| The structure,biochemistry and physiological function of goat sperm were damaged during freezing and thawing.How to minimize the damages of sperm in the freezing and thawing process has always been the focus of researches.In order to further improve the quality of cashmere goat semen after cryopreservation,semen was diluted with a Tris-based extender and we compared the different height of liquid nitrogen fumigation,and the effect of seminal plasma,egg yolk and soy lecithin on cryopreservation of Inner Mongolian cashmere goat semen,then we selected a most suitable procedure for cryopreservation of cashmere goat semen.On this foundation,the effects of oxidative phosphorylation(OXPHOS)and glycolysis on cryopreservation and after thawing were studied;and the cryopreservation action mechanism of glucose,trehalose and cholesterol on cashmere goat semen were further investigated.After frozen-thawing,the sperm motility,sperm structural integrity,ROS,Ca2+,ATP content in sperm were detected;the level of pyruvic acid and lactic acid in the suspension were detected.Sperm apoptosis and mitochondrial membrane potential were detected.The AMPK,phospho-AMPK and protein tyrosine phosphorylation in sperm were detected.The results are as follows:1.After the diluted semen was cooled to 5 ? and frozen in liquid nitrogen vapour at a height of H1(4 cm)and H2(9 cm)above the liquid nitrogen surface with 7min,the results showed that sperm motility in the group of H2(9 cm)was lower than H1(4 cm)(P<0.01),the percentage of acrosome and plasma membrane integrity was decreased in the group of H2 than H1(P<0.01),and there was no significant difference in DNA integrity between the group H1 and H2,after cryopreservation.Therefore,diluted cashmere goat semen was frozen in liquid nitrogen(LN)vapour at a height of 4 cm above the liquid nitrogen surface with 7 min achieved better cryopreservation effects.During sperm cryopreservation,removing the seminal plasma decreased sperm motility and velocity after cryopreservation(P<0.01),decreased percentage of acrosome integrity and plasma membrane integrity(P<0.05),decreased DNA integrity(P<0.01),and significantly increased the level of lipid peroxidation(P<0.01);therefore,seminal plasma is beneficial for the cryopreservation of cashmere goat semen.We compared the cryopreservation effects of the 1.5% soy lecithin and 10% egg yolk on cashmere goat semen,no significant differences were observed in sperm motility,structural integrity and the level of lipid peroxidation,so egg yolk could be replaced by soy lecithin which to protect the sperm from cryodamage.2.During the cryopreservation of cashmere goat semen,inhibition of oxidative phosphorylation(OXPHOS)and glycolysis decreased sperm motility and structural integrity(P<0.05)and decreased mitochondrial membrane potential(P<0.05),butsignificantly increased the level of lipid peroxidation(P<0.01).After thawing,inhibition of OXPHOS significantly decreased sperm motility(P<0.01)and significantly decreased the mitochondrial membrane potential(P<0.05);inhibition of glycolysis significantly decreased sperm motility(P<0.05),but not significantly effect mitochondrial membrane potential.So,during cryopreservation of cashmere goat semen,glycolysis was important as OXPHOS for sperm quality and metabolism;after thawing,both OXPHOS and glycolysis were capable of maintaining sperm motility,and OXPHOS was more effective in maintaining mitochondrial activity.3.Semen were diluted with a extender including glucose(0-112 mmol)were studied,the results indicated that in the semen extender supplemented 56 mmol glucose could increased the motility,velocity,acrosome and plasm membrane integrity of frozen-thaw sperm(P<0.05),however did not cause significant differences in DNA integrity among the groups(P>0.05).The level of ROS was significantly higher in 56 mmol glucose group than that in other groups(P<0.01),while the level of Ca2+ in 28 mmol and 56 mmol glucose group were lower than that all of the other groups(P<0.01).The ATP content in 56 mmol and 84 mmol glucose group were higher than other groups(P<0.05).After thaw,lactic acid was not detected in the low concentration glucose group(28-56mmol),but a large amount of lactic acid was detected in the 112 mmol glucose group than that all of the other groups(P<0.01).So,appropriate glucose was supplemented into the semen extender could promoted the sperm metabolism following cryopreservation;but high concentration of glucose inhibited sperm motility following cryopreservation may be due to a large amount of lactic acid was produced.4.The semen extender supplemented with trehalose and glucose did not cause significant differences in sperm motility,acrosome integrity and plasma membrane integrity,but increased DNA integrity and mitochondrial membrane potential(P<0.05),and significantly decreased the levels of ROS and Ca2+(P<0.01),and decreased the level of lipid peroxidation(P<0.05);in the group of GT-100(100mmol trehalose with 56 mmol glucose),after thawing,abundant phospho-AMPK was found in the apical part of the acrosome region,the sub-equatorial segment of the head,and the midpiece region of tail.Therefore,trehalose may exert antioxidant effects through activation of AMPK.5.Treating sperm with different concentrations of cholesterol-loaded cyclodextrin(CLC)did not cause significant differences in sperm motility after cryopreservation(P>0.05),but result in higher acrosome integrity and plasma membrane integrity(P<0.05),and significant increas the DNA integrity(P<0.01);sperm treatment with 1 mg/ml and 2 mg/ml of CLC increased mitochondrial membrane potential after cryopreservation(P<0.05),and decreased levels of ROS and Ca2+ in sperm(P<0.05);sperm treatment with 2 mg/ml of CLC significantly decreaseed the protein tyrosine phosphorylation of 70KDa;and CLC could delay the sperm acrosomal reaction response of A23187 after frozen-thawing.In short,the cashmere goat semen was diluted in Tris-based extender containing56 mmol of glucose,after semen were cooled to 5 ? and frozen in liquid nitrogen vapour at a height of 4 cm above the liquid nitrogen surface with 7 min,what was themost suitable for cryopreservation of cashmere goat semen.Glucose,trehalose and cholesterol affect the cryopreservation of cashmere goat semen through different pathways.The studies provides a theoretical basis for cryopreservation of cashmere goat semen. |
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