Biosynthesis of estradiol-17β in the ovarian follicles of the red seabream Pagrus major during vitellogenesis

ABSTRACT: The red seabream Pagrus major is a useful experimental fish for studying the endocrine control of oogenesis in teleosts. This study investigated the steroidogenic pathway for estradiol-17β (E2) biosynthesis in the ovarian follicles of red seabream. Intact follicles were isolated during vitellogenesis and incubated in vitro with different radiolabeled steroid precursors. When 17-hydroxyprogesterone (17-P), dehydroepiandrosterone (DHEA), or androstenedione (AD) were used as precursors, both testosterone (T) and estrone (E1) were synthesized by follicles, leading to estradiol-17β (E2) production. Serum steroid levels measured by enzyme-linked immunosorbent assay showed that T, E1, and E2 were present in the circulation at levels ranging from 1 ng/mL to 2 ng/mL throughout the day during the spawning season. In vitro conversion of E1 into E2, however, was 15.8-fold greater than T conversion into E2, suggesting that E2 is synthesized mainly via E1 rather than T. The results showed that E2 was synthesized from pregnenolone via 17-hydroxypregnenolone, DHEA, AD, and E1. Thus, the study demonstrated the complete steroidogenic E2 synthesis pathway in the ovarian follicles of red seabream, and revealed that E1 is the major precursor of E2.     

Effects of steroid hormones on immunoglobulin M (IgM) in rainbow trout, Oncorhynchus mykiss

The immunosuppressive effects of steroid hormones were evaluated as the response against implanted steroid hormones, cortisol (F), testosterone (T), estradiol-17 (E2), and 11- ketotestosterone (11-KT), in juvenile rainbow trout. In long term experiments (5 weeks), fish were given a single intraperitoneal implant of F or T. A clear suppressive effect of plasma IgM levels with F and T was not necessarily obtained, although mucus IgM levels were reduced corresponding to the elevated plasma steroid hormone levels. In short term experiments (1 week), intraperitoneal implantation of T, 11-KT and E2 suppressed plasma and mucus IgM levels, although the effects were not dose-dependent. When administered through diet, F and T caused a suppression of plasma IgM levels; F administration at both high and low dosages caused a significant decrease in plasma IgM levels, while only a high dose of T caused the suppression. These results suggest that sex steroid hormones, as well as F, have immunosuppressive functions in rainbow trout.     

Development of a label-free immunosensor system for detecting plasma cortisol levels in fish

Fishes display a wide variation in their physiological responses to stress, which is clearly evident in the plasma corticosteroid changes, chiefly cortisol levels in fish. In the present study, we describe a novel label-free immunosensor for detecting plasma cortisol levels. The method is based on immunologic reactions and amperometric measurement using cyclic voltammetry. For the immobilization of the antibody on the surface of sensing electrode, we used a self-assembled monolayer of thiol-containing compounds. Using this electrode, we detect the CV signal change caused by the generation of antigen–antibody complex. The immunosensor showed a response to cortisol levels, and the anodic peak value linearly decreased with a correlation coefficient of 0.990 in diluted plasma. The specificity of the label-free immunosensor system was investigated using other steroid hormones, such as 17α, 20β-dihydroxy-4-pregnen-3-one, progesterone, estriol, estradiol, and testosterone. The specific detection of cortisol was suggested by a minimal change from ?0.32 to 0.51 μA in the anodic peak value of the other steroid hormones. The sensor system was used to determine the plasma cortisol levels in Nile tilapia (Oreochromis niloticus), and the results were compared with those of the same samples determined using the conventional method (ELISA). A good correlation was obtained between values determined using both methods (correlation coefficient 0.993). These findings suggest that the proposed label-free immunosensor could be useful for rapid and convenient analysis of cortisol levels in fish plasma samples.     

Neonatal isoerythrolysis in newborn foals]

Aetiology, pathogenesis, clinical symptoms, diagnosis, prophylaxis and therapy of neonatal isoerythrolysis in foals are presented. Neonatal isoerythrolysis is caused by isoimmunisation of a brood mare to the Aa and Qa erythrocyte antigens of the foal. The disease can develop, when the mare does not possess Aa resp. Qa blood group antigens, is sensitized to the Aa or Qa erythrocyte antigens--i.e. through pregnancy, parturition, blood resp. plasma transfusions, etc.--and the foal suckles colostral antibodies to its own blood cells. Aa and Qa antibodies can cause haemagglutination and haemolysis in the foal, with a consequent decline in erythrocytes, PCV and haemoglobin resulting in several clinical symptoms. In most instances the first signs of the disease are noticed by day 2 and 3, ranging from 8 to 96 hours of life. Diagnosis is based upon clinical examination and determination of erythrocyte count, PCV and haemoglobin concentration and can be further confirmed by immunological tests. Several tests can be used to prevent the occurrence of neonatal isoerythrolysis in the newborn foal. Prior to parturition, brood mares can be typed for blood groups and tested for antibodies to Aa and Qa in order to identify mares at risk for causing neonatal isoerythrolysis in the foal. After birth, compatibility of the mare's colostrum and the foal's erythrocytes can be checked by the "jaundice foal agglutination" test. Some instructions for prophylaxis of neonatal isoerythrolysis and for the treatment by red blood cell resp. whole blood transfusions are given.     

Cortisol effects on the testicular androgen synthesizing capacity in common carp, Cyprinus carpio L.

Our previous studies on the effect of stress on pubertal development in carp have shown that repeated temperature changes caused an increase in cortisol levels and a retardation of the first waves of spermatogenesis. Identical effects, accompanied by a decrease in 11-ketotestosterone (11KT) plasma levels and the gonadosomatic index (GSI) were induced by cortisol administration via cortisol containing food pellets. The decrease in plasma 11KT is caused by a direct effect of cortisol on the steroid producing capacity of the testis, independent of luteinizing hormone (LH) levels. However, the mechanism through which cortisol interferes with testicular steroidogenesis is unknown. In the present study, we showed that in vitro physiological levels of cortisol can inhibit the conversion of 11β-hydroxyandrostenedione (OHA) into androstenetrione (OA), which is the precursor of 11KT, possibly by competing for the enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD). The same mechanism may occur in vivo. However, our results demonstrate that an elevation of plasma cortisol levels during acute cortisol treatment did not result in lower plasma levels of OA and 11KT, but we did observe an accumulation of OHA. We suggest that the previously observed decrease in 11-oxygenated androgens, as an effect of long-term cortisol treatment, is caused by a retardation of testicular development. This results in a lower steroid synthesizing capacity of the testis as a whole. Although the in vitro observed cortisol inhibition of the conversion of OHA into 11KT plays a role in the accumulation of OHA, it apparently has no effect on the final 11KT plasma concentration. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effects of dopamine 2 receptor antagonist on sex steroid levels, oocyte maturation and spawning performances in Waigieu seaperch (Psammoperca waigiensis)

The present study has investigated the effects of domperidone (DOM), a dopamine 2 receptor antagonist, on plasma steroid hormone levels and reproductive performances of a female tropical marine finfish, Waigieu seaperch (Psammoperca waigiensis), with potential for cultivation in Vietnam. We showed that oral treatment of DOM during early stage of the reproductive cycle had no significant effects on the maturation and reproductive performances of the female fish, while plasma steroid hormone (testosterone (T), 11-ketotestosterone (11-KT), 17β-estradiol (E2) and progesterone (P)) levels were modulated based on month, DOM dose and the individual hormones measured. Overall, these findings suggest that DOM may not be needed for the induction of maturation and spawning of this species under aquaculture conditions. The data in the present study are significant in highlighting practical efforts for reducing drug use, production costs and for a sustainable aquaculture in a developing country such as Vietnam.     

Diurnal rhythm of steroid biosynthesis in the testis of terminal phase male of protogynous wrasse, Pseudolabrus sieboldi, a daily spawner

The wrasse, Pseudolabrus sieboldi, is a diandric protogynous labrid fish. Spawning is performed by a terminal phase (TP) male and an initial phase (IP) female between 6:00 and 9:00 h daily during two-month-long spawning season. In the present study, to investigate the roles of steroid hormones in the diurnal spermatogenesis of the P. sieboldi TP male, all steroid hormones produced in the testis were identified and the synthetic pathways of these steroids were determined. Furthermore, the circulating levels of the major steroids produced were analyzed throughout a day at 3-hour intervals during spawning season. In the testis, 11-ketotestosterone (11-KT), estradiol-17β (E2), 17,20β-dihydoxy-4-pregnane-3-one (17,20β-P) and 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S) were synthesized as the major metabolites. In vitro steroid biosynthesis experiments showed similar results to the circulation profiles of the major steroids. This study is the first to clarify the complete steroidogenic pathways in the gonads of a diandric protogynous species throughout its life, when combined with the results of the steroidogenesis in the ovarian follicles. This is also the first report of a clear diurnal rhythm of the steroid production corresponding to the spermatogenic process in the testis of a male teleost.     

Serum steroid profiles in artificially maturing female Japanese eel,Anguilla japonica

To investigate whether steroid profiles in salmon pituitary homogenate (SPH)-induced artificially maturing Japanese eel, Anguilla japonica, resemble those in other, naturally maturing fishes, the daily changes in 11 steroids were analyzed for a 70-day period (average time needed to reach the maturational phase). Concentrations of most steroids were low and changed on a weekly basis, with maximum values 2–5 days after an SPH injection. Thus, pregnenolone, 17α-hydroxypregnenolone, dehydroepiandrosterone, progesterone, 17α-hydroxyprogesterone, 17α,20 β-dihydroxy-4-pregnen-3-one, androstenedione and estrone levels were barely or not detectable in serum throughout the experimental period, which is largely in keeping with what is known about oogenesis-related steroids in other fishes. In contrast, serum testosterone (T) levels were high, but fluctuated considerably with each SPH injection (about 0.3–8.3 ng/ml). The serum estradiol-17β (E₂) levels increased after SPH injections and gradually rose throughout the experiment, peaking at the end of the experimental period (about 0.2–7.8 ng/ml). Serum levels of 11-ketotestosterone (11-KT) before SPH treatment were higher (approximately 2 ng/ml) than those of the other steroid hormones (less than 0.5 ng/ml). 11-KT levels increased gradually over the experimental period, and, like E₂, levels peaked towards the end of the experimental period (about 15 ng/ml). The observed patterns for T, E₂ and 11-KT are unlike those in other fishes. Furthermore, the consistent elevations in levels of 11-KT, both before and after SPH treatment, are suggestive of an important role for this steroid in controlling oocyte growth.     

Effects of clove oil and MS-222 on blood hormone profiles in rainbow trout Oncorhynchus mykiss, Walbaum

Clove oil has been demonstrated to be an effective, inexpensive anaesthetic and euthanizing agent for a number of fish species, including rainbow trout, used in aquaculture and fisheries research. However, the potential for clove oil to cause perturbations in important plasma hormone concentrations has not been investigated. The effect of anaesthesia and euthanasia in trout with eugenol (the active ingredient in clove oil) on plasma cortisol, glucose, growth hormone (GH) and two thyroid hormones [tri‐iodothyronine (T₃) and thyroxine (T₄)] was compared with tricaine methanesulfonate (MS‐222) anaesthesia, and stunning by cranial concussion in two experiments. Effects on blood chemistry were different when comparing the particular anaesthetic method being used. Stunning fish significantly increased plasma cortisol and glucose levels (both P<0.05), while euthanizing fish using either clove oil or MS‐222 had no effect on these hormone levels. In contrast, the levels of GH, T₃ and T₄ hormones were unaffected regardless of whether fish were euthanized by stunning, MS‐222 or clove oil. Variation in effects between hormones were observed using clove oil eugenol. In fish sampled 10 min after anaesthetizing with 150 mg L^?1 of eugenol, cortisol levels were significantly decreased (P<0.03), while there were no differences in either glucose or GH levels. Tri‐iodothyronine and T₄ also showed significantly elevated levels (P<0.05) after 10‐min exposure to eugenol. These results highlight the importance of investigating the potential effects of any new anaesthetic or euthanizing compounds on blood plasma parameters, prior to using them in a research setting, or when comparing results to other studies which have utilized alternative anaesthetic compounds.     

Deviation of habitat salinity during seasonal gonad recrudescence affects plasma sex steroid levels and suppresses gonadal maturation in an euryhaline fish Etroplus suratensis

Impact of osmoregulation on plasma sex steroid levels and gonadal histo‐architecture was monitored to elucidate the effects of deviation from habitat salinity on gonadal recrudescence in an active reproductive season of an euryhaline fish Etroplus suratensis (pearlspot). Fish were maintained in three different salinities of 0 ppt Fresh Water (FW), 15 ppt Brackish Water (BW) and 30 ppt Sea Water (SW) for a period of 60 days. Plasma osmolality values were found to be significantly highest in SW‐acclimated fish accompanied by highest levels of plasma K? and Cl¯ ions. The progress of gonadal recrudescence was higher in BW followed by FW and SW as evident from the cellular features of gonads and increased level of plasma sex steroids, such as, in case of female and 11‐keto Testosterone and Testosterone in case of males. Plasma cortisol levels were comparatively higher in fish of both sexes in SW group. Significantly high levels of cortisol in SW suggest its role in hypo‐osmoregulation and associated stress. This study clearly reveals that salinity changes during the active reproductive phase can suppress the steroid‐mediated gonad recrudescence maximally under hypo‐osmoregulation in an euryhaline fish.     

Gonad histology and plasma steroid profiles in wild New Zealand freshwater eels (Anguilla dieffenbachii and A. australis) before and at the onset of the natural spawning migration. I. Females*

To determine steroid profiles in immature and maturing female eels from the wild, non-migratory and migratory New Zealand longfinned (Anguilla dieffenbachii) and shortfinned (A. australis) eels were caught and blood and ovarian samples collected. Plasma steroid levels were determined and related to the developmental stage of the ovary. Ovaries of non-migrants contained oogonia and previtellogenic oocytes. Vitellogenic oocytes were never observed in these groups, but instead were very common among migrants (up to 88% of oocytes). Concentrations of both androgens (androstenedione (AD), testosterone (T)) and estradiol-17 (E2) were higher in migrants than in non-migrants. Among migrants, T levels were higher in shortfins (2.27 ± 0.14 ng ml–1) than in longfins (0.82 ± 0.10 ng ml–1), whereas E2 levels were higher in longfins (mean 2.46 ng ml–1) than in shortfins. Levels of sex steroids were generally low in non-migrants. In contrast, plasma levels of 17-hydroxyprogesterone were significantly higher in non-migrants than in migrants. Similarly, cortisol levels were higher in non-migrating than in migrating shortfinned, but not longfinned, females. 17,20-Dihydroxy-4-pregnen-3-one, the putative maturation-inducing steroid in anguillids, was near minimum-detectable levels for all animals examined. Surprisingly, very high levels of 11-ketotestosterone (KT) were found in migrants, averaging nearly 3 ng ml–1 in longfins and over 20 ng ml–1 in shortfins. The identity of KT and several 5-reduced androgens was confirmed using gas chromatography - mass spectrometry. The function of KT in females is not known, but we suggest that this steroid hormone may play a role in preparing maturing animals for their spawning migration.     

Further evidence of the equivocal effects of cortisol on in vitro steroidogenesis by ovarian follicles of rainbow trout Oncorhynchus mykiss

Previous studies on salmonids have yielded equivocal results as to the role of cortisol in directly inhibiting ovarian steroidogenesis. In an effort to determine why this might be so, isolated ovarian follicles of rainbow trout were incubated with and without cortisol under varying conditions of gonadotropin or steroid precursor stimulation, incubation time and temperature. Cortisol at concentrations of 100–1000 ng ml–1 suppressed basal production of 17-estradiol (E2) in only 4 out of 20 experiments, had no effect on human chorionic gonadotropin (hCG)-stimulated production, and no suppressive effect on 17-hydroxyprogesterone (17P)-stimulated production in 14 experiments, but increased E2 production in response to 17P in 2 experiments. Cortisone had no effect on basal E2 production, suggesting that cortisol was unlikely to be exerting any effect indirectly after further metabolism. Extended incubation times at 12 °C resulted in overall decreased levels of E2 in incubation media, but this had no obvious effect on patterns generated by treatment with cortisol. Extended incubation at 18 °C did change the pattern of response to treatment with cortisol in 1 out of 3 experiments. All incubations examined produced substantial amounts of E2-glucuronide but this showed no obvious relationship to whether or not inhibition of E2 production by cortisol was observed. Effect of stress history was examined by incubating follicles from stressed or unstressed fish. In follicles from fish nearing the end of vitellogenesis, stress resulted in reduced production of both testosterone and E2 in response to hCG, but increased conversion of 17P to E2. The same effect was not observed in follicles from fish at an earlier stage of vitellogenesis. Measurement of E2 uptake by follicles from selected experiments showed that follicles contained considerable amounts of E2 and were potentially a sink for steroid produced during incubation. The experiments show that a consistent effect of cortisol on ovarian steroidogenesis remains elusive, but that stimulatory effects are as likely to occur as inhibitory effects. All responses are potentially further confounded by loss of free steroid from the medium by conjugation or absorption into the oocytes.     

The influence of reproductive status on the stimulatory action of N-methyl-D,L-aspartate on growth hormone secretion, in vitro in rainbow trout, Oncorhynchus mykiss

The glutamate agonist, N-methyl-D,L-aspartate (NMA) stimulates the secretion of growth hormone (GH) from pituitary fragments in vitro and increases plasma GH levels in vivo in rainbow trout, Oncorhynchus mykiss (Flett et al. 1994; Holloway and Leatherland 1997a,b); however gonadal steroid hormones appear to modulate this response in experimental situations. This study examines whether steroid hormones also modulate the GH-regulatory actions of NMA during the normal reproductive cycle of rainbow trout by examining the relationship between the stage of sexual maturation and the pituitary release of GH in vitro in response to an NMA (10^-8 M) challenge. NMA had no effect on mean GH release from the pituitary glands of fish that were immature (GSI <1.0), from males during early development (GSI 1.0-3.0), or from sexually mature males (with free running milt) and females (ovulated). However, NMA significantly increased GH release from pituitary glands taken from females during the early stages of gonadal growth (GSI 1.0-9.0) and from males and females sampled during the later stages of gonadal growth (males GSI 3.01-6.0; females GSI 9.01-15.0). The GH-stimulatory action of NMA in males and females progressed to a maximum effect during the late stages of gonadal growth, and disappeared in ovulated females and free running males. Moreover, in female fish, the maximal GH release in response to the NMA challenge is positively correlated with plasma 17β-estradiol levels; no such correlation was evident for plasma testosterone levels in males. Changes in the GH response to NMA during maturation while gonadal steroid levels fluctuate provides further evidence to suggest that the effects of NMA on GH secretion are intimately linked to endogenous gonadal steroid hormone levels. This revised version was published online in July 2006 with corrections to the Cover Date.     

The effects of arachidonic acid on the endocrine and osmoregulatory response of tilapia (Oreochromis mossambicus) acclimated to seawater and subjected to confinement stress

In previous studies in freshwater tilapia (Oreochromis mossambicus), dietary supplementation with arachidonic acid (ArA; 20:4n?-?6) had considerable, opposing effects on the main ion-transporting enzyme Na(+)/K(+)-ATPase in gills and kidneys and changed the release of osmoregulatory hormones, such as cortisol. The present study was performed to assess the influence of dietary ArA on (1) the osmoregulatory capacity of tilapia acclimated to seawater (SW) (34‰) and (2) the osmoregulatory imbalance associated with acute stress. The increased ambient salinity was associated with significant alterations in the tissue fatty acid composition, particularly the n?-?6 polyunsaturated fatty acids (PUFAs). Tissue levels of ArA were further increased as a result of dietary supplementation, whereas docosahexaenoic acid (DHA, 22:6n?-?3) and eicosapentaenoic acid (EPA, 20:5n?-?3) decreased in gills and kidneys. Basal plasma cortisol as well as lactate levels were elevated in the ArA-supplemented SW-acclimated tilapia compared with the control group. The 5?min of confinement (transient stress) increased plasma cortisol, glucose, and lactate levels with significantly higher levels in ArA-supplemented tilapia. Confinement was also associated with significantly elevated plasma osmolality, sodium, chloride, and potassium levels. ArA-supplemented tilapia showed markedly lower ionic disturbances after confinement, suggesting that dietary ArA can attenuate the hydromineral imbalance associated with acute stress. These results emphasize the involvement of ArA and/or its metabolites in the endocrine and osmoregulatory processes and the response to confinement stress.     

Postcoital uterine microbe colonization and endometritis in the mare

In the mare, natural breeding is associated with bacterial contamination of the reproductive tract. The purpose of this study was to examine postcoital bacterial contamination and the resulting inflammatory response of the uterus. Uterine swabs for bacteriological and cytological examination were obtained from 80 mares. Each mare was sampled once between 4 and 69 hours postbreeding. In mares which did not conceive, sampling was repeated at the following estrus. The findings were compared with those obtained prior to breeding and correlated with the breeding outcome. Bacteria were cultured from 72.5% of the postcoital swabs. There was a wide spectrum of organisms which included species known as potential causes of endometritis. Neutrophilic granulocytes were found in varying concentrations in 48.8% of cases. In 16.3% of mares both bacteriological and cytological examinations were negative. Mares with positive bacteriological and/or cytological results at the postcoital examination had better foaling rates compared to the remaining mares (p less than 0.05). The postcoital findings did not correlate with those of the prebreeding examination, or with the interval between breeding and sampling, or with the different stallions.     

Seasonal reproductive cycle of Waigieu seaperch (Psammoperca waigiensis)

Predictive and reliable parameters of reproductive status are integral aspects of sustainable fisheries and aquaculture management. These parameters are also important for an accurate evaluation of the effects of different treatments on sexual maturation in fish farming. In the present study, we have characterized the seasonal reproduction profile and described changes in sex steroids in relation to gonadal maturation and development in female and male Waigieu seaperch (Psammoperca waigiensis). The experimental period covered a full calendar year (January–December). In males and females, we observed that plasma sex steroid hormones [oestradiol‐17β (E2), testosterone (T), 11‐ketotestosterone (11‐KT) and progesterone (P)] levels showed monthly fluctuations during the spawning period. Particularly, plasma steroid hormone levels were positively associated with gonadosomatic index values. In addition, high levels of plasma steroid coincided with recruitment of oocytes into yolk accumulation in females. The main spawning period occurred between April and October in females, and between March and November in males. The non‐aromatizable androgen, 11‐KT is generally believed to be the active male‐specific androgen in teleosts, and is associated with the process of spermiation, development of secondary sexual characteristics and regulation of male reproductive behaviour in most teleost species. In this study, we found relatively high amounts of 11‐KT in females between May and December, suggesting an integral role in the maturation process, also for the females. A rapid peak in plasma P level was observed in November and suggests significant roles during post‐spawning and/or resting periods in both female and male fish. Furthermore, all oocyte developmental stages were present within the same sampling month and also within the spawning period, demonstrating the gamete group asynchronous developmental strategy. Overall, Waigieu seaperch showed strong seasonality in reproductive development with corresponding sex steroid patterns. The data presented in this study may contribute to the understanding of the reproductive endocrinology of a tropical marine finfish with increasing industrial prospects and sustainable aquaculture of this species in a developing country, such as Vietnam.     

Capture and handling stress affects the endocrine and ovulatory response to exogenous hormone treatment in snapper, Pagrus auratus (Bloch & Schneider)

Sexually mature female hatchery‐reared snapper, Pagrus auratus (Bloch & Schneider) were captured from sea cages by handline and injected at first capture (control) or 24 h after capture, transport and subsequent confinement (delayed injection) with either saline, luteinizing hormone releasing hormone analogue, human chorionic gonadotropin, or 17α‐hydroxyprogesterone. Blood was sampled before hormone treatment and again after 168 h, and fish were checked daily for ovulation. Plasma levels of 17β‐estradiol (E2), testosterone (T), 17α, 20β dihydroxy‐4‐pregnen‐3‐one (17, 20βP) and cortisol were determined by radioimmunoassay. The ovulatory response was assessed from the proportion of fish ovulating, ovulation volume, egg quality and fertility. A delay in injection resulted in significantly lower plasma E2 and T levels in response to hormone treatment, smaller ovulation volumes, and poorer egg quality than in control fish. The results are consistent with the generally inhibitory effects of stress on reproduction in fish, and confirm the requirement to treat fish with hormones designed to induce ovulation, as soon as possible after capture and disturbance.     

Development of a modified cortisol extraction procedure for intermediately sized fish not amenable to whole-body or plasma extraction methods

The corticosteroid hormone cortisol is the central mediator of the teleost stress response. Therefore, the accurate quantification of cortisol in teleost fishes is a vital tool for addressing fundamental questions about an animal’s physiological response to environmental stressors. Conventional steroid extraction methods using plasma or whole-body homogenates, however, are inefficient within an intermediate size range of fish that are too small for phlebotomy and too large for whole-body steroid extractions. To assess the potential effects of hatchery-induced stress on survival of fingerling hatchery-reared Spotted Seatrout (Cynoscion nebulosus), we developed a novel extraction procedure for measuring cortisol in intermediately sized fish (50–100 mm in length) that are not amenable to standard cortisol extraction methods. By excising a standardized portion of the caudal peduncle, this tissue extraction procedure allows for a small portion of a larger fish to be sampled for cortisol, while minimizing the potential interference from lipids that may be extracted using whole-body homogenization procedures. Assay precision was comparable to published plasma and whole-body extraction procedures, and cortisol quantification over a wide range of sample dilutions displayed parallelism versus assay standards. Intra-assay  %CV was 8.54 %, and average recovery of spiked samples was 102 %. Also, tissue cortisol levels quantified using this method increase 30 min after handling stress and are significantly correlated with blood values. We conclude that this modified cortisol extraction procedure provides an excellent alternative to plasma and whole-body extraction procedures for intermediately sized fish, and will facilitate the efficient assessment of cortisol in a variety of situations ranging from basic laboratory research to industrial and field-based environmental health applications.     

Cortisol and liver metabolism of immature American eels,Anguilla rostrata (LeSueur)

Plasma and liver composition, liver enzyme activities, and metabolite flux in isolated hepatocytes have been studied in immature American eels,Anguilla rostrata, injected daily IP with saline or cortisol (0.35 mg/kg eel). Plasma cortisol values were significantly increased above saline controls in those eels receiving cortisol at 3h and 6h following the final (tenth) injection. On day 6 and 10 of injection plasma cortisol levels were significantly below saline controls 24h following cortisol injection. Plasma glucose values were significantly depressed in the cortisol-injected eels at both 6 and 24h following the final (tenth) injection. At the 24h sampling time, plasma protein had significantly increased, but there was no change in either plasma amino acid or fatty acid levels. An increased hepatosomatic index was attributed to a major increase in total lipids, as both protein and glycogen contents were decreased. Of the liver enzymes assayed, significant activity changes occurred only for lactate dehydrogenase (decreased), mitochondrial citrate synthase (increased) and phosphoenolpyruvate carboxykinase (increased) 24h following the final (tenth) cortisol injection. Although enzyme activity changes implied increased liver gluconeogenesis, the absolute rate of lactate, alanine, and aspartate incorporation into glucose declined in viable hepatocytes isolated from cortisol-injected eels compared to the saline controls. Relative changes in metabolite flux did support a preferential increase in gluconeogenesis from amino acids. These results are consistent with the increase in amino acid gluconeogenesis as a result of cortisol administration implied in previous studies, but failed to show a definitive cortisol effect on this pathway in the eel liver. It is suggested that other hormones (e.g. thyroxine, catecholamines, glucagon) may interact in a complex way with cortisolin vivo to bring about the biochemical changes observed in this study. The rapid clearance of exogenously injected cortisol noted in this study makes causal relationships between the injected hormone and any observed metabolic effect in the intact animal difficult.     

Endocrine changes during the annual reproductive cycle of the red porgy, Pagrus pagrus (Teleostei, Sparidae)

Changes in 17-estradiol (E2), estrone (E1), testosterone (T), 11-ketotestosterone (11KT), and 17,20-dihydroxy-4-pregnen-3-one (17,20-P) levels were correlated to changes in gonadosomatic index (GSI), vitellogenin concentration (Vg), ovarian and testicular histology during the annual reproductive cycle of the red porgy, Pagrus pagrus. The production of E2, E1, T and 17,20-P was confirmed by analysis of the steroidogenic activity of ovaries. In females, the average concentration of E2 was lower than 2 ng ml^–1. E2 values first increased significantly at the stage of endogenous vitellogenesis and remained high during exogenous vitellogenesis. E1 levels were lower values than E2 (less than 300 pg ml^–1), but they increased at the beginning of exogenous vitellogenesis. Estrogens concentrations followed similar pattern to Vg and were significantly correlated. Mean levels of T were mostly lower than 1 ng ml^–1. They followed a pattern similar to that of E2 except for a further increase observed at the stage of final maturation. T and E2 levels were significantly correlated. The concentration of 11KT did not change significantly. The levels of 17,20-P ranged between 0.22 and 1.22 ng ml^–1 but changes were not related to gametogenesis. In males, the concentrations of T and 11KT fluctuated significantly during the sexual maturity stages, showing a similar pattern and were significantly correlated to GSI changes. T levels increased during spermiogenesis and spermiation stages to reach about 3 ng ml^–1. 11KT levels stayed about half those of T. The levels of estrogens showed no significant changes. Level of 17,20-P showed no significant variation related to male maturity. Results are discussed in relation to changes in plasma steroid levels during gametogenesis of other multiple spawner species.