| 超低温冷冻对俄罗斯鲟精子顶体酶活性及DNA损伤的影响 |
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| 引用本文： | 黄晓荣,张涛,冯广朋,赵峰,刘鉴毅,王妤,章龙珍,庄平.超低温冷冻对俄罗斯鲟精子顶体酶活性及DNA损伤的影响[J].海洋渔业,2016(5):487-494. |
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| 作者姓名： | 黄晓荣 张涛 冯广朋 赵峰 刘鉴毅 王妤 章龙珍 庄平 |
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| 作者单位： | 中国水产科学研究院东海水产研究所，农业部东海与远洋渔业资源开发利用重点实验室，上海 200090 |
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| 基金项目： | 公益性行业(农业)科研专项经费项目(201203065)；国家重大科技成果转化项目(ZD －2012－345－2)；国家科技基础条件平台项目 |
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| 摘 要： | 选取6 ind健康的雄性俄罗斯鲟(Acipenser gueldenstaedti),经人工催产后获得成熟的精子,研究超低温冷冻(-196℃)对俄罗斯鲟精子顶体酶活性及DNA损伤影响。结果显示:俄罗斯鲟鲜精中顶体酶的平均活性为(36.18±2.54)μIU·10-6,经过超低温冷冻后,精子顶体酶活性显著降低,添加抗冻保护液精子中顶体酶活性降至(21.55±0.79)μIU·10-6,未添加抗冻保护液精子中顶体酶活性降至(9.58±1.08)μIU·10-6,且三者间有显著性差异(P0.05)。单细胞凝胶电泳结果表明,俄罗斯鲟鲜精彗星率为(37.33±7.77)%,添加抗冻剂后冻精的彗星率为(63.67±5.13)%,未添加抗冻剂直接冷冻彗星率高达(86.00±3.61)%,三者间有显著差异(P0.05)。用CASP分析软件分析测量彗星拖尾长度(L tail)、彗星尾部DNA的相对含量(Tail DNA)、尾动量(TM)、Olive尾动量(OTM)等各项表征DNA损伤的指标,发现冻精组的各项指标均显著高于鲜精组,未添加抗冻剂直接冷冻组又高于添加抗冻剂组,3组间有显著性差异(P0.05)。本研究结果表明:超低温冷冻能导致精子顶体酶活性下降和DNA损伤,抗冻剂对精子具有保护作用。
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| 关 键 词： | 超低温冷冻 俄罗斯鲟 精子 顶体酶活性 DNA损伤 |
Effects of cryopreservation on acrosin activity and DNA damage of Russian sturgeon(Acipenser gueldenstaedti)semen |
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| Abstract: | Purpose:The study was conducted about the effects of cryopreservation on acrosin activity and DNA damage of Acipenser gueldenstaedti semen,with a view to providing references for the protection of germplasm resources and improvement of germplasm breeding of Russian sturgeon. Materials:The parents of Acipenser gueldenstaedti were collected from Qiandao lake,Zhejiang Province, P.R.China and the males were 7 aged.The average length ranged 93.6 ~108.7 cm and average weight ranged 4.27 ~6.18 kg.6 mature males were selected to extract non-polluted spermatozoa through gohopore. The sperm motility was examined by microscope,and samples above 90% of sperm motility were used for this experiment. Methods:The collected semen were randomly divided into 3 groups,and they are as follows (1)Fresh semen,namely control group;(2)original semen was diluted by 1∶1 with 23.4 mMsucrose +0.25 mMKCl+30 mMTris (pH 8.0)and addition 10% glycol as cryoprotectants and then mixtures were placed in 250 μL plastic centrifuge tube,and then samples were equilibrated for 20 min at 4 ℃,then 1 min at -20 ℃, fimally preserved in liquid nitrogen;(3)semen without diluent and cryoprotectant.The samples were placed into 250μL plastic centrifuge tube,and equilibrated for 20 min at 4 ℃,then 1 min at -20 ℃,and finally preserved in liquid nitrogen.The samples were thawed in 38 ℃ bath before determination.A portion of the samples from three groups were used to determine the acrosin activity and the rest samples were used to detect the DNA damage.The acrosin activity was determined by spectrophotometric method,and the DNA damage was studied by single cell gel electrophoresis method. Results:The average acrosin activity in fresh semen of A.gueldenstaedti was (36.18 ±2.54)μIU·10 -6 After cryopreservation,The acrosin activity declined significantly.The activity of frozen semen which cryoprotectants had been added declined to (21.55 ±0.79)μIU·10 -6 ,while the activity of frozen semen which cryoprotectants had not been added declined to (9.58 ±1.08)μIU·10 -6 .There were significant differences among the three groups(P <0.05).The results of single cell gel electrophoresis(SCGE)showed, the comet rate of fresh semen was (37.33 ±7.77 )%,while the comet rate of frozen semen which cryoprotectants had been added was (63.67 ±5.13 )%,and the comet rate of frozen semen which cryoprotectants had not been added was (86.00 ±3.61 )%.There were significant differences among the three groups(P <0.05).DNA damage indexes including L-tail,Tail DNA,TMand OTMwere analyzed with CASP software.The results showed that various indexes in frozen semen were higher than those of fresh semen and the indexes in the group without cryoprotectants were higher than those of the group with cryoprotectants. There were significant differences among the three groups(P <0.05). Conclusions:Cryopreservation could induce the decline of acrosin activity and DNA damage,but cryoprotectant could protect the semen in cryopreservation. |
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| Keywords: | cryopreservation Acipenser gueldenstaedti semen acrosin activity DNA damage |
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