2N Fish Research Articles

Enhanced Immune Response Improves Resistance to Cadmium Stress in Triploid Crucian Carp.

Previous research has indicated that triploid crucian carp (3n fish) have preferential resistance to cadmium (Cd) compared to Carassius auratas red var. (2n fish). In this article, comparative research is further conducted between the 2n and 3n fish in terms of the immune response to Cd-induced stress. Exposure to 9 mg/L Cd for 96 h changed the hepatic function indexes remarkably in the 2n fish, but not in the 3n fish. In the serum of Cd-treated 2n fish, the levels of alanine amino transferase, aspartate aminotransferase, adenosine deaminase, and total bilirubin significantly increased, while the levels of total protein, albumin, lysozyme, and anti-superoxide anion radicals decreased demonstrating hepatotoxicity. By analysis of transcriptome profiles, many immune-related pathways were found to be involved in the response of 3n fish to the Cd-induced stress. Expression levels of the immune genes, including the interleukin genes, tumor necrosis factor super family member genes, chemokine gene, toll-like receptor gene, and inflammatory marker cyclooxygenase 2 gene were significantly enhanced in the hepatopancreas of the Cd-treated 3n fish. In contrast, the expression levels of these genes decreased in the 2n fish. This research provides a theoretical basis for polyploid fish breeding and is helpful for the ecological restoration of water due to pollution.

Metabolic differentiation of diploid and triploid European sea bass juveniles

The effects of triploidy were studied on indices of growth and metabolism in juvenile European sea bass. Ploidy affected flesh quality of Dicentrachus labrax, as protein and water contents were significantly higher in triploid than in diploid fish and triploid fish exhibited significantly lower lipid content. Compared to 2n fish, triploid fish exhibited 53.4% and 28.6% more DNA and RNA, respectively, 17.2% higher RNA/DNA ratio and 28.7% more protein/DNA ratio. The activities of the aerobic metabolism enzyme CCO and the glycolytic LDH of the muscle tissue were significantly higher in the triploid fish. Nevertheless, the ratio of these two enzymes was lower in the triploids, indicating metabolic difference in the potential for aerobic metabolism. The increased activity of LDH may reflect a potential shift towards anaerobic metabolism required under demanding conditions, for example, during burst swimming, confirming the effects of ploidy on the aerobic swimming capacity of fish. The increased CCO activity of triploids observed in the present work indicates an effect of ploidy on the capacity for aerobic metabolism of triploid fish.

Effect of triploidy on digestive enzyme activity of early stages of turbot (Scophthalmus maximus).

In the present work, growth and digestive enzyme activities of total acid and alkaline proteases, pepsin, trypsin, lipase, and α-amylase, as well as partial characterization of enzyme activity, were studied in diploid and triploid turbot. Growth was similar between both groups. Acid protease activity increased consistently during the experiment, for both diploid (2n) and triploid (3n) fish. The alkaline protease activity was always higher for triploids throughout the experiment. Proteolytic acid activity (pH2) was generally higher for diploids, at all temperatures tested. Higher activity was at pH2 and 3 for 2n and 3n fish, respectively. Regarding temperature, acid and alkaline protease activity was higher at 37°C and 60°C, respectively, for both groups. The general increase in pancreatic enzymes (trypsin and amylase) before 35days after hatching (DAH) and posterior decrease until 60 DAH. There was a marked effect on enzyme activity when changing from live prey to pellets (35 DAH), especially on triploids.

Effects of steroid treatment on growth, nutrient partitioning, and expression of genes related to growth and nutrient metabolism in adult triploid rainbow trout (Oncorhynchus mykiss)

The contribution of sex steroids to nutrient partitioning and energy balance during gonad development was studied in rainbow trout. Specifically, 19-mo old triploid (3N) female rainbow trout were fed treatment diets supplemented with estradiol-17β (E2), testosterone (T), or dihydrotestosterone at 30-mg steroid/kg diet for a 1-mo period. Growth performance, nutrient partitioning, and expression of genes central to growth and nutrient metabolism were compared with 3N and age-matched diploid (2N) female fish consuming a control diet not supplemented with steroids. Only 2 N fish exhibited active gonad development, with gonad weights increasing from 3.7% to 5.5% of body weight throughout the study, whereas gonad weights in 3N fish remained at 0.03%. Triploid fish consuming dihydrotestosterone exhibited faster specific growth rates than 3N-controls (P < 0.05). Consumption of E2 in 3N fish reduced fillet growth and caused lower fillet yield compared with all other treatment groups (P < 0.05). In contrast, viscera fat gain was not affected by steroid consumption (P > 0.05). Gene transcripts associated with physiological pathways were identified in maturing 2N and E2-treated 3N fish that differed in abundance from 3N-control fish (P < 0.05). In liver these mechanisms included the growth hormone/insulin-like growth factor (IGF) axis (igf1, igf2), IGF binding proteins (igfbp1b1, igfbp2b1, igfbp5b1, igfbp6b1), and genes associated with lipid binding and transport (fabp3, fabp4, lpl, cd36), fatty acid oxidation (cpt1a), and the pparg transcription factor. In muscle, these mechanisms included reductions in myogenic gene expression (fst, myog) and the proteolysis-related gene, cathepsin-L, suggesting an E2-induced reduction in the capacity for muscle growth. These findings suggest that increased E2 signaling in the sexually maturing female rainbow trout alters physiological pathways in liver, particularly those related to IGF signaling and lipid metabolism, to partition nutrients away from muscle growth toward support of maturation-related processes. In contrast, the mobilization of viscera lipid stores appear to be mediated less by E2 and more by energy demands associated with gonad development. These findings improve the understanding of how steroids regulate nutrient metabolism to meet the high energy demands associated with gonad development during sexual maturation.

Expression of genes associated with fatty acid metabolism during maturation in diploid and triploid female rainbow trout

Abstract To study the effects of sexual maturation on fatty acid metabolism in fish fed to satiation daily, expression of thirty-five genes involved in fatty acid metabolism was determined in sexually maturing diploid (2N; fertile) and triploid (3N; sterile) female rainbow trout. Gene expression was assessed in liver, white muscle, and visceral adipose tissues for fish that were 16 to 24 M of age. Previously, we reported minimal differences in most growth measurements between maturing 2N and 3N fish, but there were disparate changes in muscle proximate composition, visceral fat stores, and fatty acid contents of energy stores at 21 M with 3N females having greater lipid stores. Here, we report that gene expression profiles of liver and white muscle corresponded to the phenotypes with significant differences in expression at 20 M. Triploid females had increased expression of genes involved in fatty acid synthesis; including gpat , srebp1 , acyl , acc , fas , and scd1 in liver and fas in muscle. Conversely, 2N muscle had increased expression of β-oxidation genes cpt1b , cpt2 , ehhadh , and acat2 and TORC1 inhibitors redd1 , erk , mo25 , and pras40 . Diploid muscle also had increased expression of pparβ along with increased expression of the fatty acid transporter gene cd36 , and β-oxidation genes cpt1a , cpt1c , aco , and acdhvl at 20 M. Additionally, 2N visceral adipose tissue had increased cpt1a expression at 21 M. Overall, data suggest that 3N females are undergoing higher levels of fatty acid synthesis while 2N females have higher levels of β-oxidation during sexual maturation. Phenotypic data supports these findings with decreasing fatty acid stores in 2N females during this time period. Additionally, changes in gene expression are associated with altered expression within the mTOR and PPARβ signaling pathways.

Effects of sexual maturation and feeding level on fatty acid metabolism gene expression in muscle, liver, and visceral adipose tissue of diploid and triploid rainbow trout, Oncorhynchus mykiss.

In many cultured fish species, such as salmonids, gonadal development occurs at the expense of stored energy and nutrients, including lipids. However, mechanisms regulating nutrient repartitioning during sexual maturation are not well understood. This study compared sexually maturing diploid (2N) and sterile triploid (3N) female rainbow trout to investigate effects of sexual maturation on expression of 35 genes involved in fatty acid metabolism, including genes within fatty acid synthesis, β-oxidation, and cofactors of the mTOR and PPAR signaling pathways, in liver, white muscle, and visceral adipose tissue. Diploid fish were fed at different rations (0.25% and 0.50% tank biomass, and satiation) to determine effects of ration on gene expression. Gene expression was affected by ration level only in white muscle; erk and acat2 had higher expression in fish fed higher rations. On the other hand, sexual maturation affected gene expression across all three tissue types. Data indicate 2N fish have higher expression of β-oxidation genes within white muscle and within visceral adipose tissue. These findings support enhanced fatty acid mobilization within these tissues during sexual maturation. Higher expression of fatty acid synthesis genes in 3N female liver is associated with higher expression of mTOR cofactors and pparγ, which reflects continued deposition of lipids in these fish. Furthermore, greater expression of genes involved in β-oxidation pathways across ration levels in 2N females suggests that sexual maturation and the associated maturation-related signals are stronger regulators of lipid metabolism-related genes rather the rations applied in the current study.

Effects of feeding level and sexual maturation on fatty acid composition of energy stores in diploid and triploid rainbow trout (Oncorhynchus mykiss)

Sexual maturation is an energy demanding, physiological process that alters growth efficiency and compromises muscle quality in many food-fish species. Lipid mobilization supplies energy required for this process. To study the effect of ration level on fatty acid composition, diploid (2N) rainbow trout, approaching ovulation, were fed at 0.25 and 0.50% of tank biomass/day and to apparent satiation (~0.75% of tank biomass/day). In addition, triploid (3N) female trout, which exhibit only minimal ovarian development, were fed at 0.50% of tank biomass/day. The primary objective of this study was to determine effects of ration level on fatty acid composition in different lipid compartments (muscle, visceral adipose tissue, liver, and gonad) during sexual maturation. Lower feeding levels produced smaller fish, but did not affect the onset of sexual maturation. Higher feeding levels resulted in fish muscle with higher relative amounts of saturated fatty acids (SFAs), but monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) were not affected by ration level. While ration level affected the fatty acid profile of each of the four tissues analyzed, the number of fatty acids affected was greatest in white muscle. An additional objective was to determine differences in the fatty acid composition of energy stores during maturation in female rainbow trout that were fed at a moderately restricted feeding level (0.50% of tank biomass/day). These differences were determined by comparing mature 2N to sterile 3N females of the same age. Diploid muscle contained higher amounts of PUFAs (44.4±1.0%) than 3N muscle (39.7±0.8%). Saturated fatty acids were in the highest concentrations in muscle and visceral adipose tissue, and 2N liver contained more PUFAs and fewer MUFAs than 3N liver; however these values are relative values. In general, fatty acids affected by ration level were not the same as fatty acids affected by ploidy. Triploid fatty acid profiles did not mimic those of the satiation fed group; which was expected if 3N fish were simply consuming excess energy. Both 2N and 3N muscle fatty acid profiles were similar to that of the diet, except muscle had lower amounts of PUFA precursors (18:3n-3 and 20:5n-3) and higher relative amounts of their product (22:6n-3). Also, 2N muscle had higher 16:1 and 3N muscle had higher 16:0 compared to the diet. It is unclear if these differences are hormonally driven or if there are other physiological dissimilarities between 2N and 3N trout causing these differences. Overall, our data suggest that 2N and 3N fatty acid metabolism is regulated differently.

Comparison of disease resistance between diploid, induced-triploid, and intercross-triploid rainbow trout including trout selected for resistance to Flavobacterium psychrophilum

All-female triploid fish are advantageous in rainbow trout (Oncorhynchus mykiss) aquaculture due to sterility and the consequent superior filet quality, growth, and feed conversion achieved at reproductive size. Triploid fish are commonly produced by pressure or temperature shock of the zygote (induced-triploids, 3NP), but can also be produced by mating a tetraploid parent with a diploid (2N) parent (intercross-triploids, 3NC). Little is known about the disease resistance of 3NC fish. In this study, we measured post-challenge survival of genetically-related 2N, 3NP and 3NC families after exposure to Flavobacterium psychrophilum the etiological agent of bacterial cold water disease (BCWD). The families used in this study originated from either an unselected commercial stock or BCWD resistant or susceptible line of rainbow trout. The overall survival of 2N fish was slightly higher than the respective 3NP and 3NC fish. Although 3NC fish tended to show higher survival than 3NP fish, this response was not sufficiently consistent to promote intercross production of triploids for increasing disease resistance. The survival of ten sets of 3NP, 3NC and 2N families, created from BCWD resistant and susceptible line parents, demonstrated overall similar inherent relative survival differences among the ploidy types both among families and between lines, although family differences in response to triploidization and triploidization strategy were observed. In summary, minimal survival difference between 3NP and 3NC families following experimental challenge suggests that innate resistance to F. psychrophilum is not substantially altered by the triploidization strategy and progress in family-based selective breeding of diploid fish for BCWD resistance will substantially translate into improved survival when triploids are derived either by shock treatment or intercross breeding.

Evaluation of long‐chain n3 fatty acid content in diploid and triploid rainbow trout

Intake of long chain n3 fatty acids (LCn3), eicosapentaenoic acid (EPA; 20:5 n3) and docosahexaenoic acid (DHA; 22:6 n3), is associated with reduced cardiovascular disease. There is growing interest in farmed fish like rainbow trout, Oncorhynchus mykiss, as sources of LCn3. The trout industry raises diploid (2N) and triploid (3N) fish. 3N females fail to sexually mature and avoid the negative effects of maturation on fillet quality. In this work we examined the LCn3 content of 2N and 3N trout. Female rainbow trout were selected from independent populations of 1 yr and 2 yr old 2N and 2 yr old 3N fish. Although each group consumed the same diet formulation, rearing conditions and feeding rates were different and only the 2 yr 2N trout were sexually mature. Boneless, skinless fillets were removed from 4 fish per group and the total fatty acid content of the fillets was determined by FAME analysis. The contents of LCn3 in the 3N 2yr trout (EPA: 1.9 ± 0.5 mg/g; DHA 7.6 ± 1.7 mg/g) were almost 2‐fold higher than in the 2N 2yr trout (EPA:0.8 ± 0. mg/g; DHA 3.9 ± 0.5 mg/g) and the 2N 1yr trout (EPA:1.0 ± 0.3 mg/g; DHA 4.0 ± 0.7 mg/g). The higher content of LCn3 was in part the result of total higher content of fatty acid in the 3N trout. The percent content of LCn3 and ratios of short chain to long chain n3 were similar in all groups. These data indicate that farm‐raised rainbow trout are a rich source of EPA and DHA. This work was supported by the USDA‐ARS.

A heart to heart on temperature: Impaired temperature tolerance of triploid rainbow trout (Oncorhynchus mykiss) due to early onset of cardiac arrhythmia

Triploid (3N) salmonids are of interest to aquaculture and sport fishing industries, however it has been shown that 3N fish have impaired tolerance to high temperatures. To test the hypothesis that poor high temperature tolerance in 3N salmonids is related to impaired O2 delivery to the body, maximum heart rate (fH) was measured in 2N (diploid) and 3N rainbow trout (Oncorhynchus mykiss) during an incremental temperature challenge. fH of both ploidies was similar at 10°C. However, a significant effect of ploidy on the response of fH to temperature from 10 to 22°C was reflected in a lower Q10 for 3N individuals. Additionally, all 3N trout developed a cardiac arrhythmia by 22°C, where 30% of 2N trout continued to maintain a rhythmic heartbeat. These findings suggest that reduced 3N high temperature tolerance could be due to early collapse of the cardiovascular system's ability to deliver O2 to the body during warming.

Effect of sexual maturation and triploidy on chemical composition and fatty acid content of energy stores in female rainbow trout, Oncorhynchus mykiss

Sexual maturation is an energy and nutrient demanding physiological process that alters growth efficiency and compromises muscle quality in many food fish species. Lipid mobilization supplies energy required for this process. To study the effect of sexual maturation on lipid mobilization in female rainbow trout on a high nutritional plane, fish growth, egg development, proximate composition, and fatty acid composition of muscle, liver, visceral fat, and gonads were determined for diploid (2N; fertile) and triploid (3N; sterile) females from July 2008 thru March 2009, when most 2N females ovulated. Previously, we reported there were minimal differences in most growth measurements between the 2N and 3N fish in this study, but there were changes in muscle proximate composition and visceral fat stores. Here, we report changes in proximate composition of liver, gonads, visceral adipose tissue, and fatty acid composition of these tissues and muscle during sexual maturation of 2N females with 3N females serving as the control. Similar and distinct patterns of change in fatty acids were observed between tissues of 2N and 3N fish, and among different tissues as the 2N fish were maturing. Highly unsaturated, long chain fatty acids appear to play an important role in gonadogenesis, either as a source of energy or for deposition in eggs to support offspring development and survival. Differences in growth and preferential mobilization of fatty acids are observed when fish are on a high nutritional plane compared to more typical, lower nutritional regimens.

Performance of Diploid and Triploid Rainbow Trout Stocked in Idaho Alpine Lakes

AbstractIncreased growth, improved survival, and genetic protection of wild stocks have been suggested as benefits of stocking triploid (i.e., sterile) salmonids for recreational fisheries. We examined the return rates and growth of mixed‐sex diploid (2N), mixed‐sex triploid (3N), and all‐female triploid (AF3N) rainbow trout Oncorhynchus mykiss across 28 alpine lakes. Equal numbers of each treatment group were stocked in 2001 and 2003 and sampled 3–4 years later. During 2004 and 2005, a total of 75 2N and 36 3N marked rainbow trout were recaptured. Taken together, the 2N fish accounted for an average of 0.68 of the total marked fish caught, and the combined proportions of test fish (including netting and angling) differed significantly between the test groups and were consistent across survey years. During 2006 and 2007, a total of 60 2N, 31 3N, and 208 AF3N marked rainbow trout were recaptured. The mean length of the test fish was similar between test groups within sampling years. Overall, the return of 3N rainbow trout to alpine lakes in Idaho was low compared with that of 2N trout, whereas AF3N trout appeared to return in higher proportions than both of the other groups. The triploid stocks studied in this evaluation did not show any growth advantages over the duration of the study. Study design limitations may have contributed in part to some of the differences in the number of 2N and 3N rainbow trout captured. However, our results suggest that fisheries managers should consider all‐female triploid rainbow trout as a low‐risk option for maintaining alpine lake fisheries while minimizing the impact on native stocks.Received January 27, 2010; accepted November 16, 2010

Establishing a base population for a breeding program in aquaculture, from multiple subpopulations, differentiated by genetic drift: I. Effects of the number of subpopulations, heritability and mating strategies using optimum contribution selection

Guidelines for constructing base populations in fish were examined using a drift model. A large number of drift differentiated, wild subpopulations, with inbreeding Fwild (Fwild∈[0.05–0.20]), and a trait with heritability htot2 (htot2∈[0.10–0.30]), were assumed. A base population was generated by sampling 2N fish from each of nsub subpopulations (nsub∈[1–8]), and then mated to produce 160 or 240 families per generation. The matings were either at random, in a structured diallel-cross, or within subpopulations to produce generation 1. Optimal contribution selection on phenotype was applied from generation 1 to11, with a rate of inbreeding of 0.5% per generation. Genetic gain (Δg) improved with nsub, the improvement being largest when going from one to two subpopulations, with diminishing returns. Most of the potential improvement was obtained with nsub=4, and was well predicted by ΔgnsubΔg1≈(1−Fwild/nsub)(1−Fwild). Initial matings across subpopulations improved Δg from the second round of selection compared to within. Consequently the effect of the mating strategy were first observed 3 generation after decisions were made and persisted over subsequent generations, but with minimal effect on the accumulated additive genetic gain in generation 11. It was concluded that sampling from at least four subpopulations was beneficial and that mating across subpopulations should start as early as possible.

Growth enhancement following dietary delivery of recombinant porcine somatotropin to diploid and triploid coho salmon (Oncorhynchus kisutch)

Diploid (2n) and triploid (3n) coho salmon were fed upon control (diet 1), recombinant porcine somatotropin (rpST)-supplemented (diet 2; 20 μg rpST/g body wt/day) or rpST/antacid/detergent-containing feed (diet 3; 20, 100 & 20 μg/g body wt/day respectively) for 16 wk. Fish were weighed and measured bimonthly and their relative performances assessed. By wk 4, 2n and 3n groups fed upon diet 2 were significantly heavier and longer than control 2n fish. At the termination of the trial, diet 3 fed animals were greater in weight than all other treatment groups. Diet 3 salmon also returned better feed conversion efficiencies than either diet 1 or 2 groups. RpST therapy induced a 28.7% and 60.2% increase in group wt for 3n diet 2/3 coho versus (-vs-) controls. Likewise, 2n rpST-treated fish increased wt -vs-2n control coho by 17% and 50% respectively over the same period. No differences were recorded between groups for body moisture, but diet 2, 3n and both diet 3 groups exhibited decreased condition factors when compared to control fish (p ≤ 0.05).

Characteristics and antagonistic activity of lactic acid bacteria isolated from chilled fish products

3n fish products, contrary to other foods, the commercial use of starter cultures is relatively unexploited. In this study, 61 isolates of lactic bacteria from lightly preserved, chilled fish products were characterized with special emphasis on their antagonistic activity and their possible use as starter cultures. The strains were phenotypically identified and characterized with regard to gas production, carbohydrate fermentations, production of off odours, H 2O 2-production, antagonistic activity and growth at 2,5 and 10°C. Growth at 10°C was detected for all strains within 4 days. At 5°C, 90% of the isolates were growing within 3–9 days. At 2°C only 33% of the strains were growing within 9 days. Fourteen strains were selected for further testing of antagonistic activity against spoilage- and pathogenic bacteria at different pH in a disc assay. Two strains, a Leuconostoc spp. (V 6) and a Lactobacillus plantarum (LKE 5), differed from the other lactic acid bacteria by a very wide inhibitory spectrum for V 6 and by very distinct inhibition zones for LKE 5. The mechanisms of their inhibitory effects are discussed.

Promotion of growth in diploid and triploid coho salmon with parenteral delivery of a recombinant porcine somatotropin

The growth response of diploid (2n ) and thermally-induced triploid (3n ) coho salmon to injected recombinant porcine somatotropin was examined over a 10 week period. All treatments were under-taken using replicate groups of 25 animals per group. Control 2n and 3n replicate groups grew at identical rates throughout the period of study, although 3n fish exhibited lower condition factors. Recombinant poreine somatotropin treated 2n and 3n fish responded positively to therapy and, by week 4 of the trial, expressed significantly greater increases in both weight and length (p < 0.01) when compared to controls. At the end of the study period, soinatotropin treated animals had gained approximately twice the amount of weight of controls. Food conversion efficiencies were greater in treated salmon, regardless of ploidy state. This relationship also extended to weight and length specific growth rates. The economic benefits of commercial application of recombinant porcine somatotropin therapy to triploid Pacific salmon culture are discussed.