studies on the regeneration and reuse of spermatophore in ... · 4.3 the regeneration of...

学校编码：10384 分类号密级

学号：22420091151165 UDC

硕士学位论文

日本囊对虾精荚的多次形成和再利用的研究

Studies on the Regeneration and Reuse of

Spermatophore in Marsupenaeus japonicus

姚东明

指导教师姓名：林琼武高级工程师

专业名称：海洋生物学

论文提交日期：2 0 1 2 年 5 月

论文答辩时间：2 0 1 2 年 6 月

学位授予日期：2 0 1 2 年 6 月

答辩委员会主席：

评阅人：

2012 年 6 月

厦门大学博硕士论文摘要库

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目录

I

目录

摘要 .................................................................................................... IX

Abstract .................................................................................................. XI

第 1 章绪论 .........................................................................................1

1.1 本研究的目的和意义 .................................................................................................. 1

1.2 日本囊对虾养殖生物学简介 .................................................................................... 2

1.2.1 日本囊对虾的分类与分布 ..................................................................... 2

1.2.2 日本囊对虾的渔业生物学特征 ............................................................. 2

1.2.3 日本囊对虾在我国对虾养殖中的地位 ................................................. 4

1.3 国内外相关的研究动态 ............................................................................................. 5

1.3.1 对虾精荚相关研究 ................................................................................. 5

1.3.2 对虾人工授精技术研究 ....................................................................... 10

1.3.3 精荚冷冻保存的研究 ........................................................................... 12

1.4 本研究的主要内容和思路 ....................................................................................... 14

1.4.1 本研究的主要内容 ............................................................................... 14

1.4.2 本研究的研究思路（技术路线图） ................................................... 15

第 2 章材料方法 ................................................................................. 17

2.1 实验材料....................................................................................................................... 17

2.1.1 亲虾来源及运输 ................................................................................... 17

2.1.2 亲虾驯养 ............................................................................................... 17

2.1.3 实验主要使用的仪器与试剂 ............................................................... 18

2.2 实验与方法 ................................................................................................................. 19

2.2.1 实验设置 ............................................................................................... 19

2.2.2 取样、样本处理及测定 ....................................................................... 23

2.2.3 几个参数的说明 ................................................................................... 24

2.2.4 数据处理 ............................................................................................... 25



II

第 3 章结果 ....................................................................................... 27

3.1 日本囊对虾精荚若干主要参数及其之间的相关性分析 ................................ 27

3.2 日本囊对虾精子和卵子在海水中不同时间的存活率 ..................................... 28

3.3 日本囊对虾精荚的多次形成 .................................................................................. 29

3.3.1 日本囊对虾精荚多次形成的次数和时间间隔 ................................... 29

3.3.2 日本囊对虾不同批次再生精荚质量的差异比较 ............................... 31

3.4 日本囊对虾精荚的再利用 ....................................................................................... 32

3.4.1 日本囊对虾死亡和蜕壳精荚再利用的可行性 ................................... 32

3.4.2 日本囊对虾四种不同授精方法的实验观测 ....................................... 34

3.4.3 日本囊对虾刚死亡雌虾体内精荚的低温保存 ................................... 35

3.4.4 日本囊对虾雄虾精荚的低温短期保存 ............................................... 36

第 4 章讨论 ....................................................................................... 41

4.1 对虾精荚有关参数及其之间的相关性 ................................................................ 41

4.1.1 对虾个体大小与精荚质量相关性 ....................................................... 41

4.1.2 雌雄亲虾体长与体重的相关性 ........................................................... 42

4.2 对虾体外人工授精过程精子和卵子在海水中相遇的最佳时段................... 43

4.3 对虾精荚的多次形成 ................................................................................................ 44

4.3.1 对虾精荚多次形成的次数和时间间隔 ............................................... 44

4.3.2 对虾再生精荚质量 ............................................................................... 44

4.3.3 不同的精荚获取方法对亲虾精荚再生能力的影响 ........................... 46

4.3.4 不同的精荚获取方法对亲虾健康及受精率的影响 ........................... 47

4.4 死亡和蜕壳雌虾精荚再利用的可行性 ................................................................ 50

4.5 对虾体外不同人工授精方法不同产生差异的问题 ......................................... 51

4.6 雌虾自然产卵法和湿法体外人工授精的应用 .................................................. 53

4.7 虾蟹精荚的冷冻保存 ................................................................................................ 54

4.7.1 不同保存液对虾蟹精荚低温保存效果的影响 ................................... 54

4.7.2 抗生素在虾蟹精荚低温保存过程的作用 ........................................... 55

第 5 章结语 ....................................................................................... 57


目录

III

5.1 本研究的主要成果 .................................................................................................... 57

5.2 本研究的创新和特色 ................................................................................................ 58

5.3 不足之处....................................................................................................................... 58

5.4 展望 ................................................................................................................................ 59

参考文献 ................................................................................................. 61

在学期间参与的科研活动和发表的文章 ............................................. 73

致谢 ..................................................................................................... 75



IV


Contents

V

Contents

Abstract(in Chinese) ............................................................................... IX

Abstract(in English) ............................................................................... XI

Chapter 1. Introduction .............................................................................1

1.1 The purpose and significance of the research ................................................... 1

1.2 Introduction of aquaculture biology of Marsupenaeus japonicus .................... 2

1.2.1 Classification and natural distribution of Marsupenaeus japonicus ....... 2

1.2.2 Fishery biological characteristics of Marsupenaeus japonicus .............. 2

1.2.3 General situation in Marsupenaeus japonicus cultivation ...................... 4

1.3 Related research of domestic and foreign ......................................................... 5

1.3.1 The research about spermatophores in shrimps and crabs ...................... 5

1.3.2 The research about the technique of artificial insemination of shrimps10

1.3.3 The research about the storage of the spermatophores in shirmps and

crabs ............................................................................................................... 12

1.4 The main contents and research idea of the present study .............................. 14

1.4.1 The main contents of the present study ................................................. 14

1.4.2 The research ideas of the present study (technical roadmap) ............... 15

Chapter 2.Materials and Methods ........................................................... 17

2.1 Materials ......................................................................................................... 17

2.1.1 Source and transportation of the broodstock ........................................ 17

2.1.2 Broodstock domestication ..................................................................... 17

2.1.3 The main equipments and reagents used in the present study .............. 18

2.2 Methods ........................................................................................................... 19

2.2.1 Experiment design ................................................................................ 19

2.2.2 Sampling, sample processing and test .................................................. 22

2.2.3 Several parameters explanation ............................................................ 23



VI

2.2.4 Data processing ..................................................................................... 24

Chapter 3. Results ................................................................................... 27

3.1 The main parameters of spermatophore in Marsupenaeus japonicas and their

correlation analysis ............................................................................................... 27

3.2 The survival rates of the sperms and eggs of Marsupenaeus japonicas at

different time in sea water ..................................................................................... 28

3.3 The regeneration of spermatophore in Marsupenaeus japonicus ................... 29

3.3.1 Frequency and time interval of spermatophore regeneration in

Marsupenaeus japonicus ............................................................................... 29

3.3.2 The differences in the quality between different batches of

regenerating spermatophore in Marsupenaeus japonicus .............................. 31

3.4 The reuse of spermatophores in Marsupenaeus japonicus ............................. 32

3.4.1 The feasibility of re-use of spermatophores from the death and

moulting female broodstock, Marsupenaeus japonicas ................................ 32

3.4.2. Experimental observation of four different methods of artificial

insemination in vitro ...................................................................................... 34

3.4.3 Cryopreservation of spermatophores in the just death female shrimps,

Marsupenaeus japonicus ............................................................................... 35

3.4.4 Cryopreservation of spermatophore in male shrimps, Marsupenaeus

japonicus ........................................................................................................ 36

Chapter 4. Discussion .............................................................................. 41

4.1 Correlations between the parameters of spermatophores in shrimps .............. 41

4.1.1 Correlations between the individual size and spermatophore quality .. 41

4.1.2 Correlations between the body length and weight of male and female

parent shrimps ................................................................................................ 42

4.2 The best meeting time of sperm and egg of shrimps in sea water during

artificial insemination in vitro ............................................................................... 43

4.3 The Regeneration of spermatophores in shrimps ............................................ 44

4.3.1 The frequency and time interval of regeneration of spermatophore in


Contents

VII

shrimps ........................................................................................................... 44

4.3.2 The quality of regeneration spermatophore in shrimps ........................ 44

4.3.3 The influence of different methods taking spermatophore on the

regeneration ability of spermatophore in shrimps ......................................... 46

4.3.4 The influence of different methods taking spermatophore on the health

of male shrimps and fertilization rate of artificial insemination in vitro ... 47

4.4 The feasibility of re-use of spermatophores from the death and moulting

female shrimps ...................................................................................................... 50

4.5 The problems about differences by different methods of artificial

insemination in vitro ............................................................................................. 51

4.6 The applications of artificial insemination in vitro by female shrimp

spawning and wet insemination ............................................................................ 54

4.7 The cryopreservation of spermatophores in shrimps and crabs ...................... 54

4.7.1 The influence of different preserving fluids on the effects of the

cryopreservation of spermatophores in shrimps and crabs ............................ 54

4.7.2 The role of antibiotics in the process of cryopreservation of

spermatophores in shrimps and crabs ............................................................ 55

Chapter 5. Epilogue ................................................................................. 57

5.1 The main results of the present research ......................................................... 57

5.2 Innovation and characteristic of the present research ..................................... 58

5.3 Shortage of the present research ..................................................................... 58

5.4 Prospects of the present research .................................................................... 59

References ............................................................................................... 61

Research activities and paper published during the learning .................. 73

Acknowledgement ................................................................................. 75



VIII


摘要

IX

摘要

日本囊对虾是我国对虾养殖重要经济种类。本研究采用自然生态学、实验

生态学、形态学、海水养殖学和个体标记等方法和技术,研究了日本囊对虾雄虾

精荚再生的次数、时间间隔、质量差异、以及亲虾群体精荚再生能力的分布，

死亡和蜕壳雌虾精荚中精子数量、存活率和受精力，不同处理的体外人工授精

方法和死亡雌虾精荚和雄虾精荚的低温短期保存。主要结果如下：

1、在水温 21±2℃，盐度 32±1，PH8.1±0.2 的条件下，雄亲虾精荚可再生 3

次，平均为 2.125 次；精荚再生的时间间隔为 5～7d，不同批次无显著差异

（P>0.05）；不同批次再生的精荚在重量、精子数量、精子合格率和受精力呈下

降趋势，差异较为显著（P<0.05）。日本囊对虾雄亲虾群体精荚多次形成的能力

分布为：100%个体 1 次，87.5%个体 2 次，80.6%个体 3 次，44.4%个体 4 次；

体长或体重与精荚重量之间的相关性显著（P<0.01），与精荚中精子合格率、受

精力和精子数量之间的相关性不显著（P＞0.05）；精子离体在海水中 30min 存

活率达 80%以上，随后快速下降，4h 后仅有 20%，卵子在海水中持续 1min 内

受精效果最好。

2、雄虾精荚精子总数为 1.10±0.23×108，精子存活率为 87.99±6.81%；蜕壳

雌虾中精荚精子总数为 0.04±0.05×108，精子存活率为 58.73±1.32%，授精能力

为 37.65±13.08%；刚死亡雌虾中精荚精子总数为 1.45±1.88×108，精子存活率为

80.96±1.60%，授精能力为 52.65±7.52%，低温保存 7d 后的授精能力为

42.76±9.96%；湿法授精是较优的体外人工授精方法，受精率为 58.68±14.78%。

分析认为废旧精荚和蜕壳后雌虾是可以再利用的，刚死亡雌虾体中精荚在精子

数量和质量均优于蜕壳雌虾中的精荚，经蜕壳的雌虾的产卵力优于初产雌虾。

3、不同保存液对精荚的保存效果有极显著地差异（P<0.01），液体石蜡中

外部形态保持得最好。精子存活率随着保存时间的增加而极显著地降低

（P<0.01）。4 种保存液的使用效果比较，液体石蜡中保存的精荚的精子寿命较

长并且有较高的存活率，林格氏溶液次之，而磷酸盐缓冲液和 0.85%NaCl 溶液

保存对虾精荚的效果欠佳。在含抗生素的液体石蜡中保存的精荚的精子存活率



X

比在不含抗生素的液体石蜡中的要稍高一些，但差异并不明显。

关键词：日本囊对虾；精荚；多次形成；再利用；人工授精


Abstract

XI

Abstract

Marsupenaeus japonicus is an important species of shrimp in China’s shrimp farming.

This research, conducted under an integrated method of natural ecology, experimental

ecology, morphology, marine aquaculture and individual markings , was aimed at an

investigation into the times, time intervals, quality and times distribution in the

population of spermatophore regeneration of male shrimps, M. japonicus；the sperm

count, the survival rate and the fertilization rate of spermatophores from death and

molting females；artificial insemination methods in vitro with different treatments；

cryopreservation of spermatophores from just death female shrimps and male shrimps.

The main results were as follows:

Ⅰ. Under the conditions of water temperature of 21±2℃, salinity of 32±1, and

PH8.1±0.2, the spermatophore of the male broodstock had 3 times multi formation

with an average of 2.125 times; the interval is ranged from 5 days to 7 days, different

batches had no significant differences (P> 0.05); different batches of regenerated

spermatophore had a downward trend in weight, sperm count, sperm fertilation rate,

the difference was notable (P <0.05). The distribution of spermatophore regeneration

times：all of the broodstock could generate one time，87.5% two times, 80.6% three

times and 44.4% four times. More than 80% of sperm survived in the seawater for

30 minutes, then declined rapidly, and four hours later only 20% remained; the

perfect time for eggs to fertilize was within one minute.

Ⅱ. The sperm quantity and sperm viability of male shrimp were 1.1.±0.23×108,

87.99±6.81%, respectively；The sperm quantity, sperm viability and fertilization

ability of moulting female shrimp were 0.04±0.05×108, 58.73±1.32%,

37.65±13.08%, respectively; The sperm quantity, sperm viability, fertilization ability,

fertilization ability after 7day’s cryogenic preservation of dead female shrimp were

1.45±1.88×108, 80.96±1.60%, 52.65±7.52%, 42.76±9.96% respectively; wet in-vitro

fertilization was the best method for in-vitro artificial insemination with its fertility

of 58.68±14.78%. In summary, the spent spermatophore and molting females could



XII

be re-used, spermatophore in the just death female shrimp were better than moulting

females in sperm quantity and quality of spermatophore, and after molting the

fecundity of the shrimp was better than primiparous females.

III. M. japonicus spermatophore in the cryopreservation experiments in different

liquid paraffin had significantly different results (P <0.01), the external characters of

spermatophore stored in morphology remains the best. Sperm viability was very

significantly reduced with the increase of retention time (P <0.01). The results

showed that, the sperm in spermatophore of M. japonicus stored in liquid paraffin

live longer and had higher survival rates, followed by Ringer's solution, and the

effect of phosphate buffer and 0.85% NaCl solution were not good enough. The

survival rate of spermatophore sperm stored in liquid paraffin containing antibiotics

was slightly higher than in antibiotic-free liquid paraffin, but the difference was not

obvious. Key words: Marsupenaeus japonicas; spermatophore; regeneration; reuse; artificial

insemination


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