Article

Holding water steroid hormones in the African cichlid fish Pseudocrenilabrus multicolor victoriae

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Abstract

Measuring hormone levels multiple times on the same individual across different life stages or treatments can facilitate our understanding of hormonal regulation of physiological and behavioral events. The conventional method of hormone measurement requires blood sampling, which is potentially lethal to small individuals. In fishes, there is an alternative non-invasive method of hormone measurement using the release of hormones across gill membranes from blood into holding water. Validation of this method is required to evaluate its application value to different species. In the present study we used the maternal mouth-brooding African cichlid fish, Pseudocrenilabrus multicolor victoriae to (i) investigate whether handling involved in using the holding water technique is a stressor by measuring excreted cortisol in male and female P. multicolor handled one or multiple times, (ii) validate use of this technique by quantifying the relationship between plasma and holding water measures of sex hormones in male P. multicolor, and (iii) demonstrate the biological relevance of this technique using excreted levels of sex hormones in female P. multicolor across different reproductive stages. Excreted cortisol and estradiol levels did not differ between fish handled one or more times, suggesting that the repeated sampling approach over the breeding cycle that we propose to use does not affect the excreted level of the hormone of interest. Measurements from plasma and holding water samples were positively related for both testosterone and estradiol, indicating that the holding water technique is a reliable index of plasma hormone levels. Excreted sex hormone levels varied with reproductive state, suggesting that the technique is a useful, non-invasive measure of sex hormone levels in P. multicolor.

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... A small size may even preclude obtaining a suf cient blood sample (e.g. FRIESEN et al., 2012), and require steroid assessments to be carried out on whole body homogenates (e.g. POTTINGER et al., 2002;KING and BERLINSKY, 2006;POTTIN-GER et al., 2011;FELIX et al., 2013). ...
... • Signifi cant correlations for cortisol in freshwater rainbow trout SCOTT and ELLIS, 2007); • Signifi cant correlation for cortisol in seawater salmon Salmo salar (ELLIS et al., 2007a); • A positive trend for 11-KT in freshwater Siamese ghting sh Betta splendens, although the authors indicate that this relationship between plasma concentration and water concentration (rather than release rate) was not signi cant due to the limited data set (DZIEWECZYNSKI et al., 2006); • Signifi cant correlations for 11-KT, androstenedione and cortisol in freshwater male threespined sticklebacks Gasterosteus aculeatus (SEBIRE et al., 2007); • Signifi cant correlation for testosterone in freshwater male three-spined sticklebacks (SEBIRE et al., 2009). However, in contrast to their previous study, this study found no relationship for androstenedione; • Signifi cant correlation for cortisol in sea-bass (FANOURAKI et al., 2008); • Signifi cant correlations for cortisol in male convict cichlids Amatitlania nigrofasciata (WONG et al., 2008); • Signifi cant correlations for testosterone and estradiol in male cichlids Pseudocrenilabrus multicolor victoriae (FRIESEN et al., 2012); • Signifi cant correlations for cortisol in male and female zebra sh Danio rerio, but not for 11-KT although a gender difference was apparent (FELIX et al., 2013). Please note that this study correlated plasma steroid concentration with water steroid concentration (rather than release rate). ...
... • Examination of androgen responsiveness (11-KT and testosterone) in a variety of cichlid species (OLIVEIRA et al., 2003;HIRSCHENHAUSER et al., 2004); • Examining the cortisol response of carp, gudgeon Gobio gobio and perch Perca fl uviatilis to boat noise (WYSOCKI et al., 2006); • Assessing the effects of nesting status and audience presence on 11-KT levels in Siamese ghting sh Betta splendens (DZIEWECZYNSKI et al., 2006); • Following seasonal changes in androgens in male stickleback in conjunction with monitoring reproductive and aggressive behaviour (SEBIRE et al., 2007(SEBIRE et al., , 2009); • Examination of cortisol responses to handling and con nement in the static container to identify stress coping style in rainbow trout families (SCOTT and ELLIS, 2007) and salmon families (KITTILSEN et al., 2009); the latter studied indicated associations between cortisol response, behaviour and disease susceptibility; • Following changes in testosterone and oestradiol levels in relation to reproductive stage in a cichlid Pseudocrenilabrus multicolor victoriae (FRIESEN et al., 2012); • Studying reproductive steroid cycles in roach Rutilus rutilus (LOWER et al., 2004;SCOTT et al., 2013). ...
Article
Full-text available
Blood sampling is arguably more stressful, injurious and proble- matic for fishes than for higher vertebrates, so the impetus to introduce non-invasive alternatives should be stronger. Over the last 15 years, studies have illustrated the potential for non-invasive monitoring of reproductive and stress steroids in fresh- and sea-water fishes. Non-invasive monitoring in fishes relies upon measurement of steroids in alternative matrices – skin mucus, the surrounding water, faeces and urine – which bear (respective) parallels with sweat, salivary, faecal and urinary methods used in higher vertebrates. All methods show promise, but differ in the extent of intrusion. Most non- invasive studies of fishes have used the water matrix. We illustrate the value of the water procedure with original time series data on cortisol release by fed and unfed fi sh in laboratory tanks. Although the water matrix has been proven under such conditions, it cannot yet be applied to free-ranging fi sh due to the uncontrolled dispersion and dilution of steroids in the water. The solution is to fi nd a ‘normaliser’ (another compound released at a constant rate, against which water steroid concentration can be expressed as a ratio). We present new data on the measure- ment and release of creatinine and melatonin as candidate norma- lisers. Our data illustrates the lability of potential normalisers, food as an exogenous source of measured excretory products, potential effects of water chemistry on metabolite recovery, and human skin as a source of steroid contamination. We emphasize the need for extensive validation of non-inva- sive assays and the value of background information to support interpretation of results.
... A small size may even preclude obtaining a sufficient blood sample (e.g. FRIESEN et al., 2012), and require steroid assessments to be carried out on whole body homogenates (e.g. POTTINGER et al., 2002;KING and BERLINSKY, 2006;POTTIN-GER et al., 2011;FELIX et al., 2013). ...
... • Significant correlations for cortisol in freshwater rainbow trout SCOTT and ELLIS, 2007); • Significant correlation for cortisol in seawater salmon Salmo salar (ELLIS et al., 2007a); • A positive trend for 11-KT in freshwater Siamese fighting fish Betta splendens, although the authors indicate that this relationship between plasma concentration and water concentration (rather than release rate) was not significant due to the limited data set (DZIEWECZYNSKI et al., 2006); • Significant correlations for 11-KT, androstenedione and cortisol in freshwater male threespined sticklebacks Gasterosteus aculeatus (SEBIRE et al., 2007); • Significant correlation for testosterone in freshwater male three-spined sticklebacks (SEBIRE et al., 2009). However, in contrast to their previous study, this study found no relationship for androstenedione; • Significant correlation for cortisol in sea-bass (FANOURAKI et al., 2008); • Significant correlations for cortisol in male convict cichlids Amatitlania nigrofasciata (WONG et al., 2008); • Significant correlations for testosterone and estradiol in male cichlids Pseudocrenilabrus multicolor victoriae (FRIESEN et al., 2012); • Significant correlations for cortisol in male and female zebrafish Danio rerio, but not for 11-KT although a gender difference was apparent (FELIX et al., 2013). Please note that this study correlated plasma steroid concentration with water steroid concentration (rather than release rate). ...
... • Examination of androgen responsiveness (11-KT and testosterone) in a variety of cichlid species (OLIVEIRA et al., 2003;HIRSCHENHAUSER et al., 2004); • Examining the cortisol response of carp, gudgeon Gobio gobio and perch Perca fluviatilis to boat noise (WYSOCKI et al., 2006); • Assessing the effects of nesting status and audience presence on 11-KT levels in Siamese fighting fish Betta splendens (DZIEWECZYNSKI et al., 2006); • Following seasonal changes in androgens in male stickleback in conjunction with monitoring reproductive and aggressive behaviour (SEBIRE et al., 2007(SEBIRE et al., , 2009); • Examination of cortisol responses to handling and confinement in the static container to identify stress coping style in rainbow trout families (SCOTT and ELLIS, 2007) and salmon families (KITTILSEN et al., 2009); the latter studied indicated associations between cortisol response, behaviour and disease susceptibility; • Following changes in testosterone and oestradiol levels in relation to reproductive stage in a cichlid Pseudocrenilabrus multicolor victoriae (FRIESEN et al., 2012); • Studying reproductive steroid cycles in roach Rutilus rutilus (LOWER et al., 2004;SCOTT et al., 2013). ...
Article
Full-text available
It was recently found that high concentrations of chicken yolk gestagens and gestagen metabolites hamper corticosterone quantification via immunoassays. However, the situation in chicken albumen is still unresolved. In addition, the ratio of steroid hormone in the yolk of wild birds might differ. To investigate these matters, corticosterone and gestagens were measured in individual fractions of high-performance liquid-chromatographic separations of chicken albumen and yolk of red jungle fowl. Similarly, yolk extracts of hens with corticosterone-releasing implants or placebos were analysed to assess the impact of elevated plasma corticosterone concentrations on authentic yolk corticosterone levels. We also compared the results of a previously used corticosterone enzyme immunoassay (EIA) to those from a commercial radioimmunoassay (RIA) kit. The analytical validations of chicken albumen, bankiva yolk and yolks from hens with or without artificially elevated plasma corticosterone levels indicated that the main share of the immunoreactivity measured via corticosterone immunoassays was caused by substances other than authentic corticosterone. In albumen, the concentration of authentic corticosterone was below the detection limit. Analysis of bankiva yolk revealed three major gestagen peaks with concentrations of up to 2000 ng per fraction and a corticosterone peak of about 0.8 ng per fraction. Both corticosterone assays found a slightly higher corticosterone peak in a corticosterone-implanted hen's yolk (EIA: 0.7 ng; RIA: 0.5 ng per fraction) compared to the shamtreated female (EIA: 0.5 ng; RIA: 0.2 ng per fraction) but both antibodies also bound to several other substances, presumably gestagens. Although a certain amount of circulating corticosterone might pass into the yolk, direct quantification of corticosterone in non-homogenized avian egg samples via immunoassays is not advisable.
... A small size may even preclude obtaining a suf cient blood sample (e.g. FRIESEN et al., 2012), and require steroid assessments to be carried out on whole body homogenates (e.g. POTTINGER et al., 2002;KING and BERLINSKY, 2006;POTTIN-GER et al., 2011;FELIX et al., 2013). ...
... • Signifi cant correlations for cortisol in freshwater rainbow trout SCOTT and ELLIS, 2007); • Signifi cant correlation for cortisol in seawater salmon Salmo salar (ELLIS et al., 2007a); • A positive trend for 11-KT in freshwater Siamese ghting sh Betta splendens, although the authors indicate that this relationship between plasma concentration and water concentration (rather than release rate) was not signi cant due to the limited data set (DZIEWECZYNSKI et al., 2006); • Signifi cant correlations for 11-KT, androstenedione and cortisol in freshwater male threespined sticklebacks Gasterosteus aculeatus (SEBIRE et al., 2007); • Signifi cant correlation for testosterone in freshwater male three-spined sticklebacks (SEBIRE et al., 2009). However, in contrast to their previous study, this study found no relationship for androstenedione; • Signifi cant correlation for cortisol in sea-bass (FANOURAKI et al., 2008); • Signifi cant correlations for cortisol in male convict cichlids Amatitlania nigrofasciata (WONG et al., 2008); • Signifi cant correlations for testosterone and estradiol in male cichlids Pseudocrenilabrus multicolor victoriae (FRIESEN et al., 2012); • Signifi cant correlations for cortisol in male and female zebra sh Danio rerio, but not for 11-KT although a gender difference was apparent (FELIX et al., 2013). Please note that this study correlated plasma steroid concentration with water steroid concentration (rather than release rate). ...
... • Examination of androgen responsiveness (11-KT and testosterone) in a variety of cichlid species (OLIVEIRA et al., 2003;HIRSCHENHAUSER et al., 2004); • Examining the cortisol response of carp, gudgeon Gobio gobio and perch Perca fl uviatilis to boat noise (WYSOCKI et al., 2006); • Assessing the effects of nesting status and audience presence on 11-KT levels in Siamese ghting sh Betta splendens (DZIEWECZYNSKI et al., 2006); • Following seasonal changes in androgens in male stickleback in conjunction with monitoring reproductive and aggressive behaviour (SEBIRE et al., 2007(SEBIRE et al., , 2009); • Examination of cortisol responses to handling and con nement in the static container to identify stress coping style in rainbow trout families (SCOTT and ELLIS, 2007) and salmon families (KITTILSEN et al., 2009); the latter studied indicated associations between cortisol response, behaviour and disease susceptibility; • Following changes in testosterone and oestradiol levels in relation to reproductive stage in a cichlid Pseudocrenilabrus multicolor victoriae (FRIESEN et al., 2012); • Studying reproductive steroid cycles in roach Rutilus rutilus (LOWER et al., 2004;SCOTT et al., 2013). ...
Article
Full-text available
Blood sampling is arguably more stressful, injurious and problematic for fishes than for higher vertebrates, so the impetus to introduce non-invasive alternatives should be stronger. Over the last 15 years, studies have illustrated the potential for non-invasive monitoring of reproductive and stress steroids in fresh- and sea-water fishes. Non-invasive monitoring in fishes relies upon measurement of steroids in alternative matrices - skin mucus, the surrounding water, faeces and urine - which bear (respective) parallels with sweat, salivary, faecal and urinary methods used in higher vertebrates. All methods show promise, but differ in the extent of intrusion. Most noninvasive studies of fishes have used the water matrix. We illustrate the value of the water procedure with original time series data on cortisol release by fed and unfed fish in laboratory tanks. Although the water matrix has been proven under such conditions, it cannot yet be applied to free-ranging fish due to the uncontrolled dispersion and dilution of steroids in the water. The solution is to find a 'normaliser' (another compound released at a constant rate, against which water steroid concentration can be expressed as a ratio). We present new data on the measurement and release of creatinine and melatonin as candidate normalisers. Our data illustrates the lability of potential normalisers, food as an exogenous source of measured excretory products, potential effects of water chemistry on metabolite recovery, and human skin as a source of steroid contamination. We emphasize the need for extensive validation of non-invasive assays and the value of background information to support interpretation of results.
... Because of the small size of the target species and the remote locations of the sampling sites the acquisition and processing of blood samples was impractical. Instead, the magnitude of the stress response was measured indirectly via the release of cortisol across the gill epithelium to the surrounding water (Scott and Ellis 2007) the rate of which varies in proportion with blood cortisol concentrations (Félix et al. 2013;Friesen et al. 2012;Gabor and Contreras 2012;Wong et al. 2008). During March 2014 five fish were transferred by handnet from a holding tank to a bucket containing 5 litres of water in which they remained for 30 min before being transferred to individual Nalgene tubs (150 ml, 6.5 cm diameter, with screw-fit lids) containing 100 ml of artificial freshwater (deionised water containing 0.33 g/l aquarium grade sea salt; Klüttgen et al. 1994). ...
... Site-specific trends in stress responsiveness are retained in female sticklebacks after translocation to an uncontaminated environment Site-dependent trends in the stress-induced rate of release of cortisol to water (WC) were evident in three-spined sticklebacks of both sexes sampled from locations downstream of WWTW effluent discharges. Overall, WC release rates, which provide a surrogate for blood cortisol concentrations (Félix et al. 2013;Friesen et al. 2012;Gabor and Contreras 2012;Wong et al. 2008), were directly proportional to the estimated concentration of WWTW effluent at these sites. Female sticklebacks translocated from the same sites 5 months previously and subsequently held in pristine aquarium conditions continued to exhibit the same site-related trends in WC release rates as their more recently wild-caught counterparts. ...
Article
Full-text available
The hypothalamic-pituitary-adrenal/interrenal (HPA/I) axis plays a key role in responding to biotic and abiotic challenges in all vertebrates. Recent studies have shown that the apical response of the HPI axis to stressors in three-spined sticklebacks varies in proportion to the concentration of wastewater treatment works (WWTW) effluent to which the fish are exposed. This study was conducted to determine whether between-site variation in stress responsiveness among WWTW effluent-exposed sticklebacks is persistent or reversible. Sticklebacks from eight sites in north-west England affected by WWTW effluent and exhibiting between-population variation in HPI axis reactivity, were moved to a clean-water aquarium environment. After five months in the contaminant-free environment the responsiveness of these fish to a standardised stressor was determined, by measuring the rate of stress-induced cortisol release across the gills, and compared with the responses of fish newly sampled from the eight original capture sites. Inter-site differences in the reactivity of the HPI axis, proportional to the effluent concentration at each site, persisted among the translocated female sticklebacks for at least 5 months. In male fish however, the direct relationship between stress responsiveness and site-specific effluent was not evident 5 months post-translocation. These results support previous observations that the HPA/I axis, a non-reproductive endocrine system, is vulnerable to modulation by anthropogenic factors in fish and show for the first time that, in female fish at least, this modulation is not transient. The mechanisms underlying these observations, and the implications for the fitness and resilience of affected populations, requires investigation.
... The flow rate of the water sample was adjusted to 5 mL/min and then washed with 5 mL of 5% methanol at 5 min intervals. Subsequently, the cartridge was dried under a nitrogen gas stream (Friesen et al., 2012). To concentrate the BPA and E 2 compounds, the cartridge was eluted with 3 mL of methanol and then injected into a HPLC for BPA and E 2 analysis. ...
Article
The occurrence of the endocrine disruptors bisphenol-A (BPA) and 17β-estradiol (E2) in cultured populations of green mussel (Perna viridis) and water samples collected from selected fresh and marine coastal environments along the eastern coast of Thailand were investigated. Analysis found high levels of BPA in mussel tissues that correlated with levels found in coastal (maximum 37.13 ng/L) and freshwater (50.7 ng/L) sites situated near industrial and densely populated areas. By comparison, high levels of E2 (62.99 ± 5.03 ng/L) were found in freshwater sites near to urban areas. Higher concentrations of BPA and E2 were found in mature green mussels (>6 mo. old; 6.40 ± 0.52 cm shell length) than levels determined in juveniles (<2–3 mo. old; 2.29 ± 0.65 cm shell length). To evaluate the potential risks associated with the consumption of green mussels, the bioconcentration factor (BCF) for BPA was determined to be 1650 for adult bivalves and 283 for juveniles. As P. viridis can accumulate BPA from the environment, this raises concerns regarding the risks posed by consuming seafood sourced from zones near to major conurbations. Whether the high levels of E2 found in green mussels is due to accumulation or to de novo synthesis as seen in other molluscs, requires further investigation. While industrial and domestic wastewater may be important sources of BPA, E2 contamination within the eastern part of the Gulf of Thailand has been linked to domestic waste. The study highlights the importance of the temporal and spatial monitoring of sentinel species, such green mussels, for environmental contaminants, the results of which can lead to the construction of regional risk maps helping to inform national strategies regarding aquaculture zoning and aquatic food safety.
... The possibility of steroid hormone accumulation was testable given the high vs. low exchange rate experimental conditions; therefore, a sub-study was conducted to assess levels of specific water-borne hormones in the WRAS water, to determine whether concentrations of target hormones were related to to expand our knowledge in this area. Newer approaches utilizing non-invasive methods to quantify hormones in water samples via radioimmunoassay (RIA) [36] and enzyme immunoassay (EIA) [37,38] techniques have facilitated research in this area, and have been validated in several commercially important fish species such as rainbow trout (Oncorhynchus mykiss) [36]. Because fish release steroid hormones into the surrounding water, either in conjugated (sulfonated or glucuronidated) forms in the urine and feces [39] or in unconjugated 'free' form through the gills [40,41], there is a potential for these compounds to gradually increase in concentration in recirculated water. ...
Article
Full-text available
A controlled seven-month study was conducted in six replicated water recirculation aquaculture systems (WRAS) to assess post-smolt Atlantic salmon (Salmo salar) performance in relation to WRAS water exchange rate. Unexpectedly high numbers of precocious sexually mature fish were observed in all WRAS toward the end of the study period; therefore, a separate investigation was conducted to quantify the levels of water-borne hormones (cortisol (C), testosterone (T), 11-ketotestosterone (11-KT), progesterone (P), and estradiol (E2)) to determine the impact of WRAS exchange rate, as well as transit through the unit processes, on soluble hormone concentrations.Triplicate water samples were collected at three separate sites in each of the six WRAS: pre-unit processes, post-unit processes, and at the makeup water influent. Water samples were concentrated and separate quantifications were carried out for each target hormone using enzyme immunoassay kits. Results indicated that among the hormones examined, only T was associated with higher concentrations in low exchange WRAS compared to high exchange WRAS. Water passage through the unit processes was associated with a significant reduction in concentration of 11-KT, in both high and low exchange WRAS. Water-borne concentrations of T, 11-KT, and E2 were significantly higher than influent makeup water; the majority of C and P concentrations were not significantly different between WRAS and makeup water samples. No significant differences were noted in the prevalence of apparently sexually mature fish or gonadosomatic indices in either sex between treatments, except a significantly higher prevalence of apparently mature female fish in low exchange WRAS. Overall, these findings suggest that, under the conditions of this study, C, P, E2, and 11-KT do not accumulate in lower exchange WRAS, and that, aside from 11-KT, the WRAS unit processes do not impact hormone concentration. Furthermore, the observed precocious sexual maturation was mostly unrelated to WRAS exchange rate.
... When employing the water-borne hormone collection technique, it is important to know whether the procedure itself induces a stress response because this would prohibit quantification of baseline hormone levels (Scott et al., 2008). Studies on other fish species have observed no stress response or acclimation to the procedure over the course of a few days (Wong et al., 2008;Friesen et al., 2012;Gabor & Contreras, 2012). Cortisol release rates in experiment 2 increased from days 1 to 3, and fell dramatically on day 4. ...
Article
This study validated a technique for non-invasive hormone measurements in California killifish Fundulus parvipinnis, and looked for associations between cortisol (a stress hormone) and 11-ketotestosterone (KT, an androgen) release rates and the density or intensity of the trematode parasites Euhaplorchis californiensis (EUHA) and Renicola buchanani (RENB) in wild-caught, naturally infected F. parvipinnis. In experiment 1, F. parvipinnis were exposed to an acute stressor by lowering water levels to dorsal-fin height and repeatedly handling the fish over the course of an hour. Neither parasite was found to influence cortisol release rates in response to this acute stressor. In experiment 2, different F. parvipinnis were exposed on four consecutive days to the procedure for collecting water-borne hormone levels and release rates of 11-KT and cortisol were quantified. This design examined whether F. parvipinnis perceived the water-borne collection procedure to be a stressor, while also exploring how parasites influenced hormone release rates under conditions less stressful than those in experiment 1. No association was found between RENB and hormone release rates, or between EUHA and 11-KT release rates. The interaction between EUHA density and handling time, however, was an important predictor of cortisol release rates. The relationship between handling time and cortisol release rates was negative for F. parvipinnis harbouring low or intermediate density infections, and became positive for fish harbouring high densities of EUHA.
... Ellis et al., 2004) and enzyme immunoassay (EIA) (e.g. Kidd et al., 2010;Friesen et al., 2012), and have been validated in cultured species such as rainbow trout (Oncorhynchus mykiss) (Ellis et al., 2004). Various non-aquaculture studies have demonstrated that ozonation has the capacity to reduce or eliminate specific waterborne steroid hormones (e.g. ...
Article
Full-text available
Steroid hormones have been shown to accumulate in recirculation aquaculture system (RAS) water over time; however, their influence on the reproductive physiology of fish within RAS remains unknown. Whether ozonation impacts waterborne hormone levels in RAS has likewise not been fully evaluated. To this end, a controlled 3-month study was conducted in 6 replicated RAS containing a mixture of sexually mature and immature Atlantic salmon Salmo salar to determine whether ozone, as typically applied in RAS to improve water quality, is associated with a reduction in waterborne hormones. Post-smolt Atlantic salmon (1,253±15g) were stocked into each RAS; 109 of 264 fish placed in each system were sexually mature males, and 5 were mature females. Water ozonation, controlled using an ORP set-point of 290–300mV, was applied with the pure oxygen feed gas within the low-head oxygenators of 3 randomly selected RAS, while the remaining 3 RAS did not receive ozone. The RAS hydraulic retention time was 6.9±0.3 days. Study fish were raised under these conditions for 12 weeks; during weeks 10 and 12, triplicate water samples were collected from the following locations in each RAS: i) culture tank, ii) makeup water, iii) pre-biofilter, iv) post-biofilter, and v) post-gas conditioning. Concentrations of 3 waterborne hormones – testosterone, 11-ketotestosterone (11-KT), and estradiol (17β-estradiol) – were quantified using enzyme immunoassays (EIA). Estradiol was significantly reduced by ozonation; testosterone and 11-KT were also reduced by ozonation, although these reductions were not observed across all sampling locations and events. Testosterone and 11-KT concentrations, however, were significantly reduced following water passage through the biofilters of both ozonated and non-ozonated RAS. The results of this study demonstrate the potential for ozone to be used in RAS as a means of preventing the accumulation of steroid hormones. Further research is required to assess whether reducing hormones in this manner impacts precocious sexual maturation in RAS-produced Atlantic salmon.
... Few studies have documented correlations between water-borne and whole-body steroid hormone concentrations (e.g., Fischer et al., 2014;Pavlidis et al., 2013;Zuberi et al., 2014), and ours is the first to examine all three methods simultaneously. Further, we add to the body of literature that has employed the water-borne technique to assess circulating steroids during distinct reproductive stages (Friesen et al., 2012;Kidd et al., 2013;Ramsey et al., 2011;Sebire et al., 2007) and expand on its utility by successfully demonstrating differences among populations of threespine stickleback with divergent evolutionary histories. We did so using a study design that would have been impossible with traditional methods, used one third of the animals that traditional methods would have required, and were able to capture within-individual variation across reproductive stages. ...
Article
Hormones play a prominent role in animal development, mediating the expression of traits and coordinating phenotypic responses to the environment. Their role as physiological integrators has implications for how populations respond to natural selection and can impact the speed and direction of evolutionary change. However, many emerging and established fish models with the potential to be ecologically or evolutionarily informative are small-bodied, making hormone sampling through traditional methods (whole-body or plasma) lethal or highly disruptive. Sampling methodology has thus restricted study design, often limiting sample sizes, and has prevented the study of at-risk/endangered populations. We utilize water-borne hormone sampling, a minimally invasive method of measuring the rate of steroid hormone release across the gills and further validate this method in a novel, evolutionary context. First, we compare water-borne hormone measures of cortisol with those quantified from plasma and whole-body samples collected from the same individuals to establish the relationship between concentrations quantified via the three methods. We then compare the release of steroid hormones in three populations of threespine stickleback to establish the sensitivity of this tool in measuring within-individual and between-individual variation in biologically relevant contexts (reproductive stages), and in assessing differences among populations with distinct evolutionary histories. We demonstrate a strong positive relationship between cortisol concentrations measured with water-borne, plasma, and whole-body collection techniques. Tracking estradiol and testosterone throughout clutch production in females produced anticipated patterns associated with growing and maturing eggs, with divergence in estradiol production in one population. Additionally, differences among populations in cortisol levels at ovulation paralleled the relative presence of a social stressor, and thus expected energetic needs within each population. We confirm that water-borne hormone sampling is sufficiently sensitive to capture biologically relevant fluctuations in steroid hormones between environmental contexts and demonstrate that among-population differences are detectable. This technique can be applied broadly to small fish to answer important ecological and evolutionary questions. By linking population variation in hormones and the multivariate phenotype, this technique will help elucidate both proximate mechanisms underlying phenotypic development and variation, and the way hormone networks alter evolutionary responses to selection.
... Our primary interest was to monitor acute fluctuations within individual fish; thus, we implemented a repeated measures design. In teleosts, hormones diffuse passively across the gills, and waterborne levels of hormone reflect the levels circulating within the individual (Ellis et al., 2005;Friesen et al., 2012). For small-bodied fishes, this is an invaluable technique allowing repeated measures when other methods would be lethal. ...
Article
Full-text available
Animal communication networks consist of multisensory signals and often include an acoustic element. Many freshwater fishes, including the blacktail shiner, Cyprinella venusta, produce specific vocalizations during courtship and agonistic behaviours. Similar to other vocal taxa, fish communication networks are vulnerable to noise pollution. The dominant frequencies of anthropogenic noise often overlap spectrally with animal acoustic signals. To investigate the role of acoustic signalling in breeding soniferous fishes in a physiological context, we conducted a series of acoustic playback trials. We measured waterborne levels of oestradiol (E2) and prostaglandin F2α (PGF2α) in gravid females exposed to courtship calls, and levels of 11-ketotestosterone (11KT) in males exposed to agonistic calls. In a separate series of trials, we exposed individuals to calls masked with bridge traffic noise. Hormone levels were quantified using enzyme-linked immunosorbent assays (ELISA) and analysed using repeated measures ANOVAs. Gravid females exhibited a significant drop in E2 (P = 0.023) and an increase in PGF2α during courtship calls (P = 0.01). Our research suggests that growl signals contribute to the onset of spawning behaviours in female C. venusta and this response may be dampened by anthropogenic noise pollution.
... In the literature, mostly immunoassays have been reported for the determination of fish endogenous compounds including SH [19,20]. Analytical procedures for the simultaneous determination of endogenous steroids like testosterone, 17βestradiol (E2), and estrone (E1) in fish plasma by SPE and gas chromatography-mass spectrometry (GC-MS) [21] and capillary separation methods [22] have been developed. ...
Article
Steroid hormones (SH) play a number of important physiological roles in vertebrates including fish. Changes in SH concentration significantly affect reproduction, differentiation, development, or metabolism. The objective of this study was to develop an in vitro high-throughput thin-film solid-phase microextraction (TF-SPME)–liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for targeted analysis of endogenous SH (cortisol, testosterone, progesterone, estrone (E1), 17β-estradiol (E2), and 17α-ethinylestradiol (EE2)) in wild white sucker fish plasma where the concentrations of the analytes are substantially low. A simple TF-SPME method enabled the simultaneous determination of free and total SH concentrations. The use of biocompatible coating allowed direct extraction of these hormones from complex biological samples without prior preparation. The carryover was less than 3%, thereby ensuring reusability of the devices and reproducibility. The results showed that TF-SPME was suitable for the analysis of compounds in the polarity range between 1.28 and 4.31 such as SH at different physicochemical properties. The proposed method was validated according to bioanalytical method validation guidelines. The limit of detection (LOD) and limit of quantification(LOQ) for cortisol, testosterone, progesterone, E1, E2, and EE2 were from 0.006 to 0.150 ng/mL and from 0.020 to 0.500 ng/mL, respectively. The recovery for the method was about 85%, and the accuracy and precision of the method for cortisol, testosterone, and progesterone were ≤ 6.0% and ≤ 11.2%, respectively, whereas those for E1, E2, and EE2 were ≤ 15.0% and ≤ 10.2%, respectively. On the basis of this study, TF-SPME demonstrated several important advantages such as simplicity, sensitivity, and robustness under laboratory conditions. Graphical abstract
... Hormones play an important role in reproductive cycles of animals particularly during maturation stages. Further, measuring blood parameters and hormone levels in fish with multiple times across developmental stages would provide better understanding of their regulations in physiology (Friesen et al, 2012). However, the levels of these parameters are species specific and synchronize during the development (Sabet et al, 2016). ...
Article
The present study investigated the basic information of the haematological and hormonal profile of high value murrel fish, Channa striatus reared under captivity. For the baseline studies; immature, matured and spent fishes of both genders were selected. The results revealed significantly lower TEC and Hb in immature fishes compared to matured male and females, and the TLC was significantly higher in spent stages than immature and matured fishes. But, no particular trend of MCV, MCH and MCHC was observed in the current study. In sexually dimorphic fishes, a dramatic increase in estrogens (E 2) compared to developmental stages was recorded, however the E 2 levels in females were significantly higher than matured males. Not surprisingly, quantity of testosterone (T) in developmental and sexually dimorphic fish was less than that of E 2 levels. A significantly higher T3 levels during early stages and in sexually dimorphic fishes were noticed compared to other developmental stages. Whereas, fluctuating trend of T4 and TSH were observed during the developmental stages and in sexually dimorphic fish. In summary, the results suggest that, the blood indices, sex steroids and growth hormones fluctuations observed in the study varied depending on the developmental and maturity stages of the fish.
... Hormones play an important role in reproductive cycles of animals particularly during maturation stages. Further, measuring blood parameters and hormone levels in fish with multiple times across developmental stages would provide better understanding of their regulations in physiology (Friesen et al, 2012). However, the levels of these parameters are species specific and synchronize during the development (Sabet et al, 2016). ...
Article
The present study investigated the basic information of the haematological and hormonal profile of high value murrel fish, Channa striatus reared under captivity. For the baseline studies; immature, matured and spent fishes of both genders were selected. The results revealed significantly lower TEC and Hb in immature fishes compared to matured male and females, and the TLC was significantly higher in spent stages than immature and matured fishes. But, no particular trend of MCV, MCH and MCHC was observed in the current study. In sexually dimorphic fishes, a dramatic increase in estrogens (E 2) compared to developmental stages was recorded, however the E 2 levels in females were significantly higher than matured males. Not surprisingly, quantity of testosterone (T) in developmental and sexually dimorphic fish was less than that of E 2 levels. A significantly higher T3 levels during early stages and in sexually dimorphic fishes were noticed compared to other developmental stages. Whereas, fluctuating trend of T4 and TSH were observed during the developmental stages and in sexually dimorphic fish. In summary, the results suggest that, the blood indices, sex steroids and growth hormones fluctuations observed in the study varied depending on the developmental and maturity stages of the fish.
... Several studies have shown that male fish have increased androgen levels during courtship and testosterone mediates aggression and courtship behavior by males (Pankhurst et al, 1999;Desjardins et al, 2007;Friesen et al, 2012). Consistent with these studies, our results describe a significant correlation between courtship behaviors and sex hormones in the male guppy. ...
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Rue (Ruta graveolens L.) is a medicinal plant that despite the anti-androgenic effects is currently used for treatment of various disorders. This study was conducted to investigate the effects of dietary supplementation of the Rue (R. graveolens L.) extract (1, 10 and 100 mg/kg in feed) on the courtship behaviors and sexual hormones of adult male guppy (Poecilia reticulate) for 30 days. Fish were fed 100 mg/kg Rue extract that showed a significant increase in 17 â-estradiol (E2) level, while the testosterone (T) level and the ratio of T/E2 were significantly decreased compared to the control group. Furthermore, Rue dietary significantly decreased the courtship behaviors in the male guppy in a dose-depended manner. The males at the highest dose group (100 mg/kg) showed less copulation attempts, in compare to control males. In addition, times spent in sigmoid display and with the females were significantly decreased in the males fed diets containing 10 and 100 mg/ kg of Rue extract. Furthermore, there was a positive correlation between courtship behaviors and T level, while negatively correlated with E2 concentrations. In conclusion, these results indicate that R. graveolens acts as anti-androgenic endocrine disruptor, and adversely affected the courtship behaviors and sex hormones of maleguppy, which can result in impaired reproduction.
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Stress in teleosts is an increasingly studied topic because of its interaction with growth, reproduction, immune system and ultimately fitness of the animal. Whether it is for evaluating welfare in aquaculture, adaptive capacities in fish ecology, or to investigate effects of human‐induced rapid environmental change, new experimental methods to describe stress physiology in captive or wild fish have flourished. Cortisol has proven to be a reliable indicator of stress and is considered the major stress hormone. Initially principally measured in blood, cortisol measurement methods are now evolving towards lower invasiveness and to allow repeated measurements over time. We present an overview of recent achievements in the field of cortisol measurement in fishes, discussing new alternatives to blood, whole body and eggs as matrices for cortisol measurement, notably mucus, faeces, water, scales and fins. In parallel, new analytical tools are being developed to increase specificity, sensitivity and automation of the measure. The review provides the founding principles of these techniques and introduces their potential as continuous monitoring tools. Finally, we consider promising avenues of research that could be prioritised in the field of stress physiology of fishes.
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The goals of this study were to examine the effect of stocking density on the stress response and disease susceptibility injuvenile rainbow trout (Oncorhynchus mykiss). Fish were sorted into one of 2 stocking densities (high density “HD”, 20-40kg/m3) or (low density, “LD”, 4-8 kg/m3) and 3 stress indices (cortisol levels in serum and water, and neutrophil: lymphocyte(N:L) ratios from blood smears) were measured at multiple time points over 21 d. Serum cortisol was significantly increased at 1 h in LD samples and at 14 d in HD samples. Water cortisol concentrations were significantly higher in LD tanks as compared with HD tanks on day 14. N:L ratios were significantly higher in HD tanks on day 14 as compared with LD tanks and with baseline. The effect of stocking density on mortality after exposure to infectious hematopoietic necrosis virus (IHNV) was compared between fish held in HD or LD conditions, with or without prior acclimation to the different density conditions. No significant differences in survival were found between HD and LD treatments or between acclimated and nonacclimatedtreatments. Cumulative results indicate that 1) 1 to 4 gram rainbow trout did not generally demonstrate significant differences in stress indices at the density conditions tested over a 21-d period, 2) independent differences were found in 3 stress indices at day 14 after sorting into LD and HD holding conditions; and 3) LD and HD stocking densities did not have a significant effect on mortality due to IHNV.
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Most animals encounter social challenges throughout their lives as they compete for resources. Individual responses to such challenges can depend on social status, sex, and community-level attributes, yet most of our knowledge of the behavioral and physiological mechanisms by which individuals respond to challenges has come from dyadic interactions between a resource holder and a challenger (usually both males). To incorporate differences in individual behavior that are influenced by surrounding group members, we use naturalistic communities of the cichlid fish, Astatotilapia burtoni, and examine resident dominant male responses to a territorial intrusion within the social group. We measured behavior and steroid hormones (testosterone and cortisol), and neural activity in key brain regions implicated in regulating territorial and social dominance behavior. In response to a male intruder, resident dominant males shifted from border defense to overt attack behavior, accompanied by decreased basolateral amygdala activity. These differences were context dependent – resident dominant males only exhibited increased border defense when the intruder secured dominance. Neither subordinate males nor females changed their behavior in response to a territorial intrusion in their community. However, neural activity in both hippocampus and lateral septum of subordinates increased when the intruder failed to establish dominance. Our results demonstrate how a social challenge results in multi-faceted behavioral, hormonal, and neural changes, depending on social status, sex, and the outcome of an intruder challenge. Taken together, our work provides novel insights into the mechanisms through which individual group members display context- and status-appropriate challenge responses in dynamic social groups.
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Fish behaviourists are increasingly turning to non-invasive measurement of steroid hormones in holding water, as opposed to blood plasma. When some of us met at a workshop in Faro, Portugal, in September, 2007, we realised that there were still many issues concerning the application of this procedure that needed resolution, including: Why do we measure release rates rather than just concentrations of steroids in the water? How does one interpret steroid release rates when dealing with fish of different sizes? What are the merits of measuring conjugated as well as free steroids in water? In the 'static' sampling procedure, where fish are placed in a separate container for a short period of time, does this affect steroid release and, if so, how can it be minimised? After exposing a fish to a behavioural stimulus, when is the optimal time to sample? What is the minimum amount of validation when applying the procedure to a new species? The purpose of this review is to attempt to answer these questions and, in doing so, to emphasize that application of the non-invasive procedure requires more planning and validation than conventional plasma sampling. However, we consider that the rewards justify the extra effort.
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To assess the possibility of thermal effects on reproductive function as a range-limiting mechanism in cottonmouths, we investigated the effects of temperature/photoperiod regimes on monthly male testosterone levels in three groups. The first was a field group, the second was a lab control group that received natural temperature/photoperiod conditions, and the third was an experimental lab group that received temperature/photoperiod conditions of a region north of the current range limit. The field and lab control groups exhibited a single testosterone peak in August (34.4 and 14.1 ng ml−1, respectively) that coincided with observed reproductive activities in Northwestern Arkansas. The experimental group also exhibited a single peak (11.7 ng ml−1), but the peak was delayed by one month. We coupled energetic calculations with environmental temperature, the timing of reproductive events, and the time available for foraging to demonstrate how pre- and postcopulation reproductive failure may serve as a sublethal range-limiting mechanism. North of their current range limit, the reduced time for energy acquisition may not allow for sufficient juvenile recruitment into the population to replace losses. Therefore, cottonmouths may be prevented from establishing a more northern range limit due to their reproductive physiology.
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In male birds, the responsiveness of androgens to sexual and territorial behaviour is predicted to vary with mating system and the degree of paternal investment ('challenge hypothesis', CH; Wingfield et al. 1990, American Naturalist, 136, 829-846). The CH predicts a higher and longer lasting 'breeding baseline' androgen level in males of polygynous species with no or only short-term paternal investment than in males of monogamous species with a high degree of paternal investment. Since the applicability of the CH to nonavian vertebrates has been unclear, we experimentally tested its predictions in several cichlid fish (Neolamprologus pulcher, Lamprologus callipterus, Tropheus moorii, Pseudosimochromis curvifrons and Oreochromis mossambicus) using a simulated territorial intruder protocol. Androgens (11-ketotestosterone: 11-KT; testosterone: T) were measured from fish-holding water. In all species sampled, the 11-KT patterns confirmed the predictions of the CH originating from the avian literature, but T patterns did not. Males of all species sampled were highly responsive to territorial intrusions; however, the magnitude and duration of this response, that is, the rapid return to baseline 11-KT levels, could not clearly be explained by the degree of paternal care. 11-KT responses to interactions with ovulating females were observed in maternal mouthbrooders but not in biparental species (e.g. Lamprologini). At the interspecific level, androgen responsiveness was greater among males of monogamous species, as predicted, but also in species with more intense pair bonding (e.g. Tropheus moorii). Thus, this study confirms the predictions of the CH in cichlid fish at both the intraspecific and the interspecific levels. (C) 2004 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
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Fish behaviourists are increasingly turning to non-invasive measurement of steroid hormones in holding water, as opposed to blood plasma. When some of us met at a workshop in Faro, Portugal, in September, 2007, we realised that there were still many issues concerning the application of this procedure that needed resolution, including: Why do we measure release rates rather than just concentrations of steroids in the water? How does one interpret steroid release rates when dealing with fish of different sizes? What are the merits of measuring conjugated as well as free steroids in water? In the 'static' sampling procedure, where fish are placed in a separate container for a short period of time, does this affect steroid release and, if so, how can it be minimised? After exposing a fish to a behavioural stimulus, when is the optimal time to sample? What is the minimum amount of validation when applying the procedure to a new species? The purpose of this review is to attempt to answer these questions and, in doing so, to emphasize that application of the non-invasive procedure requires more planning and validation than conventional plasma sampling. However, we consider that the rewards justify the extra effort.
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We used a common-garden rearing experiment to explore environmentally induced tolerance to hypoxia in the African mouth-brooding cichlid Pseudocrenilabrus multicolor. F(1) fish originating from three field populations were grown under low or high dissolved oxygen (DO), and their resting routine metabolic rate (RMR), critical oxygen tension (P(crit)), and marginal metabolic scope (MMS) were quantified. In a second rearing experiment, we compared the RMR of brooding and nonbrooding females of low-DO origin grown under low and high DO. Fish reared under low DO had a lower P(crit) than fish reared under high DO. There was also an interaction between treatment and gender; females had a higher P(crit) than males when reared under normoxia. Variation in RMR was driven primarily by population effects, and there was an interaction between treatment and population. Regardless of population or treatment, males had a higher MMS than females. Fish reared under low DO had a higher MMS than fish reared under high DO, except for the high-DO population in which there was no treatment effect. Brooding females had a higher RMR than postbrooding females regardless of the growth treatment, indicating an energetic cost to brooding. The results suggest a strong element of developmental plasticity in P(crit) across populations and both plastic and genetic components of variation in the RMR and MMS. This study also highlights the cost of parental care in mouth-brooding fishes, which may increase the fitness of the offspring at the energetic expense of the parent, a cost that may be elevated under hypoxia.
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In many territorial species androgens respond to social interactions. This response has been interpreted as a mechanism for adjusting aggressive motivation to a changing social environment. Therefore, it would be adaptive to anticipate social challenges and reacting to their clues with an anticipatory androgen response to adjust agonistic motivation to an imminent social challenge. Here we test the hypothesis of an anticipatory androgen response to territorial intrusions using classical conditioning to establish an association between a conditioned stimulus (CS = light) and an unconditioned stimulus (US = intruder male) in male cichlid fish (Oreochromis mossambicus). During the training phase conditioned males (CS-US paired presentations) showed a higher decrease in latency for agonistic response toward the intruder than unconditioned males (CS-US unpaired presentations). In the test trial, conditioned males showed an increase in androgen levels (i.e., testosterone and 11-ketotestosterone) relative to baseline, in response to the CS alone. This increase was similar to that of control males exposed to real intruders after CS, whereas unconditioned males showed a decrease in androgen levels in response to the CS. Furthermore, conditioned males were significantly more aggressive than unconditioned males during the post-CS period on test trial, even though the intruder male was not present during this period. These results reveal the occurrence of a conditioned androgen response that may give territorial males an advantage in mounting a defense to upcoming territorial intrusions, if the ability to readily elevate androgens does not co-vary with other traits that bear costs.
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Winning aggressive contests can both enhance future winning ability and change post-encounter hormones; however, it remains unclear if the context of a fight also influences such winner effects and hormone changes. We investigated this issue by using California mice (Peromyscus californicus) to test if the effect of residency status is necessary to improve future winning ability and alter post-encounter hormones. Male mice were subjected to an aggressive contest and their blood was collected 45 min after the fight. Upon contest initiation, focal mice had a 'home advantage' and three prior winning experiences, only one of these factors, or neither factor. Only individuals with a 'home advantage' and prior winning experience showed a full winner effect. Post-encounter changes in testosterone and progesterone resulted from an interaction between residency status and winning experience. These data indicate that in male California mice a 'home advantage' is necessary to produce the full winner effect and that residency status helps regulate inter-individual variation in the expression of post-encounter testosterone pulses and progesterone changes. Furthermore, these respective behavioral and physiological phenomena might be modulated in a context-specific manner, in part by the surrounding physical environment.
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In this study we have used the rainbow trout as a model animal to study the biological consequences of stress in terms of gamete quality and quantity. Groups of 30 mature male and female rainbow trout were subjected to repeated acute stress during the 9 mo prior to spawning. Time of ovulation, fecundity, and egg size were recorded in mature females, and sperm counts were carried out on the milt from the male fish, from both the stressed and control groups. Eggs from ovulated females were fertilized with milt from males subjected to the same treatment regime. Approximately 300 eggs from each female were fertilized with a sperm dilution of 10(-3) in diluent. Subsequent development of the fertilized eggs was then monitored. There were no differences in somatic weight or length between the two groups at the end of the experiment, but exposure of rainbow trout to repeated acute stress during reproductive development resulted in a significant delay in ovulation and reduced egg size in females, significantly lower sperm counts in males, and, perhaps most importantly, significantly lower survival rates for progeny from stressed fish compared to progeny from unstressed control fish. Hence, stress reduces the quality of gametes produced by rainbow trout.
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Various hormones were analyzed during the course of a reproductive cycle in the cichlid fish Oreochromis niloticus: plasma levels of the gonadal steroids 17beta-estradiol (E2), testosterone (T), 17, 20beta-OH progesterone (17,20beta-P), gonadotropin (taGtH), and plasma and pituitary concentrations of prolactin (tiPRL(I) and tiPRL(II)) and growth hormone (tiGH). Two categories of fish were sampled and sacrificed on days 1 and 3 postspawning and at 3-day intervals thereafter: typical incubating females (INC), and nonincubating females (NI), deprived of their eggs just after spawning. Such deprivation is known to suppress maternal behavior and to accelerate ovarian development and especially vitellogenesis, thus shortening the mean interspawning interval. In both groups, variations of the plasma concentrations of E2 and T appeared to depend on ovarian stages, and differences between groups appeared to reflect underlying differences in the kinetics of ovarian development. The observation of noticeable levels of 17,20beta-P in plasma before spawning, when high values of taGtH could also be detected in NI females, suggests the implication of this progestin in the control of final maturation events, as in some other teleosts. Moreover, 17,20beta-P, which was still detected a few days after spawning, but at low concentrations and only in the plasma of INC females, might play a role at the beginning of the reproductive cycle in incubating females (maternal behavior and/or slowing down of ovarian growth). The pituitary and plasma profiles of both tiPRLs isoforms appeared to depend mainly on the kinetics of ovarian development in each group of fish, suggesting a role during the beginning of vitellogenesis. However, the variance of plasma tiPRL(II), which was significantly enhanced during maternal behavior in INC females, also suggests an implication of this hormone in the control of that behavior. Concerning tiGH, comparison of the plasma profiles in INC and NI fish also suggest an influence on the control of maternal behavior, but a main effect of starvation of INC during mouthbrooding cannot be excluded.
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The 'winner effect' has been studied in a variety of species, but only rarely in mammals. We compared effects of winning three, two, one, or zero resident-intruder encounters on the likelihood of winning a subsequent aggressive encounter in the California mouse (Peromyscus californicus). During the training phase, we ensured that resident males won all encounters by staging contests with mildly sedated, smaller intruders. During the test phase, the resident male encountered an unfamiliar, more evenly matched intruder that had experience winning an encounter and was larger than the resident. Testosterone (T) plasma levels significantly increased after the final test when they had experienced two prior winning encounters, and the probability of winning a future encounter increased significantly after three prior wins independent of intrinsic fighting ability. We hypothesize a 'winner-challenge' effect in which increased T levels serve to reinforce the winner effect in male California mice.
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Measurement of fish steroids in water provides a non-invasive alternative to measurement in blood samples, offering the following advantages: zero or minimal intervention (i.e. no anaesthetic, bleeding or handling stress); results not being biased by sampling stress; repeat measurements on the same fish; the possibility of making non-lethal measurements on small and/or rare fish; integrating the response of many (or of single) fish; and allowing concurrent monitoring of behaviour or physiology. The procedure is relatively new and, although applications are still fairly limited, there are several themes and potential problem areas that are worthy of review.
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This chapter discusses the yolk formation of teleost fishes. The process of vitellogenesis in teleosts has been shown to be similar to that operating in other oviparous vertebrates. The contribution of autosynthetic processes (endogenous vitellogenesis) to the yolk mass in the teleost ovary, relative to exogenous yolk acquired by incorporation of vitellogenin, has not been estimated. Exogenous vitellogenesis can be considered to consist of two phases. The first phase involves the induction of hepatic vitellogenin production under stimulation of ovarian estrogen. During the second phase vitellogenin is taken up from the blood stream and incorporated into ovarian yolk proteins. In salmonids maturational gonadotropin occurs at high levels in plasma around spawning time but is near the lower limit of the radioimmunoassay during the phase of active incorporation of vitellogenin. There appears to be a small increase in maturational gonadotropin coincident with an increase in estradiol in trout plasma early in vitellogenesis, and antibody to maturational gonadotropin inhibits ovarian growth immediately prior to the rapid and massive increase in the ovarian weight. Therefore, there appears to be a low level of maturational hormone present when the fish resumes ovarian development after spawning, but it is sufficient to establish vitellogenin production by the liver.
Article
Plasma estradiol-17 beta and testosterone levels were assessed by radioimmunoassay during the sexual maturation of female amago salmon (Oncorhynchus rhodurus). Estradiol-17 beta levels gradually increased during vitellogenesis (June to September), reached a peak in September (about 16 ng/ml) and rapidly decreased in mature and ovulated fish (about 3-4 ng/ml) in October. The seasonal pattern of plasma testosterone levels lagged behind and followed that of estradiol-17 beta during vitellogenesis, but levels remained high in mature and ovulated fish (90-110 ng/ml). Estradiol-17 beta levels and the gonadosomatic index (GSI) values correlated well during vitellogenesis: GSI values showed a linear increase, and reached a peak (29.9 +/- 1.4) in October. Values were extremely low in ovulated fish (1.2 +/- 0.2). In vitro production of estradiol-17 beta and testosterone by ovarian follicles in response to partially purified chinook salmon gonadotropin (SG-G100) was examined monthly using 18-h incubations. Throughout the vitellogenic period SG-G100 stimulated both estradiol-17 beta and testosterone production: the steroidogenic response of follicles increased from June (about 2 ng/ml estradiol-17 beta; 0.1 ng/ml testosterone) to September (about 10 and 14 ng/ml, respectively). In October full-grown immature follicles which could be induced to mature in vitro by hormone treatment produced large amounts of testosterone (about 130 ng/ml) but not estradiol-17 beta. Postovulatory follicles also produced testosterone but the values were low (10 ng/ml) compared with full-grown immature follicles. Very low levels of estradiol-17 beta were produced by postovulatory follicles.(ABSTRACT TRUNCATED AT 250 WORDS)
Article
In this study we have used the rainbow trout as a model animal to study the biological consequences of stress in terms of gamete quality and quantity. Groups of 30 mature male and female rainbow trout were subjected to repeated acute stress during the 9 mo prior to spawning. Time of ovulation, fecundity, and egg size were recorded in mature females, and sperm counts were carried out on the milt from the male fish, from both the stressed and control groups. Eggs from ovulated females were fertilized with milt from males subjected to the same treatment regime. Approximately 300 eggs from each female were fertilized with a sperm dilution of 10-3 in diluent. Subsequent development of the fertilized eggs was then monitored. There were no differences in somatic weight or length between the two groups at the end of the experiment, but exposure of rainbow trout to repeated acute stress during reproductive development resulted in a significant delay in ovulation and reduced egg size in females, significantly lower sperm counts in males, and, perhaps most importantly, significantly lower survival rates for progeny from stressed fish compared to progeny from unstressed control fish. Hence, stress reduces the quality of gametes produced by rainbow trout.
Article
17β-oestradiol, testosterone, 11 -ketotestosterone and calcium were measured in plasma of female rainbow trout over the course of a single spawning season. The patterns of rise and fall of the levels of 17β-oestradiol and calcium during sexual maturation were similar to those demonstrated by other workers. Very high levels of testosterone were found in plasma of sexually mature fish—mean level 211 ng ml-1 in November and 115 ng ml-1 in January. Ovulation occurred from December to February.
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Cortisol is an important indicator of health and behavioral state in fishes, and is produced in response to stressors including confinement, handling and social conflict. An inherent dif-ficulty in measuring circulating cortisol is the implementation of invasive procedures that can be potent stressors. Recent studies show that cortisol can be reliably quantified from fish holding water by placing individuals in a small beaker for a predetermined collection period. We investigated whether convict cichlid fish (Amatitlania nigrofasciata) mount a significant stress response to beaker confinement and whether they habituate to the collection method. We also determined the relationship between plasma and water-borne cortisol, and changes in cortisol release rates following handling and cortisol administration. Initial beaker expo-sure induced high cortisol release rates but cichlids quickly habituated after 3–4 exposures. We revealed significant positive correlations between plasma and water-borne cortisol, and marked increases in water-borne cortisol release rates after cortisol injection that persisted for between 4 and 24 h, depending on the dosage. In conclusion, we provide convincing evidence for the utility and validity of the water-borne collection method to measure cortisol release rates in convict cichlids.
Article
A procedure previously used for sex steroids was adapted to extract free cortisol and cortisone from water samples taken from rainbow trout Oncorhynchus mykiss tanks. Both corticosteroids could be readily detected by radioimmunoassay (RIA), with cortisol being predominant. All stages of the sampling, extraction and RIA procedure were validated for cortisol. An intermittent problem with poor replication was traced to the use of diethyl ether during the extraction procedure, and was overcome by the use of ethyl acetate. Other modifications were also introduced to speed up the procedure. The concentration and time course of release of both corticosteroids were shown to be related to the degree of stress that the fish had been subjected to. It was confirmed that cortisol concentrations in water and estimated cortisol release rates increased in response to handling stress, and that both were correlated with plasma cortisol concentrations. The potential for using water cortisol concentration and release rates to assess the primary stress response of fishes as a non‐invasive alternative to blood sampling is discussed.
Article
Individual rainbow trout Oncorhynchus mykiss were held in a specially constructed tank that enabled water to be collected separately from the anterior and posterior ends of the fish. Measurement by radioimmunoassay showed that >95% of the cortisol and melatonin released into the water originated from the anterior end (dominated by the gills). High performance liquid chromatography confirmed the identity of both hormones.
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This study directly tested the hypothesis that 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P) is a goldfish preovulatory pheromone (pheromone released at peak levels during oocyte final maturation) which increases blood gonadotropin (GtH) and milt volume in males. During spontaneous ovulation, GtH and 17,20β-P in female blood and 17,20β-P released to the water increased dramatically 7–10 hr prior to ovulation, peaked 1–4 hr prior to ovulation, and then rapidly declined. Males held with these females, or exposed to their odors, had increased GtH levels and milt volumes at approximately the time when increased 17,20β-P release by ovulatory females commenced. Although these findings strongly support the hypothesis that 17,20β-P is a preovulatory female sex pheromone in goldfish which stimulates male GtH levels and milt production prior to spawning, the milt increases occurred earlier than predicted, suggesting either that preovulatory 17,20β-P release begins earlier than the data indicate or that other steroids known to have pheromonal activity are released before 17,20β-P.
Article
The effect of temperature on ovarian steroid production in the common carp, Cyprinus carpio L., has been studied in vitro with exogenous and endogenous precursors, and in fish held at three different temperatures in vivo. With radioactive testosterone as substrate, the major metabolite was testosterone glucuronide, but androstenedione and 5α-androstane-3β,17β-diol were also identified, 5α-Androstane-3β,7α,17β-triol was tentatively identified and two other polar metabolites were isolated, one of which was convertible to this triol. A significant increase in production of most metabolites occurred between 20 and 24°. Production of estradiol and testosterone from endogenous substrate under gonadotrophin stimulation in vitro showed a marked temperature dependence, but the response was closely related to ovarian maturity. Stage 4–5 ovaries produced testosterone, while late Stage 3 tissue produced only estradiol. Neither steroid was produced in significant quantities by less mature ovaries. The results indicate that the “switch off” of ovarian aromatase activity at the end of vitellogenesis is actuated by an ovarian rather than by a pituitary factor. Secretion of testosterone and estradiol showed a very significant change with temperature with the optimum at 24–29°. Profiles for individual fish show that this optimal range is extremely narrow, particularly for estradiol, where secretion may increase as much as twentyfold over 5°. The results in general correlate well with 24° as the most favourable temperature for reproduction in carp. Plasma concentrations of testosterone and estradiol closely paralleled the in vitro secretion rates of these hormones. Plasma testosterone levels were greatest in the most mature fish, whereas plasma estradiol was significantly higher in late Stage 3 fish than in those of greater or lesser ovarian maturity. More Stage 4 and 5 fish were found in the group held at 24° than at 20 or 29° for 4 weeks, but all groups contained a high proportion of early Stage 3 fish.
Article
Many aquatic species, such as teleosts, release into the water and detect multiple bioactive substances to assist in schooling, migration, alarm reactions, and to stimulate behavioral and physiological responses during reproduction and in parent–offspring interactions. Understanding the complex relationship between hormones, behavior and their function in communication requires the simultaneous examination of multiple circulating hormones. However, repeated blood sampling within a short time period is not possible in smaller animals without impacting the very behaviors under investigation. The non-invasive technique of collecting and measuring hormone values in holding water using either radioimmunoassay (RIA) or enzyme immunoassay (EIA) is becoming widely used in teleost research. Commercial assay kits in particular enable rapid and reliable data generation, yet their assay buffers are often specific and potentially incompatible with each other, which can hinder measuring multiple hormones from the same sample. We present here the validation and application of a “nested” elution technique we developed that allows for repeated sampling of multiple reproductive hormones – testosterone (T), 17β-estradiol (E2), progesterone (P), prostaglandin F2α (PGF) and 11-ketotestosterone (11KT) – from individual samples of animal holding water by using commercial EIA systems. Our results show that when using appropriate controls to account for possible technical and biological confounds, this technique provides a powerful new tool for research in aquatic endocrinology and physiology.
Article
Hormonal responses to male-male interactions have been detected in some studies of vertebrates but not others. One hypothesis that may partially explain these discrepancies is that differences in the duration of male-male interactions cause different hormonal responses. In social systems based on dominance-subordinance hierarchies interactions often last longer than if exclusive territories are maintained. Tests of the hypothesis that encounter duration explains discrepancies in hormonal responses would be facilitated by a species that shows elements of both types of social systems, such as species in which males practice alternative reproductive tactics. We compared plasma levels of corticosterone and testosterone in males of the territorial morph of the tree lizard (Urosaurus ornatus) subjected either to short (15 min) male-male encounters or to long (7 days) dominance interactions. In the long interactions, dominant males had lower levels of corticosterone than did subordinate males over the first day, but this difference subsequently disappeared. In sharp contrast, winners of short-term encounters had elevated plasma corticosterone levels which peaked the day after the encounter. Thus, males isolated after a short encounter experienced an increase in corticosterone that was apparently inhibited in males who continued interacting with other males. The delayed increase in corticosterone after a short encounter may facilitate metabolic recovery from the encounter, mobilize metabolic substrates useful in subsequent encounters, or may alter subsequent behavior. The response does not appear to be simply recovery from exercise because in a second experiment males exercised for 15 min did not show a similar delayed increase in corticosterone the following day. Testosterone levels were also monitored and did not change in any of these treatments. These results demonstrate that the duration and the outcome of male-male interactions may each independently influence hormone levels.
Article
A radioimmunoassay for the simultaneous determination of testosterone, 11β-hydroxytestosterone, 11-ketotestosterone, and their glucuronides is described. The plasma concentrations of these steroids have been determined throughout the annual reproductive cycle of the male brown trout Salmo trutta. The maximum concentration of testosterone (33.2 ± 5.4 ng/ml in late September) precedes that of 11-ketotestosterone (68.7 ± 5.5 ng/ml) which coincides with the period of spermiation in early November. Peak concentrations of testosterone glucuronide (14.5 ± 2.7 ng/ml), 11β-hydroxytestosterone glucuronide (6.8 ± 1.6 ng/ml), 11-ketotestosterone glucuronide (5.4 ± 0.7 ng/ml), and 11β-hydroxytestosterone (3.2 ± 0.7 ng/ml) were also found in November. The ratio of free:conjugated testosterone drops significantly between August and December in the male (10.7-0.66) but not in the female (6.8-5.3). Factors such as hormones and mineral ions which may affect glucuronyl transferase activity are discussed, and a possible role for glucuronides in the normal functioning of the reproductive cycle in the trout is suggested.
Article
Competition elevates plasma testosterone in a wide variety of vertebrates, including humans. The 'challenge hypothesis' proposes that seasonal peaks in testosterone during breeding are caused by social challenges from other males. However, during experimentally induced male-male conflicts, testosterone increases only in a minority of songbird species tested so far. Why is this so? Comparative evidence suggests that species with a short breeding season may not elevate testosterone levels during territory defence. These species may even be limited in their physiological capability to increase testosterone levels, which can be tested by injecting birds with gonadotropin-releasing hormone (GnRH). We studied two populations of black redstarts that differ in breeding altitude, morphology and the length of their breeding season. Unexpectedly, males of neither population increased testosterone in response to a simulated territorial intrusion, but injections with GnRH resulted in a major elevation of testosterone. Thus, black redstarts would have been capable of mounting a testosterone response during the male-male challenge. Our data show, for the first time, that the absence of an androgen response to male-male challenges is not owing to physiological limitations to increase testosterone. Furthermore, in contrast to comparative evidence between species, populations of black redstarts with a long breeding season do not show the expected elevation in testosterone during male-male challenges.
Article
The gonadal hormone testosterone (T) regulates aggression across a wide range of vertebrate species. Recent evidence suggests that the adrenal prohormone dehydroepiandrosterone (DHEA) may also play an important role in regulating aggression. DHEA can be converted into active sex steroids, such as T and estradiol (E(2)), within the brain. Previous studies show that circulating DHEA levels display diurnal rhythms and that melatonin increases adrenal DHEA secretion in vitro. Here we examined serum DHEA and T levels in long-day housed Siberian hamsters (Phodopus sungorus), a nocturnal species in which melatonin treatment increases aggression. In Experiment 1, serum DHEA and T levels were measured in adult male hamsters during the day (1200 h, noon) and night (2400 h, midnight). In Experiment 2, aggression was elicited using 5-min resident-intruder trials during the day (1800 h) and night (2000 h) (lights-off at 2000 h). Serum DHEA and T levels were measured 24 h before and immediately after aggressive encounters. In Experiment 1, there was no significant difference in serum DHEA or T levels between noon and midnight, although DHEA levels showed a trend to be lower at midnight. In Experiment 2, territorial aggression was greater during the night than the day. Moreover, at night, aggressive interactions rapidly decreased serum DHEA levels but increased serum T levels. In contrast, aggressive interactions during the day did not affect serum DHEA or T levels. These data suggest that nocturnal aggressive encounters rapidly increase conversion of DHEA to T and that melatonin may play a permissive role in this process.
Article
In this paper we attempted to investigate the existence of daily fluctuations on plasma sexual steroids (17beta-estradiol, E(2) and testosterone, T) in Senegal sole (Solea senegalensis) females. We described the monthly day/night concentrations and seasonal daily rhythms in animals reared under natural photo- and thermo-period. In addition, the influence of the natural annual fluctuation of the water temperature on the plasma concentration of these steroids was investigated, using one group of Senegal sole under a natural photoperiod, but with an attenuated thermal cycle (around 17-20 degrees C) for one year. Although no significant day/night differences were detected in monthly samplings, the existence of an annual rhythm of E(2) and T (p<0.01) with an acrophase in February was revealed by COSINOR analysis. Maximum values were reached in March for both steroids (6.1+/-1.7 ng mL(-1) at mid-dark, MD and 4.0+/-0.6 ng mL(-1) at mid-light, ML for E2 and 1.4+/-0.4 ng mL(-1) at MD and 0.8+/-0.1 ng mL(-1) at ML for T) in anticipation of the spawning season (May-June). As regards seasonal daily rhythms, the presence of daily oscillations was revealed. At the spring solstice (21st March) a daily rhythm was observed for both steroids (COSINOR, p<0.01), with an acrophase at 20:00 h (E(2)) and at 21:08 h (T). In summer, autumn and winter no daily rhythms were observed due to the low steroid levels at those seasons. When Senegal sole females were submitted to an attenuated annual thermal cycle, the steroid rhythm disappeared (there was no surge in spring, as in the control group) and these fish did not spawn, despite being subjected to natural photoperiod conditions. This result underlined the importance of the natural annual fluctuation of water temperature and photoperiod on the synchronization of the spawning season and on the onset of steroidogenesis.
Article
Rainbow trout were confined for 48 hr, during which time water quality either was allowed to deteriorate (resulting in elevated NH3, elevated free CO2, and reduced dissolved O2) or was maintained at preconfinement levels. Fish were removed and blood samples taken at 0, 2, 4, 8, 24, and 48 hr after the onset of confinement from both stressed (confined) and unstressed (unconfined) fish. Plasma cortisol and plasma prolactin (PRL) levels were determined using specific RIAs. Chronic confinement of rainbow trout, accompanied by a decline in water quality, resulted in significant elevation of plasma cortisol, maintained for the period of confinement. Plasma PRL levels were significantly lower in stressed fish, by up to 60% relative to control fish, during the first 24 hr of confinement. The stress of confinement alone, in the absence of deterioration in water quality, produced similar results, with the change in prolactin levels being less rapid but more prolonged under these conditions.
Article
This study directly tested the hypothesis that 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) is a goldfish preovulatory pheromone (pheromone released at peak levels during oocyte final maturation) which increases blood gonadotropin (GtH) and milt volume in males. During spontaneous ovulation, GtH and 17,20 beta-P in female blood and 17,20 beta-P released to the water increased dramatically 7-10 hr prior to ovulation, peaked 1-4 hr prior to ovulation, and then rapidly declined. Males held with these females, or exposed to their odors, had increased GtH levels and milt volumes at approximately the time when increased 17,20 beta-P release by ovulatory females commenced. Although these findings strongly support the hypothesis that 17,20 beta-P is a preovulatory female sex pheromone in goldfish which stimulates male GtH levels and milt production prior to spawning, the milt increases occurred earlier than predicted, suggesting either that preovulatory 17,20 beta-P release begins earlier than the data indicate or that other steroids known to have pheromonal activity are released before 17,20 beta-P.
Article
Plasma estradiol-17 beta and testosterone levels were assessed by radioimmunoassay during the sexual maturation of female amago salmon (Oncorhynchus rhodurus). Estradiol-17 beta levels gradually increased during vitellogenesis (June to September), reached a peak in September (about 16 ng/ml) and rapidly decreased in mature and ovulated fish (about 3-4 ng/ml) in October. The seasonal pattern of plasma testosterone levels lagged behind and followed that of estradiol-17 beta during vitellogenesis, but levels remained high in mature and ovulated fish (90-110 ng/ml). Estradiol-17 beta levels and the gonadosomatic index (GSI) values correlated well during vitellogenesis: GSI values showed a linear increase, and reached a peak (29.9 +/- 1.4) in October. Values were extremely low in ovulated fish (1.2 +/- 0.2). In vitro production of estradiol-17 beta and testosterone by ovarian follicles in response to partially purified chinook salmon gonadotropin (SG-G100) was examined monthly using 18-h incubations. Throughout the vitellogenic period SG-G100 stimulated both estradiol-17 beta and testosterone production: the steroidogenic response of follicles increased from June (about 2 ng/ml estradiol-17 beta; 0.1 ng/ml testosterone) to September (about 10 and 14 ng/ml, respectively). In October full-grown immature follicles which could be induced to mature in vitro by hormone treatment produced large amounts of testosterone (about 130 ng/ml) but not estradiol-17 beta. Postovulatory follicles also produced testosterone but the values were low (10 ng/ml) compared with full-grown immature follicles. Very low levels of estradiol-17 beta were produced by postovulatory follicles.(ABSTRACT TRUNCATED AT 250 WORDS)
Article
The goal of this study was to identify excretory routes of three main steroids produced by sexually mature male and female rainbow trout: 17,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P), sulfated 17,20 beta-P (17,20 beta-P-S), and testosterone glucuronide (TG). Spermiating males or maturing trout were cannulated via the dorsal aorta and urinary bladder and injected with tritiated steroids. Blood, water, and urine were sampled over the next 12 hr when the fish were killed and bile was collected. The identities of the excreted products were determined by anion-exchange chromatography, reverse-phase high-performance liquid chromatography, enzyme hydrolysis, acid solvolysis, microchemical modification, and thin-layer chromatography. Following the injection of tritiated 17,20 beta-P, 25% of the radioactivity rapidly appeared unmodified in the water; 15% appeared slowly in the urine, mainly as 17,20 beta-P-S; and 40% was recovered in the bile, mainly as 17,20 beta-P-glucuronide. 17,20 beta-P was shown to be released into the water via the gills. Over the 12-hr sampling period, 20% of the 17,20 beta-P released into the water was taken up again by the fish (also branchially). A mathematical analysis showed that 40% of the 17,20 beta-P would have been released into the water in the absence of uptake. Following the injection of tritiated 17,20 beta-P-S, 63% appeared very rapidly, in an unmodified form, in the urine, and 15% was recovered in the bile. Following the injection of tritiated TG, 9% appeared slowly, mainly untransformed, in the urine, and 59% was recovered in the bile. These results show that the three types of steroids are released into the water by three different routes: free steroids, gills; sulfated steroids, urine; and glucuronidated steroids, bile.
Article
The aim of this study was to identify the major C21 steroids produced in vivo during artificially induced final oocyte maturation and spawning in female common dentex (Dentex dentex). During the spawning season, mature females were treated with a gonadotropin-releasing hormone agonist (GnRHa)-loaded delivery system, with or without pimozide (given as a single dose at the beginning of the experiment). Blood samples were collected at various intervals during the experiment and were assayed for GnRHa, 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P), and 17,20beta,21-trihydroxy-4-pregnen-3-one (17,20beta,21-P). A higher percentage of ovulated females was observed in GnRHa-implanted fish, which produced over 10 times more eggs than controls. Relative fecundity was highest in the GnRHa + pimozide group and lowest in controls. The viability of naturally released eggs was low (2 to 15%) in all groups. Plasma concentrations of 17,20beta-P in GnRHa-implanted fish did not increase, but those in control fish decreased, such that there was a significant difference between control and treated fish between 2 and 10 days after treatment. In another experiment, ovulating common dentex were injected intramuscularly with a single dose of 50 microg kg(-1) of GnRHa in saline and were sampled for blood at 0, 3, 6, 12, and 24 h postinjection. A single water sample was taken from the tanks at 9 h postinjection, the tanks having been emptied and refilled at 6 h. Measurements were made of plasma and water concentrations of free and conjugated 17,20beta-P, 17,20beta,21-P, 17beta-oestradiol (E2), and GnRHa (plasma only). The GnRHa injection increased plasma levels of all steroids, with free 17,20beta-P reaching maximal levels within 3 h. GnRHa treatment also increased the amounts of free and conjugated steroids released into the water between 6 and 9 h.
Article
The magnitude by which plasma cortisol levels increase following exposure to a stressor is a heritable trait in rainbow trout. The relative growth in coculture of F1 lines selected for high responsiveness (HR) and low responsiveness (LR) to a confinement stressor suggested that behavioral characteristics related to food acquisition, aggression, or competitive ability might differ between the two lines. This hypothesis was tested using the F2 generation of the selected lines. The F2 lines clearly exhibited the characteristics of the F1 parents, displaying significantly divergent plasma cortisol responses to a 1-h confinement stressor and a high heritability for the trait. Behavioral differences between the lines were assessed by observing the outcome of staged fights for dominance in size-matched pairs of HR and LR fish. The identification of dominant and subordinate fish within each pair on the basis of their behavior was supported by the levels of blood cortisol in the fish attributed to each group (dominant < subordinate). Fish from the LR line were identified as dominant in significantly more trials than were HR individuals. The results suggest that behavioral attributes that affect the outcome of rank-order fights are closely linked to the magnitude of the plasma cortisol response to stress in rainbow trout. Whether the link is causal or circumstantial is not yet evident.
Article
The occurrence of intra-sexual variation in reproduction is a widespread phenomenon in teleosts. One such form of variation consists in the occurrence of alternative male types: males that invest resources in mate attraction and males that exploit the investment of the former males, by trying to sneak fertilizations during spawning. These alternative reproductive tactics can be classified according to their plasticity during the life span of the individuals (i.e., fixed vs. sequential vs. reversible). Furthermore, the differences between morphs within a given species may involve a set of different traits, including reproductive behavior, the differentiation of male morphological traits, and the patterns of gonad tissue allocation and the differentiation of gonadal accessory glands. In this paper, we review the available data on four species exhibiting different types of intra-sexual plasticity in reproduction that have been studied in our lab. The data on the proximate mechanisms, androgens and forebrain arginine-vasotocin (AVT), underlying these alternative tactics suggest that between-morph differences in androgen levels, especially in 11-ketotestosterone, are especially present in species where the alternative male types have evolved morphological traits that are tactic-specific (i.e., sexual polymorphisms) and that differences in AVT appear to be related to between-morph differences in the expression of courtship behavior. Therefore, this comparative approach leads us to propose that the different endocrine systems are involved in the differentiation of different sets of traits that make up alternative phenotypes, and that the differentiation of alternative tactics is not controlled by a single endocrine system (e.g., androgens).
Article
The relationship between androgens and paternal behavior is not straightforward, potentially because of the diversity of tasks a male must undertake to maximize reproductive success, notably alternating between courtship, aggression, and offspring care. In some species, these events are separated in time, but in others they are coincident. The endocrine profiles of species that simultaneously court, parent, and defend a nest, such as male bluebanded gobies (Lythrypnus dalli), are not well understood. We sampled a potent fish androgen, 11-ketotestosterone (KT), at different life history stages (experienced parenting males, experienced males not actively parenting, inexperienced males with their first clutch, and females), to examine this relationship. We found that experienced parenting L. dalli males have the highest KT levels of any group, while none of the other groups differed significantly. Males showed elevated KT levels when they had eggs compared to when they did not. Our data suggest that KT facilitates at least some aspects of parental care in L. dalli.
Article
We tested whether subordinate helper males of the Lake Tanganyika cooperatively breeding cichlid Neolamprologus pulcher show elevated excretion levels of the stress hormone cortisol and reduced levels of 11-ketotestosterone and testosterone when living in groups with a small breeder male, compared to similar helper males living in groups with a large breeder male, in a full-factorial repeated measures experimental design. We also measured the same hormones in breeder males with and without helper males. Previous research showed that the size difference between large male helpers and male breeders in groups of this species influences behaviour and growth decisions. Contrary to our expectation, no effect of the size-difference between helper males and breeder males on helper hormone levels was detected. Furthermore, helper males had similar hormone excretion levels to those of size-matched breeder males without helpers, and to small breeder males. There was no influence of egg laying on breeder male and helper hormone levels during the experiment. Interestingly, all three hormone levels were significantly lower in helpers showing elevated levels of submissive behaviour towards the breeders, independently of the size of the breeder males. The low cortisol levels suggest that helper males can successfully reduce stress by appeasing breeder males through submission. Furthermore, helper males showing a high level of submissive behaviour had lower levels of androgens than less submissive helpers, suggesting a lower reproductive potential in submissive helpers. We propose that helper submission may be used as an honest signal of reduced interest in reproduction towards the breeder male in this species.
Article
Chronic increases in stress hormones such as glucocorticoids are maladaptive, yet studies demonstrating a causal relationship among chronic stress, increases in glucocorticoid concentrations, and subsequent fitness costs in free-living animals are lacking. We experimentally induced chronic psychological stress in female European starlings (Sturnus vulgaris) by subjecting half of the females at our study site to a chronic stress protocol consisting of 4, 30 min stressors (loud radio, predator calls, a novel object, or predator decoys including a snake, rat, and owl) administered in random order daily for 8 days after clutch completion. Experimental females were captured at the end of the chronic stress protocol (9 days after the onset of the chronic stress protocol), and unstressed control females were captured at the same stage of the nesting cycle. Chronically stressed females had lower baseline corticosterone (CORT, the avian glucocorticoid) concentrations and lower reproductive success than unstressed females. Furthermore, surviving nestlings in experimentally stressed broods showed sensitization of the CORT response to acute stress, which is a physiological change that could persist to adulthood. Attenuation of baseline CORT concentrations in adult females is contrary to the general assumption that elevated CORT concentrations indicate stress, suggesting that more research is necessary before CORT concentrations can be used to accurately assess chronic stress in field studies.
Article
The androgen 11-ketotestosterone (11-KT) plays an important role in reproductive physiology and behaviour in male teleosts. In the three-spined stickleback, Gasterosteus aculeatus, the plasma concentrations of 11-KT are related to the breeding status of the fish. Sticklebacks are relatively small (generally less than 1g) and in order to obtain sufficient plasma for assay of 11-KT, it has been necessary in the past to sacrifice the fish. In this paper, we report on the development of a non-invasive procedure for measuring 11-KT, cortisol and androstenedione (Ad) in the three-spined stickleback. Validation of the procedure included the demonstration that the rate of release of steroids into the water was correlated to their plasma concentrations. Ten males that were kept at a low temperature and short photoperiod were moved to high temperature and long photoperiod to initiate spermatogenesis and breeding. Every two to four days, for a total of 53 days, males were removed and placed in a beaker containing 50-ml water for 30 min. The water was then processed by solid phase extraction for radioimmunoassay. Males were presented with females on days 13/14, 18/19 and 44/45. 11-KT was originally undetectable but built up gradually to reach an average release rate of between 1 and 2.5 ng/g/h between days 16 and 45 and then started to decline (but non-significantly). Ad release reached a plateau of 1 ng/g/h about day 20. However, from days 44/45 to 51, there was a highly significant rise in the rate of release of Ad to 5 ng/g/h. On days 44/45, five of the males mated successfully and five did not. However, there were no significant differences in 11-KT or Ad release rates between the two groups. Cortisol release rates fluctuated with no pattern throughout the study. The results show that it is possible to make measurements on sex and stress steroid production in sticklebacks without recourse to anaesthesia, bleeding or sacrificing the fish. The procedure is potentially a powerful tool for the study of the link between steroids and behaviour in this useful sentinel species.
Article
We combined behavioral and physiological measures to compare coping style in wild-type Betta splendens and a domesticated strain selectively bred for sports fighting. We showed previously that the fighter strain is more aggressive than the wild type during experimental conditions that most closely resemble an actual fight. We predicted that compared to the wild type, the fighter strain would show a more proactive coping style, characterized by lesser cortisol and greater sympathetic responses to non-social challenges. We introduced males to an unfamiliar environment and spatial confinement as challenges that may resemble some of those that B. splendens may encounter in its natural habitat. We developed a non-invasive stress assay that enables repeated individual measures of water-borne cortisol. We estimated sympathetic activation through opercular beat rate and recorded the duration of behavioral immobility. We found that exposure to an unfamiliar environment raised cortisol levels in the wild type but not in the fighter strain and that confinement raised cortisol levels in both. In both strains opercular beat rates were significantly reduced during the latter stages of confinement compared to during the early stages. The fighter strain, but not the wild type, adopted a behavioral strategy of immobility from the very beginning of confinement.
Article
It is a common practice to extract steroids from plasma, serum, or tissue samples prior to steroid measurement by radioimmunoassay (RIA) or enzyme immunoassay (EIA). Steroid extraction is critical because it can remove substances that interfere with the RIA or EIA. Steroid extraction is commonly achieved using organic solvents, such as diethyl ether or dichloromethane. However, organic solvent extractions can suffer from low recovery, imprecise recovery, or incomplete removal of assay interference. Here, we describe validations of a simple protocol to extract steroids (e.g., dehydroepiandrosterone, corticosterone, and estradiol) from avian plasma, serum, and brain tissue using solid phase extraction (SPE) with commercially available C18 columns. We compare various methods for (1) eluting steroids from columns, (2) drying eluates, and (3) resuspending dried eluates prior to RIA. The SPE method yields high and consistent recoveries. The SPE method also effectively separates steroids from interfering substances, even when extracting steroids from lipid-rich plasma and brain tissue. These data indicate that SPE is superior to organic solvent extraction on several measures. SPE should be broadly useful for extracting steroids from plasma or tissue samples.
Article
Given the dramatic behavioral effects of winning and losing contests, and pronounced changes in stress and sex steroid hormones post-fight, it is reasonable to suppose that these hormones also dictate future behavior. We sampled water-borne cortisol, testosterone (T), and 11-ketotestosterone (KT) before and after contests in the mangrove killifish, Kryptolebias marmoratus, to determine how endogenous steroid hormone levels might predict and respond to contest dynamics or success. Pre-fight cortisol related negatively, and pre-fight T related positively to contest initiation and winning, particularly in the smaller opponent. In the pairs where a larger fish won the contest, winners with higher pre-fight T and lower pre-fight cortisol delivered more attacks to the losers. Contest duration and escalation influenced post-fight hormone concentrations primarily in losers. Escalation significantly increased post-fight cortisol, T, and KT for losers but not for winners. However, winners that attacked losers at higher rates had higher levels of post-fight cortisol. Losers also demonstrate the most consistent post-fight hormone responses, particularly to contest escalation and duration. Despite the bidirectional relationship between hormones and contest behavior, we found no overall mean differences in pre- or post-fight cortisol, T, or KT between eventual winners and losers. Thus, it is evident that the categorical states of winner and loser cannot alone reveal the complex, reciprocal associations between endocrine systems and social behavior.
Article
We studied the role of steroid hormones for parental and alloparental brood care and social status in a cooperatively breeding fish. We measured excretion levels of testosterone and 11-ketotestosterone in males, estradiol-17beta in females and cortisol in both sexes at different stages of the breeding cycle, and compared these values to data measured in non-reproductive fish. Brood care behaviour does not seem to relate to steroid hormone excretion levels in this species. Steroid hormones varied with social status, however. Non-territorial male aggregation members, for example, showed high testosterone and low 11-ketotestosterone excretion levels, suggesting that they might pursue a "stand-by strategy" for breeding to react quickly if an occasion for breeding arises. Cortisol excretion levels are high in juvenile helpers compared to same-size aggregation members, suggesting higher stress levels in subordinate members of reproductive groups. This is the first study assessing if steroid hormone control mechanisms are involved in brood care and social roles in a cooperatively breeding fish.
  • F Zaidan
  • D L Kreider
  • S J Beaupre
F. Zaidan, D.L. Kreider, S.J. Beaupre, Testosterone cycles and reproductive energetics: Implications for Northern range limits of the cottonmouth (Agkistrodon piscivorus leucostoma), Copeia 2003 (2003) 231-240.