Effects Of Busulfan And Its Combine Treatment With High Temperature On Gonadal Development Of Yellow Drum (Nibea Albiflora)

The yellow drum(Nibea albiflora)is an important economic breeding fish in China’s coastal areas.It has the advantages of short sexual maturation period and mature breeding technology,and can be used as the recipient of fish germ cell transplantation.In fish germ cell transplantation,the preparation of sterile receptor is very important because the endogenous germ cells of the recipient compete with the donor germ cells.In order to prepare male sterile receptors,the effects of Busulfan and high temperature treatment on growth,gonad development,gene expression in germ cells and somatic cells were systematically analyzed in this study.In this study,different doses of busulfan(10 mg/kg,40mg/kg)and high temperature(30℃)were designed to treat yellow drum.The experiment was divided into five groups: control group;busulfan 10 mg/kg group(Bu10 group);busulfan 40 mg/kg group(Bu40 group);busulfan 10 mg/kg high temperature treatment group(Bu10H group);busulfan 40 mg/kg high temperature treatment group(Bu40H group).Busulfan was injected intraperitoneally twice at different doses on day 0 and 14.The results are as follows:1.First,it was found that busulfan and high temperature treatment had no significant effects on the growth performance of male and female yellow drum in each group,but the impact on the development has obvious gender differences: between groups of male gonad weight and gonadosomatic index(GSI)than the control group significantly reduced(P <0.05),but each females gonad weight and GSI have no significant difference(P < 0.05).In addition,there was almost no death in Bu10 and Bu40 groups,while the survival rate in Bu10 h and Bu40 h groups was low,indicating that the combined treatment of high temperature and busulfan had a greater impact on the survival rate of yellow drum.2.Samples were taken at the 14 th,28th,38 th and 48 th days of the experiment,and histological analysis was conducted to determine the effects of busulfan and high temperature treatment on the expression of gonadal germ cells and related genes.The results showed that on the 14 th day after busulfan injection,the germ cells apoptosised and cavities appeared in the testis of male yellow drum,and the elimination effect of germ cells in the high concentration group(Bu40 group)was more obvious than that in the low concentration group,showing a certain concentration effect.There was no cumulative effect on the germ cells of male fish treated with busulfan-high tempurature.The female ovaries were not affected by busulfan treatment and high temperature treatment,which proved that the effects of busulfan treatment on the germ cells of yellow drum were different between the sexes.3.The germline(vasa)and somatic marker genes(male: dmrt1;female: cyp19a).The fluorescence quantitative results showed that the expression of vasa gene in the gonad of male yellow drum was significantly inhibited compared with the control group on the 14 th and 28 th day after busulfan injection,and the higher the treatment concentration,the higher the degree of inhibition,while high temperature would enhance the inhibition effect,but the difference was not significant.On the 38 th and 48 th days of treatment(10 days of recovery),the expression of vasa gene in the gonadal gland of male fish in each group was still significantly inhibited compared with the control group.However,there was no significant difference in vasa gene expression between the groups and the control group.There were no significant changes in the expression of dmrt1 gene and cyp19 a gene in gonadal gland of male fish compared with control group.In situ hybridization analysis of vasa showed that vasa gene was mainly expressed in germ cells.In the control group,the expression of vasa gene was normal,mainly expressed in sperm and spermatocyte,while in the BU10,BU40 and Bu40H groups,the signal was weak,which further proved that busulfan treatment led to germ cell apoptosis.However,the expression of vasa gene in females in each group was normal and mainly expressed in the cytoplasm of oocytes.By in situ hybridization analysis of cyp19 a gene,it was found that cyp19 a gene signal existed in both control group and treatment group,and the signal existed in germ cells and surrounding somatic cells.In conclusion,busulfan treatment caused apoptosis of germ cells in testis of male yellow drum,and the gene expression of germ cells was inhibited,but had no significant effect on the germ cells and related genes of female drum.For male and female fish,busulfan treatment did not affect somatic cell related gene expression.High temperature treatment will enhance the elimination of germ cells,but it is easy to cause a high mortality,so it is not suitable to use in actual production.Based on the optimization of the above experimental results,this study concluded that the sterile yellow drum could be prepared by injecting busulfan with 40 mg/kg twice at intervals.