Regulation of Free Fatty Acid Receptor 4 on Inflammatory Gene Induced by LPS in Large Yellow Croaker (Larimichthys crocea)

Free fatty acid receptor 4 (FFAR4) plays a key role in regulating the inflammatory response in mammals. The present study aimed to investigate the function of large yellow croaker FFAR4 on inflammation. In the present study, ffar4 was widely expressed in 10 tissues of large yellow croaker including gill, head kidney and spleen. Further studies showed that treatment of head kidney macrophages with agonists (TUG891 or GSK137647A) or overexpression of ffar4 reduced the mRNA expression of pro-inflammatory genes induced by LPS, and increased the expression of pparγ. Treatment of macrophages with antagonist AH7614 increased the mRNA expression of pro-inflammatory genes induced by LPS, and decreased the mRNA expression of pparγ. In order to verify the immunomodulatory effect of PPARγ, PPARγ was overexpressed in macrophages which significantly reduced the mRNA expression of pro-inflammatory genes il6, il1β, il8, tnfα and cox2. Moreover, results of dual-luciferase assays showed that PPARγ downregulated the transcriptional activity of il6 and il1β promoters. In conclusion, FFAR4 showed anti-inflammatory effects on LPS-induced inflammation in large yellow croaker

Molecular Cloning and functional characterization of a putative Elovl4 gene and its expression in response to dietary fatty acid profiles in orange-spotted grouper Epinephelus coioides

Elongase of very long-chain fatty acids (Elovl) 4 probably plays a crucial role in marine fish species, where lack of Elovl2 has been considered as one possible reason for their low long-chain polyunsaturated fatty acids' (LC-PUFAs) biosynthetic capability. Elongase of very long-chain fatty acids 4 is the most recent member of the Elovl family that has been investigated in fish. Here, we report the molecular cloning and functional characterization of putativeelovl4cDNA isolated from marine teleost,Epinephelus coioides, and its expression in response to dietary n-3 LC-PUFA and docosahexaenoic acid (DHA) to eicosapentaenoic acid (EPA) ratio. Theelovl4cDNA of grouper was 2341bp including 301bp of 5′-untranslated region (UTR), 918bp of the coding region that encodes 305 amino acids (AA) and 1122bp of 3′UTR. Heterologous expression in yeast demonstrated that grouper Elovl4 could elongate saturated fatty acids (FA), especially 24:0 and 26:0, up to 36:0. Also, grouper Elovl4 effectively converted C20 and C22 polyunsaturated FAs to elongated polyenoic products up to C36. Tissue distribution analysis revealed that Elovl4 were widely transcribed in various tissues with the highest level in eye, brain and testis as described in other teleosts. The transcript level ofelovl4was significantly affected by dietary n-3 LC-PUFA and high LC-PUFA level repressess its expression. However, the ratio of DHA to EPA had no significant influence on its expression. These results may contribute to better understanding the LC-PUFA biosynthetic pathway in this fish species

Effects of supplemental octanoate on hepatic lipid metabolism, serum biochemical indexes, antioxidant capacity and inflammation-related genes expression of large yellow croaker (Larimichthys crocea) fed with high soybean oil diet

Dietary high soybean oil (SO) levels might cause hepatic lipid deposition, induce oxidative stress and inflammatory response in aquatic animals, while octanoate (OCT) is beneficial to metabolism and health in mammals. However, the effect of OCT has been studied rarely in aquatic animals. In this study, a 10-week feeding trial was conducted to investigate the effect of supplemental OCT on hepatic lipid metabolism, serum biochemical indexes, antioxidant capacity and inflammatory response of large yellow croaker (Larimichthys crocea) fed with high SO levels diet. The negative control diet contained 7% fish oil (FO), while the positive control diet contained 7% SO. The other four experimental diets were supplemented with 0.7, 2.1, 6.3 and 18.9 g/kg sodium octanoate (OCT) based on the positive control diet. Results showed that OCT supplementation effectively reduced the hepatic crude lipid, triglyceride (TG), total cholesterol (TC) and non-esterified free fatty acids contents, and alleviated lipid accumulation caused by the SO diet. Meanwhile, OCT supplementation decreased the serum TG, TC, alanine transaminase, aspartate transaminase and low-density lipoprotein cholesterol levels, increased the serum high-density lipoprotein cholesterol level, improved the serum lipid profiles and alleviated hepatic injury. Furthermore, with the supplementation of OCT, the mRNA expression of genes related to lipogenesis (acc1, scd1, fas, srebp1, dgat1 and cebpα) and fatty acid (FA) transport (fabp3, fatp and cd36) were down-regulated, while the mRNA expression of genes related to lipolysis (atgl, hsl and lpl) and FA β-oxidation (cpt1 and mcad) were up-regulated. Besides that, dietary OCT increased the total antioxidant capacity, activities of peroxidase, catalase and superoxide dismutase and the content of reduced glutathione, decreased the content of 8-hydroxy-deoxyguanosine and malondialdehyde and relieved hepatic oxidative stress. Supplementation of 0.7 and 2.1 g/kg OCT down-regulated the mRNA expression of genes related to pro-inflammatory cytokines (tnfα, il1β and ifnγ), and suppressed hepatic inflammatory response. In conclusion, supplementation with 0.7-2.1 g/kg OCT could reduce hepatic lipid accumulation, relieve oxidative stress and regulate inflammatory response in large yellow croaker fed the diet with high SO levels, providing a new way to alleviate the hepatic fat deposition in aquatic animals

Functional characterization and differential nutritional regulation of putative Elovl5 and Elovl4 elongases in large yellow croaker (Larimichthys crocea)

In the present study, two elongases, Elovl4 and Elovl5, were functionally characterized and their transcriptional regulation in response to n-3 LC-PUFA administration were investigated in vivo and in vitro. We previously described the molecular characterization of croaker elovl5. Here, we report the full- length cDNA sequence of croaker elovl4, which contained 1794 bp (excluding the polyA tail), including 909 bp of coding region that encoded a polypeptide of 302 amino acids possessing all the characteristic features of Elovl proteins. Functional studies showed that croaker Elovl5, displayed high elongation activity towards C18 and C20 PUFA, with only low activity towards C22 PUFA. In contrast, croaker Elovl4 could e ectively convert both C20 and C22 PUFA to longer polyenoic products up to C34. n-3 LC-PUFA suppressed transcription of the two elongase genes, as well as srebp-1 and lxrα, major regulators of hepatic lipid metabolism. The results of dual-luciferase reporter assays and in vitro studies both indicated that the transcriptions of elovl5 and elovl4 elongases could be regulated by Lxrα. Moreover, Lxrα could mediate the transcription of elovl4 directly or indirectly through regulating the transcription of srebp-1. The above ndings contribute further insight and understanding of the mechanisms regulating LC-PUFA biosynthesis in marine sh species

Effects of Dietary Squid Soluble Fractions on Growth Performance and Feed Utilization in Juvenile Snakehead(Ophiocephalus argus) Fed Practical Diets

An 8-week growth trial was conducted to investigate the effects of dietary squid soluble fractions (SSF) supplementation on growth performance and feed utilization of juvenile snakehead (Ophiocephalus argus). A fish meal diet (diet 1) with 36% fish meal (FM) was formulated. This diet was used as a control. The second diet (diet 2) was formulated by replacing 22% of the fish meal protein with poultry by-product meal based on amino acid profile of whole body of this fish. The other three diets were supplemented with 0.4% (diet 3), 0.8% (diet 4) and 1.2% (diet 5) SSF, respectively. Diets 1(control) 2, 3, 4, and 5 were isonitrogenous and isolipidic. No significant differences were observed in survival, feed intake (FI), weight gain rate (WGR), feed efficiency rate (FER) and protein productive value (PPV) between fish fed diets 1 and 2 (P>0.05). Furthermore, compared with fish fed the diet without SSF (diet 2), fish fed diets with 0.8 and 1.2% SSF (diet 4 and 5) demonstrated significantly enhanced WGR, FER, and PPV. These results suggest that 0.8% of dietary SSF is optimal for juvenile snakehead growth enhancement

Effects of Soya Saponins on Feed Intake, Growth Performance, and Cholesterol Metabolism in Juvenile Turbot (Scophthalmus maximus L)

An 8-week feeding trial was conducted to investigate the effects of soya saponins on feed intake, growth performance, and cholesterol metabolism in juvenile turbot (Scophthalmus maximus L). A control diet and two experimental diets were prepared with the supplementation of soya saponins, concentrations being as follows: 0.0% (Diet 1, Control), 0.25% (Diet 2) and 0.5% (Diet 3), respectively. The results showed that 0.25% of dietary soya saponins produced comparable growth performance with the control diet. However, the growth performance of fish fed the diet with 0.5% soya saponins was significantly lower. The selected parameters measured in plasma, liver, and feces of each group were not significantly different. These results suggested that 0.25% dietary soya saponins did not produce negative effects, but but but but 0. 5% of dietary soya saponins significantly reduced fish growth. Dietary soya saponin supplementation to FM-based diet did not significantly affect cholesterol metabolism

Effects of Dietary Corn Gluten Meal on Growth Performance and Cholesterol Metabolism in Juvenile Snakehead (Ophiocephalus argus) Fed Practical Diets

An 8-week growth trial was conducted to investigate the effects of dietary corn gluten meal (CGM) levels on growth performance and cholesterol metabolism of juvenile snakehead (Ophiocephalus argus). Six isonitrogenous (crude protein 47%) and isolipidic (crude lipid 11.5%) practical diets were formulated by replacing 0 (D1, control), 6.2 (D2), 12.3 (D3), 18.5 (D4), 22.2 (D5), and 28.4% (D6) fish meal (FM) protein with CGM protein. No significant difference in survival rate was found among dietary treatments (P>0.05). No significant differences were observed in feed intake (FI), final body weight (FBW), weight gain rate (WGR), feed efficiency rate (FER) and protein productive value (PPV) among fish fed D1-D3 (P>0.05). However, these indices significantly decreased with increasing CGM protein from 18.5%(D4) to 28.4%(D6) of diet (P<0.05). Whole-body lipid content of fish fed D6 (28.4 %) was significantly lower than that of fish fed D1 (control) (P<0.05). The plasma and liver total cholesterol, free cholesterol, cholesterol esters, LDL-C, and HDL-C levels were affected by dietary CGM protein levels. Broken line analysis based on WGR or PPV indicated that the maximum CGM protein level for the optimal growth of juvenile snakehead was 12.06% or 12.56% respectively

Dietary Histidine Requirement for Juvenile Large Yellow Croaker, Pseudosciaena crocea R.

Dietary histidine requirements for large yellow croaker (initial average weight, 6.0 ± 0.10 g) were quantified by feeding isonitrogenous (crude protein 44%) and isocaloric (20 KJ/g) amino acid test diets with graded levels of histidine [0.45% (D1), 0.66% (D2), 0.78% (D3), 0.98% (D4), 1.24% (D5) and 1.40% (D6) of dry diet]. Each diet was randomly assigned to triplicate floating sea cages (1.0 × 1.0 × 1.5 m), 60 fish/cage. At the end of the 51 day experiment, the final weight (FW) and weight gain (WG) of large yellow croaker showed a positive correlation to increasing dietary histidine content (up to 0.78%), and thereafter declined. The growth of fish fed the D6 diet was significantly lower than fish maintained on the D2 and D3 diets; however, there were no significant differences in the growth of large yellow croaker among all the dietary treatments except D6. Fish fed the D3 diet had the highest FW and WG. The shift in feed efficiency (FE) values of fish fed D1 to D5 diets increased as dietary histidine content increased, and significantly decreased in fish fed the D6 diet. Based on the second-degree polynomial regression analyses of the growth data, optimum histidine requirement for juvenile large yellow croaker was 8.7 g/kg dry diet, (18.8 g/kg–20.8 g/kg of dietary protein within 95% confidence interval)

Conventional Soybean Meal as Fishmeal Alternative in Diets of Japanese Seabass (Lateolabrax japonicus): Effects of Functional Additives on Growth, Immunity, Antioxidant Capacity and Disease Resistance

Aiming to optimize soymeal-based diets for Japanese seabass (Lateolabrax japonicas), a 105-day feeding trial was conducted to evaluate the effects of functional additives, including antioxidants (ethoxyquin, thymol and carvacrol) and chelated trace elements (Cu, Mn and Zn), on the growth, immunity, antioxidant capacity and disease resistance of fish fed diets with conventional soybean meal replacing 50% of fishmeal. Three isonitrogenous (45%) and isolipidic (11%) diets were formulated: (1) standard reference diet (FM, 42% fishmeal); (2) soymeal-based diet (SBM, 21% fishmeal and 30% conventional soybean meal); (3) SBM diet supplemented 0.0665% functional additives (FAS). Each experimental diet was randomly fed to quadruplicate groups of forty feed-trained Japanese seabass (initial average body weight = 125.6 &plusmn; 0.6 g) stocked in a saltwater floating cage. Upon the conclusion of the feeding trial, lower feed intake was observed in fish fed SBM compared to those fed FM and FAS. Fish fed FM showed the highest growth performance, estimated as the weight gain rate. Notably, FAS supported faster growth of fish than those fed SBM, indicating the optimal growth performance of dietary functional additives. The feed conversion rate showed the opposite trend among dietary treatments, with the highest value in fish fed SBM. Regarding immunity, fish fed soymeal-based diets suppressed the serum alternative complement pathway activities compared to FM, whereas the respiratory burst activity in macrophages of head kidneys showed a similar picture, but no statistical differences were observed. Further, fish fed soymeal-based diets had lower serum Cu-Zn SOD, CAT and GPx activities as well as liver vitamin E levels and scavenging rates of hydroxyl radical but higher liver MDA contents compared to the FM-fed group. Fish fed FAS had higher serum Cu-Zn SOD and GPx activities and liver vitamin E levels than those fed SBM, suggesting the enhancement of antioxidant capacity of dietary functional additives. For the disease resistance against Vibrio harveyi infection, fish fed SBM had the highest cumulative mortality, followed by the FAS and FM groups. Additionally, the biomarkers related to the immune and antioxidant capacities had a positive correlation with the relative abundance of Paracoccus and Pseudomonas, while liver MDA levels had a negative correlation with the relative abundance of Pseudomonas and Psychrobacter. Collectively, soymeal replacing 50% of fishmeal suppressed the growth, immunity, antioxidant capacity and disease resistance of Japanese seabass, while dietary supplementation of antioxidants and chelated trace elements could mitigate soymeal-induced adverse effects on growth and disease resistance through the improvement in antioxidant capacity and regulation of gut microbiota

Evaluation of Dietary Anti-nutritional Factors and Amino Acids Supplementation on Growth, Feed Efficiency Ratio, and Apparent Digestibility of Japanese Flounder (Paralichthys olivaceus) at Equal Feed Intake

A 30-day feeding trial was conducted to evaluate the anti-nutritional factors in fish meal-based diets, and limiting amino acids in soybean meal-based diets on growth, feed efficiency ratio (FER), and nutrient apparent digestibility (AD) of Japanese flounder (Paralichthys olivaceus) at equal feed intake. Four isonitrogenous and isoenergetic diets were formulated: Diet 1 contained fish meal as the main dietary protein source; Diet 2 was based on Diet 1 and supplemented with anti-nutritional factors; Diet 3 contained both fish meal and soybean meal as main protein source; Diet 4 was based on Diet 3 and supplemented with crystalline amino acids. All diets were assigned to triplicate groups of 5 fish per aquarium. Results from Diet 1 showed that force-feeding was a possible method to rear Japanese flounder with high FER and 100% survival rate. In the first three periods (4 days/period), weight gain (WG) and FER of fish fed Diets 2, 3 & 4 were significantly lower (P<0.05) than in fish fed Diet 1. After 30 days of the feeding trial, WG and FER of fish fed Diets 3 & 4 were significantly lower than in fish fed Diet 1 (P<0.05). No significant difference in WG and FER was observed either between fish fed Diets 1 & 2, or between fish fed Diets 3 & 4. The AD of dry matter and crude lipid of fish fed Diets 3 & 4 were significantly lower than those of fish fed Diet 1 (P<0.05). Results indicated that supplementation of anti-nutritional factors in a fish meal-based diet, or supplementation of crystalline amino acids in a soybean meal-based diet did not significantly affect growth performance of Japanese flounder by force feeding