白消安及其与高温联合处理对黄姑鱼(Nibea albiflora)性腺发育的影响

EFFECTS OF BUSULFAN AND ITS COMBINED TREATMENT WITH HIGH TEMPERATURE ON GONADAL DEVELOPMENT OF YELLOW DRUM(NIBEA ALBIFLORA)

黄姑鱼(Nibea albiflora)的性成熟周期短,养殖技术成熟,是合适的生殖细胞移植受体。不育受体的制备在鱼类生殖细胞移植中非常关键。为了优化不育黄姑鱼的制备方法,采用白消安和白消安-高温(30℃)对黄姑鱼进行处理,设置5个组:对照组(Con组)、白消安10 mg/kg组(Bu10组)、白消安40 mg/kg组(Bu40组)、白消安10 mg/kg高温组(Bu10H组)、白消安40 mg/kg高温组(Bu40H组),白消安以不同剂量进行2次的腹腔注射,间隔两周。采用组织学、qPCR以及原位杂交等方法分析白消安及其与高温联合处理对黄姑鱼性腺及其生殖细胞和体细胞发育的影响。研究结果表明白消安处理对黄姑鱼生殖细胞的影响具有明显的性别差异:雄鱼在处理28 d后其性腺重和GSI较对照组显著下降,进一步组织学观察发现雄鱼精巢在处理14 d时已经出现空腔,qPCR分析发现雄鱼vasa基因表达在处理期间显著降低,且在处理第48 d仍处于下调状态。然而,白消安及其与高温联合处理并未对雌鱼生殖细胞产生明显的影响。研究还发现高温处理组出现较高的死亡率,但雄鱼生殖细胞的消除效果并未显著增强。因此,白消安处理能够消除黄姑鱼雄鱼性腺内的生殖细胞,以40 mg/kg白消安2次(间隔两周)注射后消除效果最好,能够作为生殖细胞移植受体鱼。

Yellow drum(Nibea albiflora)is a suitable recipient for germ cell transplantation due to its short sexual maturation and fully-developed aquaculture technology.Sterile recipient fish is critical for the success of germ cell transplantation.To optimize the preparation method of sterile yellow drum,we use treatments of busulfan-only and busulfan-high-temperature to the yellow drums.Five groups were set up:the control(Con),groups with 10 mg/kg busulfan injection(Bu10),40 mg/kg busulfan injection(Bu40),10 mg/kg busulfan injection in high temperature(30℃)(Bu10H),and 40 mg/kg busulfan injection in high temperature(30℃)(Bu40H).Busulfan was injected intraperitoneally twice on Day 1 and Day 14,respectively,in above-mentioned experimental dosages.The histology,qPCR,and in situ hybridization were used to analyze the effects of busulfan and the high-temperature combination treatment on the development of gonads,germ cells,and somatic cells.Results show significant gender difference in the effect of busulfan treatment on the germ cells of yellow drum.On Day 28 post treatment,the gonad weight and GSI of male yellow drum decreased significantly compared with those of the control group.Histological observation revealed that cavities in the testis of male individuals were observed on Day 14 post treatment.The qPCR analysis showed that the vasa expression in males was significantly decreased during treatment and remained down-regulated on Day 48 post treatment.However,busulfan-only and busulfan-high-temperature combination treatment had insignificant effect on the germ cells of females.However,the high temperature treatment resulted in high mortalities while the elimination of male germ cells was not significantly enhanced.Therefore,the germ cells in the male gonad could be eliminated by busulfan treatment,and the best treatment was busulfan injection at 40 mg/kg in an interval of about two weeks,which is suitable for the preparation of recipient fish for germ cell transplantation.