Cortisol Implantation Research Articles

Common carp, Cyprinus carpio, prefer branchial ionoregulation at high feeding rates and kidney ionoregulation when food supply is limited: additional effects of cortisol and exercise.

This study aims to examine ionoregulatory parameters during exercise and cortisol elevation in common carp fed different food rations. Fish subjected to two different feeding regimes (0.5 or 3.0% body mass (BM) daily) received no implant or an intraperitoneal cortisol implant (250mg/kg BM) or sham, and were monitored over a 168-h post-implant (PI) period under resting, low aerobic swimming or exhaustive swimming conditions. Plasma osmolality was maintained at relatively stable levels without much influence of feeding, swimming or cortisol, especially in low feeding groups. Nevertheless, a transient hyponatremia was observed in all low feeding fish implanted with cortisol. The hyponatremia was more pronounced in fish swum to exhaustion but even in this group, Na+ levels returned to control levels as cortisol levels recovered (168h-PI). Cortisol-implanted fish also had lower plasma Cl- levels, and this loss of plasma Cl- was more prominent in fish fed a high ration during exhaustive swimming (recovered at 168h-PI). Cortisol stimulated branchial NKA and H+ ATPase activities, especially in high ration fish. In contrast, low ration fish upregulated kidney NKA and H+ ATPase activities when experiencing elevated levels of cortisol. In conclusion, low feeding fish experience an ionoregulatory disturbance in response to cortisol implantation especially when swum to exhaustion in contrast to high feeding fish.

Influence of supraphysiological cortisol manipulation on predator avoidance behaviors and physiological responses to a predation threat in a wild marine teleost fish.

The stress axis in teleost fish attempts to maintain internal homeostasis in the face of allostatic loading. However, stress axis induction has been associated with a higher predation rate in fish. To date, the physiological and behavioral factors associated with this outcome are poorly understood. The purpose of the present study was to investigate the impact of experimental cortisol elevation on anti-predator behavior and physiological responses to predator presence. We hypothesized that semi-chronic cortisol elevation would increase susceptibility to predation by increasing stress-induced risk-taking behaviors. To test this hypothesis, schoolmaster snapper were given cocoa butter implants without cortisol (sham) or with cortisol (50 mg/kg body weight) and tethered to cover. Fish were exposed to either a lemon shark or control conditions for 15-min. Space use and activity were recorded throughout and fish were terminally sampled for blood. Cortisol implantation, relative to shams, resulted in higher blood glucose and plasma cortisol concentrations with a lower plasma lactate concentration. Shark exposure, relative to controls, elicited higher blood glucose and lactate concentrations but had no effect on plasma cortisol concentration. No interactions were detected between shark exposure and cortisol treatment for any physiological trait. Behavioral metrics, including shelter use and activity, were unaffected by either cortisol implantation or shark exposure. Physiological responses to cortisol implantation likely resulted from enhanced gluconeogenic activity, whereas alterations under predator exposure may have been the product of catecholamine mobilization. Further work should address context-specific influences of stress in mediating behavioral responses to predation.

Differential expression of the corticosteroid receptors GR1, GR2 and MR in rainbow trout organs with slow release cortisol implants

The present study describes the transcriptional levels of the corticosteroid receptors (CRs) GR1, GR2 and MR in the different organs of the rainbow trout (Oncorhynchus mykiss) in response to a slow release of cortisol, throughout a 10-day period. We show that after short term (1 day after cortisol implantation), when the plasma levels of cortisol emulate an acute stress, the GR2 and MR expression levels were upregulated in the brain and head kidney tissues. This result reflects the role of these organs as regulators of the stress response. In general, the rest of the organs, especially gills, intestine, liver, muscle and spleen, showed decreased transcriptional levels of GR1, GR2 and MR, along with the highest plasma cortisol levels. At day 5 after cortisol implantation, when cortisol levels emulate a chronic stress, the most affected organs were gills and skin, where an upregulation of the CRs was found. In the recovery period, when cortisol levels were basal (day 10), we still found changes in the transcriptional levels of the CRs in gills, spleen and gonads. The cortisol increase at days 1 and 5 after implantation is accompanied by high plasma glucose concentrations, supporting the role of cortisol on carbohydrate metabolism. However, after 10days of implantation, glucose returned to control levels suggesting a trade-off on the steady state of the metabolic function. We also observed increased hematocrit and hemoglobin at day 1, indicating a cortisol-induced higher metabolic demand involving an increase in oxygen transport efficiency. Our results demonstrate that increased plasma cortisol induced by a slow-release implant of cortisol mimics the overall effects of stress and affects the expression of the three CRs, generating different transcriptional patterns in a time- and organ-specific manner.

Effects of Chronic Cortisol Administration on Global Expression of GR and the Liver Transcriptome in Sparus aurata

The present work was designed to assess the effects of artificially increased high plasma cortisol levels induced by slow-release cortisol implants on the mRNA abundance of the glucocorticoid receptor (GR) in different organs of Sparus aurata (Gilthead sea bream), as well as to evaluate global transcriptional changes in the liver, using the Aquagenomics S. aurata oligo-nucleotide microarray technology. For that purpose, groups of fish were intraperitoneally injected with implants containing two different concentrations of cortisol (50 or 200 μg/g body weight). Blood and organs were sampled after 7 and 14 days of cortisol implantation. Only fish with 200 μg/g implants exhibited a significant rise in plasma cortisol. Thus, we evaluated the expression of the GR in different organs in these fish 7 and 14 days post-implantation. GR mRNA abundance was upregulated in head kidney and heart of fish at both sampling times. In liver and muscle, GR mRNA abundance was upregulated after 14 days, whereas in gills, the GR mRNA transcript was upregulated earlier, at day 7. These results suggest that increased plasma cortisol induced by a slow-release implant of cortisol mimics the overall effects of stress and affects the expression of GR mRNA in a time- and organ-specific manner. Data obtained with the Aquagenomics S. aurata oligo-nucleotide microarray allowed the identification of a total of 491 cortisol-responsive transcripts and highlight the strong intensity of transcriptional modulation in liver of fish implanted with cortisol after 7 days, in contrast to that observed at day 14. Transcriptional remodeling highlighted a significant activity in carbohydrate metabolism mainly in the gluconeogenic pathway linked to downregulation of inflammatory and immune response processes in implanted fish.

Effects of long-term cortisol treatments on gonadal development, sex steroids levels and ovarian cortisol content in cultured great sturgeon Huso huso

The objective of this study was to examine the effect of cortisol implantations on gonadal development, sex steroid levels, and ovarian cortisol content in cultured great sturgeon Huso huso. Three groups of 5 fish for each treatment were considered. The experimental groups included: control (capsules containing cocoa butter alone), low cortisol (C5; 5mg cortisol/kg body mass+cocoa butter) and, high cortisol (C50; 50mg cortisol/kg body mass+cocoa butter). The capsules containing hormones and cocoa butter were intraperitoneally implanted into 3-year-old female fish at pre-vitellogenic stage (mean initial body mass 6809.7±73g) every 6weeks over a 6-month period from January to June. The serum levels of cortisol, glucose, cholesterol and sex steroids (testosterone and 17β-estradiol) were determined at the initial time and three weeks after each implantation. Oocyte histological characteristics (the diameter and area of the oocyte, the diameter and area of the nucleus and the ratio of the nucleus area to the oocyte area) were measured at the end of the experiment and compared to those at the initial time. Ovarian cortisol content was measured at the end of the experiment. The results showed that serum cortisol levels varied in a dose-independent manner, so that the highest cortisol concentrations were observed in C5-treated fish throughout the experiment. Serum glucose levels were significantly higher in cortisol-treated groups than those in the control group. The high dose of cortisol elicited a significant constant increase in serum cholesterol concentrations. Fish implanted with the high cortisol dose showed significant declines in serum testosterone and 17β-estradiol concentrations throughout the experiment. No significant differences were found in oocyte histological characteristics among experimental groups. The cortisol implants elicited a dose-dependent increase in ovarian cortisol content. At the end of trial, body-growth indices were the lowest in C50-implanted fish, while the low cortisol dose had no effect on growth relative to the controls. These results indicated that chronic stress induced by cortisol implantation in great sturgeon suppressed gonadal steroidogenesis and somatic growth but had no effect on ovarian growth and development.

Slow release cortisol implants result in impaired innate immune responses and higher infection prevalence following experimental challenge with infectious pancreatic necrosis virus in Atlantic salmon (Salmo salar) parr

Stress can affect the immune system and increase susceptibility to various diseases but knowledge of the underlying mechanisms is scarce. There is a complex interaction between the immune system and the endocrine system of vertebrates. In fish, cortisol is a key hormone regulating stress response and recent studies have also suggested that this hormone can affect the immune system, where cortisol is mainly regarded as an immunosuppressive factor. The aim of the present study was to examine the impact of chronically elevated levels of cortisol on the immune response and susceptibility to experimental infection with infectious pancreatic necrosis virus (IPNV). Further, the effect of IPNV challenge on circulating levels of cortisol was investigated. Atlantic salmon parr were implanted intraperitoneally with sustained-release implants of bovine of cortisol (50 μg cortisol g−1 body weight in an implant based on vegetable lipids). Vehicle implants were used as control (sham-injected). At 45 days after implantation (DAI), fish were challenged with a low virulent isolate of IPNV (by immersion). Samples of plasma, liver and head kidney was taken from fish before and 24 h, 48 h, 7 days week and 21 days post infection (DPI). Cortisol level in plasma was measured using radioimmunoassay and gene expression in liver and head kidney was analyzed with real-time PCR (RT-PCR). Infection prevalence in infected fish was assessed by virus culture and RT-PCR of head kidney samples.Cortisol implantation compared with sham-implanted fish had increased levels of plasma cortisol at 45 DAI. The relative expression of Interferon alpha-1 (IFNα-1), Myxo virus-1 Mx, Heat-shock protein 70 (HSP70), Serum amyloid A (SAA), Glucocorticoid receptor (GR) and Heat-shock protein 90 (HSP90) tends to be down-regulated by cortisol implantation. There was a higher prevalence of fish with detectable levels of IPNV, as measured by cell culture and RT-PCR, in the cortisol-implanted group challenged with IPNV (0 = 0.0305) relative to the group that received a sham implantation. Further, cortisol seems to delay the induction of the antiviral IFNα-1 pathway and Mx mRNA expression. This study shows that elevated plasma cortisol level leads to an impaired innate immune response, and higher virus (IPNV) prevalence in Atlantic salmon parr.

Translocation of infectious pancreatic necrosis virus across the intestinal epithelium of Atlantic salmon ( Salmo salar L.)

Infectious pancreatic necrosis virus (IPNV) is a viral disease that afflicts salmon aquaculture worldwide. Transmission of viral diseases may occur via the skin, gills or intestine but the main entry route for IPNV is not known. However, acute inflammation of the intestinal mucosa is an early symptom in infected fish which suggests transmission through an oral route. The main aim of this study is to determine if IPNV can translocate across the intestinal epithelium. It also aims to investigate how acute and chronic IPNV exposure as well as stress affects the physiological barrier functions of the intestinal epithelium. Possible translocation of IPNV across the intestinal mucosa as well as effects of virus on intestinal barrier function was measured in vitro, using the Ussing chamber technique. IPNV was translocated across both proximal and distal segments of the intestine with a higher rate in distal compared to proximal intestine. The translocated virions caused cytopathic effects on cultured Chinook salmon embryo cells (CHSE-214), proving viability and high virulence. Intestinal barrier function was disturbed as assessed by increased permeability for 14C-mannitol in the proximal region after exposure to IPNV in vitro. The same pattern was observed after prolonged exposure to IPNV in vivo. Further, the intestine appeared to respond with an increased mucus secretion, as indicated through increased TER after prolonged IPNV exposure. No additional effects of cortisol implantation on barrier function were observed. In conclusion, this study demonstrates that both proximal and distal intestine can be used by IPNV as a route of infection and that the intestinal barrier function is negatively affected by exposure to IPNV.

21 THE INJECTION OF CORTISOL TO UTERUS INCREASES THE IMPLANTATION RATE AND LITTER SIZE IN PIG ARTIFICIAL INSEMINATION USING CYROPRESERVED SPERMATOZOA

Cryopreserved boar spermatozoa are not routinely available to swine artificial insemination (AI) because conception and farrowing rates, along with litter size, have remained low. We have reported the positive roles of seminal plasma in frozen–thawed sperm functions (Okazaki et al. 2009 Theriogenology 71, 491–498). Moreover, the injection of seminal plasma to uterus with frozen–thawed spermatozoa significantly increased the implantation rate. Thus, the factors in seminal plasma act not only on sperm but also on uterus to induce successful fertilization and implantation in pig AI using cryopreserved spermatozoa. To test this hypothesis, we identified the factors in seminal plasma and then developed novel pig AI method using cryopreserved spermatozoa. The sperm-rich fraction was collected weekly from each boar using the gloved-hand technique. The seminal plasma was removed just after collection by centrifuge and then was frozen as described in our previous study (Okazaki et al. 2009 Theriogenology 71, 491–498). When the frozen–thawed sperm was incubated with Fluo-3/AM to determine the level of intercellular Ca2+, the level of Ca2+ was increased in a time-dependent manner, and spontaneous capacitation that was judged by tyrosine phosphorylation of sperm protein by Western blotting (Shimada et al. 2008 Development 135, 2001–2011), was also induced in post-thawed sperm. The addition of EGTA to thawing solution significantly suppressed the Ca2+-induced capacitation. Moreover, the treatment increased fertilization rate in in vitro fertilization and in vivo in artificial insemination as similar as those in sperm with seminal plasma. The same number of blastocyst was collected from uterus by AI using post-thawed sperm with EGTA. However, the pregnancy rate remained low, and the number of leukocytes in the uterus was increased. In the next experiment, we examined in seminal plasma, the level of cortisol that has been known to play an important role in controlling immune function. The results showed that cortisol (1.0 ng mL–1) was detected in seminal plasma. When the sows of natural oestrus were twice artificial inseminated with or without cortisol, the injection of cortisol (5 μg/50 mL) to uterus with sperm significantly decreased the number of leukocytes in the uterus or endometrium at 24 to 36 h after AI. The low number of leukocytes in the uterus was similar to that in uterus injected fresh semen. The cortisol injection significantly increased the implantation rate and litter size of sows as compared to AI without cortisol (implantation rate; 83% v. 51%, litter size; 10.6 v. 7.3). From these results, we concluded that the injection of cortisol with frozen–thawed spermatozoa by EGTA-containing solution was a novel method of pig AI using cryopreserved spermatozoa. This work was supported by the Programme for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry, and JST-Grant (No. 12-068 and No. 12-104).

Expression of glucocorticoid receptor in the intestine of a euryhaline teleost, the Mozambique tilapia ( Oreochromis mossambicus): Effect of seawater exposure and cortisol treatment

Cortisol plays an important role in controlling intestinal water and ion transport in teleosts possibly through glucocorticoid receptor (GR) and/or mineralocorticoid receptor. To better understand the role of GR in the teleost intestine, in a euryhaline tilapia, Oreochromis mossambicus, we examined (1) the intestinal localizations of GR; (2) the effects of environmental salinity challenge and cortisol treatment on GR mRNA expression. The mRNA abundance of GR in the posterior intestinal region of tilapia was found to be higher than that in the anterior and middle intestine. In the posterior intestine, GR appears to be localized in the mucosal layer. GR mRNA levels in the posterior intestine were elevated after exposure of freshwater fish to seawater for 7 days following an increase in plasma cortisol. Similarly, cortisol implantation in freshwater tilapia for 7 days elevated the intestinal GR mRNA. These results indicate that seawater acclimation is accompanied by upregulation of GR mRNA abundance in intestinal tissue, possibly as a consequence of the elevation of cortisol levels. In contrast, a single intraperitoneal injection of cortisol into freshwater tilapia decreased intestinal GR mRNA. This downregulation of the GR mRNA by cortisol suggests a dual mode of autoregulation of GR expression by cortisol.

Corticotropin-releasing factor and neuropeptide Y mRNA levels are modified by glucocorticoids in rainbow trout, Oncorhynchus mykiss

The primary stress response involves neuronal activation that ultimately leads to the release of glucocorticoids. Circulating glucocorticoids are thought to influence their own synthesis and release through a negative feedback mechanism that inhibits the activity of the hypothalamic and pituitary components of the stress axis. This study was designed to address the hypothesis that glucocorticoids modify corticotropin-releasing factor (CRF) and neuropeptide Y (NPY) mRNA levels in the rainbow trout ( Oncorhynchus mykiss) brain. Cortisol implantation significantly reduced CRF1 and NPY mRNA levels in fish exposed to an isolation stress. In contrast, cortisol implantation did not prevent the stress-induced elevation of CRF1 and NPY mRNA levels during confinement. Treatment with the glucocorticoid receptor antagonist RU-486 reduced CRF1 mRNA levels in both isolated and confined fish, but had no effect on NPY mRNA. Although the cytochrome P450 inhibitor metyrapone reduced ACTH-induced cortisol secretion in vitro, plasma cortisol levels were elevated in isolated trout treated with metyrapone. Nevertheless, metyrapone implantation increased CRF1 and NPY mRNA levels in confined fish. Together, these results implicate cortisol as a modulator of CRF and NPY mRNA levels in the preoptic area of the trout brain, but that cortisol is only one such regulating mechanism.

Interactive effects of cortisol treatment and ambient seawater challenge on gill Na+,K+-ATPase and CFTR expression in two strains of Atlantic salmon smolts

Abstract During peak smoltification, the interactive effects of cortisol and ambient seawater challenge were compared in two strains of Atlantic salmon ( Salmo salar ) smolts: a domesticated strain, AquaGen and a native River Imsa strain. Tissue and blood samples were taken from untreated fish on 20 May. Fish were then transferred to experimental tanks, allowed to recover for 24 h and cortisol (50 mg kg body mass −1 ), dissolved in vegetable oil, or vegetable oil alone (sham) was implanted. Samples were taken 5 days post implantation. Fish were then exposed to 24 h ambient seawater challenge (FW–SW) or freshwater to freshwater replacement (FW–FW) and sampled as before. Sham implantation had no significant impact on any of the measured parameters. Cortisol implantation significantly elevated plasma cortisol in FW–FW Imsa smolts, while no effect was observed in FW–SW Imsa smolts. Cortisol implantation had no effect on the plasma cortisol levels of AquaGen smolts regardless of FW–SW challenge. Increased plasma cortisol corresponded with significantly higher plasma glucose levels in FW–FW Imsa smolts. Plasma Na + and Cl − levels were not affected by cortisol implantation but were significantly increased in FW–SW smolts of both strains. Gill Na + ,K + -ATPase activity increased in response to cortisol implantation in only FW–FW smolts but not FW–SW smolts. Gill Na + ,K + -ATPase α-subunit mRNA levels were not affected by strain, cortisol injection or transfer protocol, while both CFTR I and CFTR II mRNA levels were significantly higher in AquaGen versus Imsa smolts regardless of treatment. CFTR I mRNA was elevated following cortisol implantation in FW–FW smolts from both strains suggesting CFTR I expression is under the control of cortisol. These findings also suggest that plasma cortisol levels are regulated differently between strains and that cortisol implantation and ambient FW–SW challenge interact, interfering with the individual effects of each of these factors.

Impaired Neutrophil Defense Activities and Increased Susceptibility to Edwardsiellosis by Cortisol Implantation in Tilapia.

In order to reveal the effect of cortisol on tilapia neutrophil defense activities in vivo, fish were given intraperitoneal injections of cortisol mixed with coconut oil as an “implant” (5 mg/0.5 mL/fish). Fish were injected with Escherichia coli into their swim bladders before 24 h prior to collecting neutrophils, which were then assessed for their chemotactic, phagocytic and respiratory burst activities. In addition, concentrations of plasma cortisol and glucose were measured. Cortisol-implanted fish showed considerably higher levels of cortisol and their neutrophil activities were significantly suppressed compared to the control group at 1 day post-implantation. Both groups of fish were challenged with Edwardsiella tarda (1 mg/fish). Cortisol-implanted fish started to die 6 days after the challenge, whereas no deaths were observed in the controls. After the challenge experiment, E. tarda was recovered from more than half of the cortisol-treated fish group and from the only one of the controls. Results from this study have demonstrated that elevated levels of cortisol in fish bodies, similar to that observed during stressful experiences, decrease neutrophil activities and increase susceptibility to bacterial pathogens.

The effect of stress on toxicant‐dependent cytochrome P450 enzyme responses in the arctic charr (Salvelinus alpinus)

In the present study we investigated the effect of stress and cortisol on cytochrome P450 (CYP) expression in Arctic charr exposed to benzo[a]pyrene (BaP). Expression of hepatic CYP1A and CYP3A was monitored 8 d after a single oral dose of BaP (10 mg/kg fish) and compared to that in unexposed fish. During this period the fish were subjected to one of the following stress regimes: no stress, no stress and cortisol implantation, 10 min of daily handling and confinement stress, and confinement stress during the last 6 h before sampling. In BaP-exposed fish daily stress resulted in significantly lower (53%) CYP1A protein levels as compared to those in unstressed fish. For CYP1A catalytic activity (measured as 7-ethoxyresorufin-O-deethylase [EROD] activity), the suppressive response to stress was less pronounced. These results contrast to previous findings of a potentiation by corticosteroids on xenobiotic-dependent CYP1A induction in vitro in cultured fish hepatic cells. No effects of high cortisol levels or BaP were found on the steroid-metabolizing CYP3A enzyme levels. The lack of any alterations in the CYP3A protein level indicates that CYP3A expression is not inducible by cortisol in the Arctic charr under the conditions used here. The conclusion was made that short-term stress associated with sampling (i.e., 6 h of confinement stress before sampling) of wild charr does not compromise the EROD activity as a reliable biomarker.

Changes in hydrolytic enzyme activities of naïve Atlantic salmon Salmo salar skin mucus due to infection with the salmon louse Lepeophtheirus salmonis and cortisol implantation.

The changes in the activities of mucus hydrolytic enzymes and plasma cortisol levels were examined following infection of Atlantic salmon Salmo salar with the salmon louse Lepeophtheirus salmonis and these changes were compared with those resulting from elevated plasma cortisol. Salmon were infected at high (Trial 1; 178 +/- 67) and low (Trial 2; 20 +/- 13) numbers of lice per fish and the activities of proteases, alkaline phosphatase, esterase and lysozyme in the mucus, as well as plasma cortisol levels were determined. At both levels of infection, there were significant increases of protease activity over time (1-way K-WANOVA; Trial 1, p = 0.004; Trial 2, p < 0.001). On several sampling days, generally on later days in the infections, the mucus protease activities of infected fish were significantly higher than control fish (Student's t-tests; p < 0.05). In addition, zymography experiments demonstrated bands of proteases at 17 to 22 kDa in the mucus of infected salmon that were absent in the mucus from non-infected fish and absent in the plasma of salmon. The intensity of these protease bands increased in the mucus over the course of both infections. However, plasma cortisol levels were elevated only in the heavily infected fish from the first trial. At high infection levels (Trial 1), alkaline phosphatase activity was higher in the mucus of infected fish at all days (t-test, p < 0.05). However, at the lower infection level (Trial 2), the mucus alkaline phosphatase activity did not differ significantly between infected and non-infected fish. Esterase and lysozyme activities were very low and did not change with time nor between non-infected and infected salmon in either challenge. Mucus enzyme activities of cortisol-implanted salmon did not change over time, nor were there any differences in activities between cortisol-implanted and control salmon. The present study demonstrates biochemical changes resulting from sea lice infection of Atlantic salmon occurring at the site of host-pathogen interaction, the mucus layer. However, the origin of these enzymes, whether host or pathogen, remains to be determined.

Metabolic Responses Associated with Confinement Stress in Tilapia: The Role of Cortisol

We examined the effect of 2- or 24-hr confinement stress and cortisol treatment on plasma cortisol, glucose, lactate and free amino acids concentration and hepatic glycogen content and activities of certain enzymes involved in the intermediary metabolism in tilapia ( Oreochromis mossambicus). Confinement of tilapia for 2 or 24 hr resulted in significantly higher plasma cortisol, glucose, lactate (2 hr), total and some of the free amino acids concentration (especially at 24 hr) and hepatic pyruvate kinase (PK), phosphoenolpyruvate carboxykinase (PEPCK; 24 hr) and lactate dehydrogenase activities (24 hr). Hepatic glycogen content was lower at 2 and 24 hr in the confined fish compared with the unstressed fish. Tilapia given cortisol implants (50 mg·kg −1 body wt) had significantly higher plasma cortisol, glucose and some of the free amino acids concentration and hepatic PEPCK and aspartate aminotransferase activities, whereas PK activity ratio was significantly lower compared with the sham group. The results suggest that glucose production 2 hr after confinement may be due to glycogenolysis, whereas the maintenance of higher glucose at 24 hr after confinement is essentially due to gluconeogenesis. Furthermore, the changes in plasma metabolites and hepatic enzyme activities with cortisol implantation suggest that cortisol plays a role in the metabolic adjustment to 24-hr confinement stress in tilapia.

The Effects of Cortisol on Hepatocyte Metabolism in Rainbow Trout: A Study Using the Steroid Analogue RU486

The objective of this study was to examine if RU486, a corticosteroid antagonist, modified hepatocyte metabolism and blocked the metabolic effects of exogenously administered cortisol in rainbow trout (Oncorhynchus mykiss). The fish were given a single intraperitoneal implant of either coconut oil alone or coconut oil containing RU486 (100 mg · kg-1),cortisol (100 mg · kg-1), or a combination of RU486 and cortisol and were sampled 7 days postimplantation. The RU486 implants had no effect on plasma cortisol and glucose concentrations, hepatocyte glycogen content, and total glucose production, but increased in vitro hepatocyte glycogen breakdown. Cortisol implantation had no effect on plasma glucose concentration, hepatocyte glycogen content, glycogen breakdown, or total glucose production, while it significantly increased alanine oxidation and gluconeogenesis in rainbow trout hepatocytes. Hepatocyte responsiveness to epinephrine and glucagon stimulation of total glucose production was not modified by either RU486 or cortisol treatment, whereas the insulin response on total glucose production was decreased with cortisol. RU486 treatment modified hepatocyte glycogen metabolism and blocked the cortisol-induced increases in alanine gluconeogenesis and glycogen mobilization for endogenous use by the hepatocytes. These results indicate that cortisol enhances the metabolic potential of hepatocytes, thereby adapting the fish to cope with stress. This study provides further validation of RU486 as a tool for studying metabolic actions of cortisol in fish.

Effects of cortisol on hepatic carbohydrate metabolism and responsiveness to hormones in the sea raven, Hemitripterus americanus

The sea raven, Hemitripterus americanus, is a sit-and-wait, low metabolic rate, marine teleost. The objective of this study was to determine i) whether cortisol implantation (50 mg. kg(-1)) for 7 days altered hepatocyte metabolism, and hepatocyte responsiveness to epinephrine, glucagon and insulin, and ii) whether 8 weeks of food-deprivation modified the above response. Cortisol implantation significantly increased hepatocyte total glucose production and oxidation from alanine compared to the sham group. There was no cortisol effect on glycogen breakdown, suggesting that the activation of other pathways, including gluconeogenesis, are required to account for the increased glucose production. Epinephrine-mediated (10(-)5M) glycogen breakdown and insulin-mediated (10(-8)M) total glucose production were enhanced in hepatocytes of cortisol implanted sea ravens, but there were no change in any glucagon (10(-7)M) effects. The enhanced glycogen breakdown in the absence of similar increases in total glucose production with epinephrine indicates mobilization of carbohydrate reserves for endogenous use by the liver.Food-deprivation for 8 weeks significantly decreased condition factor, plasma cortisol concentration and liver glycogen content in the sea raven, but had no effect on plasma glucose concentration. Hepatocyte total glucose production and flux rates from alanine increased significantly with food-deprivation. Moreover, food-deprivation increased responsiveness of total hepatocyte glucose production to the actions of glucagon and insulin, but not to epinephrine; none of these effects were modified by cortisol implantation.Our results indicate that cortisol in the sea raven exerts both a direct and an indirect or permissive effect on hepatocyte metabolism by modifying hepatocyte responsiveness to epinephrine and insulin stimulation. Cortisol implantation did not modify the effects of glucagon or food-deprivation in this species.

Retardation of ovarian growth and depression of serum steroid levels in the tilapia, Oreochromis mossambicus, by cortisol implantation

Female Oreochromis mossambicus were implanted with cortisol suspended in cocoa-butter for the evaluation of the chronic effects of cortisol on reproduction. Implant doses of 1.5 and 3.0 mg/g body weight, mimicking the effects of long-term chronic stress, suppressed various reproductive parameters over the 18-day treatment period. Most fish treated with cortisol showed reductions in body weight. Oocyte size and gonadosomatic index of treated fish became significantly lower than the control after 9 and 12 days of treatment, respectively. The retardation of oocyte growth was accompanied by depression of serum testosterone and oestradiol-17 β levels.

The effect of cortisol administration on hepatic and plasma estradiol-binding capacity in immature female rainbow trout ( Oncorhynchus mykiss)

Implantation of a cortisol-containing pellet into the peritoneal cavity of immature female rainbow trout raised plasma cortisol levels within the range commonly observed in chronically stressed fish. In cortisol-implanted fish there was a significant decline in the concentration of hepatic estradiol-binding sites relative to sham-implanted controls. This consisted of a 35% drop in cytosolic binding sites and a 29% reduction in the number of nuclear estradiol-binding sites, by 4 weeks postimplantation. Plasma estradiol-binding capacity was also influenced by cortisol treatment. After 2 weeks there was a 33% increase in plasma estradiol-binding capacity of cortisol-implanted fish. Plasma estradiol levels were unaffected by cortisol implantation, suggesting that the effects of cortisol on estradiol-binding sites were not mediated by altering the rate of estradiol secretion. The results indicate a possible mechanism by which environmental stress may suppress vitellogenesis.

The deleterious effects of cortisol implantation on reproductive function in two species of trout, Salmo trutta L. and Salmo gairdneri Richardson

Implantation of a cortisol-releasing pellet (60 mg kg −1 fish) into the peritoneal cavity of brown trout, Salmo trutta L. (sexually maturing males and females), and rainbow trout, Salmo gairdneri Richardson (maturing males and immature fish of both sexes), significantly elevated their plasma cortisol level. At 18 days postimplantation, cortisol-implanted sexually maturing male brown trout had smaller gonads, a lower plasma testosterone level, and less gonadotropin in their pituitary gland than control fish. Plasma levels of 11-ketotestosterone and gonadotropin were not significantly affected. Cortisol-implanted sexually maturing female brown trout had smaller gonads, reduced plasma levels of 17β-oestradiol, testosterone, and vitellogenin, and a lower pituitary gland gonadotropin content than control fish. The plasma gonadotropin level was unaffected. At 36 days post-implantation, cortisol treatment of maturing male rainbow trout significantly suppressed plasma gonadotropin levels. Plasma levels of testosterone, 11-ketotestosterone, and 17α,20β-dihydroxy-4-pregnen-3-one, pituitary gonadotropin content, and gonad size were not significantly affected. In sexually immature female rainbow trout, cortisol administration suppressed the level of vitellogenin in the plasma, compared to control-implanted fish. The 17β-oestradiol level was not affected. Cortisol implantation did not affect the plasma testosterone level in sexually immature male trout. These results suggest that prolonged elevation of plasma cortisol, to levels well within physiological range, can affect a wide range of reproductive parameters in both brown and rainbow trout. Further, some effects are manifest in immature as well as in mature fish. These findings are discussed in relation to the effects of cortisol treatment on the state of health of the treated fish.