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Cadmium (Cd) is a major pollutant in water, and long-term Cd exposure seriously threatens aquatic organisms. This study evaluated the effects of dietary Cd on growth performance, tissue Cd accumulation, oxidative stress, endoplasmic reticulum (ER) stress, inflammation, apoptosis, and anti-stress response in juvenile black seabream (Acanthopagrus schlegelii). Six experimental diets containing different concentration of Cd were formulated: 0.02 (Control, Cd0.02), 1.60 (Cd1.60), 24.29 (Cd24.29), 49.04 (Cd49.04), 117.26 (Cd 117.26), and 289.65 (Cd289.65) mg/kg. A feeding experiment was performed on 540 juvenile black seabreams (initial weight, 1.42 ± 0.01 g), which were conducted for ten weeks. The findings revealed that Cd in the diet lowered weight gain (WG) and specific growth rate (SGR), but had no effect on survival. Dramatically positive linear trends were found between increasing dietary Cd contents and tissue Cd accumulation, including liver, kidney, and muscle. Dietary Cd exposure increased malondialdehyde (MDA) production in both serum and liver, hence, caused oxidative stress. The endoplasmic reticulum (ER) stress-related genes, including grp78, atf6, and xbp1, were markedly increased with increased dietary Cd levels, showing ER stress was triggered. Likewise, transcriptional expression levels of genes related to anti-stress response mt2, hsp70, and hsp90 were linearly up-regulated with increased dietary Cd contents. Regarding apoptosis, the transcriptional expression levels of pro-apoptosis genes caspase7, caspase9, and bax and anti-apoptosis gene bcl-2 were all significantly promoted with the increasing dietary Cd levels. Similar patterns were found for gene expression levels linked to inflammatory response, as the dietary Cd contents increased, nuclear transcription factor nf-κb, pro-inflammatory cytokines tnfα and il-1β, and anti-inflammatory il-10 and tgfβ were dramatically elevated. In conclusion, dietary Cd supplementation suppressed growth performance, increased Cd deposition in tissues, induced oxidative stress, ER stress, apoptosis, and inflammation. In contrast, black seabream juveniles also showed substantial tolerance for dietary Cd exposure by promoting anti-stress ability.

The quality of crustacean aquatic products is affected by feed. Cholesterol (CHO), an essential element for crustacean growth, has been widely supplemented in diet, but its food quality regulation remains unclear. The study aimed to investigate the effects of different dietary CHO levels (0.12%, 1.00%, and 2.50%) on the nutritional value and flavor quality in the edible parts of female swimming crabs (Portunus trituberculatus). Results showed that dietary CHO levels significantly increased lipid content in the hepatopancreas and promoted the accumulation of n-3 long-chain polyunsaturated fatty acids (LC-PUFA) in the hepatopancreas and muscle by activating the gene expression related to biosynthesis pathways. However, with dietary CHO levels increased, protein content in muscle decreased significantly. This may be related to dietary CHO supplementation (especially 2.50% CHO level) suppressed amino acid accumulation in the hepatopancreas and muscle by downregulating the target of the rapamycin pathway and upregulating amino acid catabolism-related genes. Moreover, 1.00% CHO treatment had higher relative levels of volatiles, producing grassy, fruity, and fatty odors in muscle, which may be due to the upregulation of the branched-chain amino acid transaminase (bcat) expression level. Dietary CHO weakened nucleotide and free amino acid accumulation in hepatopancreas and muscle. Overall, this study suggests that dietary 1.00% CHO level had higher LC-PUFA and pleasing flavor substances in muscle but was not conducive to hepatopancreatic protein and flavor nucleotide deposition of swimming crab.

Cholesterol, as an indispensable nutrient, regulates molting and growth in crustacean. As crustaceans are unable to biosynthesize cholesterol de novo, it is central to understand how dietary cholesterol affects molting in crustaceans. An 8-week feeding trial was conducted to evaluate the effects of dietary cholesterol level (0.12%, 0.43%, 0.79%, 1.00%, 1.30% and 2.50%) on growth, cholesterol metabolism and expression of genes related to lipid and ecdysone metabolism in female swimming crabs (Portunus trituberculatus). A total of 192 crabs (1.41 ± 0.05 g) were randomly distributed into 192 aquaria. Each treatment had 4 replicates with each replicate containing 8 crabs. Crabs fed the 1.00% cholesterol diet showed best growth performance, and thus based on percent weight gain, the optimal dietary cholesterol requirement was calculated at 1.01%. Tissue cholesterol concentrations were positively correlated with dietary cholesterol level. The contents of functional fatty acids in hepatopancreas significantly increased as dietary cholesterol increased from 0.12% to 2.50% (P

As a functional fatty acid, Arachidonic acid (ARA) plays an important role in immunity due to its metabolites in mammals, which have been extensively studied in mammals. However, studies on the function of metabolites produced by dietary ARA in aquatic animals are not comprehensive and in-depth. Hence, an eight-week feeding trial was conducted to evaluated the regulatory effects of dietary ARA and its metabolites-eicosanoids on inflammation, oxidative stress and apoptosis in the liver of Acanthopagrus schlegelii with an initial weight of 0.99 ± 0.10 g. Six isonitrogenous and isolipidic (41% protein, 14% lipid) diets were formulated with gradient levels of ARA: 0% (ARA0.10), 0.5% (ARA0.59), 1.0% (ARA1.04), 1.5% (ARA1.42), 2.0% (ARA1.94), 2.5% (ARA2.42), respectively, and palmitic acid was used to regulate lipid levels. Results showed that the key enzyme contents of ARA metabolic pathway such as phospholipase A2 (PLA2), cyclooxygenase-2 (COX-2), lipoxygenase (LOX) cytochrome P450 (CYP450), prostaglandin E2 (PGE2), leukotriene C4 (LTC4) and 20-hydroxyeicosatetraenoic acid (20-HETE) in the liver were increased with dietary ARA levels increased. High levels of dietary ARA (1.94% and 2.42%) activated the gene expression levels of nuclear factor kappa B (nf-κb), which were confirmed by the results of immunofluorescence assay. Likewise, the expression levels of pro-inflammatory cytokines (tnf-α and il-1β) were up-regulated by ARA1.94 and ARA2.42 groups. Fish fed with excess ARA level has significantly low antioxidant capacity by decreasing the activity of related antioxidant parameters and down-regulating the expression level of related genes. The pro-apoptotic related genes (bax and caspase 7/9) were markedly up-regulated as dietary ARA levels increased from 1.04% to 2.42%. Collectively, excess dietary ARA levels (1.92% and 2.42%) produced excess eicosanoids, triggering inflammation via activating NF-κB expression and promoting its nuclear translocation, resulting in oxidative stress by reducing antioxidant parameters (SOD, GSH and GPX), inducing cell apoptosis through up-regulating pro-apoptotic gene expression levels.

An eight weeks feeding trial was conducted to evaluate the potential of cottonseed oil (CSO) as a substitute for fish oil (FO) on juvenile black seabream (Acanthopagrus schlegelii) (2.29 ± 0.01 g). Five diets were formulated with pure FO (FO) or partial CSO replacement of FO: 20% CSO (CSO20), 40% CSO (CSO40), 60% CSO (CSO60) and 80% CSO (CSO80), respectively. Results showed that fish fed CSO60 diet significantly increased growth performance compared to control group. Hepatic expression of genes related to long-chain polyunsaturated fatty acid biosynthesis were significantly up-regulated by dietary CSO60 compared to FO group. Hepatic paraffin section and serum biochemical indices showed that fish fed dietary CSO80 could increase vacuolar fat drops and cause liver damage. The expression levels of peroxisome proliferators-activated receptor alpha and its downstream lipolysis related genes were markedly up-regulated with the increasing of replacement levels of CSO, opposite results were recorded in sterol regulatory element-binding protein-1c and lipogenesis related genes. The malondialdehyde content in liver was decreased with the increasing replacement levels of CSO, whereas the antioxidant parameters were significantly higher in CSO60 group compared to FO group. Phosphatidylinositol-3 kinase and protein kinase B expression levels were significantly up-regulated by dietary CSO80, triggering inflammation by activating nuclear factor kappa b. Dietary CSO60 dramatically promote target of rapamycin expression level. With the increasing replacement levels of FO by CSO, the expression level of ribosomal protein S6 kinase 1 was significantly increased, whereas opposite result was found for the eukaryotic translation initiation factor 4E binding protein 2. Collectively, based on weight gain, double broken line model analysis showed that a maximum growth occurred at 59.13% replacement of dietary FO by CSO. These findings demonstrate that dietary CSO60 can improve growth and antioxidant capacity. However, dietary CSO80 can result in lipid deposition, liver injury and inflammation, hence, suppress growth performance.

Cholesterol, which acts as a substrate for the synthesis of ovarian sex hormones, could promote ovarian development. However, crustaceans cannot synthesize cholesterol and must obtain it from diet for growth, molt and gonadal development. Three isonitrogenous and isolipidic diets were formulated to contain 0.12%, 1.00% and 2.50% cholesterol, respectively. The results showed that dietary 1.00% and 2.50% cholesterol significantly increased percent weight gain (PWG) and specific growth rate (SGR), moreover, crabs fed diet with 1.00% cholesterol had the highest gonadosomatic index (GSI) among all treatments. Cholesterol (CHO) and triglyceride (TG) concentrations in tissues significantly increased with dietary cholesterol levels increasing from 0.12% to and 2.50%. Targeted metabolomics analyses indicated that crabs fed 1.00% cholesterol diet exhibited higher steroid hormones, such as progesterone, pregnenolone, estrone, estradiol and estriol than those fed the other diets. In addition, crabs fed the 1.00% cholesterol diet significantly up-regulated the expression of genes related to cholesterol transport, steroid hormone synthesis and ovarian development in hepatopancreas and ovary. Transcriptomics analyses revealed that DEGs (differentially expressed genes) in crabs fed 1.00% cholesterol diet were associated with “metabolism of xenobiotics by cytochrome P450″ and “progesterone-mediated oocyte maturation”. In conclusion, the results of present study demonstrated that dietary 1.00% cholesterol promoted growth and ovarian development by improving the cholesterol transport and steroid hormone synthesis for female swimming crabs.

Salinity is an important environmental factor that can affect the metabolism of aquatic organisms, while cholesterol can influence cellular membrane fluidity which are vital in adaption to salinity changes. Hence, a 4-week feeding trial was conducted to evaluate the effects of water salinity (normal 23 psu and low 5 psu) and three dietary cholesterol levels (CH0.16, 0.16%, CH1.0, 1.0% and CH1.6, 1.6%) on osmoregulation, cholesterol metabolism, fatty acid composition, long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis, oxidative stress (OS), and endoplasmic reticulum stress (ERS) of the euryhaline fish black seabream (Acanthopagrus schlegelii). The results indicated that in low salinity, fish fed with the CH1.0 diet improved ion reabsorption and osmoregulation by increased Na+ concentration in serum as well as expression levels of osmoregulation-related gene expression levels in gills. Both dietary cholesterol level and water salinity significantly affected most cholesterol metabolic parameters in the serum and tissues, and the results showed that low salinity promoted cholesterol synthesis but inhibited cholesterol catabolism. Besides, in low salinity, hepatic expression levels of LC-PUFA biosynthesis genes were upregulated by fed dietary cholesterol supplementation with contents of LC-PUFAs, including EPA and DHA being increased. Malondialdehyde (MDA) was significantly increased in low-salinity environment, whereas MDA content was decreased in fish fed with dietary CH1.0 by activating related antioxidant enzyme activity and gene expression levels. A similar pattern was recorded for ERS, which stimulated the expression of nuclear factor kappa B (nf-κb), triggering inflammation. Nevertheless, fish reared in low salinity and fed with dietary CH1.0 had markedly alleviated ERS and downregulated gene expression levels of pro-inflammatory cytokines. Overall, these findings demonstrate that cholesterol, as an important nutrient, plays vital roles in the process of adaptation to low salinity of A. schlegelii, and provides a new insight into underlying adaptive strategies of euryhaline marine fish reared in low salinity.

An eight-week feeding trial was conducted to investigate the effects of dietary lipid sources on growth performance, fatty acids composition in tissue and expression of genes related to lipid metabolism in juvenile largemouth bass (Micropterus salmoides). Four experimental diets were formulated to contain 6% coconut oil (CO), rapeseed oil (RO), linseed oil (LO) and fish oil (FO), respectively. Each diet was fed to triplicate groups of juvenile for 8 weeks. The results indicated that final body weight (FBW), percent weight gain (PWG), specific growth rate (SGR), feed efficiency (FE), survival, hepatopancreas index (HSI), viscerosomatic index (VSI) and intraperitoneal fat ratio (IPF) were not significantly influenced by dietary different lipid sources (P＞0.05). Moreover, proximate composition in whole body, dorsal muscle and liver were also not significantly affected by dietary different lipid sources (P ＞0.05). Fish fed with FO diet was significantly higher MDA concentration in hepatic and serum than those fed with CO, RO and LO diets (P

The present study was aimed at evaluating the regulatory effects of dietary lipid levels on growth performance, osmoregulation, fatty acid composition, lipid metabolism, and physiological response in Acanthopagrus schlegelii under low salinity (5 psu). An 8-week feeding trial was conducted in juvenile A. schlegelii with an initial weight of 2.27±0.05 g, and six isonitrogenous experimental diets were formulated with graded levels of lipid: 68.7 g/kg (D1), 111.7 g/kg (D2), 143.5 g/kg (D3), 188.9 g/kg (D4), 239.3 g/kg (D5), and 269.4 g/kg (D6), respectively. Results indicated that fish fed with diet containing 188.9 g/kg lipid significantly improved growth performance. Dietary D4 improved ion reabsorption and osmoregulation by increasing the concentrations of Na+, K+, and cortisol in serum and activities of Na+/K+-ATPase as well as expression levels of osmoregulation related to gene expression levels in the gill and intestine. The expression levels of long chain polyunsaturated fatty acid biosynthesis-related genes were dramatically upregulated when dietary lipid levels increased from 68.7 g/kg to 189.9 g/kg with levels of docosahexaenoic (DHA), eicosapentaenoic (EPA), and DHA/EPA ratio being highest in the D4 group. When fish fed dietary lipid levels from 68.7 g/kg to 188.9 g/kg, lipid homeostasis could be maintained by upregulating sirt1 and pparα expression levels, whereas lipid accumulation was observed in dietary lipid levels of 239.3 g/kg and over. Fish fed with high dietary lipid levels resulted in physiological stress related to oxidative stress and endoplasmic reticulum stress. In conclusion, based on weight gain, the optimal dietary lipid requirement of juvenile A. schlegelii reared at low-salinity water is 196.0 g/kg. These findings indicate that the optimal dietary lipid level can improve growth performance, n-3 LC-PUFA accumulation, and osmoregulatory ability and maintain lipid homeostasis and normal physiological functions of juvenile A. schlegelii.

The present study aimed to elucidate the mechanism of dietary betaine, as a lipid-lowering substance, on the regulation of lipid metabolism and inflammation in juvenile black seabream (Acanthopagrus schlegelii) fed a high fat diet. An 8-week feeding trial was conducted in black seabream with an initial weight of 8.39 ± 0.01g fed four isonitrogenous diets including Control, medium-fat diet (11%); HFD, high-fat diet (17%); and HFD supplemented with two levels (10 and 20 g/kg) of betaine, HFD+B1 and HFD+B2, respectively. SGR and FE in fish fed HFD+B2 were significantly higher than in fish fed HFD. Liver histology revealed that vacuolar fat droplets were smaller and fewer in bream fed HFD supplemented with betaine compared to fish fed HFD. Betaine promoted the mRNA and protein expression levels of silent information regulator 1 (Sirt1), up-regulated mRNA expression and protein content of lipid peroxisome proliferator-activated receptor alpha (pparα), and down-regulated mRNA expression and protein content of sterol regulatory element-binding protein-1(srebp-1). Furthermore, the mRNA expression levels of anti-inflammatory cytokines in liver and intestine were up-regulated, while nuclear factor kB (nf-kb) and pro-inflammatory cytokines were down-regulated by dietary betaine supplementation. Likewise, in fish that received lipopolysaccharide (LPS) to stimulate inflammatory responses, the expression levels of mRNAs of anti-inflammatory cytokines in liver, intestine and kidney were up-regulated in fish fed HFD supplemented with betaine compared with fish fed HFD, while nf-kb and pro-inflammatory cytokines were down-regulated. This is the first report to suggest that dietary betaine could be an effective feed additive to alleviate hepatic steatosis and attenuate inflammatory responses in black seabream fed a high fat diet by modulating the Sirt1/Srebp-1/Pparɑ pathway.

The present study aimed to investigate whether dietary choline can regulate lipid metabolism and suppress NFκB activation and, consequently, attenuate inflammation induced by a high-fat diet in black sea bream (Acanthopagrus schlegelii). An 8-week feeding trial was conducted on fish with an initial weight of 8·16 ± 0·01 g. Five diets were formulated: control, low-fat diet (11 %); HFD, high-fat diet (17 %); and HFD supplemented with graded levels of choline (3, 6 or 12 g/kg) termed HFD + C1, HFD + C2 and HFD + C3, respectively. Dietary choline decreased lipid content in whole body and tissues. Highest TAG and cholesterol concentrations in serum and liver were recorded in fish fed the HFD. Similarly, compared with fish fed the HFD, dietary choline reduced vacuolar fat drops and ameliorated HFD-induced pathological changes in liver. Expression of genes of lipolysis pathways were up-regulated, and genes of lipogenesis down-regulated, by dietary choline compared with fish fed the HFD. Expression of nfκb and pro-inflammatory cytokines in liver and intestine was suppressed by choline supplementation, whereas expression of anti-inflammatory cytokines was promoted in fish fed choline-supplemented diets. In fish that received lipopolysaccharide to stimulate inflammatory responses, the expression of nfκb and pro-inflammatory cytokines in liver, intestine and kidney were all down-regulated by dietary choline compared with the HFD. Overall, the present study indicated that dietary choline had a lipid-lowering effect, which could protect the liver by regulating intrahepatic lipid metabolism, reducing lipid droplet accumulation and suppressing NFκB activation, consequently attenuating HFD-induced inflammation in A. schlegelii.