Dietary sodium acetate and sodium butyrate improve high-carbohydrate diet utilization by regulating gut microbiota, liver lipid metabolism, oxidative stress, and inflammation in largemouth bass(Micropterus salmoides)

Background Adequate level of carbohydrates in aquafeeds help to conserve protein and reduce cost. However, studies have indicated that high-carbohydrate(HC) diet disrupt the homeostasis of the gut–liver axis in largemouth bass, resulting in decreased intestinal acetate and butyrate level.Method Herein, we had concepted a set of feeding experiment to assess the effects of dietary sodium acetate(SA) and sodium butyrate(SB) on liver health and the intestinal microbiota in largemouth bass fed an HC diet. The experimental design comprised 5 isonitrogenous and isolipidic diets, including LC(9% starch), HC(18% starch), HCSA(18% starch;2 g/kg SA), HCSB(18% starch;2 g/kg SB), and HCSASB(18% starch;1 g/kg SA + 1 g/kg SB). Juvenile largemouth bass with an initial body weight of 7.00 ± 0.20 g were fed on these diets for 56 d.Results We found that dietary SA and SB reduced hepatic triglyceride accumulation by activating autophagy(ATG101, LC3B and TFEB), promoting lipolysis(CPT1α, HSL and AMPKα), and inhibiting adipogenesis(FAS, ACCA, SCD1 and PPARγ). In addition, SA and SB decreased oxidative stress in the liver(CAT, GPX1α and SOD1) by activating the Keap1-Nrf2 pathway. Meanwhile, SA and SB alleviated HC-induced inflammation by downregulating the expression of pro-inflammatory factors(IL-1β, COX2 and Hepcidin1) through the NF-κB pathway. Importantly, SA and SB increased the abundance of bacteria that produced acetic acid and butyrate(Clostridium_sensu_stricto_1). Combined with the KEGG analysis, the results showed that SA and SB enriched carbohydrate metabolism and amino acid metabolism pathways, thereby improving the utilization of carbohydrates. Pearson correlation analysis indicated that growth performance was closely related to hepatic lipid deposition, autophagy, antioxidant capacity, inflammation, and intestinal microbial composition.Conclusions In conclusion, dietary SA and SB can reduce hepatic lipid deposition;and alleviate oxidative stress and inflammation in largemouth bass fed on HC diet. These beneficial effects may be due to the altered composition of the gut microbiota caused by SA and SB. The improvement effects of SB were stronger than those associated with SA.

Differential expression of miRNAs and mRNAs in the livers of largemouth bass Micropterus salmoides under heat stress

Global warming threatens freshwater ecosystems and compromises fish survival.To elucidate the role of miRNAs in the livers of heat stressed largemouth bass,juvenile fish was subject to heat stress under 37°C.Both mRNA-seq and miRNA-seq were conducted on the liver tissues under control and heat stress conditions.Differential gene expression analysis and enrichment analysis were performed on mRNA and miRNA expression profiles.A total of 406 differentially expressed genes(DEGs)were discovered,of which 212 were up-regulated and 194 were down-regulated.Most of the DEGs were significantly implicated in the regulation of“protein processing in endoplasmic reticulum”,“proteasome”,“steroid biosynthesis”,and“ornithine decarboxylase inhibitor activity”pathways.In addition,47 differentially expressed miRNAs(DEMs)were identified in largemouth bass livers under heat stress,including 21 up-regulated and 25 down-regulated miRNAs.A negatively regulated miRNA-mRNA network including 12 miRNAs and 19 mRNAs was constructed with DEMs involved in“protein degradation”,“calcium ion regulation”,“cell apoptosis”,and“lipid metabolism”.Moreover,this study indicated novel-miR-144 activated the IRE1 signaling pathway by targeting txndc5 to induce liver apoptosis in largemouth bass under heat stress.This study revealed the involvement of miRNA regulation in largemouth bass in response to heat stress.

Whole-genome resequencing reveals recent signatures of selection in five populations of largemouth bass(Micropterus salmoides)

Largemouth bass(Micropterus salmoides) is an economically important fish species in North America, Europe, and China. Various genetic improvement programs and domestication processes have modified its genome sequence through selective pressure, leaving nucleotide signals that can be detected at the genomic level. In this study,we sequenced 149 largemouth bass fish, including protospecies(imported from the US) and improved breeds(four domestic breeding populations from China). We detected genomic regions harboring certain genes associated with improved traits, which may be useful molecular markers for practical domestication, breeding, and selection. Subsequent analyses of genetic diversity and population structure revealed that the improved breeds have undergone more rigorous genetic changes. Through selective signal analysis, we identified hundreds of putative selective sweep regions in each largemouth bass line. Interestingly, we predicted 103 putative candidate genes potentially subjected to selection,including several associated with growth(psst1 and grb10), early development(klf9, sp4, and sp8), and immune traits(pkn2, sept2, bcl6, and ripk2). These candidate genes represent potential genomic landmarks that could be used to improve important traits of biological and commercial interest. In summary, this study provides a genome-wide map of genetic variations and selection footprints in largemouth bass, which may benefit genetic studies and accelerate genetic improvement of this economically important fish.

Cloning and Characterization of Largemouth Bass(Micropterus salmoides) Myostatin Encoding Gene and Its Promoter

Myostatin or GDF-8, a member of the transforming growth factor-β （TGF-β） superfamily, has been demonstrated to be a negative regulator of skeletal muscle mass in mammals. In the present study, we obtained a 5.64 kb sequence of myostatin encoding gene and its promoter from largemouth bass （Micropterus salmoides）. The myostatin encoding gene consisted of three exons （488bp, 371 bp and 1779bp, respectively） and two introns （390bp and 855 bp, respectively）. The intron-exon boundaries were conservative in comparison with those of mammalian myostatin encoding genes, whereas the size of introns was smaller than that of mammals. Sequence analysis of 1.569 kb of the largemouth bass myostatin gene promoter region revealed that it contained two TATA boxes, one CAAT box and nine putative E-boxes. Putative muscle growth response elements for myocyte enhancer factor 2 （MEF2）, serum response factor （SRF）, activator protein 1 （AP1）, etc., and muscle-specific Mt binding site （MTBF） were also detected. Some of the transcription factor binding sites were conserved among five teleost species. This infunnation will be useful for studying the tran- scriptional regulation of myostatin in fish.

Cloning,tissue distribution and effects of fasting on pituitary adenylate cyclase-activating polypeptide in largemouth bass

Pituitary adenylate cyclase activating polypeptide （PACAP） has a wide range of biological functions. We cloned the full-length cDNAs encoding PACAP and PACAP-related peptide （PRP） from the brain of largemouth bass （Micropterus salmoides） and used real-time quantitative PCR to detect PRP- PACAP mRNA expression. The PRP-PACAP cDNA has two variants expressed via alternative splicing： a long form, which encodes both PRP and PACAP, and a short form, which encodes only PACAR Sequence analysis results are consistent with a higher conservation of PACAP than PRP peptide sequences. The expression of PACAP-Iong and PACAP-short transcripts was highest in the forebrain, followed by the medulla, midbrain, pituitary, stomach, cerebellum, intestine, and kidney; however, these transcripts were either absent or were weakly expressed in the muscle, spleen, gill, heart, fatty tissue, and liver. The level of PACAP-short transcript expression was significantly higher than expression of the long transcript in the forebrain, cerebella, pituitary and intestine, but lower than that of the long transcript in the stomach. PA CAP- long and PACAP-short transcripts were first detected at the blastula stage of embryogenesis, and the level of expression increased markedly between the muscular contraction stage and 3 d post hatch （dph）. The expression of PACAP-long and PACAP-short transcripts decreased significantly in the brain following 4 d fasting compared with the control diet group. The down-regulation effect was enhanced as fasting continued. Conversely, expression levels increased significantly after 3 d of re-feeding. Our results suggest that PRP- PA CAP acts as an important factor in appetite regulation in largemouth bass.

SNP Detection of High Density Lipoprotein Binding Protein Gene (HBP) and Its Correlations with Growth Traits in Largemouth Bass(Micropterus salmoides)

High density lipoprotein binding protein （HBP） plays an important role in lipid metabolism of animals. Lipids are indispensable energy materials for fi- shes, especially for carnivorous fishes with low utilization efficiency of carbohydrates. The single nucleotide polymorphism （SNP） of HBP gene may affect the fat metabolism, thereby exerting an effect on the growth traits of largemouth bass （Micropterus salmoides）. Investigating the correlations between SNP and growth traits can provide candidate markers for molecular marker-assisted selection. In this study, partial genomic fraganents of HBP gene （ GenBank accession number： KF652241 ） were amplified based on the sequences of an available contig in the EST-SNP database of largemouth bass. Three SNP mutation loci were identified in the 3＇ non-ceding region of HBP gene by direct sequencing, including H1 （G ＋ 2782T）, 142 （T ＋ 2817C） and H3 （G ＋ 2857A）. Three SNP loci of 165 randomly selected largemouth bass individuals were detected and genotyped by SnaPshot assay. Genetic structure was analyzed by POPGENE32 software. By using spssl7.0 software, a general linear model （GLM） was established for correlation analysis between different genotypes at SNP loci of HBP gene and various growth traits. The results showed that three SNP loci were in Hardy-Weinberg equilibrium state. To be specific, loci H1 and H2 formed two haplotypes （ A and B）, and three geno- types （AA, AB, and BB） were observed; loci H1, H2 and H3 formed six diplotypes （DI, I）2, D3, D4, D5 and D6）. According to the correlations between dif- ferent genotypes and various growth traits, the body weight and total length of largemouth bass individuals with genotype BB were significantly higher than those of individuals with genotype AB （ P 〈 0.05 ） ; the body weight and total length of largemouth bass individuals with diplotype D6 were significantly higher than those of individuals with diplotype D4 （P 〈0.05）. In this study, SNP markers correlated with growth traits were obtained in the 3＇ non-coding region ofHBP gene in large-mouth bass, which could be used as candidate genetic markers for subsequent molecular marker-assisted selection breeding of largemouth bass.

Effects of Management Practices and Environmental Factors on Largemouth Bass Abundance in Illinois Inland Lakes

An understanding of the relative importance of natural factors and management practices affecting largemouth bass (Micropterus salmoides) abundance is key for enhanced angling. Standardized fish surveys, management practices, and environmental data were available from 42 man-made, inland lakes between 1960 and 1991. Management practices tested were largemouth bass stocking, lake rehabilitation, water level manipulation, aquatic vegetation controls, small fish removal, and changes in length limits of harvestable fish. Environmental factors not controlled by management were spring water influx, growing and cooling degree days, and snow depth. Lake rehabilitation (complete drainage and reflooding), changes in length limits, and aquatic vegetation controls were the only significant factors affecting largemouth bass abundance. The largest effects were due to lake rehabilitation, which increased next-year young largemouth bass numbers by 566% on average, and more restrictive limits on harvestable size, with an increase of up to 440% in adult numbers.

Availability and Timing of Fathead Minnow Supplementation Influence Largemouth Bass Survival and Production in Rearing Ponds

Adjustments to rearing practices should be justified with increases in production, stocking success, or angler satisfaction. Largemouth bass (Micropterus salmoides) production was assessed between hatchery ponds where fish were restricted to an invertebrate diet or received supplemental fathead minnow (Pimephales promelas) forage during 2015. At harvest, age-0 bass yield was 4.5 times greater and average fish length was 38 mm longer, in the pond that received fathead minnow. In 2016, a second study evaluated the timing of minnow supplementation that included earlier stockings of small fathead minnow (<30 mm) compared to delayed supplementation with larger (>30 mm) minnows. With earlier supplementation, bass yield was 2.3 times greater and fish averaged 14 mm longer at harvest. Bass survival was approximately 38% higher during 2015 when supplementation occurred and 25% higher during 2016 when minnow supplementation began earlier. Our findings show invertebrate forage was probably limiting bass production in hatchery ponds and supplementing with appropriately-sized fathead minnows increased age-0, largemouth bass production.

Evaluation of Stocking All Female Largemouth Bass (Micropterus salmoides) in Alabama (USA) Ponds

Excessive largemouth bass ( Micropterus salmoides ) recruitment in small impoundments leads to density-dependent growth depression, causing populations to have length distributions skewed towards smaller fish. To address this issue, we conducted three trials that evaluated stocking all female largemouth bass (F-LMB) in two small Alabama ponds (0.5 and 2.0 ha) where the drainage basin was isolated. Age-1 fish were individually identified for sex using a micro pipette inserted in the urogenital pore, tagged, and stocked at 38-40 F-LMB/ha into ponds that contained pre-established sunfish ( Lepomis spp.) populations. Female largemouth bass were collected with electrofishing, angling, and rotenone over a 2.5- to 5-year period among the three trials. Sex was correctly identified for 179 of the 180 F-LMB stocked. One male was detected in the first trial which resulted in successful largemouth bass reproduction and this trial was terminated after two years. Growth was rapid the first 2 years after stocking as 3-year-old fish reached an average of 429 to 459 mm and 1.37 to 1.66 kg in all 3 trials. However, 2 years after stocking, growth was nil in the second trial even though relative weights of F-LMB were generally greater than 100. In this second trial, removal of about 30% of the F-LMB inhabiting the pond and stocking small Nile tilapia Oreochromis niloticus (25 - 75 mm) resulted in average weights increasing from 1.30 to 2.34 kg in 1 year. Growth of F-LMB continued in the third trail, and average size approached 500 mm and 2.2 kg three years after stocking. Annual survival rates of F-LMB were high and ranged from 0.78 to 0.93 among the 3 trials. Stocking F-LMB offers an attractive alternative in ponds to create a low density largemouth bass population that displays fast growth and high survival when catch-and-release fishing is primarily practiced.

Arginine Intake and Carbohydrate to Lipid Ratios Affect Gene Expression of Anabolic Hormones in Largemouth Bass, Micropterus Sahnoides

Nutritional factors influence regulation of growth hormone （GH）, insulin-like growth factor-Ⅰ （IGF-Ⅰ） and insulin （INS） in fish. But so far there are no published studies describing how single indispensable amino acids and different carbohydrate to lipid ratios influence those systems. Therefore, the present study aimed to evaluate whether arginine （Arg） intake and carbohydrate to lipid ratios would affect expression of GH, IGF-Ⅰ and INS in largemouth bass.

The potential of Clostridium autoethanogenum,a new single cell protein,in substituting fish meal in the diet of largemouth bass(Micropterus salmoides):Growth,feed utilization and intestinal histology

This study investigated the effects of replacing fishmeal with Clostridium autoethanogenum protein(CAP)on growth,feed utilization and intestinal histology of largemouth bass(Micropterus salmoides).The control diet contained 350 g/kg fishmeal,and then dietary fishmeal was decreased to 300 g/kg,250 g/kg,200 g/kg,150 g/kg and 100 g/kg by CAP inclusion,respectively(FM-30,FM-25,FM-20,FM-15,FM-10).The largemouth bass(110.0±1.0 g)were fed one of the six diets for 8 weeks.The groups of FM-30,FM-25 and FM-20 showed the similar weight gain(WG)and feed conversion ratio(FCR)to the control(P>0.05),while the WG of FM-10 group was decreased by 15.9%,and FCR increased by 0.12 when compared to the control(P<0.05).FM-15 and FM-10 groups had lower protein retention,lower villus height in intestine and higher serum malondialdehyde content than the control(P<0.05).The protease activity in FM-20,FM-15 and FM-10 groups was lower than that in the control(P<0.05),but no difference was observed in crude protein digestibility among groups(P>0.05).In conclusion,CAP could replace 150 g/kg fishmeal in diet(350 g/kg fishmeal)without adverse effects on growth,feed utilization and intestinal histology of largemouth bass.

The direct and gut microbiota-mediated effects of dietary bile acids on the improvement of gut barriers in largemouth bass(Micropterus salmoides)

Fish gut barrier damage under intensive culture model is a significant concern for aquaculture industry.This study aimed to investigate the effects of bile acids(BAs)on gut barriers in Micropterus salmoides.A germ-free(GF)zebrafish model was employed to elucidate the effects of the direct stimulation of BAs and the indirect regulations mediated by the gut microbiota on gut barrier functions.Four diets were formulated with BAs supplemented at 0,150,300 and 450 mg/kg,and these 4 diets were defined as control,BA150,BA300 and BA450,respectively.After 5 weeks of feeding experiment,the survival rate of fish fed with BA300 diet was increased(P<0.05).Histological analysis revealed an improvement of gut structural integrity in the BA150 and BA300 groups.Compared with the control group,the expression of genes related to chemical barrier(mucin,lysozyme and complement 1)and physical barrier(occludin and claudin-4)was increased in the BA150 and BA300 groups(P<0.05),and the expression of genes related to immunological barrier(interleukin[IL]-6,tumor growth factorβ,IL-10,macrophage galactosetype lectin and immunoglobulin M[Ig M])was significantly increased in the BA300 group(P<0.05),but the expression of genes related to chemical barrier(hepcidin)and immunological barrier(IL-1β,tumor necrosis factor-a,IL-6 and arginase)was significantly decreased in the BA450 group(P<0.05).Gut microbiota composition analysis revealed that the abundance of Firmicutes was augmented prominently in the BA150 and BA300 groups(P<0.05),while that of Actinobacteriota and Proteobacteria showed a downward trend in the BA150 and BA300 groups(P>0.05).The results of the gut microbiota transferring experiment demonstrated an upregulation of gut barrier-related genes,including immunoglobulin Z/T(Ig Z/T),IL-6,IL-1βand IL-10,by the gut microbiota transferred from the BA300 group compared with the control(P<0.05).Feeding the BA300 diet directly to GF zebrafish resulted in enhanced expression of Ig M,Ig Z/T,lysozyme,occludin-2,IL-6 and IL-10(P<0.05).In conclusion,BAs can improve the gut barriers of fish through both direct and indirect effects mediated by the gut microbiota.

Effects of dietary vitamin K_(3) levels on growth,coagulation,calcium content,and antioxidant capacity in largemouth bass,Micropterus salmoides

An 8-week feeding trial investigated the optimum dietary vitamin K3 requirements of largemouth bass.A total of 600 healthy fish(12.96±0.07 g)were fed diets containing varying levels of vitamin K3 at 0.78(K0),5.80(K5),10.82(K10),15.84(K15),and 20.85(K20)mg/kg with four replicates per level.Results showed that dietary vitamin K3 enhanced growth,with weight gain rate and specific growth rate in K15 trials significantly higher than K0 trials(P<0.05).No significant difference was observed in the content of crude protein,crude lipid,moisture,ash,and phosphorus in muscle tissue(P>0.05).However,the supplementation of vitamin K3 significantly contributed to calcium accumulation in muscle tissues and vertebrae(P<0.05)and increased red blood cell counts(P<0.05).The blood coagulation time decreased significantly with increasing dietary vitamin K3 from 0.78 to 10.82 mg/kg(P<0.05)before stabilizing(P>0.05).The antioxidant capacity was improved with the addition of vitamin K3,which was inconsistent with the changes in malondialdehyde and MK-4(a form of menaquinone)within liver and serum,suggesting that the toxicity of excessive artificial vitamin K3 may account for this difference.Using regression analysis,the appropriate dietary vitamin K3 levels within a largemouth bass diet was determined to be in the range of 9.93-15.22 mg/kg,which will provide a reference for the preparation of vitamin premix and artificial diet for largemouth bass.

工厂化循环水养殖模式对大口黑鲈肌肉营养成分和挥发性风味物质的影响

为探究工厂化循环水养殖模式对池塘养殖的大口黑鲈肌肉营养成分和挥发性风味物质的影响,将池塘养殖的大口黑鲈成鱼移入到工厂化循环水养殖系统中进行暂养,分别于暂养第1天、第10天、第20天和第30天进行取样,测定分析大口黑鲈的肌肉营养成分和挥发性风味物质。结果显示:在工厂化循环水系统中暂养不同时间对大口黑鲈肌肉中粗蛋白质和粗脂肪含量无显著影响(P>0.05),但对肌肉中氨基酸总量以及二十碳五烯酸(EPA)和二十二碳六烯酸(DHA)的含量有显著影响(P<0.05),均在暂养第30天时含量最高,且显著高于暂养第1天、第10天和第20天时(P<0.05);采用氨基酸评分(AAS)和化学评分(CS)对大口黑鲈肌肉必需氨基酸进行营养评价,AAS模式下不同暂养时间的大口黑鲈第一限制性氨基酸均为缬氨酸,CS模式下第一限制性氨基酸均为甲硫氨酸+胱氨酸。通过构建工厂化循环水系统暂养不同时间的大口黑鲈肌肉中挥发性风味物质的指纹图谱,发现肌肉中2-庚酮、2-戊酮、2-丁酮、环戊酮、1-辛烯-3-醇、1-己醇等愉悦气味物质含量随着工厂化循环水暂养时间的延长而增加。综上可知,将池塘养殖的大口黑鲈成鱼移入到工厂化循环水系统中暂养可提高大口黑鲈肌肉营养品质及风味,是一种有效提高大口黑鲈成鱼品质的技术措施,建议在池塘养殖大口黑鲈成鱼的品质提升上推广应用。

大口黑鲈饲料营养需求及鱼粉替代研究进展

水产养殖动物的精准营养需求参数对其配合饲料的优化或者开发至关重要。文章概述了大口黑鲈蛋白质、脂肪、矿物质、碳水化合物以及维生素等主要元素需求以及蛋白源替代研究,对大口黑鲈饲料配制和降低成本等有重要的指导意义。

五倍子单宁酸对大口黑鲈生长性能和肠道健康的影响

本试验旨在研究五倍子单宁酸对大口黑鲈(Micropterus salmoides)生长性能和肠道健康的影响。选取450尾体重为(10.23±0.05)g的大口黑鲈,随机分成5个组,每组3个重复,每个重复30尾。各组分别饲喂在基础饲料的基础上添加0(F0组,作为对照组)、0.05%(F1组)、0.10%(F2组)、0.15%(F3组)、0.20%(F4组)五倍子单宁酸的试验饲料,进行为期90 d的养殖试验。结果表明:1)与F0组相比,F2和F3组的增重率(WGR)和特定生长率(SGR)显著升高(P<0.05),饲料系数(FCR)显著降低(P<0.05)。各组之间肝体比(HSI)、摄食率(FR)和存活率(SR)无显著差异(P>0.05)。2)各组之间全鱼粗脂肪、粗蛋白质、粗灰分和水分含量无显著差异(P>0.05)。3)各组之间血清酸性磷酸酶(ACP)、碱性磷酸酶(AKP)、超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量无显著差异(P>0.05)。4)各组之间肠道淀粉酶、脂肪酶和胰蛋白酶活性无显著差异(P>0.05)。5)与F0组相比,其他各组肠道绒毛增多,排列紧密;在前肠组织中,F1组和F2组的隐窝深度显著低于F0组(P<0.05)。6)与F0组相比,其他各组肠道变形菌门相对丰度均有所下降,肠道菌群稳态指数均有所升高,且F3组肠道菌群稳态指数显著高于F0组(P<0.05)。综上所述,饲料中添加五倍子单宁酸可以提高大口黑鲈的生长性能,改善肠道组织结构,优化肠道菌群,保护肠道健康;以特定生长率为评价指标,大口黑鲈饲料中五倍子单宁酸的适宜添加量为0.10%~0.15%。

基于核酸适体的高通量模型筛选抗大口黑鲈虹彩病毒药物

【目的】利用可特异性识别大口黑鲈(Micropterus salmoides)虹彩病毒(Largemouth bass virus,LMBV)的核酸适体LBVA1建立药物高通量筛选技术(Aptamer LBVA1-based high-throughput screening,LBVA1-AHTS),以快速筛选有效抗LMBV药物。【方法】用探针FAM-LBVA1检测感染不同浓度[感染复数(MOI)分别为0、0.05、0.10、0.20]LMBV 48 h,以及感染LMBV 0、6、12、24和48 h的胖头鱥(Pimephales promelas)肌肉细胞(Fathead minnow cells,FHM)荧光值,并用激光共聚焦及荧光定量PCR(RT-qPCR)验证LMBV感染情况;将利巴韦林、阿昔洛韦、加巴喷丁、茶多酚、表没食子儿茶素没食子酸酯等9种药物分别以安全工作浓度与LMBV混匀,加入FHM细胞共孵育,运用探针FAM-LBVA1及RT-qPCR技术检测各组LMBV感染情况,比较两种方法检测的药物抗LMBV感染效果,筛选出有效抗LMBV药物。【结果】探针FAM-LBVA1检测及RT-qPCR技术验证结果均表明,在感染复数(MOI)为0.20、感染时间为48 h时,对LMBV感染的检测效果最佳。9种药物抗LMBV效果筛选结果表明,LBVA1-AHTS技术与RT-qPCR技术两种筛药方法筛选出利巴韦林、加巴喷丁、金刚烷胺、卡马西平、茶多酚、表没食子儿茶素没食子酸酯和表儿茶素没食子酸酯7种药物的抗病毒效果一致,其中茶多酚、表没食子儿茶素没食子酸酯和表儿茶素没食子酸酯3种药物具有抗LMBV的效果,其余4种药物无明显抗病毒效果,两种方法一致率近80%。【结论】建立的LBVA1-AHTS模型可快速、准确、高效筛选出抗LMBV病毒药物,为快速筛选抗病毒药物提供理论和技术基础。

嗜水气单胞菌感染大口黑鲈肠炎模型的建立及评价

【目的】采用嗜水气单胞菌(Aeromonas hydrophila)感染建立稳定大口黑鲈(Micropterus salmoides)的细菌性肠炎模型,并确定导致肠炎发生的细菌浓度和作用时间。【方法】150尾健康的大口黑鲈随机分为5组:对照组和4个试验组(感染浓度分别为1×10^(8)、1×10^(7)、1×10^(6)和1×10^(5) CFU/mL),通过腹腔注射方式进行人工感染。通过临床症状、剖检病变及评分、组织学变化及评分、肠道组织超微结构变化和炎症因子基因表达量评价各试验组造模效果。【结果】攻毒后试验组表现出游动慢,摄食下降的现象,且在攻毒第2和第3天时,1×10^(8) CFU/mL攻毒组有死亡现象。剖检后可见肛门红肿、外突,腹鳍和腹部皮肤出血等症状,肠道充血、出血,部分可见肠液潴留等。各组大体病理得分与细菌的感染浓度呈正相关,与感染时间呈负相关。组织学观察可见肠道固有层充血、水肿、增厚,并伴有炎性细胞浸润,黏膜上皮损伤脱落、细胞变性、坏死、增生,杯状细胞数目明显增加;组织病理得分随细菌浓度升高而升高,随感染时间呈现先上升后下降的趋势,在感染第5天得分最高。肠道组织超微结构变化显示,攻毒组肠道微绒毛损伤脱落,形态紊乱,上皮细胞排列紊乱,线粒体水肿,出现空泡,部分细胞胞浆中细胞器溶解甚至消失,杯状细胞数目增多。各试验组炎症因子表达量的变化与细菌浓度有关,IL-15、IL-8、TNF-α和IL-1β的表达量随细菌浓度的升高而升高,且随时间呈现先上升后下降的趋势。IL-10、IL-11和TGF-β_(2)的表达量与感染浓度呈负相关,其中IL-10和IL-11的基因表达量变化显著低于对照组,TGF-β_(2)的基因表达量变化不显著。【结论】1×10^(7) CFU/mL攻毒组在整个试验过程中未出现死亡,病理学观察显示肠道有较明显的病理损伤。因此,为构建接近临床条件的细菌性肠炎模型,选择攻毒剂量为200μL 1×10^(7) CFU/mL的菌液作为肠道病理损伤模型的感染浓度。

黄连素与聚β-羟基丁酸酯对大口黑鲈生长、免疫和肠道菌群的影响

【目的】探究饲料中添加黄连素(BBR)与聚β-羟基丁酸酯(PHB)对大口黑鲈(Micropterus salmoides)反季幼苗生长、免疫和肠道菌群的影响,为BBR与PHB在大口黑鲈配合饲料中的应用和大口黑鲈反季繁育提供理论依据。【方法】以大口黑鲈秋苗(体质量130.34±0.26 mg)为试验材料,以投喂基础饲料为对照(CK),B、P和MIX组分别投喂饲料B(1‰BBR)、饲料P(1‰PHB)和饲料BP(1‰BBR+1‰PHB),BP5、BP7和BP14组分别以每5、7和14 d的频率交替投喂饲料B与饲料P,养殖30 d,测定大口黑鲈生长性能、肝脏和肠道生化指标,分析肠道菌群结构及功能。【结果】在生长性能上,与CK相比,除P和MIX组外,其余处理组终末总体质量均显著提高(P<0.05,下同),饲料系数均显著降低;BP5、BP7和BP14组增重率、存活率均显著高于CK;BP7组终末总体质量、增重率和存活率均最高,饲料系数最低。在肝脏中,B、P、BP5、BP7、BP14和MIX组的ACP和AKP活性均显著高于CK,同时GOT和GPT活性均显著低于CK。在肠道中,与CK相比,其他处理组MDA和IL-6含量均显著降低,CAT和LZM含量均显著提高;BP5和BP7组TNF含量均显著低于CK。肠道菌群物种在门分类水平分布上,与CK相比,BP5、BP7和MIX组变形菌门相对丰度明显降低、厚壁菌门相对丰度明显增加,BP7组拟杆菌门相对丰度明显增加;在属分类水平分布上,与CK相比,BP5和BP7组无色杆菌属相对丰度明显降低,乳杆菌属相对丰度明显增加。BP7组微生物功能基因在代谢通路和环境信息处理通路显著富集。【结论】饲料中添加BBR可提高大口黑鲈秋苗的生长性能,BBR和PHB的添加或混合添加均能有效提高鱼体免疫能力及抗氧化能力、改善肝脏损伤和降低肠道炎症反应,以每7 d的频率交替投喂含BBR的饲料与含PHB的饲料效果最好;推测是由于BBR和PHB改变大口黑鲈肠道菌群结构及功能,改善了肝脏及肠道健康。

桑叶低聚糖对大口黑鲈生长性能以及肠道消化酶活性、免疫功能和菌群的影响

本试验旨在探究饲料中添加桑叶低聚糖(MLO)对大口黑鲈生长性能以及肠道消化酶活性、免疫功能和菌群的影响。试验选择平均初始体重为(26.89±1.16)g的大口黑鲈幼鱼450尾,随机分成3组,每组3个重复,每个重复50尾。对照组饲喂基础饲料,试验组分别饲喂在基础饲料的基础上添加0.5%和1.0%MLO的试验饲料。试验期80 d。结果表明:1)与对照组相比,试验组大口黑鲈饲料系数(FCR)和脏体比(VSI)显著降低(P<0.05),1.0%MLO组肝体比(HSI)显著降低(P<0.05)。2)与对照组相比,试验组大口黑鲈肠道α-淀粉酶、脂肪酶和胰蛋白酶活性显著提高(P<0.05),试验组肠道绒毛高度、绒毛宽度和肌层厚度均无显著差异(P>0.05)。3)与对照组相比,试验组大口黑鲈肠道菌群Sobs指数、ACE指数和Chao1指数显著降低(P<0.05);0.5%MLO组Simpson指数显著高于对照组和1.0%MLO组(P<0.05)。在门水平上,与对照组相比,试验组大口黑鲈肠道厚壁菌门(Firmicutes)相对丰度显著降低(P<0.05),肠道变形菌门(Proteobacteria)、梭杆菌门(Fusobacteriota)和拟杆菌门(Bacteroidota)相对丰度显著提高(P<0.05),厚壁菌门/拟杆菌门值显著提高(P<0.05)。在属水平上,与对照组相比,试验组肠道支原体属(Mycoplasma)相对丰度显著降低(P<0.05),肠道不动杆菌属(Acinetobacter)、乳杆菌属(Lactobacillus)和芽孢杆菌属(Bacillus)相对丰度显著提高(P<0.05)。4)与对照组相比,试验组大口黑鲈肠道白细胞介素-8(IL⁃8)、核因子-κB(NF⁃κB)和肿瘤坏死因子-α(TNF⁃α)mRNA相对表达量显著降低(P<0.05),肠道白细胞介素-10(IL⁃10)和转化生长因子-β(TGF⁃β)mRNA相对表达量显著提高(P<0.05)。综上所述,饲料中添加MLO能够降低大口黑鲈FCR、VSI和HSI,提高肠道消化酶活性,并调节肠道菌群,改善肠道益生菌相对丰度,增强其免疫功能。