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Plant protein is the main substitution protein for fishmeal, especially rapeseed meal, and it contains anti‐nutritional factors and toxicants such as tannin. Few studies have identified clear physiological or toxicological impacts of tannin on fish. Here, our experiment evaluated the physiological and toxicological effects of tannin on grass carp (Ctenopharyngodon idellus). Different concentrations of dietary tannin (1.5, 7.2, 19.9, 39.4, 83.2 and 162.0 g kg−1 diet) were tested. Dietary tannin (≥7.2 g kg−1 diet) inhibited feed intake, and the appetite‐promoting gene expression (Grehlin, NPY, Y8a and Y8b), meanwhile, increased the appetite‐inhibiting gene expression (CCK and CART). Moreover, tannin addition reduced feed utilization (dietary nutrient retention, fat depot, activity of protease, lipase and amylase) of grass carp (p <.05). Besides, high dietary tannin (≥39.4 g kg−1 diet) lowered immunity (serum superoxide dismutase and catalase activities and the contents of total protein, albumin, complement component C3 and C4) (p <.05) and damaged the hepatic structure of grass carp, leading to attenuated growth in grass carp. This suggests that grass carp could not tolerate dietary tannin of >19.9 g kg−1. The second trial designed to test the alleviation the negative effect of tannin by tannase. After adding tannase with a dose of 1000 or 1500 U kg−1 to the diets, the weight gain of grass carp fed those diets was significantly increased compared with those fed the tannin and rapeseed meal addition groups (p <.05). Also, when high‐level tannase was supplemented to the control diet, weight gain significantly improved. The decrease in feed intake by high dietary tannin and rapeseed meal was relieved through adding tannase in diet of grass carp, especially when tannase supplemented level reached 1000 U kg−1 diet (p <.05). This study indicates that dietary tannin (≥7.2 g kg−1 diet) negatively affects the growth of grass carp through changing feed intake, feed utilization, immunity and hepatic structure, and addition of tannase reached 1000 U kg−1 diet almost alleviate adverse effects of tannin and rapeseed meal (which included high contents of tannin) on grass carp. [ABSTRACT FROM AUTHOR]

This study was conducted to examine the systemic metabolic strategies of grass carp to maintain lipid homeostasis when fed with low- or high-fat diets. The isonitrogenous diets with different fat levels (9.3, 48.7 and 107.9 g kg − 1 ) were fed to grass carp for 8 weeks. After the feeding trial, the growth rate and feed intake of grass carp fed with low-lipid (LL) diet or high-lipid (HL) diet were lower than fish fed with medium-lipid (ML) diet. Serum triglyceride (TG) and total body fat contents were significantly increased in grass carp with increasing lipid intake. Gene expression data indicated that fish increased hepatic gk and pk expressions to elevate glycolysis, and enhanced acc and fas expressions to accelerate biosynthesis of fatty acid (FA) to adapt to low lipid intake. Meanwhile, fish fed with LL diet decreased hepatic cpt1 expression to depress lipolysis, leading to low contents of serum TG and body fat. In contrast, excess lipid intake increased g6pase , pepck and pparα expressions to stimulate gluconeogenesis and β-oxidative, while decreased acc and fas expressions to reduce lipid synthesis in fish liver. Moreover, increased β-oxidative-induced FA or gluconeogenesis-induced serum glucose might induce the appetite suppression by high dietary fat through modulation of leptin expression. This study could be a reference in the systemic adaptation of lipid metabolism responding to dietary fat in fish. Statement of relevance Systemic adaptation of lipid metabolism responding to dietary lipid. [ABSTRACT FROM AUTHOR]

Ghrelin and obestatin are two gastrointestinal peptides obtained by post-translational processing of a common precursor, preproghrelin. The effect of obestatin on food intake is still controversial. The aim of the present study was to investigate the effects of ghrelin and obestatin on food intake in grass carp, Ctenopharyngodon idellus . Fish received intraperitoneal (IP) injection of saline, ghrelin (100 ng g − 1 BW), obestatin-like (25 ng g − 1 BW) and ghrelin in combination with obestatin-like. Ghrelin stimulation of food intake varied considerably among individual fish with 70.8% eliciting a robust response. In these high-responders, food intake was significantly increased by IP ghrelin within 2 h. Co-administration of ghrelin and obestatin-like resulted in a decrease in food intake, indicating that obestatin was able to antagonize the effect of ghrelin. However, IP obestatin-like alone could not regulate food intake in grass carp. RT-PCR analysis demonstrated that IP ghrelin peptide led to a significant increase in mRNA abundance of NPY, Y8a and Y8b genes compared to saline injected fish, while in combination with obestatin-like peptide decreased ghrelin-induced gene expressions of these three genes. IP sole obestatin-like peptide did not modify the expression levels of NPY, Y8a, Y8b, CART and POMC compared to the control group. Therefore, IP administration of obestatin-like peptide, partially blocking the ghrelin-induced appetite, investigated the possible involvement of obestatin as a mediator of the ghrelin stimulatory action on food intake, at least in “high-responders” grass carp. [ABSTRACT FROM AUTHOR]

A study was conducted to determine the effects of dietary non-protein energy sources on growth, tissue lipid accumulation and lipid metabolism-related genes expression of grass carp. Triplicate groups of fish were fed for 9 weeks on four isonitrogenous (300 g kg−1) experimental diets with four levels of non-protein energy (6.52 kJ g−1 control diet, 5.32 kJ g−1 high- CEL diet, 8.46 kJ g−1 high- CHO diet and 8.53 kJ g−1 high- LIP diet respectively). Increasing dietary non-protein energy source levels did not improve the growth, and the high- CEL diet reduced the growth of grass carp. The high- CHO diet tended to induce high hepatosomatic index, with high fat and glycogen content of liver. However, the high- LIP diet caused the high mesenteric fat index, but did not increase liver fat. The m RNA abundance and activities of hepatic lipogenic enzymes were significantly increased in the high- CHO diet group, whereas the opposite tendencies were observed in the high- LIP diet group. Peroxisome proliferator-actived receptor-α ( PPARα) in liver and PPARγ in mesenteric adipose tissue were up-regulated in the high- CEL diet group. Lipoprotein lipase ( LPL) gene expression was significantly increased both in liver and mesenteric adipose tissue of fish fed the high- LIP diet, while the LPL gene expression was up-regulated in liver but down-regulated in mesenteric adipose tissue of fish fed the high- CEL diet. These findings suggest that an increase in dietary non-protein energy sources alters the genes expression of lipid metabolism and increased lipid deposition. [ABSTRACT FROM AUTHOR]

Abstract: Plant protein sources have been identified to have the greatest potential to replace fish meal protein in aquafeeds. However, plant ingredients contain significant quantities of carbohydrates, and the ability of fish to utilize dietary carbohydrates as energy sources for growth is limited. Included in the carbohydrate group, cellulose is not tolerated by most fish. In the present study, the grass carp, a typical herbivorous fish, fed with duckweed was selected to study the effect of exogenous cellulase on the growth. The results of 2-month feeding experiment showed that the cellulase promoted the growth of grass carp. In addition, the cellulase increased various digestive enzyme activities, such as cellulase, amylase and protease but not the lipase activity. Meanwhile, the polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) analysis indicated that the intestinal microbiota of fish fed with the supplemental cellulase changed in bacteria species and density. Band patterns derived from control and cellulase samples showed a low degree of similarity when analyzed by cluster analysis. Some bands were unique to control samples, whereas other bands were obtained only with samples of the cellulase group. The 16S rRNA gene sequencing identified that Proteobacteria and Firmicutes were the two dominant groups, and the emergence of certain bacterial strains including Bacilli and Sphingomonas may contribute to the digestion of cellulose. The former researches and this paper results suggest that the endogenous cellulase is far from sufficient to fully digest the ingested fiber, so cellulase should be developed as a kind of aquatic additive. [Copyright &y& Elsevier]

Abstract: The R/L type pyruvate kinase (PKLR) gene was cloned from liver of grass carp (Ctenopharyngodon idella) by degenerate oligonucleotide primed PCR, including a complete open reading frame (ORF) of 1617bp. Homological protein analysis showed that the PKLR in grass carp shared a high degree of sequence identity with other known PK sequences. In addition, tissue distribution analysis showed that the highest expression of the PKLR was detected in liver. Then triplicate groups of grass carp (9 tanks) were fed twice daily to satiation with 91.8, 192.9, and 402.6gkg−1 carbohydrate diets for 8weeks, respectively. Dietary 402.6gkg−1 carbohydrate decreased growth performance, feed intake and feed utilization. With increasing dietary carbohydrates, the hepatic PK activity and mRNA expression of grass carp were up-regulated significantly. The results indicated that the PKLR play a modulation role in the adaptation of hepatic glycolysis to dietary high carbohydrates in grass carp. [Copyright &y& Elsevier]

As typical herbivorous fish, grass carp possesses greater capability to utilize carbohydrate than carnivorous and omnivorous fish. However, mechanism of carbohydrate utilization in herbivorous fish was unclear. This study is intended to investigate response of grass carp to insufficient or excessive dietary carbohydrate. Grass carp accepted isonitrogenous diets with different carbohydrate levels (15.90, 21.81 and 45.45 g 100 g −1 ) were cultured. After 8 weeks, weight gain and feed intake were highest in fish fed with medium-carbohydrate diet compared with others. Gene expression data indicated that expressions of glucose-6-phosphatase (G6Pase) were down-regulated and gene expressions of glycogen synthase (GS) were up-regulated in fish accepted lower-carbohydrate diet. In high-carbohydrate group, glucose catabolism was increased by enhanced expressions of glucokinase genes (GK). Meanwhile, fatty acid synthase (FAS) and acetyl coenzyme-A carboxylase (ACC1) genes were significantly up-regulated and lipoprotein lipase and carnitine palmitoyltransferase 1 (CPT1) genes expressions were down-regulate, inducing more lipid deposition. Subsequently, serum cholesterol and total lipid contents were significantly increased, and then induced appetite suppression through up-regulation of leptin and anorexigenic genes expressions. Furthermore, depressed food intake caused lower growth rate in grass carp with excessive carbohydrate intake. [ABSTRACT FROM AUTHOR]

Insulin receptors have been demonstrated to be involved in embryogenesis, food intake regulation and glucose metabolism in several fish, while more researchis needed for further understanding. In this study, the complete coding sequence (CDS) of insulin receptor a (insra) gene and insulin receptor b (insrb) gene in grass carp were obtained, the CDS were 4068 bp and 4514 bp in length, encoding 1355 aa protein and 1351 aa protein. Both of insra and insrb in grass carp showed high amino acid identities with other fish. Insra and insrb genes were widely expressed in all tested tissues with an overlapping but distinct expressions. The high levels of insra mRNA were distributed in hindgut and heart tissues. The insrb gene showed the highest expression levels in liver and hindgut. We also proved that two forms of grass carp insulin receptors participate in the regulation of blood glucose and might act differently. Phylogenetic analysis confirmed that different isoforms of fish insulin receptors are derived from two distinct genes, which was inconsistent with the generation of mammalian insulin receptors. Synteny analyses of insulin receptor genes showed that genes surrounding the insulin receptor genes were conserved in fish. Arhgef18, PEX11G, humanC19orf45 genes were highly conserved among mammal species. However, no conserved synteny was observed among fish, mammals, avians and amphibians. [ABSTRACT FROM AUTHOR]

Abstract: Neuropeptide Y (NPY) has been established as an important regulator of food intake in mammals. However, the involvement of NPY in the feeding behavior of fish has not yet been fully understood. The present study investigated the role of NPY in food intake in grass carp. The full-length cDNA sequence of gc-NPY was 797bp, and an ORF of 96 amino acids, including the 28-residue signal peptide and 36-residue mature peptide, which had high identities to the NPY of cyprinid fishes. Two receptors Y1 and Y5 which mediate the appetite-stimulating effects of NPY in mammals lost from some of the euteleost genomes. However, sequences of other NPY receptors Y8a and Y8b were obtained from the ESTs library of grass carp in our previous studies. NPY and both Y8 receptors were predominantly expressed in the brain. Cumulative food intake was significantly increased by intracerebroventricular administration of NPY during a 72-h observation period. For better understanding of NPY''s action in grass carp, the mRNA levels of appetite regulators, energy metabolism genes and key digestive enzymes were assessed. The results showed that the mRNA levels of Y8b were significantly increased, while the expressions of cholecystokinin (CCK), cocaine and amphetamine regulated transcript (CART), pituitary adenylate cyclase-activating peptide (PACAP), leptin, leptin receptor (leptinR) and leptin receptor overlapping transcript protein (LEPROT) were significantly decreased after NPY injection. In addition, the marked stimulation of insulin-like growth factor1 (IGF1), insulin-like growth factor 1 receptor (IGF1R), lipoprotein lipase (LPL), Stearoyl-CoA desaturase 1 (SCD1), amylase and trypsin mRNA levels in liver were caused by NPY administration, and the mRNA level of uncoupling protein 1(UCP 1) tended to decrease but not significantly. These results indicate that NPY acts as an orexigenic factor in the grass carp, and Y8b may involve in the feeding regulation. NPY not only affects food intake, but also has marked effects on energy metabolism in grass carp. Moreover, NPY seems to closely interact with other appetite regulators, and the present results provide novel insight into these complex interactions. Further understandings of appetite regulation in fish ultimately have the potential to yield methods for improving feed efficiency in aquaculture. [Copyright &y& Elsevier]

Dietary carbohydrate levels can affect gut health, but the roles played by gut microbiota and gut epithelial cells, and their interactions remain unclear. In this experiment, we investigated gut health, gut microbiota, and the gene expression profiles of gut epithelial cells in grass carp consuming diets with different carbohydrate levels. Compared to the moderate-carbohydrate diet, low-carbohydrate diet significantly increased the relative abundance of pathogenic bacteria (Ralstonia and Elizabethkingia) and decreased the abundance of metabolism in cofactors and vitamins, implying a dysregulated gut microbiota and compromised metabolic function. Moreover, low-carbohydrate diet inhibited the expression levels of key genes in autophagy-related pathways in gut epithelial cells, which might directly lead to reduced clearance of defective organelles and pathogenic microorganisms. These aforementioned factors may be responsible for the imperfect organization of the intestinal tract. High-carbohydrate diet also significantly increased the abundance of pathogenic bacteria (Flavobacterium), which directly contributed to a decrease in the abundance of immune system of the microbiota. Furthermore, the active pathways of staphylococcus aureus infection and complement and coagulation cascades, as well as the inhibition of the glutathione metabolism pathway were observed. Above results implied that high-carbohydrate diet might ultimately cause severe gut damage by affecting immune function of microbiota, mentioned immune-related pathways, and the antioxidant capacity. Finally, the correlation network diagram revealed strong correlations of the differentially immune-related gene major histocompatibility complex class I antigen (MR1) with Enhydrobacter and Ruminococcus_gnavus_group in low-carbohydrate diet group, and Arenimonas in high-carbohydrate diet group, respectively, suggesting that MR1 might be a central target for immune responses in gut epithelial cells induced by gut microbiota at different levels of dietary carbohydrate. All these results provided insight in the development of antagonistic probiotics and target genes to improve the utilization of carbohydrate. • Microbiota takes part in the process of dietary carbohydrate affecting gut health. • Inhibition of autophagy-related pathways may cause inadequate villi in LC group. • Dietary excessive carbohydrate causes oxidative stress and severe gut damage. • MR1 may be a central target for gut immune responses induced by gut microbiota. [ABSTRACT FROM AUTHOR]

Abstract: An 8-week feeding trial was conducted to investigate the effects of dietary vitamin E on growth performance and antioxidant status of juvenile grass carp. Semi-purified diets containing six levels of all-rac-α-tocopheryl acetate (0, 25, 50, 100, 200, 400mgkg−1) were assayed in triplicate. Growth performance, body composition, antioxidant activity and serum biochemistry parameter were determined. The maximum weight gain (WG), specific growth ratio (SGR) and feed intake (FI) were achieved in fish fed on a diet supplemented with 100mgkg−1 vitamin E. Vitamin E supplementation appeared to improve hepatic glutathione peroxidase (GPx) activity significantly, and higher activity of hepatic superoxide dismutase (SOD) was displayed in diets with excess vitamin E. However, serum SOD and catalase (CAT) activity were enhanced in grass carp fed 50mg vitamin Ekg−1 diet, and then decreased significantly as the content of vitamin E in the diet increased. A consistent decline in the hepatic and serum malondialdehyde (MDA) content was observed in fish fed diets with the increased supplementation of vitamin E. In addition, with the increasing level of vitamin E, serum alanine aminotransferase (ALT) was reduced. Serum alkaline phosphatase (AKP) activity was enhanced in fish fed 50 and 100mg vitamin Ekg−1 diet, whereas it decreased as the content of vitamin E in the diet increased. Based on the broken-line regression analysis of WG, 100.36mgkg−1 vitamin E was the optimal requirement of juvenile grass carp. The benefits of excess vitamin E levels need to be weighed carefully: on the one hand might enhance the antioxidant capacity, on the other hand might decrease growth. [Copyright &y& Elsevier]

Abstract: The negative effect of dietary phytic acid (PA) in feed intake is a common feature of response to stress in fish, but the regulation mechanism of feed intake is poorly understood. Our study was therefore conducted to estimate the effects of dietary PA on feed intake, feed utilization and feeding-related gene expression in juvenile grass carp. The levels of dietary PA supplementation were 0 (control), 5 (low) and 40 (high) gkg−1 diet, respectively. Triplicate groups (nine 300-L tanks) of grass carp (mean weight, 22.37±0.16g) were fed twice daily (08:00 and 16:00h) to satiation for 8weeks. Supplemental PA decreased the weight gain, feed intake, feed utilization and digestive enzyme activities of grass carp. Dietary PA supplementation decreased the apparent digestibilities of phosphorus, calcium and crude protein. The gene expression levels of cocaine- and amphetamine-regulated transcript (CART) and cholecystokinin (CCK) in the brain were enhanced with the increase of dietary PA supplementation. However, the neuropeptide Y (NPY) and ghrelin mRNA expression levels were reduced in fish fed with low PA, but increased significantly (P<0.05) in fish fed with high PA compared to the control. No significant differences were observed in the gene expressions of NPY receptors Y8a and Y8b among all the groups. The results of this study indicated that the decrease of feed utilization and the increase of CART and CCK gene expressions in the brain might be the main factors for the decrease of feed intake in grass carp caused by dietary PA supplementation. [Copyright &y& Elsevier]

Abstract: Fish species have poor capability of carbohydrate metabolism relative to mammals. Specifically, fish cannot utilize dietary glucose well. Heat shock protein 72 (Hsp72) can be induced by heat shock and prevent stress-induced activation of c-Jun amino N-terminal kinase (JNK) which then promotes the insulin signaling pathway in mammals. However, the function of Hsp72 on glucose utilization is unknown in fish. We conducted a study to understand the role played by heat shock on glucose utilization using juveniles of the grass carp fish. In this study, the juveniles of grass carp were fed with either high glucose diet or control diet. Half of the juveniles were subjected to several temporary heat shock treatments within 8weeks. The growth performance, body composition of juveniles and the expression of glucose metabolism related genes, including Hsp72 in the livers of juveniles were analyzed from each group. The heat shock treatments could improve growth performance and body composition of grass carp juveniles, and could activate JNK. Though the expression of Hsp72 increased significantly in Group GH (juveniles fed with high-glucose diet and given heat shock treatment) (P <0.05) as measured by fluorescent real time PCR and Western blot, the expression of JNK and p-JNK were not suppressed by Hsp72 in our study, which was different from some previous studies in mammals. In summary, the study showed that heat shock did not affect glucose metabolism through JNK pathway in grass carp. This may give a clue to explain why fish species have a much poorer capability of carbohydrate metabolism than mammals. [Copyright &y& Elsevier]