【作者】 袁汉文；

【导师】 龚世国；

【作者基本信息】 华中农业大学 ， 水产养殖， 2011， 博士

【摘要】 本试验以黄鳝(Monopterus albus)为研究对象。黄鳝在分类学上隶属于硬骨鱼纲、辐鳍亚纲(真口亚纲)、合鳃目、合鳃科,在我国仅发现黄鳝这个唯一种,它是东南亚地区一种重要的经济鱼类。黄鳝是一种雌性先熟雌雄同体的鱼类,在其生命发育过程中具有从功能性雌鱼向功能性雄鱼自然转变的单向发育的特性。在自然条件下,黄鳝一般在一龄以上达到性成熟,初次成熟都是雌性,产卵后开始发生性转变,三年以上可以发现雄性个体。自1944年发现黄鳝性逆转的特性以来,为了揭示黄鳝性逆转的发生机制,很多学者分别从生理生化、细胞学和分子生物学等发面做了很多努力,但到现在为止,对黄鳝性逆转的了解仍然不多,黄鳝性逆转的发生机制也不很清楚。为了进一步探讨黄鳝性逆转的发生机制,本试验研究了饲料低蛋白水平、外源性褪黑激素、外源性雌二醇、白消安和不同养殖密度对黄鳝性逆转的影响。主要研究结果如下：(1)低蛋白含量饲料对黄鳝性逆转的影响：以体重(9.50±1.50)g的黄鳝为试验对象,以白鱼粉作为主要蛋白源,按5个蛋白质水平梯度(10%、15%、20%、25%、40%)设计等能的饲料,每个试验组有三个重复,进行为期15个月的饲养试验,探讨低蛋白水平对黄鳝血清雌二醇、睾酮、性腺指数、性腺发育和性逆转的影响。结果显示,不同蛋白含量的饲料对黄鳝血清雌二醇和睾酮都有显著地影响,对照组(40%)中黄鳝血清雌二醇含量显著性地高于其他各试验组,而睾酮含量则显著性低于其他试验组。本试验中蛋白含量为10%组中,雌二醇含量在一月份达到最小值,为89.56±15.20 pg/ml；而最大值则出现在五月份蛋白含量为40%的试验组中,为1240.22±323.86 pg/ml；睾酮含量的最大值出现五月份,在蛋白含量为10%的试验组中,为804.02±34.84 pg/ml。性腺指数最小值出现在一月份蛋白含量为10%的试验组中,为0.611±0.35%；而最大值则出现在七月份蛋白含量为40%的对照组中,为11.669±3.66%。繁殖期间,投喂高蛋白含量的饲料,黄鳝性腺指数上升的速度较快。不同蛋白水平下,雌性比例有显著性差异,最高雌性比例出现在蛋白含量为40%的组中,雌性比例随着饲料蛋白含量的降低而减少。雄性比例最高出现在蛋白含量为10%的组中,蛋白含量为10%的组中雄性比例显著地高于蛋白含量为25%的组和对照组。同时,蛋白含量为10%的组中,黄鳝的成活率显著性地低于其他各组。本试验期间,各组间雌性黄鳝性腺的组织切片表明,雌性性腺发育的方式是相同的,各组之间没有差异。在间性个体中,可看见精母细胞分布在很多卵母细胞周围,在蛋白含量低的试验组中这种现象比较明显；在蛋白含量较低的试验组中可见有成熟的雄性性腺存在；七月份在蛋白含量为15%的试验组中,发现有繁殖后的功能性雄鱼。研究结果表明低蛋白含量的饲料对黄鳝的性逆转有促进作用。(2)外源性褪黑激素对黄鳝性逆转的影响：以体重(9.50±1.50)g的黄鳝为试验对象,褪黑激素按0 mg/kg(对照组)、10 mg/kg、30 mg/kg、50 mg/kg和70 mg/kg添加于饲料中,每个试验组有三个重复,进行为期15个月的饲养试验,探讨外源性褪黑激素对黄鳝性腺发育和性逆转的影响。结果显示,不同褪黑激素含量对黄鳝血清雌二醇和睾酮浓度有显著性的影响。在繁殖期结束后,黄鳝血清雌二醇和睾酮呈下降趋势,在越冬季节达到最小,产卵前迅速上升,随后下降。褪黑激素含量为10 mg/kg试验组中,黄鳝血清雌二醇浓度与对照组间没有显著性差异,但含量在30 mg/kg以上的各试验组中,血清雌二醇浓度均显著性地低于对照组；血清睾酮含量随褪黑激素含量增加而显著性下降,褪黑激素含量为10 mg/kg组与50mg/kg和70 mg/kg两组间血清睾酮含量有显著性差异。黄鳝的性腺指数只有褪黑激素含量为10 mg/kg的组与对照组间没有显著性差异,另外三组与对照组间有显著性差异,但试验组间差异性不显著。雌性比例在褪黑激素含量为10 mg/kg的试验组与对照组间没有显著性差异,含量高的各组中雌性比例显著低于对照组；间性比例中试验组间也没有显著性差异；雄性比例中,只有褪黑激素含量为70 mg/kg的试验组与对照组间有显著性差异,其他各组间均没有显著性差异。越冬期间用褪黑激素处理的黄鳝性腺中,卵母细胞间空隙较大,繁殖季节前期卵巢内可见不同发育阶段的卵母细胞,繁殖高峰季节可见部分变形的成熟卵母细胞,而间性黄鳝雄性性腺组织发育比对照组慢,雄性生殖褶相对较细；褪黑激素处理过的雄性黄鳝性腺组织比未处理过的要消瘦。本试验条件下,外源性褪黑激素对黄鳝的性腺发育有抑制作用,长时间高浓度的褪黑激素可导致黄鳝血清性类固醇激素雌二醇和睾酮含量下降,性腺指数降低,促进性逆转提前发生,但是长时间高浓度的外源性褪黑激素也会导致成活率降低。(3)外源性雌二醇对黄鳝性逆转的影响：以体重(9.50±1.50)g的黄鳝为试验对象,雌二醇按0 mg/kg(对照组)、20 mg/kg、40 mg/kg、60 mg/kg、80 mg/kg和100 mg/kg添加于饲料中,每个试验组有三个重复,进行为期15个月的饲养试验,探讨外源性雌二醇对黄鳝性逆转的影响。结果显示,添加外源性雌二醇的试验组与对照组之间,黄鳝血清雌二醇和睾酮浓度都存在显著性差异；但含量为60mg/kg以上的各试验组间血清雌二醇和睾酮浓度差异不显著。黄鳝性腺指数和肝脏指数在雌二醇试验组中与对照组间均有显著性差异。性腺指数在不同月份间具有极显著性差异,不同雌二醇含量对黄鳝性腺指数的影响也具有差异性,但它们交互作用对黄鳝性腺指数影响显示的差异性则不显著。不同性别比例中,低剂量组与对照组间没有显著性差异,高剂量组与对照组有显著性差异,试验组间差异性不显著。越冬期间用雌二醇处理的黄鳝性腺,卵母细胞间空隙较大,偶尔可见卵巢内有较大的卵母细胞,越冬后卵巢内可见不同发育阶段的卵母细胞,繁殖季节前可见处于不同发育阶段的卵母细胞均匀地排列在卵巢中,繁殖季节可见卵母细胞紧密地排列在卵巢中；雌二醇处理过的间性黄鳝性腺组织中,偶尔可见雄性性腺中包裹有较大的游离卵母细胞；而雌二醇处理过的雄性黄鳝性腺组织比较松散,不能填满整个精巢组织。本试验条件下,外源性雌二醇能够促进和维持黄鳝的雌性活性,增加血清中雌二醇的含量,利于卵巢和卵母细胞的发育成熟,增加黄鳝的性腺指数,同时也导致血清中睾酮含量降低,对雄性生殖腺的发生和发育有一定的抑制作用,使得肝脏指数上升和成活率下降。结果表明,外源性雌二醇能增加黄鳝的雌激素活性,对黄鳝的性转变有一定的延缓作用。(4)白消安对黄鳝性逆转的影响：以体重(9.50±1.50)g的黄鳝为试验对象,白消安按0 mg/kg(对照组)、10 mg/kg、30 mg/kg、50 mg/kg和70 mg/kg添加于饲料中,每组设3个平行,探讨白消安对不同时期黄鳝性腺发育和性逆转的影响。结果显示,白消安对黄鳝血清雌二醇和睾酮含量有显著性影响,但各试验组间没有显著性差异。在繁殖期结束后,雌二醇和睾酮仍处于较高水平,在越冬期最低,繁殖季节前呈上升趋势,繁殖期雌二醇和睾酮含量处于很高的水平。在各个时期,黄鳝血清雌二醇浓度均随着白消安含量的增加而呈下降趋势,而睾酮浓度均随着白消安含量的增加而略呈上升趋势。不同时期,黄鳝性腺指数的变化较大,在繁殖期达到最大值；但相同时期各试验组间,性腺指数随着白消安含量的增加呈下降趋势。雌性比例中,各试验组间均有显著性差异。间性和雄性比例中,对照组与各试验组间有显著性差异；而低含量的组间差异不显著,而白消安含量为70 mg/kg试验组中比例显著增多。经过白消安处理的试验组内,卵巢中退化的卵母细胞比例较高,而且部分细胞间有一定的空隙。间性阶段雄性生殖褶分布在稀疏的卵母细胞周围,雄性生殖腺偏消瘦,部分呈中空状态。结果表明,白消安对黄鳝的性腺发育有抑制作用,高剂量还会造成黄鳝的畸形。长时间对黄鳝进行白消安处理,可导致黄鳝血清雌二醇含量的降低和睾酮含量的增加,性腺指数降低,雌性比例减少,长时间高浓度的白消安会导致死亡率明显升高。(5)不同养殖密度对黄鳝性逆转的影响：以体重(9.88±0.70)g的黄鳝为试验对象,养殖密度分别为4、20、36、52、68、84和100尾/m2,每个试验组设三个重复,进行为期420 d的饲养试验,探讨不同养殖密度对黄鳝性逆转的影响。结果显示,不同养殖密度下,各组试验鱼的日增重和特定生长率随着养殖密度的增加不断降低,但在低密度组间没有显著性差异；净增重随着养殖密度的增加显著性地增加。各试验组间个体的肥满度随着养殖密度的增加呈下降趋势,不同密度间差异不显著；黄鳝血清雌二醇含量随着养殖密度的增加呈下降趋势,睾酮含量随着养殖密度的增加呈上升趋势,不同密度组间的差异均不显著。黄鳝性腺指数随着养殖密度的增加呈现出一种先上升后下降的趋势,性腺指数最高出现在养殖密度为52尾／m2的试验组中,各个试验组间差异性不显著。黄鳝的雌性比例随着养殖密度的增加呈下降趋势,间性比例中,高密度组和低密度组间差异性显著。雄性比例在三个高密度组间存在显著性差异。黄鳝的成活率随着养殖密度的增加呈下降趋势。雌性个体的性腺在低养殖密度试验组中,卵母细胞以第Ⅳ时相为主,卵母细胞发育较一致,随着养殖密度的增加,卵巢内可见较多的第Ⅱ时相和第Ⅲ时相的卵母细胞,个体差异较大；间性个体中不同试验组间差异性不显著。雄性性腺中,随着养殖密度的增加,雄性生殖褶在性腺内逐渐密集。本试验条件下,不同的养殖密度对黄鳝血清性类固醇和性腺指数因为受到性别比例的影响没有表现出显著性差异；而性别比例中,随着养殖密度的增加,雌性比例减少,间性比例和雄性比例呈上升趋势。更多还原

【Abstract】 This study was conducted on rice field eel, Monopterus albus. It taxonomically belongs to the teleosts, the family Synbranchidae of the order Synbranchiformes, and is also the only representative species of the group of Synbranchidae in China. This fresh-water fish is an economically important species in Southeast Asia. The rice field eel is a protogynous hermaphrodite, strictly changing its sex unidirectionally from functional female to male naturally during development. It spends one or more years reaching puberty under natural ambient conditions, which is female at first. After spawning it normally begins to change sex from female to intersex and then finally to male. It may not develop into mature functional male until it is more than 3 years old. Since the discovery of natural sex reversal in rice field eels in 1944, some efforts have been made to uncover the mechanism underlying the process in this species from both the physiological and biochemical perspective, at the cytological level and at the molecular level. Thus far, little is known about sex reversal and the detailed mechanism of sex reversal of this species remains unclear. In our study, the effects of different factors including low protein level in diets, exogenous melatonin, exogenous estradiol, busulfan and stocking density on sex reversal of rice field eel were investigated. The results were as follows:(1) Effects of low dietary protein level on sex reversal in rice field eel:Five fishmeal-based experimental diets containing various crude protein levels including 10%, 15%,20%,25% and 40% were fed to triplicate groups of 50 fish each (initial weight 9.50±1.50 g) for 15 months. Effects of low dietary protein level on serum estradiol (E2) and testosterone (T) concentrations, Gonadosomatic index (GSI), sex reversal and gonad structure at the histological level were investigated. Different dietary protein levels significantly affected both E2 and T concentrations of the rice field eels fed with the formulated feeds. E2 concentrations of the control group (40%) were significantly higher than the other treatment groups, but T concentrations of the control group were significantly lower than the other treatment groups. In our experiment, E2 concentrations achieved minimum and maximal values 89.56±15.20 pg/ml and 1240.22±323.86 pg/ml, which occurred in the 100 g/kg protein level group in January and 400 g/kg protein level group in May, respectively. T concentration achieved the maximal value of 804.02± 34.84 pg/ml in the 100 g/kg protein level group in June. The highest GSIs were observed in fish fed with the 400 g/kg protein diet. GSIs achieved the minimum and maximal values,0.611±0.35% and 11.669±3.66%, which occurred in the 100 g/kg dietary protein level group in January and 400 g/kg dietary protein level group in July, respectively. GSIs of the fish fed with higher dietary protein levels increased faster during the spawning period. Significant differences were observed in the female ratio at different dietary protein levels, the highest female ratio occurred at the 400 g/kg dietary protein level, followed by 250 g/kg,200 g/kg,150 g/kg and 100 g/kg dietary protein levels. The highest male ratio obtained at the 100 g/kg dietary protein level was significantly different compared with the 250 g/kg protein level. Meanwhile, the survival rates of the 100 g/kg dietary protein level were significantly lower than those in the other groups. Histological structure of the female gonads from all treatment groups showed the same pattern during the experimental period. In the intersex fish, some spermatocytes could be observed among the numerous oocytes in a few gonads from the lower dietary protein level groups. The mature testis were observed in lower dietary protein level treatment groups, and fortunately the spawning of the fully functional males in the 150 g/kg dietary protein level treatment group were observed in July. The results showed that low dietary protein level can promote sex reversal in rice field eel under the experimental condition.(2) Effects of exogenous melatonin on sex reversal in rice field eel:The present study investigated the effects of exogenous melatonin on gonad development and sex reversal of rice field eel (initial weight 9.50±1.50 g). Five exogenous melatonin doses of 0 mg/kg (control),10 mg/kg,30 mg/kg,50 mg/kg and 70 mg/kg were set-up for all experiments with 3 replicates per treatment for 15 months. The results showed that both serum E2 and T concentrations of the rice field eels were significantly affected by exogenous melatonin. Serum E2 and T concentrations had a decreasing tendency in each group after spawning and the lowest levels were reached during overwintering. No meaningful statistical differences were found in E2 concentrations between the 10 mg/kg treated group and the control group, but E2 concentrations of fish treated with 30 mg/kg,50 mg/kg or 70 mg/kg were significantly lower than the control group. T concentrations decreased significantly with the increasing exogenous melatonin dose, and T concentrations decreased faster in two higher doses than lower doses. GSIs were significantly higher in the control group than the treated groups, while they were not significantly different among treatments. Female ratios were significantly higher in the control group than all treated groups except 10 mg/kg treated group. Intersex ratios were not influenced by exogenous melatonin in the experiment. Male ratios were significantly higher in the 70 mg/kg treated group than the other lower treated groups and the control group. Histological structure of the female gonads showed that the empty space in ovaries became bigger with increasing exogenous melatonin doses during overwintering period. Different developing stage oocytes and some deformed mature oocytes could be seen in ovaries during the breeding period. Male gonad developed slower and male genital folds were thinner in the treated groups than the control group. These results indicated exogenous melatonin may inhibit the development of gonad and promote sex reversal in rice field eel. Serum E2 and T concentrations, GSIs and survivals decreased after the fish treated with exogenous melatonin for a long time.(3) Effects of exogenous estradiol on sex reversal in rice field eel:The present study investigated the effects of exogenous estradiol on gonad development and sex reversal of rice field eel (initial weight 9.50±1.50 g). Six exogenous estradiol doses of 0 mg/kg (control),20 mg/kg,40 mg/kg,60 mg/kg,80 mg/kg and 100 mg/kg were set-up for all experiments with 3 replicates per treatment for 15 months. Both serum E2 concentrations and T concentrations were significantly influenced by exogenous estradiol. Where the difference was not significant of E2 and T concentrations in the three treated groups of 60 mg/kg,80 mg/kg and 100 mg/kg. GSIs and HSIs were significantly higher in all estradiol treated groups than the control group. There were significant differences in GSIs of rice field eel in different month and treated with different exogenous estradiol dose, but the exogenou estradiol levels and months interaction effects on GSIs were not significant. Sex ratios were not significantly different among all treatments, there were no significant differences among the control group,20 mg/kg and 40 mg/kg treated groups, either. They were significantly affected by the higher estradiol doses of 40 mg/kg,60 mg/kg and 80 mg/kg. The empty spaces became larger between oocytes in the estradiol treated groups and developed oocytes still can be seen occasionally during the overwintering period. Different developing stage oocytes can be found in ovaries before breeding season. Sometime big free ooctyes existed in intersexual gonad. The intersex and male gonad of the estradiol-treated groups were looser than the control group, and the male genital folds can not fill the whole male testis in the treated groups. The results showed that exogenous estradiol can maintain the female activity and delay sex reversal of the rice field eel, exogenous estradiol may be benefit for the female gonad development and inhibit the male gonad development. GSIs and HSIs increased and survival decreased in the estradiol treated groups.(4) Effects of busulfan on sex reversal in rice field eel:The present study investigated the effects of busulfan on gonad development and sex reversal of rice field eel (initial weight 9.50±1.50 g). Five busulfan doses of 0 mg/kg (control),10 mg/kg,30 mg/kg,50 mg/kg and 70 mg/kg were set-up for all experiments with 3 replicates per treatment for 15 months. Both E2 and T concentrations remained high levels before and during breeding phase. They were significantly affected by busulfan, while there were no significant differences in serum steroid concentrations among treatments. Serum E2 concentrations decreased and T concentrations increased with increasing busulfan levels in the experiment. Significant differences among GSIs were detected in different season. GSIs reached maximum in breeding period and decreased quickly after spawning. GSIs decreased as the busulfan level was raised in each group. Significant differences were observed in the female ratio at different busulfan levels. Intersex and male ratioes increased significantly in all treatment groups. The highest male ratio obtained in the 70 mg/kg group was significantly different compared with the other group. The proportion of degenerated oocytes was higher and more empty space produced in the busulfan treated groups. The male genital folds were thinner and some of them were empty in the intersex and male gonad of the fish dealed with busulfan. These results indicate additive inhibitory effects of busulfan on gonad developmnet, the effect appeared to be relatee to dose. High-dose of busulfan may produce deformed individuals of rice field eel. Serum E2 concentrations and GSIs decreased, but serum T concentrations and mortality increased significantly by busulfan treated for a long time.(5) Effects of different stocking density on sex reversal in rice field eel:Fish (initial weight 9.88±0.70 g) were stocked at densities of 4,20,36,52,68,84 or 100 fish/m2 in net cages in an earthen pond, with 3 replicate cages for each density. Fish were fed a formulated diet containing 45% crude protein and cultured for 420 days. Effects of stocking density on sex reversal in rice field eel were investigated. DWG and SGR decreased with increasing density, but no statistically significant differences were found between groups of the three lowest stocking densities. NY increased with increasing stocking density significantly. CF decreased with increasing stocking density, while no meaningful statistical differences were found for CF in the groups of fish held at any stocking density. Serum T concentrations and mortality increased but E2 concentrations decreased with increasing stocking density. Steriod hormone concentrations and GSIs were not affected significantly by stocking density. The highest mean GSIs were observed for fish of the 52 fish/m2 stocking density. Female ratio and survival decreased with increasing stocking density. Statistically significant differences for intersex ratio were observed between groups of the three highest and the lowest stocking densities. There were significant differences in male ratio among the three highest stocking densities. Female gonad developed well in low stocking density groups, while male gonad developed better in high stocking density groups. No statistically significant differences were found in serum steriod concentrations and GSIs because of different sex ratio in the groups of fish at any stocking density. Intersex ratio and male ratio increased, while female ratio decreased with increasing stocking density.更多还原