Intestinal Proteases Research Articles

Dietary Lactobacillus helveticus and Gum Arabic improves growth indices, digestive enzyme activities, intestinal microbiota, innate immunological parameters, antioxidant capacity, and disease resistance in common carp

The present study aimed at determining the effects of Lactobacillus helveticus (LH), Gum Arabic (GA; natural prebiotic), and their combination as synbiotic on growth performance, digestive enzymes activity, gut microbiota, innate immunity status, antioxidant capacity, and disease resistance against Aeromonas hydrophyla in common carp, Cyprinus carpio for 8 weeks. For this, 735 common carp juveniles (Mean ± standard deviation; 22.51 ± 0.40 g) were fed with 7 different diets including basal diet (C), LH1 (1 × 107 CFU/g), LH2 (1 × 109 CFU/g), GA1 (0.5%), GA2 (1%), LH1+GA1 (1 × 107 CFU/g + 0.5%), and LH2+GA2 (1 × 109 CFU/g + 1%) for 8 weeks. Dietary supplementation with GA and/or LH significantly increased growth performance, WBC, serum total immunoglobulin, superoxide dismutase and catalase activities, skin mucus lysozyme and total immunoglobulin and intestinal lactic acid bacteria. Whereas there were significant improvements in various parameters tested in different treatments, the highest improvement in growth performance, WBC, monocyte/neutrophil percentages, serum lysozyme, alternative complement, glutathione peroxidase and malondialdehyde, skin mucosal alkaline phosphatase, protease, and immunoglobulin, intestinal total bacterial count, protease and amylase activities were observed in the synbiotic treatments, particularly LH1+GA1. After an experimental infection with Aeromonas hydrophila, all experimental treatments exhibited significantly higher survival, compared to the control treatment. The highest survival was related to the synbiotic (particularly LH1+GA1), followed by prebiotic, and probiotic treatments. Overall, synbiotic containing 1 × 107 CFU/g LH + 0.5% GA can improve growth rate and feed efficiency in common carp. Moreover, the synbiotic can improve the antioxidant/innate immune systems and dominate lactic acid bacteria in the fish intestine that may be the reasons of the highest resistance against A. hydrophila infection.

Effects of Replacing Fishmeal with the Mixture of Cottonseed Protein Concentrate and Clostridium autoethanogenum Protein on the Growth, Nutrient Utilization, Serum Biochemical Indices, Intestinal and Hepatopancreas Histology of Rainbow Trout (Oncorhynchus mykiss).

The purpose of this study was to develop the potential of cottonseed protein concentrate (CPC) and Clostridium autoethanogenum protein (CAP) in the diet of rainbow trout (Oncorhynchus mykiss) by evaluating the effects of substituting fishmeal with a CPC and CAP mixture on growth performance, nutrient utilization, serum biochemical indices, intestinal and hepatopancreas histology. In a basal diet containing 200 g/kg fishmeal (Con), the mixture of CPC and CAP (1:1) was used to reduce dietary fishmeal to 150, 100, 50 and 0 g/kg, to form five diets with the same crude protein and crude lipid contents (CON, FM-15, FM-10, FM-5 and FM-0). Then, the five diets were fed to rainbow trout (35.00 ± 0.05 g) for 8 weeks. The weight gain (WG) and feed conversion ratio (FCR) of the five groups were 258.72%, 258.82%, 249.90%, 242.89%, 236.57%, and 1.19, 1.20, 1.24, 1.28, 1.31, respectively. FM-5 and FM-0 groups showed significantly lower WG and higher FCR than the CON group (p < 0.05). In terms of whole-body composition, such as moisture, crude ash, and crude protein, no significant difference was observed among all the groups (p > 0.05), except that significantly higher crude lipid content was detected in the FM-0 group than in the CON group (p < 0.05). In the FM-5 and FM-0 groups, protein efficiency, protein retention, intestinal protease activity and amylase activity were significantly lower than in the CON group (p < 0.05). Compared to the CON group, the serum contents of glucose and total cholesterol in the FM-0 group as well as MDA in the FM-5 and FM-0 groups were significantly increased, and catalase, superoxide dismutase, and total antioxidant capacity were decreased (p < 0.05). In intestine and hepatopancreas histology, the intestinal villus height in the FM-5 and FM-0 groups and villus width in the FM-0 group were decreased significantly (p < 0.05), while no significant difference in hepatopancreas morphology was observed among all the groups except that some vacuolization was observed in the FM-0 group (p > 0.05). In summary, the mixture of CPC and CAP can effectively replace 100 g/kg fishmeal in a diet containing 200 g/kg fishmeal without adverse effects on the growth performance, nutrient utilization, serum biochemical, or intestinal and hepatopancreas histology of rainbow trout.

An innovative insecticidal approach based on plant protease inhibitor and Bt protoxins inhibits trypsin-like activity in zebrafish

The Leucaena leucocephala trypsin inhibitor (LTI) + Bacillus thuringiensis (Bt) protoxins mix has been proposed as a novel larvicide agent in order to control the vector mosquito of dengue virus, Aedes aegypti, in their aquatic breeding sites. However, use of this insecticide formulation has raised concerns about its impacts on aquatic biota. In this context, this work aimed to assess the effects of LTI and Bt protoxins, separately or in combination, in zebrafish, in regard to the evaluation of toxicity at early life stages and to the presence of LTI inhibitory effects on intestinal proteases of this fish. Results showed that LTI and Bt concentrations (250 mg/L, and 0.13 mg/L, respectively), and LTI + Bt mix (250 mg/L + 0.13 mg/L) - 10 times superior to those with insecticidal action – did not cause death nor did it induce morphological changes during embryonic and larval development (3 to 144 h post-fertilization) of zebrafish. Molecular docking analyses highlighted a possible interaction between LTI and zebrafish trypsin, especially through hydrophobic interactions. In concentrations near to those with larvicidal action, LTI (0.1 mg/mL) was able to inhibit in vitro intestinal extracts of trypsin in female and male fish by 83 % and 85 %, respectively, while LTI + Bt mix promoted trypsin inhibition of 69 % in female and 65 % in male ones. These data show that the larvicidal mix can potentially promote deleterious effects to nutrition and survival in non-target aquatic organisms, especially those with trypsin-like dependent protein digestion.

The influence of dietary protein concentration on digestive enzyme activities, growth, and body composition in juvenile bullseye snakehead (Channa marulius).

The bullseye snakehead (Channa marulius) is considered as an affordable and robust freshwater fish for farming in Asia. However, there is limited knowledge on the species' full nutritional requirements to date with extensive gaps in our knowledge and particularly in precision aspects of protein requirements. Therefore, a three-month feeding trial was conducted under semi-intensive farming conditions to determine the protein requirement of bullseye snakehead using test diets containing 40 (P40), 45 (P45), 50 (P50), and 55% (P55) crude protein levels. The growth performance results revealed that the 55% dietary protein group (P55) had the highest final mean weight (14.09 g fish-1), and net weight gain (12.82 g fish-1). When compared to other dietary treatments, the final weight (R2 = 0.921), and weight gain (R2 = 0.913), displayed a linear increasing trend as dietary protein is raised. The lowest FCR was observed in 50% (1.94±0.01) and 55% (1.97±0.01) CP diet groups compared to dietary treatments. Further analysis has shown that the body protein content also significantly increased as dietary protein was raised to 55%. Although, a reverse trend was found in body lipid levels with increasing protein in the diet. The incremental dietary protein also elevated proximal intestinal protease activity but decreased amylase and lipase activity. The overall essential and non-essential amino acids levels of snakehead fillet muscle reflected an increase in dietary protein. Overall, this study has shown that the fish fed a diet with 55% crude protein attained the highest growth performance and nutrient profile of the whole fish when compared to other dietary treatments tested. It would appear we did not obtain the maximum potential for growth under the present experimental conditions due to the upper protein constraint of 55% in the diet. Further quantitative studies are suggested.

Effects of dietary costmary (Tanacetum balsamita) essential oil on growth performance, digestive enzymes' activity, immune responses and subjected to ambient ammonia of common carp Cyprinus carpio

The present study aimed at investigating the effects of dietary costmary, Tanacetum balsamita, essential oil (CEO) on growth performance, digestive enzymes' activity, and physiological responses to ammonia exposure in common carp, Cyprinus carpio. Common carp (∼17 g) were randomly distributed in 15 tanks as five treatments receiving diets containing 0 (CTL), 100 (100E), 200 (200E), 300 (300E), and 400 (400E) mg/kg CEO for eight weeks and then exposed to 0.5 mg/l unionized ammonia nitrogen for 3 h. The results showed that dietary CEO significantly increased growth performance and feed efficiency and the best values were observed in 100E and 200E treatments. Intestinal amylase, lipase, and protease activities significantly increased in 100E, 200E, and 300E treatments, compared to CTL, and the highest amylase activity was related to 100E, and highest lipase and protease was related 100E and 200E treatments. Dietary CEO significantly decreased serum cortisol, glucose, catalase, malondialdehyde, alanine aminotransferase, aspartate aminotransferase, triglyceride, cholesterol, and low-density lipoprotein, but increased serum superoxide dismutase, glutathione peroxidase, lysozyme, complement, total immunoglobulin, high density lipoprotein, total protein, and albumin before and/or after ammonia stress, depending on CEO concentrations. Based on the present results, dietary CEO supplementation can improve digestive enzymes and growth performance of common carp. Moreover, CEO exhibits anti-stress, antioxidant, hepatoprotective, and immunostimulant effects in common carp under ammonia toxicity. Based on the results, 100–200 mg/kg CEO is recommended for common carp feed supplementation.

The protective role of vitamin C on intestinal damage induced by high-dose glycinin in juvenile Rhynchocypris lagowskii Dybowski

This study was to evaluate the mitigative effects of vitamin C (VC) on growth inhibition and intestinal damage induced by glycinin in juvenile Rhynchocypris lagowskii Dybowski. 270 healthy juvenile Rhynchocypris lagowskii Dybowski (4.65 ± 0.04 g) were randomly divided into 3 treatments, and fed with control diet, 80 g/kg glycinin diet and 80 g/kg glycinin+200 mg/kg VC diet respectively for 8 weeks. The results showed that glycinin significantly decreased the weight gain rate, specific growth rate, protein efficiency rate, feed efficiency rate and feeding rate of fish compared with the control group (P < 0.05), while VC supplementation improved the growth performance and feed utilization efficiency, and reached a level similar to the control group. Similarly, VC significantly increased the crude protein content of muscle and whole-body, and hepatopancreas and intestinal protease activities of fish fed with glycinin diet (P < 0.05). The distal intestine of fish in glycinin group showed typical damage characteristics, including breakage and atrophy of intestinal mucosal fold, and increased intestinal mucosal permeability. However, fish fed the glycinin + VC diet showed an unimpaired normal intestinal morphology. Usefully, VC supplementation could also restore impaired immune function and antioxidant capacity. VC down-regulated the mRNA levels of pro-inflammatory cytokines TNF‐α and IL-1β, and up-regulated the mRNA levels of anti-inflammatory cytokines IL-10 and TGF-β in the distal intestine of fish fed with glycinin. Furthermore, glycinin exposure could reduce the mRNA levels of HO-1, CAT and GPx by inhibiting the activation of Nrf2-Keap1 signaling pathway, while VC supplementation reversed this phenomenon and maintained the homeostasis of antioxidant defense system. Concluded, glycinin causes growth inhibition, digestive dysfunction and intestinal damage of Rhynchocypris lagowskii Dybowski, while sufficient VC intake is beneficial for fish to resist the adverse effects of glycinin.

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-1β (il-1β), 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%.

P064 High intestinal Secretory Leukocyte Protease Inhibitor (SLPI) expression correlates with abundant neutrophilic inflammation and identifies Inflammatory Bowel Disease patients with a strong IL-17 response.

Abstract Background Inflammatory bowel disease, comprising Crohn’s disease (CD) and ulcerative colitis (UC), is driven by aberrant host-microbial immune interactions. Disease heterogeneity and severity hamper effective treatment, thus new therapeutic strategies tailored to the patient’s underlying immune defect are required. In mice, upon epithelial barrier breach and microbiota driven inflammation, colonic intestinal epithelial cells increase Secretory Leukocyte Protease Inhibitor (SLPI) expression. SLPI can exert anti-microbial activity and inhibit NF-kB activation. Therefore, we hypothesized that SLPI expression is increased in the intestine of IBD patients and may differentiate a subgroup of patients with a distinctive underlying immune disease. Methods Intestinal biopsies and plasma from therapy-naive pediatric patients (age 6-18) with CD (n=41), UC (n=22) or IBD-negative controls (n=15) were collected. Intestinal SLPI protein was detected by immunohistochemistry (IHC), scored semi-quantitatively, and patients were dichotomized into ‘SLPI-high’ and ‘SLPI-low’ groups. RNA sequencing and quantitative PCR (qPCR) were performed. Plasma protein concentrations were assessed with Olink technology®. Results We observed increased SLPI mRNA and protein expression in intestinal tissue of CD and UC patients, compared to IBD-negative controls. Expression was the strongest in macroscopically inflamed tissue and more dominant in colon than in small intestinal ileum. Colonic SLPI expression was associated with endoscopic severity of inflammation, with histologic disease activity, and with clinical disease activity in CD and UC patients. Furthermore, high epithelial SLPI expression associated with abundant neutrophil infiltration and calprotectin expression. RNA-seq revealed that biopsies of SLPI-high patients had a signature of inflammatory neutrophilic inflammation including the genes OSM, IL-1B, and CXCL8, which have been associated with severe disease progression and resistance to anti-TNF treatment. Most notably, biopsies of SLPI-high patients had a signature of increased IL-17 signaling, which was supported by IHC detection of high frequencies of IL-17 producing cells in the paired biopsies and higher plasma concentrations of CCL20, MMP10 and IL-17A. Conclusion Together, these results demonstrate that intestinal SLPI expression identifies severe IBD patients with high intestinal SLPI expression and immune features of therapy resistance, who have increased IL-17A responses and subsequent neutrophilic inflammation compared to SLPI-low patients. As such, epithelial SLPI expression is a histological parameter that could support tailored treatment strategies in pediatric IBD patients at the time of diagnosis.

Impact of dietary curcumin administration along with feed-born silver nanoparticle on growth, hemato-biochemical parameters, and digestive enzyme activity of common carp (Cyprinus carpio).

This research explored the impacts of feed-born silver nanoparticles (AgNPs) on common carp (Cyprinus carpio) and whether dietary curcumin supplementation could ameliorate the impacts of AgNPs on growth, hemato-biochemical parameters and digestive enzyme activity. Nine experimental diets were prepared containing 0.00, 0.05, and 0.15 g kg-1 AgNPs, as well as 0.00, 0.75, and 1.50 g kg-1 curcumin in a factorial design. Triplicate groups of common carp (4.82 ± 0.41 g) were fed on the test diets for 60 days. The results demonstrated that AgNPs reduced growth performance and enhanced the feed conversion ratio dose-dependently. Supplementing 0.75 g kg-1 curcumin at a low AgNP level improved the growth rate, while its inclusion at a high AgNP level led to further suppression of growth performance. The highest hematocrit value, hemoglobin concentration and white blood cell count were recorded in the group receiving 0.75 g kg-1 curcumin. Serum glucose, cholesterol and triglyceride concentrations were elevated by increasing AgNP levels. However, curcumin inclusion, particularly at the lower level of AgNPs significantly decreased their values. Similarly, intestinal alkaline protease and lipase activities were progressively reduced by increasing dietary AgNP contents, but, significant improvements were observed by curcumin application at the lower AgNP level. Our results revealed that curcumin supplementation could limit the toxic effects of lower dietary AgNP contents.

Dietary Effect of Clostridium autoethanogenum Protein on Growth, Intestinal Histology and Flesh Lipid Metabolism of Largemouth Bass (Micropterus salmoides) Based on Metabolomics.

Clostridium autoethanogenum protein (CAP) is a new single-cell protein explored in aquatic feeds in recent years. This study investigated the dietary effects of CAP replacing fishmeal (FM) on the growth, intestinal histology and flesh metabolism of largemouth bass (Micropterus salmoides). In a basal diet containing 700 g/kg of FM, CAP was used to substitute 0%, 15%, 30%, 45%, 70% and 100% of dietary FM to form six isonitrogenous diets (Con, CAP-15, CAP-30, CAP-45, CAP-70, CAP-100) to feed largemouth bass (80.0 g) for 12 weeks. Only the CAP-100 group showed significantly lower weight gain (WG) and a higher feed conversion ratio (FCR) than the control (p &lt; 0.05). A broken-line analysis based on WG and FCR showed that the suitable replacement of FM with CAP was 67.1-68.0%. The flesh n-3/n-6 polyunsaturated fatty acid, intestinal protease activity, villi width and height in the CAP-100 group were significantly lower than those in the control group (p &lt; 0.05). The Kyoto Encyclopedia of Genes and Genomes analysis showed that the metabolic pathway in flesh was mainly enriched in the "lipid metabolic pathway", "amino acid metabolism", "endocrine system" and "carbohydrate metabolism". In conclusion, CAP could successfully replace 67.1-68.0% of dietary FM, while the complete substitution decreased the growth, damaged the intestinal morphology and down-regulated the lipid metabolites.

High dietary non-starch polysaccharides detrimental to nutrient digestibility, digestive enzyme activity, growth performance, and intestinal morphology in largemouth bass, Micropterus salmoides.

An 8-weeks feeding trial was carried out to evaluate the effects of different levels of dietary non-starch polysaccharide on the growth, apparent nutrient digestibility, intestinal development, and morphology of largemouth bass (Micropterus salmoides). Seven isoproteic and isolipidic experimental diets were formulated (crude protein 47.00%, crude lipid 12.50%), containing 0, 3, 6, 9, 12, 15, and 18% non-starch polysaccharides (NSPs) (named Control, NSPs3, NSPs6, NSPs9, NSPs12, NSPs15, and NSPs18), respectively. Dietary inclusion of NSPs below 9% showed no negative impacts on fish growth and feed utilization efficiency, whereas dietary NSPs inclusion level above 9% decreased weight gain rate, specific growth rate, protein efficiency, protein deposition rate, apparent digestibility of dry matter and protein, and were accompanied by a reduction in intestinal protease, Na+/K+-ATPase and alkaline phosphatase activity and an increase in feed intake and feed coefficient. The activity of lipase was significantly decreased when dietary inclusion of 15 and 18% NSPs. Moreover, the lipid deposition rate and the apparent digestibility of lipids were significantly decreased since dietary inclusion of 9% NSPs. Dietary inclusion of NSPs above 12% significantly up-regulated intestinal GLP-2 gene's expression, and was accompanied by significant changes in hindgut morphology, including increases in villus length and width, muscularis thickness and number of goblet cell, as well as a decrease in crypt depth. Additionally, dietary inclusion of NSPs above 3% significantly increased intestinal length index, and the viserosomatic index was significantly increased when dietary NSPs exceeded 15%. The linear regression analysis based on weight gain rate and feed coefficient showed that the appropriate dietary NSPs level of juvenile largemouth bass should not above 5.51%. In conclusion, high dietary NSPs adversely affects digestive enzyme activity and intestinal morphology, which in turn reduced the apparent digestibility of dietary nutrients and growth of juvenile largemouth bass.

167 Monomodular and Multi-Functional Processive Endocellulases for Improving Fibre Utilization and Reducing Environmental Footprint in Pigs

Abstract Global emission of the major greenhouse gases (GHG) from food animal production accounts for 15% of the total human-induced GHG with pork production being a significant contributor. Poor efficiency of dietary fibre utilization not only limits global pork production profit margins but also adversely affects dietary crude protein and amino acid utilization; and these collectively lead to excessive manure fibre and nitrogenous compounds, resulting in anaerobic biogenesis and emission of the major GHG from swine manure slurry storage. Two unique glycosyl hydrolase family-5 (GH5) processive endoglucanases, referred to as GH5-tCel5A1 and GH5-, have been reported. The GH5-tCel5A1 was originally identified via targeted genome sequencing of the extremely thermophilic Thermotoga maritima while the GH5-p4818Cel5_2A was screened from the porcine hindgut microbial metagenomic expression library. Both GH5-tCel5A1 and GH5-p4818_2A have been characterized to be monomodular, act as processive β-1,4-endoglucanases and have multi-functionality towards hydrolyzing dietary natural crystalline cellulose and pre-treated cellulosic substrates as well as several hemicelluloses including β-glucans, xylan, xylogulcans, mannans, galactomannans and glucomannans. We have recently overexpressed both GH5-tCel5A1 and GH5-p4818Cel5_2A in the ClearColiBL21(DE3) cell and investigated the susceptibility of both these cellulases to auto-oxidation by air-borne O2. Furthermore, we have also examined these endoglucanases’ stability under the mimicked in vitro porcine gastric and the small intestinal pH and proteases’ conditions. Further pig nutrition and growth performance studies are being conducted to investigate the in vivo efficacy of these two processive endoglucanases as newly emerging potential exogenous fibre enzymes.

Effects of a commercial feed additive (Sanacore®GM) on water quality, productive performance and blood profile of gilthead seabream (Sparus aurata)

The present study evaluated the effects of the inclusion of commercial feed additive (Sanacore® GM; SAN) in diets on growth and productive performance, digestive enzymes, intestinal microbiota, intestinal histomorphometry and blood profile of gilthead seabream (Sparus aurata). For this purpose, four diets containing 0.0% (the control), 0.1%, 0.2% and 0.4% SAN were formulated and offered to triplicate groups of fish (20–23 g) reared in cages (30 × 10 × 3 m) for ten weeks. Water quality parameters were regularly monitored during the experimental period. No marked variations (p > 0.05) were observed in water temperature, dissolved oxygen and pH values; meanwhile, substantially decreases in unionized ammonia and nitrite levels were specifically detected at the treatment of 0.4% SAN. Feeding the fish on SAN-supplemented diets confirmed significant modulations in fish growth and production, haemato-biochemical profile, intestinal microbiota and intestinal histomorphometry. Fish growth, production and feed utilization indices extensively multiplied alongside with the increase in dietary SAN levels achieving their optimum values at the treatment of 0.4% SAN in contrast to the control group (0% SAN). Intestinal α-amylase, lipase and proteases activities were notably greater in fish fed with SAN-supplemented diets in particular at the 0.4% SAN diet. Feeding gilthead seabream on SAN-containing diets markedly increased red blood cell counts, haemoglobin level and haematocrit per cent mainly at the diet of 0.4% SAN. Compared with the control group, highest values of serum total protein, albumin and globulin accompanied with lowest values of alanine and aspartate aminotransferase were significantly recorded in fish fed on the 0.4% SAN diet. Intestinal microbiota were additionally modulated due to SAN administration to gilthead seabream. However, marked reductions in counts of Vibrio sp. and Escherchia coli alongside with high Bacillus sp. counts were obtained in fish mid-intestine mainly at the 0.4% diet. Feeding gilthead seabream on SAN-enriched diets confirmed higher villi length/width, absorption area and crypt depth than the control group with optimal values at the 0.4% SAN diet. According to the outcomes observed in this study, the SAN administration to gilthead seabream, especially at the 0.4% level led to considerable enhancements in overall growth and production performance via improving the secretion of digestive enzymes and prompting the intestinal morphometry and beneficial microbiota.

Improving Growth, Digestive and Antioxidant Enzymes and Immune Response of Juvenile Grass Carp (Ctenopharyngodon idella) by Using Dietary Spirulina platensis

The present study was designed to investigate the effect of Spirulina platensis (SP) supplemented diets on the growth performance, digestive enzymes, hepatic antioxidants and innate immunity biomarkers in juvenile grass carp (Ctenopharyngodon idella). Two hundred and forty grass carp juveniles (average weight 4.81 ± 0.13 g) were divided into four treatment groups in triplicates (20 fish/replicate) and fed with diets containing 0, 1, 5, and 10% Spirulina for 90 days. A significant increase in growth (p &lt; 0.05) was observed in fish fed with diets having 1 and 5% Spirulina. Intestinal protease and lipase activities increased significantly (p &lt; 0.01) in fish fed with a diet having 5% Spirulina while intestinal amylase activity increased significantly (p &lt; 0.01) in fish fed with diets having 1, 5, and 10% Spirulina. Hepatic lipid peroxidation decreased significantly (p &lt; 0.05) in fish fed with a 1 and 5% Spirulina supplemented diet. The activity of catalase, glutathione-S-transferase, and glutathione levels increased significantly (p &lt; 0.05) in the livers of fish fed with 1% Spirulina supplemented diets while no significant difference (p &gt; 0.05) was observed for hepatic superoxide dismutase levels when compared to the control. Significant increases in the skin mucus protease (p &lt; 0.05), antiprotease (p &lt; 0.01), lysozyme (p &lt; 0.001), and peroxidase (p &lt; 0.05) activities were observed in fish fed with 5% Spirulina-supplemented diets. Gene expression analysis of head kidney showed that fish fed with a 1% Spirulina diet had significantly (p &lt; 0.01) higher expression of tnf-α, il-8, and inf-γ. In conclusion, the present study suggested that the inclusion of 5% Spirulina platensis in the diet of grass carp has positive effects on growth, digestive enzymes, antioxidants, and innate immunity.

Taurine can improve intestinal function and integrity in juvenile Rhynchocypris lagowskii Dybowski fed high-dose glycinin

The present study evaluated the protective effect and the regulatory mechanism of taurine on growth inhibition and intestinal damage induced by glycinin in juvenile Rhynchocypris lagowskii Dybowski. The control diets had no glycinin and taurine, the glycinin diets contained only 80 g/kg glycinin, and the glycinin + taurine diets contained 80 g/kg glycinin+10 g/kg taurine. Juvenile Rhynchocypris lagowskii Dybowski (4.65 ± 0.03 g/tail) were respectively fed with these 3 diets for 8 weeks. The results showed that glycinin significantly decreased the final body weight, weight gain rate, specific growth rate, protein efficiency rate, feed efficiency rate and feeding rate of fish compared with the control group (P < 0.05). While taurine supplementation improved the growth performance and feed efficiency, but final body weight, weight gain rate, specific growth rate of the glycinin + taurine group were still significantly lower than the control group (P < 0.05). Compared with the glycinin group, taurine supplementation significantly increased whole-body and muscle crude protein content, and hepatopancreas and intestinal protease activities (P < 0.05). Distal intestinal villous dysplasia and mucosal damage, and increased intestinal mucosal permeability were observed in the glycinin group, while taurine supplementation alleviated these adverse effects. Usefully, taurine supplementation could also partially restore the impaired immune function and antioxidant capacity of fish fed glycinin diets. Compared with the glycinin group, taurine supplementation down-regulated pro-inflammatory cytokines TNF‐α and IL-1β mRNA levels, and up-regulated anti-inflammatory cytokines IL-10 and TGF-β mRNA levels. Furthermore, taurine partially reversed the reduction of antioxidant genes Nrf2、HO-1, CAT and GPx mRNA levels in distal intestine induced by glycinin. Concluded, 80 g/kg glycinin led to intestinal damage, digestive dysfunction and increased intestinal mucosal permeability in juvenile Rhynchocypris lagowskii Dybowski, and these adverse effects were ultimately manifested in growth inhibition. But taurine supplementation could partially mitigate the negative effects induced by glycinin.

Effects of sea urchin feces on behaviors, digestion ability, growth, and resistance of the sea cucumber Apostichopus japonicus

Improving the aquaculture production efficiency by appropriate diets is an essential approach to meeting the increasing market demand for sea cucumbers. The feces of sea urchins, which contains various enzymes and microorganisms, is a potentially cost-effective food for sea cucumbers. To assess the usability of the fecal diet, a five-week laboratory simulation is conducted to investigate behaviors, digestion ability, growth and resistance ability of the sea cucumber Apostichopus japonicus fed with fecal diet at water temperatures of 15°C and 5°C. In the present study, A. japonicus fed with fecal diet shows an obvious preference to fecal diet rather than prepared feed at water temperatures of both 15°C and 5°C, which suggests that the feces is an applicable diet for A. japonicus. Furthermore, small A. japonicus fed with feces (group F) shows significant advantages in intestinal community richness, community diversity and intestine protease activity to A. japonicus fed with prepared feed (group S) at 15°C. These results indicate that the fecal diet provides benefits to digestion ability of small A. japonicus at 15°C. Weight gaining rate is significantly higher in the A. japonicus fed with feces than that in A. japonicus that were fed with feed or not fed with food (group C), which suggests that the direct improvement of the production efficiency at 15°C. The advantages in intestinal bacteria, protease activity, and growth are consistently found in group F compared with group S at 5°C. In addition, the composition of intestinal bacteria indicates that sea cucumbers may inherit the intestinal bacteria of sea urchins through fecal consumption. This suggests that the fecal diet enhances the digestion ability and enzyme activity at low water temperature and thus improves the growth of sea cucumbers. Furthermore, sea cucumbers fed with sea urchin feces have the highest survival rate among the three groups in exposure to an acute salinity decrease at both 5 and 15°C, indicating a better resistance to low salinity. This provides a new insight into the geographical expansion to low-salinity areas in sea cucumber aquaculture. In conclusion, the present study suggests that sea urchin feces have a great potential for the application in improving the production efficiency of sea cucumber aquaculture.

Effects of Different Viscous Guar Gums on Growth, Apparent Nutrient Digestibility, Intestinal Development and Morphology in Juvenile Largemouth Bass, Micropterus salmoides.

An 8-week feeding trial was conducted to investigate the effects of different viscous guar gums on the growth performance, apparent nutrient digestibility, intestinal development and morphology of juvenile largemouth bass. Four isoproteic and isolipidic diets (crude protein 42.5%, crude lipid 13.7%) were formulated to contain 8% cellulose (Control group), 8% low viscous guar gum with 2,500 mPa s (Lvs-GG group), 8% medium viscous guar gum with 5,200 mPa s (Mvs-GG group) and 8% high viscous guar gum with 6,000 mPa s (Hvs-GG group), respectively. Each diet was fed to quadruplicate groups of 40 fish (6.00 ± 0.01 g) per repetition. Dietary guar gum inclusion significantly decreased the weight gain rate, specific growth rate, protein efficiency ratio, protein productive value and lipid deposition rate, and these parameters decreased considerably with increasing guar gum viscous and were lowest in the Hvs-GG group. Dietary guar gum inclusion significantly decreased the apparent digestibility of dry matter, crude protein and crude lipid, and these parameters decreased considerably with increasing guar gum viscous and were lowest in the Hvs-GG group. Intestinal protease, lipase and creatine kinase activities in the guar gum groups were significantly lower than those in the control group, and intestinal protease and lipase activities decreased considerably with increased guar gum viscous. Intestinal alkaline phosphatase activity in the Hvs-GG group and intestinal Na+/K+-ATPase activity in the Mvs-GG and Hvs-GG groups were significantly lower than those in the Lvs-GG and control groups. Serum high-density lipoprotein, total cholesterol and triglyceride concentrations and superoxide dismutase activity in the guar gum groups were significantly lower than those in the control group. Intestinal villus height and muscular thickness in the guar gum groups were considerably higher than those in the control group, whereas the goblet cell relative number in the Mvs-GG and Hvs-GG groups and the microvillus height in the Lvs-GG and Hvs-GG groups were significantly lower than those in the control group. The expression level of IGF-1 in the guar gum groups and the expression level of GLP-2 in the Mvs-GG and Hvs-GG groups were significantly higher than those in the control group. These results indicated that guar gum diets adversely affected intestinal morphology, decreased intestinal digestive and absorptive enzyme activities, and caused poor nutrient digestibility and growth performance in juvenile largemouth bass. Moreover, the adverse effects of guar gum are closely related to its viscous, and high viscous guar gum produces more extreme negative impacts on juvenile largemouth bass.

Enzymatic Chicken Pulp Promotes Appetite, Digestive Enzyme Activity, and Growth in Litopenaeus vannamei.

Enzymatic chicken pulp (ECP) is an animal protein source that has been proven to be of excellent nutritional content and good quality for the majority of aquatic organisms because of its quality protein, small peptides, palatability, vitamins, and minerals. An 8-week nutritional trial was conducted to assess the effects of an ECP-based diet on the growth performance, digestive enzyme activity, and gene mRNA expression of Pacific white shrimp (Litopenaeus vannamei). Fish soluble pulp (FSP) served as the control group while in the experimental groups, and ECPs with three protein contents were used to replace FSP in equal amounts, named ECP1, ECP2, and ECP3, respectively. No significant difference in weight gain rate, specific growth rate, survival rate, or feed conversion ratio was observed (p > 0.05) between the groups. Ash content in the Pacific shrimp’s whole body was significantly higher in the ECP1 and ECP3 groups compared to the other groups (p < 0.05). Intestinal amylase and protease activities were the highest in the ECP1 and ECP2 groups, respectively (p < 0.05). With respect to gene mRNA expression, neuropeptide Y, excitatory amino acid transporter, and fatty acid transport protein 4 were significantly high in the ECP1 group (p < 0.05). In conclusion, these three ECPs have their advantages to replace FSP in shrimp feed, but ECP1 is more effective if the effects of digestive enzyme activity, appetite, and expression of growth-related genes are considered.

Effects of exogenous xylanase supplementation in plant-based diets on growth performance, nutrient digestibility and digestive enzyme activities of juvenile mori (Cirrhinus mrigala)

The present experimental study was conducted to determine the effects of xylanase supplementation on growth performance, nutrient digestibility and digestive enzyme activities of mori, Cirrhinus mrigala. Mori with an initial body weight of 8.34 ± 0.23 g were fed seven experimental diets: a diet with high fish meal content (positive control, PC), a diet with high plant content (negative control, NC), and five other diets with increasing levels of xylanase (680, 1273, 1882, 2486 and 3091 U/kg). Each diet was fed to triplicate groups of 15 fish each for 60 days. After the trial termination, supplementation of xylanase significantly improved final weight (FW), weight gain (WG) and specific growth rate (SGR) compared to NC diet. The best feed conversion ratio (FCR) was observed at 1882 U/kg of xylanase supplementation level which was non-significant (P > 0.05) to PC diet (high fish meal content). Whole body proximate composition and apparent digestibility coefficient (ADC%) for dry matter (DM), crude protein (CP), crude fat (CF), crude ash (CA) and gross energy (GE) were significantly affected by xylanase supplementation. Activities of hepatopancreatic and intestinal amylase, protease and lipase showed an increasing trend with the increase in xylanase supplementation level, peaking at 1882 U/kg of xylanase (non-significant (P > 0.05) to PC diet) and declining thereafter (2486 U/kg-3091 U/kg). Quadratic regression analysis showed that 1948 U/kg of xylanase was the optimum value for supplementation of xylanase in plant-based diet. In conclusion, exogenous xylanase supplementation improved the growth performance, nutrient digestibility and digestive enzyme activities in juvenile mori fed plant-based diet.

Dietary Clostridium butyricum Improves Growth Performance and Resistance to Ammonia Stress in Yellow Catfish (Pelteobagrus fulvidraco)

The effects of dietary Clostridium butyricum (CB) on growth performance, intestinal morphology, tight junction proteins, and immune-related gene mRNA levels in Pelteobagrus fulvidraco were investigated. The fish were fed with diets containing 0 (control, CB0), 4.8 × 10 6 (CB1), 4.5 × 10 7 (CB2), 5.1 × 10 8 (CB3), and 3.6 × 10 9 (CB4) CFU/kg Clostridium butyricum for 56 days followed by a 72 h ammonia challenge. The results showed that significantly higher final weight, specific growth rate, body length, and intestinal weight were observed in fish fed with CB diets ( P &lt; 0.05 ). The fish fed with CB1, CB2, and CB3 diets had significantly higher intestinal length, propionic acid concentration, and alkaline phosphatase activity and significantly lower feed conversion ratio than those in CB0 ( P &lt; 0.05 ). Significantly higher concentrations of butyric acid and valeric acid and significantly lower malondialdehyde content were observed in CB4 than in CB0 ( P &lt; 0.05 ). Intestosomatic index, villus length, villus width, intestinal protease, Na+/K+-ATPase, and creatine kinase activities were significantly increased in CB2 or CB3 than in CB0 ( P &lt; 0.05 ). Fish in CB2 or CB3 had significantly lower content of interleukin 1β and interleukin 6 and relative expression of interleukin 1 (Il-1), interleukin 8 (Il-8), and nuclear transcription factor-κB (Nf-κb) compared to that in CB0 ( P &lt; 0.05 ). Dietary CB significantly decreased the relative expression of myosin light chain kinase (Mlck) (P &lt;0.05). Significantly higher relative expressions of claudin-1, zonula occludens protein-1, and occludin were observed in CB2, CB3, and CB4 compared to CB0 ( P &lt; 0.05 ). Fish in CB0 had higher CMR than that in CB2, CB3, and CB4 under ammonia nitrogen stress for 48 and 72 h ( P &lt; 0.05 ). Dietary Clostridium butyricum improved growth performance and resistance to ammonia stress in yellow catfish by increasing intestinal short-chain fatty acid (SCFA) productions, upregulating genes encoding tight junction proteins, downregulating transcription of proinflammatory factors Il-1 and Il-8, and inhibiting the Mlck/Nf-κb signaling pathway.