Establishment And Application Of Detection Methods For Blood Estradiol And Testosterone In Acipenser Sinensis

Due to the deterioration of the ecological environment of the Yangtze River,the key habitat area of Acipenser sinensis has been gradually reduced,the fitness has decreased,and natural reproduction has changed from continuous to sporadic.After natural resources are destroyed,it's difficult to recover spontaneously in a short period of time,and wild resources are on the verge of extinction.The success of controlled reproduction has made it possible to preserve germplasm resources and restore wild populations.However the second sexual characteristics of A.sinensis are not obvious,it's difficult to judge the reproductive state accurately,the rate of oxytocin and the fertilization rate are low during the controlled reproduction,which affect controlled reproduction seriously.Blood steroid hormones such as testosterone and estradiol are associated with gonad development in fish,which can reflect the reproductive status of variety of fishes,and determine fish sex and gonadal development period accurately.Therefore,it's necessary to monitor the reproductive status of A.sinensis by using blood testosterone and estradiol levels continuously,and select sexual mature individual in aquaculture environment effectively.Taking A.sinensis's whole blood as test object,estradiol and testosterone were the detection targets.During pretreatment process,the sample was extracted with acetonitrile and purified by primary secondary amine?PSA?.The blood estradiol was detected by high performance liquid chromatography-fluorescence?HPLC-FLD?method and blood testosterone was detected by high performance liquid chromatography-mass spectrometry?HPLC-MS?.The results showed that the standard curve of estradiol in the range of 0.5-50 ng/mL was y=0.528x-0.1325,the correlation was good?R²=0.999?,the detection limit was 0.40 ng/mL;The standard curve of testosterone was y=0.4978x+0.0339,R²=0.999 in the range of 0.1-5.0 ng/mL,and the detection limit is 0.05 ng/mL.Two recovery rates were between 80%and 120%,the peak time were almost the same,the precision were good,and the relative standard deviation?RSD?is less than 2%.The whole blood was used to save samples and could meet the detection of blood estradiol and testosterone levels in A.sinensis.The 9-17-year-old cultured A.sinensis were used to collect the gonadal development image by B-ultrasound,and the genomic DNA sex-specific bands were amplified by extracting from fin,and the blood estradiol and testosterone levels were detected by HPLC and HPLC-MS respectively.The following results were obtained:the majority?77.66%?of gonad development among the 9-17-year-old cultured A.sinensis were in stage II,and the stage III and stage IV accounted for 22.34%;The genomic DNA specific bands appeared in female;The gonads of A.sinensis were developed to the stage III at the earliest age of 9-year-old,At same age,A.sinensises were not consistent in gonad development period completely in different genders.The level of estradiol and testosterone in the females were the same basically.The average levels of testosterone was lower in different age groups,and the levels of estradiol was higher than that of testosterone,and the highest is more than 7.000 ng/mL.The blood testosterone level in male and female blood was obviously changed,the lowest is less than 2.000 ng/mL and the highest is close to 8.000 ng/mL,estradiol was maintained at a low level?below 1 ng/mL?and basically unchanged.The breeding cycle of cultured A.sinensis and the developmental stage of specific age couldn't be determined.It's necessary to judge the development period according to a large number of samples,combined with various judgment methods,and further verify through the breeding experience.The urine of A.sinensis and A.dabryanus was used as the test object,and L-tryptophan was detected by high performance liquid chromatography-fluorescence method.The urine sample was extracted with methanol and acetonitrile,and purified by ENVI-18 solid phase extraction cartridge.The peak area of L-tryptophan standard and its mass concentration were obtained under the fluorescence wavelength?_ex=285 nm,?_em=353 nm of chromatographic conditions.The detection limit?LOD?was 0.005?g/mL,the limit of quantitation?LOQ?was 0.020?g/mL,and the recovery of L-tryptophan spiked was 80%-120%.The test results show that the method for detecting aromatic amino acids in the Acipenser of the Yangtze River is reliable and can be applied to the detection of substances with benzene ring structure in fish biological matrices.The high performance liquid chromatography and mass spectrometry established in this study can detect blood estradiol and testosterone in A.sinensis effectively,High performance liquid chromatography with fluorescence detection for L-tryptophan from urine of sturgeons.It will provide a detection method for further mastering the development of gonads in A.sinensis,so as to select sexual mature broodstock effectively.