Role of Cholecystokinin (cck) in Feeding Regulation of Largemouth Bass (Micropterus salmoides): Peptide Activation and Antagonist Inhibition.

This study investigated the role of cholecystokinin (cck) in the feeding regulation of largemouth bass (Micropterus salmoides) via peptide activation and antagonist inhibition. The results show that the cck gene was expressed in various tissues, with the highest expression level occurring in the brain. Feeding, continuous feeding, and refeeding after fasting could significantly improve the mRNA levels of cck in the brain. Moreover, the activation of cck via injecting an exogenous CCK peptide could inhibit feed intake by regulating the mRNA levels of anorexigenic and feed-promoting factors in the brain and intestine. Furthermore, the CCK peptide reduced feed intake; however, the presence of an antagonist (Ly225910-CCK1R and devazepide-CCK2R) could reverse this effect through regulating the mRNA levels of anorexigenic and feed-promoting factors in the brain and intestine. Treatment with devazepide + CCK (CCK2R) reversed feed intake more effectively than Ly225910 + CCK (CCK1R) treatment. In summary, cck could regulate the feed intake of largemouth bass through regulating feeding-related genes in the brain and intestine. In addition, cck required binding with the receptor to inhibit feed intake more effectively in largemouth bass, and the binding effect of CCK1R was better than that of CCK2R.

The effect of selenium on the intestinal health of juvenile grass carp based on the ERS-autophagy pathway.

Selenium (Se), a trace element, is vital for the maintenance of cellular redox balance, thyroid hormone metabolism, inflammation, and immunity. Aeromonas hydrophila (A. hydrophila) is a common Gram-negative conditional pathogenic bacterium in fish culture, posing a serious threat to intensive aquaculture. Our study investigated the influence of dietary Se on the intestinal immune function of grass carp (Ctenopharyngodon idella) and the related regulatory mechanisms. The 2160 healthy juvenile grass carp (9.76 ± 0.005 g) were randomly assigned to 6 test groups of 6 replicates each, and fed graded selenomethionine (0.05, 0.20, 0.40, 0.61, 0.77, 0.98 mg Se/kg diet) for 70 days and then injected with A. hydrophila for a 6-day attack test. The results indicated that appropriate Se levels (0.40 mg/kg diet) alleviated intestinal damage caused by A. hydrophila and increased intestinal immune substances C3 and C4 levels as well as the activity of acid phosphatase (ACP) and lysozyme (LZ) (P > 0.05). Appropriate levels of Se (0.40 mg/kg-0.61 mg/kg diet) decreased intestinal pro-inflammatory cytokines (IFN-γ2, IL-6, IL-12p35, IL-17 A F and IL-17D) mRNA levels (P > 0.05) and increased intestinal anti-inflammatory factors (TGF-ß1, IL-4/13A, IL-4/13B, IL-10 and IL-22) mRNA levels (P > 0.05) in juvenile grass carp. Further studies revealed that Se (0.40 mg/kg-0.61 mg/kg diet) inhibited intestinal endoplasmic reticulum stress (ERS)-related signaling pathway. Furthermore, we found that appropriate levels of Se (0.40 mg/kg-0.61 mg/kg diet) inhibited intestinal autophagy in juvenile grass carp, which may be related to ULK1, Beclin 1, ATG5, ATG12, LC3, and P62. In conclusion, appropriate levels of Se can alleviate intestinal inflammation and inhibit ERS and autophagy in juvenile grass carp. A quadratic regression analysis of intestinal ACP and LZ also indicated that the Se requirements of juvenile grass carp were 0.59 and 0.51 mg/kg, respectively.

Transcriptome-Based Analysis of the Mechanism of Action of Metabolic Disorders Induced by Waterborne Copper Stress in Coilia nasus.

To reveal the effects of waterborne copper stress on gene expression changes, molecular pathways, and physiological functions in Coilia nasus, juvenile fish were equally divided into two experimental groups, and the copper levels were 1.61 ± 0.03 mg/L (copper-exposed group) and 0 mg/L (control group), respectively. After 4 h, gill tissue samples were collected for transcript sequencing analysis, and two libraries were constructed from the copper treatment group (Cu) and the control group (C) and sequenced using Illumina sequencing technology. The results showed that approximately 40.2-46.0 M clean reads were obtained from each library, and the percentage of uniquely mapped transcripts ranged from 80.57 to 84.93%. A total of 3915 differentially expressed genes (DEGs) were identified under waterborne copper stress, among which 1300 genes were up-regulated, and 2615 genes were down-regulated. Twelve DEGs were randomly selected for quantitative RT-PCR (qRT-PCR) analysis, and the results confirmed that the transcriptome analysis was reliable. Furthermore, the DEGs were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and the results showed that most of the DEGs were involved in metabolic pathways, including steroid biosynthesis, glutathione metabolism, and peroxisome proliferator-activated receptor (PPAR) signaling pathways. Furthermore, due to the waterborne copper levels, gsk-3ß was significantly up-regulated, while other metabolism-related genes (tor, pi3k, lpl, aqp7, fabp3) were significantly down-regulated. In addition, the copper-exposed group significantly reduced the expression of some immunity genes (ifn-γ, stat1, cxcl10, and tgf-ß), and enhanced the expression of il-1ß and tnf-α. In summary, these results indicated that copper causes metabolic disorders and insufficient energy supply in the body, and induces oxidative stress, which results in reduced immune functions.

Excessive Replacement of Fish Meal by Soy Protein Concentrate Resulted in Inhibition of Growth, Nutrient Metabolism, Antioxidant Capacity, Immune Capacity, and Intestinal Development in Juvenile Largemouth Bass (Micropterus salmoides).

This study investigated the effects of replacing 0% (SPC0), 25% (SPC25), 50% (SPC50), 75% (SPC75), and 100% (SPC100) of fish meal (FM) with soy protein concentrate (SPC) on the growth, nutritional metabolism, antioxidant capacity, and inflammatory factors in juvenile largemouth bass (Micropterus salmoides) (17.03 ± 0.01 g). After 56 days of culturing, various growth parameters including FW, WGR, and SGR were not significantly different among SPC0, SPC25, and SPC50 groups; however, they were significantly higher than those in SPC75 and SPC100 groups. Conversely, significantly lower FCR were determined for the SPC0, SPC25, and SPC50 groups compared with that for the SPC100 group; specifically, no significant difference among SPC0, SPC25, and SPC50 groups was found. Moreover, compared with SPC75 and SPC100 groups, a significantly higher FI was observed in the SPC0 group, whereas a significantly lower SR was observed in SPC100 compared with that in SPC0 and SPC25 groups. Compared with the SPC0 group, significantly lower mRNA levels of tor, rps6, 4ebp1, pparγ, and fas were found in SPC75 and SPC100. Additionally, the mRNA levels of cpt were significantly higher in SPC0, SPC25, and SPC50 groups than in SPC75 and SPC100 groups. Moreover, the mRNA levels of scd and acc remained unchanged for all the groups. Replacement of FM with SPC did not significantly affect the mRNA levels of gk, pk, and pepck. Compared with the SPC0 group, significantly decreased activities of CAT were observed in the SPC50, SPC75, and SPC100 groups, and significantly decreased activities of GSH-Px were observed in the SPC75 and SPC100 groups. In addition, significantly lower activity of SOD was observed in SPC100 compared with the other groups. Moreover, compared with the other groups, the SPC75 and SPC100 groups had significantly decreased and increased contents of GSH and MDA, respectively, while significantly lower mRNA levels of nrf2, cat, sod, and gsh-px were found in SPC50, SPC75, and SPC100; however, significantly higher mRNA levels of keap1 were observed in SPC75 and SPC100 groups. Additionally, significantly higher mRNA levels of il-8 and nf-κb were found in the SPC50, SPC75, and SPC100 groups compared with the SPC0 group. Conversely, significantly lower mRNA levels of il-10 and significantly higher mRNA levels of tnf-α were found in the SPC75 and SPC100 groups compared with the other groups. Compared with the SPC0 group, mucosal thickness and villus height were significantly decreased in the SPC75 and SPC100 groups. Collectively, SPC replacing 50% FM did not affect its growth of juvenile largemouth bass. However, SPC replacing 50% or more FM might inhibit antioxidant capacity and immune capacity to even threaten the SR, resulting in impaired intestinal development in replacing FM level of 75% or more.

Unveiling the emerging role of curcumin to alleviate ochratoxin A-induced muscle toxicity in grass carp (Ctenopharyngodon idella): in vitro and in vivo studies.

BACKGROUND: Ochratoxin A (OTA), a globally abundant and extremely hazardous pollutant, is a significant source of contamination in aquafeeds and is responsible for severe food pollution. The developmental toxicity of OTA and the potential relieving strategy of natural products remain unclear. This study screened the substance curcumin (Cur), which had the best effect in alleviating OTA inhibition of myoblast proliferation, from 96 natural products and investigated its effect and mechanism in reducing OTA myotoxicity in vivo and in vitro. METHODS: A total of 720 healthy juvenile grass carp, with an initial average body weight of 11.06 ± 0.05 g, were randomly assigned into 4 groups: the control group (without OTA and Cur), 1.2 mg/kg OTA group, 400 mg/kg Cur group, and 1.2 mg/kg OTA + 400 mg/kg Cur group. Each treatment consisted of 3 replicates (180 fish) for 60 d. RESULTS: Firstly, we cultured, purified, and identified myoblasts using the tissue block culture method. Through preliminary screening and re-screening of 96 substances, we examined cell proliferation-related indicators such as cell viability and ultimately found that Cur had the best effect. Secondly, Cur could alleviate OTA-inhibited myoblast differentiation and myofibrillar development-related proteins (MyoG and MYHC) in vivo and in vitro and improve the growth performance of grass carp. Then, Cur could also promote the expression of OTA-inhibited protein synthesis-related proteins (S6K1 and TOR), which was related to the activation of the AKT/TOR signaling pathway. Finally, Cur could downregulate the expression of OTA-enhanced protein degradation-related genes (murf1, foxo3a, and ub), which was related to the inhibition of the FoxO3a signaling pathway. CONCLUSIONS: In summary, our data demonstrated the effectiveness of Cur in alleviating OTA myotoxicity in vivo and in vitro. This study confirms the rapidity, feasibility, and effectiveness of establishing a natural product screening method targeting myoblasts to alleviate fungal toxin toxicity.

Hypoxia leads to gill endoplasmic reticulum stress and disruption of mitochondrial homeostasis in grass carp (Ctenopharyngodon idella): Mitigation effect of thiamine.

Hypoxia in water environment is one of the important problems faced by intensive aquaculture. Under hypoxia stress, the effects of dietary thiamine were investigated on grass carp gill tissue damage and their mechanisms. Six thiamine diets with different thiamine levels (0.22, 0.43, 0.73, 1.03, 1.33 and 1.63 mg/kg) were fed grass carp (Ctenopharyngodon idella) for 63 days. Then, 96-hour hypoxia stress test was conducted. This study described that thiamine enhanced the growth performance of adult grass carp and ameliorated nutritional status of thiamine (pyruvic acid, glucose, lactic acid and transketolase). Additionally, thiamine alleviated the deterioration of blood parameters [glutamic oxalacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), glucose, cortisol, lactic dehydrogenase (LDH), erythrocyte fragility, and red blood cell count (RBC count)] caused by hypoxia stress, and reduced reactive oxygen species (ROS) content and oxidative damage to the gills. In addition, thiamine alleviated endoplasmic reticulum stress in the gills, which may be related to its inhibition of RNA-dependent protein kinase-like ER kinase (PERK)/eukaryotic translation initiation factor-2α (eIF2α)/activating transcription factor4 (ATF4), inositol-requiring enzyme 1 (IRE1)/X-Box binding protein 1 (XBP1) and activating transcription factor 6 (ATF6) pathways. Furthermore, thiamine maintaining mitochondrial dynamics balance was probably related to promoting mitochondrial fusion and inhibiting mitochondrial fission, and inhibiting mitophagy may involve PTEN induced putative kinase 1 (PINK1)/Parkin-dependent pathway and hypoxia-inducible factor (HIF)-Bcl-2 adenovirus E1B 19 kDa interacting protein 3 (BNIP3) pathway. In summary, thiamine alleviated hypoxia stress in fish gills, which may be related to reducing endoplasmic reticulum stress, regulating mitochondrial dynamics balance and reducing mitophagy. The thiamine requirement for optimum growth [percent weight gain (PWG)] of adult grass carp was estimated to be 0.81 mg/kg diet. Based on the index of anti-hypoxia stress (ROS content in gill), the thiamine requirement for adult grass carp was estimated to be 1.32 mg/kg diet.

Effects of replacing wheat bran with palm kernel cake or fermented palm kernel cake on the growth performance, intestinal microbiota and intestinal health of tilapia (GIFT, Oreochromis niloticus).

A nine-week feeding trial was conducted to evaluate the effects of replacing wheat bran (WB) with palm kernel cake (PKC) or fermented palm kernel cake (FPKC) on the growth performance, intestinal microbiota and intestinal health of genetically improved farmed tilapia (GIFT, Oreochromis niloticus) (initial weight 7.00 ± 0.01 g). Eleven isonitrogenous and isolipidic experimental diets were formulated by replacing 0, 20, 40, 60, 80, and 100% of dietary WB with PKC or FPKC. Replacement of WB with PKC concentrations up to 80% had no significant effect on the growth rate of tilapia or feed utilisation (p > 0.05). FPKC improved the growth performance of tilapia, with optimum growth achieved at 40% replacement level (p < 0.05). Complete replacement with PKC significantly decreased the activity of lipase and trypsin, and reduced the height of muscularis and the height of villus (p < 0.05). However, FPKC significantly increased amylase activity and villus height (p < 0.05). The apparent digestibility of dry matter and energy decreased linearly with increasing levels of PKC substitution, while FPKC showed the opposite trend (p < 0.05). PKC replacement of WB by 20% significantly reduced serum diamine oxidase activity and endothelin levels and increased intestinal tight junctions (p < 0.05). However, FPKC significantly decreased diamine oxidase activity and increased intestinal tight junctions (p < 0.05). PKC completely replaced WB, up-regulating the expression of pro-inflammatory factors (il-1ß) (p < 0.05). When 40% of WB was replaced with FPKC, the expression of pro-inflammatory factors (il-1ß and il-6) was decreased significantly (p < 0.05). Completely replacement of WB with PKC reduced the abundance of Firmicutes and Chloroflexi, while FPKC reduced the abundance of Fusobacteriota and increased the levels of Actinobacteriota. WB can be replaced with PKC up to 80% in tilapia feeds. However, the high percentage of gluten induced intestinal inflammation, impaired gut health, and reduced dietary nutrient utilisation and growth performance. Complete replacement of WB with FPKC promoted intestinal immunity. It also improved dietary nutrient utilisation and growth performance. However, the optimal growth was achieved at a 40% replacement level.

Emerging role of vitamin D3 in alleviating intestinal structure injury caused by Aeromonas hydrophila in grass carp (Ctenopharyngodon idella).

Bacterial pathogens destroy the structural integrity of functional organs in fish, leading to severe challenges in the aquaculture industry. Vitamin D3 (VD3) prevents bacterial infections and strengthens immune system function via vitamin D receptor (VDR). However, the correlation between VD3/VDR and the structural integrity of functional organs remains unclarified. This study aimed to investigate the influence of VD3 supplementation on histological characteristics, apoptosis, and tight junction characteristics in fish intestine during pathogen infection. A total of 540 healthy grass carp (257.24 ± 0.63 g) were fed different levels of VD3 (15.2, 364.3, 782.5, 1,167.9, 1,573.8, and 1,980.1 IU/kg) for 70 d. Subsequently, fish were challenged with Aeromonas hydrophila, a pathogen that causes intestinal inflammation. Our present study demonstrated that optimal supplementation with VD3 (1) alleviated intestinal structural damage, and inhibited oxidative damage by reducing levels of oxidative stress biomarkers; (2) attenuated excessive apoptosis-related death receptor and mitochondrial pathway processes in relation to p38 mitogen-activated protein kinase signaling (P < 0.05); (3) enhanced tight junction protein expression by inhibiting myosin light chain kinase signaling (P < 0.05); and (4) elevated VDR isoform expression in fish intestine (P < 0.05). Overall, the results demonstrated that VD3 alleviates oxidative injury, apoptosis, and the destruction of tight junction protein under pathogenic infection, thereby strengthening pathogen defenses in the intestine. This finding supports the rationale for VD3 intervention as an essential practice in sustainable aquaculture.

The role of vitamin E in polyunsaturated fatty acid synthesis and alleviating endoplasmic reticulum stress in sub-adult grass carp (Ctenopharyngodon idella).

Vitamin E (VE) is an essential lipid-soluble vitamin that improves the fish flesh quality. However, the underlying molecular mechanisms remain unclear. This study aimed to investigate the effects of VE on growth performance and flesh quality in sub-adult grass carp (Ctenopharyngodon idella). A total of 450 fish (713.53 ± 1.50 g) were randomly divided into six treatment groups (three replicates per treatment) and fed for nine weeks with different experimental diets (dietary lipid 47.8 g/kg) that contained different levels of VE (5.44, 52.07, 96.85, 141.71, 185.66, and 230.12 mg/kg diet, supplemented as dl-α-tocopherol acetate). Notably, the treatment groups that were fed with dietary VE ranging from 52.07 to 230.12 mg/kg diet showed improvement in the percent weight gain, special growth rate, and feed efficiency of grass carp. Moreover, the treatment groups supplemented with dietary VE level of 141.71, 185.66, and 230.12 mg/kg diet showed enhancement in crude protein, lipid, and α-tocopherol contents in the muscle, and the dietary levels of VE ranging from 52.07 to 141.71 mg/kg diet improved muscle pH24h and shear force but reduced muscle cooking loss in grass carp. Furthermore, appropriate levels of VE (52.07 to 96.85 mg/kg diet) increased the muscle polyunsaturated fatty acid content in grass carp. Dietary VE also increased the mRNA levels of fatty acid synthesis-related genes, including fas, scd-1, fad, elovl, srebp1, pparγ, and lxrα, and up-regulated the expression of SREBP-1 protein. However, dietary VE decreased the expression of fatty acid decomposition-related genes, including hsl, cpt1, acox1, and pparα, and endoplasmic reticulum stress-related genes, including perk, ire1, atf6, eif2α, atf4, xbp1, chop, and grp78, and down-regulated the expression of p-PERK, p-IRE1, ATF6, and GRP78 proteins. In conclusion, dietary VE increased muscle fatty acid synthesis, which may be partly associated with the alleviation of endoplasmic reticulum stress, and ultimately improves fish flesh quality. Moreover, the VE requirements for sub-adult grass carp (713.53 to 1590.40 g) were estimated to be 124.9 and 122.73 mg/kg diet based on percentage weight gain and muscle shear force, respectively.

High replacement of soybean meal by different types of rapeseed meal is detrimental to rainbow trout (Oncorhynchus mykiss) growth, antioxidant capacity, non-specific immunity and Aeromonas hydrophila tolerance.

A 12-week feeding trial was conducted to evaluate the effects of replacing soybean meal with different types of rapeseed meal (RSM; Chinese 95-type (oil press model) rapeseed meal [C95RM], Chinese 200-type rapeseed meal [C200RM], cold pressed rapeseed cake [CPRC], Indian rapeseed meal [IRM] and Canadian rapeseed meal [CRM]) on growth, antioxidant capacity, non-specific immunity and Aeromonas hydrophila infection tolerance in 990 fingering (average weight 12.77 ± 0.01 g) rainbow trout (Oncorhynchus mykiss). A basal diet was prepared using fishmeal and soybean meal as the main protein sources, the other 10 diets were formulated with five types of RSM at 20% (C95RM20, C200RM20, CPRC20, IRM20, CRM20) or 35% (C95RM35, C200RM35, CPRC35, IRM35, CRM35) inclusion levels to replace iso-nitrogenous soybean meal. Regardless of the RSM source, dietary inclusion of 20% RSM significantly reduced the weight gain rate (WGR) and digestive enzymes activities (except C200RM20) of fish, but increased the blood urea nitrogen (BUN) and hepatic malondialdehyde (MDA) content (except CRM20). Fish fed with CPRC20 and IRM20 exhibited relatively higher plasma cortisol and MDA content, but lower content/activities of triiodothyronine (T3), thyroxine (T4) and glutathione peroxidase (GPx) in plasma, lysozyme (LZM) and complement 3 (C3) in serum, catalase (CAT) in liver, and respiratory burst activity (RBA) of head kidney macrophages. The intestinal and hepatic tissues fed with 20% RSM were damaged to some extent, with the CPRC20 and IRM20 groups being the most severely affected. Regardless of the RSM source, dietary inclusion of 35% RSM significantly decreased WGR and digestive enzymes activities, but significantly increased plasma BUN and MDA content. The fish fed with CPRC35 and IRM35 exhibited relatively higher plasma cortisol, MDA, serum triglyceride, BUN content, but lower content/activities of T3, T4, C3, and LZM in serum, CAT, peroxidase and GPx in plasma, CAT in liver, RBA and phagocytic activity of head kidney macrophage. The hepatic and intestinal tissues damage was the worst in the IRM35 group among the 35% RSM inclusion groups. These results indicate that including ≥20% RSM in the diet, regardless of the source, reduced the growth, antioxidant capacity, immunity, and survival to Aeromonas hydrophila infection in rainbow trout.

Selenium yeast improve growth, serum biochemical indices, metabolic ability, antioxidant capacity and immunity in black carp Mylopharyngodnpiceus.

This experiment was conducted to investigate the impacts of dietary selenium yeast (SeY) on the growth performance, fish body composition, metabolic ability, antioxidant capability, immunity and inflammatory responses in juvenile black carp (Mylopharyngodn piceus). The base diet was supplemented with 0.00, 0.30 and 0.60 g/kg SeY (0.04, 0.59 and 1.15 mg/kg of selenium) to form three isonitrogenous and isoenergetic diets for juvenile black carp with a 60-day. Adequate dietary SeY (0.30 and 0.60 g/kg) could significantly increase the weight gain (WG), special growth rate (SGR) compared to the SeY deficient groups (0.00 g/kg) (P < 0.05). Meanwhile, 0.30 and 0.60 g/kg SeY elevated the mRNA levels of selenoprotein T2 (SEPT2), selenoprotein H (SEPH), selenoprotein S (SEPS) and selenoprotein M (SEPM) in the liver and intestine compared with the SeY deficient groups (P < 0.05). Adequate dietary SeY could promote glucose catabolism and utilization through activating glucose transport (GLUT2), glycolysis (GCK, HK, PFK, PK, PDH), tricarboxylic acid cycle (ICDH and MDH), glycogen synthesis (LG, GCS and GBE) and IRS/PI3K/AKT signal pathway molecules (IRS2b, PI3Kc and AKT1) compared with the SeY deficient groups (P < 0.05). Similarly, adequate dietary SeY could improve lipid transport and triglycerides (TG) synthesis through increasing transcription amounts of CD36, GK, DGAT, ACC and FAS in the fish liver compared with the SeY deficient groups (P < 0.05). In addition, adequate SeY could markedly elevate activities of antioxidant enzymes (T-SOD, CAT, GR, GPX) and contents of T-AOC and GSH, while increased transcription amounts of Nrf2, Cu/Zn-SOD, CAT, and GPX in fish liver and intestine (P < 0.05). However, adequate SeY notably decreased contents of MDA, and the mRNA transcription levels of Keap1 in the intestine compared with the SeY deficient groups (P < 0.05). Adequate SeY markedly increased amounts or levels of the immune factors (ALP, ACP, LZM, C3, C4 and IgM) and the transcription levels of innate immune-related functional genes in the liver and intestine (LZM, C3 and C9) compared to the SeY deficient groups (P < 0.05). Moreover, adequate SeY could notably reduce levels of IL-8, IL-1ß, and IFN-γ and elevate TGF-1ß levels in fish intestine (P < 0.05). The transcription levels of MAPK13, MAPK14 and NF-κB p65 were notably reduced in fish intestine treated with 0.30 and 0.60 g/kg SeY (P < 0.05). In conclusion, these results suggested that 0.30 and 0.60 g/kg SeY could not only improve growth performance, increase Se, glucose and lipid metabolic abilities, enhance antioxidant capabilities and immune responses, but also alleviate inflammation, thereby supplying useful reference for producing artificial feeds in black carp.

Tea polyphenols protect against Flavobacterium columnare-induced gill injury via suppression of oxidative stress, inflammation, and apoptosis in grass carp.

Flavobacterium columnare (F. columnare) is one of the deadliest fish pathogens causing bacterial gill rot disease in various freshwater fish species globally. Tea polyphenols (TPs) are an inexpensive product extracted from tea that have received much attention as a feed additive in aquaculture. The current study was designed to investigate the underlying mechanisms and protective effects of dietary TPs against F. columnare-induced gill injury via suppression of oxidative stress, apoptosis, and inflammation in grass carp. TPs were not supplemented to the diet (control) and were supplemented at 40, 80, 120, 160 or 200 mg/kg diet. The feeding experiment was carried out for 60 days, followed by a 3-Day F. columnare challenge test. The results showed that 120 mg/kg TPs in the diet exerted the following five protective effects in fish gill: (1) control gill-rot disease and improved histopathology, (2) inhibit excessive apoptosis, (3) enhance the activity of antioxidant enzymes and upregulate related gene expression via the Nrf2/Keap1 pathway, (4) increase the activity of immune enzymes, And (5) mediate inflammatory cytokine gene expression via the JAK/STAT3 pathway. Taken together, dietary supplementation with TPs is a compelling approach to protect the gill function of fish against F. columnare.

Effects of dietary citrus pulp level on the growth and intestinal health of largemouth bass (Micropterus salmoides).

BACKGROUND: Citrus pulp (CP) is rich in pectin, and studies have shown that pectin possesses antioxidant, anti-inflammatory, and gut microbiota-regulating properties. However, the application of CP in aquafeed is limited. In this study, the effect of dietary inclusion of CP on the intestinal health of largemouth bass (Micropterus salmoides) was investigated. Juveniles of similar size (6.95 ± 0.07 g) were fed isonitrogenous and isoenergetic diets containing different levels of CP (0%, 3%, 6%, 9%, 12%, or 15%) for 58 days. RESULTS: As the level of CP in the feed for largemouth bass increased, the fish's growth performance and intestinal health initially improved and then declined. Adding low doses of CP (≤9%) to the feed had no significant impact on the growth performance of large-mouth black bass, whereas high doses of CP (>9%) significantly reduced their growth performance. Adding 6%, 9%, or 12% of CP to that feed enhanced the expression of genes related to tight junctions, anti-inflammatory activity, anti-apoptotic activity, and antioxidant activity in the intestines of largemouth bass. It reduced intestinal inflammation and improved intestinal nutrient absorption, intestinal mucosal barrier function, and intestinal antioxidant capacity. Moreover, it improved the α-diversity, structure, and function of the intestinal flora. The addition of 6% CP had the most beneficial effect on the intestinal health of largemouth bass. On the other hand, the addition of 15% CP had adverse effects on the intestinal antioxidant capacity and intestinal mucosal barrier function of largemouth bass. CONCLUSION: Adding 6-9% CP to the feed for largemouth bass can improve their intestinal health without having a significant impact on their growth performance. CP could serve as a novel prebiotic and immunostimulant ingredient in aquafeed. © 2023 Society of Chemical Industry.

Excessive dietary soluble arabinoxylan impairs the intestinal physical and immunological barriers via activating MAPK/NF-κB signaling pathway in rainbow trout (Oncorhynchus mykiss).

Arabinoxylan (AX) has been deemed as an antinutritional factor, but limited information has addressed the effects of dietary AX on intestinal health of fish. The present study investigated the effects of dietary AX on intestinal mucosal physical and immunological barriers of rainbow trout (Oncorhynchus mykiss). Five isoproteic and isolipidic experimental diets (AXE, AX0, AX2.5, AX5 and AX10) were formulated to contain 0.03% arabinoxylanase as well as 0%, 2.5%, 5% and 10% AX, respectively. Each diet was randomly distributed to triplicate groups of 35 juvenile (average weight 3.14 ± 0.02 g) per tank in a rearing system maintained at 17 ± 1 °C for 9 weeks. Dietary AX supplementation regardless of inclusion levels significantly (P < 0.05) depressed the growth performance and feed utilization. The plasma endothelin-1 and d-lactic acid contents as well as diamino oxidase activity were significantly higher in fish fed diet AX10 compared to fish fed diet AX0. Dietary inclusion of 5-10% AX resulted in decreased intestinal villus height, goblet cell number and desmosome density, increased crypt depth, short and irregular microvilli, widened intercellular space; down-regulated the mRNA levels of occludin in hindgut, claudin3 and ZO-1 in foregut and midgut, but up-regulated the mRNA levels of claudin12 and claudin15 in midgut as well as claudin23 in foregut, midgut and hindgut. Furthermore, dietary 5-10% AX supplementation decreased the midgut and hindgut complement 3, complement 4 and sIgT contents as well as the midgut IgM and hindgut IL-10 contents. Conversely, the hindgut TNF-α and IL-6 contents increased with the rising dietary AX level. RT-qPCR demonstrated that the pro-inflammatory cytokines (IL-1ß, IL-6, IL-8, IL-12ß, IFN-γ, and TNF-α) and pIgR mRNA levels in midgut and hindgut were up-regulated by dietary AX inclusion of 5-10% AX. Meanwhile, the mRNA levels of p38 MAPK, IκBα, and NF-κB p65 in midgut and hindgut raised gradually with the increasing dietary AX content. The Western blot results showed that the protein expression levels of p38 MAPK and NF-κB generally increased with the rising dietary AX content. Dietary treatment with 0.03% arabinoxylanase did not affect the growth performance and intestinal health of rainbow trout (P > 0.05). In conclusion, excessive dietary AX inclusion (5-10%) increased the intestinal permeability and induced the intestinal inflammatory response via activating MAPK/NF-κB signaling pathway, and ultimately damaged the intestinal barrier function of rainbow trout.

Implications of vitamin D for flesh quality of grass carp (Ctenopharyngodon idella): antioxidant ability, nutritional value, sensory quality, and myofiber characteristics.

BACKGROUND: Muscle represents a unique and complex system with many components and comprises the major edible part of animals. Vitamin D is a critical nutrient for animals and is known to enhance calcium absorption and immune response. In recent years, dietary vitamin D supplementation in livestock has received increased attention due to biological responses including improving shear force in mammalian meat. However, the vitamin D acquisition and myofiber development processes in fish differ from those in mammals, and the effect of vitamin D on fish flesh quality is poorly understood. Here, the influence of dietary vitamin D on fillet quality, antioxidant ability, and myofiber development was examined in grass carp (Ctenopharyngodon idella). METHODS: A total of 540 healthy grass carp, with an initial average body weight of 257.24 ± 0.63 g, were allotted in 6 experimental groups with 3 replicates each, and respectively fed corresponding diets with 15.2, 364.3, 782.5, 1,167.9, 1,573.8, and 1,980.1 IU/kg vitamin D for 70 d. RESULTS: Supplementation with 1,167.9 IU/kg vitamin D significantly improved nutritional value and sensory quality of fillets, enhancing crude protein, free amino acid, lipid, and collagen contents; maintaining an ideal pH; and reducing lactate content, shear force, and cooking loss relative to respective values in the control (15.2 IU/kg) group. Average myofiber diameter and the frequency of myofibers > 50 µm in diameter increased under supplementation with 782.5-1,167.9 IU/kg vitamin D. Levels of oxidative damage biomarkers decreased, and the expression of antioxidant enzymes and nuclear factor erythroid 2-related factor 2 signaling molecules was upregulated in the 1,167.9 IU/kg vitamin D treatment compared to respective values in the control group. Furthermore, vitamin D supplementation activated cell differentiation by enhancing the expression of myogenic regulatory factors and myocyte enhancer factors compared to that in the control group. In addition, supplementation with 1,167.9 IU/kg vitamin D improved protein deposition associated with protein synthesis molecule (target of rapamycin) signaling and vitamin D receptor paralogs, along with inhibition of protein degradation (forkhead box protein 1) signaling. CONCLUSIONS: Overall, the results demonstrated that vitamin D strengthened antioxidant ability and myofiber development, thereby enhancing nutritional value and sensory quality of fish flesh. These findings suggest that dietary vitamin D supplementation is conducive to the production of nutrient-rich, high quality aquaculture products.

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

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

Promotion of fatty acid metabolism and glucose metabolism in the muscle of sub-adult grass carp (Ctenopharyngodon idella): The role of alpha-linoleic acid/linoleic acid (ALA/LNA) ratios.

The n6/n3 ratios improved meat quality of terrestrial animals, but alpha-linolenic acid/linoleic acid (ALA/LNA) ratios were rarely studied in aquatic animals. In this study, sub-adult grass carp (Ctenopharyngodon idella) were fed diets fed diets containing six varying ALA/LNA ratios (0.03, 0.47, 0.92, 1.33, 1.69, and 2.15) for 9 weeks and the total value of n3 + n6 (1.98) was kept constant for all six treatments. The results indicated optimal ALA/LNA ratio improved growth performance, changed fatty acid composition in grass carp muscle, and promoted glucose metabolism. Additionally, optimal ALA/LNA ratio improved chemical attributes by increasing crude protein and lipid contents, and technological attributes by increasing pH24h value and shear force in grass carp muscle. The signaling pathways related to fatty acid metabolism and glucose metabolism (LXRα/SREBP-1, PPARα, PPARγ, AMPK) might be responsible for these changes. Dietary optimal ALA/LNA ratio based on PWG, UFA and glucose contents was 1.03, 0.88 and 0.92, respectively.

Novel insights on toxicology of ochratoxin A contaminated diets to skin: Residues, mucus disruption and barrier impairment in teleost model grass carp (Ctenopharyngodon idella).

Ochratoxin A (OTA), a notorious pollutant widely present worldwide, seriously pollutes aquafeeds. This paper aims to explore the toxicity effects of OTA by the way of diet on the skin barrier in grass carp (Ctenopharyngodon idella). Results were shown as follows in the skin: (1) OTA increased the mRNA abundances of uptake transporter proteins (e.g., OAT3) and decreased efflux transporter proteins (e.g., ABCG2), which caused the accumulation of OTA in the skin of grass carp. (2) OTA upregulated the gene expression related to ROS production by enhancing the NOX (1, 2, 4) signaling pathway and decreased the ability to ROS elimination with downregulation of GPx1 (a,b), Trx by inhibiting the PGC1-α/Nrf2 signaling pathway, which caused oxidative damage to the skin. (3) OTA exacerbated apoptosis in the skin by upregulating the expression of apoptosis-related proteins mediated by ways of endoplasmic reticulum stress and mitochondrial apoptosis. Moreover, OTA down-regulated the mRNA and protein abundances of tight junction-related proteins by inhibiting the MLCK signaling pathway, which in turn disrupted the tight junctions. (4) OTA reduced the number of mucous cup cells and decreased f LZ activities and IgM contents, and finally down-regulated the mRNA abundances of mucin (2, 3), LEAP-2 (A, B), and ß-defensin (1, 2, 3), which in turn resulted in impairing skin chemical barrier. Moreover, based on the antimicrobial-related indexes (LZ activities and IgM contents), the OTA-safe upper doses were 814.827 and 813.601 µg/kg.

Aflatoxin B1 damaged structural barrier through Keap1a/Nrf2/ MLCK signaling pathways and immune barrier through NF-κB/ TOR signaling pathways in gill of grass carp (Ctenopharyngodon idella).

Aquafeeds are susceptible to contamination caused by aflatoxin B1 (AFB1). The gill of fish is an important respiratory organ. However, few studies have investigated the effects of dietary AFB1 exposure on gill. This study aimed to discuss the effects of AFB1 on the structural and immune barrier of grass carp gill. Dietary AFB1 increased reactive oxygen species (ROS) levels, protein carbonyl (PC) and malondialdehyde (MDA) contents, which consequently caused oxidative damage. In contrast, dietary AFB1 decreased antioxidant enzymes activities, relative genes expression (except MnSOD) and the contents of glutathione (GSH) (P < 0.05), which are partly regulated by NF-E2-related factor 2 (Nrf2/Keap1a). Moreover, dietary AFB1 caused DNA fragmentation. The relative genes of apoptosis (except Bcl-2, McL-1 and IAP) were significantly upregulated (P < 0.05), and apoptosis was likely upregulated through p38 mitogen-activated protein kinase (p38MAPK). The relative expressions of genes associated with tight junction complexes (TJs) (except ZO-1 and claudin-12) were significantly decreased (P < 0.05), and TJs were likely regulated by myosin light chain kinase (MLCK). Overall, dietary AFB1 disrupted the structural barrier of gill. Furthermore, AFB1 increased gill sensitivity to F. columnare, increased Columnaris disease and decreased the production of antimicrobial substances (P < 0.05) in grass carp gill, and upregulated the expression of genes involved with pro-inflammatory factors (except TNF-α and IL-8) and the pro-inflammatory response partly attributed to the regulation by nuclear factor κB (NF-κB). Meanwhile, the anti-inflammatory factors were downregulated (P < 0.05) in grass carp gill after challenge with F. columnare, which was partly attributed to the target of rapamycin (TOR). These results suggested that AFB1 aggravated the disruption of the immune barrier of grass carp gill after being challenge with F. columnare. Finally, the upper limit of safety of AFB1 for grass carp, based on Columnaris disease, was 31.10 µg/kg diet.

Cellular antioxidant mechanism of mannan-oligosaccharides involving in enhancing flesh quality in grass carp (Ctenopharyngodon idella).

BACKGROUND: Deterioration of flesh quality has bad effects on consumer satisfaction. Therefore, effects of safe mannan-oligosaccharides (MOS) on flesh quality of grass carp (Ctenopharyngodon idella) muscle were studied. A total of 540 healthy fish (215.85 ± 0.30 g) were randomly divided into six groups and fed six separate diets with graded levels of MOS (0, 200, 400, 600, 800 and 1000 mg kg-1 ) for 60 days. This study aimed at investigating the benefits of dietary MOS on flesh quality (fatty acids, amino acids and physicochemical properties) and the protection mechanism regarding antioxidant status. RESULTS: Optimal MOS could improve tenderness (27.4%), pH (5.5%) while decreasing cooking loss (16.6%) to enhance flesh quality. Meanwhile, optimal MOS improved flavor inosine 5'-monophosphate (IMP) of 11.8%, sweetness and umami-associated amino acid, healthy unsaturated fatty acid (UFA) of 14.9% and n-3 polyunsaturated fatty acids (n-3 PUFAs) especially C20:5n-3 (15.8%) and C22:6n-3 (38.3%). Furthermore, the mechanism that MOS affected pH, tenderness and cooking loss could be partly explained by the reduced lactate, cathepsin and oxidation, respectively. The enhanced flesh quality was also associated with enhanced antioxidant ability concerning improving antioxidant enzymes activities and the corresponding transcriptional levels, which were regulated through NF-E2-related factor 2 (Nrf2) and target of rapamycin (TOR) signaling. Based on pH24h , cooking loss, shear force and DHA (docosahexaenoic acid, C22:6n-3), optimal MOS levels for grass carp were estimated to be 442.75, 539.53, 594.73 and 539.53 mg kg-1 , respectively. CONCLUSION: Dietary MOS is a promising alternative strategy to improve flesh quality of fish muscle. © 2022 Society of Chemical Industry.