Variable versus constant temperature acclimation regimes: Effects on hemoglobin isomorph profile in goldfish,Carassius auratus

Goldfish,Carassius auratus, were acclimated for 2 to 3.5 weeks to three temperature regimes: [1] temporally-constant (10, 20 and 30°C), [2] diurnally-cycling (20 ± 10°C) and [3] randomly-fluctuating (± 2°C at approximately 2h intervals between extremes of 10 and 30°C). No significant differences in hematocrit were evident. Hemoglobin levels in fish at constant 30°C and under randomly fluctuating temperature were significantly elevated. Of the three hemoglobin isomorphs observed, the two minor components (G₁, G₃) tended to decrease in relative abundance with increase in constant temperature, but increased under varying temperature regimes. The converse was true of the principal hemoglobin, G₂. Extent of isomorph variation was correlated with extent of temperature variability. These observations confirm that temperature variability significantly effects thermoacclimatory response. The functional significance of changes in isomorph abundances during the acclimatory process is considered.     

Glucose-stimulated lipolysis in rainbow trout,Oncorhynchus mykiss,liver

Rainbow trout, Oncorhynchus mykiss, were used to characterize further the influence of glucose on hepatic lipolysis. Liver was removed from fed fish, cut into 1 mm³ pieces and incubated for up to 5 h in Hanks medium containing either 2 mM, 5.5 mM, 10 mM, or 25 mM glucose. Glucose-stimulated lipolysis was indicated by tissue triacylglycerol (TG) lipase activity and by medium concentrations of glycerol and fatty acids (FA). Triacylglycerol lipase activity in liver pieces incubated in the presence of higher concentrations (25 mM) of glucose was significantly higher than that in liver pieces incubated in lower concentrations (2 mM) of glucose, rising from 0.075 ± 0.002 (mean ± SEM) nmol FA released/h/mg protein to 0.092 ± 0.004 units. Similarly, higher concentrations of glucose stimulated significantly more FA release and glycerol release from liver pieces than that stimulated by lower concentrations of glucose. Glycerol release from liver pieces incubated in the presence of 10 mM glucose and 25 mM glucose was ca. 2-fold to 2.8-fold, respectively, higher than that from liver pieces incubated in the presence of either 2 mM or 5.5 mM glucose. Fatty acid release from liver pieces incubated in the presence of 10 mM or 25 mM glucose was ca. 1.8-fold higher than that from liver pieces incubated in the presence of either 2 mM or 5.5 mM glucose. Notably, increased glycerol release was not accompanied by a parallel increase in FA. Fatty acid reesterification was more pronounced in liver pieces exposed to higher glucose (10 mM and 25 mM) than in liver pieces exposed to lower glucose (2 mM and 5.5 mM). ¹⁴C-incorporation studies indicated that glucose serves as a carbon source for reesterified FA in trout liver. The route of reesterification appears to be from glucose to glycerophosphate to phosphatidic acid to diacylglycerol to TG. Increasing concentrations of glucose did not affect glycerol kinase activity, indicating that glucose-stimulated lipolysis was not accompanied by increased glycerol recycling within the liver. These results suggest that glucose stimulates fatty acid reesterification and directly enhances net lipolysis in trout liver incubated in vitro.A part of this study was presented at the Annual Meeting of the American Society of Zoologists, December 26–30, 1991, Atlanta, GA.     

Lipid-stimulated somatostatin secretion in rainbow trout,Oncorhynchus mykiss

Previous work has shown that somatostatins (SS) affect teleost lipid metabolism indirectly by inhibition of insulin (INS) and directly by stimulation of hepatic lipolysis. In the present study, rainbow trout (Oncorhynchus mykiss) were used to characterize further the lipid-SS relationship by evaluating how lipid, contributes to SS secretion bothin vivo andin vitro. In vivo hyperlipidemia was induced for up to 3 h by short-term (2 min) infusion of a triacylglycerol (TG)-rich lipid emulsion (20% Intralipid^®). Plasma total lipid concentration increased 118 and 155% over control levels 1 h and 3 h, respectively, after infusion; much of this increase was due to elevated plasma fatty acids (FA), which increased 39 and 520%, respectively, over the same time-frame. The hyperlipidemic pattern was attended by a significant increase in the plasma concentration of SS. The specific effects of fatty acids were evaluated on isolated Brockmann bodies. Palmitic acid and oleic acid stimulated SS release 378 and 82%, respectively, over baseline levels. These results indicate that lipids, and in particular fatty acids, modulate SS secretion in rainbow trout.     

Effect of ascorbic acid supplement in vitro onrainbow trout sperm viability

We evaluated motility and fertilizing ability of rainbow trout Oncorhynchus mykiss semen obtained from fish fed diets without ascorbic acid and a diet supplemented with 870 mg kg^{minus 1} of ascorbyl monophosphate. Semen was stored in vitro on ice (0 °C) during 14 days. The spermatozoa from the supplemented group had the highest motility and lowest decline in fertilizing ability after storage. Lack of a positive effect of exogenous vitamin C on semen in fish deficient in ascorbic acid (milt was supplemented with 50 mg l^–1 of ascorbic acid) suggests that the positive effect of ascorbic acid on semen quality is related to its long-term effects during spermatogenesis.     

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).     

Purification and characterization of hepatic triacylglycerol lipase isolated from rainbow trout,Oncorhynchus mykiss

Trialcylglycerol (TG) lipase was isolated and partially purified from rainbow trout liver. Triacylglycerol lipase activity was assayed by measuring¹⁴C-oleic acid release from¹⁴C-triolein.¹⁴C-oleic acid release was linear for up to two hours. Optimal activity occurred at pH 7.0 and 15°C. Most of the lipase activity was recovered in the cytosolic fraction. A 27,000-fold purification was achieved after Sepharose (Bio-gel A 0.5 M, 200–400 mesh) chromatography of a resuspended 20% ammonium sulfate fraction. The molecular weight of the trout hepatic lipase as determined by size-exclusion chromatography and by SDS-polyacrylamide gel electrophoresis was 40–43 kD. Lipase-mediated hydrolysis of TG resulted in the production of diacylglycerols, monoacylglycerols, and fatty acids. Kinetic analysis indicated that V_max=0.016 nmol/h/mg protein and that K_m=0.28 mM triolein. Lipolytic activity was enhanced in the presence of cAMP/ATP-Mg²⁺. These results suggest that the liver of trout possesses a neutral TG lipase that is responsible for mobilizing stored TG and is catalytically activated by phosphorylation.A part of this work was presented at the Annual Meeting of the American Society of Zoologists, December 26–30, 1990, San Antonio, TX.     

Stress-induced changes in the affinity and abundance of cytosolic cortisol-binding sites in the liver of rainbow trout,Oncorhynchus mykiss (Walbaum), are not accompanied by changes in measurable nuclear binding

Plasma cortisol levels and the number (N_max) and affinity (K_d) of specific hepatic cortisol-binding sites were determined in rainbow trout subjected to chronic confinement stress for 14 days. Confinement significantly elevated plasma cortisol levels to 47.3 ± 13.5 ng ml^–1 within 24h and although levels declined to 8.0 ± 3.0 ng ml^–1 after 14 days, they were significantly higher throughout than levels in unstressed control fish (< 2.0 ng ml^–1). There was a 60% reduction in cytosolic N_max in stressed fish during the first 24h of confinement (35.8 ± 7.9 cf. 129.0 ± 15.2 fmol mg^–1 protein), a decline which was sustained at 7 days after the onset of stress but, although numbers of binding sites in the liver of stressed fish were still lower than in unstressed fish, the difference was no longer significant after 14 days of confinement. There was an accompanying significant rise in the K_d of cortisol binding in stressed fish during confinement, from 4.0 ± 0.6 nM at time 0 to 8.4 ± 0.8 nM after 24 h confinement. This increment in K_d was sustained at a level significantly higher than in control fish throughout the 14 day confinement period, despite marked reductions in cortisol levels and increases in N_max in stressed fish. Throughout the study, specific binding of cortisol could not be consistently detected in high-salt nuclear extracts from stressed or unstressed fish, suggesting either that high-affinity binding sites for cortisol were absent from these preparations, that receptors were present but unable to interact with ligand because they were occupied, or that receptors were present but not being extracted. These possibilities were investigated using a range of extraction procedures, by varying the temperature of incubation, by employing dexamethasone as ligand and by examining binding in purified, intact, nuclei. Estradiol was employed as a methodological control throughout and substantial amounts of specific estradiol binding were detected in all compartments and preparations. Specific cortisol-binding sites were detected in intact nuclei of both stressed and unstressed fish, at levels an order of magnitude lower than estradiol binding in the same preparations. These data demonstrate that activation of the pituitary-interrenal axis leads to significant changes in the nature of target-tissue binding of cortisol in rainbow trout, and reveal a clear difference in the subcellular distribution of binding-sites for estradiol and cortisol, which reflects the situation in mammalian tissues.     

Intraperitoneal injection of sulfur amino acids enhance the hepatic cysteine dioxygenase activity in rainbow trout (Oncorhynchus mykiss)

As part of the investigation into cysteine metabolism in fish, sulfur amino acids and their derivatives were injected intraperitoneally to fingerling rainbow trout (Oncorhynchus mykiss) to examine how the doses of these compounds affect the hepatic cysteine dioxygenase [EC 1.12.11.20] in this species. A dose of 0.25 mmol L-cysteine per 100 g body weight induced the enzyme activity as much as 2.5 times that of the control fish within 4h after the injection. The activity increased proportionally to the increasing dose of cysteine up to the dose of 0.15 mmol per 100 g body weight. The induction was observed to be rather specific to L-cysteine. These findings suggested that the cysteine sulfinate pathway might play an important role in the metabolism of excess cysteine in rainbow trout. The dosage of L-cysteine larger than 0.50 mmol per 100g body weight led to mortality of the fish. The pathway of cysteine catabolism was considered to function to prevent toxic accumulation of cysteine in rainbow trout, as in the case of mammals.     

Growth and feed utilisation of rainbow trout subjected to changes in feed lipid concentrations

Rainbow trout Oncorhynchus mykiss grew from 44 to 326 g in 96days when held at 12 °C. Fish were fed to satiation twice dailywith either high (L₁: 30.8%, L₂:31.4%) or lower-lipid feeds (C₁: 18.8%,C₂: 21.8%). Four feeding treatments were studied.Group C₁C₂ received feed C₁ for 43 days(days 0–43) and C₂ thereafter (days 44–96).Groups L₁L₂, L₁C₂ andC₁L₂ were subjected to dietary changes asindicated by the feed designations. After a short period of feedadaptation, fish ingested similar amounts of feed energy i.e., they ateless by weight of the lipid-rich (L) feeds. Feed lipid content did notaffect growth but fish fed L-feed had reduced feed conversion ratio(FCR) compared to fish fed C-feed (0.731 vs. 0.773) during days0–43 (P < 0.01). After 96 days,L₁L₂-fish were lower in body protein(15.8%) than the C₁C₂-fish (16.8%)(P < 0.01). L-feeds also tended to increase percentage lipidand reduce percentage whole body moisture and ash. A higher net proteinutilisation (NPU) was recorded in fish fed L-feeds (43.6%)compared to fish fed C-feeds (38.8%) in days 0–43(P < 0.05). This seemed to be the result of a lower proteinintake rather than a protein-sparing effect of feed lipid. Above athreshold value of approximately 6.5 mg protein eaten·g bodywt_{minus 1}·day_–1, NPU decreased.     

Growth in Arctic charr and rainbow trout fed temporally concentrated or spaced daily meals

The effect on growth of distributing feed over a few hours compared tomore frequent meals was tested on 1+ Arctic charr (Salvelinus alpinus L.) and 1+ rainbow trout (Oncorhynchus mykiss Walbaum). Triplicate hatchery groups for each treatment were fed at a ration level of 1%/dayeither with few meals (8 times per day divided into morning and evening)or with frequent meals (32 meals equally distributed during the day). Wefound an opposite effect of meal frequency on growth in the two species.Low feeding intensity (8 meals per day) had a significantly positive effecton growth in rainbow trout but a significantly negative effect on growth inArctic charr when compared to feeding the fish frequent meals. Theopposite response to meal frequency is likely to be an effect of thedifferences in activity during feeding. Rainbow trout feed much moreaggressively than charr which can result in feeding being a more stressfulevent. In this experiment, the specific growth rate was lower and the feedconversion ratio higher for Arctic charr compared to rainbow trout.     

Protein synthesis in trout liver is stimulated by both feeding and fasting

The response of protein synthesis in the liver of the rainbow trout to feeding and fasting was investigated in 3 experiments. In the first experiment, the fractional rate of protein synthesis (k _s ), %/day) appeared to cycle with daily feeding events being increased by 46%, 123%, and 72% at 1.5h, 3h, and 6h, after a meal. In Experiment 2, liver protein synthesis fell progressively with fasting to a basal level at 4d which was only 20% of the value at 3h after feeding. Liver weight (hepatosomatic index, HSI, % body weight), total RNA and total protein also fell gradually. Between 4d and 6d, both the RNA/protein ratio and the rate of protein synthesis were significantly increased (11% and 74%). At this time, however, there was also a large loss of liver protein suggesting a concomitant increase in protein breakdown. In the last experiment, when trout were pre-fed a low ration (0.6%/d for 2 wks, LR group), the HSI and liver total RNA and protein were largely unaffected by the 6d fast (i.e., relative to the body weight). In this group, however, protein synthesis at 3h was significantly higher than in fish pre-fed a high ration (1.5%/d, HR group). In addition, at 6d after feeding, protein synthesis had increased back to fed levels in the LR group only. It is concluded that protein synthesis in the liver of the trout is influenced both by feeding events and by ration size and also by the degree to which the trout is fasted.     

Effect of expanded feed with high fish oil content on growth and fatty acid composition of rainbow trout

Rainbow trout (Oncorhynchus mykiss) were fed three different diets for 110 days — a basal dry diet with 8.4% oil content (BD8), a basal dry diet with 11.1%; oil content (BD11) a nd an expanded diet with 20.7% oil content (ED) — to investigate the influence of high fish oil exp anded diet on fatty acid composition of muscle, and to evaluate nutritional properties of edible tissue. I n fact, the experimental diets were also different in their component fatty acids, with an in creasing content of 3 highly unsaturated fatty acids (3 HUFA) from BD8 to ED. As regards biomet rics data, the condition factor and the coefficient of fatness were higher in fish fed ED in com parison with groups BD8 and BD11 (p < 0.05 ED vs. BD8). On the other hand, hepatosomatic index in group ED was markedly lower than those in groups BD8 and BD11 (p < 0.05 ED vs. BD8 and E D vs. BD11). This could be explained by the lower amount of crude protein in ED or it may indicate an excess amount of essential fatty acids (EFA) in ED. As regards fatty acid composition of fish m uscle, there were only slight differences in fatty acid composition of the edible tissue of fish wh en compared with the differences in fatty acid composition of the diets. The increased amount of fish oil in ED had a positive influence on the final weight of fish (p < 0.05 ED vs. BD8 and ED vs. BD11), but did not affect proportionately the percentage of 3 HUFA (20:53, 22:53, 22:63) and therefore the derived indices of lipid quality: so it appears possible to partially substitute fish oil in the diet with other lipid as a source of dietary fat.     

The effect of dietary coconut oil on reproductive traits and egg fatty acid composition in rainbow trout (Oncorhynchus mykiss)

Two hundred and twenty rainbow trouts (IBW: 700 g) were randomly allotted to four tanks, with a male/female ratio of 0.56. Fish were fed for 168 d with four experimental diets containing herring oil, cod liver oil and coconut oil with the following inclusion rates: diet A: 12-1-0% respectively; diet B: 6-1-6%; diet C: 0-1-12%; diet D: 0-0-13%. Irrespective of the dietary treatment, weight gains of broodstocks were high (> 3 g/d) and FCR below 2. No significant difference was observed concerning the total amount of eggs spawn, egg average weight (82.5 mg/egg) and lipid content (5.4 mg/egg). However, the fatty acid profile of eggs was significantly affected by the dietary treatments. The content of unsaturated fatty acids, particularly the n-3 fatty acid series (EPA and DHA) significantly decreased with increasing levels of coconut oil in the diet.     

Effect of feeding and fasting on plasma tryptophan and tryptophan to large neutral amino acid ratio,and on brain serotonin turnover in rainbow trout,Oncorhynchus mykiss

Two time-course experiments were conducted to determine the effect of feeding and fasting on the plasma ratio of tryptophan (trp) to the large neutral amino acids (LNAA), (trp/LNAA ratio) and brain serotonin (5-HT) turnover in rainbow trout,Oncorhynchus mykiss. Trout were fasted overnight or for 3 days and were then either fed or continued to be fasted for up to a further 3 days. Changes in plasma trp, plasma trp/LNAA ratio, brain trp, brain 5-HT, brain 5-hydroxyindoleacetic acid (5-HIAA) and brain 5-HIAA/5-HT ratio were determined over time. Feeding decreased the plasma trp/LNAA ratio, brain trp and the brain 5-HIAA/5-HT ratio. In addition, in fish sampled over 3 days, there appeared to be a rhythm in plasma trp and the brain 5-HIAA/5-HT ratio which was independent of feeding. These results indicate that in rainbow trout, feeding is a sufficient physiological event to decrease brain 5-HT turnover. Furthermore, feeding-independent changes in the brain 5-HIAA/5-HT ratio, which were evident in fasted fish sampled over 3 days, also suggest an additional, non-feeding-related modulator(s) of brain 5-HT turnover in rainbow trout.     

四川地区一株传染性造血器官坏死病毒的分离鉴定及系统发育分析

2014年5月,四川省都江堰市某虹鳟养殖场暴发一种传染性疾病,幼鱼和鱼苗死亡率分别高达40％和80％.为探究此次疾病病因和流行规律,将病料进行解剖及细菌学检查、病理组织观察、人工感染实验、病毒分离、多重RT-PCR鉴定和系统发育分析.结果显示,病鱼主要临床症状表现为腹部膨大,体表发黑,肛门拖淡黄色黏液便,解剖见鳔壁、腹膜出血,胃胀气膨大和明显肠炎;细菌学检查为阴性;组织病理学上,头肾、肾脏和脾脏造血组织广泛性变性、坏死,肠黏膜下层嗜酸性颗粒细胞浸润与坏死,肝细胞变性、坏死形成局灶性的坏死灶,并在一些肝细胞胞浆内见嗜酸性包涵体.将病鱼的肝脏、脾组织研磨过滤灭菌后,腹腔注射20尾健康虹鳟,注射组均表现为急性死亡(累积死亡率=75％),并出现与自然发病鱼相同的症状.取病鱼组织匀浆滤菌液接种到鲤上皮瘤细胞(epithelioma papulosum cyprini,EPC),盲传3代出现典型的细胞病变效应(cytopathic effects,CPE).针对IHNV、IPNV与VHSV的多重RT-PCR检测显示自然发病鱼、人工感染病鱼和病变细胞均为IHNV阳性,扩增序列与IHNV核蛋白(nucleoprotein,N)基因同源性为99.9％.对分离株的糖蛋白基因“Mid-G”区域进行系统发育分析,结果显示该分离株与亚洲分离株聚为一支,属于JRt基因型.本研究首次报道我国西南地区养殖虹鳟中IHNV感染引起的疾病.     

Isolation and partial characterization of rainbow trout (Oncorhynchus mykiss) gill mucin

Gill mucin from rainbow trout was isolated utilizing two rounds of cesium chloride density ultracentrifugation followed by gel filtration on Sepharose CL-2B. Neither density ultracentrifugation nor gel filtration alone was sufficient for purification of the mucin. Isolated gill mucin had a density of 1.5 g/ml and eluted at the void volume of the Sepharose CL-2B column. Silver-stained reducing 6% polyacrylamide gel electrophoresis of gill mucin produced a band at the origin with a smear entering the separating gel. There was no evidence of a link protein in gill mucin on reducing 12% polyacrylamide gel electrophoresis. Gill mucin had an amino acid profile similar to that of mucins in other species. Specifically, 35.1% of the total amino acids were represented by threonine and serine, while another 27.5% were alanine and proline. Gill mucin contained galactose (26.7 ± 3.2%), galactosamine (22.5 ± 4.4%), glucose (16.6 ± 8.7%), fucose (16.1 ± 1.5%), glucosamine (12.0 ± 1.9%) and mannose (5.1 ± 4.4%). Uronic acid levels from purified mucin were very low (0.7 ± 0.1%). Sialic acid was also present (0.06 g/g of mucin protein). The periodic acid-Schiff assay routinely utilized for detection of mucins was relatively insensitive for detection of gill mucin (6 × less sensitive than for pig gastric mucin) so a rabbit antiserum was raised. The antiserum produced profiles similar to the periodic acid-Schiff assay of fractions following gel filtration. Immunofluorescence of formalin-fixed rainbow trout gill tissue sections showed that the antiserum detected mucin within branchial goblet cells.     

Gluconeogenesis in trout (Oncorhynchus mykiss) white muscle: purification and characterization of fructose-1,6-bisphosphatase activity in vitro

Studies of the enzyme fructose-1,6-bisphosphatase (FBPase) of rainbow trout (Oncorhynchus mykiss) have been undertaken in order to illuminate aspects of skeletal muscle gluconeogenesis in these animals. Maximal activities in crude homogenates of several organs suggest that the liver possesses the greatest FBPase activity on a unit g^–1 tissue basis but that the white muscle, owing to its bulk, contributes substantially to whole body FBPase activity. Studies of fructose-6-phosphate-1-kinase (PFK) and FBPase in crude homogenates of several organs suggests an important role for intracellular pH in regulating the relative carbon flux through the FBPase/PFK locus in vivo. Furthermore, a three-step purification scheme is described for trout white muscle FBPase by which a stable and homogeneous (by SDS PAGE) enzyme preparation (isoelectric point = 7.2; molecular weight = 37.6 kd) was obtained. Kinetic studies of the purified enzyme were undertaken at 20°C under conditions reflective of "rest" and "exercise/recovery" intramuscular pH in vivo. Affinity for substrate (F-1,6-P₂) was increased (Km = 6.88 versus 2.44 mol 1-^–1 as was enzyme activity when pH was lowered from 7.0 to 6.5. Various inhibitor metabolites are identified including F-2,6-P₂ (mixed-type inhibitor, K_i = 0.201 mol 1^–1, pH 7.0) and AMP (non-competitive inhibitor, K_i = 0.438 mol 1^–1, pH 7.0). Inhibition by F-2,6-P₂ was strongly alleviated by a reduction in pH from 7.0 to 6.5 (I₅₀ increased from 0.14 to 0.32 mol 1^–1). AMP on the other hand was a more potent inhibitor at pH 6.5 but this inhibition was totally reversed under conditions of citrate, NH₄ ⁺ and AMP typical of muscle during recovery from exercise in vivo. In purified white muscle enzyme preparations, FBPase demonstrated maximal activity at pH 6.5 whereas the optimal pH of PFK was 7.0 or greater. Indeed, it appears from these in vitro data that regulation by metabolite levels as well as pH are required for net FBPase flux in vivo. It is concluded, therefore that trout white muscle FBPase demonstrates the potential to play an important enzymatic role in the control of intramuscular gluconeogenesis in these animals. The results are discussed in relation to present knowledge regarding the metabolic responses of trout white muscle to, and its subsequent recovery from, exhaustive exercise.