Optimizing Aeromonas hydrophila vaccine efficacy in common carp: The role of free vs. microencapsulated Lactiplantibacillus plantarum and Lactobacillus delbrueckii bulgaricus.

Common carp (Cyprinus carpio) is an economically important fish worldwide, with many of its species bred for consumption. However, there are few reports on the comprehensive comparative analysis of the muscle nutritional quality and stress resistance characteristics of different common carp species. In this study, after 15 months of feeding in the same environment, the nutritional components, serum biochemical indices, liver antioxidant and intestinal digestive enzyme activities, and muscle growth-related gene expression were determined in Songpu mirror carp (SPM; Cyprinus carpio Songpumirror), Heilongjiang wild carp (HLJ; Cyprinus carpio haematopirus), cold-resistant strain of purse red carp (CPR; Cyprinus carpio ‘Red purse cold-resistant’), Songhe carp (SH; Cyprinus carpio ‘Songhe’), and Songpu carp (SP; Cyprinus carpio Songpu). Muscle nutrient composition showed that HLJ had a significantly lower crude fat content and higher docosahexaenoic acid (DHA) + eicosapentaenoic acid (EPA) proportion than the other four common carp species (p < 0.05). The contents of lysine (Lys) and aspargine (Asp) were significantly higher in the CPR than in other species (p < 0.05). Serum biochemical parameters showed that total protein (TP), total cholesterol (T-CHO), triglycerides (TG), and low-density lipoprotein (LDL) were significantly lower in SPM than in the other species (p < 0.05). The results of tissue enzyme activity showed that the activities of superoxide dismutase (SOD) and catalase from Micrococcus lysodeikticus (CAT) in the liver were significantly higher, while the activities of lipase (LPS), trypsin (TRS), and α-amylase (α-AMS) in the intestine were significantly the lower in HLJ than in the other species (p < 0.05). In addition, the relative expression levels of growth hormone (GH), growth hormone receptor (GHR), insulin-like growth factor 1 (IGF1), insulin-like growth factor receptor (IGF1R), and myoblast determination factor (MyoD) in SP and SH were significantly higher than those in the other species, while the relative expression of myostatin (MSTN) in HLJ was significantly higher (p < 0.05). Therefore, there were significant differences in muscle nutritional quality, serum biochemical indices, liver, and intestinal enzyme activities, and muscle growth potential among the five species of common carp. This study could provide a theoretical basis for the germplasm evaluation and variety improvement of common carp.

The effects of N‐carbamylglutamate (NCG) on growth, intestinal enzyme activities, immunological and antioxidant parameters were evaluated by a 56‐d feeding trial in Pelteobagrus fulvidraco fed diets containing NCG with 0, 250, 500, 1,000 or 2,000 mg/kg, respectively. The results showed that 250 mg/kg of NCG resulted in significantly higher weight gain, intestine fold height, intestine lipase, serum lysozyme, glutathione peroxidase and total antioxidant capacity than control (p < 0.05). However, higher intestine trypsin, arginase, arginine decarboxylase, ornithine decarboxylase, diamine oxidase activities and serum nitric oxide content were observed in 500 mg/kg NCG group compared to control or 2,000 mg/kg (p < 0.05). The survival rate, intestine muscular layer thickness, serum lysozyme and superoxide dismutase activities in 2,000 mg/kg NCG group were significantly lower than those in control and 250 mg/kg group, accompanied by the higher feed conversion ratio in the same group (p < 0.05). Together, dietary NCG level at 250 or 500 mg/kg improved growth, intestinal enzyme activities, immunological and antioxidant abilities, while high NCG level of 2,000 mg/kg had a negative effect. Quadratic regression analysis on weigh growth, diamine oxidase and lysozyme activities indicated that the recommended optimum dietary NCG level was 213.48–314.50 mg/kg of the dry diet.

An 8-week feeding experiment was conducted to determine the effect of dietary methionine supplementation on intestinal microflora and humoral immune of juvenile Jian carp (initial weight of 9.9 ± 0.0 g) reared in indoor flow-through and aerated aquaria. Eight amino acid test diets (350 g kg−1 crude protein, CP), using fish meal, soybean-condensed protein and gelatin as intact protein sources supplemented with crystalline amino acids, were formulated to contain graded levels of methionine (0.6–22.0%) at a constant dietary cystine level of 3 g kg−1. Each diet was randomly assigned to three aquaria. Growth performance and feed utilization were significantly influenced by the dietary methionine levels (P < 0.05). Maximum weight gain, feed intake occurred at 12 g kg−1 dietary methionine (P < 0.05). Methionine supplementation improved hepatopancreas and intestine weight, hepatosomatic and intestine index, intestinal γ-glutamyltransferase and creatine kinase activity, Lactobacillus count, Bacillus count, lysozyme activities, lectin potency, sim-immunoglobulin M content, addiment C3,C4 contents and serum total iron-binding capacity and declined Escherichia coli and Aeromonas counts. Quadratic regression analysis of weight gain against dietary methionine levels indicated that the optimal dietary methionine requirement for maximum growth of juvenile Jian carp is 12 g kg−1 of the dry diet in the presence of 3 g kg−1 cystine.

BACKGROUND: Occurrence of resistance against antibiotics and inadequate efficacy of some vaccines necessitates studies of natural immunostimulators in aquaculture. Shrimps shell derived from Chitosan can be used as immune stimulators in fish. OBJECTIVES: In this study, the effects of oral administration of chitosan, derived from shrimp shell, on some immune responses and disease resistance in Cyprinus carpio were studied. METHODS: Three hundred healthy fish weighing 42.4+8.1 g were divided into 4 equal groups: the first group (G10) was fed with food supplemented with 10 mg kg -1 chitosan, the second (G5) and third groups (G2.5) were fed with food supplemented with 5 mg kg -1 and 2.5 mg kg -1 , respectively. The control group was fed with basal feed (without chitosan). All groups were treated for 60 days. Blood samples were taken on 0, 20, 40, and 60 days post- experiment; In addition, some immunological indices, including serum lysozyme activity, serum bactericidal activity, Nitro Blue Tetrazolium (NBT) reduction activity, serum proteins, white blood cell count (WBC), and differentiated count were measured. At the end of the treatment, fish were challenged with live Aeromonas hydrophila and mortality rate was recorded for 14 days. RESULTS: Oral administration of chitosan (0.5 and 1%) significantly enhanced NBT reduction activity and resistance to A. hydrophila infection (p=0.012). Serum lysozyme and bactericidal activity, serum total protein and globulin, WBC and leukocytes ratio showed no significant change among the groups (p>0.05). CONCLUSIONS: This study indicates that oral administration of shrimp shell chitosan may have a positive effect on some immune parameters and resistance against bacterial infection in Cyprinus carpio.

The phagocytosis of neutrophils and serum lysozyme activity were investigated in carp experimentally infected with Pseudomonas alcaligenes and Aeromonas punctata. The total leucocyte numbers, relative leucocyte counts, nitroblue tetrazolium (NBT) reduction, NBT test rate, lysozyme activity and lysozyme index were examined on days 7, 14 and 21 after injection. On days 7 and 14 there was a significant increase in total leucocyte numbers and serum lysozyme activity, but a decrease in the NBT test rate and lysozyme index. NBT reduction was unchanged compared with the control group. On day 21 the total leucocyte numbers and lysozyme activity had declined and were less than control values, but there was a significant increase of the NBT test rate, NBT reduction and lysozyme index.

Background: Probiotic bacteria as an alternative to antibiotics are necessary for aquaculture due to the prevalence of recurrent diseases. Objectives: This study investigated the effect of a probiotic combination, including endogenous bacteria such as Lactobacillus plantarum, Lactobacillus bulgaricus, Lactobacillus acidophilus, and Lactobacillus rhamnosus, on the health status of common carp. Methods: The probiotic combination was added to carp basal diets in three treatments, 1 to 3, each containing 106 CFU g-1, 107 CFU g-1, and 108 CFU g-1 probiotic combination, respectively. A fourth group (group 4) was used as a control and received no probiotic treatment for six months. After 3 months, the fish’s growth performance, biochemical indices, and innate immune responses were evaluated. Results: The growth indices, such as the daily growth rate, specific growth rate, condition conversion factor, condition factor, and protein efficiency, significantly improved in treatments 2 and 3 compared to the other groups (P &lt; 0.05). Investigation of complement, lysozyme, and serum bactericidal activity showed that probiotic treatments positively affected the immune system of common carp, with treatments 2 and 3 demonstrating a greater effect (P &lt; 0.05). Biochemical factors were also investigated, showing decreased cholesterol and triglyceride levels in the probiotic treatment group. Moreover, the probiotic treatments resulted in a significant reduction in the activity of liver enzymes AST and ALP compared to the control group (P &lt; 0.05). Conclusions: A probiotic combination with concentrations of 5 × 107 CFU g-1 and 5 × 108 CFU g-1 of food can positively impact common carp’s growth performance, immune system, and biochemical parameters. As no significant difference was seen between the two concentrations, the lower one is recommended.

Clostridium butyricum improves immune responses and remodels the intestinal microbiota of common carp (Cyprinus carpio L.)

Improvement of 3D printing age-friendly brown rice food on rough texture, swallowability, and in vitro digestibility using fermentation properties of different probiotics

Synbiotic effects of β-glucan, mannan oligosaccharide and Lactobacillus casei on growth performance, intestine enzymes activities, immune-hematological parameters and immune-related gene expression in common carp, Cyprinus carpio: An experimental infection with Aeromonas hydrophila

Recently, there has been increasing attention regarding the potential application of biodegradable nanoparticles (NPs) as adjuvants in injectable vaccines. In this study, encapsulated antigens of Aeromonas hydrophila in alginate/chitosan polymers were used as injectable vaccines in common carp. Formalin‐killed bacteria and extracellular products (ECPs) of A. hydrophila were encapsulated via emulsification method. In total, 480 juvenile common carp (32.1 ± 3.64 g, mean ± SD) were randomly divided into eight triplicate groups (20 fish for each replicate). Groups 1–3 were injected with bacterin, ECP, and bacterin + ECP, respectively. Groups 4–6 were injected with encapsulated bacterin, encapsulated ECP, and encapsulated bacterin + ECP, respectively. Groups 7 and 8 as controls were injected with alginate/chitosan and phosphate‐buffered saline (PBS), respectively. Blood samples were collected on days 0, 30, and 60 of the experiment. Immunological parameters, including antibody titer against A. hydrophila, serum lysozyme, bactericidal and complement activities, and nitro blue tetrazolium (NBT) reduction, as well as serum globulin and protein levels, were compared among the groups. Hematological parameters (red blood cell [RBC], white blood cell [WBC], hemoglobin [Hb], and hematocrit (Hct) were evaluated too. Remained fish were challenged with severe A. hydrophila, and mortality was recorded for 10 days. Results showed that anti‐A. hydrophila antibody titer, survival rate following bacterial challenge, NBT reduction, serum lysozyme activity, globulin, and protein levels were significantly higher in encapsulated bacterin and encapsulated bacterin + ECP groups compared with the control group (p &lt; 0.05). Serum bactericidal and complement activity showed no significant change in different groups of sampling points. Then, it can be concluded that the encapsulation of bacterin and bacterin + ECP of A. hydrophila using chitosan/alginate NPs enhanced the protection and immune responses in common carp and this method is a promising candidate for a vaccine adjuvant in fish.

Fermented soya bean meal (FSBM) is believed to have improved nutritional qualities compared with soya bean meal (SBM) and is also cheaper than soya protein concentration (SPC) and fish meal (FM). Therefore, the present study was conducted to compare the effects of FSBM replacing SBM, SPC and FM in diets on growth performance, serum biochemistry profile, short-chain fatty acid concentrations in digesta, intestinal mucosal enzyme activities, intestinal proinflammatory cytokine concentrations and morphology in weaned piglets. One hundred and twenty 28-day-old piglets (Duroc×Landrace×Yorkshire, body weight: 6.73±1.14kg) were randomly allocated to four treatment diets (six replicate pens with five piglets per pen) containing SBM, SPC, FM or FSBM as the protein source, respectively. Dietary FSBM supplementation improved average daily gain (p<0.05), gain to feed ratio (p<0.05), and digestibility of dry matter, gross energy, crude protein and organic matter (p<0.05) in pigs compared with those fed SBM during 0-14days and reduced diarrhoea rate (p<0.05) compared with those fed SBM and FM during 0-14days. Moreover, pigs fed FBSM had greater IgA and IgM contents and antioxidase activities than those provided SBM and SPC on day 14. In addition, the butyrate concentration in the cecum of pigs fed FSBM was greater than those fed the other diets (p<0.05), and the trypsin activity in duodenum and jejunum of pigs provided FSBM was greater than those fed SBM (p<0.05). Moreover, higher villus height (p<0.05) and villus height to crypt depth ratio (p<0.05) and lower crypt depth (p<0.05) in the duodenum of pigs fed FSBM were observed, and pigs fed FSBM had a lower (p<0.05) TNF-α concentration in jejunum compared with those fed SBM. In conclusion, dietary FSBM supplementation to replace SBM, SPC and FM could improve piglets' growth performance, intestinal health and immune function.

Background: The application of probiotics to aquaculture is rather new. Probiotics affect the intestinal microbial flora of fish and subsequently modulate its immune response and growth performance. OBJECTIVES: This study was conducted to evaluate the effect of food supplementation with L.plantarum and L.bulgaricus on growth performance and gut microbiota of Cyprinus carpio. METHODS: For this purpose, 480 juveniles of C. carpio (40.2 ±6.3 gr Mean ±SD) were randomly divided into three equal groups (each group in three replicates) and fed with diet containing 5×107 cfu g_1 of Lactobacillus plantarum (group A), Lactobacillus bulgaricus (group B) and control diet (group C) for 60 days. To evaluate the persistent presence of the bacteria and their effects on the microbiota of the digestive system, remained fish of each group were fed with free probiotic diet from day 60 to 75. RESULTS: Results showed that most growth indices of probiotic treated groups were increased compared to control group in all sampling points. Although FCR decreased significantly in Groups A (2.9±0.43) and B (2.75±0.37) compared to control (3.88±0.52), SGR, WGP and DWG increased only in Group B compared to control group (P 0.05). Intestinal lactobacillus ratio at days 30 and 60 was significantly higher than the control group (P<0.05). Group B showed the highest lactobacillus rate among the groups at day 30. Total intestinal bacteria count on day 30 and 60 were significantly higher in probiotic-treated fish compared to the control group (P<0.05). CONCLUSIONS: It can be concluded that L.bulgaricus can promote growth indices and intestinal Lactic acid bacterial proportion in common carp. Then it can be a proper candidate for a probiotic in common carp after more trials in farm scale.

Nucleotides from yeast RNA were evaluated for their ability to enhance the non‐specific immune responses in carp, Cyprinus carpio. Oral administration of nucleotides to fish daily for 3 days resulted in enhanced responses of phagocytic and nitroblue tetrazolium (NBT) activities in kidney phagocytic cells. This activation of kidney cells was observed for at least 10 days post‐treatment. The serum complement and lysozyme activities also increased in fish treated with nucleotides. Furthermore, the number of Aeromonas hydrophila in nucleotide‐treated fish significantly decreased in the blood, kidney and liver after intraperitoneal injection. Thus yeast nucleotides appear to enhance non‐specific immune responses in fish.

The present study aimed to investigate the effects of dietary supplementation of Lactobacillus delbrueckii (L. delbrueckii) on growth performance, body composition, intestinal enzyme activities and gene expression of Cyprinus carpio Huanghe var. Fish (mean 1.05 ± 0.03 g in triplicate) were conducted for 8 weeks with five treatments in triplicate. The fish in the control were fed a basal diet, and those in D2, D3, D4 and D5 were fed basal diet containing L. delbrueckii at 1 × 10 CFU/g, 1 × 10 CFU/g, 1 × 10 CFU/g and 1 × 10 CFU/g. The growth performance and the hepatic IGF‐I expression of fish increased significantly as L. delbrueckii level increased from 0 to 1 × 10 CFU/g, but decreased significantly with further increasing L. delbrueckii levels. The whole body protein, lipid contents, intestinal digestive and absorptive enzyme activities were significantly affected by dietary L. delbrueckii levels with the highest values observed in fish fed diet containing 1 × 10⁶ CFU/g L. delbrueckii. The carbohydrate content showed the opposite trend. The results suggest that supplementation of L. delbrueckii as probiotic in the diet at approximately 1 × 10 CFU/g can improve growth performance, intestinal enzyme activities and the growth‐related gene expression in Cyprinus carpio Huanghe var.

This study investigated the effects of valine on growth, intestinal enzyme activities and microflora in juvenile Jian carp (Cyprinus carpio var. Jian). A total of 1200 fish with an average initial weight of 9.67 ± 0.03 g were fed diets containing 5.3 (unsupplemented control), 8.7, 11.8, 14.9, 18.7 and 20.1 g valine kg−1 diet for 60 days. Results indicated that the specific growth rate, feed efficiency, body protein and lipid content of fish were significantly improved by the dietary valine (P < 0.05). The hepatopancreas weight and activities of trypsin, amylase, lipase, chymotrypsin, glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) took the similar trends. Similarly, the optimum levels of dietary valine induced increases in the intestinal length, weight, folds height and activities of alkaline phosphatase, gamma-glutamyl transpeptidase and creatine kinase. In contrast, the trends of muscle GOT activity and plasma ammonia content were opposite. Intestinal Aeromonas, Escherichia coli, Lactobacillus and Bacillus were changed by dietary valine supplementations. The dietary valine requirement for Jian carp (9.67–76.4 g) based on SGR was 13.7 g valine kg−1 diet (4.0 g valine 100 g−1 CP). Together, these results indicated that valine improved fish growth, digestive and absorptive ability.