Effects of Aeromonas infection on the immune system, physical barriers and microflora structure in the intestine of juvenile grass carp (Ctenopharyngodon idella).

Grass carp (Ctenopharyngodon idella) is an intensively cultured and economically important herbivorous fish species in China, but its culture is often impacted by Aeromonas pathogens such as Aeromonas hydrophila and Aeromonas veronii. In this study, healthy grass carp were separately infected with A. hydrophila or A. veronii for 12, 24, 48 or 72 h. The results showed that the mRNA expression levels of intestinal inflammatory factors (tnf-α, il-1ß and il-8), complement factors (c3 and c4), antimicrobial peptides (hepcidin, nk-lysin and ß-defensin-1), immunoglobulins (igm and igt), and immune pathway-related signaling molecules (tlr1, tlr2, tlr4, myd88, irak4, irak1, traf6, nf-κb p65 and ap-1) were differentially upregulated in response to A. hydrophila and A. veronii challenge. Additionally, the expression levels of the intestinal pro-apoptotic genes tnfr1, tnfr2, tradd, caspase-8, caspase-3 and bax were significantly increased, whereas the expression of the inhibitory factor bcl-2 was significantly downregulated, indicating that Aeromonas infection significantly induced apoptosis in the intestine of grass carp. Moreover, the expression of intestinal tight junction proteins (occludin, zo-1, claudin b and claudin c) was significantly decreased after infection with Aeromonas. Histopathological analysis indicated the Aeromonas challenge caused severe damage to the intestinal villi with adhesions and detachment of intestinal villi accompanied by severe inflammatory cell infiltration at 12 h and 72 h. The 16S rRNA sequencing results showed that Aeromonas infection significantly altered the structure of the intestinal microflora of the grass carp at the phylum (Proteobacteria, Fusobacteria, Bacteroidetes and Firmicutes) and genus (Proteus, Cetobacterium, Bacteroides, and Aeromonas) levels. Take together, the findings of this study revealed that Aeromonas infection induces an intestinal immune response, triggers cell apoptosis, destroys physical barriers and alters microflora structure in the intestine of juvenile grass carp; the results will help to reveal the pathogenesis of intestinal bacterial diseases in grass carp.

Microalgae as fishmeal alternatives in aquaculture: current status, existing problems, and possible solutions.

Fishmeal is an indispensable ingredient for most aquatic animals. However, the finite supply and escalating price of fishmeal seriously limit its use in aquaculture. Thus the development of new, sustainable protein ingredients has been a research focus. Microalgae are potential fishmeal alternatives owing to their high protein content and balanced amino acid profile. Studies suggest that suitable replacement of fishmeal with microalgae is beneficial for fish growth performance, but excessive replacement would induce poor growth and feed utilization. Therefore, this paper aims to review research on the maximum substitutional level of fishmeal by microalgae and propose the main issues and possible solutions for fishmeal replacement by microalgae. The maximum replacement level is affected by microalgal species, fish feeding habits, quality of fishmeal and microalgal meals, and supplemental levels of fishmeal in the control group. Microalgae could generally replace 100%, 95%, 95%, 64.1%, 25.6%, and 18.6% fishmeal protein in diets of carp, shrimp, catfish, tilapia, marine fish, and salmon and trout, respectively. The main issues with fishmeal replacement using microalgae include low production and high production cost, poor digestibility, and anti-nutritional factors. Possible solutions to these problems are recommended in this paper. Overall, microalgae are promising fishmeal alternatives in aquaculture.

Insights into Dietary Different Co-Forms of Lysine and Glutamate on Growth Performance, Muscle Development, Antioxidation and Related Gene Expressions in Juvenile Grass Carp (Ctenopharyngodon idellus).

The study aimed to compare the effects of crystalline L-lysine and L-glutamate (CAA), Lys-Glu dipeptide (KE) on the growth and muscle development of grass carp (Ctenopharyngodon idellus), and related molecular mechanisms. Five experimental diets (CR, 0.5% CAA, 1.5% CAA, 0.5% KE, 1.5% KE) containing Lys and Glu as free (Lys and Glu, CAA) dipeptide (Lys-Glu, KE) forms were prepared, respectively. A total of 450 juvenile grass carp with an initial weight of 10.69 ± 0.07 g were randomly assigned to 15 cages, and 5 treatments with 3 replicates of 30 fish each for 61 days of feeding. The results showed that the group of 0.5% KE exhibited the best growth performances according to the indicator's weight gain rate (WGR) and specific growth rate (SGR), although no statistically significant occurred among all groups; diet supplemented with 0.5% CAA significantly elevated the condition factor (CF) and viscerasomatic index (VSI) of juvenile grass carp. Diet supplemented with different Lys and Glu co-forms at different levels promoted the muscle amino acid content compared with those of CR group. Comparing with the CR group and other groups, the hardness of 0.5% CAA group significantly increased, and the springiness of 0.5% KE group excelled. Both the muscle fiber diameter and density of 0.5% KE group showed significant difference with those of the CR group, and a negative correlation between them was also observed. To uncover the related molecular mechanism of the differences caused by the different co-forms of Lys and Glu, the effect of different diets on the expressions of protein absorption, muscle quality, and antioxidation-related genes was analyzed. The results suggested that comparing with those of CR group, the dipeptide KE inhibited the expressions of genes associated with protein metabolism, such as AKT, S6K1, and FoxO1a but promoted PCNA expression, while the free style of CAA would improve the FoxO1a expression. Additionally, the muscle development-related genes (MyoD, MyOG, and Myf5) were significantly boosted in CAA co-form groups, and the expressions of fMYHCs were blocked but fMYHCs30 significantly promoted in 0.5% KE group. Finally, the effect of different co-forms of Lys and Glu on muscle antioxidant was examined. The 0.5% CAA diet was verified to increase GPX1a but obstruct Keap1 and GSTP1 expressions, resulting in enhanced SOD activity and reduced MDA levels in plasma. Collectively, the different co-forms of Lys and Glu influenced the growth of juvenile grass carp, and also the muscle development and quality through their different regulation on the protein metabolism, muscle development- and antioxidative-related genes.

Physiological and Transcriptome Analysis Reveal the Regulation Mechanism Underlying the Muscle Quality Effect of Dietary Schisandra chinensis in Triploid Crucian Carp (Carassius auratus).

Schisandra chinensis (sc) is generally demonstrated to improve antioxidant and immune functions in mammal. The present study through physiological and transcriptome analysis revealed alterations in muscle metabolisms of triploid crucian carp (Carassius auratus) cultured at different concentrations of S. chinensis diets (sc0, sc0.125%, sc0.25%, sc0.5%, sc1%, sc2%) after 8 weeks. The serum antioxidant enzyme activities analysis showed that dietary S. chinensis could reduce oxidative stress and increase organismic antioxidant capacity. Meanwhile, the detected results of muscle components presented that the amino acids and two flavor nucleotides of GMP and IMP significantly elevated while muscle crude lipid significantly reduced in S. chinensis feeding groups. In addition, springiness, chewiness, and fiber density in S. chinensis feeding groups muscle were significantly upregulated while muscle fiber diameter and area showed an opposite trend. By comparative transcriptome analysis of the muscles, functional enrichments of differentially expressed genes showed that multiple terms were related to purine metabolism, glycerolipid metabolism, regulation of actin cytoskeleton, and peroxisome. Finally, some key hub genes such as egln, gst, ggct, su1b, pi3kr4, myh9, lpl, gcdh, mylk, and col4a were identified by weighted gene co-expression network analysis. Taken together, our findings facilitate the understanding of the molecular basis underlying the muscle quality effect of dietary S. chinensis in triploid crucian carp, which provides valuable insights into the nutritional strategies of the aquaculture industry.

Intervention of Dietary Protein Levels on Muscle Quality, Antioxidation, and Autophagy in the Muscles of Triploid Crucian Carp (Carassius carassius Triploid).

The aim of this study is to investigate the effect of dietary protein levels on flesh quality, oxidative stress, and autophagy status in the muscles of triploid crucian carp (Carassius carassius triploid), and the related molecular mechanisms. Six experimental diets with different protein levels (26%, 29%, 32%, 35%, 38%, 41%) were formulated. A total of 540 fish with an initial weight of 11.79 ± 0.09 g were randomly assigned to 18 cages and six treatments with three replicates of 30 fish each for 8 weeks feeding. It could be found that the whole-body ash content significantly increased in high protein level groups (p < 0.05). The 29% dietary protein level group exhibited the highest muscle moisture, although there was an inconspicuous decrease in the chewiness of the muscles when compared with the other groups. The dietary protein level influenced the content of free amino acids and nucleotides, especially the content of flavor amino acids, which exhibited an increasing tendency along with the increasing protein level, such as alanine and glutamic acid, while the flavor nucleotides showed different fluctuation trends. Moreover, the genes related to muscle development were shown to be influenced by the dietary protein level, especially the expression of MRF4, which was up-regulated with the increasing dietary protein levels. The 29% dietary protein level promoted the majority of analyzed muscle genes expression to the highest level when compared to other dietary levels, except the Myostain, whose expression reached its highest at 38% dietary protein levels. Furthermore, the effect of dietary protein levels on antioxidant signaling pathway genes were also examined. High protein levels would boost the expression of GSTα; GPX1 and GPX4α mRNA expression showed the highest level at the 32% dietary protein group. The increasing dietary protein level decreased both mRNA and protein expressions of Nrf2 by up-regulating Keap1. Autophagy-related gene expression levels reached the peak at 32% dietary protein level, as evidenced by a similar change in protein expression of FoxO1. In summary, muscle nutritional composition, antioxidative pathways, and autophagy levels were affected by the dietary protein levels. A total of 29-32% dietary protein level would be the appropriate level range to improve muscle quality and promote the antioxidant and autophagy capacity of triploid crucian carp muscles.

Effect of Tributyrin on Growth Performance and Pathway by which Tributyrin Regulates Oligopeptide Transporter 1 in Juvenile Grass Carp (Ctenopharyngodon idellus).

The nutritional functions of tributyrin (TB) have been extensively studied, but questions remain regarding its influence on the growth of juvenile grass carp (Ctenopharyngodon idellus) and the regulation pathway to PepT1 in the intestine of grass carp. To answer the remaining questions, feeding trials, cell trials, and peritoneal injection trials were conducted in this study. The results showed that an appropriate level of TB (0.5 g/kg and 1.0 g/kg) supplementation in feed significantly promoted the growth performance of juvenile grass carp. The expressions of intestine genes (CDX2, SP1 and PepT1) related to oligopeptide transportation increased in the 0.5 g/kg TB group of feeding trials and both the 5 mM and 10 mM TB groups of the intestine cell trials, respectively. Subsequently, the injection trials of inhibitors CDX2 and SP1 demonstrated that the inhibition of CDX2 or SP1 decreased the mRNA expression of PepT1. Finally, the results of independent or combined treatments of TB and the inhibitors suggested that CDX2/SP1 mediated TB regulation on PepT1. These findings may help us to better understand the functions of TB on growth and PepT1 oligopeptide transportation, which could be modulated by dietary TB through the CDX2/SP1-PepT1 pathway in juvenile grass carp.

Molecular characterization of adenosine monophosphate deaminase 1 and its regulatory mechanism for inosine monophosphate formation in triploid crucian carp.

Inosine monophosphate (IMP) is the main flavoring substance in aquatic animal, and adenosine monophosphate deaminase1 (AMPD1) gene is a key gene in IMP formation. At present, the research on the mechanism of AMPD1 regulating IMP formation in aquatic animal is still blank. In this study, in order to study the mechanism of AMPD1 regulating IMP formation in fish, the full open reading frame (ORF) of AMPD1 which was 2160bp was obtained for the first time in triploid crucian carp (Carassius auratus). It encoded 719 amino acids with a molecular mass of 82.97 kDa, and the theoretical isoelectric point value was 6.31. The homology analysis showed that the homology of triploid crucian carp and diploid Carassius auratus was the highest, up to 99%. And the phylogenetic tree showed that triploid crucian carp was grouped with diploid Carassius auratus, Culter alburnus, and Danio rerio. And real-time fluorescence quantitative results showed that AMPD1 was expressed specifically in muscle of triploid crucian carp (p < 0.05). The results of detection the localization of AMPD1 in cells indicated that the AMPD1 was mainly localized in cytoplasm and cell membrane. Further, we examined the effects of glutamate which was the promotor of IMP formation on the expression of AMPD1 and the formation of IMP in vivo and in vitro experiments, the results showed that 3% glutamate and 2 mg/ml glutamate could significantly promote AMPD1 expression and IMP formation in triploid crucian carp muscle tissue and muscle cells (p < 0.05). Then we inhibited the expression of AMPD1 in vivo and in vitro experiments, we found the formation of IMP in muscle tissue and muscle cells of triploid crucian carp all were inhibited and they affected the gene expression of AMPK-mTOR signaling pathway. The all results showed that AMPD1 mediated glutamate through AMPK-mTOR signaling pathway to regulate the formation of fish IMP.

Molecular Characterization of LKB1 of Triploid Crucian Carp and Its Regulation on Muscle Growth and Quality.

Liver Kinase B1 (LKB1) is a serine/threonine kinase that can regulate energy metabolism and skeletal muscle growth. In the present study, LKB1 cDNA of triploid crucian carp (Carassius auratus) was cloned. The cDNA contains a complete open reading frame (ORF), with a length of 1326 bp, encoding 442 amino acids. Phylogenetic tree analysis showed that the LKB1 amino acid sequence of the triploid crucian carp had a high sequence similarity and identity with carp (Cyprinus carpio). Tissue expression analysis revealed that LKB1 was widely expressed in various tissues. LKB1 expressions in the brain were highest, followed by kidney and muscle. In the short-term LKB1 activator and inhibitor injection experiment, when LKB1 was activated for 72 h, expressions of myogenic differentiation (MyoD), muscle regulatory factor (MRF4), myogenic factor (MyoG) and myostatin 1 (MSTN1) were markedly elevated and the content of inosine monophosphate (IMP) in muscle was significantly increased. When LKB1 was inhibited for 72 h, expressions of MyoD, MyoG, MRF4 and MSTN1 were markedly decreased. The long-term injection experiment of the LKB1 activator revealed that, when LKB1 was activated for 15 days, its muscle fibers were significantly larger and tighter than the control group. In texture profile analysis, it showed smaller hardness and adhesion, greater elasticity and chewiness. Contrastingly, when LKB1 was inhibited for 9 days, its muscle fibers were significantly smaller, while the gap between muscle fibers was significantly larger. Texture profile analysis showed that adhesion was significantly higher than the control group. A feeding trial on triploid crucian carp showed that with dietary lysine-glutamate dipeptide concentration increasing, the expression of the LKB1 gene gradually increased and was highest when dipeptide concentration was 1.6%. These findings may provide new insights into the effects of LKB1 on fish skeletal muscle growth and muscle quality, and will provide a potential application value in improvement of aquaculture feed formula.

Functional characterization of MEKK3 in the intestinal immune response to bacterial challenges in grass carp (Ctenopharyngodon idella).

Mitogen-activated protein kinase kinase kinase 3 (MEKK3) is an evolutionarily conserved Ser/Thr protein kinase of the MEKK family that is essential for the host immune response to pathogen challenges in mammals. However, the immune function of MEKK3s in lower vertebrate species, especially in bony fish, remains largely unknown. In this study, a fish MEKK3 (designated CiMEKK3) gene was cloned and identified from grass carp (Ctenopharyngodon idella). The present CiMEKK3 cDNA encoded a 620 amino acid polypeptide containing a conserved S-TKc domain and a typical PB1 domain. Several potential immune-related transcription factor-binding sites, including activating protein 1 (AP-1), nuclear factor kappa B (NF-κB) and signal transducer and activator of downstream transcription 3 (STAT3), were observed in the 5' upstream DNA sequence of CiMEKK3. A phylogenetic tree showed that CiMEKK3 exhibits a close evolutionary relationship with MEKK3s from Cyprinus carpio and Carassius auratus. Quantitative real-time PCR analysis revealed that CiMEKK3 transcripts were widely distributed in all selected tissues of healthy grass carp, with a relatively high levels observed in the gill, head kidney and intestine. Upon in vitro challenge with bacterial pathogens (Aeromonas hydrophila and Aeromonas veronii) and pathogen-associated molecular patterns (PAMPs) (lipopolysaccharide (LPS), peptidoglycan (PGN), L-Ala-γ-D-Glu-mDAP (Tri-DAP) and muramyl dipeptide (MDP)), the expression levels of CiMEKK3 in the intestinal cells of grass carp were shown to be significantly upregulated in a time-dependent manner. In vivo injection experiments revealed that CiMEKK3 transcripts were significantly induced by MDP challenge in the intestine; however, these effects could be inhibited by the nutritional dipeptides carnosine and Ala-Gln. Moreover, subcellular localization analysis and luciferase reporter assays indicated that CiMEKK3 could act as a cytoplasmic signal-transducing activator involved in the regulation of NF-κB and MAPK/AP-1 signaling cascades in HEK293T cells. Taken together, these findings strongly suggest that CiMEKK3 plays vital roles in the intestinal immune response to bacterial challenges, which will aid in understanding the pathogenesis of inflammatory bowel disease in bony fish.

Dietary Supplementation With Hydroxyproline Enhances Growth Performance, Collagen Synthesis and Muscle Quality of Carassius auratus Triploid.

An eight-week experiment was undertaken to examine the effect of dietary hydroxyproline (Hyp) supplementation on growth performance, collagen synthesis, muscle quality of an improved triploid crucian carp (Carassius auratus Triploid) (ITCC). Six isonitrogenous (340 g/kg diet), isolipidic (60 g/kg diet) and isocaloric (17.80 MJ/kg diet) diets were formulated containing a certain amount of Hyp: 0.09% (the control group), 0.39, 0.76, 1.14, 1.53 and 1.90%. Each diet was randomly assigned to three tanks and each group was fed two times daily until apparent satiation. The results showed that growth performance and feed utilization of ITCC were significantly improved with the dietary Hyp level was increased from 0.09 to 0.76%. Crude protein, threonine and arginine content in the dorsal muscle in 0.76% hydroxyproline group were significantly higher than those in basic diet group (p < 0.05). The muscle textural characteristics increased remarkably with the amount of Hyp in the diet rising from 0.09 to 1.53% (p < 0.05). Meanwhile, the contents of type I collagen (Col I) and Pyridinium crosslink (PYD) in the muscle of fish were significantly increased by dietary Hyp (p < 0.05). The muscle fiber diameter and density of the fish were significantly increased when fed with 0.76% Hyp (p < 0.05). Furthermore, dietary supplementation with an appropriate concentration of Hyp substantially increased the expression of genes involved in collagen synthesis (col1a1, col1a2, p4hα1, p4hß, smad4, smad5, smad9, and tgf-ß) and muscle growth (igf-1, tor, myod, myf5, and myhc) (p < 0.05). In conclusion, dietary supplementation of Hyp can enhance fish growth performance, collagen production, muscle textural characteristics and muscle growth of ITCC. According to the SGR broken-line analysis, the recommended supplementation level of Hyp was 0.74% in the diet for ITCC, corresponding to 2.2% of dietary protein.

Grass carp MAP3K4 participates in the intestinal immune response to bacterial challenge.

Mitogen-activated protein kinase kinase kinase 4 (MAP3K4) is a multifunctional mediator of the conserved MAPK signaling pathway that plays essential roles in the regulation of immune responses in mammals. However, the function of teleost MAP3K4s in innate immunity, especially in the intestinal immune system, is still poorly understood. In the current study, we identified a fish MAP3K4 homolog (CiMAP3K4) in Ctenopharyngodon idella as well as its immune function in intestine following bacterial infection in vivo and in vitro. The open reading frame (ORF) of CiMAP3K4 encodes putative peptide of 1544 amino acids containing a predicted serine/threonine protein kinase (S_TKc) domain with high identity with other fish MAP3K4s. Phylogenetic analysis revealed the CiMAP3K4 belonged to the fish cluster and showed the closest relationship to Pimephales promelas. Quantitative real-time PCR (qRT-PCR) analysis revealed that CiMAP3K4 transcripts were widely distributed in all tested tissues, especially with high expression in the muscle and intestine of healthy grass carp. In vitro, CiMAP3K4 gene expression was upregulated by bacterial PAMPs (lipolysaccharide (LPS), peptidoglycan (PGN), L-Ala-γ-D-Glu-meso-diaminopimelic acid (Tri-DAP) and muramyl dipeptide (MDP)) and pathogens (Aeromonas hydrophila and Aeromonas veronii) in primary intestinal cells. In vivo, the mRNA expression levels of CiMAP3K4 in the intestine were significantly induced by bacterial MDP challenge in a time-dependent manner; however, this effect could be inhibited by the bioactive dipeptides ß-alanyl-l-histidine (carnosine) and alanyl-glutamine (Ala-Gln). Moreover, CiMAP3K4 was located primarily in the cytoplasm, and its overexpression increased the transcriptional activity of AP-1 in HEK293T cells. Collectively, these results suggested that CiMAP3K4 might play an important role in the intestinal immune response to bacterial infections, which paves the way for a better understanding of the intestinal immune system of grass carp.

JAK2 Mediates the Regulation of Pept1 Expression by Leptin in the Grass Carp (Ctenopharyngodon idella) Intestine.

Oligopeptide transporter 1 (Pept1) is located on the brush border membrane of the intestinal epithelium and plays an important role in dipeptide and tripeptide absorption from protein digestion. In this study, we cloned and characterized the cDNA sequence of Janus kinase 2 (JAK2) from Ctenopharyngodon idella. The expression patterns of JAK2 in various tissues and developmental stages were characterized by quantitative real-time PCR (qRT-PCR). The mRNA expression levels of JAK2 and Pept1 regulated by leptin in the intestine were also analyzed in vitro and in vivo. The cDNA sequence of JAK2 is 3378 bp in length, and the mRNA of JAK2 was broadly expressed in all tissues and embryonic stages of C. idella analyzed. In addition, we found that leptin regulated expression of JAK2 and Pept1 in the intestine; Pept1 expression was down-regulated by the JAK2 inhibitor AG490 in vivo and in vitro. Furthermore, luciferase experiments showed that overexpression of the JAK2 gene significantly upregulated the activity of the Pept1 5' regulatory sequence in C. idella. In conclusion, these results may help in elucidating the regulatory effect of the leptin-mediated JAK2 pathway on intestinal Pept1 expression in C. idella and the molecular mechanism of peptide transport by the intestinal transporter Pept1 in fishes.

p38 mitogen-activated protein kinases (MAPKs) are involved in intestinal immune response to bacterial muramyl dipeptide challenge in Ctenopharyngodon idella.

The p38 mitogen-activated protein kinases (MAPKs) are essential cytoplasmic signal molecules of innate immune pathways that play a vital role in host immune defense responses to pathogenic challenges. In this study, two fish p38 genes (Cip38α and Cip38ß) were characterized for the first time from the grass carp Ctenopharyngodon idella. Similar to other reported p38MAPKs, both Cip38α and Cip38ß contained a conserved phosphorylation motif (Thr-Gly-Tyr, TGY) and a substrate binding site (Ala-Thr-Arg-Trp, ATRW) in the serine/threonine protein kinase (S_TKc) domain. Expression profile analysis showed that Cip38α and Cip38ß mRNAs were broadly expressed in all of the examined tissues and developmental stages of C. idella. In addition, in vivo injection experiments directly revealed that Cip38α and Cip38ß showed strong responsiveness to Aeromonas hydrophila and muramyl dipeptide (MDP) challenges, and their expression levels were significantly upregulated in the intestine of grass carp. Additionally, the MDP-induced expression levels of intestinal inflammatory cytokines (TNF-α and IL-15) and an antimicrobial peptide (ß-defensin) were significantly inhibited by the p38MAPK-specific inhibitor SB203580. Moreover, the nutritional dipeptide carnosine and Ala-Gln were found to significantly suppress the bacterial MDP-induced expression of p38MAPK pathway genes and inflammatory cytokines in the intestine of grass carp. Finally, overexpression analysis demonstrated that Cip38α and Cip38ß could act as efficient activators in the regulation of AP-1 signaling pathways through interaction with CiMKK6. Altogether, this study provided experimental evidence of the presence of a functional p38 pathway in grass carp, which revealed its involvement in the intestinal immune response to bacterial challenges in bony fish.

Effects of dietary yeast culture on growth performance, immune response and disease resistance of gibel carp (Carassius auratus gibelio CAS â ¢).

A 50-day feeding trial was carried out to evaluate the partial replacement of fishmeal by yeast culture (YC) on growth performance, immune response and resistance against Aeromonas hydrophila in gibel carp CAS Ⅲ (Carassius auratus gibelio). Four isonitrogenous and isoenergetic practical diets including a basal diet (the control diet containing 10% fish meal, D0) and three yeast culture diets (substituting 20%, 40%, 60% of the fishmeal in the basal diet, D20, D40 and D60, respectively) were formulated. Each diet was randomly allocated to quadruplicate fish groups (average initial body weight: 28.70 ±â€¯0.03 g) reared in a recirculating system. After the growth trial, bacterial challenge test was conducted. The results showed that no noteworthy variations in feed intake, growth performance and morphology indices were found among groups (P > 0.05). YC Supplemented diet exerted little significant influence on plasma parameters including triglyceride, glucose, creatinine, total protein and urea nitrogen compared with the control group (P > 0.05). No obvious variations were found in activities of plasma lysozyme, IgM, MPO and SOD before challenge test among dietary treatments (P > 0.05), whereas considerable higher value of the foresaid indicators was discovered in D40 after bacteria challenge (P < 0.05). Transcriptional levels of Toll like receptor 2 (TLR2), myeloid differentiation factor 88 (MyD88), Toll/IL-1 receptor domain-containing adaptor protein (TIRAP) and interleukin-1ß (IL-1ß) in spleen after challenge were significantly up-regulated in D40 compared with D0 (P < 0.05). Cumulative survival rate in D40 and D60 were significantly higher than those in D0 and D20 (P < 0.05). Taken together, yeast culture could be a suitable fishmeal alternative in diets of gibel carp and dietary inclusion of 4 g YC per 100 g diet enhanced the immunity and disease resistance of gibel carp partly via TLR2 pathway.

Replacement of fishmeal by spirulina Arthrospira platensis affects growth, immune related-gene expression in gibel carp (Carassius auratus gibelio var. CAS III), and its challenge against Aeromonas hydrophila infection.

The present study examined the effect of dietary spirulina, Arthrospira platensis on growth performance, blood physiological indices, immune-related gene expressions and resistance of juvenile gibel carp against Aeromonas hydrophila infection. Four isonitrogenous (360 g kg-1) and isolipidic (90 g kg-1) diets were formulated with containing different levels of spirulina powder of 0 g (SP0, the control diet), 3.38 g (SP3.38), 6.76 g (SP6.76) and 13.52 g (SP13.52) per 100 g diet to replace 0%, 25%, 50% and 100% of fishmeal protein, respectively. And each diet was randomly assigned to triplicate tanks (150-L capacity per each) and each tank was stocked with 22 fish (15.37 ±â€¯0.06 g). Fish were fed one of the tested diets up to satiation twice a day for 46 days. A challenge test was carried out after the feeding trial by injecting Aeromonas hydrophila intraperitoneally for 7 days. The results showed that fish growth, feeding rate in groups SP3.38 and SP6.76 were significantly higher than those of groups SP0 and SP13.52 (P < 0.05). Feed efficiency and protein retention rate had no significant difference among all tested groups. Plasma superoxide dismutase and phagocyte activity of blood leukocytes significantly increased in the spirulina-fed fish groups at 12-h post the bacterial challenge (P < 0.05). Both pre and post challenge test, plasma lysozyme activities in spirulina-fed groups were significantly higher than that in the control group (P < 0.05). Plasma malondialdehyde got the lowest value in the SP13.52 group before and after the challenge test. The transcriptional levels of TLR2 (Toll like receptor 2), myeloid differentiation factor 88 (MyD88), Toll/IL-1 receptor domain-containing adaptor protein (TIRAP), interleukin-1ß (IL-1ß) and tumor necrosis factor-α1 (TNF-α1) in spleen and kidney significantly increased post the bacterial challenge compared to the pre challenge. And the relative expressions of the immune-related genes of spirulina-fed fish groups were higher than those of the control group before and after the challenge test. The 7-day cumulative survival rate after the bacterial challenge was highest in the SP3.38 group (P < 0.05). The present results indicated that low dietary inclusion of spirulina significantly enhanced the immune response of gibel carp partly through TLR2 pathway and 3.38% of dietary spirulina was recommended for the juveniles based on the growth and immune response.

Effects of dietary Tenebrio molitor meal on the growth performance, immune response and disease resistance of yellow catfish (Pelteobagrus fulvidraco).

This study evaluated the influence of diets containing mealworm (Tenebrio molitor) meal in partial substitution of fishmeal on growth performance and immune responses of juvenile yellow catfish (Pelteobagrus fulvidraco). Four diets were formulated to contain 0 (the control diet), 9, 18 and 27 g mealworm meal per 100 g diet with 0%, 25%, 50% and 75% of fishmeal replacement, respectively. Yellow catfish were randomly divided into 4 groups with 3 replicates in each group. The fish in each group were fed with one of the four experimental diets for 5 weeks. Growth performance, plasma parameters (SOD, MDA, IgM, C3, lysozyme) and immune related genes (MHC II, IL-1, CypA, IgM, HE) of yellow catfish were determined at the end of the feeding trial, as well as 24 h post bacterial (Edwardsiella ictaluri) challenge. The present results showed that dietary inclusion of mealworm meal (MW) had no negative effects on the growth performance of the juvenile yellow catfish, compared to the control group. At the end of the feeding trial, plasma MDA contents of MW supplemented groups were significant lower than the control group. Plasma SOD activities increased significantly with the increasing dietary MW contents at the end of feeding trial (pre-challenge) and 24 h post challenge with E. ictaluri. Significant increase of plasma lysozyme activity was found in MW supplemented groups compared to the control group 24 h post bacterial challenge. Plasma IgM levels increased significantly with the increasing dietary MW contents at the end of feeding trial. Compared with the control group, the immune related genes of MHC II, IL-1, IgM and HE of the fish in the MW supplemented groups significantly upregulated pre-challenge or 24 h post bacterial challenge. Finally, it was observed that the survival rate of the 27% MW group was significant higher (P < 0.05) than the control group but was not significantly differed from the 18% MW group. The present results indicated that dietary inclusion level of at least 18% MW could improve the immune response and the bacterial resistance of yellow catfish without any negative growth effects.