/L)was added to the cryopreservation dilution.The results showed that 1 mmol/L leucine had a positive effect on the quality of cashmere goat sperm after frozen-thawed.The average path speed after frozen-thawed was54.6 μm/s,straight-line speed 58.7 μm/s,curve-line speed 93.7 μm/s.Phenylalanine with different concentrations(0.01,0.1,1,10 mmol/L)was added to the cryopreservation dilution.The results showed that the sperm motility after frozen-thawed with 10 mmol/L phenylalanine added in the dilution solution was 48.4 %,sperm viability was 24.4 %,The average path speed after frozen-thawed was 29.7 μm/s,straight-line speed 26.9 μm/s,curveline speed 51.9 μm/s,all significantly lower than the control group(P<0.05).Therefore,the addition of phenylalanine to the cryopreservation dilutions did not have a positive effect on cashmere goat sperm after frozen-thawed,but instead exhibited a negative effect with increasing phenylalanine concentrations.2.The antioxidant index of frozen-thawed sperms was determined due to the protective effect of glutamine addition on the viability and structural integrity of frozen-thawed sperm.The results showed that 4 mmol/L glutamine had a positive effect on the antioxidant index,the total antioxidant capacity was 1.59 mmol/L,CAT activity was 374.93 U/mgprot,SOD activity was 92.95 U/mgprot and GPx activity was 1390.77 m U/mgprot were significantly higher than those of the control group(P<0.05).The ROS level after frozen-thawed was 603 and the MDA content was 18.2 μmol/mgprot were significantly lower than that of control group(P<0.05).In conclusion,the addition of 4 mmol/L glutamine to the cryopreservation dilution solution could reduce the oxidative stress that spermatozoa are subjected to during freezing,leading to an improvement in the cryopreservation effect of cashmere goat semen.3.The energy metabolism index of frozen-thawed sperms was tested because the addition of leucine had a positive effect on the motional parameters of sperms after frozenthawed.The results showed that 1 mmol/L leucine had a positive effect on the energy metabolism of frozen-thawed sperms of cashmere goats.ATP content 0.42 μmol/m L,mitochondrial membrane potential was 2.82 ΔΨM,the activity of Mitochondrial Respiratory Chain Complex I was 197.32 U/mgprot,and the activity of Mitochondrial Respiratory Chain Complex II was 109.26 U/mgprot,were significantly higher than that of the control group(P<0.05).Therefore,the addition of 1 mmol/L leucine to the cryopreservation extender improved the energy metabolism of sperm and subsequently improved the cryopreservation of cashmere goat semen.In summary,this study showed that the addition of 4 mmol/L glutamine to the freezeextender of inner Mongolian cashmere goat semen could reduce oxidative stress during sperm freezing,and the addition of 1 mmol/L leucine could improve energy metabolism of sperm,thereby improving the freeze-preservation effect of cashmere goat semen. |
