The Variations Of Sex Steroid Hormones Content And The Sperm Density During Artificial Reproduction Of The Male Japanese Eel(Anguilla Japonica)

Japanese eel(Anguilla japonica), affiliated to the Osteichthyes eel Anguilliformes, Anguillidae, Anguilla is Asia’s important economic farmed fish,widely distributed in China.Due to overfishing and habitat destruction, the japanese eel’s dwindling natural resources, and the japanese eel artificial repoduction technology has not yet been commercialized, aquaculture on wild fishing all the eel seedling supply, exacerbating the germplasm resources.So to carry out the entire river eel cultured is very important.In our experiment ultra-performance liquid chromatography coupled with electronspray ionization-triple quadrupole mass spectrometry(UPLC/MS/MS) method was applied to determine the content and changes of 7 steroid hormones in the ovaries of female farmed marbled eels during artificially induced maturation through carp pituitary extract and h CG injection. The results showed that the chromatographic separation of 7 gonad steroid hormones, namely testosterone(T), progesterone(P),estrone(E1),estradiol-17β(E2), estriol(E3), 17α,20β-dihydroxy-4-pregnen-3-one(DHP),and 17α-hydroxyprogesterone(17α-OHP) was well performed using an x Bridge C18 column. The linear regression coefficients(R) of T、P、E1、E2、E3、DHP、17α-OHP were above 0.99 in the range of 0~200ng/ml and the limits of detection were above0.1~0.2 ng/g. Average recoveries of T, P, E1,E2, E3, DHP, 17α-OHP also reached more than 89.00%, and relative standard deviations were less than 7.3%. The results indicated that the 7 steroid hormones increased with the development of gonad maturation, and the UPLC/MS/MS method is a sensitive and stable approach with high recovery rate in simultaneously quantifying 6 gonad steroid hormones in the fish.The results showed that the contents of progesterone(P),estrone(E1),estradiol-17β(E2) and estriol(E3) was so little that almost can not be detected.The contents oftestosterone(T),17,20-dihydroxy-4-pregnen-3-one(DHP) and 17-hydroxyprogesteron-e(17α-OHP) was well performed.In the control group the contnet of T,DHP and17α-OHP was 0、2.9±0.5、0ng/m L(in blood),0.21±0.30、0、2.32±1.65ng/g(in testis).In the group Ⅱ(mature group) the content of T,DHP and 17α-OHP was 0.74±0.23、6.75±2.21、15.6±2.97ng/m L,0.9±0.22、8.24±2.47、17.91±2.53ng/g respectively.In the group Ⅲ(after spermiation) the content was 0、7.87±0.6、5.39±0.66ng/m L,0.87±0.03、0、7.41±0.41ng/g respectively.The research indicates that the content of T、DHP and 17α-OHP was low in the control group,and increased obviously during spermiation and then decrease after spermiation in male eel.By the secretory action analysis of hypohysis gonadotropic hormone(Gt H) cell, it was verified there were many secretory granule,secretory gloube, mitochondria and endoplasmic reticulum in the Gt H cell of group Ⅱ(spermiation group).The Gt H cell metabolic activity was very strong in the group Ⅱ(spermiation group).The study found the control group male anatomy when seminal testis thin and less red meat, refined lobular small semicircle. Are mainly in the testes spermatogonial cells, spermatogonial cell sizes, the average diameter of 10μm, the presence of small interior hollow structure, testes more connective tissue, testis in the first stage Ⅰ : spermatogonia before proliferating. Just ejaculation group male pectoral black eel, larger bust, abdomen soft, gently squeeze the fish abdomen milky semen. After the autopsy found large and gonadal hypertrophy, lobular fine milky white, interlobular white semen. The majority of the testis sperm, a small amount of secondary spermatocytes; Seminiferous wall thinning, decreased testis connective tissue, hollow disappear; sperm motility and volume are large, in a spiral-like motion,part free to students sperm seminal vesicles, testis in the first period ⅴ: fully mature sperm; eel male ejaculation 10 d group who began to appear black spots, belly is not full, there is little squeeze abdomen semen, and occasionally a small amount of yellow liquid outflow. Almost entirely within the testis sperm, but a small number of sperm capsule born; from histological testis in the first period Ⅴ: fully mature sperm.Transmission electron microscopy observed ejaculation group Gt H eel pituitary cells secrete pellets increased efflux portion formed vacuoles. Cells in a very large number of mitochondria, endoplasmic reticulum, and more significantly, the cell metabolism strong. Gt H cell cellular metabolic control and ejaculation 10 d group eel is relatively weak.In this paper, spectrophotometer, studied six wavelengths(380,450,530,600,680 and 780nm), the relationship between the eel(Anguilla japonica) sperm density(D)and the absorbance(A) between. It was found that, at six different wavelengths to eel sperm density of 1 × 107 cells / m L as the lower limit of detection, absorbance increases sperm density increases; fit regression equation based on sperm density and the absorbance was found two times better fit regression equation, which is the optimum detection wavelength of 680 nm, the regression equation was A680 =-0.0007D2 + 0.2579D-0.3615(r = 0.9928). The results showed that the spectrophotometric rapid determination of eel sperm concentration, sperm count standardization program has some role in promoting.In summary, this paper focused on the sex steroid hormones in japanese eel development content changes in different periods and found that some sex steroids with the developmental stages of their content increased significantly; for eel was with a group using the same method artificial propagation, why such a big gap effect,mainly due to their gonadal sex steroids on the content of a large gap, which may be analyzed teleost ovary development has a very important role, especially in promoting oocyte but this article does not conduct in-depth study of its way to transforming the key factor enzyme, therefore, research in this area needs to be continued; the growth and maturation of cells.