Pedro Gómez-Requeni

Protein growth performance, amino acid utilisation and somatotropic axis responsiveness to fish meal replacement by plant protein sources in gilthead sea bream (Sparus aurata)

Partial or total replacement of fish meal by a mixture of plant protein sources (corn gluten meal, wheat gluten, extruded peas, rapeseed meal) balanced with indispensable amino acids (IAA) was examined in juvenile gilthead sea bream (Sparus aurata) over the course of a 12-week growth trial. A diet with fish meal (FM) as the sole protein source was compared to diets with 50%, 75% and 100% of replacement (PP50, PP75, PP100). Protein retention was improved with more plant protein supply, and just a slight decrease in the final weight gain was found in fish fed PP50 and PP75 diets. However, in the PP100 group, weight gain was depressed up to 30% mainly as the result of a marked reduction of feed intake. These fish also showed a lower fat gain along with a marked hypocholesterolemic effect. Dietary treatment did not alter the hepatic activity of amino acid catabolising enzymes (alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), glutamate dehydrogenase (GDH)), although the size of the total muscle free amino acid (FAA) pool was increased by more plant protein supply. The activity of the somatotropic axis also varied among experimental groups, and the up-regulation of circulating growth hormone (GH) levels with a high plant protein supply followed the decrease in growth rates, plasma levels of insulin-like growth factor-I (IGF-I), and liver mRNA transcripts of IGF-I and GH receptors. This catabolic feature evidenced a liver desensitisation to the anabolic action of GH in the PP100 group, and to a lesser extent in the PP75 group. Taken together all these findings, up to 50-75% of fish meal replacement seems to be feasible with IAA supplementation, but further research is needed to fully identify the responsible factors for the depressed feed intake in order to achieve a full replacement in a fish species having high dietary protein requirements.

Effects of dietary amino acid profile on growth performance, key metabolic enzymes and somatotropic axis responsiveness of gilthead sea bream (Sparus aurata)

Abstract Juvenile gilthead sea bream were fed to visual satiety with isonitrogenous diets based on fish meal and different plant ingredients (33–35% replacement) supplemented with free amino acids to meet the desired indispensable amino acid (IAA) profile and dispensable amino acid (DAA) content. In diets M and WB, IAA profile and DAA content resemble that of the muscle or whole body, respectively. In diets MGlu and WBGlu, DAA content was increased by adding l -glutamic acid (Glu) and thus the IAA/DAA ratio varied from 1.13 (diet M) to 0.80 (diet WBGlu). Growth rates were not significantly different among experimental groups, but feed conversion ratio and nitrogen retention were impaired by the decrease of dietary IAA/DAA ratio. Postprandial ammonia excretion increased with the increase of dietary DAA content irrespective of IAA profile. Conversely, hepatic activity of glutamate dehydrogenase (GDH) was lower in fish fed diet WBGlu than in fish fed diet M. Hepatic growth hormone (GH) binding was not significantly affected by the dietary treatment, but circulating levels of insulin-like growth factor-I (IGF-I) and GH were, respectively, down- and up-regulated in fish fed diet WBGlu, which suggests some defect in the transmission of GH receptor signal. Fat retention and hepatic activities of lipogenic enzymes (glucose-6-phosphate dehydrogenase, G6PD; malic enzyme, ME) were decreased in fish fed diet MGlu. Key metabolic enzymes of hepatic glycolysis (glucokinase, GK) and gluconeogenesis (phosphoenolpyruvate carboxykinase, PEPCK) were also altered in this group of fish. Since soybean meal concentration was highest in diet MGlu, results on lipid and carbohydrate metabolism can be primarily attributed to this component of the diet. In contrast, data on growth performance, ammonia excretion and GH axis mainly reflect changes in the dietary amino acid profile, which reveals that a muscle IAA profile and a high IAA/DAA ratio are important in feeds for gilthead sea bream.

Overview of fish growth hormone family. New insights in genomic organization and heterogeneity of growth hormone receptors

Growth hormone (GH), prolactin (PRL) and somatolactin (SL) are single chain proteins structurally and functionally related. Fish PRL and GH receptors (PRLR, GHR) have been characterized in several fish species. There is limited evidence of fish PRLR isoforms, but emerging data support the existence of different GHR variants. In gilthead sea bream, black sea bream, turbot and fugu, but not in zebrafish, GHR has retained an exclusive fish intron (10/10A). In gilthead sea bream and turbot, this intron is not alternatively spliced, but the black sea bream intron is either removed or retained during mRNA processing, resulting in a long GHR isoform with a 31 amino acid insertion that does not alter the open reading frame. This or any other GHR variant are not found in gilthead sea bream, but a truncated anchored form has been reported in turbot. The latter GHR isoform comprises extracellular and trans-membrane domains, the first 28 amino acids of the intracellular domain and 21 divergent amino acids before a stop codon. This GHR variant is the result of alternative splicing, being the 3′ UTR and the divergent sequence identical to the sequence of the 5′ end of the 9/10 intron. The physiological significance of different fish GHR isoforms remains unclear, but emerging data provide suitable evidence for season and nutrition related changes in the somatototropic axis activity. The up-regulation of circulating GH together with the decrease of plasma titres of insulin-like growth factor-I (IGF-I), an altered pattern of serum IGF binding proteins and a reduced expression of hepatic IGF-I and GHRs represent a mechanism conserved through vertebrate evolution. It secures the preferential utilization of mobilized substrates to maintain energy homeostasis rather than tissue growth. Somatolactin also changes as a function of season, ration size, dietary amino acid profile and dietary protein source creating opposite plasma GH and SL profiles. There is now direct evidence for a lipolytic effect of fish SL, acting at the same time as an inhibitory factor of voluntary food intake. Indeed, long-term feeding restriction results in the enlargement of the summer GH peak, whereas the SL rise coincident with shortened day length is delayed in juvenile fish until late autumn. These findings agree with the idea that SL may act as a marker of energy surplus, priming some particular process such as puberty onset. However, it remains unclear whether SL works through specific receptors and/or dimers or heterodimers of GH and PRL receptors.

Regulation of the somatotropic axis by dietary factors in rainbow trout (Oncorhynchus mykiss).

The activity of the somatotropic axis was analysed in juvenile rainbow trout (Oncorhynchus mykiss) fed either a fishmeal-based diet (FM) or graded levels of plant proteins to replace 50% (PP50 diet), 75% (PP75 diet) or 100% (PP100 diet) of the fishmeal protein. For this purpose, partial cloning and sequencing of the gene encoding rainbow trout growth hormone receptor (GHR) was first accomplished by RT-PCR, using degenerate primers based on the sequences of non-salmonid fish GHR. Growth rates and energy retention were lowered by the PP75 and PP100 diets and a concurrent and progressive increase in plasma levels of growth hormone (GH) was found. However, no changes in hepatic GH binding and total plasma insulin-like growth factor (IGF)-I levels were observed among the four experimental groups. This fact agrees with the lack of changes in hepatic measurements of GHR and IGF-I transcripts. No consistent changes in IGF transcripts were found in peri-visceral adipose tissue and skeletal muscle, but GHR mRNA was up-regulated in the peri-visceral adipose tissue of fish fed the PP75 and PP100 diets, which would favour the lipolytic action of GH. Two specific bands (47 and 33 kDa) of IGF-binding proteins were found in the plasma of all analysed fish, but the sum of the two integrated areas increased progressively with plant protein supply, which might reflect a reduced free IGF availability. Therefore, in our experimental model, the growth impairment could be due, at least in part, to a lowered availability of biologically active IGF (free IGF fraction) rather than to liver GH desensitization or defect in IGF synthesis and release at the systemic and/or paracrine-autocrine level.

In vitro effect of leptin on somatolactin release in the European sea bass (Dicentrarchus labrax): dependence on the reproductive status and interaction with NPY and GnRH

The aim of the present work was to investigate the neuroendocrine control of pituitary somatolactin (SL) release using dispersed pituitary cell culture obtained from male European sea bass (Dicentrarchus labrax) at different stages of sexual development. The effect of mouse recombinant leptin, sea bream gonadotropin releasing-hormone (sbGnRH) and porcine neuropeptide Y (pNPY) and their potential interaction on the SL release were investigated. High doses of leptin (10(-8)-10(-6)M) were differentially effective in inducing SL release depending on the sexual developmental stage. Porcine NPY alone was not effective on basal SL release, but it dose-dependently (0.1 and 1 nM) enhanced SL release induced by leptin (10(-6) and 10(-8)M) in late pre-pubertal but not in post-pubertal stages. No effect of sbGnRH in association or not with leptin was observed on SL release. These findings are the first evidences that leptin and pNPY can play an important role in the neuroendocrine control of pars intermedia function and SL release in fish. In addition, the sensitivity of SL producing cells to leptin and NPY only in prepubertal and pubertal stages, provides the potential role of SL in the nutritional control of the onset of puberty.