| Eel is one of the most important edible and cultivated fish species.But the artificial breeding of eel is still a worldwide problem.Through basic reaearch of the reproductive biology to solve the problem,is the fundamental way to solve eel cultivated.In this study,using exogenous hormones to induce eels ovarian mature.Morphological indexes were detected in the ripening process,and changes of serum and gonad steroid levels of the eels on different stages of ovarian development were also detected.This study provide the method of ultro high performance liquid chromatography-tandem mass spectrometry for target steroid quantitation in the process of the eel maturation.The liquid phase parameters and sample pretreatment method was optimized.This method for the determination of steroid hormones in eel gonad and serum were applicated,which has characteristics of sensitive,high recovery rate,accuracy and stability.The main results were show below:1?Eel can be induced ovarian maturation by repeated injection of CPE(carp pituitary,20mg/kg)and HCG(human chorionic gonadotropin,300IU/kg).Among them,the gonadal maturity coefficient control group unchanged,only the highest(2.25±0.23)%,without gonadal development;the gonads of experimental group mature gradually,and the GSI increased significantly,which highest up to(23.14±3.87)%,the effect of hormones is obvious.2?UPLC–MS/MS method were developed and validated to simultaneously determine 6 steroids hormones in serum samples: Testosterone(T),17?-hydroxyprogesterone(17?-OHP),17?,20?-dihydroxy-4-pregnen-3-one(DHP),estrone(E1),estradiol-17?(E2),estriol(E3).The results showed that the linear range of 6 steroid hormones between 12.5~125 ng/mL,linear relationship is good(r>0.99);The average recoveries obtained from serum samples was 86.9%~98%;The respectively relative standard deviations is 1.7%~9.5%;The detection limit(S/N>10)was 0.1ng/mL.With the increase of the hormone injection times(before spawning),steroid hormones in serum of experimental groups eel were gradually incrensed.Among them,levels of 5 serum hormones of experimental groups were highest when it spawn,expecting for E3(highest when it dystocia).Levels of serum hormones of experimental groups were higher than control group(E1,E3 were not detected).Levels of T,17?-OHP,DHP and E2 were remain at a relatively low level,and the average content were(3.65±0.63),(2.53±0.49),(2.27±0.24),(2.46±0.47)ng/mL.3?The application of UPLC–MS/MS method to simultaneously determine 4 steroids hormones in gonad tissue samples: T,DHP,E2,E3.The results showed that the linear range of 4 steroid hormones between 25~250ng/mL;The average recoveries obtained from gonadal tissue samples was 73.6%~88.1%;The detection limit(S/N>10)was 0.1ng/g.With the increase of the hormone injection times,steroid hormones in gonad of experimental groups eel showed an gradually incrensed trend,and its content is higher than the control group.While the content of E2 and DHP in gonad tissue of experimental groups was highest when it dystocia.The conent of E2 and E3 in gonad of control groups eel were not detected throughout the experimental period.4?Gonad steroid hormone levels were detected by the ELISA.The results showed that levels of gonads P and 17?-OHP were relatively high of the controls(2.83±0.28,19.07±3.03 ng/g),and levels of gonads T,DHP,E3,E1 and E2 were relatively low of the controls(1.07±0.14,0.35±0.08,0.33±0.08 ng/g,33.45±3.46 pmol/L,55.2±4.69 ng/L);With the increase of the hormone injection times,levels of gonads T,P,17?-OHP and DHP increases gradually,while E2 gradually decreased,E1 showed a trend of first increased and then decreased.Among them,levels of gonads P,DHP and E3 were relatively highest in the dystocia group(3.76±0.25,2.41±0.14,1.69±0.23 ng/g);T had the highest hormone level in the spawning group(3.26±0.45 ng/g);Levels of gonads E2,E1 and E2 reached the highest hormone levels after hormone injection at 3,7,11 of experiental group(60.09±2.26 ng/L,46.37±4.37 pmol/L,27.17±1.53 ng/g). |
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