Effects of dietary lipid source on fatty acid composition,expression of genes involved in lipid metabolism and antioxidant status of grass carp (Ctenopharyngodon idellus)

This study determined the effect of different lipid sources on growth, feed use, lipid metabolism and antioxidant status of grass carp (Ctenopharyngodon idellus). Juvenile fish (56.9 ± 4.7 g) were divided into four triplicate groups and fed diets containing 30 g/Kg of fish oil (FO), olive oil (OO), peanut oil (PO) and linseed oil (LO), respectively, for 60 days. Weight gain and feed conversion ratio were not significantly different between the dietary groups, but we observed changes in the fatty acid composition of muscle and intraperitoneal fat reflecting the fatty acid profile of the dietary lipid source. In the hepatopancreas, the highest mRNA level of fatty acid translocase CD36 (FAT/CD36) and carnitine palmitoyl transferase (CPT‐1A) was both observed in the FO group. In muscle, the expression of FAT/CD36 and CPT‐1A in the LO group was significantly higher than that in other groups, except for CPT‐1A in the PO group. In addition, the lowest and highest content of malondialdehyde in serum was observed in OO and FO groups, respectively. In summary, dietary lipid source altered the fatty acid composition, potential uptake (FAT/CD36) and oxidation (CPT‐1A) of fatty acids, and antioxidant status of grass carp, which should be considered when selecting a lipid source.     

草鱼ATGL基因的表达及饲喂n-3 HUFAs对其的影响

实验获得了草鱼脂肪组织甘油三酯水解酶(ATGL) 部分cDNA序列(GenBank 登录号为HQ845211), 并进行了序列同源性分析; 采用实时定量反转录聚合酶链式反应(qRT-PCR)方法, 检测了ATGL基因在草鱼不同组织的表达状况; 研究了投喂n-3高不饱和脂肪酸(n-3 HUFAs)对草鱼肝胰脏ATGL基因时序表达的影响。结果显示, 所获得的草鱼ATGL基因部分cDNA序列长度为687 bp, 与人、牛、小鼠、长腭泥虎鱼、大黄鱼等物种的同源性为65%~75%; 该基因在草鱼心脏、肝胰脏、脾脏、鳃、肾脏、肌肉、腹腔脂肪组织、脑、小肠、精巢10个组织中均有表达, 其中在腹腔脂肪组织中表达丰度最高, 在肝胰脏和肌肉中表达丰度次之。处理组草鱼摄食n-3 HUFAs饲料后, ATGL基因的表达水平在第1周和第2周显著高于对照组, 第3周后, 该基因的表达水平在处理组与对照组间无显著差异。研究首次克隆得到草鱼ATGL基因部分cDNA序列, 并发现该基因在草鱼脂质蓄积及代谢较旺盛的组织中表达水平较高, 且其在肝胰脏中的表达受到n-3 HUFAs的影响, 其规律为先被诱导升高, 然后回复到正常水平。     

Dietary α‐linolenic acid affects lipid metabolism and tissue fatty acid profile and induces apoptosis in intraperitoneal adipose tissue of juvenile grass carp (Ctenopharyngodon idella)

A 56‐day feeding trial was conducted to elucidate the effects and mechanism action of dietary α‐linolenic acid (ALA, 18:3n‐3) on lipid accumulation and fatty acid profile of muscle, hepatopancreas and intraperitoneal fat (IPF) in juvenile grass carp using three isonitrogenous and isoenergetic semi‐purified diets containing 0.0% (control group), 1.0% and 2.0% ALA, respectively. The lowest intraperitoneal fat (IPF) ratio was found in 2.0% group. In the muscle, hepatopancreas and IPF, docosahexaenoic acid (DHA, 22:6n‐3) and eicosapentaenoic acid (EPA, 20:5n‐3) contents increased with the increase in dietary ALA. In the IPF, caspase 3, caspase 8 and caspase 9 showed the highest activities in 2.0% group, while the value of Bcl‐2/Bax (B‐cell leukaemia 2/Bcl‐2‐associated X protein) reached the lowest. Meanwhile, swelling of the IPF mitochondria was observed in 2.0% group. The gene expressions of fatty acid desaturase (FAD) and fatty acid elongase (ELO) in the hepatopancreas and muscle showed significantly higher levels in the treatment groups, whereas an opposite trend was existed in the IPF. Fatty acid synthase (FAS), sterol regulatory element binding protein‐1c (SREBP‐1c) in the IPF and hepatopancreas reached the lowest in 2.0% group. Overall, dietary ALA could promote n‐3 highly unsaturated fatty acids (HUFAs) synthesis and suppress the accumulation of lipid by decreasing the expression of related genes and promoting the apoptosis in IPF.     

Effects of different dietary lipid sources on growth performance,body composition and lipid metabolism‐related enzymes and genes of juvenile golden pompano,Trachinotus ovatus

Three groups of juvenile golden pompano, Trachinotus ovatus (54.75 ± 0.25 g), were each fed one of three diets containing different lipid sources: fish oil (FO), soybean oil (SO) and lard oil (LO). Fish were reared in sea cages for 8 weeks, and the fish fed the FO diet had significantly higher specific growth rate (SGR) but lower condition factor (CF) than the other treatments. The fatty acid (FA) composition of whole‐body lipids was closely correlated with those in the diets. Although no differences can be found in hepatic fatty acid synthase (fasn) activity, the carnitine palmitoyl transferase 1 (cpt1) activity in fish fed the FO diet was significantly higher compared with other treatments. In addition, the relative gene expression of lipid metabolism‐related enzymes, such as cpt1, fas, apolipoprotein B100 (apoB100), delta‐6 fatty acyl desaturase (fadsd6) and fatty acid‐binding protein 1 (fabp1), was also influenced by the different dietary lipid sources. Serum triglyceride (TG) and glucose content in fish fed the LO and FO diets were significantly higher than those in the SO group. Accordingly, it can be concluded that FO could not be completely replaced by SO or LO in golden pompano diets. The lipid sources of a diet could impose significant influence on body condition factor and hepatic lipid metabolism of golden pompano.     

Optimal dietary alpha‐linolenic acid/linoleic acid ratio improved digestive and absorptive capacities and target of rapamycin gene expression of juvenile grass carp (Ctenopharyngodon idellus)

Growth performance, digestive and absorptive capacities and target of rapamycin (TOR), ribosomal protein S6 kinase 1 (S6K1) and eIF4E‐binding protein (4E‐BP) gene expression in the hepatopancreas and intestine of juvenile grass carp (Ctenopharyngodon idellus) fed graded ratios of dietary alpha‐linolenic acid/linoleic acid (ALA/LNA) (0.01, 0.34, 0.68, 1.03, 1.41, 1.76 and 2.15) for 60 days were investigated. The results showed that ALA/LNA ratio of 1.03 significantly improved (i) per cent weight gain (PWG) and feed efficiency, (ii) hepatopancreatic trypsin, chymotrypsin, lipase, amylase and intestinal creatine kinase (CK) activities, (iii) hepatopancreatic trypsinogen‐2 and chymotrypsinogen mRNA levels. Meanwhile, fish fed with ALA/LNA ratio of 0.68 significantly enhanced, (iv) Na⁺/K⁺‐ATPase and γ‐glutamyl transpeptidase activities in whole intestine, and alkaline phosphatase activities in the proximal intestine (PI) and distal intestine, (v) amylase, intestinal Na⁺/K⁺‐ATPase alpha‐subunit isoform 1, Na⁺/K⁺‐ATPase alpha‐subunit isoform 8 and CK mRNA abundances, (vi) TOR and S6K1 gene expression in the hepatopancreas and intestine of juvenile grass carp. Based on the quadratic regression analysis of PWG, cholecystokinin and leptin contents in the PI, optimal dietary ALA/LNA ratio of juvenile grass carp (8.78–72.00 g) was estimated to be 1.08, 1.19 and 1.05, respectively.     

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.     

An evaluation of increasing linolenic acid level in the diet of Macrobrachium nipponense: Lipid deposition,fatty acid composition and expression of lipid metabolism‐related genes

To determine the effects of linolenic acid (LNA, 18:3n‐3) in oriental river prawn (Macrobrachium nipponense), an 8‐week feeding experiment was conducted using six isonitrogenous and isoenergetic semi‐purified diets containing 0.07 g/kg (control), 7.3 g/kg, 16.6 g/kg, 20.2 g/kg, 27.3 g/kg and 36.3 g/kg LNA. The hepatopancreas lipid content decreased significantly when dietary LNA content was >20.2 g/kg. Fatty acid analysis revealed that the percentage of 18:3n‐3 in the hepatopancreas significantly increased with increasing dietary LNA levels, while 20:5n‐3, 22:5n‐3 and 22:6n‐3 levels in the hepatopancreas decreased in a curvilinear manner as dietary LNA increased. Additionally, qRT‐PCR results revealed that hepatopancreas mRNA expression of acetyl‐CoA carboxylase (ACC) decreased with increasing dietary LNA, while the greatest carnitine palmitoyl transferase‐1(CPT1) mRNA expression was observed in the 2.73 g/kg and 36.3 g/kg groups. Furthermore, hepatopancreas mRNA expression of acyl‐CoA delta‐9 desaturase (SCD) and fatty acyl elongase 6(elovl6) was downregulated when prawns fed the diets containing >20.2 g/kg LNA. These results indicate that dietary 18:3n‐3 could decrease lipid deposition through increased fatty acid β‐oxidation and modulated fatty acid synthesis, and alter fatty acid composition by regulating fatty acyl elongase and fatty acyl desaturase mRNA expression in the M. nipponense.     

Effects of different dietary lipids on growth,body composition and lipid metabolism‐related enzymes and genes in juvenile largemouth bass,Micropterus salmoides

This study investigates the effects of different lipids on growth, body composition and lipid metabolism of largemouth sea bass fish Micropterus salmoides. A total of 360 juvenile M. salmoides (mean ± SD mass = 33.83 ± 0.15 g) were randomly stocked into 12 tanks of 0.5 m³ volume for 8 weeks. Four replicates were made in each group, which were fed one of three diets containing fish oil (FO), soybean oil (SO) or lard oil (LO). The weight gain rate and specific growth rate did not differ among the groups (p > 0.05). Fish oil fish had the lowest condition factor (p < 0.05) and highest serum glucose content (p < 0.05). Crude lipid contents in the whole body and in the liver and muscle of FO fish were significantly lower than in the SO and LO groups (p < 0.05). The fatty acid composition of whole‐body lipids was closely correlated with that of the diet. The carnitine palmitoyltransferase 1 (cpt1) activity in the FO group was significantly higher than those in the SO and LO groups (p < 0.05). No significant differences in fatty acid synthase (fasn) activity were observed among the groups (p > 0.05). The Cpt1 and fasn gene expression levels in the FO group were significantly higher than those of the SO and LO groups (p < 0.05). The apolipoprotein B100 gene expression level was significantly higher in the SO group than in the FO group (p < 0.05). Fatty acid‐binding protein 1 gene expression levels in the FO and SO groups were not different (p > 0.05) but were both higher than that of the LO group (p < 0.05). The delta‐6 fatty acyl desaturase gene expression level in the LO group was significantly higher than that in the FO group (p < 0.05), but lower than that in the SO group (p < 0.05). It can be concluded that FO can be completely replaced by SO or LO in the M. salmoides diet, at least within the 8‐week culture period. Different types of dietary lipids significantly affect body condition and hepatic lipid metabolism in M. salmoides.     

Dietary ratios of n‐3/n‐6 fatty acids do not affect growth of Nile tilapia at optimal temperatures (28°C) nor at temperatures that simulate the onset of winter (22°C)

Nile tilapia (Oreochromis niloticus) juveniles were fed diets containing 13 g/kg total polyunsaturated fatty acids (PUFAs) at different n‐3/n‐6 dietary ratios (0.2, 0.5, 0.8, 1.3 and 2.9) for 56 days, at 28°C. Subsequently, fish were submitted to a winter‐onset simulation (22°C) for 33 days. PUFA n‐3/n‐6 dietary ratios did not affect fish growth at either temperature. At 28°C, tilapia body fat composition increased with decreasing dietary PUFA n‐3/n‐6. Winter‐onset simulation significantly changed feed intake. The lowest dietary n‐3/n‐6 ratio resulted in the highest feed intake. At both temperatures, body concentrations of α‐linolenic acid, docosahexaenoic acid, eicosatrienoic acid and docosapentaenoic acid decreased as dietary n‐3/n‐6 decreased. Body concentrations of eicosapentaenoic acid (EPA, 20:5 n‐3) increased with decreasing concentrations of dietary EPA. The n‐6 fatty acids with the highest concentrations in tilapia bodies were linoleic acid and arachidonic acid (ARA, 20:4 n‐6). At 28°C, SREBP1 gene expression was upregulated in tilapia fed the lowest n‐3/n‐6 diet compared to tilapia fed the highest n‐3/n‐6 ratio diet. Our results demonstrate that a dietary PUFA of 13 g/kg, regardless of the n‐3/n‐6 ratio, can promote weight gains of 2.65 g/fish per day at 28°C and 2.35 g/fish per day at 22°C.     

Dietary α‐Ketoglutarate supplementation alleviates harmful effects of high environmental ammonia on grass carp,Ctenopharyngodon idella

Current intensive fish farming usually causes high environmental ammonia (HEA) in ponds that is toxic to fish. α‐Ketoglutarate (α‐KG) can be rapidly transaminated to glutamic acid and further aminated to glutamine. Therefore, we hypothesized that dietary α‐KG supplementation would alleviate HEA toxicity to fish. To test the hypothesis, 270 healthy grass carp (Ctenopharyngodon idella) juveniles were randomly assigned to control, HEA (18.37 mg/L ammonia) and HEA + α‐KG (0.75% of α‐KG) groups. Ammonia and free amino acid content in plasma and brain, liver glutamic pyruvic transaminase and glutamic oxaloacetic transaminase activity, and urea and glycogen content were measured on the first, seventh and 42nd days. Our results showed short‐term HEA exposure (1 day) led to a significant ammonia accumulation in the brain and plasma and significantly decreased glutamic and aspartic acid content in the brain and increased glutamine content in the brain and plasma. The long‐term HEA exposure (42 days) caused significant reductions in glycine and arginine content in the brain tissue. In most cases, dietary α‐KG supplementation alleviated the fluctuations in FAA content in the brain and plasma. Our results suggested dietary α‐KG alleviated HEA toxicity to grass carp.     

Dietary soybean β‐conglycinin suppresses growth performance and inconsistently triggers apoptosis in the intestine of juvenile grass carp (Ctenopharyngodon idella) in association with ROS‐mediated MAPK signalling

The current study aimed to investigate the effects of dietary soybean β‐conglycinin on growth performance and intestine apoptosis in juvenile grass carp (Ctenopharyngodon idella). For fish fed with the 80 g β‐conglycinin/kg diet for 7 weeks, the specific growth rate and feed intake were decreased. In the proximal intestine, dietary β‐conglycinin did not induce DNA fragmentation, tended to decrease the reactive oxygen species (ROS) content, and decreased ROS‐generating enzyme (NADPH oxidase [NOX]) activity. Subsequently, in the mid‐intestine, dietary β‐conglycinin caused DNA fragmentation, tended to increase the ROS content, increased caspase‐3, caspase‐8 and caspase‐9 activities, upregulated the mRNA levels of proapoptotic molecules (apoptotic protease‐activating factor‐1 [Apaf1] and Bcl‐2‐associated X protein [BAX]) and mitogen‐activated protein kinase (MAPK)‐related signal molecules (Jun N‐terminal kinase (JNK) and p38 MAPK) and increased the protein levels of p38 MAPK and phospho‐p38 MAPK. Moreover, in the distal intestine, dietary β‐conglycinin induced DNA fragmentation, elevated NOX activity and the ROS content and increased caspase‐3, caspase‐8 and caspase‐9 activities, death ligand (TNF‐α) mRNA expression level, and p38 MAPK and phospho‐p38 MAPK protein levels. In summary, dietary soybean β‐conglycinin suppressed fish growth and inconsistently caused apoptosis among the different intestinal segments which was partially associated with ROS‐mediated MAPK signalling.     

Effects of dietary n‐3HUFA on juvenile white shrimp,Litopenaeus vannamei: Growth,feed utilization,antioxidant enzymes activities and fatty acid compositions

Present study investigates the effects of n‐3 high‐unsaturated fatty acid (n‐3HUFA) levels on growth performance, antioxidant enzymes activities and fatty acid compositions of juvenile Litopenaeus vannamei. These represented seven iso‐nitrogenous and iso‐lipidic diets. Analysed n‐3HUFA concentrations were 0.16% (control), 0.48%, 0.74%, 1.39%, 2.39%, 2.92% and 3.44% respectively. A total of 840 juvenile L. vannamei were randomly stocked into 21 0.5 m³ tanks for 56 days. A significant increase (p < 0.05) was observed from 0.16% to 0.74% n‐3HUFA and a decrease when n‐3HUFA was above these levels in weight gain rate (WGR) and specific growth rate. Total cholesterol, triglyceride and low‐density lipoprotein in serum showed a significant decrease, high‐density lipoprotein showed a significant increase (p < 0.05). Phenoloxidase activity in serum and sodium‐potassium adenosine triphosphatase activity in gill were significantly affected by dietary n‐3HUFA (p < 0.05), both of them showing a downward trend after upward. Malic dehydrogenase and superoxide dismutase activities in serum were also significantly affected by dietary n‐3HUFA (p < 0.05), which rose first and then decreased in general, both of them have a maximum in 2.39% group. No significant differences of the activities of aspartate transaminase and alanine transaminase were observed among all groups (p > 0.05). With dietary n‐3HUFA increase, both ∑HUFA and n‐3HUFA contents gradually increased in hepatopancreas and tail muscles (p < 0.05). Based on broken‐line regression analysis of WGR, the optimal n‐3HUFA requirement is 0.89% for juvenile L. vannamei with initial weight of 0.50 ± 0.01 g.     

Effects of dietary n‐3HUFA on different growth stages of the white shrimp,Litopenaeus vannamei: Growth,haematological characteristics,enzyme activities and fatty acid profiles

This study investigated the effects of n‐3 high unsaturated fatty acid (n‐3HUFA) levels on the growth performance, antioxidant enzyme activities and fatty acid profiles of both subadult and adult Litopenaeus vannamei (L. vannamei). Seven iso‐nitrogenous and iso‐lipidic diets were used, containing n‐3HUFA concentrations of 1.6 (control), 4.8, 7.4, 13.9, 23.9, 29.2 and 34.4 g/kg, respectively. Two 8‐week feeding trials were conducted to determine the dietary n‐3HUFA requirements of L. vannamei with an initial body weight of 4.25 ± 0.00 g (subadults) and 8.50 ± 0.01 g (adults). The results showed that the dietary n‐3HUFA level significantly affected the weight gain rate (WGR), specific growth rate, the feed conversion ratio and the hepatosomatic index (HSI) (p < 0.05), but did not significantly affect the survival rate (p > 0.05). At appropriate level, dietary n‐3HUFA improved growth performance and HSI of both subadult and adult L. vannamei. Both subadults and adults showed significant differences in body composition (p < 0.05), except for moisture and crude ash (p > 0.05). Cholesterol and low‐density lipoprotein significantly decreased with increasing dietary n‐3HUFA both in subadults and adults (p < 0.05); however, triglyceride showed no significant change (p > 0.05). High‐density lipoprotein (HDL) in subadults was significantly affected by dietary n‐3HUFA (p < 0.05), but followed no apparent regularity; HDL significantly changed in adults and showed an upward trend followed by a downward trend (p < 0.05). There was no significant effect on aspartate transaminase (AST) activity in subadults, but AST in adults and alanine transaminase (ALT) in subadults and adults were significantly affected (p < 0.05). Dietary n‐3HUFA significantly affected serum polyphend oxidase, malic dehydrogenase, alkaline phosphatase, superoxide dismutase and sodium‐potassium adenosine triphosphatase enzyme activities in gills (p < 0.05). The fatty acid composition of the shrimp tissue was associated with the fatty acid composition of the diet. Dietary n‐3HUFA supplementation significantly improved the contents of tissue ∑HUFA and n‐3HUFA, increased the n‐3/n‐6 ratio in the tail muscle and decreased the contents of tissue polyunsaturated fatty acid and saturated fatty acid (p < 0.05). Based on the WGR, the broken‐line equations indicated that the optimum requirements of dietary n‐3HUFA were determined to be 9.0 and 5.1 g/kg for subadult and adult L. vannamei, respectively.     

Effects of dietary protein and lipid levels on growth performance,fatty acid composition and antioxidant‐related gene expressions in juvenile loach Misgurnus anguillicaudatus

Dojo loach (Misgurnus anguillicaudatus) is one of the most important cultured freshwater fish in several East Asian countries. However, a little information is available in its nutritional requirements. Thus, this study was conducted to evaluate the effects of feeding varying levels of dietary protein and lipid on growth, fatty acid composition and antioxidant‐related gene expressions in juvenile loach. Six practical diets at three levels of protein (30%, 40% and 50%) and two levels of lipid (6% and 12%) were fed to loach juveniles (initial weight 0.40 g) in triplicated groups (20 fish per replicated) for a period of 8 weeks. Results showed that regardless of lipid level, body weight gain of fish was significantly increased with incremental dietary protein level. Meanwhile, feed conversion ratio was significantly decreased by dietary protein levels, and the lowest value was observed in fish fed dietary protein levels of 50%, regardless of dietary lipid level. Moreover, the percentage of 22:6n‐3 in viscera was significantly increased by different protein levels. The expression level of catalase was significantly increased with incremental dietary protein level with both lipid levels. Meanwhile, the expression level of hepatic nuclear factor erythroid 2‐related factor 2 (Nrf2) was downregulated with incremental dietary protein level with 6% of lipid level, but the expression was upregulated with incremental dietary protein level with 12% of lipid level. In conclusion, these data suggested that 6% lipid and 50% protein in diet was optimal for loach during early development stage.