葡萄糖对绒山羊精子冷冻保存及代谢的影响
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摘要
旨在研究葡萄糖对绒山羊精子冷冻保存及代谢的影响。本研究将混合后的精液样品,在含有不同浓度葡萄糖(0、28、56、84和112mmol·L~(-1))的冷冻稀释液进行冷冻-解冻,利用计算机辅助精液分析系统(CASAS)、流式细胞仪和酶标仪检测精子的运动性能、精子DNA完整性、顶体完整性、质膜完整性和精子中ROS和Ca2+水平,以及精子中ATP浓度、精子悬浮液中乳酸和丙酮酸浓度。结果表明,在冷冻稀释液中添加56mmol·L~(-1)葡萄糖可以显著提高绒山羊精子冷冻-解冻后的精子活力、完整顶体和完整质膜的精子比率(P<0.05),但各组间完整DNA的精子比率差异不显著(P>0.05)。56mmol·L~(-1)葡萄糖组精子细胞中ROS水平极显著高于其它各组(P<0.01);28和56mmol·L~(-1)葡萄糖组精子细胞中Ca2+水平极显著低于其它各组(P<0.01);与其它各组相比,56和84mmol·L~(-1)葡萄糖组精子细胞中ATP浓度极显著升高(P<0.01)。精液解冻后,在低葡萄糖浓度组(28~56mmol·L~(-1))的精子悬浮液中未检测到乳酸,而112mmol·L~(-1)组检测到大量乳酸(P<0.01);当冷冻稀释液中添加低浓度的葡萄糖(28~84mmol·L~(-1)),葡萄糖对精子悬浮液中丙酮酸的产生存在剂量依赖性作用,而高浓度(112mmol·L~(-1))组中丙酮酸产生减少(P<0.05)。综上表明,在冷冻稀释液中添加56mmol·L~(-1)葡萄糖可以促进绒山羊精子冷冻-解冻过程中的代谢,提高其冷冻保存效果。
The purpose of this study was to investigate the effect of glucose on cryopreservation and metabolism of cashmere goats semen.In this study,the mixed semen samples were diluted in extender containing different concentrations of glucose(0,28,56,84 and 112 mmol·L~(-1))and frozen-thawed.After thawing,sperm motility,the percentage of sperm with intact DNA,intact acrosome and intact plasm membrane,level of ROS and Ca2+in sperm and concentration of ATP in sperm,and concentrations of lactic acid and pyruvic acid in sperm suspension were assessed byusing computer-assisted sperm analysis system(CASAS),flow cytometry and microplate reader,respectively.The results showed that the semen extender supplemented with 56 mmol·L~(-1) glucose could increase sperm motility,the percentage of intact acrosome and plasm membrane in frozen-thaw cashmere goats semen(P<0.05),however no significant differences were observed in the percentages of intact DNA among the groups(P>0.05).The group of 56 mmol·L~(-1) glucose could extremely significantly increase the level of ROS(P<0.01),while the groups of 28 and 56 mmol·L~(-1) glucose could extremely significantly decrease the level of Ca2+(P<0.01);the group of 56 and 84 mmol·L~(-1) glucose extremely significantly increase the ATP concentration,in comparison to the other concentration groups(P<0.01).After thawing,lactic acid was not detected in the low concentration glucose groups(28-56 mmol·L~(-1)),but a large amount of lactic acid was detected in the 112 mmol·L~(-1) glucose group(P<0.01).Glucose had a dose-dependent effect on pyruvic acid production in sperm suspension when semen extender supplemented with low concentration glucose(28-84 mmol·L~(-1)),but high concentration glucose(112 mmol·L~(-1))significantly decreased pyruvic acid production(P<0.05).In summary,the semen extender supplemented with56 mmol·L~(-1) glucose could promote metabolism of cashmere goat sperm during freeze-thawing and improve cryopreservation effect.
The purpose of this study was to investigate the effect of glucose on cryopreservation and metabolism of cashmere goats semen.In this study,the mixed semen samples were diluted in extender containing different concentrations of glucose(0,28,56,84 and 112 mmol·L~(-1))and frozen-thawed.After thawing,sperm motility,the percentage of sperm with intact DNA,intact acrosome and intact plasm membrane,level of ROS and Ca2+in sperm and concentration of ATP in sperm,and concentrations of lactic acid and pyruvic acid in sperm suspension were assessed byusing computer-assisted sperm analysis system(CASAS),flow cytometry and microplate reader,respectively.The results showed that the semen extender supplemented with 56 mmol·L~(-1) glucose could increase sperm motility,the percentage of intact acrosome and plasm membrane in frozen-thaw cashmere goats semen(P<0.05),however no significant differences were observed in the percentages of intact DNA among the groups(P>0.05).The group of 56 mmol·L~(-1) glucose could extremely significantly increase the level of ROS(P<0.01),while the groups of 28 and 56 mmol·L~(-1) glucose could extremely significantly decrease the level of Ca2+(P<0.01);the group of 56 and 84 mmol·L~(-1) glucose extremely significantly increase the ATP concentration,in comparison to the other concentration groups(P<0.01).After thawing,lactic acid was not detected in the low concentration glucose groups(28-56 mmol·L~(-1)),but a large amount of lactic acid was detected in the 112 mmol·L~(-1) glucose group(P<0.01).Glucose had a dose-dependent effect on pyruvic acid production in sperm suspension when semen extender supplemented with low concentration glucose(28-84 mmol·L~(-1)),but high concentration glucose(112 mmol·L~(-1))significantly decreased pyruvic acid production(P<0.05).In summary,the semen extender supplemented with56 mmol·L~(-1) glucose could promote metabolism of cashmere goat sperm during freeze-thawing and improve cryopreservation effect.
引文
[1]KUCUK N,AKSOY M,UCAN U,et al.Comparison of two different cryopreservation protocols for freezing goat semen[J].Cryobiology,2014,68(3):327-331.
[2]MARTIN G,SABIDO O,DURAND P,et al.Cryopreservation induces an apoptosis-like mechanism in bull sperm[J].Biol Reprod,2004,71(1):28-37.
[3]KONYALI C,TOMAS C,BLANCH E,et al.Optimizing conditions for treating goat semen with cholesterol-loaded cyclodextrins prior to freezing to improve cryosurvival[J].Cryobiology,2013,67(2):124-131.
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[5]SIEME H,OLDENHOF H,WOLKERS W F.Sperm membrane behaviour during cooling and cryopreservation[J].Reprod Domest Anim,2015,50(S3):20-26.
[6]SHAFIEI M,FOROUZANFAR M,HOSSEINI S M,et al.The effect of superoxide dismutase mimetic and catalase on the quality of postthawed goat semen[J].Theriogenology,2015,83(8):1321-1327.
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[9]NAING S W,WAHID H,AZAM K M,et al.Effect of sugars on characteristics of Boer goat semen after cryopreservation[J].Anim Reprod Sci,2010,122(1-2):23-28.
[10]RODRIGUEZ-GIL J E.Mammalian sperm energy resources management and survival during conservation in refrigeration[J].Reprod Domest Anim,2006,41(S2):11-20.
[11]邱建华.山羊精液液态保存过程中精子葡萄糖代谢的研究[D].泰安:山东农业大学,2011.QIU J H.Investigation of goat sperm metabolism during long term liquid storage of goat semen[D].Taian:Shandong Agricultural University,2011.(in Chinese)
[12]KAMP G,BUSSELMANN G,LAUTERWEIN J.Spermatozoa:models for studying regulatory aspects of energy metabolism[J].Experientia,1996,52(5):487-494.
[13]SCOTT T W,WHITE I G,ANNISON E F.Glucose and acetate metabolism by ram,bull,dog and fowl spermatozoa[J].Biochem J,1962,83(2):398-404.
[14]BARBAS J P,MASCARENHAS R D.Cryopreservation of domestic animal sperm cells[J].Cell Tissue Bank,2009,10(1):49-62.
[15]赵娜,甄林青,胡启蒙,等.超低温冷冻过程引起猪精子产生“似凋亡”变化[J].畜牧兽医学报,2013,44(11):1766-1774.ZHAO N,ZHEN L Q,HU Q M,et al.Cryopreservation procedure induces the alterations of“apoptosislike”of boar sperm[J].Acta Veterinaria et Zootechnica Sinica,2013,44(11):1766-1774.(in Chinese)
[16]朱士恩.家畜繁殖学[M].5版.北京:中国农业出版社,2011.ZHU S E.Reproduction in farm animals[M].5th ed.Beijing:China Agriculture Press,2011.(in Chinese)
[17]SALAMON S,RITAR A J.Deep freezing of Angora goat semen:effects of diluent composition and method and rate of dilution on survival of spermatozoa[J].Aust J Biol Sci,1982,35(3):295-303.
[18]WILLIAMS A C,FORD W C L.The role of glucose in supporting motility and capacitation in human spermatozoa[J].J Androl,2011,22(4):680-695.
[19]DU PLESSIS S S,AGARWAL A,MOHANTY G,et al.Oxidative phosphorylation versus glycolysis:what fuel do spermatozoa use?[J].Asian J Androl,2015,17(2):230-235.
[20]FERRAMOSCA A,ZARA V.Bioenergetics of mammalian sperm capacitation[J].Biomed Res Int,2014,2014:902953.
[21]RAMIO-LLUCH L,YESTE M,FERNANDEZ-NOVELL J M,et al.Oligomycin A-induced inhibition of mitochondrial ATP-synthase activity suppresses boar sperm motility and in vitro capacitation achievement without modifying overall sperm energy levels[J].Reprod Fertil Dev,2014,26(6):883-897.
[22]LOSANO J D A,ANGRIMANI D S R,DALMAZZO A,et al.Effect of mitochondrial uncoupling and glycolysis inhibition on ram sperm functionality[J].Reprod Domest Anim,2017,52(2):289-297.
[23]GALLOWAY C A,YOON Y.Perspectives on:SGP symposium on mitochondrial physiology and medicine:what comes first,misshape or dysfunction?The view from metabolic excess[J].J Gen Physiol,2012,139(6):455-463.
[24]MATSUZAKI M,MIZUSHIMA S,HIYAMA G,et al.Lactic acid is a sperm motility inactivation factor in the sperm storage tubules[J].Sci Rep,2015,5:17643.
[25]ST JOHN J C,BOWLES E J,AMARAL A.Sperm mitochondria and fertilisation[J].Soc Reprod Fertil Suppl,2007,65:399-416.
[26]NASCIMENTO J M,SHI L Z,TAM J,et al.Comparison of glycolysis and oxidative phosphorylation as energy sources for mammalian sperm motility,using the combination of fluorescence imaging,laser tweezers,and real-time automated tracking and trapping[J].J Cell Physiol,2008,217(3):745-751.
[27]GIBB Z,AITKEN R J.The impact of sperm metabolism during in vitro storage:the stallion as a model[J].Biomed Res Int,2016,2016:9380609.
[28]THOMSON L K,FLEMING S D,AITKEN R J,et al.Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis[J].Hum Reprod,2009,24(9):2061-2070.
[29]AITKEN R J.Reactive oxygen species as mediators of sperm capacitation and pathological damage[J].Mol Reprod Dev,2017,84(10):1039-1052.
[30]ZHU Z D,FAN X T,LV Y H,et al.Vitamin E analogue improves rabbit sperm quality during the process of cryopreservation through its antioxidative action[J].PLoS One,2015,10(12):e0145383.
[31]DAVILA M P,MUNOZ P M,TAPIA J A,et al.Inhibition of mitochondrial complex I leads to decreased motility and membrane integrity related to increased hydrogen peroxide and reduced ATP production,while the inhibition of glycolysis has less impact on sperm motility[J].PLoS One,2015,10(9):e0138777.
[32]POTTS R J,NOTARIANNI L J,JEFFERIES T M,et al.Seminal plasma reduces exogenous oxidative damage to human sperm,determined by the measurement of DNA strand breaks and lipid peroxidation[J].Mutat Res,2000,447(2):249-256.
[2]MARTIN G,SABIDO O,DURAND P,et al.Cryopreservation induces an apoptosis-like mechanism in bull sperm[J].Biol Reprod,2004,71(1):28-37.
[3]KONYALI C,TOMAS C,BLANCH E,et al.Optimizing conditions for treating goat semen with cholesterol-loaded cyclodextrins prior to freezing to improve cryosurvival[J].Cryobiology,2013,67(2):124-131.
[4]AISEN E G,MEDINA V H,VENTURINO A.Cryopreservation and post-thawed fertility of ram semen frozen in different trehalose concentrations[J].Theriogenology,2002,57(7):1801-1808.
[5]SIEME H,OLDENHOF H,WOLKERS W F.Sperm membrane behaviour during cooling and cryopreservation[J].Reprod Domest Anim,2015,50(S3):20-26.
[6]SHAFIEI M,FOROUZANFAR M,HOSSEINI S M,et al.The effect of superoxide dismutase mimetic and catalase on the quality of postthawed goat semen[J].Theriogenology,2015,83(8):1321-1327.
[7]AMIDI F,PAZHOHAN A,SHABANI N M,et al.The role of antioxidants in sperm freezing:a review[J].Cell Tissue Bank,2016,17(4):745-756.
[8]PURDY P H.A review on goat sperm cryopreservation[J].Small Rumin Res,2006,63(3):215-225.
[9]NAING S W,WAHID H,AZAM K M,et al.Effect of sugars on characteristics of Boer goat semen after cryopreservation[J].Anim Reprod Sci,2010,122(1-2):23-28.
[10]RODRIGUEZ-GIL J E.Mammalian sperm energy resources management and survival during conservation in refrigeration[J].Reprod Domest Anim,2006,41(S2):11-20.
[11]邱建华.山羊精液液态保存过程中精子葡萄糖代谢的研究[D].泰安:山东农业大学,2011.QIU J H.Investigation of goat sperm metabolism during long term liquid storage of goat semen[D].Taian:Shandong Agricultural University,2011.(in Chinese)
[12]KAMP G,BUSSELMANN G,LAUTERWEIN J.Spermatozoa:models for studying regulatory aspects of energy metabolism[J].Experientia,1996,52(5):487-494.
[13]SCOTT T W,WHITE I G,ANNISON E F.Glucose and acetate metabolism by ram,bull,dog and fowl spermatozoa[J].Biochem J,1962,83(2):398-404.
[14]BARBAS J P,MASCARENHAS R D.Cryopreservation of domestic animal sperm cells[J].Cell Tissue Bank,2009,10(1):49-62.
[15]赵娜,甄林青,胡启蒙,等.超低温冷冻过程引起猪精子产生“似凋亡”变化[J].畜牧兽医学报,2013,44(11):1766-1774.ZHAO N,ZHEN L Q,HU Q M,et al.Cryopreservation procedure induces the alterations of“apoptosislike”of boar sperm[J].Acta Veterinaria et Zootechnica Sinica,2013,44(11):1766-1774.(in Chinese)
[16]朱士恩.家畜繁殖学[M].5版.北京:中国农业出版社,2011.ZHU S E.Reproduction in farm animals[M].5th ed.Beijing:China Agriculture Press,2011.(in Chinese)
[17]SALAMON S,RITAR A J.Deep freezing of Angora goat semen:effects of diluent composition and method and rate of dilution on survival of spermatozoa[J].Aust J Biol Sci,1982,35(3):295-303.
[18]WILLIAMS A C,FORD W C L.The role of glucose in supporting motility and capacitation in human spermatozoa[J].J Androl,2011,22(4):680-695.
[19]DU PLESSIS S S,AGARWAL A,MOHANTY G,et al.Oxidative phosphorylation versus glycolysis:what fuel do spermatozoa use?[J].Asian J Androl,2015,17(2):230-235.
[20]FERRAMOSCA A,ZARA V.Bioenergetics of mammalian sperm capacitation[J].Biomed Res Int,2014,2014:902953.
[21]RAMIO-LLUCH L,YESTE M,FERNANDEZ-NOVELL J M,et al.Oligomycin A-induced inhibition of mitochondrial ATP-synthase activity suppresses boar sperm motility and in vitro capacitation achievement without modifying overall sperm energy levels[J].Reprod Fertil Dev,2014,26(6):883-897.
[22]LOSANO J D A,ANGRIMANI D S R,DALMAZZO A,et al.Effect of mitochondrial uncoupling and glycolysis inhibition on ram sperm functionality[J].Reprod Domest Anim,2017,52(2):289-297.
[23]GALLOWAY C A,YOON Y.Perspectives on:SGP symposium on mitochondrial physiology and medicine:what comes first,misshape or dysfunction?The view from metabolic excess[J].J Gen Physiol,2012,139(6):455-463.
[24]MATSUZAKI M,MIZUSHIMA S,HIYAMA G,et al.Lactic acid is a sperm motility inactivation factor in the sperm storage tubules[J].Sci Rep,2015,5:17643.
[25]ST JOHN J C,BOWLES E J,AMARAL A.Sperm mitochondria and fertilisation[J].Soc Reprod Fertil Suppl,2007,65:399-416.
[26]NASCIMENTO J M,SHI L Z,TAM J,et al.Comparison of glycolysis and oxidative phosphorylation as energy sources for mammalian sperm motility,using the combination of fluorescence imaging,laser tweezers,and real-time automated tracking and trapping[J].J Cell Physiol,2008,217(3):745-751.
[27]GIBB Z,AITKEN R J.The impact of sperm metabolism during in vitro storage:the stallion as a model[J].Biomed Res Int,2016,2016:9380609.
[28]THOMSON L K,FLEMING S D,AITKEN R J,et al.Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis[J].Hum Reprod,2009,24(9):2061-2070.
[29]AITKEN R J.Reactive oxygen species as mediators of sperm capacitation and pathological damage[J].Mol Reprod Dev,2017,84(10):1039-1052.
[30]ZHU Z D,FAN X T,LV Y H,et al.Vitamin E analogue improves rabbit sperm quality during the process of cryopreservation through its antioxidative action[J].PLoS One,2015,10(12):e0145383.
[31]DAVILA M P,MUNOZ P M,TAPIA J A,et al.Inhibition of mitochondrial complex I leads to decreased motility and membrane integrity related to increased hydrogen peroxide and reduced ATP production,while the inhibition of glycolysis has less impact on sperm motility[J].PLoS One,2015,10(9):e0138777.
[32]POTTS R J,NOTARIANNI L J,JEFFERIES T M,et al.Seminal plasma reduces exogenous oxidative damage to human sperm,determined by the measurement of DNA strand breaks and lipid peroxidation[J].Mutat Res,2000,447(2):249-256.