The Protective Effect of a Dietary Extract of Mulberry (Morus alba L.) Leaves against a High Stocking Density, Copper and Trichlorfon in Crucian Carp (Carassius auratus).

This study was designed to examine the protective effects of the extract of mulberry (Morus alba L.) leaves (EML) on crucian carp (Carassius auratus) against a high stocking density, Cu exposure and trichlorfon exposure, which adversely impact fish growth performance, feed intake and fish locomotion. High stocking densities decreased the activities of amylase, lipase, trypsin, Na+/K+-ATPase and alkaline phosphatase (AKP), and increased the content of malonaldehyde (MDA) in fish digestive organs, indicating an impairment of the digestive function and a disturbance of the antioxidant status. Cu exposure increased the activities of glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) in fish digestive organs, suggesting the activation of amino acid metabolism. Furthermore, trichlorfon exposure reduced the activities of lactate dehydrogenase (LDH), glutathione reductase (GR), GOT and GPT, and the capacities of the anti-superoxide anion (ASA) and anti-hydroxyl radical (AHR) in fish muscles, indicating a disruption of the bioenergetic homeostasis and antioxidant status. Our present study indicates that dietary EML supplementation relieved the detrimental effects induced by these stressors.

Scoparia dulcis L. Extract Relieved High Stocking Density-Induced Stress in Crucian Carp (Carassius auratus).

The objective of this study was to investigate the effects of Scoparia dulcis extract (SDE) on stress induced by high stocking density and Cu and trichlorfon exposure in crucian carp (Carassius auratus). The results showed that these stressors exerted detrimental effects in fish, such as inhibition of growth performance, reduced feed intake, and interruption of fish locomotion. Under high stocking density, dietary SDE supplementation increased the content of reduced glutathione (GSH) and the activities of amylase, catalase (CAT), and glutathione reductase (GR) and decreased the content of malonaldehyde (MDA) in the intestine of crucian carp. A similar trend was presented in the hepatopancreas under Cu exposure. Dietary SDE supplementation enhanced the activities of CAT, superoxide dismutase (SOD), glutathione peroxidase (GPx), lactate dehydrogenase, glutamate-oxaloacetate transaminase, and glutamate-pyruvate transaminase in the muscle of crucian carp under trichlorfon exposure. The optimum dietary SDE supplementation levels were 4.07, 4.33, and 3.95 g kg-1 diet based on the recovery rate of weight gain (RWG), feed intake (FI), and inhibitory rate of rollover (IR) for crucian carp under high stocking density and Cu and trichlorfon exposure, respectively. Overall, dietary supplementation with SDE may be a useful nutritional strategy for relieving these stresses in aquatic animals.

The Extract of Angelica sinensis Inhibits Hypoxia-Reoxygenation and Copper-Induced Oxidative Lesions and Apoptosis in Branchiae and Red Blood Corpuscles of Fish.

The study explored the effects of Angelica sinensis extract (AsE) on oxidative lesions and apoptosis in branchiae and red blood corpuscles in hypoxia-reoxygenation (HR) and Cu-treated carp (Cyprinus carpio var. Jian). After feeding trial for 30 days, the carp were exposed to HR and CuSO4. The results indicated that dietary AsE increased the durative time, decreased the oxygen consumption rate, suppressed ROS generation and cellular component oxidation, decreased enzymatic antioxidant activity and reduced glutathione (GSH) levels in red blood corpuscles and branchiae in carp under hypoxia. Moreover, dietary AsE avoided the loss of Na+,K+-ATPase, metabolic and antioxidant enzyme activities, ROS generation and cellular component oxidation, as well as the increase in caspase-8, 9, and 3 activities in the branchiae of the carp and inhibited ROS generation. It furthermore avoided the loss of Na+,K+-ATPase and metabolic enzyme activities, the decrease in GSH levels and hemoglobin content, the increase in the activities of caspase-8, 9, and 3 and the increase in the levels of cytochrome c and phosphatidylserine exposure in the red blood corpuscles of Cu-exposed carp. The present results suggested that dietary AsE improved hypoxia tolerance and inhibited HR or Cu-triggered oxidative lesions and apoptosis. Therefore, AsE can be utilized as a natural inhibitor of Cu and HR stress in fish.

[Study on wavelength overlapping HPLC fingerprint and multi-component content determination of Danshen].

This project was launched to and establish the wavelength overlapping HPLC fingerprint for Danshen and determine the contents of 7 component in Danshen. The chromatographic fingerprints were built by using Agilent Eclipse Plus C_(18)( 4. 6 mm×100 mm,3. 5 µm) as stationary phase and 0. 1% formic acid solution( A)-acetonitrile( B) as mobile phase with gradient elution( 0-5 min,10%-20% B; 5-20 min,20%-30% B; 20-25 min,30%-50% B; 25-40 min,50%-65% B; 40-45 min,65%-80% B; 45-46 min,80%-10% B; 46-50 min,10% B) at a flow rate of 1 mL·min~(-1). The column temperature was maintained at 30 ℃ and the detection wavelength was set at 250,280,310 and 340 nm. The technique of wavelength overlapping fingerprint was established and the contents of 7 indicative compounds have been determined in this method. The results of wavelength overlapping HPLC fingerprint showed all-around information of the fingerprints at 250,280,310 and 340 nm,and the similarity among 17 batches of Danshen was over 0. 828-0. 936. In wavelength overlapping HPLC fingerprint,15 common peaks were selected as the common peaks,and 7 contents of them were indentified as rosmarinic acid,salvianolic acid B,salvianolic acid A,phenolic acid,dihydrotanshinone Ⅰ,tanshinone Ⅰ and tanshinone ⅡA. The results of methodological study demonstrated that the method met the requirements of the determination. The method established in this study is simple,convenient and durable,which can provide a scientific basis for the quality control of Danshen.

Jian carp (Cyprinus carpio var. Jian) intestinal immune responses, antioxidant status and tight junction protein mRNA expression are modulated via Nrf2 and PKC in response to dietary arginine deficiency.

This study investigated the effect of dietary arginine on the immune response, antioxidant status and tight junction mRNA expression in the intestine of juvenile Jian carp (Cyprinus carpio var. Jian). A total of 1200 juvenile Jian carp with an average initial weight of 6.33 ± 0.03 g were fed graded levels of arginine (9.8-24.5 g kg(-1) diet) for nine weeks. The study showed that arginine deficiency up-regulated interleukin 1, interleukin 8 and transforming growth factor-ß and down-regulated tumour necrosis factor α gene expression (P < 0.05). Additionally, arginine deficiency increased malondialdehyde (MDA), protein carbonyl (PC) and glutathione contents and decreased the activities of copper/zinc superoxide dismutase (SOD1), glutathione peroxidase (GPx), catalase (CAT) and glutathione reductase (GR) and glutathione-S-transferase (GST) (P < 0.05). Meanwhile, arginine deficiency significantly increased claudin 7, occludin, protein kinase C, NF-E2-related factor 2 and Kelch-like-ECH- associated protein 1 mRNA expression and decreased SOD1, CAT and GR mRNA expression (P < 0.05). All of these results indicated that arginine deficiency impaired intestinal immune function via the regulation of mRNA expression of cytokines, tight junction proteins, antioxidant enzymes, Nrf2/Keap1 and PKC in fish intestine.

Effect of dietary arginine on the immune response and gene expression in head kidney and spleen following infection of Jian carp with Aeromonas hydrophila.

This study was conducted to test the hypothesis that elevated dietary arginine enhances immunity of juvenile Jian carp (Cyprinus carpio var. Jian). Fish were fed graded levels of dietary arginine for 9 weeks. Subsequently, a challenge test was conducted by injection of Aeromonas hydrophila. Head kidney and spleen weights, as well as erythrocyte and leukocyte counts were significantly influenced by dietary arginine levels. A similar trend was also observed for hemagglutination titre, serum lysozyme activity, IgM concentration, C3 and C4 content. The highest survival rates following A. hydrophila infection were obtained in fish fed the diets containing arginine at 16.1-21.9 g/kg diet. Phagocytic activity of leukocytes was significantly enhanced by dietary arginine supplementation. In contrast, acid phosphatase activity significantly decreased with dietary arginine levels. Dietary arginine levels did not have a significant effect on the total iron-binding capacity. Gene expression of TNF-α and TGF-ß in head kidney significantly increased with dietary arginine levels up to 21.9 g/kg diet, and decreased thereafter. Fish fed the basal diet exhibited the highest IL-10 mRNA expression level. Gene expression of IL-1ß and TOR increased with dietary arginine addition, reaching a plateau at 18.5 and 21.9 g arginine/kg diet, respectively. In spleen, higher IL-1ß and TNF-α gene expressions were obtained in fish fed the diets containing 24.5 g arginine/kg diet than in fish fed the other dietary treatments. TGF-ß mRNA expression levels were significantly lower in fish fed the diets containing ≤21.9 g arginine/kg diet. IL-10 and TOR mRNA expression levels were lower in fish fed 16.1 g arginine/kg diet, while 4E-BP mRNA expression levels increased with dietary arginine levels up to 12.7 g/kg diet and decreased thereafter. Our results indicate that arginine has beneficial effects on regulating mRNA expression of inflammatory cytokines, as well as TOR and 4E-BP and improving humoral and cellular immunity, therefore enhancing disease resistance of fish.

Effect of choline on antioxidant defenses and gene expressions of Nrf2 signaling molecule in the spleen and head kidney of juvenile Jian carp (Cyprinus carpio var. Jian).

The present work evaluates the effects of various levels of dietary choline on antioxidant defenses and gene expressions of Nrf2 signaling molecule in spleen and head kidney of juvenile Jian carp (Cyprinus carpio var. Jian). Fish were fed with six different experimental diets containing graded levels of choline at 165 (choline-deficient control), 310, 607, 896, 1167 and 1820 mg kg(-1) diet for 65 days. At the end of the feeding trail, fish were challenged with Aeromonas hydrophila and mortalities were recorded over 17 days. Dietary choline significantly decreased malondialdehyde and protein carbonyl contents in spleen and head kidney. However, anti-superoxide anion and anti-hydroxyl radical activities in spleen and head kidney also decreased. Interestingly, activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and glutathione reductase (GR) in spleen, GPx activity in head kidney, and glutathione contents in spleen and head kidney were decreased with increase of dietary choline levels up to a certain point, whereas, activities of SOD, GST and GR in head kidney showed no significantly differences among groups. Similarly, expression levels of CuZnSOD, MnSOD, CAT, GPx1a, GPx1b and GR gene in spleen and head kidney were significantly lower in group with choline level of 607 mg kg(-1) diet than those in the choline-deficient group. The relative gene expressions of Nrf2 in head kidney and Keap1a in spleen and head kidney were decreased with increasing of dietary choline up to a certain point. However, the relative gene expression of Nrf2 in spleen were not significantly affected by dietary choline. In conclusion, dietary choline decreased the oxidant damage and regulated the antioxidant system in immune organs of juvenile Jian carp.

Effect of dietary lysine on growth, intestinal enzymes activities and antioxidant status of sub-adult grass carp (Ctenopharyngodon idella).

The dietary lysine requirement of sub-adult grass carp (460 ± 1.5 g) was assessed by feeding diets supplemented with grade levels of lysine (6.6, 8.5, 10.8, 12.9, 15.0 and 16.7 g kg(-1) diet) for 56 days. The test diets (28% CP) contained fish meal, casein and gelatin as sources of intact protein, supplemented with crystalline amino acids. Weight gain (WG), feed intake and feed efficiency were significantly improved with increasing levels of lysine up to 12.9 g kg(-1) diet and thereafter declined (P < 0.05). Quadratic regression analysis of WG at 95% maximum response indicated lysine requirement was 10.9 g kg(-1) diet. Activities of trypsin, chymotrypsin, lipase, Na(+), K(+)-ATPase and alkaline phosphatase in intestine, creatine kinase activity in proximal and mid-intestine responded similar to WG (P < 0.05). In addition, lipid and protein oxidation decreased with increasing levels of lysine up to certain values and increased thereafter (P < 0.05); the anti-hydroxyl radical capacity, dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione-S-transferase (GST) activities and glutathione content were increased with increasing dietary lysine levels up to certain values in the detected tissues, except for hepatopancreatic GST. Requirement estimated on the basis of malondialdehyde content in intestine and hepatopancreas was 10.6 and 9.53 g lysine kg(-1) diet, respectively.

Antioxidant status of serum, muscle, intestine and hepatopancreas for fish fed graded levels of biotin.

Lipid peroxidation, protein oxidation and antioxidant activities of muscle, intestine, hepatopancreas and serum in juvenile Jian carp (Cyprinus carpio var. Jian) were investigated after feeding graded levels of biotin (0.010, 0.028, 0.054, 0.151, 0.330, 1.540 and 2.680 mg kg(-1) diet) for 63 days. Both malondialdehyde and protein carbonyl content in all studied tissues and serum were the lowest in fish fed diets containing 0.151-0.330 mg biotin kg(-1) diet and then increased in fish fed the diet with 2.680 mg biotin kg(-1) diet (P < 0.05). Similarly, glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase activities in serum significantly decreased with biotin levels up to 0.151 mg kg(-1) diet (P < 0.05). Conversely, capacities of anti-hydroxyl radical (AHR) and anti-superoxide anion (ASA) in the detected tissues and serum significantly improved with biotin levels up to 0.054-1.540 mg kg(-1) diet and then decreased in 2.680 mg biotin kg(-1) diet group for muscle and intestinal AHR as well as hepatopancreas ASA (P < 0.05). Activities of superoxide dismutase in all studied tissues and serum significantly elevated with biotin levels up to 0.330 mg kg(-1) diet and then decreased when fish fed the diet with 2.680 mg biotin kg(-1) diet, except intestine (P < 0.05). Meanwhile, activities of catalase, glutathione peroxidase, glutathione-S-transferase and glutathione reductase and total thiol content in all studied tissues and serum showed the upward trend with biotin supplementations (P < 0.05). These results indicated that biotin improved antioxidant status and depressed lipid peroxidation and protein oxidation in all studied tissues and serum.

Effects of dietary histidine on antioxidant capacity in juvenile Jian carp (Cyprinus carpio var. Jian).

In the present study, we tested the hypothesis that dietary histidine could improve antioxidant capacity of juvenile Jian carp (Cyprinus carpio var. Jian). A total of 1,200 juvenile Jian carp were fed graded levels of histidine at 2.3 (unsupplemented control), 4.4, 6.3, 8.6, 10.8 and 12.7 g/kg diet for 60 days. Results showed that the content of malondialdehyde (MDA) and protein carbonyl (PC) in serum and all tissues apparently decreased with increasing histidine levels up to an optimal level and increased thereafter. Anti-superoxide anion (ASA) capacity, glutathione peroxidase (GPX) activities and glutathione (GSH) content in serum and all tissues, anti-hydroxyl radical (a-HR) capacity, catalase (CAT) and glutathione-S-transferase (GST) activities in serum, muscle and intestine, superoxide dismutase (SOD) activities in serum and intestine, as well as glutathione reductase (GR) activity in serum, muscle and hepatopancreas were improved by dietary histidine. Fish fed diet with 8.6 g/kg histidine had lower serum glutamate-pyruvate transaminase (GPT) activity than that fed with control diet, whereas pattern of glutamate-oxaloacetate transaminase (GOT) activity was opposite. The present results suggested that histidine could improve antioxidant capacity and inhibit lipid peroxidation and protein oxidation of juvenile Jian carp.

Effect of dietary arginine on growth, intestinal enzyme activities and gene expression in muscle, hepatopancreas and intestine of juvenile Jian carp (Cyprinus carpio var. Jian).

The present study was conducted to test the hypothesis that dietary arginine promotes digestion and absorption capacity, and, thus, enhances fish growth. This improvement might be related to the target of rapamycin (TOR) and eIF4E-binding protein (4E-BP). A total of 1200 juvenile Jian carp, Cyprinus carpio var. Jian, with an average initial weight of 6.33 (SE 0.03) g, were fed with diets containing graded concentrations of arginine, namely, 9.8 (control), 12.7, 16.1, 18.5, 21.9 and 24.5 g arginine/kg diet for 9 weeks. An real-time quantitative PCR analysis was performed to determine the relative expression of TOR and 4E-BP in fish muscle, hepatopancreas and intestine. Dietary arginine increased (P < 0.05): (1) glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase activities in muscle and hepatopancreas; (2) intestine and hepatopancreas protein content, folds height, and trypsin, chymotrypsin, lipase, Na⁺/K⁺-ATPase, alkaline phosphatase, γ-glutamyl transpeptidase and creatine kinase activities in intestine; (3) Lactobacillus counts; (4) relative expression of TOR in the muscle, hepatopancreas and distal intestine (DI); (5) relative expression of 4E-BP in proximal intestine (PI) and mid-intestine (MI), as compared with the control group. In contrast, dietary arginine reduced (P < 0.05): (1) plasma ammonia content; (2) Aeromonas hydrophila and Escherichia coli counts; (3) relative expression of TOR in PI and MI; (4) relative expression of 4E-BP in the muscle, hepatopancreas and DI. The arginine requirement estimated by specific growth rate using quadratic regression analysis was found to be 18.0 g/kg diet. These results indicate that arginine improved fish growth, digestive and absorptive ability and regulated the expression of TOR and 4E-BP genes.