A delayed and unsynchronized ovary development as revealed by transcriptome of brain and pituitary of Coilia nasus.

Coilia nasus is an anadromous fish that has been successfully domesticated in the last decade due to its high economic value. The fish exhibits a delayed ovary development during the reproductive season, despite breeding and selection for five to six offspring. The molecular mechanism of the delayed ovary development is still unknown, so the obstacles have not been removed in the large-scale breeding program. This study aims to investigate the key genes regulating ovarian development by comparing the transcriptomes of ovarian-stage IV and stage II brain/pituitary of Coilia nasus. Ovarian stages were validated by histological sections. A total of 75,097,641 and 66,735,592 high-quality reads were obtained from brain and pituitary transcriptomes, respectively, and alternatively spliced transcripts associated with gonadal development were detected. Compared to ovarian Ⅱ- brain, 515 differentially expressed genes (DEGs) were upregulated and 535 DEGs were downregulated in ovarian Ⅳ- brain, whereas 470 DEGs were upregulated and 483 DEGs were downregulated in ovarian Ⅳ- pituitary compared to ovarian Ⅱ- pituitary. DEGs involved in hormone synthesis and secretion and in the GnRH signaling pathway were screened. Weighted gene co-expression network analysis identified gene co-expression modules that were positively correlated with ovarian phenotypic traits. The hub genes Smad4 and TRPC4 in the modules were co-expressed with DEGs including Kiss1 receptor and JUNB, suggesting that ovarian development is controlled by a hypothalamic-pituitary-gonadal axis. Our results have provided new insights that advance our understanding of the molecular mechanism of C. nasus reproductive functions and will be useful for future breeding.

DL-Methionyl-DL-Methionine/DL-Methionine Supplementation Alleviated the Adverse Effects of Dietary Low Fishmeal Levels on Growth and Intestinal Health of Micropterus salmoides.

DL-methionyl-DL-methionine (AQUAVI® Met-Met) (Met-Met) (0.10%, 0.20%, 0.30%, and 0.40%) or DL-methionine (DL-Met) (0.10%, 0.20%, 0.30%, and 0.40%) were added to a low-fishmeal diet in an attempt to reduce fishmeal in the diet of Micropterus salmoides (M. salmoides). The fish were randomly allocated into ten experimental groups (n = 100), each with 4 replicates of 25 fish (16.39 ± 0.01 g) each. Compared to 25% FM, 0.40% of DL-Met and 0.10% of Met-Met promoted growth, and 0.10% of Met-Met decreased FCR. Compared to 25% FM, the supplementation of Met-Met or DL-Met improved the intestinal antioxidant capacity by upregulating the NF-E2-related factor 2-mediated antioxidant factors and enzyme activities and nuclear factor kappa-B-mediated anti-inflammatory factors while downregulating the pro-inflammatory factors, thereby exerting anti-inflammatory effects. Moreover, 0.10% of the Met-Met diet affected the Firmicutes-to-Bacteroidota ratio, increased the levels of Proteobacteria, changed the composition of intestinal flora (Roseburia, Lachnospiraceae_NK4A136_group, and unclassified_Oscillospiraceae), and enhanced intestinal dominant bacteria (Caldicoprobacter, Pseudogracilibacillus, and Parasutterella), leading to improved gut health. In summary, the supplementation of DL-Met or Met-Met alleviated the adverse effect of fishmeal reduction (from 40 to 25%) on the growth performance and intestinal health of M. salmoides.

Different Starch Sources Affect the Growth Performance and Hepatic Health Status of Largemouth Bass (Micropterus salmoides) in a High-Temperature Environment.

The experiment was designed to investigate the effects of different starch types on the growth performance and liver health status of largemouth bass in a high-temperature environment (33-35 °C). In this study, we designed five diets using corn starch (CS), tapioca starch (TS), sweet potato starch (SPS), potato starch (PS), and wheat starch (WS) as the starch sources (10%). We selected 225 healthy and uniformly sized largemouth bass (199.6 ± 0.43 g) and conducted the feeding experiment for 45 days. The results showed that the WS group had the highest WGR, SGR, and SR and the lowest FCR. Among the five groups, the WS group had the highest CAT activity, SOD activity, and GSH content, while the SPS group had the highest MDA content. Furthermore, oil red O staining of liver samples showed that the TS group had the largest positive region, indicating high lipid accumulation. Lastly, the gene expression results revealed that compared with the WS group, the CS, TS, and SPS groups showed suppressed expression of nrf2, keap1, cat, sod, gpx, il-8, and il-10. Therefore, our results demonstrated the effect of different starch sources on largemouth bass growth performance and hepatic health in a high-temperature environment.

Establishment of a Coilia nasus Spermatogonial Stem Cell Line Capable of Spermatogenesis In Vitro.

The process by which spermatogonial stem cells (SSCs) continuously go through mitosis, meiosis, and differentiation to produce gametes that transmit genetic information is known as spermatogenesis. Recapitulation of spermatogenesis in vitro is hindered by the challenge of collecting spermatogonial stem cells under long-term in vitro culture conditions. Coilia nasus is a commercially valuable anadromous migrant fish found in the Yangtze River in China. In the past few decades, exploitation and a deteriorating ecological environment have nearly caused the extinction of C. nasus's natural resources. In the present study, we established a stable spermatogonial stem cell line (CnSSC) from the gonadal tissue of the endangered species C. nasus. The cell line continued to proliferate and maintain stable cell morphology, a normal diploid karyotype, and gene expression patterns after more than one year of cell culture (>80 passages). Additionally, CnSSC cells could successfully differentiate into sperm cells through a coculture system. Therefore, the establishment of endangered species spermatogonial stem cell lines is a model for studying spermatogenesis in vitro and a feasible way to preserve germplasm resources.

Sodium chloride alleviates oxidative stress and physiological responses induced by extreme winter cold in genetically improved farmed tilapia (GIFT; Oreochromis niloticus).

A 6-week trial was designed to investigate the effects of dietary sodium chloride supplementation on physiological, metabolic, and molecular stress response parameters. The findings showed that (1) there were no significant differences between sodium chloride supplementation groups (0.05S, 0.1S, and 0.15S) and the control group (P > 0.05), except for the 0.2S diet, which showed better final body weight, weight gain rate, specific growth rate, and feed conversion ratio than the control group (P < 0.05). (2) The hypothermic stress experiment results showed that the survival rates in the 0.1S and 0.15S diets were significantly higher than the control group (P < 0.05). (3) Transcription results showed that these enriched pathways in the gill were mainly energy metabolism and apoptosis pathways, while the major enrichment pathways in the liver were mainly amino acid metabolism and carbohydrate metabolism. (4) The plasma parameter results showed, compared to the control group, the 0.15S diet significantly increased the plasma GLU, TG contents, and Na+ and K+ concentrations and decreased the plasma ALT activity (P < 0.05). In addition, the 0.1S diet increased the plasma ALB content and Cl- concentration (P < 0.05). The gill Na+/K+-ATPase activity decreased markedly when the fish were fed the 0.1S and 0.15S diets (P < 0.05). The antioxidant enzyme activity results showed that the 0.1S and 0.15S diets significantly increased the T-SOD activities (P < 0.05). Gene expression results showed that compared to the control group, the 0.1S and 0.15S diets up-regulated the expression of gys, hsp70, mlcp, mlc, myosin, tnt mRNA, and down-regulated the akt, gk, and erk mRNA expression. Based on the regression analysis, the optimum dietary sodium chloride levels range from 0.10 % to 0.13 % of the diet, which could facilitate energy regulation, improve the immune response, and ultimately strengthen the cold resistance of GIFT.

Phenylalanine Plays Important Roles in Regulating the Capacity of Intestinal Immunity, Antioxidants and Apoptosis in Largemouth Bass (Micropterus salmoides).

This experiment was planned to explore the role of dietary phenylalanine levels in intestinal immunity, antioxidant activity and apoptosis in largemouth bass (Micropterus salmoides). Six iso-nitrogen and iso-energy diets with phenylalanine levels of 1.45% (DPHE1), 1.69% (DPHE2), 1.98% (DPHE3), 2.21% (DPHE4), 2.48% (DPHE5) and 2.76% (DPHE6) were designed. Juvenile largemouth bass were fed the experimental diet for 8 weeks. In this study, the DPHE5 group increased the expression of intestinal antioxidant genes in largemouth bass (p < 0.05), and the increase of antioxidant enzyme activities and content of related substances was most concentrated in the DPHE3 and DPHE4 groups (p < 0.05). The results of plasma biochemistry were similar to that of enzyme activity. The expression of genes related to the TOR signalling pathway mainly increased significantly in the DPHE5 group (p < 0.05). Similarly, the expression of inflammatory factors, as well as apoptotic factors, also showed significant increases in the DPHE5 group (p < 0.05). In conclusion, unbalanced phenylalanine in the diet could lead to a decrease in intestinal immune and antioxidant capacity and also cause a decline in the aggravation of intestinal cell apoptosis.

Enzymatically Hydrolyzed Poultry By-Product Supplementation, Instead of Fishmeal, Alone Improves the Quality of Largemouth Bass (Micropterus salmoides) Back Muscle without Compromising Growth.

This study was designed to investigate the effects of enzymatically hydrolyzed poultry by-products (EHPB) on the growth and muscle quality of largemouth bass. Different concentrations of EHPB (0.00, 3.10, 6.20, 9.30, and 12.40%) were added to replace fishmeal (0.00 (control), 8.89 (EHPB1), 17.78 (EHPB2), 26.67 (EHPB3), and 35.56% (EHPB4)), respectively, in dietary supplementation. The results revealed that the growth performance and muscle amino acid and fatty acid remained unaltered in EHPB1 (p > 0.05). EHPB1 showed significant reduction in muscle hardness, gumminess, chewiness, and muscle fiber count and exhibited a significant increase in muscle fiber volume. The decrease in muscle hardness, gumminess, and chewiness means that the muscle can have a more tender texture. The expression of protein metabolism-related genes reached the highest levels in EHPB1 and EHPB2 (p < 0.05). The mRNA levels of s6k and igf-1 in EHPB2 and EHPB1 were significantly lower than those in the control group. Compared to the control group, the expression of muscle production-associated genes paxbp-1 was higher in EHPB1, and myod-1, myf-5, and syndecan-4 were higher in EHPB2. The mRNA levels of muscle atrophy-related genes, in EHPB4 and EHPB2, were significantly lower than those in the control group. Therefore, the EHPB1 group plays a role in promoting the expression of genes related to muscle formation. In summary, replacing 8.89% of fishmeal with EHPB in feed has no effect on growth and may improve back muscle quality in largemouth bass.

Dietary Supplementation of Chlorella vulgaris Effectively Enhanced the Intestinal Antioxidant Capacity and Immune Status of Micropterus salmoides.

An M. salmoides fish meal diet was supplemented with 0 (CHL0, Control), 38 (CHL38), 76 (CHL76), 114 (CHL114), and 152 (CHL152) mg/kg C. vulgaris for 60 days, and their serum and intestinal samples were analyzed. The results showed that the albumin (ALB) and total protein (TP) contents were observably enhanced in the CHL76 group compared with the Control group. The intestinal glutathione (GSH) and glutathione peroxidase (GSH-Px) contents were enhanced significantly in the CHL76 group, while the total antioxidant capacity (T-AOC) was enhanced in the CHL38 group, compared with the Control group. However, supplementation of >76 g/kg C. vulgaris significantly inhibited the superoxide dismutase (SOD) activity in the intestines of M. salmoides. Moreover, the malondialdehyde (MDA) content was observably dropped in the CHL-supplemented groups compared with the Control group. Transcriptome analysis of the CHL76 and Control groups displayed a total of 1384 differentially expressed genes (DEGs). KEGG analysis revealed that these DEGs were enriched in apoptosis, cytokine-cytokine receptor interaction, tight junction (TJ), and phagosome signaling pathways, which were associated with improved intestinal immunity in the CHL76 group. Additionally, the DEGs enriched in the above pathways were also correlated with the antioxidant parameters, such as catalase (CAT), GSH, GSH-Px, SOD, T-AOC, and MDA. Therefore, our study found that dietary supplementation of C. vulgaris effectively enhanced the intestinal antioxidant capacity of M. salmoides by increasing antioxidant enzyme activity and decreasing MDA content. Additionally, dietary supplementation of C. vulgaris improved the intestinal immune status of M. salmoides by reducing proapoptotic and proinflammatory factors, increasing intestinal TJs- and phagosome-related genes expressions, and increasing the serum ALB and TP contents. Lastly, quadratic regression analysis of the serum biochemical indices (ALB and TP) and intestinal antioxidant parameters (GSH-Px and GSH) revealed that the optimal supplemental level of C. vulgaris in the M. salmoides diet was 58.25-77.7 g/kg.

The Role of Algae Extract (Ulva lactuca and Solieria chordalis) in Fishmeal Substitution in Gibel Carp (Carrassius auratus gibeilo).

The function of algae extract (AE) in fishmeal (FM) substitution with plant proteins in the diets of Gibel carp (Carrassius auratus gibeilo) was investigated during a 56-day trial. Diets 1 and 2 contained 10% FM, Diets 3 and 4 contained 5% FM, and Diet 5 and 6 contained 0% FM. In contrast, Diets 2, 4, and 6 were supplemented with 0.2% AE. The results showed that FM reduction inhibited growth performance, while AE supplementation alleviated growth inhibition. FM reduction significantly decreased the crude protein levels of the whole body, while the contents of whole-body lipids were significantly decreased with AE supplementation. There were no significant changes in ALB, ALP, ALT, AST, TP, GLU, GLU, and TC in plasma. FM reduction with AE supplementation mitigated the decrease in antioxidant capacity by heightening the activity of antioxidant enzymes and related gene expressions, which mitigated the decrease in immune capacity by affecting the expression of inflammatory factors. In summary, AE supplementation could alleviate the negative effects of FM reduction in Gibel carp.

Effects of Enzymatic Cottonseed Protein Concentrate as a Feed Protein Source on the Growth, Plasma Parameters, Liver Antioxidant Capacity and Immune Status of Largemouth Bass (Micropterus salmoides).

This study appraised the impact of enzymatic cottonseed protein concentrate (ECP) as a fish meal (FM) substitute on the growth and health of largemouth bass (Micropterus salmoides) (initial weight 14.99 ± 0.03 g). Five diets with equal nitrogen, fat, and energy were designed to replace 0%, 7.78%, 15.56%, 23.33%, and 31.11% FM by adding 0%, 3.6%, 7.2%, 10.8%, and 14.4% ECP, named ECP0, ECP3.6, ECP7.2, ECP10.8, and ECP14.4, respectively. We fed 300 fish with five experimental diets for 60 days. The results revealed that weight gain rate (WGR) and specific growth rate (SGR) did not notably reduce until the addition of ECP exceeded 7.2%. The proximate composition of fish was not affected by the amount of ECP added in diets. Plasma total protein (TP), albumin (ALB), and high-density lipoprotein (HDL) concentrations increased with the increase of ECP dosage, while the triglyceride (TG) and low-density lipoprotein (LDL) concentrations and alkaline phosphatase (ALP) activity showed an opposite trend. For hepatic antioxidant capacity, the hepatic total superoxide dismutase (T-SOD) and catalase (CAT) activities, glutathione (GSH) content, and the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), superoxide dismutase (SOD), and CAT were increased by ECP, while the hepatic malondialdehyde (MDA) content and the expression of kelch-like-ECH-associated protein 1 (Keap1) were decreased. With regard to inflammation, the expression of nuclear factor-kappa B (NF-κB), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α) were inhibited by ECP. In summary, the amount of ECP added to diet can reach 7.2% to replace 15.56% FM without hampering the growth of largemouth bass, and ECP can improve the antioxidant and immune capacity.

Histidine Deficiency Inhibits Intestinal Antioxidant Capacity and Induces Intestinal Endoplasmic-Reticulum Stress, Inflammatory Response, Apoptosis, and Necroptosis in Largemouth Bass (Micropterus salmoides).

This 56-day study aimed to evaluate the effects of histidine levels on intestinal antioxidant capacity and endoplasmic-reticulum stress (ERS) in largemouth bass (Micropterus salmoides). The initial weights of the largemouth bass were (12.33 ± 0.01) g. They were fed six graded levels of histidine: 0.71% (deficient group), 0.89%, 1.08%, 1.26%, 1.48%, and 1.67%. The results showed that histidine deficiency significantly suppressed the intestinal antioxidant enzyme activities, including SOD, CAT, GPx, and intestinal level of GSH, which was supported by significantly higher levels of intestinal MDA. Moreover, histidine deficiency significantly lowered the mRNA level of nrf2 and upregulated the mRNA level of keap1, which further lowered the mRNA levels of the downstream genes sod, cat, and gpx. Additionally, histidine-deficiency-induced intestinal ERS, which was characterized by activating the PEPK-signalling pathway and IRE1-signalling pathway, including increased core gene expression of pepk, grp78, eif2α, atf4, chopα, ire1, xbp1, traf2, ask1, and jnk1. Dietary histidine deficiency also induced apoptosis and necroptosis in the intestine by upregulating the expressions of proapoptotic genes, including caspase 3, caspase 8, caspase 9, and bax, and necroptosis-related genes, including mlkl and ripk3, while also lowering the mRNA level of the antiapoptotic gene bcl-2. Furthermore, histidine deficiency activated the NF-κB-signalling pathway to induce an inflammatory response, improving the mRNA levels of the proinflammatory factors tnf-α, hepcidin 1, cox2, cd80, and cd83 and lowering the mRNA levels of the anti-inflammatory factors tgf-ß1 and ikbα. Similarly, dietary histidine deficiency significantly lowered the intestinal levels of the anti-inflammatory factors TGF-ß and IL-10 and upregulated the intestinal levels of the proinflammatory factor TNF-α, showing a trend similar to the gene expression of inflammatory factors. However, dietary histidine deficiency inhibited only the level of C3, and no significant effects were observed for IgM, IgG, HSP70, or IFN-γ. Based on the MDA and T-SOD results, the appropriate dietary histidine requirements of juvenile largemouth bass were 1.32% of the diet (2.81% dietary protein) and 1.47% of the diet (3.13% dietary protein), respectively, as determined by quadratic regression analysis.

Appropriate dietary phenylalanine improved growth, protein metabolism and lipid metabolism, and glycolysis in largemouth bass (Micropterus salmoides).

The purpose of this study was aimed to determine the appropriate level of dietary phenylalanine and explored the influences of phenylalanine on target rapamycin (TOR) signaling and glucose and lipid metabolism in largemouth bass. Six isonitrogenous/isoenergetic diets with graded phenylalanine levels (1.45% (control group), 1.69%, 1.98%, 2.21%, 2.48%, and 2.76%) were designed. Experimental feed was used to feed juvenile largemouth bass (initial body weight 19.5 ± 0.98 g) for 8 weeks. The final body weight, specific growth rate (SGR), feed efficiency ratio (FER), and weight gain (WG) reached their highest values in the 1.98% dietary phenylalanine group and then declined with increasing phenylalanine addition. No significant difference was found in the whole-body composition of largemouth bass between different dietary phenylalanine groups. Compared with the control group, 1.69% dietary phenylalanine significantly reduced the contents of plasma glucose (GLU) and total protein (TP), and total cholesterol (TC) contents increased significantly in the 1.98% dietary phenylalanine group (P < 0.05). The key gene expressions of TOR signaling pathway and lipid metabolism was significantly inhibited by 2.21% dietary phenylalanine (P < 0.05). The 1.98% dietary phenylalanine group showed significantly increased expression of genes related to insulin signaling pathway and factors involved in fatty acid synthesis (P < 0.05). Furthermore, 2.76% dietary phenylalanine group inhibited glucose metabolism by lowering the key gene expressions of glucose metabolism (P < 0.05). According to quadratic regression analyses based on the WG and FER, the appropriate level of dietary phenylalanine for largemouth bass were 2.00% and 2.02% of the diet (4.23% and 4.27% dietary protein), respectively, with a constant amount of tyrosine (1.33%). Hence, the total aromatic amino acid requirements were 3.33% and 3.35% of the diet (equivalent to 7.03% and 7.09% of the protein content), which may provide a theoretical basis for the development of largemouth bass feed formulas. Therefore, the growth and metabolism of largemouth bass could be promoted by controlling the content of phenylalanine in the diet, or the imbalance of phenylalanine can form a specific pathological model.

Dietary Supplementation with Eucommia ulmoides Leaf Extract Improved the Intestinal Antioxidant Capacity, Immune Response, and Disease Resistance against Streptococcus agalactiae in Genetically Improved Farmed Tilapia (GIFT; Oreochromis niloticus).

A 7-week rearing trial was designed to investigate the effects of Eucommia ulmoides leaf extract (ELE) on growth performance, body composition, antioxidant capacity, immune response, and disease susceptibility of diet-fed GIFT. The results showed that dietary ELE did not affect growth performance or whole-body composition (p > 0.05). Compared with the control group, plasma ALB contents increased in the 0.06% dietary ELE group (p < 0.05), and plasma ALT and AST activities decreased in the 0.08% dietary ELE group (p < 0.05). In terms of antioxidants, compared with GIFT fed the control diet, 0.06% dietary ELE upregulated the mRNA expression levels of Nrf2 pathway-related antioxidant genes, including CAT and SOD (p < 0.05), and 0.06% and 0.08% dietary ELE upregulated the mRNA levels of Hsp70 (p < 0.05). In terms of immunity, 0.06% dietary ELE suppressed intestinal TLR2, MyD88, and NF-κB mRNA levels (p < 0.05). Moreover, the mRNA levels of the anti-inflammatory cytokines TGF-ß and IL-10 were upregulated by supplementation with 0.04% and 0.06% dietary ELE (p < 0.05). In terms of apoptosis, 0.06% and 0.08% ELE significantly downregulated the expression levels of FADD mRNA (p < 0.05). Finally, the challenge experiment with S. agalactiae showed that 0.06% dietary ELE could inhibit bacterial infection, and significantly improve the survival rate of GIFT (p < 0.05). This study demonstrated that the supplementation of 0.04−0.06% ELE in diet could promote intestinal antioxidant capacity, enhance the immune response and ultimately improve the disease resistance of GIFT against Streptococcus agalactiae.

Establishment of a Coilia nasus Gonadal Somatic Cell Line Capable of Sperm Induction In Vitro.

Coilia nasus is an important economic anadromous migratory fish of the Yangtze River in China. In recent years, overfishing and the deterioration of the ecological environment almost led to the extinction of the wild resources of C.nasus. Thus, there is an urgent need to protect this endangered fish. Recently, cell lines derived from fish have proven a promising tool for studying important aspects of aquaculture. In this study, a stable C. nasus gonadal somatic cell line (CnCSC) was established and characterized. After over one year of cell culture (>80 passages), this cell line kept stable growth. RT-PCR results revealed that the CnGSC expressed some somatic cell markers such as clu, fshr, hsd3ß, and sox9b instead of germ cell markers like dazl, piwi, and vasa. The strong phagocytic activity of CnGSC suggested that it contained a large number of Sertoli cells. Interestingly, CnGSC could induce medaka spermatogonial cells (SG3) to differentiate into elongated spermatids while co-cultured together. In conclusion, we established a C. nasus gonadal somatic cell line capable of sperm induction in vitro. This research provides scientific evidence for the long-term culture of a gonadal cell line from farmed fish, which would lay the foundation for exploring the regulatory mechanisms between germ cells and somatic cells in fish.

Dietary leucine supplementation improves growth performance, metabolic responses of liver via GCN2/ATF4, and insulin signaling pathways in largemouth bass (Micropterus salmoides).

An 8-week growth experiment was conducted to investigate the effects of dietary leucine on growth performance, body composition, and gene expression of hepatic nutrient metabolism in the largemouth bass (Micropterus salmoides). Six isonitrogenous (49.87%) diets with graded leucine levels (2.62, 3.07, 3.60, 3.87, 4.20, 4.71% of dry diet) were fed to triplicate groups with 20 juvenile fish (20.00 ± 0.13 g). The results revealed that the specific growth rate (SGR) and weight gain (WG) increased significantly with increasing dietary leucine levels, reached their maximal value in the Leu-4.20% groups, and then decreased slightly. Although the feed conversion ratio (FCR) showed decreasing trends, no significant difference was detected. Leucine supplementation significantly improved the content of body protein and total plasma protein (TP). Additionally, a higher expression level of target of rapamycin (TOR) and ribosomal protein S6 (S6) mRNA was observed in the Leu-3.87% and Leu-4.20% diets, whereas the GCN2 (general control nonderepressible2 kinase) and AFT4 (activating transcription factor 4) mRNA expression levels were suppressed. The lipid content of the body was not influenced by leucine levels, whereas the content of total triglyceride (TG) first decreased significantly with increasing dietary leucine levels from 2.62 to 3.87% and then increased with increasing leucine levels (4.20% to 4.71%). The total cholesterol (TC) and low-density lipoproteins (LDL) trended in a similar direction but did not achieve statistical significance (P > 0.05). The expression of insulin receptor substrate 1 (IRS-1) was significantly elevated by dietary leucine levels, while protein kinase B (AKT) and phosphatidylinositol 3-kinase (PI3K) expression was inconsistently upregulated. Furthermore, leucine supplementation decreased plasma glucose and hepatic glycogen contents, and the expression levels of glucokinase (GK), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6pase) were significantly inhibited at 4.20% and 4.71% leucine diets. Analyses of the change in SGR and FCR using the quadratic regression model estimated that the optimum dietary leucine requirement of juvenile largemouth bass was 4.42% and 4.63% of the dry diet (8.86% and 9.28% of dietary protein), respectively.

Methionine played a positive role in improving the intestinal digestion capacity, anti-inflammatory reaction and oxidation resistance of grass carp, Ctenopharyngodon idella, fry.

A study was carried out to appraisal the function of methionine on intestinal digestion and the health of grass carp (Ctenopharyngodon idella) fry (initial weight 0.36 ± 0.01 g). The fry were fed graded dietary methionine levels (0.33%-1.20% dry matter) in 18 recirculatory tanks (180 L). After an 8-week breeding experiment, the results revealed that 0.71%-1.20% dietary methionine levels markedly upregulated the mRNA levels of intestinal digestion including trypsin, amylase, chymotrypsin and AKP, and 0.71%-0.87% dietary methionine level significantly increased intestinal trypsin activities compared with the 0.33% dietary methionine level. For inflammation, 0.71%-1.20% dietary methionine levels downregulated the mRNA levels of NF-κBp65, IL-1ß, IL-6, IL-8, IL-15 and IL-17D, whereas upregulated the mRNA levels of anti-inflammatory cytokines, including IL-4/13B, IL-10 and IL-11. In terms of antioxidants, although dietary methionine levels had no significant effect on the expression of most core genes of the Nrf2/ARE signaling pathway, such as Nrf2, Keap 1, GPx4, CAT, Cu/Zn-SOD. Furthermore, dietary methionine levels had no significant effect on the expression of p38MAPK, IL-12p35, TGF-ß2 and IL-4/13A. 0.71%-1.20% dietary methionine levels still increased the mRNA levels of GPx1α, GSTR and GSTP1. Furthermore, higher intestinal catalase activity and glutathione contents were also observed in fry fed 0.71%-1.20% diets. In summary, 0.71%-1.20% dietary methionine levels played a positive role in improving the intestinal digestion capacity of digestion, anti-inflammatory reaction and oxidation resistance of grass carp fry. This study provided a theoretical basis for improving the survival rate and growth of grass carp fry.

Salinity levels affect the lysine nutrient requirements and nutrient metabolism of juvenile genetically improved farmed tilapia (Oreochromis niloticus).

This 62-d research aimed to evaluate the effects of dietary lysine levels (DLL) and salinity on growth performance and nutrition metabolism of genetically improved farmed tilapia (GIFT) juveniles (Oreochromis niloticus). Six diets with lysine supplementation (1·34, 1·70, 2·03, 2·41, 2·72 and 3·04 % of DM) were formulated under different cultured salinities in a two-factorial design. The results indicated that supplemental lysine improved the specific growth rate (SGR) and weight gain (WG) and decreased the feed conversion ratio (FCR). Meanwhile, the fish had higher SGR and WG and lower FCR at 8 ‰ salinity. Except for moisture, the whole-body protein, lipid and ash content of GIFT were increased by 8 ‰ salinity, which showed that DLL (1·34 %) increased the whole-body fat content and DLL (2·41 %) increased whole-body protein content. Appropriate DLL up-regulated mRNA levels of protein metabolism-related genes such as target of rapamycin, 4EBP-1 and S6 kinase 1. However, 0 ‰ salinity reduced these protein metabolism-related genes mRNA levels, while proper DLL could improve glycolysis and gluconeogenesis mRNA levels but decrease lipogenesis-related genes mRNA levels in liver. 0 ‰ salinity improved GLUT2, glucokinase and G6 Pase mRNA levels; however, sterol regulatory element-binding protein 1 and fatty acid synthase mRNA levels were higher at 8 ‰ salinity. Moreover, 8 ‰ salinity also increased plasma total protein and cholesterol levels and decreased glucose levels. These results indicated that the recommended range of lysine requirement under different salinity was 2·03-2·20 % (0 ‰) and 2·20-2·41 % (8 ‰) and 8 ‰ salinity resulted in higher lysine requirements due to changes in the related nutrient metabolism, which might provide useful information for designing more effective feed formulations for GIFT cultured in different salinity environment.

Dietary chlorella (Chlorella vulgaris) supplementation effectively improves body color, alleviates muscle inflammation and inhibits apoptosis in largemouth bass (Micropterus salmoides).

Muscle quality, antioxidant status, and inflammatory and apoptotic molecule expression were investigated in juvenile largemouth bass fed five levels of Chlorella for 60 days. The results showed that muscle quality can be improved by increasing the muscle crude protein content, muscle and skin brightness value (L*), redness value (a*) and yellowness value (b*) in Chlorella-supplemented diets without affecting the growth and muscle fiber development of fish. Chlorella supplementation did not cause oxidative stress in muscle, but optimal Chlorella administration alleviated the muscle inflammatory response by downregulating the nuclear factor κB (NF-κB)-mediated proinflammatory factors such as interleukin 1ß (IL-1ß) and interleukin 8 (IL-8). Moreover, anti-apoptotic effects were induced by upregulation of anti-apoptotic genes, such as b cell lymphoma-2 (bcl-2) and myeloid cell leukemia-1 (mcl-1), and downregulation of pro-apoptotic genes, including bcl2-associated x (bax) and caspase3. In conclusion, Chlorella improved muscle quality, alleviated muscle inflammation and resisted muscle apoptosis.

Effects of replacing fishmeal with methanotroph (Methylococcus capsulatus, Bath) bacteria meal (FeedKind®) on growth and intestinal health status of juvenile largemouth bass (Micropterus salmoides).

A ten-week feeding trial evaluated the feasibility of methanotroph (Methylococcus capsulatus) bacteria meal (FeedKind®, FK) as a fishmeal substitute in largemouth bass (Micropterus salmoides) diets. Six isonitrogenous and isoenergetic diets with different inclusion levels of FK (0 (fishmeal group), 43, 86, 129, 172 and 215 g/kg) were formulated to replace 0, 50, 100, 150, 200 and 250 g/kg fishmeal, respectively. The results showed that FK inclusion level could reach 129 g/kg without significantly affecting growth or feed coefficient rate (P > 0.05), while growth performance was decreased and feed coefficient rate increased when FK inclusion levels exceeded 129 g/kg (P < 0.05). Increase in FK inclusion levels tended to reduce plasma total cholesterol and total triglyceride whilst plasma total protein, albumin, alanine aminotransferase and aspartate aminotransferase in FK treatment groups were unchanged compared with fishmeal group (P > 0.05). FK inclusion levels at 43 g/kg and 86 g/kg were not detrimental to intestinal morphology whilst it was unfavourable when FK inclusion levels exceeded 86 g/kg as the total length of intestinal wall thickness and villus height, villus height were obviously decreased compared with fishmeal group (P < 0.05). As regards to inflammatory cytokine genes, FK instead of fishmeal increased the expression levels of TLR2, RelA, TNF-α, IL-1ß, IL-10 and TGF-ß, 43 g/kg and 86 g/kg FK decreased the expression level of Caspase-3 (P < 0.05). In conclusion, 129 g/kg FK can replace 150 g/kg fishmeal without negative effects on the growth performance, and replacing 100 g/kg fishmeal with 86 g/kg FK is more beneficial to intestinal health.