Effect of extracts from heshiko , a fermented mackerel product, on cholesterol metabolism in Wistar rats

Heshiko extract was separated into peptide and amino acid fractions by ion-exchange column chromatography. Heshiko extract and these fractions were administered to rats in a diet enriched with lipid and cholesterol for 30 days. In the heshiko extract group, increase of triglyceride, total cholesterol, and low-density lipoprotein (LDL)-cholesterol levels in plasma and accumulation of total lipids in the liver were suppressed, while amounts of both lipid and cholesterol excreted to feces were increased. Administration of heshiko extract tended to increase fecal bile acids and promoted the activity of cholesterol 7α-hydroxylase, the rate-limiting enzyme in the synthesis of bile acid from cholesterol in the liver. However, activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the cholesterol synthesis system in the liver, decreased due to regulation by the feedback of lipid transportation from diet to the liver. The same effect was observed in the peptide and free amino acid fraction groups. These data suggest that both excretion of cholesterol and bile acid to feces and promotion of cholesterol 7α-hydroxylase activity are related to the hypocholesterolemic effects of heshiko extract and that both peptides and amino acids contribute to that effect.     

Effect of extracts from <Emphasis Type="Italic">narezushi</Emphasis>, a fermented mackerel product,on cholesterol metabolism in Wistar rats

Narezushi extract was separated into peptide and nonpeptide fractions by ion-exchange column chromatography. The narezushi extract and fractions were administered to rats in a diet enriched with lipid and cholesterol for 30 days. In the narezushi extract and nonpeptide fraction groups, increases in triglyceride, total cholesterol, and low-density lipoprotein-cholesterol levels in the plasma and accumulation of total lipids and triglyceride in the liver were suppressed, while both lipid and cholesterol fecal excretion were increased. In the peptide fraction group, these effects were also observed, except for the suppressing effect on liver lipid accumulation. Narezushi extract administration tended to increase fecal bile acids and promoted the activity of cholesterol 7α-hydroxylase, the rate-limiting enzyme in the synthesis of bile acid from cholesterol in the liver. However, the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the cholesterol synthesis system in the liver, decreased due to regulation by the feedback of lipid transportation from diet to the liver. These results suggest that both the increase in cholesterol and bile acid fecal excretion and the promotion of cholesterol 7α-hydroxylase activities are related to the hypocholesterolemic effects of narezushi extract. Amino acids and organic acids, which are abundantly contained in the nonpeptide fraction, seemed to have more intensive hypocholesterolemic effects than peptides existing in the peptide fraction.     

Free radical scavenging ability and structure of a 90-kDa protein from the scallop shell

We have previously reported that the scallop shell extract possesses free radical scavenging activity. In this study, we isolated a glycoprotein with a molecular weight of approximately 90 kDa (named 90-kDa protein) which showed 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and superoxide anion radical scavenging, reducing, and ferrous ion-chelating activities. Amino acid composition analysis showed that the 90-kDa protein was rich in Asx (Asp or Asn), Ser and Gly residues. A BLAST search of partial amino acid sequences determined by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry revealed that the 90-kDa protein is a novel protein. The 90-kDa protein is a yellow protein that contains fluorescent substances. To the best of our knowledge, this is the first report of a radical scavenging protein from the scallop shell.     

Effect of Dietary Lecithin in Combination with Casein or Crab Protein on Cholesterol Uptake and Transport in the Lobster Homarus americanus

Purified diets, formulated with and without lecithin and containing either casein or a protein isolated from whole crab, were fed to juvenile lobsters. In order to examine the role of these dietary factors on cholesterol uptake and transport, levels of cholesterol were determined in serum, lipo-protein fractions, tissues, and feces of juvenile lobsters fed the experimental diets. Lobsters fed diets supplemented with lecithin had significantly higher levels of serum and lipoprotein cholesterol than lobsters fed diets without supplemental lecithin regardless of the proteins used. Levels of fecal total cholesterol also were generally greater for lobsters fed lecithin-supplemented diets. Tissue, serum, and fecal levels of intubated radiolabeled cholesterol, however, were not significantly different among diet groups, and apoprotein electrophoretic mobility was not affected by the choice of dietary protein.     

The effect of dietary protein source (fishmeal vs. plant protein) and non‐starch polysaccharide level on fat digestibility and faecal bile acid loss in rainbow trout (Oncorhynchus mykiss)

This study investigated in rainbow trout (Oncorhynchus mykiss) if diet composition and feeding level affect faecal bile acid loss, and whether this reflects on the apparent digestibility coefficient (ADC) of fat. Six diets were formulated with either fishmeal or plant protein as main protein source. This created a contrast in the supply of bile acids, the bile acid precursor cholesterol, taurine and the taurine precursors (methionine + cysteine) involved in bile acid conjugation. For both protein sources, three diets were formulated with increasing inclusion of a non‐starch polysaccharide (NSP)‐rich ingredient mixture (0.0, 82.0 and 164.2 g/kg diet). This aimed at enhancing faecal bile acid loss. Fish were fed both restrictively (1.2% BW/day) and to satiation. A similar fat ADC was found when substituting fishmeal with a plant protein mixture, suggesting that the lower content of bile acids, cholesterol, taurine, methionine and cysteine in the plant‐based diets did not limit fat digestion. Faecal bile acid loss increased alongside dietary NSP level, however, only during satiation feeding and most strongly for fish fed the fishmeal‐based diets. Enhanced faecal bile acid loss was not caused by NSP‐bile acid binding/entrapment, but by an increase in faeces production. During satiation feeding, fat ADC negatively correlated with faecal bile acid loss. From this it is concluded that bile acid availability/synthesis can become limiting for fat digestion in rainbow trout under conditions that enhance faecal bile acid loss (i.e. dietary NSP level and feeding level).     

Glycoproteins isolated from scallop shells inhibit differentiation of 3T3-L1 preadipocyte cells

We previously demonstrated that the organic components isolated from scallop shells (scallop shell extract) inhibit the differentiation of 3T3-L1 preadipocyte cells. In this study, we show that a pronase-treated scallop shell extract inhibited differentiation, but degradation of sugar chains in the scallop shell extract with trifluoromethane sulfonic acid resulted in the loss of the differentiation-inhibiting activity, suggesting that the sugar chain modifications were responsible for the inhibitory activity. To identify the bioactive substance in the scallop shell extract, we isolated glycoproteins from the scallop shell extract via affinity chromatography using concanavalin A (Con A), wheat germ agglutinin, lens culinaris agglutinin (LCA), and ricinus communis agglutinin. LCA and Con A binding fractions inhibited the differentiation of 3T3-L1 preadipocyte cells. In addition, a glycoprotein with a molecular weight of 16 kDa that was purified from the LCA binding fraction reduced lipid accumulation in 3T3-L1 cells during differentiation. The glycoprotein inhibited differentiation-associated mitotic clonal expansion and suppressed the expression of an adipocyte-specific protein, fatty acid binding protein. These results suggest that the sugar chains of glycoproteins in the scallop shell extract inhibit the differentiation of 3T3-L1 preadipocyte cells.     

Effects of Yucca shidigera Extract on Growth, Nitrogen Retention, Ammonia Excretion, and Toxicity in Channel Catfish Ictalurus punctatus and Hybrid Tilapia Oreochromis mossambicus × O. niloticus

Nitrogenous waste control in aquaculture systems is necessary to maintain healthy animals and to meet effluent standards. Decreased ammonia production has been attained in traditional livestock production systems by adding extracts of Yucca shidigera to the feed or storage lagoon system. An extract of Yucca shidigera was fed to post yolk-sac and juvenile channel catfish Ictalurus punctatus and hybrid tilapia Oreochrornis mossambicus × O. niloticus to determine the effects on growth, nitrogen retention, ammonia excretion, and toxicity. Post yolk-sac channel catfish fry fed the 1.0-g yucca extract/kg as an agar coating were the only group of experimental organisms that had significantly greater weight gains compared to the control and 0.5-g yucca extract/kg diet groups after 12 wk. Significantly lower fecal nitrogen concentrations were obtained from catfish fed diets containing yucca extract compared to the control groups, although no differences between fecal nitrogen concentrations for catfish fed diets with either 0.5-g or 1.0-g yucca extract/kg diet occurred. Ammonia excretion rates were significantly lower for catfish fed diets containing yucca extract compared to the control group. Accordingly, fish fed diets containing the yucca extract had significantly greater whole-body protein composition, while lipid levels were significantly lower. No significant differences in proximate composition occurred for fish fed any of the diets containing yucca extract, though growth rates were significantly higher than the controls. This study demonstrated that a yucca extract can be used in the diet of channel catfish, and increased growth and decreased ammonia concentrations can be obtained in rearing systems.     

Effects of alcohol extract of defatted soybean meal on growth performance and digestive physiology of yellowtail <Emphasis Type="Italic">Seriola quinqueradiata</Emphasis>

The effects of alcohol extract of soybean meal on growth performance, digestibility, and pancreatic enzyme and bile acid status of yellowtail Seriola quinqueradiata were studied. Fish were fed one of the following for 8 weeks: a fish meal (FM) control diet (FMD), a defatted soybean meal diet (SBMD), an alcohol-extracted SBM diet (ExSBMD), an ExSBMD with alcohol-extract diet (ExSBM + ExtD), and an FMD with alcohol-extract diet (FM + ExtD). Growth performance was significantly inferior in fish fed with SBM, ExSBM + ExtD, and FM + ExtD compared to FMD- or ExSBMD-fed fish. Total bile acid levels, and trypsin and lipase activities in the anterior intestine were significantly lower in fish fed with SBM, ExSBM + ExtD, and FM + ExtD than in fish fed with ExSBMD and FMD, despite similar values in gallbladder or pyloric caeca. Cholecystokinin mRNA levels of the former diet-fed fish were significantly lower than those of FMD- or ExSBMD-fed fish. Lipid and protein digestibility of fish fed with SBMD, ExSBM + ExtD, and FM + ExtD was significantly reduced in comparison with that of fish fed with FMD or ExSBMD. These findings indicate that the alcohol extract of soybean meal inhibited the secretion of bile acids and pancreatic enzymes by a decrease of cholecystokinin stimulation. This inhibition seemed to be responsible for a low growth performance through impairment of the assimilation of lipid and protein.     

饲料中添加胆汁酸对军曹鱼生长及体组成的影响

在4组等氮等能的军曹鱼（Rachycentron canadum）实用饲料中分别添加不同梯度水平的胆汁酸（0,0.015%,0.030%和0.045%）,连续投喂初始体质量为（5.4±0.12）g的军曹鱼8周后,通过测定军曹鱼增重率（WGR,%）、特定生长率（SGR,%）和饲料系数（FCR）,并进行肝脏成分分析和血清分析,比较各组军曹鱼生长和体组成的影响。结果表明,添加水平为0.03%组的增重率高于对照组和0.015%添加组,饲料系数低于上述2组;军曹鱼血清胆固醇和肝脏脂肪的质量分数均与饲料中胆汁酸质量分数呈负相关;4组的成活率、特定生长率、全鱼和肌肉灰分、水分质量分数没有显著性差异。由此可见,饲料中添加胆汁酸能够促进军曹鱼的生长,并能降低脂肪在军曹鱼体内中的沉积。     

Organic components from scallop shell increase expression of keratinocyte growth factor in human skin fibroblast

We observed previously that scallop shells contain organic components that have various useful applications for skin. The organic components from scallop shell (scallop shell extract) enhance the turnover rate of the rat skin epidermal layer and increase the rate of recovery of UVB-induced injury to rat dorsal skin. We investigated the effect of scallop shell extract on the expression of keratinocyte growth factor (KGF) from human skin fibroblasts because it has been reported that KGF promotes wound healing and reduces the generation of UVB-induced free radicals in skin. Scallop shell extract increased the mRNA expression level of KGF in skin fibroblasts and the secretion of KGF from the fibroblasts at the protein level. These results suggest that the scallop shell extract may promote recovery from UVB-induced injury in skin through an increase in the secretion of KGF from the fibroblast cell.     

Effect of dietary inclusion of various sources of green tea on growth,body composition and blood chemistry of the juvenile olive flounder, <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

This study was performed to determine the effect of the dietary inclusion of various sources of green tea on growth, body composition and blood chemistry of the olive flounder. Twenty-five juvenile fish were distributed into each of 15 180 l flow-through tanks. Five experimental diets with triplicates were prepared: control, raw leaves, dry leaves, by-product and extract. The 5% various sources (raw leaves, dry leaves and by-product) of green tea were included in the experimental diets at the expense of 5% wheat flour. The extract was diluted with water and mixed with the ingredients to maintain 5% green tea at the same concentration as in the other diets. Weight gain and specific growth rate of fish fed the control diet and experimental diet containing extract were higher than those of fish fed the diets containing raw and dry leaves and by-product. Feed and protein efficiency ratio for fish fed the control diet and diet containing extract was higher than for fish fed the diets containing raw leaves and by-product. The level of low-density lipoprotein (LDL) cholesterol of fish fed the control diet was higher than that of fish fed the other diets containing various sources of green tea. The glutamic oxaloacetic transaminase (GPT) concentration of fish fed the diets containing raw leaves and extract was lower than that of fish fed the control diet. Crude lipid content of liver in fish fed the control diet was higher than that in fish fed the diets containing raw and dry leaves and by-product but was not significantly different from that of fish fed the diet containing extract. Dietary inclusion of extract was the most effective way to improve growth and feed utilization of the olive flounder among the various sources of green tea, and all sources of green tea were effective in lowering serum LDL cholesterol and GPT of the fish.     

Effects of dietary supplements on the availability of minerals in fish meal; preliminary observations

Preliminary studies were conducted to determine if several feed supplements with the potential to improve dietary mineral availabilities in fish meal had any measurable effect in fish feeds. In the first study with rainbow trout, 11 supplements were tested: citric acid; sodium citrate; potassium chloride; sodium chloride; histamine dihydrochloride; EDTA disodium salt; sodium bicarbonate; a mixture of amino acids; ascorbic acid; a mixture of inositol and choline; and cholecalciferol. Apparent availability of calcium, phosphorus, magnesium, sodium, iron, manganese and strontium in fish meal-based diets was determined using both yttrium oxide (Y₂O₃) and chromium oxide (Cr₂O₃) as inert dietary markers. Apparent availability was expressed as the fractional net absorption (%) of minerals from diets. After a 7-day acclimation period with test diets, fecal samples were collected for five consecutive days using passive collection systems. Apparent availability of calcium, phosphorus, magnesium, iron, manganese and strontium was increased by citric acid supplementation. Apparent availability of manganese also was increased by EDTA and sodium citrate. The other supplements had no measurable effect on the apparent availability of minerals in fish meal. In the second study, the effect of supplemental citric acid was further investigated using monogastric (rainbow trout) and agastric fish (goldfish). Fish were fed for 5 weeks (rainbow trout) or 3 weeks (goldfish) with fish meal-based diets containing either 0% (control), 2% or 5% citric acid on a dry basis. Feces were collected by settling and by stripping. Apparent availabilities of calcium and phosphorus were greatly affected by citric acid supplementation in rainbow trout but not in goldfish. Phosphorus levels in feces of fish fed a diet with 5% citric acid were approximately half of that of fish fed the control diet (0% citric acid) in the rainbow trout trial. This pattern was consistent during the 5-week feeding trial. A dietary supplement of citric acid as high as 5% did not reduce feed intake or appetite of rainbow trout. Conversely, this level of dietary acidification led to a marked reduction of feed intake in goldfish. Dietary supplementation of citric acid at 2% level did not reduce feed intake of goldfish; however, this level of dietary acidification had little effect on the apparent availability of major minerals in fish meal-based diet. Levels of non-fecal excretion of calcium and phosphorus, inorganic phosphorus in urine, and citric acid in feces were increased in rainbow trout fed 5% citric acid. The pH values of the feces and urine were decreased in rainbow trout fed citric acid. Plasma bicarbonate, plasma calcium and phosphorus, and blood pH of rainbow trout tended to increase by a 5% dietary supplementation of citric acid. The soluble inorganic phosphorus content increased in the diets and decreased in the feces of rainbow trout by supplementing the diet with 5% citric acid. Feces samples of rainbow trout collected by stripping provided similar availability values to data collected by settling for most elements except sodium, which had negative values in all dietary treatments.     

Nutrient Concentrations of Catfish Feces and Practical Diets After Immersion in Water

A study was conducted to quantify changes in the proximate components of fish feces and feeds when immersed in water for varying time periods. Catfish feces, a floating catfish diet, and a sinking trout diet were immersed for 5, 10, 20, 40, or 80 minutes. Dry matter losses were noted for all samples after immersion in water. There was a disproportionate loss of crude protein, ash, and nitrogen free extract (NFE) from the catfish fecal samples, ash and ether extract from the floating catfish feed, and NFE from the sinking trout feed. The losses noted would change crude protein, ether extract, NFE, and energy digestion coefficients by less than 7%, but ash and crude fiber digestion coefficients could change by as much as 23 and 35%, respectively. The ash concentration of unimmersed catfish feces indicated that this component may be a reliable indicator of nutrient digestibility in catfish.     

The effects of scallop shell extract on collagen synthesis

ABSTRACT:   Previous observations showed that scallop shells contain organic components that have various useful applications for skin. In this study, the effect of the organic components of scallop shell (scallop shell extract) on collagen metabolism is investigated. Collagen metabolism is tightly controlled by the collagen degrading matrix metalloproteinases (MMP) and their tissue inhibitors (TIMP). Treatment of human skin fibroblast cells with the scallop shell extract increased the mRNA expression levels of type I collagen, MMP-1 and TIMP-1, suggesting that the scallop shell extract may activate collagen metabolism in skin fibroblast cells. Sirius red staining and the colorimetric quantification of collagen in fibroblast cells demonstrated that the scallop shell extract increased collagen content by approximately 1.3-fold. In vivo studies also revealed that the topical application of the scallop shell extract to rat dorsal skin increased the collagen content in the skin tissue section. These results suggest that the scallop shell extract may be effective for the treatment of photoaged and aging skin, which undergo collagen loss.     

Scallop shell extract promotes recovery from UV-B-induced damage in rat skin epidermal layer

ABSTRACT:   It has been previously reported that scallop shell extract can protect keratinocyte cells against UV-B-induced damage in vitro by acting as an antioxidant and a growth promoter. To establish the growth promoting activity of scallop shell extract, the effect was investigated in vivo. Exposure of rat dorsal skin to UV-B caused the formation of erythema and eschar. Either the scallop shell extract or vehicle (water) was applied once a day for 5 days to the injured dorsal area and the area of the erythema and eschar was estimated using National Institute of Health (NIH) image software. Wound healing of the erythema and eschar was clearly promoted after application of scallop shell extract when compared to application of the vehicle control. Histological studies indicated that scallop shell extract promoted the recovery of the epidermal layer. These results suggest that scallop shell extract activates rat keratinocyte cells and promotes the turnover of skin stratum corneum. This conclusion was also supported by measurement of the turnover rate of the stratum corneum. Therefore, scallop shell extract may be effective in protecting skin against UV-B.     

Poor utilization of dietary free amino acids by white sturgeon

Three separate experiments were conducted to investigate the utilization of dietary free amino acids by white sturgeon,Acipenser transmontanus. In the first experiment, sturgeon were fed for 8 weeks with isoenergetic and isonitrogenous diets containing either intact protein, an amino acid mixture, or a neutralized amino acid mixture of similar composition. Sturgeon fed the two amino acid diets grew significantly (p<0.05) less, mainly because of reduced feed intake. In the second experiment, postprandial changes in plasma free amino acids of sturgeon force-fed the above diets were monitored at 2, 4, 6, 8, 12, and 24 h Total plasma amino acids in sturgeon force-fed the intact protein diet showed a gradual increase and reached peak levels at 8 h. In contrast, plasma amino acids of fish force-fed the amino acid diets showed a rapid increase and reached maximal levels at 2 h. In the third experiment, free amino acid diets showed a urine of sturgeon force-fed the three diets was monitored at 4, 8, 12, 16, 20, and 24 h. In all dietary groups, peak excretion of indispensable amino acids was detected at 8 h after feeding but with significantly higher levels from fish force-fed the amino acid diets. Sturgeon force-fed the neutralized amino acid diet excreted 35% less amino acids compared to fish force-fed the non-neutralized diet. Sturgeon force-fed the intact protein diet excreted significantly higher amounts of ammonia. Under amino acid feeding conditions, sturgeon excreted more amino acids in its urine, but the urinary excretion of amino acids was not a major route in dealing with the rapid absorption of dietary free amino acids.     

Amino Acid Availability of Four Practical Feed Ingredients Fed to Striped Bass Morone saxatilis

Four dietary protein sources were bio-assayed for amino acid availability, as estimated by true digestibility, when fed to striped bass Morone saxatilis . Diets were formulated to contain either herring fish meal, soybean meal, corn gluten meal or peanut meal as the sole source of dietary protein. A fifth diet, containing no protein, was fed to estimate the level of endogenous amino acids for calculation of true digestibility. The five dietary treatments were randomly assigned to ten tanks of striped bass having an average weight of 150 g per fish. All fish received the assigned diet fed at a rate 1.5% of the biomass per day for a period of 10 d. Fecal samples were collected from anesthetized fish by gentle, manual stripping of the lower digestive tract. Diets and feces were analyzed for dry matter, chromium, nitrogen and amino acid concentrations. There were no statistical differences (P > 0.05) among the protein sources for apparent dry matter digestibility or availability of arginine, threonine, valine and nonessential amino acids with the exception of cysteine. Corn gluten meal had a significantly lower availability coefficient for lysine, and peanut meal had significantly lower availability coefficients for histidine, isoleucine, leucine, and lysine when compared to herring fish meal and soybean meal. Statistically there were no differences between soybean meal and herring fish meal for any nutrient tested. These data suggest that in terms of amino acid availability and overall protein quality, soybean meal could be used to spare herring fish meal in striped bass diets, with corn gluten meal being equally as useful when supplemented with lysine or complemented with other proteins.     

Influence of dietary soy protein and peptide products on bile acid status and distal intestinal morphology of rainbow trout Oncorhynchus mykiss

To investigate the influence of dietary soy protein size on the bile acid status and the distal intestinal morphology in rainbow trout Oncorhynchus mykiss, diets based on soybean meal (SBM), soy protein isolate (SPI) and 4 soy peptide products having different molecular sizes were fed to trout (initial BW, 19?g) for 10?weeks. A diet based on fish meal (Cont) and a diet including the smallest-peptide and low-saponin product (Hi-Nute AM) and supplemented with soya saponin (AMS) were also fed. In fish fed diets SBM and SPI, the gallbladder was atrophied, the proportion of biliary cholyltaurine decreased, and highly vacuolated epithelial cells of mucosal folds and proliferation of connective tissues in the submucosa of the distal intestine were observed. These parameters tended to improve in fish fed soy peptide diets and especially those of fish fed diet AM were similar to fish fed diet Cont. Although the gallbladder of fish fed diet AMS was smaller than fish fed the unsupplemented diet AM, no morphological abnormalities were observed in the distal intestine. These results suggest that soy proteins/peptides with relatively large sizes and with soya saponin affect the bile acid status and distal intestinal morphology of rainbow trout.     

Effect of an alcohol extract from a defatted soybean meal supplemented with a casein-based semi-purified diet on the biliary bile status and intestinal conditions in rainbow trout Oncorhynchus mykiss (Walbaum)

A feeding experiment was conducted to examine the effect of a supplemental ethanol extract from a defatted soybean meal on the biliary bile status and intestinal conditions of rainbow trout Oncorhynchus mykiss . A semi-purified control diet based on casein (Cont) and three diets supplemented with the extract (ES), bovine bile salts (BS) and their combination (ESBS) were fed to trout (10 g) for 6 weeks. The growth, feed efficiency ratio, fat digestibility and gallbladder- and hepato-somatic indices of fish fed diet ES decreased. Compared with this group, these parameters were improved in fish fed diet ESBS. The total biliary bile salt content was the lowest in fish fed diet ES and this group had a high proportion of chenodeoxycholyltaurine. The inclusion of the bile salts to diet ES (diet ESBS) increased the total biliary bile salt content. A similar trend was observed in the bile salt concentration of intestinal digesta. Although morphological changes occurred in epithelial cells of the distal intestine of fish fed diet ES, the histological features of fish fed diet ESBS were similar to those of fish fed diet Cont. These results indicate that alcohol-soluble substances of soybean meal cause physiological changes in the biliary bile and intestine of rainbow trout.