Marco Saroglia

Dynamics of collagen indicating amino acids, in embryos and larvae of sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata), originated from broodstocks fed with different vitamin C content in the diet

The dynamics of hydroxyproline–proline (HYP–PRO) and hydroxylysine (HYL), as amino acids indicators of collagen, has been studied in fertilised eggs, embryos and fasting larvae of sea bass (Dicentrarchus labrax L.) and gilthead sea bream (Sparus aurata L.). Broodstocks of the two species were fed every second day with two types of diets: one containing sufficient ascorbate for a normal growth and the other with an extra addition of a very high dose (2000 mg kg−1 feed) of coated l-ascorbic acid (AA). The collagen hydroxylation expressed as the HYP-PRO ratio, hydroxylysine (HYL1 and HYL2) and total ascorbate (TAA) concentrations were analysed in several embryonic and larvae developmental stages. TAA concentrations in the newly fertilised eggs and larvae resulted significantly higher in offspring of broodstocks fed an additional dose of 2000 mg AA/kg feed than in those fed a normal diet (P<0.05 or P<0.01). The differences in HYP–PRO ratio values between the groups fed AA-supplemented and unsupplemented diets, were significant for all the analysed stages, being higher in the supplemented groups. The values of HYL were higher, although not always significantly, for all the analysed stages, in offspring of the supplemented group, compared to the offspring of the unsupplemented one. In conclusion, a vitamin C dose of 2000 mg kg−1 feed, delivered every second day to sea bass and gilthead sea bream broodstock, increases the collagen synthesis in embryos and fasting larvae, in comparison with a diet containing vitamin C at the recommended concentration for growth.

Influence of environmental temperature and water oxygen concentration on gas diffusion distance in sea bass (Dicentrarchus labrax ,L.)

Gas diffusion distance (GDD) of sea bass was measured in fish bred under farm conditions, at different dissolved oxygen concentrations (DO): normoxia condition (80–100% of the saturation value) and `mild hyperoxia condition (120–130% of the saturation value). Measures were carried out two times in a year (beginning of summer and autumn) in order to evaluate the effect of water temperature on GDD at the two different dissolved oxygen concentrations. There was a significant influence of both dissolved oxygen concentration (p ≤0.001) and environmental temperature (p ≤0.001) on GDD. In summertime it was 1.75xa0μm and 2.31xa0μm for fish reared under normoxia and hyperoxia, respectively, and in autumn 2.51xa0μm and 2.96xa0μm for fish reared under normoxia and hyperoxia, respectively. When DO was reduced at the higher temperatures, GDD decreased as well. Results lead to the conclusion that GDD increased with the increasing of DO, both due to reduced water temperature and to the mild oxygen hypersaturation following application of pure oxygen. The advantage for fish may be found in the compromise between maximising O2 diffusion at the gills and ions/water intake/loss, known as `osmoregulatory compromise.

Molecular biology and fish welfare: A winning combination

The issue of animal welfare in aquaculture is of growing interest and there is an increasing consumer demand for documentation of safe and ethically defendable food production. In this context, we have looked for molecular markers among those genes whose expression is modified by the different farming conditions. We have compared gene expression of sea bass farmed at different population densities by differential display, and we have obtained six bands differentially expressed whose sequences have been deposited in the public databases; two of them were suppressed by high population density, while four were induced by the treatment. These genes can be used as biomarkers, and together with a panel of stress-related genes of sea bass (D. labrax) that we have already obtained, could allow the rapid diagnosis of the welfare status of a fish using RT-PCR. We are certain that the new molecular techniques will find their place in the everyday management of fish farming. On the other hand, we are also aware that the scarcity of genomic resources for some fish species, in spite of their economical interest, will retard the beneficial effects that modern biotechnology could bring to aquaculture industry. Therefore, an effort should be made to reduce, as far genomic resources are concerned, the gap that separates farmed species from “model organisms” such as Danio rerio and Fugu rubripes.

Ascorbate dynamics in embryos and larvae of sea bass and sea bream, originating from broodstocks fed supplements of ascorbic acid

The transfer of L-ascorbic acid (AA) from broodstock to the fertilised eggs and its dynamics in embryos and fasting larvae, has been studied in sea bass (Dicentrarchus labrax L.) and sea bream (Sparus aurata L.). Two types of diets were fed to the broodstocks: one containing sufficient ascorbate for normal growth and the other with an extra addition of a very high dose (2,000 mg/kg feed) of ethyl-cellulose-coated L-ascorbic acid.The concentration of AA and total ascorbate (L-ascorbic acid plus dehydroascorbic acid) was detected through several embryo and larval development stages, including larvae just before feeding. In sea bass and sea bream, the mean (SD) concentration of total ascorbate in fertilised eggs originating from broodstock fed AA supplemented diet, was 218.5 (17.7) and 122.4 (5.1) μg/g wet weight respectively. This was significantly higher (P < 0.01) than the unsupplemented groups, which contained only 155.9 (6.9) and 103.9 (3.5) μg/g wet weight respectively. A diet with a vitamin C content adequate for normal growth, may not be sufficient for broodstock when the goal is transfer of TAA to embryos.

An improved method for assay of vitamin C in fish feed and tissues

SummaryA method was devised to assay four forms of vitamin C: L-ascorbic acid (AA), dehydroascorbic acid (DHA), ascorbate-2-mono- and polyphosphate (AMP, APP), as well as ascorbate-2-monosulphate (AMS), in sample series of different fish tissues and feed. Direct and indirect detection were combined. Sample extractions were carried out with 0.2 mol L−1 sodium acetate buffer (pH 4.8) and extracts were deproteinized after different chemical or enzymatic reactions, with perchloric acid. The DHA was reduced to AA with dithioerythritol (DTE). Ascorbate oxidase enzyme was used for the detection of background and an acidic phosphatase enzyme for the hydrolysis of different phosphate esters. Ascorbate-2-sulphate was detected directly with help of coinjection of the compound. Chromatographic analysis was carried out with a single column isocratic reverse phase method. The mobile phase was an aqueous buffer of 0.04 M sodium-acetate, 0.05 mM EDTA, 0.5 mM tetrabutylammonium dihydrogen phosphate (TBA) adjusted to pH 3.76 with 85% H3PO4 and with 24 mL methanol added to 1000 mL. C-18 columns were used with 0.6 mL min−1 flow rate at 23°C. The vitamin C forms were detected by UV absorption at 250 nm. The determination limit was 1.0–5.0 μg g−1 in AA equivalent. The standard deviations were between 1–6% and depended on the concentrations of vitamin C forms and tissues. Recoveries were between 90–96% in samples.

Cloning and expression analysis of myostatin, fibroblast growth factor 6, insulin-like growth factor I and II in liver and muscle of sea bass (Dicentrarchus labrax, L.) during long-term fasting and refeeding

Abstract The exceptionally fast growth that fish experience after periods of fasting has been called “compensatory growth”. This phenomenon has been studied in intensive aquaculture as a means of enhancing growth rates, but the mechanisms by which food intake activates an increase in somatic growth, and especially in muscle growth, are complex and not yet fully understood. In the present paper, we describe the molecular cloning and sequencing of sea bass (Dicentrarchus labrax) myostatin (MSTN) and fibroblast growth factor 6 (FGF6), which have been shown to be major genetic determinants of skeletal muscle growth, together with insulin-like growth factor I (IGF-I) and IGF-II, which are potent mitogens known to play important roles in growth and development. We then report the pattern of expression of the four aforementioned genes, in liver and myotomal muscle in response to prolonged fasting and refeeding. Nutritional status significantly influenced the expression of IGF-I, IGF-II and MSTN, whereas the muscular FGF6 expression levels were not affected by the feeding status of the animals. Taken together these data indicate that IGF-I, IGF-II and MSTN are involved in the sea bass muscle compensatory growth induced by refeeding, whereas FGF6 probably has not a role in this phenomenon.

EST projects in aquaculture: sea bass, red tuna and perch

Riassunto Progetto EST in acquacoltura: spigola, tonno rosso e persico. Queste tre specie stanno richiamando un sempre maggior interesse. Si è reso quindi necessario effettuare un programma che permetta di valutare le abitudini comportamentali dei pesci di allevamento e gli eventuali problemi nelle varie fasi del ciclo biologico indagando, per esempio, l’espressione di geni coinvolti nella maturazione dei gameti e nella fecondazione e/o quelli che hanno un ruolo nella risposta di tipo biologico agli stressori ambientali. A tal fine abbiamo dato il via a questo progetto preparando una library di cDNA di fegato di branzino e sequenziando alcune centinaia di cloni il 55% dei quali risultano essere sequenze singole, mentre i restanti vanno a formare dei contig. Contemporaneamente è in corso uno studio sulle gonadi di tonno rosso e sul fegato di persico. L’ obiettivo di questo progetto è quello di creare database di sequenze di cDNA di queste tre specie di pesci ad interesse commerciale. Di tali library verranno sequenziati circa 5.000 cloni; le sequenze ottenute, depositate in banca dati, saranno disponibili via Internet a tutta la comunità scientifica che le potrà utilizzare per le ricerche più disparate.

Molecular cloning and real-time quantification of a glucocorticoid receptor in sea bass (Dicentrarchus labrax, L) exposed to stress

Riassunto Clonaggio molecolare di un recettore glucocorticoide nella spigola (Dicentrarchus labrax, L) e quantificazione via real-time PCR della sua espressione in soggetti esposti a stress. Nell’ambito di una ricerca sui marker molecolari descrittori della risposta allo stress cronico in pesci di rilevante interesse per l’acquacoltura è stato clonato e sequenziato un recettore per l’ormone glucocorticoide (GR) con l’obiettivo di quantificarne l’espres sione tramite la tecnica della real-time RT-PCR. I teleostei sembrano utilizzare un solo ormone, il cortisolo, sia per la regolazione dei carboidrati che per la regolazione dell’equilibrio idro-salino, presumibilmente con il GR come unico recettore. Quest’ultimo è un recettore nucleare ed agisce come un “ligand-dependent transcription factor” con trollando e regolando l’espressione genica. Benché le sequenze nucleotidiche che codificano per il GR siano state clo nate in diverse specie ittiche, non esisteva in banca genica la sequenza relativa alla spigola (Dicentrarchus labrax L.) che in seguito a questo studio è stata depositata (Accession N° AY549305). I dati della real-time RT-PCR dimo strano chiaramente che lo stress da alte densità di allevamento influenza i livelli di espressione del gene del GR. L quantità del mRNA in questo caso diminuisce con l’aumentare dei livelli plasmatici del cortisolo.