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Methionine is one of the most limited factors in plant-based feed for fish. It plays critical roles in fish growth performance and feed utilization. However, little is known about its effects on amino acid metabolism and intestinal homeostasis in turbot. In this study, an 8-week feeding trial was conducted to examine the nutritional physiology of turbot fed with diets containing deficient (0.85%, LM), adequate (1.82%, MM) or excess (2.80%, HM) levels of methionine respectively. After the feeding trial, fish growth and feed efficiency were significantly reduced by LM diet while no significant difference was observed between MM group and HM group. Methionine deficiency diet also led to a reduction of free Met, Cys, Thr, Arg and His levels in plasma, while an elevation of Gly, Lys and Ala concentrations. The Asp and Gly levels in HM group were significantly lower than other groups. The mRNA expression of cystathionine β-synthase (cbs) and s-adenosylmethionine decarboxylase (samdc) in LM group were up-regulated while the expression of methionine synthase (ms) in LM group was depressed. The expression of ornithine decarboxylase (odc) was not affected by dietary methionine levels. Furthermore, methionine deficiency decreased the height of intestinal villus and microvilli, as well as the number of goblet cells. The mRNA expression of proliferating cell nuclear antigen (pcna) and mucin-2 (muc-2), as well as the content of glutathione, were also induced with the methionine level in the diets. These results provide important new clues in methionine function in teleost.

In the present study, Lactobacillus plantarum P8 was used to ferment soybean meal (SBM), resulting in a significant reduction in the indigestible oligosaccharides (sucrose, raffinose, and stachyose) and anti-nutritional factors (tannin, trypsin inhibitors, glycinin, and β-conglycinin) in SBM. Nine isonitrogenic and isoenergetic experimental diets were formulated in which SBM or L. plantarum P8 fermented SBM (LPFSBM) replaced fish meal (FM) protein with 0 (control); 15% by SBM (Diet 1), 30% by SBM (Diet 2), 45% by SBM (Diet 3), 60% by SBM (Diet 4), 15% by LPFSBM (Diet 5), 30% by LPFSBM (Diet 6), 45% by LPFSBM (Diet 7), and 60% by LPFSBM (Diet 8). Triplicate groups of 30 fish were fed to apparent satiation twice daily for 66 d in an indoor recycling seawater system. No significant differences were found in survival rate among the different groups. However, 45% SBM, 60% SBM, and 60% LPFSBM (Diets 3, 4, and 8) diets significantly reduced growth performance and feed utilization. The LPFSBM diets did not affect moisture, crude protein, crude lipid, or crude ash contents of whole-body measurements. However, 60% SBM (Diet 4) significantly decreased crude protein and lipid content while increasing moisture and crude ash contents of the whole-body. The activity of serum lysozyme was significantly decreased in the group fed with 60% SBM (Diet 4). Total antioxidant capacity was significantly increased in the group fed the 45% LPFSBM (Diet 7). Apparent digestibility coefficient (ADC) of protein was significantly lower in fish fed diets with 45% and 60% of protein from SBM and 60% of protein from LPFSBM than in the control group. When the substitution level of SBM was equal to or above 45%, the activities of protein metabolism enzymes (alanine aminotransferase, ALT and aspartate aminotransferase, AST) were significantly lower than those in the control group. The level of LPFSBM did not affect the activities of ALT and AST. SBM-induced pathological changes seen in the distal intestine of fish fed 45% and 60% SBM (Diets 3 and 4) were not pronounced in groups fed LPFSBM. The results of the present study suggest that protein from SBM could substitute up to 30% of FM protein, with protein from LPFSBM being able to replace up to 45% of FM protein. Statement of relevance The present study was conducted to investigate the effects of Lactobacillus plantarum P8 fermentation on soybean meal and evaluate LPFSBM as a partial replacement for fish meal in diets of turbot by examining growth, activities of protein metabolism enzymes, intestinal morphology, and immune responses. Our results would be helpful to develop cost effective and sustainable dietary formulations for turbot. The results of this study indicate that LPFSBM could replace up to 45% of fish meal protein in juvenile turbot diet. The ANFs are important factors that affect the growth performance of turbot, damaging the normal structure of intestine and a depression of digestion and absorption function. LPFSBM reduced SBM-induced pathomorphological changes in the distal intestine which partly accounted for the L. plantarum P8 fermentation reduces indigestible oligosaccharides and anti-nutritional factors in soybean meal and enhanced the apparent digestibility coefficients (ADCs) of dry matter and crude protein of soybean meal. The results are reliable and of both theoretical and practical importance. The work described has not been submitted elsewhere for publication, in whole or in part, and all the authors listed have approved the manuscript that is enclosed. I have read and have abided by the statement of ethical standards for manuscript submitted to Aquaculture.

There is an increasing trend to replace fish oil (FO) in fish diets with vegetable oils (VO), driven by the short supply of FO derived from wild fish stocks. However, little is known about its influences on fish health and metabolism. In the present study, FO was increasingly replaced by linseed oil (LO) in juvenile turbot's diet, with substitution levels at 0, 33.3%, 66.7% and 100%. The 66.7% FO could be replaced by LO without affecting fish growth and feed efficiency. Turbot in three LO replacing groups (33.3%, 66.7% and 100%LO) showed decreased body protein content compared to 100% FO group, while 33.3% LO group showed the highest body lipid content. In 100% LO group, both fatty acid desaturase and sterol O-acyltransferase2 gene expression significantly increased, while carnitine palmitoyltransferase 1α gene expression significantly decreased ( P P transferrin gene expression increased in 33.3% LO group but decreased in 100% LO group, which might be the reason of slight anaemia in 100% LO group. Further studies are still needed to illustrate the inner mechanism of transferrin regulation. Statement of Relevance In commercial aquaculture, fish oil replacement has been widely conducted but less is known about its effect on lipid metabolism and immunity. Our research found that fish oil could be replaced by linseed oil at 66.7% in juvenile turbot's diet without affecting fish growth performance and immunity.

Our previous study showed that dietary soybean saponin (SA) consumption reduced growth performance and damaged the morphology of the intestinal mucosa in zebrafish (Danio rerio). Glutamine (Gln) plays an important role in ameliorating intestinal atrophy. Therefore, the present study tested the hypothesis that dietary alanine-glutamine (Ala-Gln) supplementation ameliorates the growth suppression and intestinal injury induced by dietary SA in zebrafish. Five experimental diets were prepared by supplementing the basal diet with either SA at 0, 5 or 10 g/kg, and Ala-Gln at 0 or 10 g/kg, with adjustment of Ala-Ala to maintain equal nitrogen levels among all diets. Fish with average initial body weight of (9.00 ± 0.13) mg were fed five diets for 4 weeks. The growth performance increased significantly and intestinal morphology was improved by supplementation of Ala-Gln at 10 g/kg. Ala-Gln supplementation improved growth via regulation of the growth hormone (GH)–insulin like growth factor I (IGF I) axis. The experimental diets significantly affected the mRNA expression levels of amino acid and peptide transporters. Dietary Ala-Gln upregulated the mRNA expression levels of glycolysis and fat synthesis-related factors, significantly reduced the activity of AMP-activated protein kinase (AMPK), but stimulated the activity of target of rapamycin (TOR). Overall, Ala-Gln might be broken down into free amino acids, with Gln being absorbed by the intestinal mucosa cells. Dietary Ala-Gln supplementation ameliorated the intestinal injury induced by SA, and promoted the breakdown of carbohydrates and fats for energy, thereby improving the growth performance of zebrafish.

Hydroxyproline (hyp) is one of the bioactive molecules rich in fishmeal but low in plant protein sources. With increased utilization of plant proteins in aquafeeds, a better understanding is warranted for the necessity of hyp supplementation in high plant protein based diets. In the present study, isonitrogenous and isoenergetic turbot diets were formulated with 40%, 50% and 60% fishmeal substituted by plant proteins, with (or without) addition of 0.6% hyp. After an 8-week feeding trial in juvenile turbot, hyp supplementation significantly improved specific growth rate (SGR) and feed efficiency ratio (FER) in fish fed diets with 50% or higher fishmeal replaced, but not in the group with 40% fishmeal replaced by plant proteins. Hyp levels in plasma and tissues were reduced after plant protein substitutions and replenished by dietary supplementation. Dietary hyp significantly increased muscle hardness, springiness and chewiness of fish ( P

Taurine is essential for development and homeostasis in various fish species. However, very limited information was available on the regulation of taurine biosynthesis in fish. In the present study, the effects of dietary sulfur amino acid supplementation on taurine biosynthesis were examined in juvenile turbot. Fish were fed with a casein-based semi-purified basal diet (CON) and diets supplemented with methionine at 0.5% (MM) and 1.5% (HM), cysteine at 0.3% (MC) and 0.6% (HC), or taurine at 1.5% (MT) and 2.5% (HT) separately, twice per day for 2 weeks. Growth performance, taurine concentration in multiple tissues, hepatic activities of cysteine dioxygenase (CDO) and cysteine sulfinic acid decarboxylase (CSD) were measured. Methionine, cysteine and taurine supplementations promoted growth performance in turbot. Methionine and cysteine supplementations stimulated hepatic CDO but not hepatic CSD activities. Dietary supplementation of methionine, cysteine and taurine increased taurine concentration in the liver. These results suggest the possibility that juvenile turbot has taurine biosynthesis capability, but may not be enough for its endogenous requirement.

Long chain polyunsaturated fatty acid (LC-PUFA) biosynthesis is limited in many marine species, normally due to lack of Δ5 fatty acyl desaturase (Fad) activity. Among exceptions, abalone possesses considerable LC-PUFA conversion ability from C18 precursors. However, its characterization and relevant enzyme are not well characterized. Here we successfully cloned and characterized two Δ5 Fads in abalone (Haliotis discus hannai Ino). Two Δ5 Fad mRNA transcripts, Hdhfad1 (GQ 470626) and Hdhfad2 (GQ 466197), were found in abalone sharing 96.82% similarity for cDNA sequence and 96.58% similarity for their deduced amino acid sequences. Hdhfad1 cDNA is 1530 bp in length with an opening reading frame (ORF) coding for 438 amino acids. Hdhfad2 cDNA is 1525 bp in length with an ORF encoding for 439 amino acids. Both have characteristic features of front–end desaturase, including three histidine boxes, an N-terminal cytochrome b5 domain with heme-binding motif and transmembrane regions. Both Hdhfad1 and Hdhfad2 were expressed in tissues of abalone, especially in hepatopancreas and intestine. HdhFad1 possessed higher Δ5 desaturase activity but expressed at a lower level than HdhFad2. Both isoforms preferred 20:4n-3 than 20:3n-6 as substrate. These results should provide valuable information on the molecular evolution of Fads and better understanding of LC-PUFA biosynthesis in abalone.