Growth hormone receptors in ovary and liver during gametogenesis in female rainbow trout (Oncorhynchus mykiss).

Changes of growth hormone receptivity in the ovary during the reproductive cycle were studied in rainbow trout (Oncorhynchus mykiss). A method for characterizing growth hormone receptors in crude ovary homogenate was required for this. Binding of radiolabelled recombinant rainbow trout growth hormone (125I-labelled rtGH) to crude ovary preparation was dependent on ovarian tissue concentration. The sites were specific to growth hormone, with no affinity for prolactins and gonadotrophins. Similar high affinities for 125I-labelled rtGH were obtained with crude ovary (4.2 x 10(9) +/- 0.3 mol l-1) and crude liver preparations (4.9 x 10(9) +/- 0.1 mol l-1) at all stages of ovogenesis, and with ovarian membrane preparations (8.2 x 10(9) mol l-1) tested at the beginning of vitellogenesis. Ovarian growth hormone receptor concentration was highest during the early phases of follicular development (endogenous vitellogenesis: 315-310 fmol g-1 ovary) and decreased regularly during oocyte and follicular growth (exogenous vitellogenesis) to reach a minimal value at oocyte maturation (42 fmol g-1 ovary). In postovulated fish, binding was at a similar level (297 fmol g-1 ovary) to that found in endogenous vitellogenesis. Conversely, the absolute binding capacity of the whole ovary was low from immaturity to early exogenous vitellogenesis (0.1-0.6 pmol per pair of gonads), increased slowly during vitellogenesis and more markedly during rapid oocyte growth and at the time of final maturation (10.8 pmol per pair of gonads). In postovulated fish, the absolute binding capacity decreased partially (4.4 pmol per pair of gonads). Mean hepatic growth hormone receptor concentration did not vary with the reproductive stage for most of the cycle (3.0-4.5 pmol g-1 liver) except in endogenous vitellogenesis where significantly higher concentrations were observed (6.7 pmol g-1 liver). Individual ovarian growth hormone receptor concentrations were correlated with hepatic growth hormone receptor concentrations, indicating that they are regulated in a similar way. We conclude that growth hormone receptors are present in the ovary during the entire ovarian cycle in rainbow trout, probably mainly in somatic cells as indicated by the same concentration of binding sites in immature and in postovulated fish. Growth hormone is potentially important during oocyte recruitment in vitellogenesis and initiation of growth and during final follicular maturation.     

Patterns of transgene inheritance in rainbow trout (Oncorhynchus mykiss).

There have been very few studies of the inheritance of introduced genes (transgenes) in fish. We have followed the inheritance of the mammalian fusion gene MTrGH from founder generation transgenics (originating from eggs microinjected with the MTrGH DNA) to offspring in crosses with control fish. Initial screening of the founder generation transgenics was by analysing DNA from blood samples. Only three out of six fish which carried the novel gene in blood DNA transmitted it to their offspring, despite the presence of the gene in DNA extracted from the sperm of all four male fish in this group. The frequency of transgenics in the progeny groups from the three fish which transmitted the gene varied widely: in one of these groups more than one type of MTrGH restriction pattern was found. These results suggest widespread mosaicism in founder generation transgenics.     

Gonadal sex reversal in triploid rainbow trout (Oncorhynchus mykiss).

Sex determination in salmonids is primarily governed by sex chromosomes; however, phenotypic expression and successful development of the gonads may be influenced by additional factors. Exposure to exogenous steroids during the critical period of gonadal differentiation will reverse the expected phenotypic sex of both female and male trout. Triploidy, a viable condition in rainbow trout (RBT), alters the degree of gonadal development in a gender-specific manner. Males produce testes with similar morphology and function as diploid fish, but females produce underdeveloped ovaries devoid of growing oocytes. One possible explanation for this observed gender difference is that the timing of meiotic initiation may influence ovarian/testicular development in triploid RBT. To determine whether the early entrance of germ cells into meiosis results in the lack of ovarian development in triploid females, the objective of this study was to sex-reverse genotypic triploid female RBT (XXX) into phenotypic males and genotypic triploid male RBT (XXY) into phenotypic females. Male fish were exposed to estradiol-17beta (E(2)) and females were exposed to the non-aromatizable androgen 17alpha-methyldihydrotestosterone (MDHT). Over 90% of the male fish treated with exogenous E(2) developed gonadal structures indistinguishable from the gonads of triploid females. Triploid female RBT treated with MDHT developed testes; however, not all fish treated with this androgen were completely sex reversed. The results of this investigation are consistent with the hypothesis that the failure of ovarian development in triploid RBT is due to the early onset of meiosis and does not appear to be due to genotypic sex. J. Exp. Zool. 284:466-472, 1999.     

Endocrine and orexigenic actions of growth hormone secretagogues in rainbow trout (Oncorhynchus mykiss)

The effects of growth hormone secretagogues (GHSs) on the teleost somatotropic axis are poorly understood, particularly with respect to insulin-like growth factor-I (IGF-I) and the IGF-binding proteins (IGFBPs). To assess the endocrine and orexigenic responses of rainbow trout (Oncorhynchus mykiss) to GHS treatment, animals were injected with human GHRH(1-29)-amide, KP-102 or rat ghrelin at 0, 1 or 10 pmol/g body mass. Feed intake was tested at 2 and 5 h post-injection and plasma levels of growth hormone (GH), IGF-I and the IGFBPs were determined at 3, 6 and 12 h post-injection. Feed intake was significantly elevated by all of the GHSs tested at both post-injection time points. All GHSs elevated plasma GH levels in a time-dependent manner. Plasma IGF-I levels were elevated by all GHSs at 3 h post-injection, whereas those animals treated with KP-102 and ghrelin exhibited depressions at 6 h. Four IGFBPs were identified in the plasma by western blotting. Levels of the 20 kDa IGFBP decreased over the sampling time. Levels of the 32 kDa IGFBP were significantly depressed by all GHSs tested. Levels of the 42 kDa IGFBP were significantly elevated by all GHSs tested. Plasma levels of the 50 kDa IGFBP was decreased in some treatment groups at 3 h, but elevated by 6 h in the ghrelin-treated groups and elevated in all treatment groups by 12 h post-injection. The endocrine and orexigenic responses demonstrate that GHSs influence the teleost neuroendocrine system beyond short-term actions (<3 h post-injection) on GH release and the responses of the IGFBPs to GHS treatment support this notion and clarify their identification as functional homologues to mammalian IGFBPs.     

Effects of human insulin-like growth factor-I on release of growth hormone by rainbow trout (Oncorhynchus mykiss) pituitary cells.

A recombinant human IGF-I (rhIGF-I) was applied to primary cultures of rainbow trout pituitary cells. In wells containing 3 x 10(4) and 6 x 10(4) cells/well, rhIGF-I inhibited basal GH release both in short (6 h) and long (12 and 24 h) exposures. The decline in GH release was dose-dependent over the range of 0.01 and 100 mM. The combination of rhIGF-I and low concentrations of synthetic somatostatin (SRIF) enhanced the inhibitory effect of rhIGF-I in an additive manner. Any appreciable effect of rhIGF-I on PRL release was not evidenced. To our knowledge, this report demonstrates for the first time the participation of IGFs on the inhibitory component of fish GH regulation.     

Circannual rhythms of gonadal maturation in female rainbow trout (Oncorhynchus mykiss).

Individually identified, 2-year-old female rainbow trout were maintained for up to 51 months on a constant schedule of 6 hr light and 18 hr darkness (LD 6:18), constant temperature (8.5-9.0 degrees C), and constant feeding rate. The fish exhibited free-running circannual rhythms of gonadal maturation and ovulation, which were self-sustaining for up to three cycles. The periodicity of the rhythm showed variation between fish and in successive cycles for the same fish, ranging from approximately 11 to 15 months.     

Liver 3-hydroxyanthranilic acid oxygenase activity in rainbow trout (Oncorhynchus mykiss).

1. In rainbow trout, 3HAA activity was comparable with those of terrestrial animals; 3HAA:PC activity ratio suggests ineffective conversion of tryptophan to niacin. 2. Inactivation as well as reactivation under different conditions was investigated. 3. Some characteristics of the enzyme extract were studied with the aim of optimizing assay in fish.     

The effect of thyroid hormone and glucocorticoids on carp growth hormone-releasing factor (GRF)-induced growth hormone (GH) release in rainbow trout (Oncorhynchus mykiss).

1. The effect of thyroid hormone and glucocorticoids on carp growth hormone-releasing factor (GRF)-induced growth hormone (GH) secretion was studied on rainbow trout using a dispersed pituitary cell culture system. 2. A combined administration of lower doses (0.01 microM) of 3,5,3'-triiodo-L-thyronine (T3) and dexamethasone (Dex) significantly increased spontaneous as well as carp GRF-induced GH release. 3. Lower doses of Dex alone had no effect, and T3 had a marginal effect on GH release. Higher doses of either Dex or T3 potentially reduced GH release. 4. This study indicates an important role of thyroid hormone and/or glucocorticoids in the hypothalamic regulation of GH secretion in fish.     

Ubiquitin genes in rainbow trout (Oncorhynchus mykiss)

Ubiquitin is a small protein involved in intracellular proteolysis. It is highly conserved throughout eukaryotic phyla and has been detected in such diverse species as yeast, barley, Drosophila and man. A previous study showed that chromatin of rainbow trout testis contains free ubiquitin with a sequence similar to that of other phyla. In the present study, which focused on rainbow trout but included eleven other species, it is shown that fish ubiquitin genetic organisation and expression are similar to those of other phylogenetic groups through the following set of observations: (a) Multiple loci were detected, (b) These loci encode repeats of ubiquitin, (c) Although the DNA sequences are not conserved, the encoded amino acid sequences are fully conserved, (d) The expression of ubiquitin was influenced by cell culture conditions and viral infection.     

Relationships among growth and different NOR phenotypes in a specific stock of rainbow trout (Oncorhynchus mykiss).

Growth is one of the most important aspects in the genetic improvement of cultured fish species. Consequently, genetic parameters related to this feature and their response to selection have been the focus of most research in this area. Such research indicates that, in general, there is enough additive genetic variance related to growth, justifying the use of selection. Based on the usefulness of cytogenetic and molecular markers in the fish culture, the aim of the present work was to analyze the possible relationships among cytogenetic characteristics, specifically the NOR phenotypes, and the increase in length and weight in specimens of the rainbow trout (Oncorhynchus mykiss), resultant from directed mating between homozygous females and heterozygous males according to their NOR phenotypic patterns. The equations of the relationship between length and weight of the analyzed specimens followed the model Wt = a Lt(b), showing b values higher than 3, determinant of a positive allometric growth. The results showed that the different NOR phenotypes were not related with the growth values for length and weight in any statistical test.     

Growth hormone stimulates hepatic lipid mobilization in rainbow trout,Oncorhynchus mykiss

Rainbow trout were used to characterize the direct influence of growth hormone on hepatic lipid mobilization in vitro. Liver was removed from fish fasted 72 h, sliced into 1-mm³ pieces and incubated in Hanks' medium containing ovine or salmon growth hormone (0.28 ng·ml^-1–28 g·ml^-1). Lipid mobilization, resulting from triacylglycerol hydrolysis, was evaluated by fatty acid and glycerol release into culture medium. Control rates of fatty acid release and glycerol release were 0.95±0.16 (mean ± SE) and 0.88±0.28 mol·l^-1·mg fresh weight, respectively. Both ovine growth hormone (28 ng·ml^-1) and salmon growth hormone (28 ng·ml^-1) stimulated fatty acid release into culture medium, increasing rates by 112% and 97%, respectively, during the course of a 24-h incubation. Glycerol release was similarly augmented by growth hormone treatment. Growth hormone-stimulated metabolite release became evident within 12 h and persisted for up to 72 h. The presence of amino acids in the culture medium potentiated the lipolytic action of growth hormone. Ovine growth hormone (28 ng·ml^-1) in the presence of amino acids stimulated a 53% increase in fatty acid, and a 108% increase in glycerol, release over rates observed in the absence of amino acids. Salmon growth hormone (28 ng·ml^-1) in the presence of amino acids stimulated a 53% increase in fatty acid, and a 44% increase in glycerol, release over rates observed in the absence of amino acids. Ovine growth hormone (28 ng·ml^-1) also stimulated gluconeogenesis, as indicated by a 75% increase in phosphoenolpyruvate carboxykinase activity in liver pieces incubated in the presence of amino acids. These results indicate that growth hormone directly stimulates lipid breakdown in the liver of trout and that amino acids potentiate growth hormone-stimulated lipolysis.Abbreviations AA amino acid(s) - dGDP deoxy-guanosine diphosphate - ED ₅₀ 50% effective dose - FA fatty acid(s) - fw fesh weight - GH growth hormone - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid - MS-222 tricaine methanesulfonate - MEM minimum essential medium - oGH ovine growth hormone - PEPCK phosphoenolpyruvate carboxykinase - PK_c protein kinase C - rpm revolutions per minute - sGH salmon growth hormone - TG triacylglycerol - w/v weight per volume This paper was presented in abstract form at the Annual Meeting of the American Society of Zoologists, Dec. 26–30, 1991, Atlanta, GA     

Effects of hypoxia on the venous circulation in rainbow trout (Oncorhynchus mykiss)

Hypoxia in fish is generally associated with bradycardia while cardiac output (Q) remains unaltered or slightly increased due to a compensatory increase in stroke volume (SV). Rainbow trout (Oncorhynchus mykiss) were subjected to severe (P(W)O2=7.3+/-0.2 kPa) or mild (P(W)O2=11.5+/-0.2 kPa) hypoxia. Central venous pressure (P(ven)), dorsal aortic pressure (P(da)), heart rate (f(H)) and Q, were recorded in vivo. Both levels of hypoxia triggered a significant increase in P(ven). Severe hypoxia was associated with bradycardia and unaltered Q, whereas mild hypoxia was associated with a small but significant increase in Q and no bradycardia. These findings indicate that an increase in P(ven) promotes an increase in SV during hypoxia. Since mild hypoxia increased P(ven), Q and SV without bradycardia or reduced systemic resistance (R(sys)), we hypothesize that an active increase in venous tone serving to mobilize blood to the central venous compartment in order to increase cardiac preload and consequently SV, is an important cardiovascular trait associated with hypoxia. Pharmacological pre-treatment with prazosin (1 mg kg(-1)) did not conclusively reveal the underlying mechanisms to the observed changes in P(ven). This study discusses the influence of venous pooling, reduced R(sys) and altered venous tone on changes in P(ven) observed during hypoxia.     

Compliance and smooth muscle reactivity of rainbow trout (Oncorhynchus mykiss) vessels in vitro

Systemic veins have a profound influence on cardiac output in mammals. Venoregulatory mechanisms have not been adequately studied in fish and their existence has been questioned. In the present study, two characteristics of vascular mechanics, compliance and agonist-induced tension development, were investigated in rainbow trout vessels in vitro. Rapid compliance in the anterior cardinal vein and efferent branchial artery was calculated from step-wise changes in the volume-pressure curve of isolated vessel segments. Agonist-induced tension development was examined in four veins; anterior and posterior cardinals, intestinal and duct of Cuvier. Venous compliance was not altered in response to epinephrine, norepinephrine or angiotensin II, while efferent branchial artery compliance was decreased by 10^-6 mol·l^-1 epinephrine and norepinephrine but not angiotensin II. The ratios of venous to arterial compliance in vessels from two rainbow trout strains were similar (21:1 and 32:1) and consistent with the ratio reported for mammalian viens (24:1). Trout veins contracted in response to agonists in both an, agonist- and vesselspecific manner. The greatest tension per vessel wet weight was produced in anterior cardinal vein. The response pattern of anterior cardinal vein and duct of Cuvier were similar; acetylcholine, arginine vasotocin, epinephrine and norepinephrine, and the thromboxane A₂ agonist, U-44,069, produced approximately identical contractions, whereas angiotensin II was virtually ineffective. Conversely, angiotensin II was more potent than epinephrine in posterior cardinal vein. In cumulative dose-response experiments, epinephrine was equipotent in anterior cardinal vein and duct of Cuvier, whereas the latter was less sensitive to acetylcholine. Both atrial natriuretic peptide and sodium nitroprusside relaxed precontracted veins. This is the first study to determine compliance in fish vessels and the contractile nature of different rainbow trout veins. These findings suggest that venous tone and therefore cardiac output in fish may be regulated by neural or humoral mechanisms.Abbreviations ACH acetylcholine - ACV anterior cardinal vein - ANG II salmon asn¹-val⁵ angiotensin II - ANP rat atrial natriuretic peptide - AVT arginine vasotocin - DNR Department of Natural Resources - DOC duct of Cuvier - EBA efferent branchial artery - EC₅ threshold dose producing 5% maximal contraction - EC₅₀ dose producing 50% maximal contraction - EPI epinephrine - HI K⁺ 80 mmol·l^-1 - KCl IV, intestinal vein - NEPI norepinephrine - PBS phosphate buffered saline - PCV posterior cardinal vein - SNP sodium nitroprusside - U-44,069 thromboxane A₂ agonist     

Effects of ambient colour on colour preference and growth of juvenile rainbow trout Oncorhynchus mykiss (Walbaum)

Colour preference of individual juvenile rainbow trout Oncorhynchus mykiss was tested at 1 and 12° C, and also at 12° C after a 42 day growth experiment under white, blue, green, yellow or red ambient colour. All experiments were carried out under controlled laboratory conditions and the preference was assessed by the location of the fish in a preference tank with four chambers. Rainbow trout showed a preference for blue and green at 1° C and for green at 12° C. After the growth experiment the fish reared in blue tanks preferred blue and green but green was the most preferred colour for the fish reared in green, yellow and red tanks. Yellow and especially red chambers were avoided, irrespective of the ambient colour during the growth trial. The final mass of fish reared in the red aquaria was significantly smaller than that of the fish in green tanks. In addition, when the data of the preference tests were correlated with the data of the growth experiment using mean values of the four tested colours, a very good linear relationship was observed between the preference ( i.e. visit frequency in coloured compartments) and growth rate as well as food intake. When considering the results both from the preference and growth trials it is suggested that green is the best environmental colour for rearing juvenile rainbow trout while rearing in a red environment cannot be recommended.     

Domestication shapes morphology in rainbow trout Oncorhynchus mykiss

In this study, clonal lines from North American resident and migratory populations of rainbow trout Oncorhynchus mykiss adapted to different geographical conditions and with different domestication histories were characterized morphologically. Lines reared in a common‐garden experiment were characterized for external shape and meristic values, searching for a general pattern of morphological variation due to exposure to captive conditions. A sharp distinction was identified between wild and captive lines. The body profile was deeper in captive lines, with longer dorsal and anal fins and shorter and deeper caudal peduncles. Highly significant differences were also identified in meristic values among the lines but no consistent relation between meristic values and domestication status was detected. This morphological characterization will facilitate the selection of lines with divergent phenotypes for subsequent quantitative trait loci analysis, aimed at identifying genome regions linked with morphological adaptive response to captive conditions.     

Antibacterial proteins in rainbow trout, Oncorhynchus mykiss

Antibacterial proteins are an important part of the innate immune system for all animals. They have been extensively studied in mammals, amphibians and invertebrates, but have received only scant attention in fish. Their expression and processing, however, provide a way of monitoring defence vigour during development or with seasonal changes in physiology. The aim of the present work was to identify and characterise antibacterial proteins in rainbow trout. In vitro analyses of extracts of the peripheral blood leucocytes, head kidney leucocytes and mucus from adult unstimulated (non-immune) fish showed marked antibacterial activity against Gram positive bacteria. Fractionation by ion exchange chromatography and RP-HPLC of head kidney extracts showed the presence of two forms of lysozyme but no constitutively expressed antimicrobial proteins of < 10 kDa. By contrast, chromatographic analyses of mucus revealed at least four antibacterial proteins. Two are conventional lysozymes, a third is an unusual lysozyme-like protein with a low isoelectric point, and the fourth is a highly hydrophobic, cationic peptide of c. 3 kDa.     

虹鳟对时间和地点的学习记忆(英文)

为确定虹鳟(Oncorhynchus mykiss)是否能将日投放两次食物的不同时间和地点联系起来,在30天内,于09:00-10:00将食饵投放在水族箱一侧,于15:00-16:00将食饵投放在水族箱另一侧.在第21天和28天,不放食饵以确定虹鳟对时间-地点的学习记忆.结果表明,虹鳟将不同时间和地点联系起来以获取食物,并且在水族箱两个投放食饵之处均表现出预取食活动,提示该物种具有控制时间-地点学习任务的内在机制.     

Testis transplantation in male rainbow trout (Oncorhynchus mykiss)

The objective of the present study was to establish a procedure for the transplantation of an intact testis from one male rainbow trout (Oncorhynchus mykiss) to another individual and evaluate the reproductive function of the transplanted testis at sexual maturity. Isogenic (cloned) male rainbow trout were produced by crossing a completely homozygous male (YY) with a homozygous female (XX) to eliminate any problem of tissue rejection. Transplantation was performed on four pairs of sexually immature animals (n = 8); each served both as a donor and recipient. The left testis was removed by making a ventral midline incision to expose the body cavity and gonads. The left testis was disconnected at the anterior and posterior points of attachment and transferred to the recipient fish where it was placed in position adjacent to the pyloric cecae. The right testis was left intact. After 4 wk, the fish were injected (i.p.) twice weekly for 8 or 9 wk with salmon pituitary extract (1.5 mg/kg) to induce precocious sexual maturation. A similar number of untreated fish were maintained as controls. Following this treatment, all the fish were killed, and the right (intact) and left (transplanted) testes were removed, weighed, and sampled for sperm. Although the mean weights of the left, transplanted testes were significantly (P: < 0.05) smaller than the intact testes (transplants = 1.2 g; intact = 3.9 g), transplanted testes were present in all animals, had increased in mass, and were sexually mature containing sperm. The mean fertility, as measured by the proportion of eggs completing first cleavage, of sperm derived from transplanted testes (92%) was no different from the sperm obtained from intact testes (84%). Similarly, there was no difference in the number of embryos attaining the eyed stage of development, after 18 days of incubation, that were derived from transplanted (84%) or intact testes (85%).     

Resistance to beta-lactam antibiotics in Aeromonas hydrophila isolated from rainbow trout (Oncorhynchus mykiss).

Bacterial infections caused by members of the genus Aeromonas, with a relatively high antibiotic resistance, are among the most common and troublesome diseases of fish raised in ponds with recirculation systems. In this study, carried out at an experimental aquaculture station in northern Portugal, 51 strains identified as belonging to the genus Aeromonas were isolated from 20 rainbow trout (Oncorhynchus mykiss) skin and kidney samples, as well as from raceway water samples. Macro- and microscopic examination of the fish tissues revealed lesions or cellular alterations in skin and kidney that seemed to correlate with the presence of those isolates. The sensitivity of all isolated strains to different groups of beta-lactam antibiotics (penicillins, cephalosporins, monobactams and carbapenems) was evaluated using the disc diffusion method. The highest rates of resistance were to amoxicillin, carbenicillin and ticarcillin. Unexpected resistance to imipenem, an antibiotic of clinical usage, was also detected, which suggests that resistance may have been transferred to the Aeromonas population from the environment.