The effect of dietary folic acid on biochemical parameters and gene expression of three heat shock proteins (HSPs) of blunt snout bream (Megalobrama amblycephala) fingerling under acute high temperature stress.

The effects of dietary folic acid on biochemical parameters and gene expression of three heat shock proteins (HSPs) of blunt snout bream (Megalobrama amblycephala) fingerling under acute high temperature stress. Six dietary folic acid groups (0.0, 0.5, 1.0, 2.0, 5.0, and 10.0) mg/kg diets were designed and assigned into 18 tanks in three replicates each (300 l/tank) and were administered for 10 weeks in a re-circulated water system. The fingerlings with an initial weight of 27.0 ± 0.03 g were fed with their respective diets four times daily. At the end of the experiment, samples were collected before challenge, 0, 24, 72 h, and 7 days. Serum total protein (TP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), cortisol, glucose, complement C3 (C3), complement C4 (C4, immunoglobulin M (IgM) hepatic superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and the expression of heat shock protein 60 (HSP60), 70 (HSP70), and 90 (HSP90) were studied. The results showed that fish fed with dietary folic acid between 1.0, 2.0, and 5.0 mg/kg significantly (P < 0.05) increased serum TP, C3, C4 hepatic SOD, CAT, and the expression of HSP60, HSP70, and HSP90 before and after temperature challenge of 32 °C. Also, serum ALP, cortisol, glucose, and hepatic MDA were significantly (P < 0.05) reduced by supplementation of dietary folic acid level 1.0, 2.0, and 5.0 mg/kg before and after the same temperature challenge of 32 °C. Before stress, 0, 24, 72 h, and 7 days significantly (P < 0.05) affects serum biochemical parameters, immune and antioxidant capacities, and expression level of three HSPs. Furthermore, there was no statistical evidence to show that dietary folic acid inclusion level and temperature duration have significant interactive effect on serum biochemical parameters, antioxidant parameters, and gene expression level (P > 0.05) of the three HSPs. However, there were statistical significant interactive effect between dietary folic acid inclusion level and temperature duration on serum C3 and C4 (P < 0.05) except IgM (P > 0.05). The present results indicate that supplementation of basal diet from 1.0 mg/kg; 2.0 and 5.0 mg/kg can enhance acute high temperature resistance ability in M. amblycephala fingerling to some degree and improve physiological response, immune and antioxidant capacities, and expression level of three HSPs.

Threonine modulates immune response, antioxidant status and gene expressions of antioxidant enzymes and antioxidant-immune-cytokine-related signaling molecules in juvenile blunt snout bream (Megalobrama amblycephala).

A 9-week feeding trial was conducted to investigate the effects of graded dietary threonine (Thr) levels (0.58-2.58%) on the hematological parameters, immune response, antioxidant status and hepatopancreatic gene expression of antioxidant enzymes and antioxidant-immune-cytokine-related signaling molecules in juvenile blunt snout bream. For this purpose, 3 tanks were randomly arranged and assigned to each experimental diet. Fish were fed with their respective diet to apparent satiation 4 times daily. The results indicated that white blood cell, red blood cell and haemoglobin significantly responded to graded dietary Thr levels, while hematocrit didn't. Complement components (C3 and C4), total iron-binding capacity (TIBC), immunoglobulin M (IgM), superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) increased with increasing dietary Thr levels up to 1.58-2.08% and thereafter tended to decrease. Dietary Thr regulated the gene expressions of Cu/Zn-SOD, Mn-SOD and CAT, GPx1, glutathione S-transferase mu (GST), nuclear factor erythroid 2-related factor 2 (Nrf2), heat shock protein-70 (Hsp70), tumor necrosis factor-alpha (TNF-α), apolipoprotein A-I (ApoA1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and fructose-bisphosphate aldolase B (ALDOB); while the gene expression of peroxiredoxin II (PrxII) was not significantly modified by graded Thr levels. These genes are involved in different functions including antioxidant, immune, and defense responses, energy metabolism and protein synthesis. Therefore, this study could provide a new molecular tool for studies in fish immunonutrition and shed light on the regulatory mechanisms that dietary Thr improved the antioxidant and immune capacities of fish.

Threonine affects digestion capacity and hepatopancreatic gene expression of juvenile blunt snout bream (Megalobrama amblycephala).

The present study conducted a 9-week feeding trial to investigate the effects of threonine (Thr) on the digestion capacity and hepatopancreas gene expression of juvenile blunt snout bream (Megalobrama amblycephala). For this purpose, three tanks (300 litres/tank) were randomly arranged and assigned to each experimental diet. Juvenile fish were fed with diets containing graded Thr levels (0·58, 1·08, 1·58, 2·08 or 2·58 % of the diet) to apparent satiation four times daily. At the end of the feeding trial, the results indicated that hepatopancreas weight, hepatosomatic index, hepatopancreatic protein content, intestinal weight, intestosomatic index and intestinal protein content increased with increasing dietary Thr levels up to 1·58 % and thereafter decreased (P< 0·05). The activities of chymotrypsin, trypsin, amylase and lipase elevated as dietary Thr levels increased up to 1·58 % (P< 0·05), while these activities decreased in most cases after 1·58 % dietary Thr except for chymotrypsin and trypsin in the hepatopancreas (plateau 1·58-2·08 % Thr). The relative gene expression levels of chymotrypsin, trypsin, amylase, lipase, target of rapamycin and insulin-like growth factor-I were up-regulated, and the highest values were observed with 1·58 % dietary Thr or 1·58 and 2·08 % dietary Thr, whereas the relative gene expression levels of eukaryotic translation initiation factor 4E-binding protein 2 gradually decreased (P< 0·10) as dietary Thr levels increased up to 1·58 % and thereafter significantly increased (P< 0·05), which could explain that about 1·58 % dietary Thr could improve the growth and development of digestive organs and activities of digestive enzymes of juvenile blunt snout bream.

Effect of High Dietary Carbohydrate on the Growth Performance, Blood Chemistry, Hepatic Enzyme Activities and Growth Hormone Gene Expression of Wuchang Bream (Megalobrama amblycephala) at Two Temperatures.

The effects of high carbohydrate diet on growth, serum physiological response, and hepatic heat shock protein 70 expression in Wuchang bream were determined at 25°C and 30°C. At each temperature, the fish fed the control diet (31% CHO) had significantly higher weight gain, specific growth rate, protein efficiency ratio and hepatic glucose-6-phosphatase activities, lower feed conversion ratio and hepatosomatic index (HSI), whole crude lipid, serum glucose, hepatic glucokinase (GK) activity than those fed the high-carbohydrate diet (47% CHO) (p<0.05). The fish reared at 25°C had significantly higher whole body crude protein and ash, serum cholesterol and triglyceride, hepatic G-6-Pase activity, lower glycogen content and relative levels of hepatic growth hormone (GH) gene expression than those reared at 30°C (p<0.05). Significant interaction between temperature and diet was found for HSI, condition factor, hepatic GK activity and the relative levels of hepatic GH gene expression (p<0.05).

A deficiency or an excess of dietary threonine level affects weight gain, enzyme activity, immune response and immune-related gene expression in juvenile blunt snout bream (Megalobrama amblycephala).

A feeding trial was conducted to investigate the impacts of deficient and excess dietary threonine levels on weight gain, plasma enzymes activities, immune responses and expressions of immune-related genes in the intestine of juvenile blunt snout bream. Triplicate groups of fish (initial weight 3.01 ± 0.01 g, 30 fish per tank) were fed with deficient (0.58%), optimum (1.58%) and excess (2.58%) threonine level diets to near satiation four times a day for 9 weeks. A mixture of l-amino acids was supplemented to simulate the whole body amino acid pattern of blunt snout bream, except for threonine. The results showed that both deficiency and excess threonine level diets significantly (P < 0.05) reduced the weight gain of blunt snout bream. Excess dietary threonine level triggered plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities (P < 0.05); whereas superoxide dismutase (SOD) activity was not significantly influenced by imbalanced-dietary threonine level (P > 0.05). Plasma complement component 3 (C3) and component 4 (C4) concentrations were significantly depressed by the deficiency of dietary threonine (P < 0.05). Dietary threonine regulated the target of rapamycin (TOR), eukaryotic translation initiation factor 4E-binding protein 2 (4E-BP2), tumour necrosis factor alpha (TNF-α) and copper-zinc superoxide dismutase (Cu/Zn-SOD) gene expressions in the intestine of blunt snout bream, which may go further to explain the adverse effects of a deficient and/or an excess dietary threonine level on growth, immunity and health of fish. Furthermore, the present study also suggests that an optimum dietary threonine could play an important role in improving growth, enhancing immune function and maintaining health of fish.

Effects of emodin and vitamin E on the growth and crowding stress of Wuchang bream (Megalobrama amblycephala).

This study aimed to investigate the effects of dietary emodin, high-dose vitamin E and their combination on the growth of Megalobrama amblycephala and its resistance to acute crowding stress. The fish were randomly divided into four groups: a control group fed with basal diet, and three treatment groups fed with basal diet supplemented with 60 mg/kg emodin (the emodin group), 500 mg/kg vitamin E (the vit E group), and 60 mg/kg emodin together with 500 mg/kg vitamin E (the combination group). After 60 days, the fish were exposed to acute crowding stress for 24 h. The results showed that the weight gain of the vit E group, specific growth rate of the vit E group, total serum protein concentration (TP) of the vit E group, serum lysozyme activity of the emodin group, serum superoxide dismutase (SOD) activity of the emodin group, hepatic heat shock protein 70 (HSP70) levels of the vit E group and the emodin group, and serum alanine aminotransferase (ALT) activity of the combination group significantly increased while the weight gain and specific growth rate of the combination group significantly decreased compared with the control group before stress. After crowding stress, the vit E group had improved serum TP 12 h post-stress, hepatic SOD activity 24 h post-stress, and hepatic HSP70 mRNA levels 12 and 24 h post-stress while the emodin group had enhanced serum SOD activity 12 and 24 h post-stress and hepatic HSP70 mRNA levels 12 and 24 h post-stress, as compared with the control. However, the serum cortisol content of the three treatment groups 12 and 24 h post-stress, ALT activity in the vit E group and emodin group 24 h post-stress, and serum alkaline phosphatase and liver catalase activity in the combination group 24 h post-stress were lower than those in the control group. The cumulative mortality was lower in the emodin, vit E, and combination group after Aeromonas hydrophila infection compared with the control group. Therefore, dietary supplementation with 60 mg/kg emodin or 500 mg/kg vitamin E can improve HSP70 mRNA levels and antioxidant capabilities, resistance to crowding stress, and growth in M. amblycephala. However, the combination of emodin and vit E does not have a synergistic effect in M. amblycephala.

The influence of various feeding patterns of emodin on growth, non-specific immune responses, and disease resistance to Aeromonas hydrophila in juvenile Wuchang bream (Megalobrama amblycephala).

The present study was conducted to evaluate the effect of various feeding patterns of emodin on growth, non-specific immune response, and disease resistance to Aeromonas hydrophila in juvenile Wuchang bream. Healthy Megalobrama amblycephala (initial weight: 3.47 ± 0.032) were grown in a circulating water system for 8 weeks. Five groups were studied: one control group was fed with a basal diet for eight weeks (Pattern 1, P1), and three treatment groups were fed with a trial diet of 30 mg emodin kg(-1) at one-week (Pattern 2, P2), two-week (Pattern 3, P3), four-week (Pattern 4, P4) intervals. The final treatment group maintained the trial diet for the entire eight-week study duration (Pattern 5, P5). Results indicated that different feeding patterns of emodin significantly influenced the weight gain rate of Wuchang bream (P < 0.05). Fish in the P4 treatment group had significantly higher rates of weight gain (WG) than those in other treatment groups. There were no significant differences in survival rates or feed conversion ratios (FCR) between treatment groups and the control group. White blood cell count (WBC), respiratory burst activity, superoxide dismutase (SOD) activity, myeloperoxidase (MPO) activity and tumor necrosis factor-α (TNF-α) activity were shown to increase at first and then decrease from P3 condition to P5 condition. Fish under P4 treatment showed the most significant improvement of all tested parameters compared to control. Significantly higher levels (P < 0.05) of plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity were observed in P2 and P4 treatment groups when compared with the control group, while no significant differences were observed in the AST and ALT activity of fish in P2, P3, P4 and P5 treatment groups. In a bacterial challenge experiment with A. hydrophila, fish under P4 and P5 treatment showed lower cumulative mortality than the control group. The results of this study suggest that an initial 4-week feeding interval is recommended for the economic and practical culture of M. amblycephala.

Effects of anthraquinone extract from Rheum officinale Bail on the growth performance and physiological responses of Macrobrachium rosenbergii under high temperature stress.

In order to study the effects of anthraquinone extract from Rheum officinale Bail on Macrobrachium rosenbergii under high temperature stress, freshwater prawns were randomly divided into five groups: a control group was fed with basal diet, and four treatment groups fed with basal diet supplemented with 0.05%, 0.1%, 0.2%, and 0.4% anthraquinone extracts for 10 weeks, respectively. Then, freshwater prawns were exposed to high temperature stress at 35 degrees C for 48h. The growth, changes in haemolymph total protein, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lysozyme, nitrogen monoxide (NO) and hepatic catalase (CAT), superoxide dismutase (SOD) and malondialdehyde (MDA) were investigated. The results showed that compared the control group, the specific growth rates, feed conversion efficiency, haemolymph ALP and lysozyme activities, total protein contents, hepatic CAT and SOD activities increased while haemolymph AST, ALT and hepatic MDA contents decreased in treatment groups before the stress, but their levels did not correlate with the doses of anthraquinone extracts. The specific growth rate (SGR), feed conversion efficiency and haemolymph lysozyme activity significantly increased but haemolymph AST activity decreased in 0.1% dose group; whereas haemolymph ALP activity and feed conversion efficiency increased but ALT activity and hepatic MDA contents significantly decreased in 0.2% dose group before the stress compared with the control. After high temperature stress, 0.1-0.2% anthraquinone extract also could improve the haemolymph total proteins, lysozyme and ALP activities, hepatic catalase, and superoxide dismutase, and reduce haemolymph ALT and AST activities, hepatic malondialdehyde contents. The cumulative mortality in the control was about 100% at 48h after high temperature stress while the cumulative mortality in the treatment groups supplemented with 0.1-0.2% anthraquinone extract were about 48-65%. The artificial infection with Vibrio anguillarum also showed the cumulative mortality in the control was about 100% while the cumulative mortality in the treatment groups supplemented with 0.1-0.2% anthraquinone extracts were about 57-80%. The present study suggested that ingestion of a basal diet supplemented with 0.1-0.20% anthraquinone extracts could prevent high temperature stress and promote the growth of prawns.