Immunohistochemical localization of rainbow trout androgen receptors in the testis

SUMMARY: We examined the distribution of two rainbow trout androgen receptors (rtAR: rtAR-α and rtAR-β) in the testis immunohistochemically using a specific antibody to clarify the target cells of androgen in spermatogenesis. Positive rtAR immunoreactivity in paraffin-embedded sections was revealed using microwave treatment, and was detected in the nuclei of Sertoli cells, Leydig cells, and other interstitial cells. The presence of rtAR in Leydig cells suggested that fish androgens regulate Leydig cell activity in an autocrine fashion similar to mammalian androgens. In addition, we found that not all Leydig cells exhibited rtAR immunoreactivity in the mature testis by double staining using anti-3β-hydroxysteroid dehydrogenase (3β-HSD) antibody. Furthermore, rtAR immunoreactivity was also detected in the nuclei of spermatogonia, spermatocytes, and spermatids. The intensity of rtAR immunoreactivity in the nuclei of spermatogonia seemed to be weaker than those of spermatocytes and spermatids. These results suggested that androgens act directly on both germ cells and somatic cells in the regulation of spermatogenesis in the rainbow trout.     

Effect of partial replacement of dietary fish meal by soybean meal with betaine attractant supplementation on growth performance and fatty acid profiles of juvenile rainbow trout (Oncorhynchus mykiss)

This study was conducted to investigate the effects of dietary incorporation of soybean meal for fish meal replacement and supplementation of betaine as an attractant on growth performance and fatty acid profiles of rainbow trout (Oncorhynchus mykiss). Juvenile trout were fed practical diets, with increasing soybean levels and betaine supplementations. The experimental diets consisted of a control diet with fish meal as the sole protein source (control) and no attractant, 25% soybean‐1% betaine diet (SBM25‐B1), 50% soybean‐2% betaine diet (SBM50‐B2), and 50% soybean‐0% betaine diet (SBM50‐B0). Average body weight was 12.69 ± 0.16 g at the beginning of experiments. Following 54 days feeding programme with experimental diets, average body weights reached 47.45 ± 1.22 g, 58.11 ± 1.77 g, 56.34 ± 1.87 g and 53.76 ± 1.74 g in the control, SBM25‐B1, SBM50‐B2 and SBM50‐B0 groups respectively. As compared with control treatment, significant differences were observed in weight gain, specific growth rate and feed intake of 1% betaine treatment at 25% soybean‐meal‐incorporated diet (p < .05), but no differences were observed in feed conversation ratio and survival rates (p > .05). Compared with the control treatment, betaine‐supplemented groups had significantly higher total saturated fatty acid contents (p < .05). On the other hand, the control treatment showed a significantly higher level of monounsaturated fatty acid than the betaine‐supplemented groups (p < .05). Significant differences were observed in fatty acid profile of 1% betaine‐supplemented group (p < .05) compared with the control. Present findings revealed that 1% betaine supplementation with dietary incorporation of soybean meal at 25% level positively influenced growth performance, feed utilization and fatty acid profiles of rainbow trout juveniles.     

Control of gallbladder motility in the rainbow trout,Salmo gairdneri

Immunohistochemistry revealed nerves containing VIP-like and 5-HT-like material in both gallbladder wall and bile duct of the rainbow trout, while endocrine cells containing gastrin/CCK-like and substance P-like material were present in the mucosa of the bile duct and the duodenum. Fluorescence histochemistry showed adrenergic nerves close to the muscle layer of the gallbladder.Sulphated CCK8, caerulein and non-sulphated CCK8 (in this order of potency), 5-HT and acetylcholine were excitatory on isolated strip preparations, while VIP and adrenergic agonists were inhibitory. The adrenergic drugs were probably actingvia a beta-adrenergic receptor, while the effects of 5-HT and cholinergic drugs were antagonized by methysergide and atropine respectively.Electrical stimulation of the gallbladder nervesin situ failed to show any effect or under certain conditions induced a rebound effect.It is concluded that the motility control of the rainbow trout gallbladder may involve an inhibitory innervation by adrenergic and possibly VIP-releasing nerves, while 5-HT, acetylcholine and a CCK-like substance may be involved in the excitatory control.     

A study of gross,histological and blood biochemical changes in rainbow trout,Oncorhynchus mykiss (Walbaum), with rainbow trout gastroenteritis (RTGE)

The mechanisms behind the pathogenesis of rainbow trout gastroenteritis (RTGE) are still unknown. This study examined the macroscopic and microscopic changes in trout with RTGE (RTGE+), as well as the blood chemistry. A total of 464 rainbow trout were sampled from 11 sites in the UK, comprising 152 RTGE+ fish and 330 random, apparently healthy fish. A case definition for RTGE was assessed by the analysis of its agreement with three laboratory tests: histopathology, packed cell volume and kidney bacteriology. Cluster analysis indicated the presence of three distinct presentations within the population of RTGE+ fish. Cluster A included gross signs associated with moribund RTGE+ fish, and clusters B and C identified gross signs consistent with concurrent diseases, notably furunculosis, enteric redmouth and proliferative kidney disease. The information gained was used to select RTGE+ fish without concurrent disease for the analysis of RTGE pathogenesis with blood biochemistry. This analysis revealed a severe osmotic imbalance and a reduced albumin/globulin ratio as indicatives of selective loss of albumin. These findings are compatible with a protein losing enteropathy.     

Morphological, physiological and biochemical parameters characterizing the over-ripening of rainbow trout eggs

In rainbow trout (Oncorhynchus mykiss) freshly ovulated eggs and over-ripened eggs which had been retained in the coelomic cavity for 7, 14 and 21 days were investigated in aspects of morphology, physiology and biochemistry. Egg viability was significantly reduced from 85.9±16.4% in freshly ovulated eggs to 25.1±21.9% in over-ripened eggs which had been retained in the coelomic cavity for 21 days. Further during over-ripening in the ovarian fluid the pH significantly decreased, while the levels of proteins, of esterified and non esterified fatty acids and the activities of aspartate aminotransferase and acid phosphatase significantly increased. Also egg parameters changed: the wet weight of the unhardened eggs increased, the weight increase during hardening and the levels of esterified and of non esterified fatty acids significantly decreased. In freshly ovulated eggs the yolk consisted of a homogenous mass and the perivitelline space was small, but in over-ripened eggs the yolk was non homogenous with numerous vesicular inclusions and the perivitelline space was enlarged. When freshly ovulated eggs were incubated in water the cortical reaction was detectable within 5 min, in over-ripened eggs hardly no extrusion of cortical vesicles was visible and the width of the perivitelline space was very irregular.For the investigated freshly ovulated and over-ripened samples the egg viability significantly correlated with ovarian fluid parameters (pH, protein, non esterified fatty acids, esterified fatty acids, aspartate aminotransferase, acid phosphatase) and egg parameters (weight increase during hardening, weight of the hardened eggs).     

Hypothalamic control of prolactin release in the rainbow trout,Salmo gairdneri: in vitro studies

Hypothalamic control of prolactin (PRL) release in immature rainbow troutSalmo gairdneri was investigated using anin vitro perifusion system of the rostral pars distalis. Hypothalamic extract of trout induced a dose-dependent stimulation of PRL release. A similar effect was observed when infusing the medium from a 24h static incubation of the hypothalamus. Extracts from different control tissues (muscle, liver, gut) did not changein vitro release, thus confirming the specificity of this stimulatory effect. Hypothalamic extract from adult male rat, known to contain PRL release inhibiting factors, stimulatedin vitro PRL secretion in rainbow trout. This suggests that PRL cells are predominantly influenced by PRL releasing factors. Measurement of TRH and serotonin content in trout hypothalamus indicated consistent physiological levels of these two factors. HPLC studies of hypothalamic extract showed that immunoreactive — TRH eluted at the same place as labelled TRH standard. Moreover, pizotifen, a serotonin antagonist, partially inhibited the stimulation observed with trout hypothalamic extract. These results suggest that, in immature rainbow trout, PRL release is under stimulatory hypothalamic control and that serotonin and probably TRH play a major role in this control.     

Antidiuretic action of vasoactive endothelins in the in situ perfused rainbow trout kidney

The present studies examined, for the first time, the renal actions of endothelin-1 (ET-1) and Sarafotoxin S6b (SRTX-6b) (an endothelin-like peptide from snake venom) at 10^-11 M and 10^-9 M, using the in situ perfused kidney of the rainbow trout, Oncorhynchus mykiss. In further studies ET-1 (10^-9 M) was accompanied by Captopril (5 × 10^-4 M) to inhibit angiotensin II formation and determine whether the newly-identified intrarenal renin-angiotensin system (RAS) in the trout kidney was involved in ET-1's actions. These studies demonstrated that ET-1 and SRTX-S6b constrict the trout trunk vasculature, increasing vascular resistance and decreasing perfusate flow rates. Captopril did not affect this response and therefore angiotensin II is not implicated in the vascular responses. Direct action of endothelins on vascular receptors is indicated which, in vivo, is likely to be involved in regulation of renal vascular tone. Both ET-1 and SRTX-6b induced immediate decreases in glomerular filtration rates (GFR) reaching 30% and 34% decrease with 10^-11 M ET-1 and SRTX-6b respectively and 50% and 57% decrease with 10^-9 M ET-1 and SRTX-6b respectively. Urine flow rates decreased to a slightly lesser extent because of decreased tubular reabsorption of water; relative free water clearances increased from approximately 17% to 21% while urine/plasma inulin concentration ratios decreased slightly. This significant depressed urine osmolarity. Captopril completely blocked the effects of endothelins on tubular water reabsorption suggesting intrarenal RAS involvement in this action, although a kinin-mediated effect cannot, at this stage, be excluded. The glomerular antidiuretic action of ET-1 and SRTX-6b partially reflected a decreased population of filtering nephrons (41% filtering in control kidneys, 32–36% filtering in the presence of 10^-11 M M ET-1/SRTX-6b, 31–32% filtering in the presence of 10^-9 M ET-1/SRTX-6b). In addition, 25–40% reductions of single nephron filtration rates were estimated. Glomerular actions of endothelins were partially inhibited by Captopril, suggesting either that renal endothelins have both direct renal actions and secondary effects through activation of the renal RAS, or that kinins can modulate the renal actions of endothelins.     

Supplemental citric acid and particle size of fish bone-meal influence the availability of minerals in rainbow trout Oncorhynchus mykiss (Walbaum)

Juvenile rainbow trout Oncorhynchus mykiss (Walbaum) were fed six low-phosphorus (P) diets supplemented with two different sizes of ground fish bone-meals (fine, 68 μm or less; coarse, 250–425 μm) and a coarse bone-meal diet containing four levels of citric acid (0, 4, 8 or 16 g kg⁻¹ diet) to investigate the effects of pH and bone particle size on P bioavailability. The basal diet provided 3.4 g P   kg⁻¹ and bone-meal increased P contents to 5.4–6.0 g P   kg⁻¹. Coarse bone-meal diets supplemented with 0, 4, 8 or 16 g kg⁻¹ of citric acid had pH values of 6.0, 5.7, 5.4 and 5.0, respectively. Weight gain and whole-body water, protein and lipid contents were not influenced by bone-meal supplementation. Supplementing the basal diet with both coarse and fine bone-meal significantly increased whole-body ash content. Fish fed no bone-meal were hypophosphataemic compared with fish fed with either fine or coarse bone-meals. Phosphorus in fine bone-meal had higher availability than P in coarse bone-meal. Bone-meal supplementation significantly decreased whole-body manganese content from 8.9 μg g⁻¹ in fish fed no bone-meal to 2.3 and 4.5 μg g⁻¹ in fish fed with fine and coarse bone-meals, respectively. The concentration of magnesium increased but zinc concentration was not affected by bone-meal supplements. Citric acid increased whole-body ash content but the influence of citric acid on the body P content was not significant ( P  = 0.07). Dietary acidification by citric acid significantly increased whole-body iron in a linear fashion. The bioavailability of dietary P can be improved by fine grinding the bone in fish meals.     

Replacement of fish meal with two fermented soybean meals in diets for rainbow trout (Oncorhynchus mykiss)

The present study investigated the effect of fish meal (FM) replacement with fermented soybean meal (FSM) on growth and feed utilization of rainbow trout. Two FSM products, FSM1 (more fermentation with more small peptide and acid than FSM2) and FSM2 were used to replace 20%, 40% and 60% of FM in control diet (250 g/kg FM), respectively (FSM1‐20, FSM1‐40, FSM1‐60, FSM2‐20, FSM2‐40 and FSM2‐60). Then the seven diets were fed to rainbow trout (18.1 g) for 8 weeks. Weight gain (WG), feed conversion ratio (FCR) and digestibility of crude protein and dry matter showed no significant difference among the groups of FSM1‐20, FSM1‐40, FSM2‐20, FSM2‐40 and the control, but WG significantly decreased and FCR increased when 60% FM was replaced by both FSMs (p < .05). The replacement of 40%, 60% FM resulted in lower villus height than the control (p < .05), and intestinal protease activity was lower in FSM2‐40, FSM2‐60 and FSM1‐60 groups than the control group (p < .05). In addition, the activity of alkaline phosphatase and superoxide dismutase increased with increasing levels of FSM (p < .05). In conclusion, dietary fish meal could be replaced by 40% with both FSMs without adverse effects on growth and feed utilization of rainbow trout based on an eight weeks feeding trial.     

Citric acid substituted the inclusion of inorganic phosphorus in diet of rainbow trout (Oncorhynchus mykiss)

This study was conducted to evaluate the effects of citric acid (CA) supplementation in diet without inorganic phosphorus (P) on growth, muscle and bone composition, proteolytic activities and serum antioxidant property of rainbow trout. Six diets were designed as the negative diet without monocalcium phosphate (MCP) supplementation, the positive diet containing 10 g kg^?1 MCP and CA supplementation diets with 4, 8, 12, 16 g kg^?1 CA supplementation in negative diet, and then were fed to rainbow trout (113.6 g) for 60 days. Results showed that the fish fed 8 g kg^?1 CA, 12 g kg^?1 CA diet had higher weight gain, higher contents of crude ash and P in bone, and lower feed conversion ratio than those of fish fed negative diet (P < 0.05), and showed the similar levels as those of fish fed positive diet (P > 0.05). The proximate composition and P level of muscle were not affected by dietary CA and MCP. The proteolytic activity in intestine, but not in stomach and gastric digesta, was significantly improved by dietary CA and MCP (P < 0.05), when compared with negative control. The activities of serum superoxide dismutase of 12 g kg^?1 CA and 10 g kg^?1 MCP groups were significantly higher, and the malondialdehyde of 8 g kg^?1 CA and 12 CA g kg^?1 groups were significantly lower than those of negative control (P < 0.05). The above results indicated that the supplementation of CA could substitute the inclusion of MCP in rainbow trout diet and the supplementation level was suggested to be 8–12 g kg^?1.     

Characterization of putative steroid receptors in the membrane, cytosol and nuclear fractions from the olfactory tissue of brown and rainbow trout

Specific binding sites for testosterone have been detected in three compartments of olfactory tissue from brown and rainbow trout. Binding of³H-testosterone to the membrane fraction of olfactory tissue is of high affinity (K_d = 0.5–1.9 nM) and limited capacity (N_max = 30–60 fmol mg⁺¹ protein). Binding is reversible, and is eliminated by protease treatment. The membrane binding site exhibits a high degree of ligand specificity; 11β-hydroxytestosterone, 11-ketotestosterone, 17α-hydroxyprogesterone, 17α,20β-dihydroxy-4-pregnen-3-one, cortisol, and estradiol-17β all fail to displace testosterone at 20-fold excess while testosterone itself competes successfully. These attributes are consistent with the presence of specific steroid receptor proteins. Binding of testosterone within the cytosol is of moderate affinity (K_d = 9.0–23.0 nM) and high capacity (N_max = 0.5–2.9 pmol mg⁺¹ protein) and is more readily displaced by a number of steroid competitors than is the case for the membrane site. The rate of association and dissociation of testosterone from the cytosolic binding site is markedly more rapid than the equivalent processes in the membrane fraction. Binding of testosterone to the nuclear extract is of high affinity (K_d ∼3.0 nM) and limited capacity (N_max ∼50 fmol mg⁺¹ protein). There are no substantial differences between species or between sexes in the affinity or capacity of testosterone-binding sites in nuclear extract or membrane fraction. However, cytosolic testosterone-binding sites are three- to four-fold more abundant in rainbow trout than in brown trout, and female rainbow trout have more cytosolic binding sites than male rainbow trout, but a lower affinity for testosterone than male sites. Preliminary evidence supports the involvement of the membrane-associated testosterone-binding site in olfactory processes. Rainbow trout display an EOG response to testosterone at a concentration (≥ 10⁺⁹ M) which is consistent with the equilibrium dissociation constant (K_d) of the membrane-associated testosterone-binding site. Binding of³H-testosterone to the membrane-associated site shows a pH dependency which is comparable to the effects of pH on the EOG response to testosterone in intact fish. The attributes of the intracellular testosterone-binding sites are common to testosterone receptors in other fish tissues which are known androgen target tissues. This suggests that the development and/or function of salmonid olfactory tissue may be susceptible to influence by endogenous testosterone.     

虹鳟受精卵第一次卵裂组织学观察及四倍体制备最佳时机的研究

采用组织切片技术对虹鳟受精卵第一次卵裂进行观察,且利用热休克和静水压处理受精卵,诱导四倍体。其结果显示,静水压诱导效果较好,在受精后8h进行诱导四倍体的出现率最高,达到25～28%。热休克诱导未出现四倍体。受精卵在受精后400min(积温43.5℃.h)处于第一次卵裂前期,受精后490min(积温52.5℃.h)处于第一次卵裂中期,受精后640min(积温66.8℃.h),处于第一次卵裂后期,670min(积温69.3℃.h)处于第一次卵裂末期,700min(积温71.8℃.h)以后形成卵裂沟,并穿过整个受精卵,产生两个均等大小的两个卵裂球(blastomeres)。     

Effects of egg aging on the metabolites of ovarian fluid in rainbow trout,Oncorhynchus mykiss

Nuclear magnetic resonance ‐based metabolomics was applied to study effects of egg aging on ovarian fluid metabolites in rainbow trout, Oncorhynchus mykiss. The eggs of three females were pooled and then assigned to three plastic vials for 18 days in vitro storage at 4°C. Ovarian fluid samples were taken 0, 6, 12 and 18 days after storage. Three groups of metabolites including amino acids, osmolytes and energy metabolites were found to change during storage period. The glucose levels of ovarian fluid showed significant decreases on days 12 and 18 after storage (p < .05). For acetoacetate and acetate, significant increases were observed, respectively, on days 12 and 18 after storage (p < .05). The creatine levels of ovarian fluid increased significantly on days 12 and 18 after storage (p < .05). Lactate levels in ovarian fluid elevated during storage period (p < .05). Glycerol levels showed a significant increase on days 12 and 18 after storage (p < .05). The values of osmolytes in ovarian fluid (e.g., betaine, taurine, trimethylamine‐N‐oxide, N,N‐dimethylglycine) showed a decreasing trend on day 12 which continued until the end of storage on day 18 (p < .05). Almost all amino acids elevated on days 12 and 18 after storage (p < .05). After an apparent elevation in isoleucine levels on days 6 and 12, this amino acid decreased on day 18 after storage (p < .05). The osmolytes might act as antioxidant against free‐radicals produced as a result of over‐ripening. Glucose can be used as energy resource for eggs and bacteria during ova storage. Also, change pattern of amino acids indicate hydrolysis of proteins as the time of storage increases.     

Role of exogenous glutathione in teleost fish and its effects on antioxidant defense responses in rainbow trout exposed to 3,3',4,4'-tetrachlorobiphenyl

Intraperitoneal injection of glutathione (GSH) is shown to effectively increase tissue glutathione content in two teleost species, the rainbow trout and the American eel. GSH uptake into teleost tissues is a distinct piscine feature of GSH biochemistry compared with mammalian species where little or not uptake occurs of exogenously added GSH. Enhancement and depletion of tissue GSH status was used as a tool to examine the effects of altered GSH content on other cellular antioxidants in 3,3,4,4-tetrachlorobiphenyl (TCB) exposed rainbow trout. -Tocopherol content was unchanged and thiobarbituric acid reactive substances (TBARS), an indicator of lipid peroxidation, changed little in liver and muscle of trout exposed to TCB and the various GSH treatments. However, -tocopherol levels in muscle and superoxide dismutase activities in the various tissues were significantly higher in controls and TCB-saline treated fish than in GSH deficient and lipoate supplemented TCB exposed trout. GSH status thus appears to affect some cellular antioxidant defenses in TCB exposed trout.     

Distribution and induction of cytochrome P450 1A1 in the rainbow trout brain

Cytochrome P450 (CYP) 1A1 participates in the activation as well as detoxification of environmental pollutants such as aromatic hydrocarbons. This CYP form is also efficiently induced by aromatic hydrocarbons. The presence of CYP 1A1 in the brain might thus be of physiological and toxicological importance. In the present investigation on rainbow trout, the distribution of 7-ethoxyresorufin-O-deethylase (EROD) activity, a cytochrome CYP 1A1 catalyzed reaction, was measured in whole tissue homogenates from brain parts. In control fish, a relatively high activity was found in the rainbow trout olfactory bulb compared to the other brain parts. Although an EROD induction (3 to 7-fold) by β-naphthoflavone (BNF) was recorded in all brain parts from the rainbow trout, the highest induced activity was measured in the olfactory bulbs. To ascertain the distribution of EROD activity in cells, whole brain tissue was subfractionated by differential centrifugation. The fractionation scheme separated mitochondria (P2 fraction) and microsomes (P3 fraction) as determined by marker enzymes and electron microscopy. In control rainbow trout, a low EROD activity could be measured in the P2 fraction. BNF induced the EROD activity in both P2 and P3 fractions. Western blotting showed the induction by BNF of a protein band in the P2 and P3 fractions with a molecular mass around 58,000 when highly specific anti-cod CYP 1A1 antibodies were used. ELISA measurements confirmed the induction of CYP 1A1 protein in the rainbow trout brain subcellular fractions.     

The degree of carotenoid esterification influences the absorption of astaxanthin in rainbow trout, Oncorhynchus mykiss (Walbaum)

The absorption of astaxanthin from diets (30 mg kg^?1 inclusion) supplemented with either unesterified astaxanthin; isolated astaxanthin monoesters, diesters or a cell‐free carotenoid extract from Haematococcus pluvialis were studied in rainbow trout (>200 g). No significant differences (P > 0.05) were recorded in the apparent digestibility coefficients (ADC) (≈60–65%) between astaxanthin sources. However, following consumption of a single meal, peak serum astaxanthin levels at 32 h (≈1.0–1.6 μg mL^?1) were significantly higher (P < 0.05) in fish fed unesterified astaxanthin and astaxanthin monoester, compared to fish fed astaxanthin diester and the cell free extract. However, no significant differences (P > 0.05) were recorded in serum astaxanthin uptake rates between sources of astaxanthin. Results suggest that the extent of carotenoid esterification negatively influences the peak serum levels of astaxanthin in rainbow trout.