Effects Of Different Exogenous Factors On Sex Reversal Of Monopterus Albus

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.