Effects of supplementing coated methionine in a high plant-protein diet on growth, antioxidant capacity, digestive enzymes activity and expression of TOR signaling pathway associated genes in gibel carp, Carassius auratus gibelio.

This study explored the impacts of supplementation of different levels of coated methionine (Met) in a high-plant protein diet on growth, blood biochemistry, antioxidant capacity, digestive enzymes activity and expression of genes related to TOR signaling pathway in gibel carp (Carassius auratus gibeilo). A high-plant protein diet was formulated and used as a basal diet and supplemented with five different levels of coated Met at 0.15, 0.30, 0.45, 0.60 and 0.75%, corresponding to final analyzed Met levels of 0.34, 0.49, 0.64, 0.76, 0.92 and 1.06%. Three replicate groups of fish (initial mean weight, 11.37 ± 0.02 g) (20 fish per replicate) were fed the test diets over a 10-week feeding period. The results indicated that with the increase of coated Met level, the final weight, weight gain (WG) and specific growth rate initially boosted and then suppressed, peaking at 0.76% Met level (P< 0.05). Increasing dietary Met level led to significantly increased muscle crude protein content (P< 0.05) and reduced serum alanine aminotransferase activity (P< 0.05). Using appropriate dietary Met level led to reduced malondialdehyde concentration in hepatopancreas (P< 0.05), improved superoxide dismutase activity (P< 0.05), and enhanced intestinal amylase and protease activities (P< 0.05). The expression levels of genes associated with muscle protein synthesis such as insulin-like growth factor-1, protein kinase B, target of rapamycin and eukaryotic initiation factor 4E binding protein-1 mRNA were significantly regulated, peaking at Met level of 0.76% (P< 0.05). In conclusion, supplementing optimal level of coated Met improved on fish growth, antioxidant capacity, and the expression of TOR pathway related genes in muscle. The optimal dietary Met level was determined to be 0.71% of the diet based on quadratic regression analysis of WG.

Roles of ß-catenin in innate immune process and regulating intestinal flora in Qi river crucian carp (Carassius auratus).

In mammals, ß-catenin participates in innate immune process through interaction with NF-κB signaling pathway. However, its role in teleost immune processes remains largely unknown. We aimed to clarify the function of ß-catenin in the natural defense mechanism of Qi river crucian carp (Carassius auratus). ß-catenin exhibited a ubiquitous expression pattern in adult fish, as indicated by real-time PCR analysis. Following lipopolysaccharide (LPS), Polyinosinic-polycytidylic acid (polyI: C) and Aeromonas hydrophila (A. hydrophila) challenges, ß-catenin increased in gill, intestine, liver and kidney, indicating that ß-catenin likely plays a pivotal role in the immune response against pathogen infiltration. Inhibition of the ß-catenin pathway using FH535, an inhibitor of Wnt/ß-catenin pathway, resulting in pathological damage of the gill, intestine, liver and kidney, significant decrease of innate immune factors (C3, defb3, LYZ-C, INF-γ), upregulation of inflammatory factors (NF-κB, TNF-α, IL-1, IL-8), and downregulation of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) activities, increase of Malondialdehyde (MDA) content. Following A. hydrophila invasion, the mortality rate in the FH535 treatment group exceeded that of the control group. In addition, the diversity of intestinal microflora decreased and the community structure was uneven after FH535 treatment. In summary, our findings strongly suggest that ß-catenin plays a vital role in combating pathogen invasion and regulating intestinal flora in Qi river crucian carp.

Ginger polysaccharide alleviates the effects of acute exposure to carbonate in crucian carp (Carassius auratus) by regulating immunity, intestinal microbiota, and intestinal metabolism.

Alkaline stress poses a significant challenge to the healthy growth of fish. Ginger polysaccharide (GP) is one of the main active substances in ginger and has pharmacological effects, such as anti-oxidation and immune regulation. However, the physiological regulatory mechanism of GP addition to diet on alkalinity stress in crucian carp remains unclear. This study aimed to investigate the potential protective effects of dietary GP on antioxidant capacity, gene expression levels, intestinal microbiome, and metabolomics of crucian carp exposed to carbonate (NaHCO3). The CK group (no GP supplementation) and COG group (NaHCO3 stress and no GP supplementation) were set up. The GPCS group (NaHCO3 stress and 0.4% GP supplementation) was stressed for seven days. Based on these data, GP significantly increased the activities of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX), acid phosphatase (ACP), and alkaline phosphatase (AKP) in carp under alkalinity stress (p < 0.05) and decreased the activity of malon dialdehyde (MDA) (p < 0.05). GP restored the activity of GSH-PX, ACP, and AKP to CK levels. The expression levels of tumor necrosis factor ß (TGF-ß), tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), and interleukin 8 (IL-8) genes were decreased, and the expression levels of determination factor kappa-B (NF-κB) and interleukin 10 (IL-10) genes were increased (p < 0.05). Based on 16 S rRNA high-throughput sequencing, GP improved the changes in the intestinal microbial diversity and structural composition of crucian carp caused by NaHCO3 exposure. In particular, GP increased the relative abundance of Proteobacteria and Bacteroidetes and decreased the relative abundance of Actinobacteria. The metabolic response of GP to NaHCO3 exposed crucian carp guts was studied using LC/MS. Compared to the COG group, the GPCS group had 64 different metabolites and enriched 10 metabolic pathways, including lipid metabolism, nucleotide metabolism, and carbohydrate metabolism. The addition of GP to feed can promote galactose metabolism and provide an energy supply to crucian carp, thus alleviating the damage induced by alkalinity stress. In conclusion, GP can mitigate the effects of NaHCO3 alkalinity stress by regulating immune function, intestinal flora, and intestinal metabolism in crucian carp. These findings provide a novel idea for studying the mechanism of salt-alkali tolerance in crucian carp by adding GP to feed.

Comparative Effect of Chemical and Green Zinc Nanoparticles on the Growth, Hematology, Serum Biochemical, Antioxidant Parameters, and Immunity in Serum and Mucus of Goldfish, Carassius auratus (Linnaeus, 1758).

Recently, nano feed supplement research has great attention to improving healthy aquatic production and improving the aquatic environment. With the aims of the present study, chemical and green synthesized nanoparticles are characterized by various instrumentation analyses, namely UV-Vis spectrophotometry (UV-Vis), X-ray diffraction (XRD), Fourier transform infra-red (FTIR) spectroscopy, and scanning electron microscope (SEM). After characterization analysis of these nanoparticles utilized in aquatic animals, the composition ratio is as follows: controls (without ZnO-NPs (0 mg/L)), T1 (0.9 mg/L ZnO-NPs), T2 (1.9 mg/L ZnO-NPs), T3 (0.9 mg/L GZnO-NPs), T4 (1.9 mg/L GZnO-NPs). SEM investigation report demonstrates that the structure of the surface of green synthesized zinc oxide nanoparticles (GZnO-NPs) was conical shape and the size ranging was from 60 to 70 nm. Concerning hematological parameters, the quantity of hemoglobin increased in different doses of green zinc nanoparticles, but the values of MCV and MCH decreased somewhat. However, this decrease was the highest in the T2 group. Total protein and albumin decreased in T2 and triglyceride, cholesterol, glucose, cortisol, creatinine, and urea increased, while in T3 and T4 groups, changes in biochemical parameters were evaluated as positive. Mucosal and serum immunological parameters in the T2 group showed a significant decrease compared to other groups. In zinc nanoparticles, with increasing dose, oxidative damage is aggravated, so in the T2 group, a decrease in antioxidant enzymes and an increase in MDA were seen compared to other groups. In this regard, the concentration of liver enzymes AST and ALT increased in the T2 group compared with control and other groups. This can confirm liver damage in this dose compared with control and other groups. This research work suggests that green synthesized form of zinc nanoparticles in higher doses have less toxic effects in comparison to the chemical form of zinc nanoparticles and can act as suitable nutrient supplements in aquatic animals.

Goldfish or aquatic turtle? Impact of two animal assisted interventions on children's pain, anxiety, and fear during IV catheterization: A randomized controlled trial.

PURPOSE: This study aimed to determine the effect of goldfish and aquatic turtle nursing interventions on children's pain, anxiety, and fear during IV catheter insertion. DESIGN AND METHODS: The randomized controlled trial included 98 children (control group = 32, goldfish group = 33, aquatic turtle group = 33) aged 5-6 years, attending outpatient clinic for blood sampling and catheterisation procedures. Pain was measured using the Wong Baker Facial Pain Scale, anxiety was measured using the Child Anxiety Scale-State, and child fear was measured using the Child Fear Scale, before, during, and after catheterization. RESULTS: When analyzing the pain, anxiety, and fear findings, no statistical difference was found between the scores of the mother, child, and researcher before the procedure. During the catheterization procedure, the pain, anxiety, and fear scores of the children in the fish and turtle groups increased statistically less than those in the control group. When analyzing the post-procedure findings, the pain, anxiety, and fear scores of the control group were similar to those before the procedure. In the fish and turtle groups, anxiety and fear scores were statistically the lowest (p < 0.05). CONCLUSION: It can be concluded that animal-assisted practices reduce anxiety and fear during and after the catheterization procedures while diverting attention from pain. PRACTICE IMPLICATIONS: Goldfish or aquatic turtle intervention improve children's attitudes toward pain, anxiety, and fear during IV catheter insertion. Pediatric nurses could consider incorporating, such as the animal assisted intervention, into their clinical practice to reduce the pain, anxiety, and fear experienced by children during medical/nursing procedures.

α-lipoic acid ameliorates hepatotoxicity induced by chronic ammonia toxicity in crucian carp (Carassius auratus gibelio) by alleviating oxidative stress, inflammation and inhibiting ERS pathway.

High environment ammonia (HEA) poses a deadly threat to aquatic animals and indirectly impacts human healthy life, while nutritional regulation can alleviate chronic ammonia toxicity. α-lipoic acid exhibits antioxidative effects in both aqueous and lipid environments, mitigating cellular and tissue damage caused by oxidative stress by aiding in the neutralization of free radicals (reactive oxygen species). Hence, investigating its potential as an effective antioxidant and its protective mechanisms against chronic ammonia stress in crucian carp is highly valuable. Experimental fish (initial weight 20.47 ± 1.68 g) were fed diets supplemented with or without 0.1% α-lipoic acid followed by a chronic ammonia exposure (10 mg/L) for 42 days. The results revealed that chronic ammonia stress affected growth (weight gain rate, specific growth rate, and feed conversion rate), leading to oxidative stress (decreased the activities of antioxidant enzymes catalase, superoxide dismutase, glutathione peroxidase; decreased total antioxidant capacity), increased lipid peroxidation (accumulation of malondialdehyde), immune suppression (decreased contents of nonspecific immune enzymes AKP and ACP, 50% hemolytic complement, and decrease of immunoglobulin M), impaired ammonia metabolism (reduced contents of Glu, GS, GSH, and Gln), imbalance of expression of induced antioxidant-related genes (downregulation of Cu/Zu SOD, CAT, Nrf2, and HO-1; upregulation of GST and Keap1), induction of pro-apoptotic molecules (transcription of BAX, Caspase3, and Caspase9), downregulation of anti-apoptotic gene Bcl-2 expression, and induction of endoplasmic reticulum stress (upregulation of IRE1, PERK, and ATF6 expression). The results suggested that the supplementation of α-lipoic acid could effectively induce humoral immunity, alleviate oxidative stress injury and endoplasmic reticulum stress, and ultimately alleviate liver injury induced by ammonia poisoning (50-60% reduction). This provides theoretical basis for revealing the toxicity of long-term ammonia stress and provides new insights into the anti-ammonia toxicity mechanism of α-lipoic acid.

Roundup® disrupts tissue architecture, attenuates Na+/K+-ATPase expression, and induces protein oxidation/nitration, cellular apoptosis, and antioxidant enzyme expressions in the gills of goldfish, Carassius auratus.

Extensive agricultural activities to feed the growing population are one major driving force behind aquatic pollution. Different types of pesticides are used in farmlands to increase crop production and wash up into water bodies. Glyphosate-based herbicide Roundup® is one of the most used pesticides in the United States; however, its effects on teleost species are still poorly understood. This study focused on the effects of environmentally relevant concentrations of Roundup exposure (low- and high-dose: 0.5 and 5 µg/L for 2-week) on Na+/K+-ATPase (NKA, a biomarker for sodium­potassium ion pump efficacy), cytochrome P450-1A (CYP1A, a monooxygenase enzyme), 2,4-dinitrophenyl protein (DNP, a biomarker for protein oxidation), 3-nitrotyrosine protein (NTP, a biomarker for protein nitration), superoxidase dismutase (SOD, an antioxidant enzyme), catalase (CAT, an antioxidant enzyme) expressions, and cellular apoptosis in the gills of goldfish. Histopathological and in situ TUNEL analyses showed widespread tissue damage, including lamellar fusion, loss of gill architecture, club shape of primary lamellae, mucous formation, and distortion in the epithelium layer, as well as apoptotic nuclei in gills. Immunohistochemical and qRT-PCR analyses provided insights into the expressions of molecular indicators in gills. Fish exposed to Roundup exhibited a significant (P < 0.05) downregulation of NKA expression in gills. Additionally, we observed upregulation of CYP1A, DNP, NTP, SOD, and CAT expressions in the gills of goldfish. Overall, our results suggest that exposure to Roundup causes disruption of gill architecture, induces protein oxidation/nitration and cellular apoptosis, and alters prooxidant-antioxidant homeostasis in tissues, which may lead to reduced fitness and survivability of teleost species.

Oral administration of wild plant-derived minerals and red ginseng ameliorates insulin resistance in fish through different pathways.

Many kinds of fish are characterized by a limited efficiency to use carbohydrates. For this reason, raw fish and mixed feed containing a lot of fish meal have been used as feed for fish farming. However, continuing to use high-protein diets not only increases the cost of fish farming, but may also fuel animal protein shortages. Furthermore, carbohydrates are added to improve the texture of the feed and act as a binding agent and are usually contained at 20% in the feed. It makes sense, therefore, to find ways to make good use of carbohydrates rather than wasting them. The physiological mechanisms of glucose intolerance in fish are not yet well understood. Therefore, we investigated the glucose utilization of fish, omnivorous goldfish Carassius auratus and carnivorous rainbow trout Oncorhynchus mykiss. Furthermore, the effects of oral administration of wild plant-derived minerals and red ginseng on the glucose utilization in these fish muscle cells were investigated. As a result, we found the following. (1) An extremely high insulin resistance in fish muscle and the symptom was more pronounced in carnivorous rainbow trout. (2) Administration of wild plant-derived minerals promotes the translocation of the insulin-responsive glucose transporter GLUT4 to the cell surface of white muscle via activation of the PI3 kinase axis, whereas administration of red ginseng not only promotes GLUT4 transfer and translocation to the cell surface of white muscle via AMPK activation as well as promoting glucose uptake into muscle cells via a pathway separate from the insulin signaling system. (3) In fish, at least goldfish and rainbow trout, both PI3K/Akt and AMPK signaling cascades exist to promote glucose uptake into muscle cells, as in mammals.

The Comprehensive Effects of Carassius auratus Complex Formula against Lipid Accumulation, Hepatocarcinogenesis, and COVID-19 Pathogenesis via Stabilized G-Quadruplex and Reduced Cell Senescence.

Carassius auratus complex formula (CACF) is a traditional Chinese medicine known for its antidiabetic effects. Hepatocellular carcinoma (HCC) is a major cause of cancer-related deaths worldwide, and there are currently no effective therapies for advanced HCC. This study explores the comprehensive effects and possible mechanisms of CACF on HCC. The results show that CACF reduces the viability of hepatoma cells in vitro, while benefiting normal hepatocytes. In addition, CACF inhibits hepatoma cell growth in a zebrafish xenotransplantation model and decreases lipid accumulation, represses inflammation and cell proliferation markers in fatty acid translocase (CD36) transgenic zebrafish, and inhibits the expression of cell proliferation and ß-catenin downstream targets in telomerase (tert) transgenic zebrafish models. Ingenuity Pathway Analysis reveals that CACF exerts multiple functions, including reduction of inflammation and inhibition of lipid transporter and PPAR signaling pathway. Surprisingly, CACF also regulates the expression of genes and reduces coronavirus infection and pathogenesis in a zebrafish model. CACF treatment is validated to regulate the expression of genes for anti-coronavirus activity. Mechanistically, CACF stabilizes G-quadruplex and reduces cell senescence associated ß-galactosidase activity. In summary, CACF may be a promising therapeutic agent with multiple functions including anticancer, anti-inflammation, and anti-microorganisms in a zebrafish model.

The role of visfatin/ NAMPT in the regulation of feeding in goldfish (Carassius auratus).

The protein NAMPT (nicotinamide phosphoribosyltransferase, encoded by the NAPMT gene) is present in two forms. The intracellular form of NAMPT (iNAMPT) is the rate-limiting enzyme in a major nicotinamide adenine dinucleotide (NAD) biosynthetic pathway and regulates cellular metabolism. NAMPT is also secreted by cells in the extracellular milieu, and referred to as extracellular NAMPT (eNAMPT or visfatin). In mammals, visfatin has been linked to various metabolic disorders. However, the role of visfatin in regulating energy homeostasis in fish is not known. In this study, we assessed the effects of nutritional status on NAMPT mRNA expression and the effects of visfatin peripheral injections on food intake and the expression of appetite regulators in goldfish. Our results show that NAMPT is widely expressed in peripheral tissues and brain. Fasting induced increases in NAMPT expression in liver but had no effect on either brain or intestine NAMPT expression levels. Intraperitoneal injections of visfatin (400 ng/g) induced an increase in food intake and in expression levels of hepatic leptin and sirtuin1. Visfatin injections decreased intestine CCK and PYY, and telencephalon (but not hypothalamic) orexin and NPY expression levels. Visfatin did not affect plasma glucose levels, intestine ghrelin or brain CART, POMC and AgRP expressions. These data suggest that visfatin/NAMPT might be involved in the regulation of feeding and energy homeostasis in goldfish.

Dietary supplementation of probiotics fermented Chinese herbal medicine Sanguisorba officinalis cultures enhanced immune response and disease resistance of crucian carp (Carassius auratus) against Aeromonas hydrophila.

Aeromonas hydrophila, a Gram-negative bacterium, is one of the major pathogens causing bacterial sepsis in aquatic animals due to drug resistance and pathogenicity, which could cause high mortality and serious economic losses to the aquaculture. Sanguisorba officinalis (called DiYu in Chinese, DY) is well known as herbal medicine, which could inhibit the growth of pathogenic bacteria, hemostasis and regulate the immune response. Moreover, the active ingredients in DY could remarkably reduce drug resistance. In this study, we investigated the effects of probiotic fermentation cultures on A. hydrophila through in vitro and in vivo experiments. Three lactic acid bacteria, including Lactobacillus rhamnosus (LGG), Lactobacillus casei (LC) and Lactobacillus plantarum (LP), were selected to ferment the Chinese herbal medicine DY. The assays of antagonism showed that all three fermented cultures could influence the ability of A. hydrophila growth, among which L. rhamnosus fermented DY cultures appeared to be the strongest inhibitory effect. In addition, the biofilm determination revealed that L. rhamnosus fermented DY cultures could significantly inhibit the biofilm formation of A. hydrophila compared to the other groups. Furthermore, protease, lecithinase and urease activities were found in the three fermentation cultures. Three probiotics fermented DY cultures were orally administration with crucian carp to evaluate the growth performance, immunological parameters and pathogen resistance. The results showed that the three fermentation cultures could promote the growth performance of crucian carp, and the immunoglobulins, antioxidant-related enzymes and immune-related genes were significantly enhanced. Besides, the results showed that crucian carp received L. rhamnosus (60.87%), L. casei (56.09%) and L. plantarum (41.46%) fermented DY cultures had higher survival rates compared with the control group after infection with A. hydrophila. Meanwhile, the pathological tissue results revealed that the probiotic fermented cultures could largely improve the tissues damage caused by the pathogenic bacteria. In conclusion, this study proved that the fermentation cultures of three probiotics could effectively inhibit the growth of A. hydrophila, regulate the level of immune response and improve the survival rate against A. hydrophila in crucian carp. The present data suggest that probiotic fermented Sanguisorba officinalis act as a potential gut-targeted therapy regimens to protecting fish from pathogenic bacteria infection.

Individual and combined effects of the dietary Spirulina platensis and Bacillus licheniformis supplementation on growth performance, antioxidant capacity, innate immunity, relative gene expression and resistance of goldfish, Carassius auratus to Aeromonas hydrophila.

This study evaluated the individual and combined effects of the dietary Spirulina platensis (SP) and probiotic bacterium Bacillus licheniformis (BL) on the growth performance, immune responses, and disease resistance in goldfish (Carassius auratus). A total of 216 fish (3.39 ± 0.24 g) were randomly distributed in 12 tanks with 18 fish per tank (4 treatments with 3 replications) and fed with diets containing 0% S. platensis and B. licheniformis (T0), 108 CFU/g B. licheniformis (T1), 2.5% S. platensis (T2), and 108 CFU/g B. licheniformis + 2.5% S. platensis (T3(. There were no significant differences in growth parameters. The alternative complement pathway (ACH50) and lysozyme activity were significantly increased in T2 and T3 treatments. No marked differences were observed in total immunoglobulin and protease activity among treatments (P > 0.05). The relative expression of IGF-1 was not affected by experimental diets (P > 0.05). Ghrelin gene showed significantly higher mRNA levels in fish fed with SP and BL (P < 0.05). The relative expression of catalase (CAT), and glutathione reductase (GSR) significantly increased in fish fed with the SP and BL (P < 0.05). No marked difference in glutathione peroxidase (GPX) gene expression was seen between the treatments (P > 0.05). The mRNA levels of lysozyme, IL6, IL-1ß, TGF, and TNF2 transcription were higher in fish fed with SP and BL (P < 0.05). No notable difference was observed in TNF1 and IL10 gene expression between treatments (P > 0.05). Moreover, the result of the challenge test with A. hydrophila showed that goldfish fed with SP and BL had a lower mortality rate than the control. In conclusion, the supplementation of SP and BL can be used as feed additives to enhance disease resistance against A. hydrophila infection by stimulating the immune system in goldfish.

Boosted growth performance, immunity, antioxidant capacity and disease resistance of crucian carp (Carassius auratus) by single or in combination dietary Bacillus subtilis and xylo-oligosaccharides.

In this study, a total of 420 healthy crucian carp (9.77 ± 0.04 g) were randomly divided into CK, B·S, XOS and B·S + XOS group, and cultured for 8 weeks. Results showed that the dietary Bacillus subtilis (B. subtilis) and xylo-oligosaccharides (XOS) can significantly increased the final weight, weight gain, specific growth rate, feed efficiency, protein efficiency and survival rate of crucian carp. Dietary B. subtilis and XOS can significantly increased the activities of catalase, glutathione, superoxide dismutase and total antioxidant capacity, significantly decreased the contents of malondialdehyde, and significantly increased the activities of alkaline phosphatase, acid phosphatase, lysozyme and the contents of complement component 3,4 and immunoglobulin M in crucian carp serum. In addition, compared with CK group, the expression levels of TGF-ß and IL-10 in B·S, XOS and B·S + XOS group were significantly increased, and the expression levels of TNF-α, HSP90, IL-1ß, TLR4 and MyD88 were significantly decreased. Supplementation of B. subtilis and XOS can also improve the intestinal tissue morphology of crucian carp. After injection of 1 × 107 CFU/mL Aeromonas hydrophila (A. hydrophila), compared with CK group, the survival rates of the B·S group, the XOS group and the B·S + XOS group were increased by 13.98%, 10.56% and 30.74%, respectively. These results show that dietary B. subtilis and XOS can significantly improve the growth performance, antioxidant capacity, immunity and resistance to A. hydrophila of crucian carp, and the combined effect is better than that of single addition.

Enhancement of growth, antioxidative status, nonspecific immunity, and disease resistance in gibel carp (Carassius auratus) in response to dietary Flos populi extract.

This study investigated the effects of dietary Flos populi extract (FPE) on the growth, antioxidation capability, innate immune response, and disease resistance in gibel carp. A total of 480 fish were fed with five different diets containing 0, 0.5, 1.0, 1.5, or 2.0 g kg-1 FPE (designated as control, D0.5, D1.0, D1.5, or D2.0 groups) for 45 days. The fish were challenged with A. hydrophila after the feeding trial. Compared with the control, the feed efficiency (FE), weight gain (WG), final body weight (FBW), and specific growth rate (SGR) were significantly improved in groups D1.0 and D1.5. Dietary FPE significantly increased serum superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) activities, as well as glutathione (GSH) content. The contents of protein carbonyl (PCC) and malondialdehyde (MDA) in serum decreased significantly. Additionally, FPE supplementation in diets resulted in significant improvement in serum lysozyme (LZM) and myeloperoxidase (MPO) activities, as well as immunoglobulin M (IgM) and complement 3 (C3) concentrations. The hepatic antioxidant enzymes (CAT and SOD) activities increased, whereas content of MDA decreased in fish treated with dietary FPE than those of control both pre- and post-challenged. After 12 h-challenge, an obvious downregulation of hepatic Kelch-like-ECH-associated protein 1 (Keap1), splenic tumor necrosis factor-α (TNF-α), interleukin (IL)-8, IL-1ß, and toll-like receptor 2 (TLR2) mRNA levels was observed in fish treated with dietary FPE, whereas hepatic Nrf2 transcription level was upregulated compared to the control. Furthermore, compared to group D0.5, higher relative percent survival (RPS) was observed in gibel carp fed dietary 1.0-2.0 g/kg FPE. Our results reveal that FPE supplemented diet has a stimulatory effect on antioxidant capacity and nonspecific immune response, along with improved growth performance and enhanced resistance against A. hydrophila infection in juvenile gibel carp.

Ononin: A candidate anti-parasitic drug isolated from Spatholobi caulis against infections of Dactylogyrus intermedius (Monogenea).

Dactylogyrus is a common parasitic pathogen, which causes high mortality of fish when presents in large numbers, resulting in serious economic losses. Herbal medicines contain myriad of bioactive compounds is a valuable reserve for developing safe and effective anti-parasite drugs. Here, we conducted bioassay-guided fractionation to isolate and identify the anti-parasitic constituents from Spatholobi caulis. Among five extraction solvents (petroleum ether, chloroform, ethyl acetate, methanol and water), S. caulis methanolic extract had the highest parasiticide activity in Carassius auratus, and therefore subjected to further separation and purification using multiple chromatography methods. One compound exhibiting the strongest parasiticidal activity was obtained and identified as ononin by analyzing its spectral data (NMR and ESI-MS). The EC50 value of ononin against Dactylogyrus was 0.655 mg/L and showed 100% parasiticide activity with 3.0 mg/L. The 24, 48, 72, 96 h LC50 for goldfish were 4.691 (the 95% CI of 4.526-4.873) mg/L, 4.612 (4.441-4.800) mg/L, 4.472 (4.345-4.607) mg/L, 4.288 (4.155-4.428) mg/L, respectively. The present results discovered for the first time that ononin had potent parasiticidal activity and have the potential to be developed as new anti-parasitic drug for the control of Dactylogyrus.

Boesenbergia pandurata application in Goldfish ( Cyprinus carpio) Feed to Enhancing Fish Growth, Immunity System, and Resistance to Bacterial Infection.

Background: This study investigated how the inclusion of Boesenbergia pandurata extract (BPE) in goldfish feed affects fish growth, immunity, and resistance to infection by Aeromonas hydrophila and Pseudomonas fluorescens.   Methods: Four fish feeds, were prepared by adding BPE at the concentrations of 0 (control), 2, 4, and 6 g kg-1, respectively, and 120 goldfish (Cyprinus carpio; initial weight 5 g) were separated into 12 boxes and fed with specific pellets and examined thrice. The experiment lasted 12 weeks, beginning with the different feeds, fish growth was measured at Weeks 4 and 8 after the feeding period. Moreover, a challenge test with pathogen bacteria to assay disease resistance was administered at Week 8 after the feeding period, and the survival rate and relative percentage of survival were quantified at Week 12.   Results: At Week 8, the goldfish that were fed BPE-containing feeds were significantly heavier than the fish that received the control feed (pellet without BPE), and the highest weight gain, reaching 72.44 g, was obtained with Pellet 3; accordingly, the specific growth rate after BPE treatment (5.7%) was higher than that after control treatment. Conversely, the feed conversion ratio in the control group, 2.03, was higher than the ratios in the BPE groups, which were decreased to 0.55-0.90. Lastly, BPE treatment consistently enhanced the immunity parameters of goldfish (relative to control treatment) at weeks 4 and 8, and following BPE treatment, the rate of resistance against bacterial infection, 68.3%-77.0%, was higher than that after control treatment.   Conclusions: BPE addition in goldfish feed clearly produces a positive effect by enhancing fish growth, immunity, and resistance to infection by pathogenic bacteria, and 4 g kg-1 is the optimal BPE concentration in feed prepared for goldfish.

Appropriate leucine supplementation promotes glucose metabolism and enhances energy homeostasis in juvenile crucian carp (Carassius auratus gibelio var. CAS III).

In order to characterize the molecular mechanisms by which leucine regulates carbohydrate metabolism and energy homeostasis, juvenile crucian carps (Carassius auratus gibelio var. CAS III) fed with a high carbohydrate diet were supplemented with different levels of dietary leucine: 0% (Leu0), 0.4% (Leu4), 0.8% (Leu8), 1.2% (Leu12), 1.6% (Leu16), 2.0% (Leu20), and 5.0% (Leu50). After 8 weeks, RNA sequencing was performed on samples collected from the Leu0, Leu8, Leu12 and Leu50 groups. Differentially expressed genes were then detected and analyzed. The results showed a total of 91.6 Gb of clean bases were generated. Moreover, a total of 1131, 5254, and 1539 DEGs were detected in Leu8, Leu12, and Leu50 compared with Leu0, respectively, encompassing 161 common DEGs. STEM analysis elucidated four significant clusters of DEGs that were associated with "glycerophospholipid metabolism," "glycerolipid metabolism," "PPAR signaling pathway," and "adipocytokine signaling pathway." Moreover, the mRNA expression levels of acyl-CoA synthetase long chain family member 5 (ACSL5), choline kinase beta (CHKB), cryptochrome-1 (CRY1), lon protease homolog 2, peroxisomal isoform X2 (LONP2), lipin 1 (LPIN1), membrane bound O-acyltransferase domain containing 2 (MBOAT2), phosphoenolpyruvate carboxykinase 1 (PEPCK), and uridine-cytidine kinase 2b (UCK2b) were then further investigated in all leucine treatment groups at starvation times of 0 h, 24 h, and 48 h. The results revealed that the expression levels of UCK2b and MBOAT2 were negatively correlated with the addition of leucine, whereas CHKB, LONP2, CRY1, PEPCK, and LPIN1 were positively correlated. In conclusion, dietary leucine supplementation below 1.2% enhanced carbohydrate metabolism in juvenile crucian carp fed with a high-carbohydrate diet, whereas concentrations above 2.0% is a better choice for energy homeostasis under starvation.

Nesfatin-1 stimulates the hypothalamus-pituitary-interrenal axis hormones in goldfish.

Stress in vertebrates is mediated by the hypothalamus-pituitary-adrenal (in mammals)/interrenal (in fish) (HPA/I) axis, which produces the corticotropin-releasing factor (CRF), adrenocorticotropic hormone (ACTH), and corticosteroids, respectively. Nesfatin-1, a novel anorexigenic peptide encoded in the precursor nucleobindin-2 (NUCB2), is increasingly acknowledged as a peptide that influences the stress axis in mammals. The primary aim of this study was to characterize the putative effects of nesfatin-1 on the fish HPI axis, using goldfish (Carassius auratus) as an animal model. Our results demonstrated that nucb2/nesfatin-1 transcript abundance was detected in the HPI tissues of goldfish, with most abundant expression in the pituitary. NUCB2/nesfatin-1-like immunoreactivity was found in the goldfish hypothalamus, pituitary, and interrenal cells of the head kidney. GPCR12, a putative receptor for nesfatin-1, was also detected in the pituitary and interrenal cells. NUCB2/nesfatin-1-like immunoreactivity was observed in ACTH-expressing pituitary corticotrophs. Acute netting and restraint stress upregulated nucb2/nesfatin-1 mRNA levels in the forebrain, hypothalamus, and pituitary, as well as crf and crf-r1 expression in the forebrain and hypothalamus. Intraperitoneal and intracerebroventricular administration of nesfatin-1 increased cortisol release and hypothalamic crf mRNA levels, respectively. Finally, we found that nesfatin-1 significantly stimulated ACTH secretion from dispersed pituitary cells in vitro. Collectively, our data provide the first evidence showing that nesfatin-1 is a stress responsive peptide, which modulates the stress axis hormones in fish.

Effects of thyroxine and propylthiouracil on feeding behavior and the expression of hypothalamic appetite-regulating peptides and thyroid function in goldfish (Carassius auratus).

There is poor evidence for an association between thyroidal state, feeding and appetite regulation in fish. We assessed how an altered thyroid state influences feeding behavior, food intake and expression of hypothalamic appetite-regulating peptides (Klotho-α and Klotho-ß; orexin, OX; cholecystokinin, CCK; agouti-related peptide, AgRP; cannabinoid receptor 1, CB1) in goldfish. We also measured the expressions of hypothalamic, pituitary and liver transcripts that regulate the thyroid [thyrotropin-releasing hormone (TRH), thyrotropin-releasing hormone receptor (TRH-R) type 1, thyroid stimulating hormone beta (TSHß), deiodinases (DIO2, DIO3), UDP-glucuronosyltransferase (UGT1A1), thyroid receptor alpha and beta (TRα, TRß)], and circulating levels of total thyroxine (tT4) and total triiodothyronine (tT3). Goldfish were implanted with propylthiouracil (PTU) or T4 osmotic pumps for 12 days. T4- treatment increased feeding behavior but not food intake, increased central TSHß and DIO2, and hepatic DIO2 transcript expression and increased central DIO3 mRNA. Under hyperthyroid conditions, hypothalamic Klotho and CCK expressions were downregulated, suggesting an increased metabolic state and a hypothalamic response to regulate energy balance. AgRP, OX and CB1 were not affected by T4 treatment. PTU had no effect on any of the parameters examined, suggesting it is not a sensitive thyroid inhibitor in fish. Overall, we show that unlike in mammals, hyperthyroid conditions in goldfish do not lead to an increased desire or need to consume food, furthering evidence for a weak link between the thyroid and appetite.

Toxicity and deleterious impacts of selenium nanoparticles at supranutritional and imbalance levels on male goldfish (Carassius auratus) sperm.

BACKGROUND: Selenium has a major role in male reproduction and antioxidative mechanisms. Although deficiency of this element can result in damages to the body's organs, this metalloid can induce deleterious effects in organisms by causing oxidative stress. This study assessed the spermatotoxicity of selenium nanoparticles (SeNPs) in goldfish (Carassius auratus) based on genotoxicity, antioxidant status, sperm quality, and histopathology. METHODS: The fish with an average weight of 70 g (n = 288) were divided into four experimental groups (three replicates) and fed three times a day with SeNPs at different levels of 0, 0.1, 0.5, and 1 mg kg diet for 30 and 60 days. RESULTS: After 30 and 60 days of feeding trial, compared to the control group, spermatocrit percentage markedly decreased at 1 mg kg SeNPs on day 30 as well as at 0.5 and 1 mg kg on day 60 (p < 0.05). Computer-assisted sperm analysis parameters especially VCL, VSL, and VAP decreased in response to SeNPs (p < 0.05). Percentage of fast speed progressive sperm cells was highest in fish fed with 0.1 mg kg SeNPs following the dietary experiment and significantly reduced in a SeNPs dose-dependent manner (p < 0.05). In addition, the levels of Malondialdehyde and Glutathione peroxidase were significantly elevated in seminal plasma of all SeNPs-treated groups (p < 0.05). On day 60, DNA damage of sperm was greatly increased at 1 mg kg SeNPs (p < 0.05). Moreover, the highest percentage of spermatocyte and spermatid were observed at the highest dose of SeNPs while the highest percentage of spermatozoa was recorded at the lowest and moderate SeNPs doses. CONCLUSION: These findings suggested that non-optimal doses of SeNPs could reduce sperm quality, induce oxidative stress, and DNA damage in sperm, and disrupt testis development.