Differential expression of lipid metabolism‐related genes and miRNAs in Ctenopharyngodon idella liver in relation to fatty liver induced by high non‐protein energy diets

To explore the mechanism of fatty liver formation induced by high non‐protein energy diets in grass carp (Ctenopharyngodon idella), basal diet and high‐energy diets were fed to juvenile grass carp for 9 weeks. The experimental groups fed on high‐energy diets which included a high‐lipid diet (H‐LIP), a high‐carbohydrate diet (H‐CHO) and a high‐lipid and carbohydrate diet (H‐CL). The control group fed on basal diet. Growth performance, liver fat accumulation, serum biochemical indexes and the expression levels of lipid metabolism‐related genes (SREBP‐1, PPARγ, FAS, ACC1, and LPL) and miRNAs (miR‐33, miR‐122, and miR‐370) were examined at the end of the feeding trial. There were no significant differences in growth rate and feed efficiency among the four groups. However, significant increase in mesenteric and liver fat contents, and lipid droplets in the liver was induced by high‐lipid and high‐carbohydrate diets. There were significant differences in serum biochemical indicators such as AST/ALT, GLB, TG and TP, and liver fatty acid composition between the control and experimental groups. The expression levels of SREBP‐1, PPARγ, FAS, ACC1 and LPL were upregulated, while CPT‐1 was downregulated with the high‐energy treatments. Additionally, the expression levels of miR‐33, miR‐122 and miR‐370 in the liver were higher in the three high‐energy treatments than those in the control (P < 0.05). The results suggest that modifications of lipid metabolism‐related genes and miRNAs may be involved in fatty liver formation induced by high non‐protein energy diets in grass carp.     

Effects of a dietary administration of purple coneflower (Echinacea purpurea) on growth,antioxidant activities and 8 miRNAs expressions in crucian carp (Carassius auratus)

Echinacea purpurea (EP), a globally popular herbal medicine, has been used to treat various diseases in human and animals. However, little has been reported about its effects in fish. In this study, crucian carp (Carassius auratus) were selected to evaluate the effects of EP on growth performance and antioxidant response and the expressions of microRNAs. The results showed EP could stimulate the growth of crucian carp with the best effect was observed at dose of 4 g kg^?1. In serum, the content of hydroxyl radical (·OH) and malondialdehyde (MDA) decreased by EP supplementation, whereas the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) increased. Similarly, the content of ·OH decreased, and the activities of CAT, GPX and GR increased in liver of crucian carp. Furthermore, in livers of crucian carp, EP supplements upregulated the expressions of the microRNAs (miR‐16, miR‐21, miR‐125b, miR‐146a, miR‐155, miR‐181a and miR‐223), which had been confirmed to participate in regulating antioxidant and immune function in mammals. Our results suggest EP supplements in diets stimulated growth performance and antioxidant response of crucian carp. In liver, the upregulation of specific miRNAs may participate in the antioxidant effects of EP diets.     

Evidence of rapid transfer and bioaccumulation of Microcystin‐LR poses potential risk to freshwater prawn Macrobrachium rosenbergii (de Man)

Microcystins accumulate in aquatic organisms and can be transferred to higher trophic levels, eventually affecting vector animals and consumers. We examined three levels of an aquatic food chain (Microcystis aeruginosa, Daphnia magna and Macrobrachium rosenbergii) to identify the transfer efficiency and risk of microcystin on prawns. Samples were analysed using ultra performance liquid chromatography‐mass spectrometry (MS)/MS and microcystin‐LR (MC‐LR) distributions in prawn tissues were studied. The results showed that prawns accumulate MC‐LR both directly from M. aeruginosa and indirectly through D. magna which was pre‐exposed to M. aeruginosa. MC‐LR was detected in the gills, digestive tracts and hepatopancreas of the prawns 2 h after exposure. MC‐LR accumulated in prawns to 0.49 ± 0.04 μg g^?1 dry weight in hepatopancreas within 24 h, while it was not detected in muscle samples, and rarely appeared in blood samples in such a short period. Although MC‐LR was not detected in muscle, the head including hepatopancreas of the prawns accumulated troublesome amounts of MC‐LR. These results demonstrate that microcystis blooms in prawn farming potentially pose a risk to human consumers, although prawns may be exposed to the bloom for a very short time, hence regular monitoring of blue green algae population is recommended.     

Effects of dietary Bacillus natto supplementation on growth performance and the growth‐related gene/microRNA expression in the skeletal muscle of grass carp (Ctenopharyngodon idella)

In this study, Bacillus natto NT was evaluated for use as a probiotic supplement in the feeds on the growth performance and the growth‐related genes' and microRNAs' (miRNAs) expression in the skeletal muscle of grass carp (Ctenopharyngodon idella). Grass carps (ave. wt. 43.96 ± 0.27 g) were fed diets supplemented with 1.87 × 10⁹ (Bn1), 3.73 × 10⁹ (Bn2), 5.60 × 10⁹ (Bn3), 7.47 × 10⁹ (Bn4) and 9.33 × 10⁹ (Bn5) B. natto NT cells per 100 g feed for 56 days. The control group (BnC) was not supplemented with the B. natto NT. The fish of Bn3, Bn4 and Bn5 groups displayed better growth performance and lower feed conversion ratio (FCR) than the other groups (P < 0.05). Compared with the fish of BnC group, miR‐1a, miR‐181a, miR‐23a and miR‐206 expressions increased (P < 0.05), and the myostatin and myocyte enhancer factor C (MEF2C) mRNAs were down‐regulated (P < 0.05) in the fish of Bn4 and Bn5 groups. The specific growth rate analysis and apparent expressional regulation of the growth‐related miRNAs and genes stimulated by B. natto suggest the potential application of B. natto in improving the growth performance on the grass carps.     

Screening and identification of microRNAs during larval metamorphic development of Chinese mitten crab Eriocheir sinensis

MicroRNAs (miRNAs) represent a class of endogenous small non‐coding RNAs with 18–25 nucleotides that are involved in diverse biological processes. In the present study, differentially expressed miRNAs in four different metamorphic stages of Eriocheir sinensis were comprehensively detected and validated from high‐throughput sequencing miRNA data sets. We identified and characterized 597 known miRNAs and 901 novel miRNAs from the four small RNA libraries. We also demonstrated the evolutionary conservation of miRNAs from Chinese mitten crab with those from other species. Analyses of miRNA expression profiles during brachyurization development of the Chinese mitten crab revealed that 86 mature miRNAs were dysregulated, 47 of which were significantly differentially expressed. The results of real‐time quantitative PCR analyses for a few selected miRNAs were similar to those of deep sequencing analyses. Intriguingly, the results of Gene Ontology annotation and KEGG pathway analyses indicated that miRNA gene clusters and families were consistently dysregulated at four different metamorphic stages, although they were enriched at various levels. Functional enrichment analysis revealed the versatile physiological roles (over six signal pathways) of these miRNAs. The results suggest that these miRNAs might play potential key roles in the regulation of gene expression in the brachyurization development of the Chinese mitten crab. Particularly, miR‐2a‐3p, miR‐305‐5p, miR‐263a‐5p and miR‐7‐5p may play major regulatory roles in the metamorphosis of E. sinensis.     

Optimal dietary carbohydrate‐to‐lipid ratios for silvery‐black porgy (Sparidentex hasta) juveniles

Four isonitrogenous diets containing different carbohydrate:lipid (CHO:L) ratios (0.3, 0.6, 1.1 and 1.8) were tested in triplicate groups (16 fish per replicate) of silvery‐black porgy juveniles for 8 weeks. Growth performance was not affected by different dietary CHO:L ratios (P > 0.05); however, the viscerosomatic index, the intraperitoneal fat, whole‐body lipid, energy and n‐3 long chain polyunsaturated fatty acids levels increased with decreasing dietary CHO:L ratios (P < 0.05). Fish fed with 1.8 CHO:L diet had the lowest apparent digestibility coefficients of protein and lipid, as well as the lowest plasma haemolytic and lysozyme activities (P < 0.05). Red blood cell counts and plasma glucose levels were higher in fish fed with 1.1 and 1.8 CHO:L ratio diets than in the other groups (P < 0.05). Plasma alkaline phosphatase and alanine aminotransferase, as well as the level of lipid peroxidation and total antioxidant capacity in the liver and plasma increased as dietary CHO:L ratios decreased (P < 0.05). The results of the current study indicated that the diets with CHO:L ratios between 0.6 and 1.1 are optimal for silvery‐black porgy, whereas higher ratios may result in hyperglycaemia and immune suppression, and lower CHO:L ratios may lead to oxidative stress and liver dysfunction.     

Acute effects of ammonia exposure on the plasma and haematological parameters and histological structure of the juvenile blunt snout bream,Megalobrama amblycephala,and post‐exposure recovery

In this study, we investigated the toxic effects of ammonia‐N on the plasma and haematological parameters and histological structure of blunt snout bream (Megalobrama amblycephala) juveniles. The fish (initial weight, 14.79 ± 0.01 g) were randomly sorted into six tanks (200 L), and each tank was stocked with 40 fish for culture. The juveniles were exposed to two ammonia‐N levels—0 mg/L (control group) and 25 mg/L (experimental group)—and sampled at 0, 6, 12, 24, 48 and 72 hr, and then they underwent 96 hr of post‐exposure recovery. The results showed that ammonia‐N had significant effects on the plasma and haematological parameters. The treatment group showed increased cortisol, plasma ammonia and haematocrit levels and white blood cell count with increasing exposure time, up to 24 hr, and then the levels and count decreased. A significantly higher plasma glucose level was observed in the treatment group at 12 hr. After 96 hr of post‐exposure recovery, all parameter levels decreased to the control levels. The fish displayed histopathological alterations in the gills, liver and kidney. The results indicate that the severity of the lesions clearly differed among the organs, with the liver showing the most extensive damage, followed by the gills and kidney. Adverse effects to physiological indicators and histological structure increased with increasing exposure time before 24 hr. The fish showed self‐regulation; however, the histological structure could not recover fully, the gill tissue showed irreversible changes and the kidney tissue exhibited the worst recovery ability.     

Enhanced haematological and immune responses in common carp Cyprinus carpio fed with olive leaf extract‐supplemented diets and subjected to ambient ammonia

The aim of the present study was to evaluate the potential effects of olive leaf (Olea europea L.) extract (OLE) on common carp (Cyprinus carpio) health in response to ambient ammonia toxicity. Fish were fed with experimental diets containing OLE (0, 1, 5 and 10 g/kg) for 60 days followed by a 3‐hr challenge with ammonia (0.5 mg/L unionized ammonia‐N). Results showed that haemoglobin levels, differential leucocyte counts and mean corpuscular haemoglobin concentration were not affected by dietary OLE levels and ammonia challenge. Ammonia challenge significantly increased red blood cells and decreased blood mean corpuscular volume, mean corpuscular haemoglobin, plasma lysozyme, complement and bactericidal activities. Plasma lysozyme, complement and total immunoglobulin values improved by 5 and/or 10 g OLE/kg supplementation. Before the ammonia exposure, the 5 and 10 g OLE/kg treatment significantly increased blood white blood cell, but there was no significant difference among the treatments after the ammonia exposure. Ammonia exposure significantly increased plasma malondialdehyde levels; however, 1 and 5 g OLE/kg treatments had significantly lower values compared to the other treatments. The present results suggest that 1 g OLE/kg diet may improve fish health and reduce the adverse effects of ammonia toxicity on common carp.     

Effects of short‐term starvation on the rhythmic expression of microRNAs in skeletal muscle of goldfish (Carassius auratus)

Molecular oscillators exist in peripheral tissues such as in skeletal muscles and diet is a dominant factor to affect Zeitgeber for peripheral clocks. MicroRNAs (miRNAs) play an important role in muscle cell proliferation and differentiation. However, the knowledge of starvation effects on miRNA rhythmic expression remains limited in teleost. In this study, the circadian expression pattern of miRNAs was investigated in goldfish muscle upon restricting feeding treatment. The data showed that 15 miRNAs exhibited a daily rhythmicity among the 70 miRNAs assayed in muscles of normally fed goldfish. While after 7‐day and 15‐day fasting treatment, 23 and 18 miRNAs showed circadian rhythmicity in goldfish muscles respectively. Only 4 miRNAs (miR‐23a, miR‐29, miR‐199a‐3p and miR‐455) exhibited a daily rhythmicity in all three groups of different nutrient treatments. Correlation analysis of the circadian‐miRNAs indicates different feeding stimulation could alter the circadian‐miRNA components and their expression profile in skeletal muscle upon short‐term feed deprivation. These miRNAs may play important roles in regulating circadian expression of genes involved in muscle cell differentiation and growth by nutritional alteration, thus having a potential application in fish aquaculture.     

Dietary copper requirement of fingerling Heteropneustes fossilis for formulating copper‐balanced commercial feeds

Dietary copper requirement of Heteropneustes fossilis (6.74 ± 0.03 g) was determined by feeding purified diets containing same protein (400 g/kg) and gross energy (17.89 kJ/g) but different levels of copper for 12 weeks. Graded amount of CuSO₄.5H₂O (0, 1.96, 3.93, 5.89, 7.86, 9.82, 11.79 mg/kg) was supplemented to basal diet to attain desired dietary copper levels (0, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/kg). Analysed dietary copper concentrations were 4.28, 4.63, 5.28, 5.70, 6.19 and 6.69 mg/kg. Absolute weight gain, feed conversion ratio and protein gain improved with the increasing levels of dietary copper up to 5.28 mg/kg. Further inclusion of copper at a level of 5.70 mg/kg did not improve the above parameters. Significantly higher (p < .05) plasma ceruloplasmin, liver copper‐zinc superoxide dismutase, catalase activities and lower thiobarbituric acid reactive substances were evident in fish receiving diets with 5.28 and 5.70 mg/kg copper compared to other groups. Whole body and liver copper concentrations increased significantly (p < .05) with increasing dietary copper levels. Quadratic regression analysis of absolute weight gain, feed conversion ratio, protein gain and broken‐line regression analysis of plasma ceruloplasmin activity and liver TBARS value against the variable dietary copper levels depicted the dietary copper requirements for fingerling H. fossilis in the range of 5.24–5.68 mg/kg.     

Growth performance,robustness against stress,serum insulin,IGF‐1 and GLUT4 gene expression of red tilapia (Oreochromis sp.) fed diet containing graded levels of creatine

The experimental diets containing five levels of creatine supplementation, namely control (N = 0 g/kg creatine), B1 (1.00 g/kg glycine), B2 (0.40 g/kg creatine), B3 (0.80 g/kg creatine) and B4 (1.20 g/kg creatine) were tested to red tilapia up to apparent satiation 3 times a day for 56 days. After the feeding trial, fish were stressed by air exposure for challenge test against stress. The results described that creatine supplementation improved final body weight, feed conversion ratio (FCR) and protein retention. Creatine supplementation decreased blood glucose, cholesterol and triglyceride levels (p < .05), indicating more effective usage of non‐protein energy. Furthermore, creatine supplementation also increased plasma insulin levels and upregulated IGF‐1 and GLUT4 gene expressions in the liver significantly; it also improved robustness against stress, which was shown by higher superoxide dismutase, higher postchallenge survival and plasma cortisol, malondialdehyde and lower glucose levels. In conclusion, creatine supplementation at a dose of 0.289–0.389 g/kg diet escalated fish growth and improved robustness against stress via improving the SOD activity in red tilapia.     

Effects of sodium butyrate stimulation on immune‐related mRNA‐miRNA network in intestine of grass carp

Sodium butyrate is one of the most popular feed additives in animal husbandry. In recent years, sodium butyrate has been increasingly used as supplement in aquaculture. The present study is to investigate the intestinal mRNA and microRNA response to diet with sodium butyrate in grass carp (Ctenopharyngodon idella), an important aquaculture species in China. mRNA and microRNA profiles of intestine of grass carp fed with diet contained 0, 1.0, 2.5, 5.0, 7.5 and 10.0 g/kg sodium butyrate were obtained by RNA‐seq using Illumina Hiseq 2,500 platform. The feeding trial was performed using 18 individuals of 1‐year‐old grass carp (n = 3 for each group) and lasted for 40 days in tanks in laboratory. A total of 349,860,852 sequence reads were generated from six intestinal libraries. Functional analysis of differentially expressed genes showed that genes participated in immune pathways tend to be activated by sodium butyrate supplementation. A total of 700 microRNAs were obtained, including 275 conserved microRNAs and 425 novel microRNAs which are potentially involved in regulating 14,300 genes. Spearman's correlation analysis identified 18 pairs of microRNA‐mRNA associated with immune pathways (p < .01 and R<?0.5). The potential genes targeted by microRNAs include CXCL12, AKT1S1, Cab39 and MHCII which are important genes associated with intestinal immune pathways. To our knowledge, this is the first integrated profiling of both mRNA and microRNA in intestine with supplementation of sodium butyrate in grass carp. The present results suggest that sodium butyrate affects intestinal immune system by regulating microRNA‐mRNA interaction in fish.     

Dietary β‐carotene improves growth performance and antioxidant status of juvenile Piaractus mesopotamicus

This study was conducted to investigate the effects of dietary synthetic β‐carotene on growth performance, haematological and immunological parameters, energy reserves and antioxidant status of juvenile fish Piaractus mesopotamicus. Two hundred and ten fish (12.24 ± 2.98 g) were fed with a control extruded feed (CEF) and the same diet supplemented with β‐carotene (BEF) for 90 days. A higher weight gain and specific growth rate were found in fish fed BEF in comparison with those fed CEF. In addition, BEF‐fed fish showed lower liver somatic index and a higher percentage of eosinophils. No statistical differences were found in energy reserves, except for plasma triglycerides which decreased in fish fed with BEF. Regarding oxidative stress markers, decreased activities of antioxidant enzymes were found in intestine and gills of BEF‐fed fish (SOD and GST, respectively). In the liver, both LPO levels and CAT activity decreased in fish fed with BEF. Additionally, lower brain LPO levels without changes in the antioxidant enzymes were observed in BEF‐fed fish. The inclusion of dietary synthetic β‐carotene improved growth and antioxidant status, and had a plasma triglyceride‐lowering effect in juvenile P. mesopotamicus.     

Molecular cloning,characterization, tissue expression and nutritional regulation of O‐GlcNAc transferase gene in hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂)

O‐GlcNAc transferase gene (OGT) was considered as the sole rate‐limiting enzyme in the O‐GlcNAc modification. In the present study, the OGT gene of hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂) was cloned and characterized, and its expression in response to dietary carbohydrate level and acute glucose treatment was investigated. The full‐length of OGT (GenBank accession no. KY656469 ) was 4,063 bp, including a 302 bp 5′untranslated terminal region (UTR), a 3,165 bp coding region that encoded 1,054 amino acids residues and a 596 bp 3′ UTR. The highly conservation of OGT gene between fish and mammals was also observed through multiple sequences alignment and phylogenetic analysis. O‐GlcNAc transferase gene was ubiquitously expressed in all detected tissues with highest expressions in brain and liver, to a lesser degree, in eye, heart, kidney and intestine. The increasing dietary carbohydrate from 8.02% to 16.08% had no significant effect on the mRNA expression of OGT. However, the expression of OGT was slightly elevated at 6 hr post‐glucose injection, and the elevation became significant at 24 hr time‐point. These data may enhance our understanding on the nutritional regulation of OGT and O‐GlcNAc modification in fish species.     

Plasma proteins,hepatic enzymes,thyroid hormones and liver histopathology of Cyprinus carpio (Linnaeus, 1758) exposed to an oxadiazin pesticide,indoxacarb

Indoxacarb is a relatively new pesticide from oxadiazin class, which is used near carp ponds for agricultural purposes. Thus, the aim of this study was to determine indoxacarb effects on common carp (Cyprinus carpio). The fish were exposed to 0 (control), 0.75, 1.5 and 3 ppm of indoxacarb over 21 days and plasma biochemical characteristics and liver histopathology were examined. Exposure to indoxacarb induced fall in total protein after 21 days. Globulin increased after 7 days and then decreased after 14‐ and 21‐day exposure. Plasma alanine transaminase and aspartate transaminase activities increased in 1.5 and 3 ppm treatments after 7‐day exposure. Indoxacarb exposure significantly decreased plasma alkaline phosphatase after 7 days with no change at the second and third samplings. After 7 days, plasma T₃ levels had no significant change; however, it decreased after 14 days in the 1.5 and 3 ppm treatments and also reduced after 21‐day exposure to the 3 ppm treatment compared to the control group. Plasma T₄ level only decreased significantly in the 3 ppm treatment compared to the control group after 21 days. Different histopathological symptoms such as necrosis, hyperaemia, sinusoidal space extension, pyknotic nuclei, leucocyte infiltration and melanomacrophage aggregates were observed after 21‐day exposure to indoxacarb. The symptoms intensity was dependent on indoxacarb concentration. In conclusion, the present results show that indoxacarb exposure adversely affects common carp health and welfare, which consequently may induce serious problems in this species aquaculture.     

Modulation of growth,immunity and antioxidant‐related gene expressions in the liver and intestine of juvenile Sillago sihama by dietary vitamin C

An 8‐week feeding experiment was implemented to study the effect of dietary vitamin C (VC) on growth, immunity, oxidation resistance and relevant gene expressions in juvenile S. sihama (2.33 ± 0.02 g). Fish were fed six diets containing VC level of 5, 16, 27, 65, 122 and 233 mg/kg, respectively. VC‐deficient signs including ascites syndrome, skin decay and haemorrhage were found in the fish fed basal diets. Appropriate VC supplement in diets (a) increased weight gain (WG), specific growth rate (SGR), feed intake (FI) and protein efficiency ratio (PER); (b) increased contents of hepatic total immunoglobulin (IgM), complement 3 (C3) and complement 4 (C4) and activity of alkaline phosphatase (AKP); (c) increased hepatic and intestinal copper–zinc superoxide dismutase (CuZnSOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione S‐transferase (GST) activities, but decreased hepatic MDA content; and (d) up‐regulated gene expressions of NF‐E2‐related factor 2 (Nrf2), Kelch‐like ECH‐associated protein 1 (Keap1), CAT, GST, GPx, glutathione reductase (GR), CuZnSOD and manganese superoxide dismutase (MnSOD) in liver and intestine. These results indicated that dietary VC significantly influenced the growth, immunity and oxidation resistance at enzymatic and genic levels. Based on regression analysis for PER, IgM, intestinal CuZnSOD and hepatic VC concentration, the optimal requirement of dietary VC in juvenile S. sihama was estimated to be 98.33, 139.03, 104.23 and 143.69 mg/kg.     

Estimation of dietary vitamin K requirement of juvenile Chinese soft‐shelled turtle,Pelodiscus sinensis

A 10‐week feeding trial was conducted to estimate the dietary vitamin K requirements of juvenile Chinese soft‐shelled turtles, Pelodiscus sinensis, using menadione sodium bisulphite (MSB) as the vitamin K source. Juvenile soft‐shelled turtles with 6.1 ± 0.1 g body weight were individually reared and fed diets containing seven levels of MSB (0.0, 5.0, 8.9, 12.3, 16.6, 20.7 and 41.0 mg/kg). Turtles fed with an MSB‐free diet exhibited the lowest feed utilization, carapace Ca concentrations, carapace strength or liver menaquinone (MK‐4) concentrations among all dietary groups. Weight gain (WG) in the turtles tended to increase with increase in dietary MSB until a dietary MSB level of 16.6 mg/kg, where WG levelled off. Total plasma prothrombin concentrations, an indicator for blood clotting function, in the turtles fed on MSB supplemented diets were significantly (p < 0.05) lower than those in turtles fed on MSB supplemented diets. Liver MSB concentrations increased with increase in dietary MSB. Using broken‐line or sigmoidal regression model, the vitamin K requirements of juvenile Chinese soft‐shelled turtles based on WG, total plasma prothrombin concentrations and liver vitamin K were 21.5, 25.8 and 29.9 mg MSB per kg, respectively.