The tolerance of fish to fasting offers a model to study the regulatory mechanisms and changes produced when feeding is restored. Gilthead sea bream juveniles were exposed to a 21-days fasting period followed by 2 h to 7-days refeeding. Fasting provoked a decrease in body weight, somatic indexes, and muscle gene expression of members of the Gh/Igf system, signaling molecules (akt, tor and downstream effectors), proliferation marker pcna, myogenic regulatory factors, myostatin, and proteolytic molecules such as cathepsins or calpains, while most ubiquitin-proteasome system members increased or remained stable. In bone, downregulated expression of Gh/Igf members and osteogenic factors was observed, whereas expression of the osteoclastic marker ctsk was increased. Refeeding recovered the expression of Gh/Igf system, myogenic and osteogenic factors in a sequence similar to that of development. Akt and Tor phosphorylation raised at 2 and 5 h post-refeeding, much faster than its gene expression increased, which occurred at day 7. The expression in bone and muscle of the inhibitor myostatin (mstn2) showed an inverse profile suggesting an inter-organ coordination that needs to be further explored in fish. Overall, this study provides new information on the molecules involved in the musculoskeletal system remodeling during the early stages of refeeding in fish.conditions has been characterized and the ratio of the circulating levels of Gh and Igf1 is considered a good marker of growth quality in terms of its relation with body indexes or muscle fibers composition 12,13 .Muscle growth is also controlled by myogenic regulatory factors (MRFs) (MyoD, Myf5, Myog and Mrf4), and the negative endogenous regulator myostatin, as well as other factors that control sequentially the process of development and growth 6,14,15 . The effects of fasting and refeeding on myogenesis have been studied in diverse fish species 6,10,16 demonstrating the function of the different MRFs during the metabolic arrest caused with food limitation and the subsequent activation when feeding is restored. Furthermore, during fasting and refeeding, proteolytic molecules play a remarkable role to adapt to the changes in amino acids supply. This is more important in fish that have a specific and enhanced protein metabolism. The main endogenous proteolytic systems, each one performing specific degradative or regulatory functions according to the nutritional conditions are, calpains, cathepsins and ubiquitin-proteasome, all of which are well-known in fish, especially salmonids [17][18][19] , but also in gilthead sea bream [20][21][22] .In addition to muscle, bone is also an important tissue for skeletal and locomotion functions, but also, as a reservoir of minerals that is clearly affected when nutrition is restricted. Essential during development, synchronicity between bone and muscle is required for proper musculoskeletal growth 23,24 . Besides to being induced by the Gh/Igf system, the process of osteogenesis is also regulated by skeleton-derived factors that...

World population is expected to increase to approximately 9 thousand million people by 2050 with a consequent food security decline. Besides, climate change is a major challenge that humanity is facing, with a predicted rise in mean sea surface temperature of more than 2°C during this century. This study aims to determine whether a rearing temperature of 19, 24, or 28°C may influence musculoskeletal development and muscle lipid metabolism in gilthead sea bream juveniles. The expression of growth hormone (GH)/insulin-like growth factors (IGFs) system-, osteogenic-, myogenic-, and lipid metabolism-related genes in bone and/or white muscle of treated fish, and the in vitro viability, mineralization, and osteogenic genes expression in primary cultured cells derived from bone of the same fish were analyzed. The highest temperature significantly down-regulated igf-1, igf-2 , the receptor igf-1ra , and the binding proteins igfbp-4 and igfbp-5b in bone, and in muscle, igf-1 and igf-1ra , suggesting impaired musculoskeletal development. Concerning myogenic factors expression, contrary responses were observed, since the increase to 24°C significantly down-regulated myod1 and mrf4 , while at 28°C myod2 and myogenin were significantly up-regulated. Moreover, in the muscle tissue, the expression of the fatty acid transporters cd36 and fabp11 , and the lipases lipa and lpl-lk resulted significantly increased at elevated temperatures, whereas β-oxidation markers cpt1a and cpt1b were significantly reduced. Regarding the primary cultured bone-derived cells, a significant up-regulation of the extracellular matrix proteins on, op , and ocn expression was found with increased temperatures, together with a gradual decrease in mineralization along with fish rearing temperature. Overall, these results suggest that increasing water temperature in this species appears to induce unfavorable growth and development of bone and muscle, through modulating the expression of different members of the GH/IGFs axis, myogenic and osteogenic genes, while accelerating the utilization of lipids as an energy source, although less efficiently than at optimal temperatures.

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Gut microbiota plays a fundamental role in maintaining host's health by controlling a wide range of physiological processes. Administration of probiotics and manipulation of photoperiod have been suggested as modulators of microbial composition and are currently undergoing an extensive research in aquaculture as a way to improve health and quality of farmed fish. However, our understanding regarding their effects on physiological processes is still limited. In the present study we investigated whether manipulation of photoperiod and/or probiotic administration was able to alter microbial composition in zebrafish larvae at hatching stage. Our findings show that probiotic does not elicit effects while photoperiod manipulation has a significant impact on microbiota composition. Moreover, we successfully predicted lipid biosynthesis and apoptosis to be modulated by microbial communities undergoing continuous darkness. Interestingly, expression levels of caspase 3 gene (casp3) and lipid-related genes (hnf4a, npc1l1, ppar, srebf1, agpat4 and fitm2) were found to be significantly overexpressed in dark-exposed larvae, suggesting an increase in the occurrence of apoptotic processes and a lipid metabolism impairment, respectively (p<0.05). Our results provide the evidence that microbial communities in zebrafish at early-life stages are not modulated by a short administration of probiotics and highlight the significant effect that the dark photoperiod elicits on zebrafish microbiota and potentially on health.Recently, Deaver et al., observed Ruminococcus torques, a bacterial species known to negatively affect gut barrier integrity, and Lactobacillus johnsonii, known to help maintaining the intestinal epithelial cell layer, to respectively increase and decrease their abundance in mice undergoing a 4-week period of constant 24 h light [17]. Thus, it is of paramount importance for the microbiota to follow regular diurnal oscillation in order to protect against homeostasis impairment and, consequently, diseases.

The dietary inclusion of plant-based products in fish feeds formulation is required for the sustainable development of aquaculture. Moreover, considering functional diets, hydroxytyrosol, one of the major phenolic compounds found in olives (Olea europaea), has been identified as a potential candidate to be used in the aquafeeds industry due to its health promoting abilities. The aim of this study was to evaluate the effects of the inclusion of an olive juice extract rich in hydroxytyrosol as an additive (0.52 g HT/kg feed) in a high-fat (24% lipids) diet in gilthead sea bream (Sparus aurata) juveniles. Moreover, the experimental diets, with or without the extract, were administered daily at a standard (3% of total biomass in the tank) or restricted ration (40% reduction) for 8–9 weeks. Growth and biometric parameters, insulin-like growth factor 1 (IGF-1) plasma levels and growth hormone/IGF axis-, myogenic- and osteogenic-related genes expression in liver, white muscle and/or bone were analyzed. Moreover, in vitro cultures of vertebra bone-derived cells from fish fed the diets at a standard ration were performed at weeks 3 and 9 to explore the effects of hydroxytyrosol on osteoblasts development. Although neither body weight or any other biometric parameter were affected by diet composition after 4 or 8 weeks, the addition of the hydroxytyrosol-rich extract to the diet increased IGF-1 plasma levels, regardless of the ration regime, suggesting an anabolic condition. In muscle, the higher mRNA levels of the binding protein igfbp-5b and the myoblast fusion marker dock5 in fish fed with the hydroxytyrosol-rich diet suggested that this compound may have a role in muscle, inducing development and a better muscular condition. Furthermore in bone, increased osteogenic potential while delayed matrix mineralization after addition to the diet of the olive juice extract was supported by the upregulated expression of igf-1 and bmp4 and reduced transcript levels of osteopontin. Overall, this study provides new insights into the beneficial use of hydroxytyrosol as a dietary additive in gilthead sea bream functional diets to improve muscle-skeletal condition and, the aquaculture industry.

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Soybeans are one of the most used alternative dietary ingredients in aquafeeds. However, they contain phytoestrogens like genistein (GE), which can have an impact on fish metabolism and health. This study aimed to investigate the in vitro and in vivo effects of GE on lipid metabolism, apoptosis, and autophagy in rainbow trout (Oncorhynchus mykiss). Primary cultured preadipocytes were incubated with GE at different concentrations, 10 or 100 μM, and 1 μM 17β-estradiol (E2). Furthermore, juveniles received an intraperitoneal injection of GE at 5 or 50 µg/g body weight, or E2 at 5 µg/g. In vitro, GE 100 μM increased lipid accumulation and reduced cell viability, apparently involving an autophagic process, indicated by the higher LC3-II protein levels, and higher lc3b and cathepsin d transcript levels achieved after GE 10 μM. In vivo, GE 50 µg/g upregulated the gene expression of fatty acid synthase (fas) and glyceraldehyde-3-phosphate dehydrogenase in adipose tissue, suggesting enhanced lipogenesis, whereas it increased hormone-sensitive lipase in liver, indicating a lipolytic response. Besides, autophagy-related genes increased in the tissues analyzed mainly after GE 50 µg/g treatment. Overall, these findings suggest that an elevated GE administration could lead to impaired adipocyte viability and lipid metabolism dysregulation in rainbow trout.