Effects Of Co-treatment With High Temperature And Methyltestosterone On Sex Differentiation Of Nile Tilapia

The Nile tilapia(Oreochromis niloticus)are the third most important aquaculture fish.Because the growth rate of males of Nile tilapia is over 30% faster than that of females,and the breeding cycle is short,the offspring of breeding will greatly reduce the growth rate of the female parent.Carrying out single male breeding can improve breeding efficiency.Exogenous androgen treatment can induce female Nile tilapia to reverse sex into males,However,androgens are endocrine disrupting substances in the environment,which will cause great harm to the ecological environment.In addition,sex differentiation of Nile tilapia is also affected by temperature changes,high temperatures can induce the sex reversal of Nile tilapia females into pseudo-males.The purpose of this experiment was to explore how much androgen use can be compensated by high temperature treatment and to clarify the effects of high temperature and methyltestosterone treatment on the sex differentiation process of Nile tilapia.In the critical period of tilapia sex determination,we co-treated fry by high temperature and adding different concentrations of exogenous androgen-containing feeds,and counted the male rate of each group to select the least level of exogenous-androgen under the assistance of high temperature.We also analyzed the changes of estrogen and androgen levels in tilapia during sexual reversal and identified the changes in mRNA and protein expression levels of key genes Cyp19a1 a and Dmrt1 that are involved in sex differentiation under high temperature and exogenous androgen treatment.These provide a scientific basis for the whole male aquaculture production of Nile tilapia,and provide a theoretical basis for the cultivation of parthenogenic fry.The specific results are:1)The statistical results of a male rate under the combined treatment of high temperature and methyltestosterone(MT)show that as the MT concentration in the feed increases,the male rate increased significantly in both control temperature group(28?)and high temperature group(36?).The study also showed that the male ratio of the treatment group and the high temperature group fed the same MT concentration was significantly higher than that of the control temperature group;Under the co-treatment of high temperature and MT,when the MT concentration in the feed was 10 mg/kg,the male rate can reach 100%,but the value in the control temperature group was 20 mg/kg,which indicates that high temperature reduced the amount of hormone use.2)The results of endogenous estrogen levels showed that after MT treatment,the level of estradiol(E2)in females was significantly reduced regardless of the control temperature group(28?)or high temperature group(36?);With the increase of MT concentration in feed,the tendency of E2 level to decrease was not significant;the effect of high temperature on the E2 reduction was more significant than that of the MT treatment.After measuring endogenous androgen levels,the results showed that the levels of androgens in normal fish were extremely low,and as the MT concentration in the feed increased,whether in the control temperature group(28?)or the high temperature group(36?),the 11-KT(ketotestosterone)level was significantly increased in tilapia fry.In the treatment group fed with the same MT concentration,the 11-KT level in the high-temperature group was significantly higher than that in the control temperature group,which indicates that both high-temperature and methyltestosterone treatment can cause up-regulation of 11-KT.3)The results showed that under the co-treatment of high temperature and methyl testosterone(MT),the expression level of Cyp19a1 a gene in the control female group was significantly lower than that in the control male group,which was close to that of the control male group;IHC results showed that Cyp19a1 a protein positive signal was detected in the gonads of the control female group,but the positive signal was weaker after high temperature treatment.For two groups with a male rate of 100%,Cyp19a1 a mRNA expression was completely suppressed,and no positive signal of protein was detected in the gonads.4)The analysis results of the male sex differentiation key gene Dmrt1 showed that under high temperature and methyl testosterone(MT)treatment,compared with the control female group,the Dmrt1 gene expression level in the high temperature treatment group was significantly increased or even higher than that in the control male fish group.There was a weaker Dmrt1 protein positive signal in the gonads of the control female group,and the positive signal increased significantly after high temperature treatment.In the control female group,only weak Dmrt1 protein positive signal was found in the gonads,and the positive signal was significantly enhanced after high temperature treatment.In two groups with a male rate of 100%,the Dmrt1 gene expression level was higher than that of the high temperature treatment group,and a significant Dmrt1 protein positive signal was detected in the gonads.In summary,both high temperature and methyltestosterone treatment can induce sexual reversion of female Nile tilapia into pseudo-males.In inducing all females to reverse sex into males,,assisted treatment with high temperature can reduce the used concentration of methyltestosterone in the feed.In the process of females undergoing sexual reversal under the influence of high temperature and methyltestosterone,the levels of endogenous estrogens decreased and the levels of endogenous androgens increased.The expression of key genes for male sex differentiation in the gonads was increased,and the expression of key genes for female sex differentiation was suppressed.