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This study aimed to assess the effects of dietary increasing concentrations of Aloe vera (AV) powder of 0.5%, 2.5% and 5% on the growth performance, hepatic oxidative status, histology, and lipid metabolism and cellular signaling pathways-related genes' expression in gilthead sea bream (Sparus aurata). The preliminary phytochemical analysis revealed the richness of the dried AV extract on total phenol content, total flavonoid content, and condensed tannins when compared to the lyophilized sample. The dried extract showed a good DPPH-radical scavenging activity and its profiling by HPLC-DAD-ESI-MS revealed the presence of anthraquinones namely aloin A, aloin B and their hydroxyl (7-hydroxyaloin A and 7-hydroxyaloin B) and methyl-hydroxy (8-O-methyl-7-hydroxyaloin A and 8-O-methyl-7-hydroxyaloin B) derivatives as well as aloeresin A and B. The AV supplementation in fish diet did not affect growth performance (WG, WGR, and SGR) and feed utilization (FI, FCR, FER), and HSI indexes. However, the hepatic insulin-like growth factors (IGF-I and II) levels were significantly enhanced. Genes' expression levels of enzymes or transcription factors involved in lipolysis (lpl, hsl, and atgl), beta-oxidation (pparα, hadh), fatty acid transporters (cd36, fabp11) and lxrα were significantly down-regulated by the two high concentrations of AV powder. In contrast, fatty acid synthase (fas), a key gene of lipogenesis was significantly up regulated by dietary AV 5% powder supplementation. The induction of fas together with the down-regulation of peroxisome proliferator-activated receptor (pparα) and hydroxyacyl-coenzyme A dehydrogenase (hadh) could explain the lipid accumulation resulting in hepatic steatosis, which was confirmed by histological analysis, since the diets at the two higher concentrations (AV 2.5% and AV 5%) induced a significant increase in the number and diameter of hepatic lipid vacuoles in a dose dependent manner. Moreover, the mRNA levels of protein kinase B named (akt), mammalian target of rapamycin (mtor) and extracellular regulated kinase (erk1/2) involved in cell survival and proliferation were decreased by all AV powder supplemented diets. AV 5% increased catalase and glutathione S transferase activities suggesting a cellular strategy to fight against reactive oxygen species (ROS) accumulation. In conclusion, dietary supplementation with AV 0.5% is recommended for gilthead sea bream feed formulation, as it stimulates the igf-i expression. However, higher levels of AV should be avoided as they might cause lipid metabolism disruption, oxidative stress and liver steatosis.

International audience; Adipogenesis is a tightly regulated process, and the involvement of autophagy has been recently proposed in mammalian models. In rainbow trout, two well-defined phases describe the development of primary cultured adipocyte cells: proliferation and differentiation. Nevertheless, information on the transcriptional profile at the onset of differentiation and the potential role of autophagy in this process is scarce. In the present study, the cells showed an early and transient induction of several adipogenic transcription factors genes' expression (i.e., cebpa and cebpb) along with the morphological changes (round shape filled with small lipid droplets) typical of the onset of adipogenesis. Then, the expression of various lipid metabolism-related genes involving the synthesis (fas), uptake (fatp1 and cd36), accumulation (plin2) and mobilization (hsl) of lipids, characteristic of the mature adipocyte, increased. In parallel, several autophagy markers (i.e., atg4b, gabarapl1 and lc3b) mirrored the expression of those adipogenic-related genes, suggesting a role of autophagy during in vitro fish adipogenesis. In this regard, the incubation of preadipocytes with lysosomal inhibitors (Bafilomycin A1 or Chloroquine), described to prevent autophagy flux, delayed the process of adipogenesis (i.e., cell remodelling), thus suggesting a possible relationship between autophagy and adipocyte differentiation in trout. Moreover, the disruption of the autophagic flux altered the expression of some key adipogenic genes such as cebpa and pparg. Overall, this study contributes to improve our knowledge on the regulation of rainbow trout adipocyte differentiation, and highlights for the first time in fish the involvement of autophagy on adipogenesis, suggesting a close-fitting connection between both processes

β2-adrenoceptors are a subtype of G-protein coupled receptors whose activation leads to increased protein synthesis and decreased degradation in mammalian skeletal muscle, causing hypertrophy. In this study, we compared the effects of the classical β2-agonist noradrenaline (NA) with two representatives of a new generation of agonists (formoterol, FOR and salmeterol, SALM) on growth and metabolism of primary cultured muscle cells of gilthead sea bream. Activation of signaling pathways, cell development and expression of relevant genes were analyzed in day 4 myocytes. The three agonists increased either cAMP levels or PKA phosphorylation, plus TOR phosphorylation, and the proportion of proliferating cell nuclear antigen (PCNA)-positive cells, in parallel with pcna mRNA levels. Thus, demonstrating that these cells are β2-agonists-responsive, and supporting enhanced cell proliferation. The expression of the myogenic factor myf5 was significantly down-regulated, suggesting that the cells were already destined to the muscular linage; while insulin-like growth factors (igf-1 and igf-2) transcript levels were up-regulated, proposing an additional anabolic effect through their local production. Furthermore, SALM treatment up-regulated expression of the lipases (hsl and lipa) and the β-oxidation marker cpt1a, and all three agonists increased mitochondrial dehydrogenase hadh mRNA levels. These data correspond with a situation of enhanced lipolytic and β-oxidation capacity, a fact supported by the higher glycerol released into the media induced by the agonists. Overall, these results suggest a hyperplastic growth condition and a favorable protein/fat ratio profile upon these treatments; consequently, β2-agonists (especially SALM) may be considered good candidates to optimize the growth in this aquaculture species.

The upward trend of seawater temperature has encouraged improving the knowledge of its consequences on fish, considering also the development of diets including vegetable ingredients as an approach to achieve a more sustainable aquaculture. This study aims to determine the effects on musculoskeletal growth of: (1) a high-water temperature of 28 &deg, C (versus 21 &deg, C) in gilthead sea bream juveniles (Sparus aurata) fed with a diet rich in palm oil and, (2) feeding the fish reared at 28 &deg, C with two other diets containing rapeseed oil or an equilibrated combination of both vegetable oils. Somatic parameters and mRNA levels of growth hormone-insulin-like growth factors (GH-IGFs) axis-, osteogenic-, myogenic-, lipid metabolism- and oxidative stress-related genes in vertebra bone and/or white muscle are analyzed. Overall, the data indicate that high-water rearing temperature in this species leads to different adjustments through modulating the gene expression of members of the GH-IGFs axis (down-regulating igf-1, its receptors, and binding proteins) and also, to bone turnover (reducing the resorption-activity genes cathepsin K (ctsk) and matrix metalloproteinase-9 (mmp9)) to achieve harmonic musculoskeletal growth. Moreover, the combination of palm and rapeseed oils seems to be the most beneficial at high-water rearing temperature for both balanced somatic growth and muscular fatty acid uptake and oxidation.

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.

This study aimed to evaluate the effect of dietary protein source (fishmeal, FM; or plant-feedstuffs, PF) and dietary protein/carbohydrate (P/CH) ratio on gilthead seabream appetite regulation and intermediary metabolism. Additionally, the effect of sampling 5 h after feeding (AF) compared to 24 h AF was also evaluated. Four isolipidic diets were formulated having as major protein sources FM or PF (20% FM and 80% PF), and P/CH ratios of 50/10 or 40/20, being the pregelatinized maize starch the main carbohydrate source (diets FM-P50/CH10; FM-P40/CH20; PF-P50/CH10; PF-P40/CH20). Diets were fed until satiation to 140 g gilthead seabream for 41 days. The expression of appetite regulation genes was assessed at 5 and 24 h AF, while other evaluated parameters were assessed only at 5 h AF. Liver leptin expression was higher at 5 h AF, and brain leptin receptor (lepr) expression was higher at 24 h AF. Brain expression of cocaine- and amphetamine-regulated transcript (cart), leptin and ghrelin receptor (ghrr)-a and liver ghrr-b were also affected by sampling time, but the effects were dependent of the diet provided. FM-based diets promoted the expression of brain cart and leptin (at 24 h AF), and liver growth hormone receptor (ghr)-ii, and increased plasma cholesterol and total lipids levels. Fish fed the PF-based diets had higher liver glycogen content, number and size of adipocytes, and expression of hepatic leptin (at 24 h AF), fatty acid synthase, glucokinase, and target of rapamycin. Regarding dietary P/CH ratio, fish fed the P50/CH10 diets presented higher feed efficiency, plasma triglycerides, and expression of intestine cholecystokinin (at 5 h AF), liver ghrr-b (at 24 h AF), glutamate dehydrogenase and ghr-ii. The protein efficiency ratio, hepatosomatic and visceral indices, plasmatic glucose level, and brain lepr expression (at 5 h AF) were higher in fish fed the P40/CH20 diets. The majority of appetite regulation related-genes were not affected by the use of PF-based diets, while the higher dietary CH seemed to lead to a shorter satiety sensation. PF-based diets promoted liver lipid deposition, hypocholesterolemia, and the activation of glycogenesis pathway, while higher CH content induced an increase in plasma glucose that appeared to be stored as lipids. In conclusion, PF-based diets with up to 20% of CH can be used in gilthead seabream without compromising growth performance and FI, and only slightly modifying appetite and metabolic parameters.

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 harvested 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, pparγ, 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

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.

Leptin, ghrelin, and insulin influence lipid metabolism and thus can directly affect adipose tissue characteristics, modulating the organoleptic quality of aquaculture fish. The present study explored gilthead seabream (Sparus aurata) cultured preadipocytes development, and the regulation of adipogenesis by those three hormones. Preadipocytes presented a fibroblast-like phenotype during the proliferation phase that changed to round-shaped with an enlarged cytoplasm filled with lipid droplets after complete differentiation, confirming the characteristics of mature adipocytes. peroxisome proliferator-activated receptor-γ (pparγ) expression was higher at the beginning of the culture, while fatty acid synthase and 3-hydroxyacyl-CoA dehydrogenase gradually increased with cell maturation. The expression of lipoprotein lipase-like, lysosomal acid lipase (lipa), fatty acid translocase/cluster of differentiation-36 (cd36), and leptin receptor (lepr) were not affected during cell culture development; and undetectable expression levels were observed for leptin. Concerning regulation, leptin inhibited lipid accumulation significantly reducing pparγ and cd36 gene expression, both in early differentiating and mature adipocytes, while ghrelin decreased the expression of pparγ in the early differentiating phase but did not reduce intracellular lipid content significantly. Additional insulin past the onset of adipogenesis did not affect lipid accumulation either. In conclusion, at present culture conditions leptin has an anti-adipogenic function in differentiating preadipocytes of gilthead seabream and continues exerting this role in mature adipocytes, while ghrelin and insulin do not seem to influence adipogenesis progression. A better understanding of leptin, ghrelin, and insulin impact on the adipogenic process could help in the prevention of fat accumulation, improving aquaculture fish production and quality.

The aquaculture sector needs to develop new tools to optimally increase production for feeding, while facing future environmental changing conditions. One of the major improvements in this industry has been reducing the usage of fishmeal and fish oil (FO) in diet formulations to promote a more sustainable production. In this line and considering global warming, in the present study, juvenile gilthead sea bream were fed a diet with a 60% of FO substitution by vegetable oils (VO) containing mainly palm oil (P) (80% of the VO content) and maintained at 21 or 28 °C. Furthermore, in the condition of elevated temperature, fish were fed with two other diets with the same level of FO substitution and containing either 80% of the VO content of rapeseed oil (R) or an equilibrated combination of both VO (named PR). High temperature induced a significant increase in growth, but a reduction in the hepatosomatic and viscerosomatic indexes, and circulating triglycerides. Otherwise, significant differences were not found among the fish fed the three distinct diets at 28 °C for any of the somatic or plasma parameters analyzed. In the adipose tissue, the higher number of adipocytes of smaller size in fish maintained at 28 °C compared to fish at 21 °C, suggested an activation of adipogenesis despite the gene expression of peroxisome proliferator-activated receptor gamma (pparg) was significantly decreased. Furthermore, lipid droplets of smaller area and downregulation of the fat translocase cd36 and the fatty acid binding protein fabp1 were found in the liver of fish reared at 28 °C compared to those at 21 °C. Concerning differences among dietary groups, fish fed R diet showed in the liver a more extensive surface area occupied by lipid droplets of bigger size, while contrarily, fish fed PR diet had the smallest area filled with smaller lipid droplets. In adipose tissue, PR diet-fed fish had also adipocytes within the smaller size range. These histological data, together with the significant upregulation of several transcription factors (i.e. ppars) and genes related to lipid transport in the two tissues, indicated that the PR diet could be the most equilibrated for gilthead sea bream, to grow at a high rearing temperature while avoiding excessive fat accumulation. Thus, this study highlights the need of fine-tuning the substitution of FO by VO in the elaboration of feeds for this species to assure proper growth and physiological status in a future global warming scenario.

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