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1 脊尾白虾精子体外保存的研究 1, 2, 2, 2, 1, 2 (1., ; 2., ) 摘要 : 采用精子存活率和顶体酶活力两种评价精子质量方法, 研究了不同稀释剂与抗冻保护剂对脊尾白虾 (Exopalaemon carinicauda) 精子体外保存效果的影响结果表明 : 稀释剂 Ⅰ- 壬氏液为脊尾白虾精子体外保存的最佳稀释剂, 4 下保存 4 d 后, 稀释剂 Ⅰ 组精子存活率为 %, 顶体酶活力为 μ IU/10 6, 两种指标值均高于其他实验组, 各组精子存活率与顶体酶活力之间相关性显著 γ=0.846(p<0.05); 在液氮 -196 下保存 2 d 后, 稀释剂 Ⅰ 组精子存活率达 %, 顶体酶活力为 μiu/10 6, 两种指标值均高于其他各组以及 4 下稀释剂 Ⅰ 组同期保存效果, 各组精子存活率与顶体酶活力之间相关性极显著, γ=0.862 (P<0.01) 15%DMSO 为脊尾白虾精子超低温保存的最佳抗冻保护剂, 此条件下超低温保存 2 d 后, 精子存活率为 81.32%, 顶体酶活力为 1.385μIU/10 6, 各组精子存活率与顶体酶活力之间相关性极显著, 相关系数 γ=0.864(p<0.01) 关键词 : 脊尾白虾 (Exopalaemon carinicauda); 精子 ; 体外保存 ; 稀释剂 ; 抗冻剂中图分类号 : Q 文献标识码 : A 文章编号 : (2013) (Exopalaemon carinicauda), (Crustacea), (Malacostraca), (Eumalacostraca), (Decapoda), (Palaemonidae),, 3,,,,,, (Fenneropenaeus chinensis)(penaeus japonicus),, [1] [2-3] [3] [4-6] [5-6] [7],,,,,,,, 1 材料与方法 1.1 脊尾白虾来源及精子的采集 ,, ( )cm, ( )g,, (: NaCl g, NaOH 0.19 g, KCl 1.12 g, MgSO 4 7H 2 O 4.93 g, H 3 BO g, ml),, 200, r/min 5 min,, 主要试剂 Ficoll : 0.12 mol/l NaCl, mol N-2 -N-2- (Hepes) (Ficoll), ph % Ficoll : mol/l NaCl, mol/l : ; : : (2011BAD13B03) : (1987-),,,,, puyunhui@yahoo.com.cn;,, yanbinlun@yahoo.com.cn Marine Sciences / Vol. 37, No. 3 /

佳稀释剂, 4 下保存 4 d 后, 稀释剂 Ⅰ 组精子存活率为 71.50 %, 顶体酶活力为 1.803 μ IU/10 6, 两种指标值均高于其他实验组, 各组精子存活率与顶体酶活力之间相关性显著 γ=0.846(p<0.

2 Hepes 0.01 Triton X-100, ph 8.0, 0.1% 500 mmol 2 : 23 mmol/l Nα- -DL- -P- (BAPNA), (DMSO), : BAPNA/DMSO 9: 1 : 1 表 1 稀释剂成分 Tab. 1 The composition of diluting solutions ( ml) : [8] NaCl 7.8 g, KCl 0.2 g, CaCl g, NaHCO g : Kurokura extender [9] NaCl 7.5 g, KCl 0.2 g, CaCl g, NaHCO g : D-20 [10] NaCl 8.0 g, KCl 1.0 g, 15 g [11] [12] [13] [14] NaCl g, NaOH 0.19g, KCl 1.12 g, MgSO4 7H 2 O 4.93 g, H 3 BO g NaCl 25.0 g, Na 2 SO g, KCl 0.37 g, MgCl2 6H 2 O 10.7g, NaHCO g NaCl 25.0, KCl 0.6 g, CaCl g, MgCl g, NaHCO g NaCl 8.7 g, KCl 7.2 g, CaCl g, MgCl2 0.1 g, NaHCO g, NaH 2 PO g, 1g 1.3 方法 , /ml, 4, d , 10%DMSO,,, d 1.3.3, DMSO, 10 : ~ (V/ V) 5% 10% 15% 20%; ~ (Dimethyl sulfoxide, DMSO) (V / V) 5% 10% 15% 20%; DMSO 10%; DMSO 20%, 4 30 min, :, 40, ,, 2% B () 3~4 min,,, 200, 4, 50, Kennedy [15], (1) 1.5 ml, 250 L; (2), 0.5 m L Ficoll 1.5 ml, Ficoll ; (3), r/min 30 min; (4) Ficoll, 100 L Ficoll ; (5) 100 L, ; (6)( ) 1 ml, ; (7) 25 3 h, ; (8) 3 h, 100 μl ( ); (9) r/min 30 min, ; (10) 410 nm ; (11) :, 25 1 mol Nα- -DL- -P- (Nα-Benzoyl- DL-arginine-P-nitroanilide hydrochloride, BAPNA) /min : IU /10 6 = 平均 ( OD实验 - OD 对照 ) 10 6 {1485 精子数 (10 )} 6 96 / 2013 / 37 / 3

63 g, NaOH 0.19g, KCl 1.12 g, MgSO4 7H 2 O 4.93 g, H 3 BO 3 0.53 g NaCl 25.0 g, Na 2 SO 4 3.9 g, KCl 0.37 g, MgCl2 6H 2 O 10.7g, NaHCO 3 0.23 g NaCl 25.0, KCl 0.6 g, CaCl 2 0.25 g, MgCl2 0.

3 1.4 数据处理 3,, SPSS16.0, (One-way ANOVA), LSD(Least Significant Difference) Duncan 2 结果 2.1 不同稀释剂对精子低温保存的影响 4 d, %,, 4 30 min,, 85%,, 89.63%,, 76.86%, 1 2 d,,,, 4 d, 71.50%,,,, 41.71% 表 2 不同稀释剂对低温保存的脊尾白虾精子存活率的影响 Tab. 2 Survival rate of spermatozoa of Exopalaemon carinicauda under hypothermic preservation with different diluting solutions (%) 93.18± ± ± ± ± ± ± min (%) e b cd c de cde a 1 d (%) f b b c e d a 2 d (%) e a a b d c a 3 d (%) f b c d e d a 4 d (%) f b c c e d a (%) : (One-way ANOVA) Duncan, (P<0.05), 3 4 1,, 4 30 min, Ⅰ 1 Fig.1 Acrosin activities of spermatozoa of Exopalaemon carinicauda under hypothermic preservation with different diluting solutions, Ⅴ Ⅵ, Ⅶ 4 d, Ⅰ, μiu/10 6, 2 1,,,, Ⅴ, Ⅶ4 d,, γ=0.846 (P<0.05) 2.2 稀释剂对精子超低温保存的影响 %,, 4,, 85%,, 90.23%,, 86.53%, 1 d, 2 d, Marine Sciences / Vol. 37, No. 3 /

18±0.46 93.18±0.46 93.18±0.46 93.18±0.46 93.18±0.46 93.18±0.46 93.18±0.46 4 30 min (%) 89.63 0.62 e 82.11 1.12 b 87.74 0.70 cd 87.16 0.69 c 88.95 0.40 de 88.42 0.69 cde 76.86 1.63 a 1 d (%) 82.85 0.

4 , 83.50%,, 55.66%,,,, 18.63% 4 表 3 不同稀释剂对超低温保存的脊尾白虾精子存活率的影响 Tab. 3 Survival rate of spermatozoa of Exopalaemon carinicauda under cryopreservation with different diluting solutions (%) (%) a b ab ab b b b ab 1 d (%) a c c c c bc bc b 2 d (%) a c b b b b b b (%) ,, 4 30 min, Ⅰ, 3.760μIU/10 6, Ⅴ Ⅵ, Ⅶ 2 d, Ⅰ, 2.507μIU/10 6,, 0.919μIU/ Fig. 2 Acrosin activities of spermatozoa of Exopalaemon carinicauda under cryopreservation with different dilution solutions 3 2,,, Ⅶ, 4, γ=0.862 (P<0.01) 2.3 抗冻剂对精子超低温保存的影响 %,, 1 d,,, 85.11%, 2 d,,, 81.32%,, 42.84% 4,,, 20%, DMSO 5% 15%,, 15%, 20% 10% 10%DMSO 3 30 min,,, 1 d,,,, 2 d 1 d, IU/ ,, DMSO,,,, γ=0.864(p<0.01) 98 / 2013 / 37 / 3

54 0.60 (%) 83.23 4.38 a 90.23 1.39 b 87.37 1.49 ab 87.61 2.76 ab 88.09 1.93 b 89.16 2.60 b 88.44 1.46 b 86.53 1.35 ab 1 d (%) 73.45 2.92 a 87.52 1.62 c 86.52 1.84 c 85.07 1.55 c 85.37 4.48 c 83.05.4.86 bc 81.

5 表 4 不同抗冻剂对低温保存的脊尾白虾精子存活率的影响 Tab. 4 Survival rate of spermatozoa of Exopalaemon carinicauda under cryopreservation with different cryoprotectants (%) (%) a a bc d b 86.59±0. 30 bc 87.93±0. 68 cd 89.78±0. 78 d bc bc a 1d (%) a b d d c de e f d de b 2d (%) a b d d c de e f d de b (%) Fig. 3 Acrosin activities of spermatozoa of Exopalaemon carinicauda under cryopreservation with different cryoprotectants 3 讨论 3.1 精子保存质量的评价方法 [16],, [17-19],, 2% B,,,,,,,,, 3.2 稀释剂对精子保存效果的影响 [17,19-21], 7, ph Na + K + Ca 2+ Mg 2+, K + Na + [22] K + Ca 2+ K + Na +, Ca 2+ [23-24] [25] 7 (),, 3 NaCl KCl NaHCO 3 3,,, K +,,,,, [23-24] D-20() Marine Sciences / Vol. 37, No. 3 /

63 c 80.56 0.82 de 81.18 2.93 e 85.11 0.78 f 77.81 1.22 d 80.23 1.03 de 62.23 1.39 b 2d (%) 42.84 0.63 a 56.40 1.22 b 73.84 0.61 d 74.59 0.90 d 71.08 1.19 c 76.45 1.45 de 77.81 0.66 e 81.32 2.01 f 71.

6 ,,,,, D-20() 3.3 抗冻保护剂对精子保存效果的影响 Chow [26] 10%, [27] 6%DMSO [20] 10%DMSO 5%~10%, 60%,, 59%, DMSO DMSO,,, ;, [28] DMSO, DMSO, DMSO DMSO (0~60 ) [29] [30] DMSO DMSO, 35, DMSO,,,, 40, DMSO,, 15%DMSO, 20%,,,, : [1],,,. [J]., 2010, 34 (10): [2],,,. [J]., 2004, 25(2): [3]. Exopalaemon carinicauda(holthuis) [J]., 2005, 20(5): [4],,,. [J]., 2008, 28(4): [5],,. [J]., 2010, 38(7): , [6],,. [J]., 2006, 27(3): [7],,,. [J]., 2008, 25(5): 1-3. [8] Chen S L, Ji X S, Yu G C, et al. Cryopreservation of spermatozoa from turbot ( Scophthalmus maximus ) and application to large-scale fertilization[j]. Aquaculture, 2004, 236: [9] Routray P, Choudhary A K, Dash S N, et al. Cryopreservation of dead fish spermatozoa several hours after death of Indian major carp, Labeo rohita and its successful utilization in fish production[j]. Aquaculture, 2006, 261: [10] Maria A N, Viveiros A T M, Freitas R T F, et al. Extenders and cryoprotectants for cooling and freezing of piracanjuba (Bryconor bignyamus)semen,an endangered Brazilian teleost fish[j]. Aquaculture, 2006, 260: [11] Leung T J R, Lawrence A L. Observation on the decline in sperm quality of Penaeus setiferus under laboratory conditions[j]. Aquaculture, 1987, 65 : [12] Tereza G C V, Halccrow K. Influence of some external factors on the acrosome reation in the spermatozoa of Homarus americanus Milne Edwards 1837[J]. Journal of Crustacean Biology, 1988, 8(3): [13] Chao N H. Cryopreservation of finfish and shellfish sperms[j]. Taiwan Fisheries Research, 1996, 4(2): [14]. [J]., 1999, 19(4) : [15] Kennedy W P, Kaminski J M, Van der Ven H H, et al. A simple, clinical assay to evaluate the acrosine activity of human spermatozoa[j]. Journal of Andrology, 1989, 10: [16],. [J]., 1997, 21(1): [17] Anchordoguy T J, Crogwe J H, Griffin F J, et al. Cryopreservation of sperm from the marine shrimp Sicyonia ingentis[j]. Cryobiology, 1988, 25: [18] Wang Q Y, Misamore M, Jiang C Q, et al. Egg water induced reaction and biostain assay of spermfrom marine shrimp Penaeus vannamer :dietary effects on sperm quality[j]. Journal of the World Aquaculture Society, 1995, 26: [19] Bhavanishankar S, Subramoniam T. Cryopreservation of spermatozoa of the edible mud crab Scylla serrata (Forskal)[J]. Journal of Experimental Zoology, 1997, 277: / 2013 / 37 / 3

20%,,,, : [1],,,. [J]., 2010, 34 (10): 53-56. [2],,,. [J]., 2004, 25(2): 39-40. [3]. Exopalaemon carinicauda(holthuis) [J]., 2005, 20(5): 23-26. [4],,,. [J]., 2008, 28(4): 34-37. [5],,. [J]., 2010, 38(7): 3512-3515, 3544.

7 [20],. [J]., 1996, 27(2): [21] Bray W A, Lawrence A L. Male viability determinations in Penaeus vannamei: evaluation of short-term storage of spermatophores up to 36h and comparison of Ca 2+ -free saline and seawater as sperm homogenate media[j]. Aquaculture, 1998, 160: [22],,,. [J]., 1992, 2: [23],,. [J]., 2004, 39(1): [24],,,. 鱥 [J]., 2004, 24(1): 7-8. [25],,,. [J]. ( ), 2007, 4: [26] Chow S, Tam Y, Ogasawara Y. Cryopreservation of spermatophore of the fresh water shrimp, Macrobrachium rosenbergii[j]. Biol Bul, 1985, 168: [27],,,. [J]., 1992, 92: 3-7. [28],,,. [J]., 1994, 18(4): [29]. [J]., 1987, 39(1): [30],,. [J]., 1987, 5: Study on in vitro preservation of spermatozoa of Exopalaemon carinicauda PU Yun-hui 1,2, XU Xing-hong 2, GAO Huan 2, LIU Zhao-pu 1, YAN Bin-lun 2 (1. College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing , China; 2. Jiangsu Key Laboratory of Marine Biotechnology, Huaihai Institute of Technology, Lianyungang , China) Received: Dec., 20, 2012 Key words: Exopalaemon carinicauda; spermatozoa; in vitro preservation; diluting solution; cryoprotectants Abstract: In this paper, the effect of different diluting solutions and cryoprotectants on in vitro cryopreservation of spermatozoa was studied by evaluation of spermatozoa quality using survival rate and acrosin activity assays. The results showed that Ringer s extender was the best diluting solution of spermatozoa in vitro preservation. After a preservation for 4 d at 4 with Ringer s extender, the survival rate was 71.50%, and the acrosin activity reduced to 1.803μIU/10 6. Both of these two values were higher than those of other experimental groups. The survival rate was significantly correlated with acrosin activity in all the experimental groups, R=0.846 (P<0.05). After a preservation for 2 d in liquid nitrogen with Ringer s extender, the survival rate was 83.50% and the acrosin activity reduced to 2.507μIU/10 6. Similarly, both of them were higher than other groups, as well as the values obtained at 4. There existed a highly significant correlation between survival rate and acrosin activity, R=0.862 (P<0.01). 15% DMSO was the best cryoprotectant for cryopreservation. After a 2 d preservation, the survival rate was 81.32%, and the acrosin activity reduced to 1.385μIU/10 6. There existed a highly significant correlation between survival rate and acrosin activity, R=0.864 (P<0.01). ( 本文编辑 : 谭雪静 ) Marine Sciences / Vol. 37, No. 3 /

Aquaculture, 1998, 160: 63-67. [22],,,. [J]., 1992, 2: 10-12. [23],,. [J]., 2004, 39(1): 16-20. [24],,,. 鱥 [J]., 2004, 24(1): 7-8. [25],,,. [J]. ( ), 2007, 4: 86-94. [26] Chow S, Tam Y, Ogasawara Y.

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32期中国农学通报 2014,30(32):38-42 Chinese Agricultural Science Bulletin 中草药饲料添加剂对黄鳝性激素含量的影响 1,2 1,2 1,2 2 赵庆凯, 丁炜东, 邴旭文, 王建新 ( 1 南京农业大学无锡渔业学院, 江苏无锡 214081; 2 中国水产