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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.

As a kind of biologically active molecule, hydroxyproline (Hyp) is abundant in fish meal (FM) but not in plant protein sources. It remains very challenging to replace FM with plant protein sources, particularly for carnivorous fish. This study aims to investigate the effect of dietary Hyp supplementation on Chinese perch (Siniperca chuatsi) fed with FM partially replaced by fermented soybean meal (FSM), with 46% FM as the positive control and 31% FM plus 15% FSM as the negative control. Based on the negative control diet, 0.5%, 1%, 1.5% and 2% of dietary crystal L-Hyp was supplemented to formulate four experimental diets, respectively. Although the replacement of 15% FM with FSM in the feed resulted in a significant decline in growth performance of Chinese perch, dietary supplementation of 1% Hyp could improve the growth performance with high mRNA levels of the gh and igf1 genes. Piecewise regression analysis revealed that a dietary level of 2.27% Hyp could achieve the maximum weight gain rate. With high mRNA levels of orexigenic genes (npy and agrp in the brain, and ghrelin in the gut) and low mRNA levels of anorexigenic genes (pomc and cart in the brain, and leptin-A in the liver), an appropriate level of Hyp could be added to the diet of Chinese perch as a food attractant to promote the appetite and increase food intake. Besides, partial replacement of FM with FSM might promote the deposition of amino acids in the muscle, which may not be affected by dietary supplementation of Hyp. Furthermore, dietary supplementation of Hyp affected the activity of prolyl hydroxylase (PHD) and the mRNA levels of type I collagen regulatory genes and coding genes of three prolyl 4-hydroxylase (P4H) isozymes in the muscle of Chinese perch to some extent. In addition, supplementation of an appropriate level of Hyp could significantly improve the collagen content and muscle quality (hardness, chewiness and springiness) of Chinese perch, which may greatly enhance its edible and nutritional values.

Host-probiotics of many fish species have not been applied due to the difficulty in isolation and functional verification, and some exogenous probiotics were confirmed to promote the growth performance of fish. Therefore, this study aims to screen the exogenous probiotics that can be used in the culture of Chinese perch and to explore their mechanism of action. Chinese perch were respectively fed with basal diet (CK) supplemented with Clostridium butyricum CB1 (C), Enterococcus faecalis EF1 (E), Bacillus subtilis BS1 (B), and Lactobacillus plantarum LP1 (L) at 1.0 × 108 CFU/g with equal amount for 8 weeks. After feeding, B group presented significantly higher abundance of Bacillus, Lactobacillus, and Paenibacillus, and L group displayed significantly higher abundance of Lactobacillus than CK group. Above increased probiotics species led to higher intestinal lipase activity in B group, intestinal protease activity in L group, and length of intestinal villi and thickness of intestinal wall in B and L groups through fermentation to reduce pH value and secretion of active substances, thus facilitating the nutrient absorption to improve the weight gain (WG). Additionally, B and L groups respectively exhibited higher abundance level of the infectious diseases-related pathway and lower abundance of Aeromonas, which was responsible for higher expression of pro-inflammatory cytokines (p65-NF-κB, IL-Iβ, TNFα, and PAI) and higher blood immune parameter levels (globulin (GLB), total protein (TP), and lysozyme activity (LZM)), eventually improving survival rate. E group significantly inhibited microbial diversity and abundance of Energy metabolism pathway and Amino acid metabolism pathway, which might the reason for the decrease in crude protein content in whole-fish. C groups significantly reduced the microbial diversity and the length of intestinal villi to affect the host's nutrient absorption efficiency, thus causing a decrease in growth performance. Moreover, the imperfection of intestinal villi may allow more LPS to penetrate into the blood, thereby down-regulating the immune status and inducing inflammation, ultimately reducing survival rate. Above results indicated that Bacillus subtilis BS1 and Lactobacillus plantarum LP1 might be used in aquaculture, and probiotics may affect growth performance and gut health by regulating gut microbiota.

Taste receptors play important roles in perception of L-amino acids and feeding behavior. However, convincing evidence providing causal links between taste receptors and feeding habits is lacking. We generated two mutant lines with t1r1 deletion in zebrafish by CRISPR/Cas9 technology. t1r1 deletion eliminated sensitivity to alanine in zebrafish. Additionally, it did not affect feeding behavior of zebrafish supplied with habitual food such as Artemia salina, while significantly enhanced their acceptance of rejective food such as duckweed and plant-protein source diets. Furthermore, replacement of fish meal with plant protein in diets significantly suppressed transcript levels of orexigenic gene npy and increased mRNA expression levels of anorexigenic gene pomca in wild-type zebrafish but had no effect in t1r1−/− zebrafish. In addition, when fed with fish meal diet, transcript levels of npy and pomca were significantly different in t1r1−/− zebrafish compared with wild-type zebrafish. Overall, these findings suggested that T1R1 was indispensable for amino acid perception and significantly affected the feeding preference and habit in zebrafish.

Few studies have investigated the effects of long-term low-concentration nitrite exposure and detoxification on growth and physiology of fish. Considering this, the present study focuses on the effect of nitrite exposure and detoxification on Chinese perch. Chinese perch were exposed to water with 0 mg/L (C group) and 0.3 mg/L nitrite (N group) for 30 and 60 days, respectively (C30, C60, N30, N60). No significant differences in the average daily growth (ADG), food intake (FI), feed conversion ratio (FCR), and histology were observed between C30 group and N30 group, while N30 group exhibited significantly lower protein retention and crude protein content and significantly higher levels of blood urea nitrogen (BUN) and higher activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) than C30 group. The ADG, FI, plasma glucose, total triglyceride, total cholesterol, the activities of SOD, CAT and GSH-Px were significantly decreased in N60 group, whereas the levels of methemoglobin, BUN, and immune responses-related indicators (globulin, aspartate aminotransferase, and alanine aminotransferase) were significantly increased. The damages to the gill and liver of fish and the increased expressions of pro-inflammatory cytokines genes (IL-Iβ, p65-NF-κB, TNFα and PAI) were observed in N60 group. In addition, fish exposed to nitrite for 30 and 60 days were detoxified in nitrite-free water for 30 days (DN30 and DN60). The detoxification significantly increased the protein retention and protein content. However, detoxification did not improve the appetite or fully restore histopathological injury in DN60 group. Those results indicated that 30-day exposure to 0.3 mg/L of nitrite promoted protein catabolism for providing energy to activate the defense, and that the detoxification could completely reverse the effects. The 60-day exposure to nitrite could disrupt defense system, cause histopathological damage, and affect energy metabolism. Moreover, nitrite exposure significantly decreased appetite. Above factors might be responsible for growth inhibition. The detoxification post 60-day nitrite exposure only restored partial physiological functions. Our findings provide a reference for the concentration control of nitrite in feed farming of Chinese perch.

Social learning plays pivotal roles in gaining new foraging skills and food preferences. However, the molecular mechanism underlying the effects of social learning on feeding habits is unclear. Chinese perch, as a demersal piscivore, has a unique feeding habit. Once the fry of Chinese perch start feeding, they only accept live prey fish in the wild and refuse dead prey fish or artificial diets. In the present study, we used the inhibitor of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) signaling pathway KN-93 in vivo and in vitro, to explore how social learning can affect the transition from live prey fish to dead prey fish in Chinese perch. Analysis of gene expression, protein phosphorylation, success rate, learning times, and food intake allowed us to gain insights into the molecular mechanism of social learning in acquiring new feeding habits in Chinese perch. Our results showed that the p-CaMKII, phospho-cAMP-response element binding (p-CREB) and proto-oncogenes c-fos (c-fos), and melanin-concentrating hormone (mch) expression levels were significantly lower with KN-93 treatment than levels in the control condition, whereas the expression of the anorexigenic gene proopiomelanocortin (pomc) was significantly higher in vitro. The increased success rate and food intake of Chinese perch induced by social learning, were diminished after the injection of the CaMKII inhibitor KN-93 in vivo. Inhibited signaling of CaMKII was observed in Chinese perch after KN-93 treatment. We also found that the expression levels of the c-fos, mch or pomc genes were increased or decreased in the learning group and then decreased or increased in the learning group with KN-93 injection, respectively. These results indicate that the higher expression of the orexigenic mch gene and the decreased expression of the anorexigenic pomc gene might contribute to the learning of eating dead fish prey in Chinese perch. Altogether, our results showed that social learning could promote the feeding habit transition from live prey fish to dead prey fish through the CaMKII - c-fos - pomc/mch signaling pathway in Chinese perch.

The mTOR signaling pathway integrates energy status with hormonal signals to regulate not only protein synthesis and growth, but also food intake and energy balance. However, the involvement of TOR signaling pathway in food intake regulation is not clearly characterized in fish. To investigate whether TOR signaling is integrated with both hypothalamic neuronal activity and peripheral signaling in Chinese perch (Siniperca chuatsi), intracerebroventricular administration of branched-chain amino acid (BCAA: leucine, valine and isoleucine) and the inhibitor of TOR signaling rapamycin were performed to detect the regulation of TOR signaling on food intake. The results showed that only leucine could stimulate the TOR signaling, but valine and isoleucine inhibit the TOR signaling. The three BCAA also showed different effects on the food intake and the expression of appetite control genes (hypothalamic neuronal signaling: npy, agrp, pomc, and peripheral hormone: ghrelin, leptin). The up-regulation or down-regulation of TOR signaling by leucine or valine, might suppress or stimulate the food intake through modulating the protein levels of anorexigenic POMC, respectively. We also found the same effects of leucine, valine or isoleucine on the TOR signaling and appetite gene expression in the primary brain cells of Chinese perch. In addition, the different regulation of BCAA on TOR signaling could be attributed to the regulation of BCAA on the interaction between SESN2 and GATOR. These results indicate that three BCAA exhibit the different regulatory roles on the TOR signaling and food intake in fish, unlike in mammals, BCAA activate mTOR signaling.

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, 400 mg kg− 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 100 mg kg− 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 50 mg vitamin E kg− 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 100 mg vitamin E kg− 1 diet, whereas it decreased as the content of vitamin E in the diet increased. Based on the broken-line regression analysis of WG, 100.36 mg kg− 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.

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) g kg− 1 diet, respectively. Triplicate groups (nine 300-L tanks) of grass carp (mean weight, 22.37 ± 0.16 g) were fed twice daily (08:00 and 16:00 h) to satiation for 8 weeks. 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

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.

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 1617 bp. 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 402.6 g kg − 1 carbohydrate diets for 8 weeks, respectively. Dietary 402.6 g kg − 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.

Young grass carp (mean total length and weight, 5.5 cm and 1.8 g respectively) at the food transition stage from zooplankton or benthos to aquatic macrophytes, were fed with either duckweed or chironomid larvae for 60 days. Fish fed with duckweed had significantly higher gut growth than those fed with chironomid larvae in terms of length or weight. Consistent with this, the mRNA expression of epidermal growth factor ( Egf ) was higher in fish fed with duckweed than those fed with chironomid larvae, whereas the Igf II mRNA level of fish fed with duckweed was significantly lower than those fed with chironomid larvae. Consistent with higher appetite of fish fed with plant diets, the mRNA levels of neuropeptide Y ( Npy ), neuropeptide Y receptor Y8a ( Npy Y8a ), neuropeptide Y receptor Y8b ( Npy Y8b ) and leptin receptor ( Ob-r ) in fish fed with duckweed were simultaneously higher than those fed with chironomid larvae, and leptin receptor gene related protein ( Ob-rgrp ) expression was lower. The differential expressions might potentially contribute to gut growth of young grass carp fed with duckweed and adapted to herbivory. The mRNA levels of trypsin and amylase in fish fed with duckweed were significantly higher than those fed with chironomid larvae, and lipoprotein lipase expression was significantly lower. Concordant changes in enzyme activities of trypsin and lipase were observed. Our results suggested that in young grass carp, plant food not only stimulated gut growth possibly through Egf , but also up-regulated expressions of Npy , Npy Y8a , Npy Y8b and Ob-r as well as trypsin and amylase to increase the intake and digestion of plant food.