| Tilapia(Oreochromis)is native to tropical and subtropical regions.It is an international fish mainly promoted by FAO and one of the most important freshwater species.Tilapia has the characteristics of early sexual maturity,short reproductive cycle and reproductive habits of nesting,hatching and juvenile protection.Mixed-rearing tilapia consumes a lot of energy,which limits its growth,especially adult tilapia grows much more slowly.Reproductive behavior leads to high density of pond population,disparity in bodyweight,low commodity rate and high feed coefficient,which greatly reduces fish production and quality.At present,all-male tilapia are commonly used for aquaculture,but their sexual precocity and reproductive habits still exist.In order to eliminate the adverse effects of sexual maturity,it is an ideal choice to produce a population composed of sterile individuals.Active immunization with gonadotropin-releasing hormone(GnRH)can inhibit the development of animal sexual organs,promote animal growth,improve meat quality and improve production performance.We found the existence of sterile tilapia in the course of culture,which has the characteristics of fast growth,high fullness and degeneration of gonad(ovary).In this study,all female GIFT stra:in of Nile Tilapia(Oreochromis niloticus)were immunized with GnRH-A antigen to study the effects on reproductive function and its regulation mechanismIn this paper,the differences of growth rates,gonad development and hormone levels between infertile tilapia and common tilapia(male and female)were studied.All female tilapia families were bred as experimental materials.GnRH-A antigen was synthesized and prepared for active immunization.The growth,sex ratio,GnRH antibody,serum hormone content(GnRH,FSH,LH,E2,T)were measured by immunizing with different doses in all female tilapia.The optimal dose of GnRH-A antigen was used for active immunization,and the immunohistochemics,transcriptomics and proteomic studies of tilapia were studied.The main research contents and results are as follows.1.It was found that the body weight and length of sterile tilapia were significantly larger than female tilapia(P<0.05)and also higher than that of male tilapia(P>0.05).The order of relative fatness was infertile tilapia>male tilapia>female tilapia,but the difference was not significant.The serum GH and T contents of sterile tilapia were the highest,and the serum E2 content was significantly lower than that of male and female tilapia.The serum FSH,GnRH and LH levels were between male and female tilapia.2.Three generations of pseudo-male and all-female GIFT tilapia families were selected by hormone-induced sex control.The female rate of natural breeding tilapia in F3 generation was 99.9%,and six families are all female(100%).6 theoretical pseudo-male tilapia families were obtained by methyltestosterone induction in all female families.The average weight of F3 families increased by 22.97%,and the average weight of the best families(F3-13)increased by 34.32%compared with the control group.Pseudo-male and all-female tilapia are good materials for breeding and experimental research.3.According to the amino acid sequence of GnRH in tilapia,the amino acid sequence(Pyr-His-Trp-Ser-Tyr-{D-Ala}-Leu-Arg-Pro-NHEt)prepared by GnRH-A antigen was designed.Purpose GnRH-A(purity:98.38%)was synthesized and purified.Taking carbon Diimide hydrochloride(EDC.HCL)as coupler,GnRH-A was connected with bovine serum albumin(BSA)to form a complex,antigen emulsion was made by adding complete or incomplete Freulld's adjuvant.The CFU-BSA-GnRH-A and IFU-BSA-GnRH-A antigen emulsions have the characteristics of stable,sterile and harmless to tilapia.4.All females were immunized with GnRH-A complete adjuvant emulsified antigen at doses of 200,100,50 and 25?g/tail.14days later,the same dose of GnRH-A incomplete adjuvant emulsified antigen was used to enhance immunization.After vaccination,the sex ratio,growth traits and organ index of tilapia were measured.The results showed that:GnRH-A vaccine immunization had a long-term and sustained effect on the reproductive system of tilapia.Until the end of the experiment,the decreasing trend of the female rate in each group had not been slowed down,and the effect of sex change was the best when treated with 100 ?g/tail dose.In the early stage of the experiment,the growth rate of tilapia was inhibited,,but they basically returned to the level of the control group at the end of the experiment.On the 70th day,the relative fatness of tilapia in 200 ?g/tail treatment group was significantly higher than that in control group(P<0.05),and the relative fatness of tilapia in 100 ?g/tail treatment group was also higher(P>0.05).The gonad coefficient and spleen index of 200?g/tail treatment group were significantly higher than those of control group(P<0.05),while the gonad and liver index of 50 ?g/tail treatment group were significantly lower than control group(P<0.05).5.After the first immunization of GnRH-A antigen,GnRH antibody was rapidly produced in tilapia,After strengthening immunization,the antibody content continued to increase,and maintained a significantly higher level than that of the control group.The serum GnRH content of treatment group decreased and remained stable after vaccination.The GnRH content of 200,100 and 50?g/tail treatment groups were significantly lower than that of control group,and that of 25 ?g/tail treatment group was lower than that of control group from 28 days.The contents of FSH and LH in serum of each treatment group increased first and then decreased or remained stable,at the end of the experiment,the contents of FSH and LH in 200 and 100 ?g/tail treatment groups had no significant difference with those in control group,but the contents of FSH and LH in low dose group were significantly higher than those in control group.The serum E2 level in treatment group and control group showed an increasing trend.Except for Day28 and 42 of 200 ?g/tail treatment group,the E2 level in each treatment group was significantly higher than that in control group at different time.During the experiment,there was no significant difference in serum T content between the treatment groups and the control group.6.After 70 days of immunization with four different doses of GnRH-A antigens,the pituitary and gonad of tilapia were harvested.The gene expression of the pituitary was determined by real-time quantitative PCR,and the gonad GnRH was localized and analyzed by immunohistochemical method.The results showed that,there was no significant difference in the expression of GnRHR in pituitary between each treatment group and control group;the expression of LH-? in pituitary of tilapia in 100 ?g/tail treatment group was significantly lower than that in control group(P<0.05);the expression of LH-? of tilapia in 50 and 25 ?g/tail treatment group was very significantly higher than that in control group(P<0.01),and there was no significant difference between 2 high dose treatment groups and control group.The expression of GnRHR in pituitary was highly positively correlated with LH-beta gene expression(0.899*),the expression of FSH-beta gene in pituitary was highly correlated with serum FSH content(0.972**),the correlation between serum GnRH content and other indicators was very low(P>0.05).The immunoreactive products of GnRH in the gonads of tilapia were yellow or brown.The number of GnRH positive cells in the control group was small,and the colar was light.The GnRH positive cells in the gonads of infertile tilapia are more abundant and darker than female in the treatment groups and the control group.The histochemical scores in 100 and 50?g treatment groups were significantly higher than those in the control group(P<0.05),but there was no significant difference between the other groups and the control group.7.Tilapia were immunized with 100?g/tail GnRH-A antigen,and gonads were collected 70 days after first immunization.Transcriptomics and proteomics of tilapia immunized with GnRH-A antigen were studied by RNA-seq and DIA techniques.The results showed that,644 differentially expressed genes were obtained by gonadal transcriptome analysis after immunization,including 80 up-regulated genes and 564 down-regulated genes.1150 differentially expressed proteins were obtained by proteome analysis,of which 351 were up-regulated and 799 were down-regulated.There were 209 genes with identical differential expression patterns between transcriptome and proteome,of which 9 were up-regulated and 200 were down-regulated.The results showed that GnRH antigen inhibited the expression of gonad genes in tilapia.The downregulated gene functions mainly focus on single-organism process.binding,cellular process,metabolic process and catalytic activity,and the pathways involved include ECM-receptor interaction,Focal adhesion,Cardiac muscle contraction,oxidative phosphorylation.The 6 differentially expressed genes involved in GnRH signal transduction pathway were all down-regulated.In addition,several important functional genes related to gonadal development after GnRH-A antigen immunization were screened.This study demonstrates the effectiveness of GnRH-A vaccine in suppressing gonad development of tilapia at molecular level,and lays a foundation for elucidating the mechanism of GnRH-A antigen immunity. |
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