Changes in plasma levels of gonadal steroids and putative gonadotropin in association with spawning and brooding behaviour of male demoiselles

Nesting male demoiselles Chromis dispilus were captured and blood sampled under water by divers, and plasma levels of reproductive hormones correlated with behavioural status at the time of capture. Plasma gonadotropin-II (dGtH-II) was measured using a heterologous assay based on carp GtH-II standards and an antibody to the β -subunit of cGtH-II. Plasma samples diluted parallel to standards in the assay, and there was a significant transient increase in dGtH-II in response to treatment with gonadotropin releasing hormone analogue (GnRHa). Male demoiselles had elevated plasma levels of dGtH-II during periods of reproductive display, but low levels of dGtH-II during periods of brooding, irrespective of egg stage. Plasma testosterone levels followed a similar pattern whereas plasma 11-ketotestosterone and 17,20 β -dihydroxy-4-pregnen-3-one levels tended to remain elevated during the early phases of egg brooding. The results are consistent with earlier studies showing that reproductive hormone levels in demoiselles are behaviourally mediated, and support the view that behavioural alterations of reproductive endocrine status in fishes are mediated through the action of GnRH.     

Relationship between stress, feeding and plasma ghrelin levels in rainbow trout, Oncorhynchus mykiss

Sexually immature rainbow trout were acclimated to small-volume (1 m³) holding tanks and then exposed to short-term stress to examine the relationship between feeding, stress, plasma ghrelin levels and other plasma stress parameters. Plasma ghrelin levels showed an increase 24 h after a single feed, plasma lactate and glucose levels decreased over the same period and plasma cortisol levels were low and constant. One hour of confinement stress resulted in elevations of plasma cortisol, glucose and lactate and depression of plasma ghrelin levels. In a separate experiment, 2 h of confinement stress also depressed feeding immediately after stress, concomitant with increases in plasma cortisol, lactate and glucose; however, in this case there was no change in plasma ghrelin concentrations. A repeat of the 2-h confinement experiment using fish that had not been acclimated to small-volume holding tanks produced a more marked elevation in plasma cortisol and a stronger suppression of feeding post-stress but in this case also, there was no change in plasma ghrelin levels. The results of this study confirm that feeding in rainbow trout is suppressed by confinement stress although the effect is transitory in this domesticated stock. Similar to that in other fishes, plasma ghrelin levels appear to be modulated by feeding status and may be influenced by stress, suggesting an orexigenic role for ghrelin in rainbow trout.     

Poststress levels of lysozyme and cortisol in adult rainbow trout: heritabilities and genetic correlations

Lysozyme response in stressed rainbow trout was compared with measurements of poststress cortisol activity. Estimates of heritability of lysozyme and cortisol were both moderate-to-high and both traits displayed positive genetic correlations in pair-wise comparisons of stress exposures. Genetic correlations between lysozyme and cortisol in stressed rainbow trout tended to be negative, although insignificant. Neither lysozyme, nor cortisol exhibited significant correlations with serum haemolytic activity. It is concluded that the data do not confirm earlier suggestions that lysozyme should be superior to cortisol in consistency of stress response in rainbow trout.     

Effects of exogenous estradiol-17β on early growth and gonadal development of diploid and triploid female rainbow trout (Oncorhyncus mykiss)

Triploidy is a viable condition in teleosts. However, in many salmonids, the triploid condition in the female results in sterility as gametogenesis appears to be disrupted. Although the underlying mechanisms regulating the gonadal development of teleosts have not been clearly elucidated, the reversal of phenotypic sex by the administration of the appropriate exogenous steroid during early development supports the argument that gonadal steroids play a pivotal role in sexual differentiation and subsequent gonad development in these fish. To determine whether the failure of normal ovarian development in triploid female rainbow trout (Oncorhynchus mykiss) is due to an absence or reduction of endogenous sex steroids, ovarian morphology was compared between diploid and triploid juvenile rainbow trout treated with exogenous estradiol-17β (E₂). The ovaries of both untreated and E₂ treated diploid fish, at 145 days post-fertilization, contained synchronously developing oocytes in the perinucleolar stage, whereas ovaries from untreated and estradiol-treated triploid fish of the same age were considerably smaller and devoid of developing oocytes. No differences in the ovaries of triploid untreated fish and triploid fish treated with E₂ were observed. It is reported that exposure to exogenous E₂ during the period of gonadal differentiation is not sufficient to induce oocyte development in triploid rainbow trout. © 1996 Wiley-Liss, Inc.     

Successful shoreline spawning of rainbow trout in two Canadian alpine lakes

Successful shoreline spawning of rainbow trout Oncorhynchus mykiss has occurred for several decadal periods without hatchery stocking in Joe and Harry Lakes, two small (5–6 ha) alpine waters at >2000 m elevation in south-central British Columbia. Redds are most concentrated near groundwater inflows, in Joe Lake mainly on sand and gravel, in Harry Lake mainly over rocks and boulders.     

Branchial release of free cortisol and melatonin by rainbow trout

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.     

Effects of aluminium and cortisol on in vitro calcium deposition on otoliths in rainbow trout

Aluminium (1–100 μM) reduced in vitro calcium deposition on otoliths concentration-dependently in rainbow trout, but cortisol (1 and 10 μg ml⁻¹) had no effect.     

Effects of experimental anaemia on intra-erythrocytic phosphate levels in rainbow trout, Oncorhynchus mykiss

The concentrations of intra-erythrocytic adenylates (ATP, ADP and AMP) and guanylates (GTP, GDP and GMP) were determined in rainbow trout subjected to 10% blood removal every 12 h for 96 h. Haemoglobin concentration, [Hb], decreased from 6·043±0·617 to 0·957 ± 0·195 g dl⁻¹. This decrease in [Hb] was followed by a continuous increase in total organic phosphates, e.g. adenylates plus guanylates. Intra-erythrocytic NTP (ATP plus GTP) levels increased significantly after 48 h when haemoglobin concentration was 2·427 ± 0·256 g dl⁻¹. Although a significant increase in GDP levels in animals with [Hb] less than 1·677 ± 0·235 g dl⁻¹ was observed, the general increase in guanylate level was mainly due to the GMP which increased about 85-fold during the experimental period. It is suggested that the erythrocytes of anaemic rainbow trout have the capacity to increase NTP/Hb₄ ratios which may represent an advantage for anaemic fish.     

Milt characteristics and plasma levels of gonadal steroids in greenback flounder Rhombosolea tapirina following treatment with exogenous hormones

Fish were treated with exogenous hormones, and milt and blood samples were collected for up to 96 h post‐treatment. Blood plasma samples were assayed for the gonadal steroids testosterone (T), 11‐ketotestosterone (11KT) and 17,20ß‐dihydroxy‐4‐pregnen‐3‐one (17.20ßP). Milt volume, spermatocrit and sperm motility were measured from milt samples. Non‐spermiated fish showed increased plasma T and 11KT in response to human chorionic gonadotropin (hCG) but not luteinising hormone releasing hormone analogue (LHRHa). Fish did not become spermiated in response to treatment with hCG, LHRHa, 11KT, 17‐hydroxyprogesterone (17P) or 17,20ßP. Spermiated fish showed an increase in milt volume in response to hCG and LHRHa but not exogenous steroids. Sperm motility declined to zero over 120 s and was not affected by hormone treatment or sampling time. Increased milt volume was accompanied by increased plasma T and 11KT, but not 17.20ßP levels. In a separate experiment, LHRHa delivered by injection or pellet was equally effective at increasing milt volume but had no effect on plasma steroid levels. Spermatocrit declined with stripping but was not affected by hormone treatment, nor was sperm motility. Co‐treatment of fish with 17P plus LHRHa had no additive effect on plasma steroid concentrations or milt volume. The results suggest that as in other teleosts, gonadotropin mediates milt production in greenback flounder.     

Effect of salmon gonadotropic hormone on sex steroids in male rainbow trout: plasma levels and testicular secretion in vitro

Summary Male rainbow trout were treated with salmon gonadotropic hormone (GTH) at different stages of the circannual reproductive cycle; spawning fish were also treated with an antiserum against salmon GTH. Injection of GTH led to a several-fold increase of plasma sex steroid levels during spermatogenesis and in the spawning season but was without effect at early stages of testicular development. GTH neutralization during the spawning season was followed by a several-fold decrease of plasma sex steroid levels. During spermatogenesis and in the spawning season, both treatment regimes resulted in an increased sensitivity of testicular explants in response to a subsequent stimulation of steroid secretion in vitro. This up-regulatory response may facilitate and maintain the high sex steroid plasma levels observed during the spawning season. It may also be necessary to allow for concomitant peak values of plasma GTH and sex steroids in the spawning season, a situation difficult to understand within the negative feedback concept. The adaptive capacities of the testicular steroidogenic system indicate that it is not only an effector site for GTH but also an active part of the endocrine system controling reproduction.Abbreviations BSA bovine serum albumin - bw body weight - E₂ 17-estradiol - GnRH gonadotropin releasing-hormone - GTH gonadotropic hormone - LH luteinizing hormone - OHT 11-hydroxytestosterone - OT 11-ketotestosterone - 17-20P 17-hydroxy, 20-dihydroprogesterone - PE pituitary extract - raGTH rabbit anti-GTH antiserum - rPS rabbit preimmune serum - T testosterone     

Anthropogenic influences on population sizes, age and growth of naturalized rainbow trout, Oncorhynchus mykiss, in Kenya

Riverine fishery in Kenya has witnessed profound changes since the 1950s, especially after independence (in 1963) when watersheds on the south‐eastern slopes of Mt Kenya were opened up for settlement. In particular, rainbow trout populations have declined essentially resulting from anthropogenic changes through over‐exploitation and/or degraded habitats. While there still are self‐sustaining rainbow trout populations in this stream among others they are in decline an indication that changes that have occurred on the watershed have altered their sizes, age and growth. The study was carried out in the Sagana, a third‐order stream, which rises at about 4000 m altitude on the south‐eastern slope of Mt Kenya, to obtain and document information on population sizes, age and growth of rainbow trout populations and to relate them with those recorded in the 1950s when trout streams were pristine (with little human influence). Stations were fished bi‐monthly from 1996 to 1998 and later for 6 months in 2002. Information on age and growth was obtained by use of annual marks, tagging fish of known age, and by validating their age using captive fish of known age. The minimum mean back‐calculated length at age for age one rainbow trout was 13.09 cm in upstream station and 15.10 cm for downstream stations. However, there was no significant difference in mean back‐calculated lengths at age for all years between fish in upstream and downstream stations (t‐test, t = −0.01, P = 0.99). Although female fish showed higher mean annual back‐calculated length increments than males, there was no significant difference in mean back‐calculated lengths at age between sexes (t‐test, t = −0.27, P = 0.80). The rate of growth in length was rapid for 1‐year‐old fish and declined in the second and third years. This study observed that most of the fish were small with only a few reaching more than 2 years of age because of overfishing. There are good reasons for optimism about the future of trout populations in this stream but concerted efforts are required to rehabilitate them. If trout populations are to increase, a management strategy is required to reduce fishing pressure and to maintain stream fishery against competing needs for resources in the catchments.     

Influence of growth rate on white muscle dynamics in rainbow trout and brook trout

In trout, fast growth stimulated white muscle fibre hypertrophy ( P 0·001) and hyperplasia ( P <0·01) in outer fibres but not in deep fibres. Glycogen was most prevalent in outer fibres ( P <0·01) and in brook trout ( P <0·01) that on average had three to four times larger fibres than rainbow trout.     

Analysis of the association between spawning time QTL markers and the biannual spawning behavior in rainbow trout (Oncorhynchus mykiss)

The rainbow trout is a salmonid fish that occasionally exhibits broodstocks with biannual spawning behavior, a phenomenon known as a double annual reproductive cycle (DARC). Spawning time quantitative trait loci (SPT-QTLs) affect the time of the year that female rainbow trout spawn and may influence expression of the DARC trait. In this study, microsatellite markers linked and unlinked to SPT-QTLs were genotyped to investigate the underlying genetics of this trait. SPT-QTLs influenced the DARC trait since in two case-control comparisons three linked markers (OmyFGT12TUF, One3ASC and One19ASC) had significant levels of allelic frequency differentiation and marker-character association. Furthermore, alleles of One3ASC and One19ASC had significantly higher frequencies in populations that carried the DARC trait.     

Effect of hypoxia on in vivo thromboxane and prostacyclin levels in arterial blood of rainbow trout, Oncorhynchus mykiss (Walbaum)

Endogenous levels of TXB₂ and 6-keto PGF_1a are reported in dorsal aortic blood from rainbow trout. Acute hypoxia induced an increase in TXB₂ levels whereas 6-keto PGF_1a was unaffected. We suggest that enhanced thromboxane synthesis might have a role in microcirculation of various organs in fish hypoxia.     

Relationship of plasma steroids to germ cell development and the presence of protamine mRNA in rainbow trout during the induction of spermatogenesis with partially purified salmon gonadotropin

The gonadosomatic index (GSI) of pre–pubertal male rainbow trout, which had been injected biweekly with partially purified salmon gonadotropin (sG–G100, 50 μ mUg kg⁻¹ body weight), increased from 0.05 to 1.85 over 21 weeks from injection, while control GSI remained below 0.05. Plasma testosterone (T) increased from 2 to 11.34 ng ml⁻¹ by week 21 in injected fish, while control level remained below 1.5 ng ml⁻¹. In injected fish plasma 11–ketotestosterone (KT) and 17,20–dihydroxyprogesterone (17α20βP) levels increased from 20.2 to 41.9 and 8.9 to 219.7 ng ml⁻¹ respectively. Plasma T, 11–KT, and 17α20βP were all correlated with the GSI ( P <0–001) in injected fish. The most advanced stage of germ cells present in the control fish were spermatogonia. However, in injected fish spermatozoa were present by week 21. Eggs fertilized at this time with spermatozoa from injected fish achieved a 78% fertilization rate, whereas the testicular homogenate was incapable of fertilizing eggs.     

A non‐invasive stress assay based upon measurement of free cortisol released into the water by rainbow trout

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.     

The effects of capture, handling, confinement and ectoparasite load on plasma levels of cortisol, glucose and lactate in the coral reef fish Hemigymnus melapterus

Tropical labrids Hemigymnus melapterus sampled underwater had low plasma levels of cortisol, glucose, and lactate. Plasma cortisol levels were elevated by capture stress within 5–6 min, while glucose and lactate levels were not. Plasmalevels of cortisol and glucose increased after 2–4 h of handling and transport to the laboratory. Levels of cortisol and glucose fell with laboratory acclimation back to values similar to those found in wild fish. Parasitism by gnathiid isopods across an order of magnitude of isopod numbers had no effect on plasma levels of cortisol or glucose. Thus, H. melapterus has a stress response similar to that shown by temperate species, and relatively high parasite loads are not apparently stressful to fish in the wild. This may be related to the counterproductive effects of physiological stress responses on the immune system or behaviour-modulated processes that counter parasitic invasion.     

Urine of reproductively mature female rainbow trout, Oncorhynchus mykiss (Walbaum), contains a priming pheromone which enhances plasma levels of sex steroids and gonadotrophin II in males

Urine of reproductively mature female rainbow trout was shown to contain a priming pheromone which raised the levels of 17 a ,20β-dihydroxy-4-pregnen-3-one (17,20β-P), testosterone and gonadotrophin II in the blood plasma of reproductively mature male rainbow trout. Milt volumes, however, were unaffected.
Because it had been established in a previous study that the sulphated form of 17,20β-P is abundant in the urine of spawning rainbow trout, synthetic 17 a ,20β-dihydroxy-4-pregnen-3-one 20-sulphate was also tested for pheromonal activity. It was found to have only a smalt and inconsistent priming effect on steroid levels and did not alter the orientation or spawning activity of males.
It was also shown that, in the majority of experiments, there was a significant drop, over the course of 24 h, in the levels of 17,20β-P and testosterone in the control groups of males.     

Involvement of cortisol and sirtuin1 during the response to stress of hypothalamic circadian system and food intake-related peptides in rainbow trout,Oncorhynchus mykiss

Stress is conditioning animal welfare by negatively affecting a wide range of physiological and behavioral functions. This may be applied to circadian physiology and food intake. Cortisol, the stress-related hormone, may mediate such effect of stress, but other indirect mediators might be considered, such as sirtuin1. Then, either the independent modulatory effect or the existence of any interaction between mediators may be responsible. The circadian system is the main modulator of several integrative mechanisms at both central and peripheral levels that are rhythmically presented, thus influencing different processes such as food intake. In this way, food intake is controlled by the circadian system, as demonstrated by the persistence of such rhythms of food intake in the absence of environmental external cues. Our study aimed to evaluate the daily profile of hypothalamic mRNA abundance of circadian clock genes (clock1a, bmal1, per1 and rev-erbβ-like), and food intake regulators (crf, pomc-a1, cart, and npy) in rainbow trout (Oncorhynchus mykiss), the impact of stress on such rhythms, and the involvement of cortisol and sirtuin1 as mediators. Four cohorts of trout were subjected to 1) normal stocking density (control group), 2) high stocking density for 72 hours (stress group), 3) normal stocking density and implanted with mifepristone, a glucocorticoid receptors antagonist, and 4) mifepristone administered and stressed for 72 hours. Fish from each group were sampled every 4-h along the 24-h LD cycle, and cortisol, glucose and lactate plasma levels were evaluated. Hypothalamic mRNA abundance of clock genes, food intake regulators, glucocorticoid receptors and sirtuin1 were qPCR assayed. Our results reveal the impact of stress on most of the genes assayed, but different mechanisms appear to be involved. The rhythm of clock genes displayed decreased amplitude and averaged levels in stressed trout, with no changes of the acrophase being observed. This effect was not prevented by mifepristone. On the contrary, the effect of stress on the daily profile of crf, pomc-a1, and npy was totally prevented by mifepristone administration. Accordingly, cortisol appears to mainly mediate the effect of stress on food intake regulators through binding to specific glucocorticoid receptors within trout hypothalamus, whereas sirtuin1 is apparently mediating such effects on the circadian system in the same brain region. Further research must be performed to clarify those mechanisms through which stress influences food intake and the circadian oscillator within the same brain region, hypothalamus, in rainbow trout, and the interaction among them all.