【作者】 储张杰；

【导师】 龚世园；

【作者基本信息】 华中农业大学 ， 水产养殖， 2008， 博士

【摘要】 黄鳝（Monopterus albus Zuiew）,俗称鳝鱼、田鳗和长鱼等,它是一种淡水硬骨鱼类,广泛分布于中国、印度、马来西亚和印度尼西亚。已在中国中南部开展大面积的养殖。中国目前的养殖总产量已经达到18万吨,由于其生长快,成活率高以及对网箱养殖条件的适应性好,它被当作中国水产养殖的首选品种之一。鳝肉质细嫩,鲜美可口,营养价值高,具有滋补强身和药用功能,是人们喜爱的滋补水产品,深受中国消费者的青睐。然而,中国的黄鳝养殖还停留在初级阶段,养殖依然依赖捕捞野生苗种,随着养殖规模和捕捞强度的加大,野生苗种资源急剧下降,成功地解决黄鳝的人工繁殖技术将成为发展其规模养殖的先决条件。但黄鳝具有特殊的性逆转现象:它是一种雌雄同体鱼类,在其生命的早期发育阶段为雌性,产卵后变为间性个体,然后进入雄性阶段,在进行黄鳝苗种人工繁育过程中,存在的主要问题是黄鳝具有性逆转的特性,即刚成熟的个体为雌性,但随着黄鳝的生长和繁殖,又逐步转为雄性,使得在黄鳝繁殖群体中,雌性个体小,怀卵量少,繁殖力很低;亲鱼选配时,雌雄鳝的比例和大小还难以搭配协调,亲鳝难选,繁殖数量难以提高,极大地限制了黄鳝人工繁殖规模和水平,必须进行黄鳝性逆转的研究。本研究主要是通过营养、外源性激素以及环境因子等,使用组织学、细胞学、内分泌学和放射免疫学等相关学科知识,探讨黄鳝性逆转的人工调控技术,希望能够找到减少性逆转比例的途径,延长性逆转时间,大幅度提高黄鳝的怀卵量及繁殖力,解决黄鳝苗种的规模化繁殖的关键问题,而黄鳝性逆转调控研究所取得的进展,必将给黄鳝的人工繁殖和养殖带来新的突破,促进我国黄鳝养殖业的可持续发展。1.黄鳝体长、体重、年龄与性比和怀卵量关系在湖北省汉川市汈汊湖水域,随机采取12个月的黄鳝样品,总数258尾黄鳝,雌性152尾,占58.91%;间性44尾,占17.05%;雄性62尾,占24.04%。在总体黄鳝标本中,其体长范围为9.4-76.8cm,其中雌性体长为9.4-60.5cm,平均体长为39.6cm,20cm以下全部为雌性;雌雄间体体长为27.1-63.3cm,平均体长为44.5cm;雄性体长范围为29.4-76.8cm,平均体长为51.63cm,其中70cm以上全部为雄性。雌性分布最多的区域是20-50cm,占雌性总数的93.42%,雌雄间体分布最多的区域是30-50cm,占间体总数的72.73%。雄性分布最多的区域是40cm以上,占雄性总数的75.81%。结果表明:在汉川市汈汊湖水域体长25cm左右是黄鳝性逆转的起点。其体重范围为0.48-753.60g,其中雌性体重为250g以下,平均体重为58.46g,23.70g以下全部为雌性;雌雄间体体重范围25.40g-206.30g,平均体重为76.45g;雄性体重范围为29.65-753.60g,平均体重为144.38g,其中250g以上全部为雄性。雌性分布最多的区域是20-100g,占雌性总数的75.00%,雌雄间体分布最多的区域是40-100g,占间体总数的70.45%。雄性分布最多的区域是100g以上,占雄性总数的72.58%。结果表明在汈汊湖水域体重25g左右是黄鳝性逆转的起点。其年龄范围为0-Ⅶ龄,其中Ⅱ龄以下雌性占92.17%,Ⅰ龄以下全部为雌性;雌雄间体年龄范围Ⅱ-Ⅴ龄,Ⅲ-Ⅳ龄占间体总数的70.45%,Ⅲ-Ⅳ龄为黄鳝性逆转的高峰期;雄性年龄范围为Ⅱ-Ⅶ龄,Ⅱ龄以下雄性只占其年龄段总数的2.00%,Ⅳ-Ⅶ龄占雄性总数的77.42%,其中Ⅵ龄以上全部为雄性。结果表明:黄鳝的性逆转的起点时间应该略早于Ⅱ周年。黄鳝怀卵量范围是41-1267粒/尾,20-30cm的体长段怀卵率最高,随着体长的增长,黄鳝的怀卵率急剧下降,绝对怀卵量以30-40cm体长段的黄鳝较高,平均怀卵强度随着体长的增长而增长。由于大个体黄鳝怀卵率较少,但平均怀卵强度较大,如:60-70cm之间,怀卵率只有2.5%,但最大个体怀卵量达到1267粒/尾。2.饲料中不同蛋白质含量对黄鳝性逆转的影响本实验从营养的角度探讨了不同的饲料蛋白水平（25%、35%、45%、55%）对雌性黄鳝性腺发育及性逆转的影响,试验鱼体长26.5±2.6cm,体重21.93±2.05g,试验鱼都为雌性（在繁殖季节挑选）研究发现黄鳝的性腺发育与饲料蛋白水平密切相关。在适宜的蛋白质水平的范围内,黄鳝的怀卵量随着饲料蛋白水平的增加而升高,蛋白质25%的试验组黄鳝的平均怀卵量最低,只有390.67±58.71粒/尾,而45%的试验组黄鳝的平均怀卵量最高,达到1331.67±287.77粒/尾;但55%（663.00±71.86）试验组的怀卵量与35%试验组（736.33±49.52）的怀卵量无显著性差异。通过长时间投喂不同蛋白质组饲料对黄鳝的性逆转比例也存在较大的差异,25%的蛋白组转为雄性和间体的比例分别为38.65±1.34%,17.61±1.47%;35%的蛋白组转为雄性和间体的比例分别为24.15±6.55%,11.20±1.45%;而45%的蛋白组只有7.61±1.43%和5.21±1.25%的黄鳝转为雄性和雌雄间体,有87.18±1.21%的黄鳝仍然保持雌性。55%的蛋白组转为雄性和间体的比例分别为14.21±5.52%,7.18±0.96%,其他三组雌性个体比例都少于45%的蛋白组,说明45%的蛋白质饲料能较好地维持黄鳝的雌性发育。通过对黄鳝血清雌二醇（E₂）影响的研究表明:随着蛋白质水平的升高（在45%蛋白质含量范围内）,雌鳝个体的E₂水平显著升高,其中蛋白质45%的饲料组各月的E₂水平最高。产卵前三个月蛋白质35%、45%的饲料组E₂水平显著高于25%、55%饲料组,在6月四组的E₂水平达到最大值分别为435.05±46.15pg/ml、539.76±29.26pg/ml、944.5±449.25pg/ml、526.72±60.93 pg/ml,说明适宜的饲料蛋白质含量能够维持较高的黄鳝血清E₂的分泌,而较高的E₂水平是维持雌性发育的先决条件。试验表明:饲料蛋白含量（45%）能够较好地维持黄鳝的雌性发育,提高黄鳝的怀卵量,并且能够在一定程度上延缓黄鳝的性逆转。3饲料中不同脂肪含量对黄鳝性逆转的影响用鱼油作为脂肪源,按照添加后脂肪占饲料重量的5%、10%、15%的比例投喂黄鳝（蛋白质都是35%）12个月,结果表明:10%的鱼油添加量能够维持较高的E₂的分泌,15%脂肪水平能促进黄鳝血清睾酮的分泌,5%脂肪水平能使睾酮值维持较低的水平。较高的饲料脂肪含量能使黄鳝的肝脏指数迅速升高,而4、5、6三个月的肝脏指数的降低,说明黄鳝的性腺发育在卵黄积累和卵细胞成熟阶段,需要大量动用肝脏中储存的营养物质和能量。15%的脂肪组转为雄性和间体的比例分别为30.52±3.66%,12.31±1.05%;10%的脂肪组转为雄性和间体的比例分别为26.46±2.12%,9.18±3.06%;而5%的脂肪组转为雄性和间体的比例分别为13.24±1.25%,6.37±1.64%,有80.39±1.65%的黄鳝仍然保持雌性。说明在蛋白质相同的情况下,随着饲料脂肪含量的升高黄鳝的性逆转程度上升,较低的脂肪饲料（5%）能较好地维持黄鳝的雌性发育。脂肪15%的试验组黄鳝的平均怀卵量最低,只有391.27±47.61粒/尾,而5%的试验组黄鳝的平均怀卵量最高,达到875.39±143.50粒/尾,但10%试验组的怀卵量与15%试验组的怀卵量无显著性差异,说明过高的脂肪含量对提高黄鳝的怀卵量是十分不利的,5%左右的饲料脂肪含量能够较好地维持黄鳝的雌性发育,并能够相应地提高雌鳝的怀卵量。4.外源丙酸睾酮对黄鳝性腺发育及性逆转的影响饲料中添加0mg/kg、10mg/kg和30mg/kg丙酸睾酮,投喂黄鳝12个月,结果表明:30mg/kg中有18.51%的雌鳝转变为雄黄鳝,高于0mg/kg、10mg/kg组14.81%的转雄比例,但三组的雄性和雌雄间体并无显著性差异,三组保持雌性的比例分别为74.07%、70.37%和70.37%,说明在本实验条件下,不同的丙酸睾酮对雌性黄鳝的性逆转无显著的影响。5.外源戊酸雌二醇对黄鳝性逆转的影响本试验研究了外源戊酸雌二醇对雌性黄鳝性类固醇激素的分泌以及延缓黄鳝性逆转的影响,试验鱼体长26.5±2.6cm,体重21.93±2.05g,它们都为雌性,用含不同浓度（0mg/kg,10 mg/kg,50 mg/kg）的戊酸雌二醇饲料投喂试验鱼12个月,试验鱼喂养在固定于池塘中的网箱（2m×1m×1.5m）。结果表明:黄鳝的性腺发育对不同的外源戊酸雌二醇反应存在季节性差异,不同的戊酸雌二醇对黄鳝性类固醇激素的分泌也存在差异,低浓度（10 mg/kg）的戊酸雌二醇对黄鳝的性逆转存在一定的抑制作用,但仍然存在性逆转个体,雌雄间体和雄性个体分别为12.00±1.25%、10.05±0.85%。高浓度的戊酸雌二醇能促进血清雌二醇的分泌,降低睾酮的分泌,2龄试验鱼在经历12个月体重达到168.81±42.81g时仍然保持雌性,而对照组雌雄间体和雄性个体分别为16.90±1.35%,28.75±1.65%。并且已经性逆转的雄性个体的精巢发育完善,试验表明较高浓度（50 mg/kg）的戊酸雌二醇能延缓黄鳝的性逆转。6.外源复方炔诺酮对黄鳝生长及性逆转的影响饲料中添加0片/kg、10片/kg和30片/kg复方炔诺酮,投喂黄鳝12个月,结果表明:在饲料中添加复方炔诺酮对黄鳝的体长和体重无显著性影响。高剂量能在4、5、6三个月降低血清E₂和T的水平,性逆转比例低于对照组。7.生态环境胁迫—无栖息巢对黄鳝性逆转的影响试验开始第一个月无栖息巢组的E₂水平急剧下降,自第二个月与对照组下降的幅度无显著性差异,无栖息巢组在第二年4月就达到其最大值618.18±30.05pg/ml显著高于对照组262.26±18.75 pg/ml,对照组在5月底才达到其最大值。本研究表明环境胁迫—无栖息巢能显著影响黄鳝血清E₂的分泌,与对照组相比,开始时下降速度较快,至翌年比对照组整整提早1各月达到最大值。无栖息巢组的T值在翌年5月就达到最大值6.75±0.85 ng/ml,对照组的T值要推迟1个月才达到最大值5.25±0.31 ng/ml,研究表明无栖息巢能够显著影响黄鳝血清T的分泌。表现在秋季对睾酮的分泌产生抑制,早春迅速促进血清睾酮值上升。另外,不管是体长还是体重,有栖息巢都显著快于无栖息巢组。历经12个月,无栖息巢组有38.85±0.85%的黄鳝转为雄性,只有47.14±3.50%保持雌性;而对照组只有24.6±1.37%的黄鳝转为雄性,有61.18±2.31%的黄鳝还是保持雌性,两者的间性比例无显著性差异,说明环境胁迫—无栖息巢能显著促进黄鳝从雌性向雄性转变。在黄鳝性逆转启动的时间方面,通过1000多个性腺组织切片的证实,在雌性黄鳝卵细胞发育到第Ⅳ期,潜伏在雌性性腺生殖褶上的雄性原始精原细胞开始发育并逐渐形成精小囊,散布在卵细胞的间隙中间,也就是说在黄鳝排卵前雄性生殖细胞已经开始启动发育（国内学者普遍认为黄鳝的性逆转在产卵后才启动）,同时卵母细胞从第Ⅳ期逐渐发育到第Ⅴ期。黄鳝产卵后,卵母细胞不断退化,位于生殖褶上的多个精小囊内的生精细胞发育成初级精母细胞,这时生殖囊内还可见到Ⅱ、Ⅲ时相的卵母细胞;随着雄性生殖细胞的发育,卵细胞的进一步退化,进入雄性发育阶段。并且,在鱼类性腺中首次发现了卵黄卵（Ⅳ期）和成熟精子细胞并存的现象。更多还原

【Abstract】 The rice field eel（Monopterus albus） can be found in China,India,Malaysia and Indonesia,and has been popularly cage cultured in central and southern China.The total output of China has reached 180,000 tons.It was a popular and highly appreciated fish on the Chinese market,and it has been selected as a target species for Chinese aquaculture due to its rapid growth,good survival rate and easy adaptation to cage culture conditions.However,Chinese rice field eel aquaculture was still at an elementary stage.Farming activities still rely on the capture of wild juveniles.With the increased fishing,the wild juvenile resources declined sharply.The achievement of controlled reproduction techniques of this species remains an essential prerequisite for domestication and development of farming activities for rice field eel.However,it has a special phenomenon of sex reversal:It was a hermaphroditical fish,early stages of development of life were females.After spawning,it became intersex,then entered male stage.The mainly spawning period was from May to June.When body length reached 35-45cm,it began to change its sex.Because of sex reversal,individuals of female were smaller. Absolutely fecundity was lower.Large-scale artificial breeding has not solved,therefore, the study of sex reversal played a very important role in solving the artificial breeding of the fish.The present study aimed to find the way of the manual control technology of sex reversal in M.albus,by using nutrition,exogenous hormone and environmental factors. The study could bring new breakthrough in breeding and promote the sustainable development of aquaculture industry. 1.The studies on the relations of body length,weight,age and sex ratio and fecundity in M.albusDuring 12 months,samples were collected in Diao-cha lake,Hubei province.The total number was 258.The number of females was 152（accounted for 58.91%）,intersexes 44 （17.05%）,males 62（24.04%）.Total body length was 9.4-76.8 cm.Body length of female was 9.4-60.5 cm,average 39.6 cm.Those below 20cm were all females.Intersexual body length was 27.1-63.3 cm, average 44.5 cm.Body length of male was 29.4-76.8 cm,average 51.63 cm.Those 70 cm above were all males.The results show that 25 cm was the starting point of body length of sex reversal in M.albus.Total body weight was 0.48-753.60 g.The average weight of females was 58.46 g. Those below 23.70g were all females.Intersexual body weight was 25.40 g-206.30g, average 76.45 g.The weight of males was 29.65-753.60g,average 144.38 g.Those 250 g above were all males.The results show that 25 g was the starting point of body weight of sex reversal in M.albus.The number of females below 2 years accounted for 92.17%of the total females. Intersexual individuals were 2-5 years,and 3-4 years were the peak period.Males were 2-7 years.The number of males,2 years,only accounted for 2.00%of the total males. Those above 6 years were all males.The results show that 2 years was the starting point of the age of sex reversal in M.albus.The number of impregnated eggs was 41-1267.Those 20-30 cm had the highest impregnated rate.2.Effects of different protein level diets on sex reversal of M.albusThe study was conducted to investigate the effect of different protein levels（25%, 35%,45%and 55%） on sex reversal of M.albus.The study indicated that the gonadal development of M.albus was closely related with the protein levels in diets.In a short period（one month）,the GSI of different groups was not significantly different.In the end of June next year,the GSI was significantly different.They were 1.45±1.05%,3.00±2.36%,7.71±1.67%,2.35±0.81%,respectively.GSI was closely related with the protein levels in diets.Within the appropriate context,the absolute intensity was increased with the protein levels in diets.The ratios of sex reversal individuals in different groups were different.25%protein levels groups,38.65±1.34%changed its sex to male,and 17.61±1.47%to intersex. 35%protein levels groups,24.15±6.55%changed its sex to male,and 11.20±1.45%to intersex.45%protein levels groups,7.61±1.43%changed its sex to male,5.21±1.25% to intersex,and 87.18±1.21%maintain female.55%protein level groups,14.21±5.52%changed its sex to male,about 7.18±0.96%to intersex,and the ratio of female in the groups was less than that of 45%groups.The study indicated that 45%protein level in diets could be favorable for gonadal development of female M.albus.In the studies on section of serum estradiol（E₂）,E₂ of 45%protein level groups was the highest.E₂ of 35%and 45%groups were significantly higher than that of 25%,55% groups in April,May and June,and reached their peak value in June.The study indicated that it high section of serum E₂ could be maintained with the suitable protein levels in diets.It could delay the sex reversal of M.albus in some degree.3.Effects of different lipid level diets on sex reversal of M.albusThe experiment was conducted to investigate the effect of different lipid levels（5%, 10%and 15%） on sex reversal of M.albus.The study indicated that the ratio of sex reversal of M.albus was increased with fat level in the same protein level diets.5%fat level in diets could be favorable for gonadal development of female M.albus and increase the absolute intensity.4.Effects of different concentrations of exogenous testosterone propionate （TP） diets on sex reversal of M.albusThe study aimed to investigate the effect of different dose of TP（0 mg / kg,10mg / kg and 30 mg / kg） on sex reversal of M.albus.18.51%individuals in 30 mg / kg groups changed its sex to male,higher than that of 0 mg / kg and 10mg / kg groups,but the total ratio of male and intersual was no significant difference.The study indicated that different dose of TP has no significantly effects on sex reversal of the fish. 5.Effects of different concentrations of exogenous estradiol valerate diets on sex reversal of M.albusFishes were treated with different concentrations of estradiol valerate in diets,0mg/kg, 10mg/kg and 50mg/kg,for 12 months.Lower concentrations（10mg/kg） of estrsdiol valerate in diets could inhibit the sex reversal of the rice-field eel in some degree.Higher concentration of estradiol valerate in diets could reduce the secretion of T while the concentration of serum E₂ kept higher levels.The sex steroid hormonal profiles and experimental data lead to the conclusion that treatment of diets of estradiol valerate （50mg/kg） could delay the process of sex reversal in rice-field eel.6.Effects of different concentrations of exogenous Compound norethindrone diets on sex reversal of M.albusThe study aimed to investigate the effect of different dose of compound norethindrone （0 piece/kg,10 pieces/kg and 30 pieces/kg） on sex reversal of M.albus.The study indicated that treatment of diets of compound norethindrone could delay the process of sex reversal in rice-field eel.It has no significantly effects on body-length and body-weight of the fish.7.Effects of Eco-environmental stress（no habitat nest） on the sex reversal of M.albusThe E₂ levels without habitat nest groups were declined sharply in the first month.It reached peak value（618.18±30.05pg/ml） in April of the next year and significantly higher than that of controls.This study showed that environmental stress-no nest habitat could significantly affect the secretion of serum E₂ of M.albus.T levels without habitat nest groups reached its peak value（6.75±0.85 ng/ml） in May of the next year while controls reached its peak value（5.25±0.31 ng/ml） in June of the next year.The study indicated that no habitat nest could significantly affect the T secretion.Besides,the study showed that environmental stress-no habitat nest can significantly affect the growth of body-length,weight and the sex reversal of M.albus.After 12 months,38.85±0.85%individuals of the group without habitat nest changed their sex to male,47.14±3.50%maintained female.24.6±1.37%of the controls changed their sex to male,61.18±2.31%maintained female.It was confirmed that the starting time of sex reversal in the fish was at stageⅣthrough more than 1,000 biopsy of gonadal tissue.The original spermatogonia hidden in female gonads gradually began to develop a small testis capsule and spread in the gap among oocytes.Then oocytes from the stageⅣgradually developed intoⅤ.Oocytes degenerated after spawning.Spermatogenic cells developed into primary spermatocytes and reproductive capsule could be seen with oocytes ofⅡ,Ⅲstages.With the development of male reproductive cells,oocytes degradated and started the male developmental stage.The phenomenon that eggs with yolk（Ⅳstage） and mature sperm cells co-existence in gonads was first found in fish.更多还原