Insulin-like growth factor binding protein-3 (igfbp-3) and igfbp-5 in yellowtail kingfish (Seriola lalandi): molecular characterization and expression levels under different nutritional status and stocking density.

Insulin-like growth factor-binding proteins (IGFBPs) play important roles in regulating growth and development by binding to IGF, where IGFBP-3 and IGFBP-5 are the main binding carriers of IGF in the circulation system. In the present study, the gene sequences of igfbp-3, igfbp-5a, and igfbp-5b were cloned from the liver of yellowtail kingfish (Seriola lalandi). The ORF sequences of igfbp-3, igfbp-5a, and igfbp-5b were 888, 801, and 804 bp in length, which encoded 295, 266, and 267 amino acids, respectively. The above three genes were widely expressed in yellowtail kingfish tissues, with igfbp-3 being the most highly expressed in the heart, brain, and gonads, while igfbp-5a and igfbp-5b were both most highly expressed in the liver and kidney. The expression levels of igfbp-3, igfbp-5a, and igfbp-5b were detected throughout the embryonic and larval stages, suggesting their roles in early development and growth regulation of yellowtail kingfish. Besides, igfbp-3 and igfbp-5a were significantly up-regulated in the liver under food deprivation and high-density rearing conditions, which was exactly opposite to the growth performance of yellowtail kingfish, implying that they may serve as biomarkers of adverse culture conditions. Overall, the above results initially identified the molecular characteristics of igfbp-3/-5a/-5b in yellowtail kingfish and implied that they might play important roles in the growth and development, providing a basis for further research on underlying regulatory mechanisms.

Dietary methanotroph bacteria meal alleviates soybean meal-induced enteritis by improving immune tolerance and intestinal flora profile of juvenile turbot (Scophthalmus maximus L.).

An 8-week growth trial was performed to investigate the protective effects of methanotroph bacteria meal (MBM) produced from methane against soybean meal-induced enteritis (SBMIE) in juvenile turbot (Scophthalmus maximus L.). Five isonitrogenous and isolipidic diets were formulated: fishmeal-based diet (FM, the control group); FM with approximate 50% of fishmeal substituted by 399.4 g/kg soybean meal (SBM); SBM supplemented with 63.6, 127.2 and 190.8 g/kg MBM (named MBM1, MBM2 and MBM3), each diet was randomly assigned to triplicate fibreglass tanks. Results showed that fish fed with SBM exhibited enteritis, identified by reduced relative weight of intestine (RWI), as well as expanded lamina propria width and up-regulated gene expression of pro-inflammatory cytokines (tnf-α, il-6 and il-8) in intestine. While the above symptoms were reversed when diet SBM supplemented with MBM at the levels of 63.6 and 127.2 g/kg, as well as characterized by up-regulated gene expression of anti-inflammatory cytokines (tgf-ß and il-10) and tight junction protein (claudin3, claudin4 and claudin7) in intestine. Intestinal transcriptome analysis showed that the differentially expressed genes between groups FM and SBM predominantly enriched in the JAK-STAT signaling pathway, and the enrichment of differentially expressed genes between groups SBM and SBM supplemented with 63.6 g/kg MBM was in the inflammatory bowel disease (IBD) and JAK-STAT signaling pathway. To be specific, the expression of jak1, jak2b, stat1 and stat5a was significantly up-regulated when fish fed with SBM, suggested the activation of JAK-STAT signaling pathway, while the expression of these above genes was depressed by providing MBM to diet SBM, and the gene expression of toll-like receptors tlr2 and tlr5b showed a similar pattern. Moreover, intestinal flora analysis showed that community richness and abundance of beneficial bacteria (Cetobacterium and acillus_coagulans) were improved when fish fed with SBM supplemented with 63.6 g/kg MBM. Overall, methanotroph bacteria meal may alleviate SBMIE by regulating the expression of tight junction protein, toll-like receptors and JAK-STAT signaling pathway, as well as improving intestinal flora profile, which would be beneficial for enhancing the immune tolerance and utilization efficiency of turbot to dietary soybean meal.

Optimal dietary lipid levels alleviated adverse effects of high temperature on growth, lipid metabolism, antioxidant and immune responses in juvenile turbot (Scophthalmus maximus L.).

Fish physiological health is often negatively impacted by high-temperature environments and there are few studies on how dietary lipids affect fish growth and physiology when exposed to heat stress. The main objective of this research was to examine the impact of dietary lipid levels on growth and physiological status of juvenile turbot (Scophthalmus maximus L.) and determine if dietary lipid concentration could alleviate the possible adverse effects of heat stress. Five diets containing 6.81%, 9.35%, 12.03%, 14.74%, and 17.08% lipid, respectively, were formulated and fed to turbot (initial weight 5.13 ± 0.02 g) under high-temperature conditions (24.0-25.0 °C). Meanwhile, the diet with 12.03% lipid (considered by prior work to be an optimal dietary lipid level) was fed to turbot of the same size at normal temperature. Results suggested that, among the different dietary lipid levels under high-temperature conditions, fish fed the optimal lipid (12.03%) exhibited better growth compared to non-optimal lipid groups, as evidenced by higher weight gain and specific growth rate. Simultaneously, the optimal lipid diet may better maintain lipid homeostasis, as attested by lower liver and serum lipid, along with higher liver mRNA levels of lipolysis-related genes (pgc1α, lipin1, pparα, lpl and hl) and lower levels of synthesis-related genes (lxr, fas, scd1, pparγ, dgat1 and dgat2). Also, the optimal lipid diet might mitigate oxidative damage by improving antioxidant enzyme activity, decreasing malondialdehyde levels, and up-regulating oxidation-related genes (sod1, sod2, cat, gpx and ho-1). Furthermore, the optimal lipid may enhance fish immunity, as suggested by the decrease in serum glutamic-oxalacetic/pyruvic transaminase activities, down-regulation of pro-inflammatory genes and up-regulation of anti-inflammation genes. Correspondingly, the optimal lipid level suppressed MAPK signaling pathway via decreased phosphorylation levels of p38, JNK and ERK proteins in liver. In summary, the optimal dietary lipid level facilitated better growth and physiological status in turbot under thermal stress.

Effects of Tributyrin Supplementation on Growth Performance, Intestinal Digestive Enzyme Activity, Antioxidant Capacity, and Inflammation-Related Gene Expression of Large Yellow Croaker (Larimichthys crocea) Fed with a High Level of Clostridium autoethanogenum Protein.

An 8-week growth experiment was conducted to investigate effects of tributyrin (TB) supplementation on growth performance, intestinal digestive enzyme activity, antioxidant capacity, and inflammation-related gene expression of juvenile large yellow croaker (Larimichthys crocea) (initial weight of 12.90 ± 0.02 g) fed diets with high level of Clostridium autoethanogenum protein (CAP). In the negative control diet, 40% fish meal was used as the major source of protein (named as FM), while 45% fish meal protein of FM was substituted with CAP (named as FC) to form a positive control diet. Based on the FC diet, grade levels of 0.05%, 0.1%, 0.2%, 0.4%, and 0.8% tributyrin were added to formulate other five experimental diets. Results showed that fish fed diets with high levels of CAP significantly decreased the weight gain rate (WGR) and specific growth rate (SGR) compared with fish fed the FM diet (P < 0.05). WGR and SGR were significantly higher than in fish fed diets with 0.05% and 0.1% tributyrin that fed the FC diet (P < 0.05). Supplementation of 0.1% tributyrin significantly elevated fish intestinal lipase and protease activities compared to FM and FC diets (P < 0.05). Meanwhile, compared to fish fed the FC diet, fish fed diets with 0.05% and 0.1% tributyrin showed remarkably higher intestinal total antioxidant capacity (T-AOC). Malondialdehyde (MDA) content in the intestine of fish fed diets with 0.05%-0.4% tributyrin was remarkably lower than those in the fish fed the FC diet (P < 0.05). The mRNA expressions of tumor necrosis factor α (tnfα), interleukin-1ß (il-1ß), interleukin-6 (il-6), and interferon γ (ifnγ) were significantly downregulated in fish fed diets with 0.05%-0.2% tributyrin, and the mRNA expression of il-10 was significantly upregulated in fish fed the 0.2% tributyrin diet (P < 0.05). In regard to antioxidant genes, as the supplementation of tributyrin increased from 0.05% to 0.8%, the mRNA expression of nuclear factor erythroid 2-related factor 2 (nrf2) demonstrated a trend of first rising and then decreasing. However, the mRNA expression of Kelch-like ECH-associated protein 1 (keap1) was remarkably lower in fish fed the FC diet than that fed diets with tributyrin supplementation (P < 0.05). Overall, fish fed tributyrin supplementation diets can ameliorate the negative effects induced by high proportion of CAP in diets, with an appropriate supplementation of 0.1%.

Long noncoding RNA lincsc5d regulates hepatic cholesterol synthesis by modulating sterol C5 desaturase in large yellow croaker.

Although long noncoding RNA (lncRNA) plays a vital role in cholesterol metabolism, very little information is available in fish. Thus, a 10-week feeding experiment was performed to estimate the effects of lncRNA on cholesterol metabolism in large yellow croaker fed with fish oil (FO), soybean oil (SO), olive oil (OO), and palm oil (PO) diets. Results showed that fish fed with OO and PO diets had higher liver total cholesterol (TC) and cholesterol ester (CE) contents compared with fish fed with FO diets. Analysis of the KEGG pathway showed that the steroid biosynthesis pathway was enriched in comparisons FO vs SO, FO vs OO, and FO vs PO. Meanwhile, sterol C5 desaturase (SC5D), a cholesterol synthase, was up-regulated in the steroid biosynthesis pathway. SC5D was widely expressed in all tissues examined, and the highest expression of SC5D was detected in brain. More importantly, a novel lncRNA associated with sc5d gene was identified by RNA sequencing and named as lincsc5d. The tissue distribution of lincsc5d was similar to that of sc5d. A nuclear/cytoplasmic RNA separation assay showed that lincsc5d was a nucleus-enriched lncRNA. qRT-PCR results demonstrated that lincsc5d was markedly up-regulated in the SO, OO, and PO groups. Furthermore, the results of TC content and the lincsc5d and sc5d expression in hepatocytes agreed with in vivo results. In conclusion, this study indicated that vegetable oils, especially OO and PO, increased hepatic cholesterol levels by promoting cholesterol synthesis, and lncRNA lincsc5d and sc5d might be involved in cholesterol synthesis.

Replacement of dietary fish meal with Clostridium autoethanogenum meal on growth performance, intestinal amino acids transporters, protein metabolism and hepatic lipid metabolism of juvenile turbot (Scophthalmus maximus L.).

Clostridium autoethanogenum meal (CAM) is a novel single-cell protein, which is produced from bacteria using carbon monoxide (CO) as sole carbon source. To evaluate the efficiency of CAM as an alternative for dietary fish meal, a 56-days growth experiment was performed on juvenile turbot (Scophthalmus maximus L.) with initial average weight of 9.13 ± 0.02 g. Six iso-nitrogenous (crude protein, 51.0%) and iso-lipidic (crude lipid, 11.5%) diets were formulated with 0%, 15%, 30%, 45%, 60% and 80% dietary fish meal protein substituted by CAM protein, which were designated as CAM0 (the control group), CAM15, CAM30, CAM45, CAM60 and CAM80, respectively. Results showed that no significant differences were observed in survival rate (over 97.50%) among different dietary treatments (p > 0.05). The specific growth rate (SGR) was not significantly affected when replacement levels of dietary fish meal with CAM were less than 45% (p > 0.05). The feed intake (FI) was significantly linear reduced with increasing dietary CAM (p < 0.05), whereas no significant differences were observed in feed efficiency ratio (FER), protein efficiency ratio (PER) and protein retention (PR) among different dietary treatments (p > 0.05). With increasing dietary CAM, lipid retention (LR) and carcass lipid tended to be increased in both significantly linear and quadratic patterns (p < 0.05). The apparent digestibility coefficient (ADC) of crude protein and some essential amino acids, including threonine, valine, lysine, histidine and arginine, showed significantly linear increase with increasing dietary CAM (p < 0.05). Furthermore, with the increase of dietary CAM, the gene expression of intestinal peptide and amino acids transporters was first up-regulated and then down-regulated with significantly quadratic pattern (p < 0.05), peaking in fish fed with diets CAM30 or CAM45, which was similar to the expression of genes related protein degradation in muscle. For genes related to protein metabolism in liver and muscle, the expression of mammalian target of rapamycin (mtor) was not significantly affected by dietary CAM, while the general control nonderepressible 2 (gcn2) tended to be first up-regulated and then down-regulated with significantly quadratic pattern (p < 0.05). Apart from that, the lipid metabolism of turbot was also affected by high dietary CAM, evidenced by increased expression of hepatic genes related to lipogenesis as well as reduced expression of genes related to lipid oxidation and lipid transport. In conclusion, CAM can replace up to 45% fish meal protein in diet for juvenile turbot without significantly adverse effects on growth performance. But excessive dietary CAM would result in significant growth reduction, and excessive lipid deposition may also occur in fish fed diets with high levels of CAM.

Effects of dietary lysolecithin on growth performance, serum biochemical indexes, antioxidant capacity, lipid metabolism and inflammation-related genes expression of juvenile large yellow croaker (Larimichthys crocea).

A 70-day feeding trial was conducted to investigate effects of dietary lysolecithin on growth performance, serum biochemical indexes, antioxidant capacity, lipid metabolism and inflammation-related genes expression of juvenile large yellow croaker (Larimichthys crocea) with initial weight of 6.04 ± 0.08 g. A formulated diet containing approximately 42% crude protein and 12.5% crude lipid was used as the control diet (CON). The other three experimental diets were formulated with supplementation of 0.2%, 0.4% and 0.6% lysolecithin based on the control diet, respectively. Results showed that weight gain rate (WGR) and specific growth rate (SGR) significantly increased in fish fed diets with lysolecithin compared with those in the control diet (P < 0.05). Fish fed diets with 0.4% and 0.6% lysolecithin had notably higher lipid content in muscle than that in the control diet (P < 0.05). When fish were fed diets with lysolecithin, serum high-density lipoprotein cholesterol (HDL-c) content was notably higher than that in the control diet (P < 0.05), while fish fed the diet with 0.6% lysolecithin had a significant lower serum low-density lipoprotein cholesterol (LDL-c) content than that in the control diet (P < 0.05). Meanwhile, serum aspartate transaminase (AST) and alanine transaminase (ALT) activities in fish fed diets with lysolecithin were remarkably lower than those in the control diet (P < 0.05). With the increase of dietary lysolecithin from 0.2% to 0.6%, mRNA expression of stearoyl-coenzyme A desaturase 1 (scd1), diacylglycerol acyltransferase 2 (dgat2) and sterol-regulatory element binding protein 1 (srebp1) showed decreasing trends. Furthermore, mRNA expression of carnitine palmitoyl transferase 1 (cpt1) and lipoprotein lipase (lpl) among each dietary lysolecithin treatment were significantly higher than those in the control diet (P < 0.05). In terms of inflammation, mRNA expression of tumor necrosis factor α (tnf-α) and interleukin-1 ß (il-1ß) were significantly down-regulated in fish fed diets with lysolecithin compared with those in the control diet (P < 0.05), while the mRNA expression of interleukin-10 (il-10) was significantly higher than that in the control diet (P < 0.05). In conclusion, dietary lysolecithin could promote the growth performance, improve hepatic lipid metabolism and regulate inflammation response in juvenile large yellow croaker, and the optimal supplement level of lysolecithin was approximately 0.4% in this study.

Replacement of Dietary Fishmeal Protein with Degossypolized Cottonseed Protein on Growth Performance, Nonspecific Immune Response, Antioxidant Capacity, and Target of Rapamycin Pathway of Juvenile Large Yellow Croaker (Larimichthys crocea).

A 70-day feeding experiment was carried out to assess the replacement of dietary fishmeal (FM) protein with degossypolized cottonseed protein (DCP) on large yellow croaker (Larimichthys crocea) with initial body weight (13.09 ± 0.50 g). Five isonitrogenous and isolipidic diets replaced fishmeal protein with 0%, 20%, 40%, 60%, and 80% DCP were formulated and named as FM (the control group), DCP20, DCP40, DCP60, and DCP80, respectively. Results displayed that weight gain rate (WGR) and specific growth rate (SGR) in the DCP20 group (263.91% and 1.85% d-1) were significantly increased compared with the control group (194.79% and 1.54% d-1) (P < 0.05). Furthermore, fish fed the diet with 20% DCP significantly increased the activity of hepatic superoxide dismutase (SOD) compared with the control group (P < 0.05). Meanwhile, the content of hepatic malondialdehyde (MDA) in the DCP20, DCP40, and DCP80 groups was significantly lower than that in the control group (P < 0.05). The activity of intestinal trypsin in the DCP20 group was significantly degraded compared with that in the control group (P < 0.05). The transcription of hepatic proinflammatory cytokine genes (interleukin-6 (il-6); tumor necrosis factor-α (tnf-α); and interferon-γ (ifn-γ)) in the DCP20 and DCP40 groups was significantly upregulated compared with that in the control group (P < 0.05). As to the target of rapamycin (TOR) pathway, the transcription of hepatic target of rapamycin (tor) and ribosomal protein (s6) was significantly up-regulated, while the transcription of hepatic eukaryotic translation initiation factor 4E binding protein 1 (4e-bp1) gene was significantly downregulated in the DCP group compared with the control group (P < 0.05). In summary, based on the broken line regression model analysis of WGR and SGR against dietary DCP replacement levels, the optimal replacement level was recommended to be 8.12% and 9.37% for large yellow croaker, respectively. These results revealed that FM protein replaced with 20% DCP could promote digestive enzyme activities and antioxidant capacity and further activate immune response and the TOR pathway so that growth performance of juvenile large yellow croaker was improved.