Evaluation of sturgeon follicle-stimulating hormone, luteinizing hormone, estradiol, progesterone and testosterone in Acipenser persicus serum to identify fertile ...
J . Appl. Ichthyol. 15 (1999), 196-198 Q 1999 Blackwell Wissenschafts-Verlag, Berlin ISSN 0175-8659
Evaluation of sturgeon follicle-stimulating hormone, luteinizing hormone, estradiol, progesterone and testosterone in Acipenser persicus serum to identify fertile broodstock By S.H. Safi’, A. Mojabi’, G. Azari Takami’, 1. Nowrouzian’ ,M. Mahmoodi4and S. Bokaei’ ’Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahid Bahomar Universily of Kerman, Kerman, Iran. 2Department of Clinical, Faculty of Veterinary Medicine. University of Tehran, Teheran, Iran, ’Department of Health, Hygenie and Aquatic Diseases, Faculty of Veterinary Medicine, University of Teheran, Teheran, Iran “Department of Epidemiology and Biostatistics, School of Public Health, Teheran University of Medical Sciences, Teheran, Iran
Summary Various methods have been proposed for the selection of suitable sturgeon broodstock for artificial reproduction. In this study, serum levels of reproductive hormones, namely sturgeon homologues of follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T), estradiol (E2) and progestrone (P), were measured by radioimmunoassay (RIA) in 36 mature broodstock before and after injection of pituitary extract, and the results correlated with sex, age and oocyte polarization index (p). Mature males were significantly younger than females, and showed significant differences in serum P and T levels following pituitary extract injection, although not before that. A significant difference was found between LH, E2 and T before and after injection in the female group. There was also a significant difference between serum levels of T and P in the fertile and infertile groups. Stepwise discriminant function analysis was used to discriminate between fertile and infertile groups. The function had 93,8% overall correct classification, i.e. amongst 100 female broodstock fish, almost 94% of them would be correctly allocated to the relevant group. So the proposed model provides a reliable method for selecting suitable broodstock fish. Introduction Water pollution and illegal catches of sturgeon in the Caspian basin have caused a drastic decline in valuable sturgeon populations, obliging the authorities to culture valuable species in hatcheries. Under these artificial conditions, pituitary injections are used to bring broodstock to sexual maturity. Among the different techniques that can be used to determine the maturity of the fish, histological study of ovaries such as measuring the oocyte polarisation index (i.e. the oocyte‘s germinal vesicle position) is apparently simple and precise (Ginzburg and Dettlaff, 1969) but, in practice, often leads to discrepant results. During the period when the nucleus migrates towards the animal pole of ova there is a measurable increase in the level of gonadotropin hormones in the blood of broodstock, and the completion of maturity stages of ova are under the influence of these hormones (Yaron, 1995). Thus, measurement of FSH and LH like hormones and steroid hormones (P, E2 and T) in serum may be a precise method to determine the time of sexual maturity and selection of the best broodstock for artificial propagation. In this study, these hormones were measured by radio-immunoassay in the serum of wild-caught Persian sturgeon (Acipenser persicus) broodstock, before and after standard injections of fish pituitary extract.
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Material and methods Fish The Acipenser persicus broodfish used in this study were all randomly selected from wild-caught stock from the Caspian Sea, which were transferred to the Sturgeon International Research Institute (Rasht, Iran) for artificial propagation. Upon their arrival, male and female fish were segregated and acclimated to local natural water temperatures (11 to 17.5”C from May to June) and photoperiod in kurinski ponds (120 m length, varying from 30 to 10 m width along their length, 2.5 meters in depth), ponds that are specifically designed to simulate natural conditions and thereby promote maturation of sturgeon broodstock. All brood fish selected for the experiment were individually tagged. Fish were fed natural feed. Blood sampling Blood sampling lasted from June 1995 until May 1996. Sampling was done in two steps: the first blood sampling was taken on the day after transferring the fish to the ponds, before pituitary extract injection. This injection followed standard methods (Ginsburg and Dettlaff, 1969). The second sample was collected after the time required for sexual maturity, which varied from 20 to 36 hours after hypophysation depending on the water temperature (Ginsburg and Dettlaff, 1969). Blood samples (10ml) were collected from the caudal vein without anaesthesia, and serum was separated by centrifugation (lOmin at 1500 rpm) and stored at -70°C until analysis. Radioirnrnunoassays Serum samples were assayed for FSH, LH, progesterone, estradiol, and testosterone using radioimmunoassay procedures with commercial kits and according to the instructions provided by the manufacturers (Amerlex-M, Amersham, UK). All assays were based on antibodies against human hormones. The mean recoveries for FSH, LH, progesterone, estradiol, and testosterone were 107Y0, 100.9%, 83,4%, 101.5%, 93% respectively. The minimum detectable FSH, LH, progesterone, estradiol, and testosterone levels were 0.54 Ul/ml, 0.62 Ul/ml, 0.08 ng/ml, 4.7 pg/ml, 0.08ng/ml, respectively. The RIA results for steroid hormones can be regarded as valid but the degree of homology and hence RIA cross-reactivity between human and sturgeon FSH and LH has not been determined and so these RIA results should at present be regarded with some caution. Nonetheless, although the absolute values may not be correct, the results provide an estimate of the concentration of FSH and LH like hormones before and after pituitary extract injection in the two studied groups, and comparisons between and within the groups remain valid.
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Evaluation af sturgeon hormones to identify fertile broodstock Oocyte polarization index (p) A few ova were collected at the same time as the first blood sample and fixed in 5% formaldehyde after Smin in boiling water. Sections were prepared along the polar axis of the ovum as it crossed the germinal vesicle. The oocyte polarization index was calculated by dividing the distance between germinal vesicle and the membrane of the ovum by the diameter of the ovum along the animal-vegetal axis according to Ginzburg and Dettlaff ( 1969). Age determination The pectoral fin ray sections were used for age estimates, according to standard methods. Statistical analysis Student t’test was used to identify the significant differences between groups of fish in the studied parameters and Fischer’s linear discriminant function was used to discriminate between the fertile and infertile fish.
Results As shown in Table 1, the levels of P and T after pituitary extract injection showed a highly significant difference between male and female brood fish (p
