温度和溶解氧对金乌贼精子能量代谢和活性的影响
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摘要
为了解金乌贼(Sepiaesculenta)精子对外界环境变化的响应和存活机制,分析测定了不同温度(16℃、20℃、24℃)和不同溶解氧(1 mg/L、3 mg/L、6 mg/L)条件下保存36 h后精荚能量物质含量、糖脂代谢关键酶活性以及精子激活后的活力变化。结果显示,温度和溶氧处理36h后精荚糖原和甘油三酯含量均显著低于未处理组(P<0.05),但蛋白质含量与未处理组无显著差异(P>0.05)。当温度从16℃升高至24℃时,精荚糖原含量逐渐下降,甘油三酯的含量则先降后稳;己糖激酶(HK)、乳酸脱氢酶(LDH)、丙酮酸脱氢酶(PDH)和琥珀酸脱氢酶(SDH)活性随温度升高而升高,脂肪酶(LIP)活性先升后稳;精子激活初期运动速度和不运动率随温度升高无显著变化,而快速运动率则先稳后降。当海水溶解氧从1 mg/L升至6 mg/L时,精荚糖原含量逐渐下降,甘油三酯含量先稳后降; HK、PDH、SDH活性随溶解氧上升呈逐渐上升趋势, LDH先升后降, LIP则先稳后升;精子激活初期运动速度和快速运动率随溶解氧升高呈先升后稳的变化趋势,而不运动率则呈先降后稳趋势。上述结果表明,精荚中的糖原和甘油三酯是精子的主要供能物质,精子较低的能量物质消耗为其长期存活提供了保障,低温和低氧环境可抑制精子的耗能,更利于其在精荚中长期存活。
In order to study the response and survival mechanism of spermatozoa in the external environment, the contents of energy substances, the activities of key glycolipid metabolizing enzymes in the spermatophore, and sperm motility after a 36-h preservation were examined under different temperatures(16℃, 20℃, 24℃) and dissolved oxygen levels(1 mg/L, 3 mg/L, 6 mg/L). The results showed that the glycogen and triglyceride contents in the spermatophore after the 36-h treatment were significantly lower than those before the 36-h treatment, but the protein content showed no significant differences before and after the 36-h treatment. When the water temperature increased from 16℃ to 24℃, the glycogen content decreased, while the triglyceride content first decreased and then stabilized. The activities of hexokinase(HK), lactate dehydrogenase(LDH), pyruvate dehydrogenase(PDH), and succinate dehydrogenase(SDH) increased when the water temperature increased, while the activity of lipase(LIP) first increased and then stabilized. The initial velocity and immotile rate after activation had no significant changes, but the progressive motility rate was first stable and then decreased when the water temperature increased. When the dissolved oxygen content increased from 1 mg/L to 6 mg/L, the glycogen content decreased, while the triglyceride content was first stable and then decreased. The activities of HK, PDH, and SDH increased, while the activity of LDH first increased and then decreased when the dissolved oxygen content increased. The activity of LIP was first stable and then increased, while the initial velocity and progressive motility rate after activation first increased and then stabilized, while the immotile rate first decreased and then stabilized. These results suggested that glycogen and triglycerides in the spermatophore were the main energy substances of the spermatozoa. Low energy substance consumption was the foundation for the long-term survival of the sperm. Low temperature and hypoxia, which reduced the energy consumption of the spermatozoa, were beneficial for its long-term survival.
In order to study the response and survival mechanism of spermatozoa in the external environment, the contents of energy substances, the activities of key glycolipid metabolizing enzymes in the spermatophore, and sperm motility after a 36-h preservation were examined under different temperatures(16℃, 20℃, 24℃) and dissolved oxygen levels(1 mg/L, 3 mg/L, 6 mg/L). The results showed that the glycogen and triglyceride contents in the spermatophore after the 36-h treatment were significantly lower than those before the 36-h treatment, but the protein content showed no significant differences before and after the 36-h treatment. When the water temperature increased from 16℃ to 24℃, the glycogen content decreased, while the triglyceride content first decreased and then stabilized. The activities of hexokinase(HK), lactate dehydrogenase(LDH), pyruvate dehydrogenase(PDH), and succinate dehydrogenase(SDH) increased when the water temperature increased, while the activity of lipase(LIP) first increased and then stabilized. The initial velocity and immotile rate after activation had no significant changes, but the progressive motility rate was first stable and then decreased when the water temperature increased. When the dissolved oxygen content increased from 1 mg/L to 6 mg/L, the glycogen content decreased, while the triglyceride content was first stable and then decreased. The activities of HK, PDH, and SDH increased, while the activity of LDH first increased and then decreased when the dissolved oxygen content increased. The activity of LIP was first stable and then increased, while the initial velocity and progressive motility rate after activation first increased and then stabilized, while the immotile rate first decreased and then stabilized. These results suggested that glycogen and triglycerides in the spermatophore were the main energy substances of the spermatozoa. Low energy substance consumption was the foundation for the long-term survival of the sperm. Low temperature and hypoxia, which reduced the energy consumption of the spermatozoa, were beneficial for its long-term survival.
引文
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[4]Wang Q,Zhao Y L,Chen L Q.Biochemical composition and sperm metabolism in the reproductive system of the male,Eriocheir sinensis[J].Journal of Fisheries of China,2002,26(5):411-416.[王群,赵云龙,陈立侨.中华绒螯蟹雄性生殖系统生化组成及精子代谢[J].水产学报,2002,26(5):411-416.]
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[8]Gomendio M,Roldan E R S.Implications of diversity in sperm size and function for sperm competition and fertility[J].The International Journal of Developmental Biology,2008,52(5):439-447.
[9]Cosson J,Groison A L,Suquet M,et al.Marine fish spermatozoa:racing ephemeral swimmers[J].Reproduction,2008,136(3):277-294.
[10]Lahnsteiner F,Patzner R A,Weismann T.Energy resources of spermatozoa of the rainbow trout Oncorhynchus mykiss(Pisces,Teleostei)[J].Reproduction Nutrition Development,1993,33(4):349-360.
[11]Lahnsteiner F,Berger B,Weismann T,et al.Determination of semen quality of the rainbow trout(Oncorhynchus mykiss)by sperm motility,seminal plasma parameters,and spermatozoal metabolism[J].Aquaculture,1998,163(1-2):163-181.
[12]Alavi S M H,Cosson J.Sperm motility in fishes.I.Effects of temperature and pH:a review[J].Cell Biology International,2005,29(2):101-110.
[13]Dziewulska K,Domaga?a J.Effect of pH and cation concentrations on spermatozoan motility of sea trout(Salmo trutta m.trutta L.)[J].Theriogenology,2013,79(1):48-58.
[14]Lahnsteiner F,Mansour N.The effect of temperature on sperm motility and enzymatic activity in brown trout Salmo trutta,burbot Lota lota and grayling Thymallus thymallus[J].Journal of Fish Biology,2012,81(1):197-209.
[15]Morisawa M,Suzuki K,Morisawa S.Effects of potassium and osmolality on spermatozoan motility of salmonid fishes[J].Journal of Experimental Biology,1983,107(11):105-113.
[16]Cosson J,Groison A L,Fauvel C,et al.Description of hake(Merlucius merlucius)spermatozoa:flagellar wave characteristics and motility parameters in various situations[J].Journal of Applied Ichthyology,2010,26(5):644-652.
[17]Wada T,Takegaki T,Mori T,et al.Sperm displacement behavior of the cuttlefish Sepia esculenta(Cephalopoda:Sepiidae)[J].Journal of Ethology,2005,23(2):85-92.
[18]Wang L.Reproductive behavior and reproductive strategy of Sepia esculenta[D].Qingdao:Ocean University of China,2016:38-41.[王亮.金乌贼繁殖行为和繁殖策略研究[D].青岛:中国海洋大学,2016:38-41.]
[19]Lahnsteiner F,Berger B,Weismann T,et al.Motility of spermatozoa of Alburnus alburnus(Cyprinidae)and its relationship to seminal plasma composition and sperm metabolism[J].Fish Physiology and Biochemistry,1996,15(2):167-179.
[20]Wang Q,Zhao Y L,Chen L Q.Review of male nutrition reproduction and sperm metabolism,in the decapod crustacea[J].Chinese Journal of Zoology,2002,37(5):78-82.[王群,赵云龙,陈立侨.虾蟹精子发育的营养及代谢研究概况[J].动物学杂志,2002,37(5):78-82.]
[21]Adiyodi K G,Adiyodi R G.Reproductive Biology of Invertebrates.Vol.Ⅱ:Spermatogenesis and Sperm Function[M].New York:Wiley,1983:692-718.
[22]Jeyalectumie C,Subramoniam T.Biochemistry of seminal secretions of the crab Scylla serrata with reference to sperm metabolism and storage in the female[J].Molecular Reproduction and Development,1991,30(1):44-55.
[23]Anilkumar G,Sudha K,Anitha E,et al.Aspects of sperm metabolism in the spermatheca of the brachyuran crab Metopograpsus messor(Forskal)[J].Journal of Crustacean Biology,1996,16(2):310-314.
[24]Lahnsteiner F,Caberlotto S.Motility of gilthead seabream Sparus aurata spermatozoa and its relation to temperature,energy metabolism and oxidative stress[J].Aquaculture,2012,370-371:76-83.
[25]Hirohashi N,Tamura-Nakano M,Nakaya F,et al.Sneaker male squid produce long-lived spermatozoa by modulating their energy metabolism[J].Journal of Biological Chemistry,2016,291(37):19324-19334.
[26]Ru X S,Gao T X,Liu S L,et al.Effects of temperature on digestive and metabolic enzymes activities of sea cucumber(Apostichopus japonicus)broodstock[J].Marine Sciences,2015,39(3):1-6.[茹小尚,高天翔,刘石林,等.温度对刺参繁殖期消化酶和代谢酶活力的影响[J].海洋科学,2015,39(3):1-6.]
[27]Zheng D B,Pan L Q,Jin C X.The effect of dissolved oxygen on hemocyanin,osmolality and ion-transport enzyme activities of shrimp Litopenaeus vannamei[J].Periodical of Ocean University of China,2010,40(11):35-38.[郑德斌,潘鲁青,金彩霞.溶解氧对凡纳滨对虾血淋巴血蓝蛋白、渗透压和鳃丝离子转运酶活力的影响[J].中国海洋大学学报(自然科学版),2010,40(11):35-38.]
[28]Wang C L,Wu D H,Dong T Y,et al.Oxygen consumption rate and effects of hypoxia stress on enzyme activities of Sepiella japonica[J].Chinese Journal of Applied Ecology,2008,19(11):2420-2427.[王春琳,吴丹华,董天野,等.曼氏无针乌贼耗氧率及溶氧胁迫对其体内酶活力的影响[J].应用生态学报,2008,19(11):2420-2427.]
[29]Islam M S,Akhter T.Tale of fish sperm and factors affecting sperm motility:a review[J].Advances in Life Sciences,2011,1(1):11-19.
[30]Lahnsteiner F.Spermatozoa of the teleost fish Perca fluviatilis(perch)have the ability to swim for more than two hours in saline solutions[J].Aquaculture,2011,314(1-4):221-224.
[31]Williot P,Kopeika E F,Goncharov B F.Influence of testis state,temperature and delay in semen collection on spermatozoa motility in the cultured siberian sturgeon(Acipenser baeri Brandt)[J].Aquaculture,2000,189(1-2):53-61.
[32]Caldwell G S,Fitzer S,Gillespie C S,et al.Ocean acidification takes sperm back in time[J].Invertebrate Reproduction&Development,2011,55(4):217-221.
[33]Romagosa E,Souza B E,Sanches E A,et al.Sperm motility of Prochilodus lineatus in relation to dilution rate and temperature of the activating medium[J].Journal of Applied Ichthyology,2010,26(5):678-681.
[34]Mehlis M,Bakker T C M.The influence of ambient water temperature on sperm performance and fertilization success in three-spined sticklebacks(Gasterosteus aculeatus)[J].Evolutionary Ecology,2014,28(4):655-667.
[35]Levitan D R.The importance of sperm limitation to the evolution of egg size in marine invertebrates[J].The American Naturalist,1993,141(4):517-536.
[36]Levitan D R.Sperm Limitation,Gamete Competition,and Sexual Selection in External Fertilizers[M].San Diego:Academic Press,1998:173-215.
[37]Krzyzosiak J,Molanb P,Vishwanatha R.Measurements of Bovine sperm velocities under true anaerobic and aerobic conditions[J].Animal Reproduction Science,1999,55(3-4):163-173.
[38]Naud M,Havenhand J N.Sperm motility and longevity in the giant cuttlefish,Sepia apama(Mollusca:Cephalopoda)[J].Marine Biology,2006,148(3):559-566.
[39]Austin C R,Lutwak-Mann C,Mann T.Spermatophores and spermatozoa of the squid Loligo pealii[J].Proceedings of the Royal Society,1964,161(983):143-152.
[2]Qi Z Y.Economic Mollusca of China[M].Beijing:China Agriculture Press,1998:293-294.[齐钟彦.中国经济软体动物[M].北京:中国农业出版社,1998:293-294.]
[3]Zheng Y J,Chen X Z,Cheng J H.Biological Resources and Environment of the Continental Shelf in the East China Sea[M].Shanghai:Shanghai Scientific&Technical Publishers,2003:722-727.[郑元甲,陈雪忠,程家骅.东海大陆架生物资源与环境[M].上海:上海科学技术出版社,2003:722-727.]
[4]Wang Q,Zhao Y L,Chen L Q.Biochemical composition and sperm metabolism in the reproductive system of the male,Eriocheir sinensis[J].Journal of Fisheries of China,2002,26(5):411-416.[王群,赵云龙,陈立侨.中华绒螯蟹雄性生殖系统生化组成及精子代谢[J].水产学报,2002,26(5):411-416.]
[5]Han S,Zheng X D,Li Q,et al.Ultrastructure of Sepia esculenta(Cephalopoda:Sepiida)spermatozoa and its damage by low temperature[J].Oceanologia et Limnologia Sinica,2010,41(4):483-488.[韩松,郑小东,李琪,等.金乌贼(Sepia esculenta)精子超微结构和低温损伤电镜观察[J].海洋与湖沼,2010,41(4):483-488.]
[6]Liu C L,Zou J,Liu J G,et al.Ultrastructure of the spermatozoon of Sepia esculenta Hoyle[J].Progress in Fishery Sciences,2011,32(3):1-6.[刘长琳,邹健,刘君刚,等.金乌贼精子的超微结构[J].渔业科学进展,2011,32(3):1-6.]
[7]Lahnsteiner F,Berger B,Weismann T,et al.Physiological and biochemical determination of rainbow trout,Oncorhynchus mykiss,semen quality for cryopreservation[J].Journal of Applied Aquaculture,1996,6(4):47-73.
[8]Gomendio M,Roldan E R S.Implications of diversity in sperm size and function for sperm competition and fertility[J].The International Journal of Developmental Biology,2008,52(5):439-447.
[9]Cosson J,Groison A L,Suquet M,et al.Marine fish spermatozoa:racing ephemeral swimmers[J].Reproduction,2008,136(3):277-294.
[10]Lahnsteiner F,Patzner R A,Weismann T.Energy resources of spermatozoa of the rainbow trout Oncorhynchus mykiss(Pisces,Teleostei)[J].Reproduction Nutrition Development,1993,33(4):349-360.
[11]Lahnsteiner F,Berger B,Weismann T,et al.Determination of semen quality of the rainbow trout(Oncorhynchus mykiss)by sperm motility,seminal plasma parameters,and spermatozoal metabolism[J].Aquaculture,1998,163(1-2):163-181.
[12]Alavi S M H,Cosson J.Sperm motility in fishes.I.Effects of temperature and pH:a review[J].Cell Biology International,2005,29(2):101-110.
[13]Dziewulska K,Domaga?a J.Effect of pH and cation concentrations on spermatozoan motility of sea trout(Salmo trutta m.trutta L.)[J].Theriogenology,2013,79(1):48-58.
[14]Lahnsteiner F,Mansour N.The effect of temperature on sperm motility and enzymatic activity in brown trout Salmo trutta,burbot Lota lota and grayling Thymallus thymallus[J].Journal of Fish Biology,2012,81(1):197-209.
[15]Morisawa M,Suzuki K,Morisawa S.Effects of potassium and osmolality on spermatozoan motility of salmonid fishes[J].Journal of Experimental Biology,1983,107(11):105-113.
[16]Cosson J,Groison A L,Fauvel C,et al.Description of hake(Merlucius merlucius)spermatozoa:flagellar wave characteristics and motility parameters in various situations[J].Journal of Applied Ichthyology,2010,26(5):644-652.
[17]Wada T,Takegaki T,Mori T,et al.Sperm displacement behavior of the cuttlefish Sepia esculenta(Cephalopoda:Sepiidae)[J].Journal of Ethology,2005,23(2):85-92.
[18]Wang L.Reproductive behavior and reproductive strategy of Sepia esculenta[D].Qingdao:Ocean University of China,2016:38-41.[王亮.金乌贼繁殖行为和繁殖策略研究[D].青岛:中国海洋大学,2016:38-41.]
[19]Lahnsteiner F,Berger B,Weismann T,et al.Motility of spermatozoa of Alburnus alburnus(Cyprinidae)and its relationship to seminal plasma composition and sperm metabolism[J].Fish Physiology and Biochemistry,1996,15(2):167-179.
[20]Wang Q,Zhao Y L,Chen L Q.Review of male nutrition reproduction and sperm metabolism,in the decapod crustacea[J].Chinese Journal of Zoology,2002,37(5):78-82.[王群,赵云龙,陈立侨.虾蟹精子发育的营养及代谢研究概况[J].动物学杂志,2002,37(5):78-82.]
[21]Adiyodi K G,Adiyodi R G.Reproductive Biology of Invertebrates.Vol.Ⅱ:Spermatogenesis and Sperm Function[M].New York:Wiley,1983:692-718.
[22]Jeyalectumie C,Subramoniam T.Biochemistry of seminal secretions of the crab Scylla serrata with reference to sperm metabolism and storage in the female[J].Molecular Reproduction and Development,1991,30(1):44-55.
[23]Anilkumar G,Sudha K,Anitha E,et al.Aspects of sperm metabolism in the spermatheca of the brachyuran crab Metopograpsus messor(Forskal)[J].Journal of Crustacean Biology,1996,16(2):310-314.
[24]Lahnsteiner F,Caberlotto S.Motility of gilthead seabream Sparus aurata spermatozoa and its relation to temperature,energy metabolism and oxidative stress[J].Aquaculture,2012,370-371:76-83.
[25]Hirohashi N,Tamura-Nakano M,Nakaya F,et al.Sneaker male squid produce long-lived spermatozoa by modulating their energy metabolism[J].Journal of Biological Chemistry,2016,291(37):19324-19334.
[26]Ru X S,Gao T X,Liu S L,et al.Effects of temperature on digestive and metabolic enzymes activities of sea cucumber(Apostichopus japonicus)broodstock[J].Marine Sciences,2015,39(3):1-6.[茹小尚,高天翔,刘石林,等.温度对刺参繁殖期消化酶和代谢酶活力的影响[J].海洋科学,2015,39(3):1-6.]
[27]Zheng D B,Pan L Q,Jin C X.The effect of dissolved oxygen on hemocyanin,osmolality and ion-transport enzyme activities of shrimp Litopenaeus vannamei[J].Periodical of Ocean University of China,2010,40(11):35-38.[郑德斌,潘鲁青,金彩霞.溶解氧对凡纳滨对虾血淋巴血蓝蛋白、渗透压和鳃丝离子转运酶活力的影响[J].中国海洋大学学报(自然科学版),2010,40(11):35-38.]
[28]Wang C L,Wu D H,Dong T Y,et al.Oxygen consumption rate and effects of hypoxia stress on enzyme activities of Sepiella japonica[J].Chinese Journal of Applied Ecology,2008,19(11):2420-2427.[王春琳,吴丹华,董天野,等.曼氏无针乌贼耗氧率及溶氧胁迫对其体内酶活力的影响[J].应用生态学报,2008,19(11):2420-2427.]
[29]Islam M S,Akhter T.Tale of fish sperm and factors affecting sperm motility:a review[J].Advances in Life Sciences,2011,1(1):11-19.
[30]Lahnsteiner F.Spermatozoa of the teleost fish Perca fluviatilis(perch)have the ability to swim for more than two hours in saline solutions[J].Aquaculture,2011,314(1-4):221-224.
[31]Williot P,Kopeika E F,Goncharov B F.Influence of testis state,temperature and delay in semen collection on spermatozoa motility in the cultured siberian sturgeon(Acipenser baeri Brandt)[J].Aquaculture,2000,189(1-2):53-61.
[32]Caldwell G S,Fitzer S,Gillespie C S,et al.Ocean acidification takes sperm back in time[J].Invertebrate Reproduction&Development,2011,55(4):217-221.
[33]Romagosa E,Souza B E,Sanches E A,et al.Sperm motility of Prochilodus lineatus in relation to dilution rate and temperature of the activating medium[J].Journal of Applied Ichthyology,2010,26(5):678-681.
[34]Mehlis M,Bakker T C M.The influence of ambient water temperature on sperm performance and fertilization success in three-spined sticklebacks(Gasterosteus aculeatus)[J].Evolutionary Ecology,2014,28(4):655-667.
[35]Levitan D R.The importance of sperm limitation to the evolution of egg size in marine invertebrates[J].The American Naturalist,1993,141(4):517-536.
[36]Levitan D R.Sperm Limitation,Gamete Competition,and Sexual Selection in External Fertilizers[M].San Diego:Academic Press,1998:173-215.
[37]Krzyzosiak J,Molanb P,Vishwanatha R.Measurements of Bovine sperm velocities under true anaerobic and aerobic conditions[J].Animal Reproduction Science,1999,55(3-4):163-173.
[38]Naud M,Havenhand J N.Sperm motility and longevity in the giant cuttlefish,Sepia apama(Mollusca:Cephalopoda)[J].Marine Biology,2006,148(3):559-566.
[39]Austin C R,Lutwak-Mann C,Mann T.Spermatophores and spermatozoa of the squid Loligo pealii[J].Proceedings of the Royal Society,1964,161(983):143-152.