L.M.P. Valente

Dietary lipid level affects growth performance and nutrient utilisation of Senegalese sole (Solea senegalensis) juveniles

Over the last few years, several aspects of Senegalese sole (Solea senegalensis) culture have been developed and optimised but the dietary lipid level for optimal growth has never been determined. Hence, five isonitrogenous diets (56 % dietary protein) with increasing dietary lipid levels (4, 8, 12, 16 and 20 % DM) were fed to satiation to triplicate groups of twenty fish (mean initial weight 10 g). Fifteen tanks were randomly assigned one of the five diets. Feed was distributed using automatic feeders, and fish were fed over a 16-week period. At the end of the experiment the fish fed on diets containing the two lowest dietary lipid levels (4 and 8 %) showed a 3-fold body-weight increase with a significantly higher daily growth index than fish fed higher lipid levels (1.2 v. 0.8). Moreover, these fish displayed a significantly lower dry feed intake (12 g/kg per d) and feed conversion ratio (1.0) compared with fish fed higher lipids levels (16-19 g/kg per d; feed conversion ratio 2.0). Low dietary lipid levels ( < 12 %) significantly improved nutrient retention and gain and hence growth, without major effects on whole-body composition. Despite the slight alteration in n-3 PUFA muscle content in the fish fed low-fat-diets, this fish fed low dietary lipid still remains a rich n-3 PUFA product and generally maintained its nutritional value. These results evidenced a low lipid tolerance of Senegalese sole juveniles and suggest a maximal dietary inclusion level of 8 % lipids for both optimal growth and nutrient utilisation without compromising flesh quality.

Dietary lipid levels have a remarkable impact on the expression of growth-related genes in Senegalese sole (Solea senegalensis Kaup)

SUMMARY In Senegalese sole (Solea senegalensis Kaup), growth is negatively correlated to dietary lipid levels. To understand the molecular basis of this effect a molecular toolbox of 12 genes, including fgf6, fst, mstn1, myf5, mrf4, myod1, myod2, myog, myHC, mylc2, igf1r and insr, was developed. The expression profiles of these genes were investigated in white muscle and liver of fish fed with three dietary lipid levels (4%, 12% and 20%). The expression of igf-I and igf-II was also examined. MRFs and myosins were only expressed in the muscle and, except for myf5, the general trend was a decrease in expression with an increase in dietary lipids. Fgf6 was identified for the first time in liver and its expression augmented in hepatic tissues with increasing dietary lipid levels. A similar tendency was observed for mstn1 and igf-I. The opposite was observed for igf1r expression in muscle and liver. Myog, mrf4, mylc2 and igf1r were highly correlated with growth and nutrient utilisation indices. In addition to its practical implications, this work provides a valuable contribution towards our understanding of the genetic networks controlling growth in teleosts.

Molecular evolution of zebrafish dnmt3 genes and thermal plasticity of their expression during embryonic development

DNA reprogramming by DNA (cytosine-5)-methyltransferases (dnmts) after fertilisation is a dynamic mechanism that is essential for early development. Amongst the three types of dnmt genes in vertebrates, dnmt3 is the one involved in de novo methylation and comprises three related genes, termed dnmt3a, dnmt3b and dnmt3L in mammals. Zebrafish (Danio rerio) has six dnmt3 paralogues, which have hitherto been termed dnmt3 to dnmt8. Bayesian inference of phylogeny and synteny analysis revealed that dnmt6 and dnmt8 are in fact duplicated dnmt3a genes, whereas the other paralogues are closely related to dnmt3b. Hence, we propose a revised nomenclature that more accurately reflects the relationship amongst zebrafish dnmt3 genes. Both dnmt3a genes were ubiquitously expressed in adult tissues, whilst the various dnmt3b paralogues were differentially expressed, with notably high expression levels in the gonads. The influence of embryonic temperature on dnmt3 expression was investigated, since it is known to have a significant impact in early development and a long-term effect on growth in some teleost species. Embryos were incubated at 23, 27 or 31°C and samples collected at six developmental stages from blastula until protruding mouth. Dnmt3 expression during early development was remarkably dynamic. In particular, mRNA levels of the two dnmt3a genes showed a marked increase throughout development and several significant differences in dnmt3a and dnmt3b transcript levels were found between temperatures at the same developmental point. Taken together, our data indicate that dnmt3 paralogues are diverging and that dnmt3a and dnmt3b may play different roles in thermal epigenetic regulation of gene expression during early development.

Feed intake and growth of fast and slow growing strains of rainbow trout (Oncorhynchus mykiss) fed by automatic feeders or by self-feeders

Abstract Rainbow trout (8.5–9.5 g) of two strains (C and M) differing in growth potential were compared with respect to feeding motivation and feeding rhythms, over a 65-day experimental period, employing self-feeding or automatic feeding. Growth rate, feed gain ratio, feed intake and pattern of feeding activity of fish fed with self-feeders, were recorded, as was body composition of both strains. The final weight of fish of the fast-growing strain, strain C, fed using self-feeders, was significantly higher (82.6 g) than that observed for fish of the slow-growing strain, strain M (69.3 g). When the automatic feeders were used, no significant differences were found between the strains in terms of body weight gain (65 g). Results observed for feed gain ratio were also similar between the two strains. Although the voluntary feed intake (VFI) did not vary significantly with the genetic origin of the fish, strain C displayed a consistently higher VFI compared to strain M. The retention efficiency of nutrients and energy were similar between strains but significantly different between feeding systems. With regard to body composition, when fish were fed by means of self-feeders, no significant differences were found between the two rainbow trout strains. However, when fed automatically, dry matter and lipid content were highest in the strain M fish. The energy and protein content of the whole fish were not significantly influenced either by the feeding system or by the strain of the fish used.

Conjugated linoleic acid in diets for large-size rainbow trout ( Oncorhynchus mykiss ): effects on growth, chemical composition and sensory attributes

The effects of graded levels (0 %, 0.5 %, 0.75 and 1 %) of dietary conjugated linoleic acid (CLA) were assessed on 97 g rainbow trout. Fish were fed to satiation twice a day for 12 weeks. At the end of the experiment, all groups of fish weighed more than 250 g and no significant differences were detected in growth performance, feed conversion, nutrient or energy utilisation or body composition between treatments. A decrease in liver lipid content resulted from including CLA and was accompanied by a reduction in malic enzyme activity. The muscle saturated acid and PUFA content did not vary between dietary treatments, despite the increasing concentration of stearic acid and CLA. In the liver, however, both fractions increased significantly with dietary CLA. Moreover, the MUFA decreased significantly in both muscle and liver. CLA was incorporated into tissue lipids, with levels in flesh (2.1-4.2 %) being 2-fold higher than in liver (0.8-1.9 %). In muscle, the percentage of cis-9, trans-11 isomer ranged from 39.5 % to 41.8 % and that of trans-10, cis-12 isomer from 31.4 % to 33.4 % of total CLA. The incorporation of CLA isomers in the liver varied with dietary treatment, and the cis-9, trans-11 isomer seemed to be more efficiently incorporated than trans-10, cis-12. Sensory data indicated slight-to-moderate differences between the trout fed with and without CLA. The present results suggest that 250 g rainbow trout can incorporate CLA in both muscle and liver, contributing to the production of a functional food.

Advances in research on the prenatal development of skeletal muscle in animals in relation to the quality of muscle-based food. I. Regulation of myogenesis and environmental impact

Skeletal muscle development in vertebrates - also termed myogenesis - is a highly integrated process. Evidence to date indicates that the processes are very similar across mammals, poultry and fish, although the timings of the various steps differ considerably. Myogenesis is regulated by the myogenic regulatory factors and consists of two to three distinct phases when different fibre populations appear. The critical times when myogenesis is prone to hormonal or environmental influences depend largely on the developmental stage. One of the main mechanisms for both genetic and environmental effects on muscle fibre development is via the direct action of the growth hormone-insulin-like growth factor (GH-IGF) axis. In mammals and poultry, postnatal growth and function of muscles relate mainly to the hypertrophy of the fibres formed during myogenesis and to their fibre-type composition in terms of metabolic and contractile properties, whereas in fish hyperplasia still plays a major role. Candidate genes that are important in skeletal muscle development, for instance, encode for IGFs and IGF-binding proteins, myosin heavy chain isoforms, troponin T, myosin light chain and others have been identified. In mammals, nutritional supply in utero affects myogenesis and the GH-IGF axis may have an indirect action through the partitioning of nutrients towards the gravid uterus. Impaired myogenesis resulting in low skeletal myofibre numbers is considered one of the main reasons for negative long-term consequences of intrauterine growth retardation. Severe undernutrition in utero due to natural variation in litter or twin-bearing species or insufficient maternal nutrient supply may impair myogenesis and adversely affect carcass quality later in terms of reduced lean and increased fat deposition in the progeny. On the other hand, increases in maternal feed intake above standard requirement seem to have no beneficial effects on the growth of the progeny with myogenesis not or only slightly affected. Initial studies on low and high maternal protein feeding are published. Although there are only a few studies, first results also reveal an influence of nutrition on skeletal muscle development in fish and poultry. Finally, environmental temperature has been identified as a critical factor for growth and development of skeletal muscle in both fish and poultry.

Temperature affects methylation of the myogenin putative promoter, its expression and muscle cellularity in Senegalese sole larvae

Myogenin (myog) encodes a highly conserved myogenic regulatory factor that is involved in terminal muscle differentiation. It has been shown in mammals that methylation of cytosines within the myog promoter plays a major role in regulating its transcription. In the present study, the Senegalese sole (Solea senegalensis) myog putative proximal promoter was identified and found to be highly conserved among teleosts. Therefore, it is plausible that it plays a similar role in controlling myog expression. Cytosine methylation of the myog promoter in skeletal muscle of Senegalese sole larvae undergoing metamorphosis was influenced by rearing temperature. A lower temperature (15°C) significantly increased myog promoter methylation in skeletal muscle, particularly at specific CpG sites, relatively to higher rearing temperatures (18 and 21°C). Myog transcription was downregulated at 15°C, whereas expression of dnmt1 and dnmt3b was upregulated, consistently with the higher myog methylation observed at this temperature. Rearing temperature also affected growth and fast muscle cellularity, producing larger fibers at 21°C. Taken together, our data provide the first evidence of an epigenetic mechanism that may be underlying the temperature-induced phenotypic plasticity of muscle growth in teleosts.

Incubation temperature induces changes in muscle cellularity and gene expression in Senegalese sole (Solea senegalensis)

Fertilised eggs of Senegalese sole were incubated at 15, 18 or 21 °C, and after hatching all larvae were reared at 21 °C until 30 days post-hatch. By this point larvae from the 18 or 21 °C temperature groups had 11 and 9% more muscle fibres than those from 15 °C, respectively. Hyperplastic growth during metamorphosis was higher in larvae from 18 °C. Embryonic temperature induced gene expression changes, albeit with a variable pattern throughout development. Myf5, myod2, myHC and fst mRNA levels were significantly higher at several stages prior to hatching in embryos incubated at 21 °C, whereas hsp90AB and hsp70 transcripts were present at higher levels in the 15 °C group. Myf5, myod1, myod2, pax7, myog, fst, igf-II, igf1r, hsp90AA and hsp90AB were expressed at higher levels during early development, particularly during somitogenesis. In contrast, mrf4, myHC, mylc2, igf-I, mstn1 and hsp70 were up-regulated at later stages of larval development, namely during and after metamorphosis. This study is the first example of thermal plasticity of myogenesis with prolonged effect in a flatfish.

Lipid digestion, absorption and uptake in Solea senegalensis.

Dietary lipids are the major energy source for metabolic purposes in most fish species, and improve dietary protein utilization for growth. In a previous study we have reported a low tolerance of Senegalese sole juveniles to dietary lipid levels and suggested a maximal dietary inclusion level of 8% lipids for both optimal growth and nutrient utilization. The mechanisms behind this apparent poor utilization of the dietary lipids are still to be elucidated. The primary aim of the present study was to investigate the overall process of digestion and lipid absorption in relation to dietary lipid levels. Triplicate groups of twenty fish (mean initial mass 29g) were fed two isonitrogenous diets (54% of protein dry matter basis) with different lipid levels (L4 and L17, 4 and 17% lipids dry matter basis), for 88days. Protein and lipid apparent digestibility coefficients as well as lipase activity were similar in both groups suggesting that Solea senegalensis has the ability to digest equally well a low fat or a high fat diet. Plasma triglyceride concentrations were significantly higher 5 and 16h after feeding in fish fed the L17 compared to those fed L4, following dietary lipid supply, demonstrating effective lipid absorption. Expression of proteins related to lipid transport (microsomal triglyceride transfer protein), trafficking (Fatty acid binding protein 11) and fatty acid uptake (VLDL-r) was significantly higher in liver of fish fed the high fat diet 16h after the meal, but remained unchanged in muscle. In conclusion, it seems that high fat diets do not impair lipid digestion and absorption.

Biochemical growth characterization of fast and slow-growing rainbow trout strains: effect of cell proliferation and size

Concentrations of RNA and DNA, determined in two rainbow trout strains, Cornec and Mirwart, over a period of 9 months, were used to assess and characterize growth potential. More rapid growth and higher muscle nucleic acid concentrations in the Cornac strain trout, suggest a more metabolically active strain with higher rates of protein synthesis. On the other hand, the higher concentration of muscle DNA, in the Cornec strain, suggests a smaller cell size and a larger number of cells per unit weight issue. The faster decrease of DNA concentration, with increasing body weight, in the Mirwart strain, reflected a lower percentage of hyperplasia and consequently a lower capacity for growth.