A reference growth curve for nutritional experiments in zebrafish (Danio rerio) and changes in whole body proteome during development.

Zebrafish is one of the most used vertebrate model organisms in molecular and developmental biology, recently gaining popularity also in medical research. However, very little work has been done to assess zebrafish as a model species in nutritional studies in aquaculture in order to utilize the methodological toolbox that this species represents. As a starting point to acquire some baseline data for further nutritional studies, growth of a population of zebrafish was followed for 15 weeks. Furthermore, whole body proteome was screened during development by means of bi-dimensional gel electrophoresis and mass spectrometry. Fish were reared under best practice laboratory conditions from hatching until 103 days post-fertilization (dpf) and regularly fed ad libitum with Artemia nauplii from 12 dpf. A growth burst occurred within 9-51 dpf, reaching a plateau after 65 dpf. Fork length and body weight were significantly lower in males than in females from 58 dpf onwards. Proteomics analysis showed 28 spot proteins differently expressed through development and according to sex. Of these proteins, 20 were successfully identified revealing proteins involved in energy production, muscle development, eye lens differentiation, and sexual maturation. In summary, zebrafish exhibited a rapid growth until approximately 50 dpf, when most individuals started to allocate part of the dietary energy intake for sexual maturation. However, proteomic analysis revealed that some individuals reached sexual maturity earlier and already from 30 dpf onwards. Thus, in order to design nutritional studies with zebrafish fed Artemia nauplii, it is recommended to select a period between 20 and 40 dpf, when fish allocate most of the ingested energy for non-reproductive growth purposes.

Nutritional and hormonal control of lipolysis in isolated gilthead seabream (Sparus aurata) adipocytes.

We examined the effects of diet composition and fasting on lipolysis of freshly isolated adipocytes from gilthead seabream (Sparus aurata). We also analyzed the effects of insulin, glucagon, and growth hormone (GH) in adipocytes isolated from fish fed with different diets. Basal lipolysis, measured as glycerol release, increased proportionally with cell concentration and time of incubation, which validates the suitability of these cell preparations for the study of hormonal regulation of this metabolic process. Gilthead seabream were fed two different diets, FM (100% of fish meal) and PP (100% of plant protein supplied by plant sources) for 6 wk. After this period, each diet group was divided into two groups: fed and fasted (for 11 days). Lipolysis was significantly higher in adipocytes from PP-fed fish than in adipocytes from FM-fed fish. Fasting provoked a significant increase in the lipolytic rate, about threefold in isolated adipocytes regardless of nutritional history. Hormone effects were similar in the different groups: glucagon increased the lipolytic rate, whereas insulin had almost no effect. GH was clearly lipolytic, although the relative increase in glycerol over control was lower in isolated adipocytes from fasted fish compared with fed fish. Together, we demonstrate for the first time that lipolysis, measured in isolated seabream adipocytes, is affected by the nutritional state of the fish. Furthermore, our data suggest that glucagon and especially GH play a major role in the control of adipocyte lipolysis.

Production and characterization of recombinantly derived peptides and antibodies for accurate determinations of somatolactin, growth hormone and insulin-like growth factor-I in European sea bass (Dicentrarchus labrax).

A specific radioimmunoassay (RIA) for European sea bass (Dicentrarchus labrax) growth hormone (GH) was developed and validated. For this purpose, a stable source of GH was produced by means of recombinant DNA technology in a bacteria system. The identity of the purified protein (ion exchange chromatography) was demonstrated by Western blot and a specific GH antiserum was raised in rabbit. In Western blot and RIA system, this antiserum recognized specifically native and recombinant GH, and it did not cross-react with fish prolactin (PRL) and somatolactin (SL). In a similar way, a specific polyclonal antiserum against the now available recombinant European sea bass SL was raised and used in the RIA system to a sensitivity of 0.3 ng/ml (90% of binding of tracer). Further, European sea bass insulin-like growth factor-I (IGF-I) was cloned and sequenced, and its high degree of identity with IGF-I peptides of barramundi, tuna, and sparid fish allowed the use of a commercial IGF-I RIA based on barramundi IGF-I antiserum. These assay tools assisted for the first time accurate determinations of SL and GH-IGF-I axis activity in a fish species of the Moronidae family. Data values were compared to those found with gilthead sea bream (Sparus aurata), which is currently used as a Mediterranean fish model for growth endocrinology studies. As a characteristic feature, the average concentration year round of circulating GH in growing mature males of European sea bass was higher than in gilthead sea bream. By contrast, the average concentration of circulating SL was lower. Concerning to circulating concentration of IGF-I, the measured plasma values for a given growth rate were also lower in European sea bass. These findings are discussed on the basis of a different energy status that might allowed a reduced but more continuous growth in European sea bass.