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 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

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 ± 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

Effects of Replacing Fish Meal with Soybean Meal or Fermented and Phytase-Treated Soybean Meal Respectively, on Growth Performance, Feed Utilization, and Apparent Digestibility in Juvenile Turbot (Scophthalmus maximus L.)

The objective of this study was to evaluate the effect of fish meal (FM) substitution with gradient soybean meal (SBM) or fermented and phytase-treated soybean meal (PHSBM) in the diets of turbot (Scophthalmus maximus). A 9 week feeding trial was conducted using juvenile turbots (Scophthalmus maxima) fed seven experimental diets. The seven isonitrogenous (approximately 50% crude protein) and isoenergetic (approximately 21.0 kJ/g diet of gross energy) diets were formulated to include FM protein substitution with corresponding amounts of protein from soybean meal and phytase-treated soybean meal sources. Results showed that survival rate and feed intake did not differ significantly between the FM diet and any plant protein incorporated diets. Compared with the FM diet, final body weight and SGR were significantly reduced by the SBM2, SBM3, and PHSBM3 diets. Except for the PHSBM1 diet, feed efficiency ratio in the other SBM or PHSBM incorporated diets was much lower than in the FM diet. Body ash content was not affected by gradient PHSBM incorporated diets compared with the FM diet, while SBM incorporated diets (SBM2 and SBM3) showed a higher ash content than the FM diet (P<0.05). Body crude protein was significantly reduced when fishmeal protein was replaced by soybean meal up to 60% (SBM3). There was no significant difference in the crude lipid and moisture contents among different treatments. Fish meal replaced by gradient PHSBM did not affect the apparent digestibility coefficients (ADC) of dry matter and crude protein, while the ADC of dry matter was markedly reduced in all the soybean meal incorporated diets compared with the FM diet. These results show that 30% fish meal protein could be replaced by soybean meal in the diet of turbot, while PHSBM could be substituted for up to 45% dietary fish meal

Apparent Digestibility of Selected Feed Ingredients in Juvenile Turbot (Scophthalmus maxima L.)

In aquaculture, replacing dietary fishmeal with other protein sources is challenging. In order to select substitute sources, it is necessary to determine the apparent digestibility coefficients, many of which have not been well characterized for fish species. We investigated the apparent digestibility of eight protein sources fed to juvenile turbot (Scophthalmus maxima L.). These were Peruvian super red fishmeal (FM), peanut meal (PM), corn gluten meal (CGM), dehulled solvent extracted soybean meal (SBM), wheat gluten (WG), Australian beef meat and bone meal (MBM), spray-dried (pork) hemoglobin meal (SDHM) and American pet-food grade poultry byproduct meal (PBM). The apparent digestibility coefficients of dry matter (DM), crude protein (CP), gross energy (GE), and amino acids were analyzed. Results indicated that turbot utilized high-protein feedstuffs better than high-carbohydrate or high-fiber feedstuffs. This study provides valuable information on ingredient selection and evaluation of feed for turbot

Effects of Dietary Soy Protein Concentrate on Growth Performance, Digestion, and Protein Metabolism of Juvenile Darkbarbel Catfish Pelteobagrus vachelli

A 10-week feeding trial in a recirculation rearing system was conducted to investigate the effects of dietary soy protein concentrate (SPC) levels on survival, growth, digestion, and protein metabolism of juvenile darkbarbel catfish Pelteobagrus vachelli. The results demonstrated that survival and whole-body composition were independent of dietary treatments. Feed intake (FI), specific growth rate (SGR), feed conversion ratio (FCR), apparent digestibility coefficients, digestive enzymes (pepsin and trypsin) activity and protein metabolism enzymes (alanine aminotransferases) decreased with increasing dietary SPC. The diet with 60% SPC was least cost effective. Results suggest that SPC could replace 60% or less fish meal protein without negatively influencing the growth of juvenile Pelteobagrus vachelli