Why Do Some Rainbow Trout Genotypes Grow Better With a Complete Plant-Based Diet? Transcriptomic and Physiological Analyses on Three Isogenic Lines.

Within the context of a growing aquaculture production coupled with a plateau of the production in the main components of aquafeeds (fish oil and fishmeal), recent studies have typically focused on replacing these feedstuffs with terrestrial plant ingredients for cultured carnivorous aquatic species, such as rainbow trout (Oncorhynchus mykiss). Substitution rates without adverse effects have, however, reached their limit. One potential way forward would be to take advantage of the genetic variability that exists in the salmonid population. However, to date, little is known about the underlying molecular mechanisms responsible for this genetic variability. The aim of the present research was to understand why some genotypes are better able to utilize plant-based diets devoid of marine resources. In this regard, three isogenic lines of rainbow trout (R23h, AB1h, and A22h), with similar growth when fed marine resources-based diets and which differ greatly in their responses to a plant-based diet, were fed with either a complete plant-based diet (V diet) or a marine resources-based diet (M diet) since first-feeding. Fish traits and the hepatic transcriptome of these three genotypes were compared after 5 months of feeding. First, differences in the ability to grow with the V diet observed amongst genotypes was not due to higher feed intake, but instead due to differences in feed efficiency. The comparison of transcriptome profiles revealed 575 (R23h vs. AB1h), 1,770 (R23h vs. A22h), and 2,973 (AB1h vs. A22h) probes differentially expressed amongst the three genotypes when fed the V diet. Interestingly, R23h and AB1h fish, which were the least affected by the V diet, exhibited the highest growth. These results demonstrate that these fish were able to maintain a high level of energy production and protein synthesis. Moreover, these genotypes were also able to activate pathways linked to lipid and cholesterol metabolisms, such as the biosynthesis of long-chain polyunsaturated fatty acids. Finally, as previously, immunity seems to also play an important role in the ability of fish to use the V diet, and further studies are needed to understand the mechanisms by which immunity interacts with growth.

Variable impacts of L-arginine or L-NAME during early life on molecular and cellular markers of muscle growth mechanisms in rainbow trout.

Two experiments were conducted to test if manipulations of the Arginine-Nitric oxide pathway during the early life of rainbow trout would act on its early myogenic process. In experiment 1, trout embryos were immersed at 72° days post-fertilization (°dpf) or 104°dpf in water alone (control treatment, C) or containing 2 mM/L L-Arg (treatment A) or 1 mM/L of L-NAME, a NOS inhibitor (treatment N). We observed the beginning of expression of myf5 and fmhc genes at 72°dpf and 96°dpf, respectively. "A" treatment doubled the free Arg content of eggs but did not affect either the pattern of expression of myf5 and fmhc, nor white muscle cross-sectional area and number of white muscle fibres at hatching, nor embryo survival and fry growth. "N" treatment also did not affect these markers. In experiment 2, trout fry were fed from first feeding onwards and during 20 days either a control diet (C) or the same diet supplemented with L-NAME (0.1 g/100 g diet, N-diet). In C-fed fry, distribution of a single meal after overnight fasting induced changes in pcna, myod1, myog, fmhc, inos, nnos and ctsd gene expressions. N-feeding decreased fry growth but did not change their growth trajectory or survival. Twenty days of N-feeding led, compared to C-feeding, to changes in kinetics of transcription of pcna, myod1, myog, fmhc, inos, nnos, ctsd genes and to decreased white muscle cross-sectional area, total number of white muscle fibres, and number of large muscle fibres. L-NAME feeding thus decreased fry muscle growth by altering both hyperplasia and hypertrophy.

Muscle growth mechanisms in response to isoenergetic changes in dietary non-protein energy source at low and high protein levels in juvenile rainbow trout.

This study investigates muscle growth mechanisms in juvenile rainbow trout in response to isoenergetic changes in dietary non-protein energy (NPE) source (F, fat vs. C, carbohydrates) at two levels of digestible protein to digestible energy (DP/DE) ratio. Fish (initial weight 32.4 g) were fed four diets having similar DE levels (~18 kJ g-1) with a high (HP/E~26 mg kJ-1) vs. low (LP/E~14 mg kJ-1) DP/DE ratio using F or C as major NPE source (7 week-experiment). The lowering of dietary DP/DE ratio increased myoblast determination protein 1a (myod1a) and decreased myostatin 1b (mstn1b) and cathepsin D (ctsd) muscle mRNA levels. The isoenergetic change in dietary NPE from F to C decreased myod1a and proliferative cell nuclear antigen (pcna) muscle mRNA levels. An interaction between DP/DE ratio and NPE source was observed in muscle transcript levels of myogenic factor 6 (mrf4/myf6), fast myosin heavy chain (fmhc) and fast myosin light chain 2 (fmlc2). White muscle total cross-sectional area decreased at low dietary DP/DE ratio and also when NPE source changed from F to C, linked i) to a decreased total number of white muscle fibres, indicating that low dietary DP/DE restricted muscle hyperplasia and that dietary carbohydrate were less efficiently used than fat to sustain muscle hyperplasia, and ii) to decreased percentage of large muscle fibres, indicating limited fibre hypertrophy. Not only the DP level or the DP/DE ratio, but also the isoenergetic change in dietary NPE source (fat vs carbohydrates) thus appears as a potent regulator of muscle hyperplasia and hypertrophy.

Detection of new pathways involved in the acceptance and the utilisation of a plant-based diet in isogenic lines of rainbow trout fry.

To meet the growing demand of fish feed for aquaculture, an increasing proportion of marine ingredients are being replaced by blends of plant products. However, the total replacement of marine ingredients in salmonid diets impairs fish performance. This is particularly true during the early fry stage and this stage is therefore considered of particular importance. In rainbow trout (RBT), the existence of a genetic variability to survive and grow with plant-based diets devoid of marine ingredients has now been proved, but the mechanisms behind are little studied especially at early stage. To investigate these, we analysed the whole transcriptome of three isogenic lines of RBT fry, which have similar growth when fed a marine resources-based diet (M diet) but which highly differ in their responses to a plant-based diet (V diet). Analysis of transcriptomes profiles revealed 1740, 1834 and 246 probes differentially expressed among the three genotypes when fed the V diet. The use of these lines led to the discovery of potential molecular markers linked to plant-based diet utilisation, some of them belonging to new pathways, never described before. An important number of genes was related to immunity, but further investigations are needed to better understand the difference between the genotypes in their immune status response to V diet exposure. Finally, differences in expression of genes related to feed intake and sensory perception among genotypes suggested that the mechanisms underlying the differences in growth on plant-based diet are closely linked to diet acceptance. Research on plants components affecting feed intake should be thus further explored.

Effects of dietary oxidized fish oil supplementation on oxidative stress and antioxidant defense system in juvenile rainbow trout (Oncorhynchus mykiss).

The objective of the study was to characterize the response of the antioxidant defense system against dietary prooxidant conditions in rainbow trout juveniles. Fish (initial mean weight: 62 ±â€¯1 g) were fed three fishmeal and plant-derived protein-based diets supplemented with 15% fresh fish oil (CTL diet), 15% fresh fish oil from tuna by-products (BYP diet) or 15% autooxidized fish oil (OX diet) over a 12-week growth trial at 17.5 ±â€¯0.5 °C. No significant differences in growth performance were recorded between dietary groups. Muscle lipid content was reduced and n-6 PUFA levels were increased in rainbow trout fed diets BYP and OX compared to CTL. After 12 weeks of feeding, the level of lipid peroxidation products in muscle was not affected whereas the 8-isoprostane content in liver was increased in fish fed diet OX as well as plasma total and oxidized glutathione contents. The hepatic and muscle contents for α-tocopherol were decreased in fish fed BYP and OX. Hepatic antioxidant enzyme activities and mRNA levels were not affected after 12 weeks of feeding, except for catalase and glutathione peroxidase 1b2 mRNA levels that were decreased in trout fed diet OX. Fish fed diet OX and BYP displayed also reduced cytosolic Nrf2 and both cytosolic and nuclear NF-κB protein levels in liver. The present work indicates that feeding rainbow trout juveniles with fresh fish oil from by-products or moderately oxidized lipid appears not to be detrimental to the growth performance of fish. The mechanisms beyond the control of the antioxidant defense system by moderately oxidized lipid require further investigations in rainbow trout juveniles.

Postprandial kinetics of gene expression of proteins involved in the digestive process in rainbow trout (O. mykiss) and impact of diet composition.

The impact of increased incorporation of plant ingredients on diets for rainbow trout was evaluated in terms of gene expression of gastric (gastric lipase, pepsinogen) and intestinal (prolidase, maltase, phospholipase A2) digestive enzymes and nutrient transporters (peptide and glucose transporters), as well as of postprandial levels of plasma glucose, triglycerides and total free amino acids. For that purpose, trout alevins were fed from the start of exogenous feeding one of three different experimental diets: a diet rich in fish meal and fish oil (FM-FO), a plant-based diet (noFM-noFO) totally free from fish meal and fish oil, but containing plant ingredients and a Mixed diet (Mixed) intermediate between the FM-FO and noFM-noFO diets. After 16 months of rearing, all fish were left unfed for 72 h and then given a single meal to satiation. Blood, stomach and anterior intestine were sampled before the meal and at 2, 6 and 12 h after this meal. The postprandial kinetics of gene expression of gastric and intestinal digestive enzymes and nutrient transporters were then followed in trout fed the FM-FO diet. The postprandial profiles showed that the expression of almost all genes studied was stimulated by the presence of nutrients in the digestive tract of trout, but the timing (appearance of peaks) varied between genes. Based on these data, we have focused on the molecular response to dietary factors in the stomach and the intestine at 6 and 12 h after feeding, respectively. The reduction in FM and FO levels of dietary incorporation induced a significant decrease in the gene expression of gastric lipase, GLUT2 and PEPT1. The plasma glucose and triglycerides levels were also reduced in trout fed the noFM-noFO diet. Consequently, the present study suggests a decrease in digestive capacities in trout fed a diet rich in plant ingredients.

Looking at the metabolic consequences of the colchicine-based in vivo autophagic flux assay.

Monitoring autophagic flux in vivo or in organs remains limited and the ideal methods relative to the techniques possible with cell culture may not exist. Recently, a few papers have demonstrated the feasibility of measuring autophagic flux in vivo by intraperitoneal (IP) injection of pharmacological agents (chloroquine, leupeptin, vinblastine, and colchicine). However, the metabolic consequences of the administration of these drugs remain largely unknown. Here, we report that 0.8 mg/kg/day IP colchicine increased LC3-II protein levels in the liver of fasted trout, supporting the usefulness of this drug for studying autophagic flux in vivo in our model organism. This effect was accompanied by a decrease of plasma glucose concentration associated with a fall in the mRNA levels of gluconeogenesis-related genes. Concurrently, triglycerides and lipid droplets content in the liver increased. In contrast, transcript levels of ß-oxidation-related gene Cpt1a dropped significantly. Together, these results match with the reported role of autophagy in the regulation of glucose homeostasis and intracellular lipid stores, and highlight the importance of considering these effects when using colchicine as an in vivo "autophagometer."

Dietary carbohydrate and lipid source affect cholesterol metabolism of European sea bass (Dicentrarchus labrax) juveniles.

Plant feedstuffs (PF) are rich in carbohydrates, which may interact with lipid metabolism. Thus, when considering dietary replacement of fishery by-products with PF, knowledge is needed on how dietary lipid source (LS) and carbohydrates affect lipid metabolism and other metabolic pathways. For that purpose, a 73-d growth trial was performed with European sea bass juveniles (IBW 74 g) fed four diets differing in LS (fish oil (FO) or a blend of vegetable oils (VO)) and carbohydrate content (0 % (CH-) or 20 % (CH+) gelatinised starch). At the end of the trial no differences among diets were observed on growth and feed utilisation. Protein efficiency ratio was, however, higher in the CH+ groups. Muscle and liver fatty acid profiles reflected the dietary LS. Dietary carbohydrate promoted higher plasma cholesterol and phospholipids (PL), whole-body and hepatic (mainly 16 : 0) lipids and increased muscular and hepatic glycogen. Except for PL, which were higher in the FO groups, no major alterations between FO and VO groups were observed on plasma metabolites (glucose, TAG, cholesterol, PL), liver and muscle glycogen, and lipid and cholesterol contents. Activities of glucose-6-phosphate dehydrogenase and malic enzyme - lipogenesis-related enzymes - increased with carbohydrate intake. Hepatic expression of genes involved in cholesterol metabolism was up-regulated with carbohydrate (HMGCR and CYP3A27) and VO (HMGCR and CYP51A1) intake. No dietary regulation of long-chain PUFA biosynthesis at the transcriptional level was observed. Overall, very few interactions between dietary carbohydrates and LS were observed. However, important insights on the direct relation between dietary carbohydrate and the cholesterol biosynthetic pathway in European sea bass were demonstrated.

Early decrease in dietary protein:energy ratio by fat addition and ontogenetic changes in muscle growth mechanisms of rainbow trout: short- and long-term effects.

As the understanding of the nutritional regulation of muscle growth mechanisms in fish is fragmentary, the present study aimed to (1) characterise ontogenetic changes in muscle growth-related genes in parallel to changes in muscle cellularity; (2) determine whether an early decrease in dietary protein:energy ratio by fat addition affects the muscle growth mechanisms of rainbow trout (Oncorhynchus mykiss) alevins; and (3) determine whether this early feeding of a high-fat (HF) diet to alevins had a long-term effect on muscle growth processes in juveniles fed a commercial diet. Developmental regulation of hyperplasia and hypertrophy was evidenced at the molecular (expression of myogenic regulatory factors, proliferating cell nuclear antigen and myosin heavy chains (MHC)) and cellular (number and diameter of white muscle fibres) levels. An early decrease in dietary protein:energy ratio by fat addition stimulated the body growth of alevins but led to a fatty phenotype, with accumulation of lipids in the anterior part, and less caudal muscle when compared at similar body weights, due to a decrease in both the white muscle hyperplasia and maximum hypertrophy of white muscle fibres. These HF diet-induced cellular changes were preceded by a very rapid down-regulation of the expression of fast-MHC. The present study also demonstrated that early dietary composition had a long-term effect on the subsequent muscle growth processes of juveniles fed a commercial diet for 3 months. When compared at similar body weights, initially HF diet-fed juveniles indeed had a lower mean diameter of white muscle fibres, a smaller number of large white muscle fibres, and lower expression levels of MyoD1 and myogenin. These findings demonstrated the strong effect of early feed composition on the muscle growth mechanisms of trout alevins and juveniles.

Dietary cholecalciferol regulates the recruitment and growth of skeletal muscle fibers and the expressions of myogenic regulatory factors and the myosin heavy chain in European sea bass larvae.

The aim of this study was to determine whether dietary cholecalciferol affects the recruitment and growth of axial skeletal muscle fibers in first-feeding European sea bass. Larvae were fed diets containing 0.28 (VD-L, low dose), 0.69 (VD-C, control dose), or 3.00 (VD-H, high dose) mg cholecalciferol/kg from 9 to 44 d posthatching (dph). Larvae were sampled at 44 dph for quantification of somatic growth, muscle growth, and muscle growth dynamics and at 22 and 44 dph for the relative quantification of transcripts encoded by genes involved in myogenesis, cell proliferation, and muscle structure. The weight increase of the VD-L-fed larvae was less than that of the VD-H-fed group, whereas that of VD-C-fed larvae was intermediate. The level of expression of genes involved in cell proliferation (PCNA) and early myogenesis (Myf5) decreased between 22 and 44 dph, whereas that of the myogenic determination factor MyoD1 and that of genes involved in muscle structure and function (myosin heavy chain, myosin light chains 2 and 3) increased. Dietary cholecalciferol regulated Myf5, MyoD1, myogenin, and myosin heavy chain gene expression, with a gene-specific shape of response. The maximum hypertrophy of white muscle fibers was higher in larvae fed the VD-C and VD-H diets than in larvae fed the VD-L diet. White muscle hyperplasia was highly stimulated in VD-H-fed larvae compared to VD-L- and VD-C-fed ones. These findings demonstrate a dietary cholecalciferol effect on skeletal muscle growth mechanisms of a Teleost species.

Production of a functional chicken single-chain variable fragment antibody derived from caecal tonsils B lymphocytes against macrogamonts of Eimeria tenella.

Avian coccidiosis is due to a protozoan intracellular parasite belonging to the genus Eimeria which multiplies in the intestine of the host. In order to identify Eimeria antigens which reflect the natural avian humoral immune response, chicken hybridomas were produced by fusion of myeloma MuH1 with B lymphocytes from Eimeria tenella infected chicken. B lymphocytes used for fusions were isolated from tonsils at the basis of caeca where the parasite develops. One of the clones (G1F5) recognised oocyst antigens and the macrogamont stage of the parasite in ELISA and immunofluorescence assay. A single-chain variable fragment (scFv) antibody was cloned from the light chain variant region (VL) and heavy chain variant region (VH) genes of the hybridoma. This recombinant antibody (scFv G1F5) exhibited antigen binding specificity to oocysts and macrogamonts of E. tenella equivalent to the mAb produced by the clone G1F5. Nucleotide sequence analysis of VL genes from scFv G1F5 compared to the germ-line revealed vestiges of gene conversion. scFv derived from chicken B lymphocytes isolated from the gut-associated lymphoid tissue following experimental infection can reveal specific antigens recognised by the avian immune response.