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1 长期超低温保存后真鲷精子的质量变化陈亚坤 1, 2, 刘清华 1, 赵春彦 1, 肖志忠 1, 徐世宏 1, 马道远 1, 李军 1 (1.,, ; 2., ) 摘要 : 通过对精子运动率受精率和超微结构的观察, 研究了 2003~2009 年分 5 批冷冻保存的真鲷 (Pagrus major) 精子经 1~73 个月保存后质量变化情况冷冻精子在 40 水浴中解冻, 大约 100~110 s 转化为液态后取出, 用海水激活后观察精子运动率, 人工受精 6~8 h 后观察受精率, 同时精子用 2.5% 戊二醛前固定, 经处理后用扫描电镜和透射电镜观察精子超微结构结果表明, 冷冻精子的最高运动率 (87.67%±2.52%) 和受精率 (71.33%±8.84%) 都是 2009 年保存 1 个月的精子, 保存 1, 13, 26 个月的精子运动率和受精率差异不显著 (P<0.05), 保存 48 个月后的精子运动率和受精率显著低于保存 1 个月的精子 (P<0.05), 2003 年保存的精子 (73 个月 ) 运动率 (50.67%±5.31%) 和受精率最低 (39.56%±0.69%) 精子的超微结构损伤主要包括精子头部损伤, 线粒体损伤以及精子质膜的损伤关键词 : 低温保存 ; 真鲷 (Pagrus major); 精子 ; 运动率 ; 受精率中图分类号 : Q331 文献标识码 : A 文章编号 : (2010) , 1953 Blaxter [1] (Clupea harengus), 50,, 200, [2],,,, 200~ a [3],, 2 a, [4~6],, (Cyprinus carpio) 342 d 14 d [7] ; (Clarias gariepinus) 14 d 51%, 16 41% [8], [9~11] (Pagrus major),, 15%~18% (dimethylsulfoxide, DMSO) [12], 2003~ %DMSO 1~73,,, 1 材料和方法 1.1 亲鱼培养及配子采集 (4, 6 ; 3~4 kg, 10, 9~10 ) ( : 3 m 2.5 m 2 m), 16~18 3 5,, 0.003% 3~5min,,, : ; : : (Y Q) : (1982-),,,,, yakunchen@163.com;,, : , junli@qdio.ac.cn 50 / 2010 / 34 / 6

2 5, 90% , 100, 100, ( 95% ), 1.2 药品及仪器 DMSO SIGMA, Hanks (8 g/l NaCl, 0.4 g/ L KCl, 0.14 g/ L CaCl 2, 0.1 g/ L MgSO 4 7H 2 O, 0.1 g/ L MgCl 2 6H 2 O, 0.06 g/ L Na 2 HPO 4 12H 2 O, 0.06 g/ L KH 2 PO 4, 1g/ L, 0.35 g/ L NaHCO 3 ) Kryo ; Nikon-YS-100; YDS-30B 真鲷精子的冷冻保存 15%DMSO 1 3, ( 1.6 ml) 2 ml, : 0 5 min, 20 /min 0 150,, 3, 真鲷精子长期超低温保存后运动率观察和受精实验 2003~ ~73, ~110 s, 1 250, [12], 500 1,, 6~8 h,, ( / ) 真鲷精子超低温保存后超微结构变化 15%DMSO 2.5% (0.2 mol/l, ph=7.4, ) (scanning electron microscope, SEM), (0.2 mol/l, ph=7.4), 1%,,,, (KYKY-1000B) (transmission electron microscopy, TEM),, 1%,, Epon812,,, ( H-7000) 1.6 统计分析 SPSS 16.0 (SPSS Inc. Chicago, Illinois, USA) ±SD, One-Way ANOVA, SNK(Student-Newman-Keuls test) 2 结果 2.1 冷冻精子的运动率和受精率 2003~2009 5, 1 表 1 冷冻精子的运动率和受精率 Tab.1 Motility and fertilization rate of post-thaw sperm ( ) (%) (%) 2009 (1 ) 87.67±2.52 a 71.33±8.84 a 2008 (13 ) 86.67±2.08 a 69.22±1.02 a 2007 (26 ) 87.33±2.08 a 60.33±2.33 a 2005 (48 ) 75.33±1.53 b 47.22±3.89 b 2003 (73 ) 50.67±5.31 c 39.56±0.69 c : (P<0.05), , 1, 13, 26 (P>0.05), 48 1, 13, 26 (P<0.05), 2003 (73 ) 2.2 真鲷精子超低温保存后超微结构变化情况 1 2 Marine Sciences / Vol. 34, No. 6 /

1 ( 5μm) Fig. 1 The SEM photos of fresh and post-thaw sperm 1-1. ; 1-2. ; 1-3. ; 1-4. 1-1. fresh sperm; 1-2. sperm with broken head; 1-3.

,, ;2-3. ; 2-4., 2-1. fresh sperm, nu: nucleus; ne: nuclear envelope; mi: mitochondrion; pm: plasmalemma; 2-2.

3 1 ( 5μm) Fig. 1 The SEM photos of fresh and post-thaw sperm 1-1. ; 1-2. ; 1-3. ; fresh sperm; 1-2. sperm with broken head; 1-3. sperm with mitochondria flaking off; 1-4. sperm with membrane damage 2 ( 1 μm) Fig. 2 TEM photographs of fresh and post-thaw sperm 2-1, nu: ; ne: ; mi: ; pm: ; 2-2.,, ;2-3. ; 2-4., 2-1. fresh sperm, nu: nucleus; ne: nuclear envelope; mi: mitochondrion; pm: plasmalemma; 2-2. sperm with nuclear envelope and plasmalemma loss and mitochondria in the process of dissolving; 2-3. sperm with mitochondrial loss; 2-4. sperm with disappeared nuclear envelope and broken plasmalemma, 1.3~1.4 μm,, 3, ( ), : 52 / 2010 / 34 / 6

4 ( 1-2), ( ,2-3), ( ,2-4) 3 讨论 ( 196 ),,,, Suquet [4] (Psetta maxima) 9, 1a (Pseudosiaena crocea) [5] 1 1a (Siniperca chuatsi), [6], 2003~2009,, 1, 13, 26, 43 1, 13, 26, Kerby [13] (Morone saxatilis), 342 d 14d [7] d [8], Smith [5], 36,, [11] 3a (Penaeus monodon) [9], 30, 60, 90, 120, 150, 180, 210 d, 60 d, 90~120 d,,, Walters [14],, (reactive oxygen species, ROS) ROS [15] ROS,, [15~17],, ROS ROS,,,,,,,, ROS : [1] Blaxter J H S. Sperm storage and cross fertilization of Spring and Autumn spawning herring[j]. Nature, 1953, 172: [2] Gwo J C. Cryopreservation of sperm of some marine fishes[a].tiersch T R, Mazik P M. Cryopreservation in aquatic species[c]. Baton Rouge, Louisiana, USA: Wor1d Aquaculture Society, [3] Ashwood-Smith M J. Low temperature preservation of cells, tissues and organs[a]. Ashwood-Smith M J, Frrant J. Low temperature preservation in medicine and biology[c]. Turnbridge Well: Pitman Medical Itd, [4] Suquet M, Dranno C, Petton B, et al. Long-term effect of the cryopreservaton of turbot (Psetta maxima) spermatozoa [J]. Aquatic Living Resources, 1998, 11: [5], [J], 2002, 21: [6] Ding S Y, Ge J C, Hao C, et al. Long-term cryopreservation of sperm from Mandarin fish Siniperca chuatsi[j]. Animal Reproduction Science, 2009, 113: [7] Hisahi K, Reijiro H, Masakatsu T, et al. Cryopreservation of carp sperm [J]. Aquaculture, 1984, 37: [8] Steyn G J, Van Vuren J H J. The fertilizing capacity of cryopreserved sharptooth catfish (Clarias gariepinus) sperm[j]. Aquaculture, 1987, 63: [9] Vuthiphandchai V, Nimrat S, Kotcharat S, et al. Development of a cryopreservation protocol for long-term storage of black tiger shrimp (Penaeus monodon) spermatophores[j]. Theriogenology, 2007, 68: [10] Smith K D, Stultz D R, Steinberger E. Survival of Marine Sciences / Vol. 34, No. 6 /

5 spermatozoa in a human sperm bank. Effects of long-term storage in liquid nitrogen[j]. Journal of the American Medical Association, 1973, 223: 774. [11],. [J]., 2001, 21: [12] Liu Q H, Li J, Zhang S C, et al. An efficient methodology for cryopreservation of spermatozoa of red seabream, Pagrus major, with 2-mL Cryovials[J]. The World Aquaculture Society, 2006, 37: [13] Kerby J H. Cryogenic preservation of sperm from striped bass[j]. Transactions of the American Fisheries Society, 1983,112: [14] Walters C, Wheeler L, Stanwood P C. Longevity of cryogenically stored seeds[j]. Cryobiology, 2004, 48: [15] Kadirvel G, Kumar S, Kumaresan A. Lipid peroxidation, mitochondrial membrane potential and DNA integrity of spermatozoa in relation to intracellular reactive oxygen species in liquid and frozen-thawed buffalo semen[j]. Animal Reproduction Science, 2009, 114: [16] Numan-Bucak M, Atessahin A, Yüce A. Effect of anti-oxidants and oxidative stress parameters on ram semen after the freeze-thawing process[j]. Small Ruminant Research, 2008,75: [17] Numan-Bucak M, Sarıōzkan S, Barbaros-Tuncera P, et al. Effect of antioxidants on microscopic semen parameters, lipid peroxidation and antioxidant activities in Angora goat semen following cryopreservation[j]. Small Ruminant Research, 2009, 81: Quality change of Pagrus major sperm after long-term cryopreservation CHEN Ya-kun 1,2, LIU Qing-hua 1, ZHAO Chun-yan 1, XIAO Zhi-zhong 1, XU Shi-hong 1, Ma Dao-yuan 1, LI Jun 1 (1. Institute of Oceanology, the Chinese Academy of Sciences, Qingdao , China; 2. Graduate University, the Chinese Academy of Sciences, Beijing , China) Received: Mar., 22, 2010 Key words: cryopreservation; Pagrus major; sperm; motility; fertilization rate Abstract: After cryopreservation (1-73 months), the quality of Pagrus major sperm, which was cryopreserved from 2003 to 2009 in 5 batches, was examined by observing motility, fertilization rate and ultrastructures. The cryopreserved sperm were thawed in 40 o C water bath for 100~110 s. After activation with seawater, the motility of the sperm was estimated; 6-8 h after artificial fertilization, fertilization rate was examined; and after being fixed with by 2.5% glutaraldehyde, the ultrastructure of sperm was studied with scanning electron microscope and transmission electron microscope. The highest motility (87.67%±2.52%) and fertilization rate (71.33%± 8.84%) were obtained in sperm after a 1-month cryopreservation, even though they were not significantly different from those of the sperm after a 13-month or and 26-month cryopreservation (P>0.05). After a 48-month cryopreservation, the motility and fertilization rate decreased significantly in comparison with those of sperm after a 1-month cryopreservation (P <0.05). The lowest motility (50.67% ± 5.31%) and fertilization rate (39.56% ± 0.69%) were obtained in sperm cryopreserved for 73 months; main ultrastructural damage was observed in the head, mitochondria and membranes of sperm. ( 本文编辑 : 谭雪静 ) 54 / 2010 / 34 / 6

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NONE 中国农业大学学报 1999, 4(3): 109~113 Journal of China Agricultural University 小鼠桑棍胚玻璃化冷冻前后生物频谱处理对体外发青的影晌 1 朱士恩 2 王海彦曾申明王永胜张忠诚 ( 中国农业大学动物科技学院 ) 周林陈银 (