饲料中补充蜡样芽孢杆菌对凡纳滨对虾生长及其肠道微生物组成的影响

为了研究外源补充蜡样芽孢杆菌对凡纳滨对虾生长及对其肠道微生物组成的影响,以基础饲料为空白组,在基础饲料中添加蜡样芽孢杆菌活菌作为实验组(活菌含量为108 CFU/g),每组4个平行,在养殖场对凡纳滨对虾自幼苗至成虾进行一个完整养殖周期的养殖实验。选取养殖场中8个光照、位置等条件基本相同的养殖池分为2组,空白组投喂基础饲料,实验组投喂免疫饲料,养殖周期95 d。期间在第45、52、59、66、73天采样,称重并计算生长速率,并取其肠道内容物提取DNA,用16S r DNA序列V4区高通量测序方法检测对虾肠道内微生物群落的结构及变化情况。结果显示:(1)投喂免疫饲料的实验组对虾生长速度比空白组平均快15.2%。(2)健康的凡纳滨对虾肠道微生物以变形菌门和拟杆菌门含量高,厚壁菌门、疣微菌门、浮霉菌门、放线菌门含量较低。(3)空白组变形菌门平均含量(68.30%),明显高于实验组平均含量(57.94%);空白组拟杆菌门平均含量(23.58%)明显低于实验组平均含量(30.06%)。(4)变形菌门中以弧菌属、Anaerospora、小红卵菌属为主,空白组弧菌属平均占总含量的5.40%,实验组平均占总含量的1.94%;拟杆菌门中以异养硝化菌属、粘着杆菌属为主,实验组异养硝化菌属平均占总含量的8.12%,空白组平均占含量的5.56%。研究表明,饲料中添加蜡样芽胞杆菌投喂凡纳滨对虾后可改变对虾肠道的微生物组成,提高对虾生长速度。     

益生菌对凡纳滨对虾免疫功能及肠道菌群的影响

以类芽孢杆菌和双歧杆菌为益生菌,添加到凡纳滨对虾(平均体质量3.45g)的饲料中,饲养28d(水温25~27℃,盐度25~26,pH 7.8~8.1,溶解氧6mg/L),采用南京建成试剂盒和变性梯度凝胶电泳技术,研究益生菌对凡纳滨对虾免疫功能及肠道菌群的影响。结果表明,益生菌能够显著提高对虾血清过氧化物酶(对照组43.22U/mL,试验组76.33U/mL)、超氧化物歧化酶(对照组60.61U/mL,试验组101.18U/mL)、一氧化氮合酶(对照组7.54U/mL,试验组12.44U/mL)、酸性磷酸酶(对照组0.0446U/mL,试验组0.0574U/mL)和碱性磷酸酶(对照组0.0183U/mL,试验组0.0236U/mL)的活性(P0.01),明显改善对虾肠道的菌群组成,从而提高对虾抵抗疾病的能力。     

养殖水体中添加蜡样芽孢杆菌PC465 对凡纳滨对虾抗病力的影响

将蜡样芽胞杆菌(Bacillus cereus)PC465添加到凡纳滨对虾(Litopenaeus vannamei)养殖水体中至终浓度分别为10~4、10~5和10~6 CFU/mL,以无益生菌添加的养殖组为对照组,定期检测实验对虾肠道和养殖水体中的细菌总数、弧菌总数以及水体中氨氮含量和亚硝酸氮含量。实验结果表明,水体中添加益生菌能降低凡纳滨对虾肠道内细菌数量,且与对照组相比差异显著(P0.05);高浓度的益生菌处理组可以明显降低养殖水体内弧菌数量(P0.05),但是益生菌并没有显著影响水体中的氨氮含量和亚硝酸氮含量。养殖4周后进行WSSV投喂感染实验,感染实验表明,实验组H组(益生菌浓度为10~6 CFU/mL)和M组(益生菌浓度为10~5 CFU/mL)凡纳滨对虾的累计死亡率分别为63.9%和74.6%,显著低于对照组100%的累计死亡率(P0.05)。感染实验期间采用荧光定量PCR方法测定了凡纳滨对虾3种免疫相关基因的表达情况,统计数据显示,益生菌处理组的脂多糖-β-1,3-葡聚糖结合蛋白(lipopolysaccharide-β-1,3-glucan-binding protein,LGBP)、β-1,3-葡聚糖结合蛋白-脂蛋白(beta-1,3-glucan-binding protein-lipoprotein,βGBP-HDL)、热激蛋白70(heat shock proteins,Hsp70)mRNA的表达量在WSSV感染后呈显著上调趋势。实验结果提示,水体中添加蜡样芽孢杆菌PC465可以提高凡纳滨对虾抗WSSV感染能力,其作用机制可能是降低对虾肠道和水体中的细菌和弧菌数量,或调节免疫相关基因的表达水平。     

不同养殖模式对凡纳滨对虾肠道微生物群落的影响

为了探究不同养殖模式下凡纳滨对虾肠道微生物群落组成的差异,本研究采用16S rRNA扩增子测序技术对4种不同养殖模式[淡水池塘养殖模式(FWP)、海水池塘养殖模式(SP)、高位池养殖模式(HP)和生物絮团养殖模式(BFP)]下凡纳滨对虾的肠道微生物多样性和群落结构进行了研究.结果 显示,FWP和HP下凡纳滨对虾肠道菌群...     

蜡样芽孢杆菌对凡纳滨对虾幼体变态的影响

研究了1株蜡样芽孢杆菌zou8对数期细胞对凡纳滨对虾无节幼体Ⅲ期到溞状幼体Ⅱ期幼体变态的影响;并通过zou8与弧菌的平板拮抗、共培养试验探讨其促进幼体变态的机制是否与拮抗病原弧菌有关。结果表明:高数量zou8细胞对凡纳滨对虾无节幼体Ⅲ期到溞状幼体Ⅱ期幼体的变态具有极显著(P<0.01)促进作用,尤其是多次加菌;zou8促进幼体变态作用的发挥并非通过产生拮抗物质直接抑制病原弧菌,而是通过其他可能机制。     

枯草芽孢杆菌改善水质提高凡纳滨对虾幼体抗逆性的研究

投放不同浓度梯度的枯草芽孢杆菌于凡纳滨对虾幼体养殖环境中,监测不同枯草芽孢杆菌使用浓度下与养殖环境相关的水质因子（氨氮、亚硝酸盐氮、化学需氧量）的变化;观察不同枯草芽孢杆菌使用浓度下凡纳滨对虾幼体对人工制造的胁迫环境的抗逆性;观测不同枯草芽孢杆菌使用浓度下凡纳滨对虾幼体的成活率与体重增长率。研究枯草芽孢杆菌对水质改善和凡纳滨对虾幼体抗逆性的影响。结果表明,枯草芽孢杆菌能显著（P〈0.05）降低化学需氧量、氨氮含量,抑制亚硝酸盐氮的产生;当枯草芽孢杆菌投放浓度为1.25×10^4cfu/ml时,水体中化学需氧量、氨态氮、亚硝酸盐氮含量均值比对照组分别降低了65.30%、59.70%、88.64%,成活率比对照组提高了10.00%,体重增长率是对照组的2.44倍;当枯草芽孢杆菌使用浓度为1.25×10^4～1.25×10^6cfu/ml时,对虾抗逆性显著（P〈0.05）增强,对虾在氨氮、亚硝酸盐氮、高锰酸钾、甲醛胁迫下24小时的成活率均值分别比对照组提高了16.94%、24.44%、44.17%、18.61%。     

一种赖氨酸芽孢杆菌对凡纳滨对虾生长和水质影响

为探讨一种赖氨酸芽孢杆菌对凡纳滨对虾生长、存活和养殖水质的影响。设1个试验组和1个对照组,每组3个平行,125尾对虾/桶,初始体质量(5.18±0.13) g、体长(9.40±0.18) cm、水温(26±2)℃、盐度30、试验时间10 d。试验组定期添加浓度为0.463×10⁹ CFU/mL的赖氨酸芽孢杆菌菌液,试验组和对照组NH⁺₄-N质量浓度变化范围分别为(0.05±0.01)～(5.51±0.09) mg/L、(0.05±0.01)～(6.07±0.22) mg/L。NO^-₂-N质量浓度变化范围分别为(0.07±0.01)～(0.13±0.01) mg/L、(0.07±0.01)～(0.19±0.01) mg/L。NO^-₃-N质量浓度变化范围分别为(0.07±0.01)～(0.81±0.07) mg/L、(0.07±0.01)～(1.26±0.07) mg/L。pH的变化范围分别为(7.10±0.01)～(7.74±0.01)...     

高通量测序法分析两株益生菌对凡纳滨对虾肠道菌群结构的影响

为分析荚膜红细菌和蜡样芽孢杆菌2株益生菌对凡纳滨对虾肠道菌群结构的影响,本实验对凡纳滨对虾进行为期30 d的养殖饲喂实验,饲喂后期利用高通量测序凡纳滨对虾肠道微生物的16S rDNA V4区,来分析不同益生菌饲喂后凡纳滨对虾肠道菌群的结构特征,并结合对虾体质量增加率和攻毒后累计死亡率的宏观指标来进行分析。结果显示：①空白组（CK）、荚膜红细菌组（Rc）和蜡样芽孢杆菌组（Bc）样品OTU范围为374~506,其中CK组对虾肠道菌群OTU数量最低,饲喂益生菌后的2组对虾肠道菌群中OTU数量相对较高;②在门分类水平上,3组的变形菌门数均为最多,CK组主要为变形菌门和少量拟杆菌门,Bc组主要有变形菌门、拟杆菌门、无壁细菌门、厚壁细菌门和假单胞菌,Rc组主要是变形菌门、拟杆菌门、厚壁菌门、酸杆菌门、放线细菌、梭杆菌门和蓝细菌;③稀释曲线和Shannon指数结果可见,CK组样品物种丰度和复杂度最低,且Bc组样品丰度和复杂度相对较高;④PCA分析发现,Rc组和CK组样品微生物组成较为接近,结合宏观对虾体质量增加率和攻毒后累计死亡率的结果分析,可见相较于荚膜红细菌,蜡样芽孢杆菌对凡纳滨对虾肠道微生物菌群影响更显著,且益生效果更佳。研究表明,饲喂益生菌可以扩增对虾肠道微生物菌群丰度,并能抑制弧菌属等有害菌群的生长,提高对虾体质量增加率并降低死亡率,从而达到益生效果,其中以饲喂蜡样芽孢杆菌菌株效果更佳。     

枯草芽孢杆菌对水质及凡纳滨对虾幼体免疫指标影响的研究

研究了不同浓度的枯草芽孢杆菌（Bacillus substilis）对养殖环境水质和凡纳滨对虾（Penaeus vannamei）幼体抗病力相关酶活性的影响。结果表明,枯草芽孢杆菌能显著（P〈0．05）降低化学需氧量（COD）和氨氮（NH3-N）含量,抑制亚硝酸氮（NO2-N）的产生;当枯草芽孢杆菌投放浓度为1．25×10^4cfu·mL^-1时,水体中COD、NH3-N和NO2-N含量均值比对照组分别降低了65．30％、59．70％和88．64％;20d后测定对虾的碱性磷酸酶（AKP）、过氧化物酶（POD）、酚氧化酶（PO）、超氧化物歧化酶（SOD）、抗菌和溶菌活力,各值相对于对照组分别提高了3．67、1．64、0．45、3．06、2．57和1．14倍;成活率比对照组提高了10％,增重率是对照组的2．44倍。因此,一定浓度的枯草芽孢杆菌能改善凡纳滨对虾幼体的养殖水质进而提高凡纳滨对虾幼体的抗病力。     

密度和饲料种类对凡纳滨对虾Litopenaeus vannamei争胜行为和生长的影响

在水温(23.0±1.0)℃和盐度(26±1.0)‰下,将体长(46.1±3.2)mm的凡纳滨对虾Litopenaeus vannamei饲养在0.54m×0.36m×0.28m的塑料水槽中,密度为40尾/m~2(LLD)、80尾/m~2(MLD)、120尾/m~2(MHD),和160尾/m~2(HHD),投喂人工配合饲料;同时在密度50尾/m2下分别投喂人工配合饲料(ACF)、菲律宾蛤仔肉(RPF)和冰鲜杂鱼(FFF),测定投喂前1h、投喂时、投喂后1h对虾间的争斗数量、争斗时间、争斗发起方、胜利次数等指标,以探讨密度和饲料种类对凡纳滨对虾争胜行为和生长性能的影响。61d的饲养结果表明:凡纳滨对虾个体间的争斗次数和胜利次数随密度的升高而增加;不同密度间平均优势指数差异不显著(P0.05);饱食情况下凡纳滨对虾对三种饲料的选择无倾向性。     

Effects of the probiotic Bacillus subtilis (BP11 and BS11) on the growth and survival of Pacific white shrimp,Litopenaeus vannamei

The effect of Bacillus S11 (BS11)‐ and/or Bacillus P11 (BP11)‐supplemented feeds on the growth performance, survival, immunoenhancement and disease resistance of cultured Pacific white shrimp, Litopenaeus vannamei, was evaluated. Four feeding treatments of (i) regular feed (control), (ii) BS11‐supplemented feed, (iii) BP11‐supplemented feed and (iv) BS11‐and BP11‐supplemented feed were prepared and used for shrimp cultivation in closed recirculating cement tanks (~400 L) in two trials, one for juvenile and PL‐30 shrimp at 60 and 90 days, respectively. The results showed that BS11 gave a higher probiotic potential than BP11 for both age groups of L. vannamei in cultivation, because the average weight and survival of shrimp fed BS11‐supplemented feed were significantly higher (P < 0.05) than those of the control and the other two groups. The survival of shrimp fed either BS11‐or both BS11‐and BP11‐supplemented feed was significantly higher (P < 0.05) than that of the control group. In addition, the highest total haemocyte and granular haemocyte counts and phenoloxidase activity were found in shrimp fed with the BS11‐supplemented feed. After challenge with Vibrio harveyi 639 (~10⁷ CFU mL^?1) by immersion, the lowest cumulative death (%) and disease resistance were clearly found in shrimp fed with the BS11‐supplemented feed.     

Effect of dietary probiotics on apparent digestibility coefficients of nutrients of white shrimp Litopenaeus vannamei Boone

Apparent digestibility coefficients (ADCs) of dry matter (DM), crude protein (CP), lipid, phosphorus (P), essential amino acids (EAA), non‐essential amino acids (NEAA) and fatty acids were determined for white shrimp (Litopenaeus vannamei). A probiotic (Bacillus sp.) was used at 0% (diet 1), 0.5% (diet 2), 1.0% (diet 3), 3.0% (diet 4) and 5.0% (diet 5) inclusion rates in experiment diets. Apparent digestibility coefficients was determined using 0.01% Y₂O₃ as an indicator. A total of 300 shrimp (initial mean body weight 11.5±0.6 g) were randomly stocked into fifteen 500‐L outdoor concrete tanks. Faeces were collected from three replicate groups of shrimp three times a day. The ADCs of five diets were: DM, 66.1–69.6%; CP, 80.4–84.7%; lipid, 82.2–84.9%; P, 25.4–28.5%; EAA, 82.0–85.4%; NEAA, 81.5–85.1%; saturated fatty acids (SFA), 79.6–82.4%; monounsaturates (MUFA), 81.6–84.2% and polyunsaturates (PUFA)+highly unsaturates (HUFA), 83.4–86.6%. Apparent digestibility coefficients of DM, CP, lipid, P, amino acids and fatty acids in diet 1 were significantly lower than in other diets (P<0.05). Apparent digestibility coefficient of P in diets increased as probiotic increased. Apparent digestibility coefficient of lipid in diet 3 was the highest, but the difference was not significant among diets. In general, ADCs of most amino acids and fatty acids were over 80% in all diets, and PUFA+HUFA>MUFA>SFA. The results showed that ADCs of diets supplemented with 10, 30 and 50 g probiotic kg^?1 basal diet were higher than in other diets except lipid, and the difference was significant.     

芽孢杆菌合生素在对虾集约化养殖中的应用

将芽孢杆菌合生素作为饲料添加剂应用于凡纳滨对虾(Litopenaeus vannamei)的集约化养殖中,试验组A、B的合生素添加量分别为0·3%和0·5%。结果发现,试验组与对照组相比对虾的单位产量显著提高(P<0·05),其中A组增产35·18%,B组增产42·31%,饲料系数则明显降低(P<0·05)。在对虾的成活率和增重率方面,A组分别提高了15·57%和53·34%,B组则分别提高24·02%和49·28%。以对虾的肥满度(体重/体长×100)作为衡量对虾品质的一项指标,发现A组和B组比对照组分别提高了41·60%和42·48%。由于芽孢杆菌合生素的使用,两试验组的排换水量和渔药使用量比对照组均有所减少,进而降低了养殖风险和生产成本。但两试验组之间的对虾产量、饵料系数、成活率、增重率、肥满度和生产成本等各项指标均无显著差异(P>0·05)。     

饲料中补充蜡样芽孢杆菌(Bacillus cereus)生物膜对凡纳滨对虾(Litopenaeus vannamei)生长、抗病力及其肠道微生物组成的影响

为了研究外源补充蜡样芽孢杆菌(Bacillus cereus)生物膜对凡纳滨对虾(Litopenaeus vannamei)生长、抗病力及对其肠道微生物组成的影响,以基础饲料为空白组,在基础饲料中添加蜡样芽孢杆菌游离态活菌作为游离态组(活菌含量为108 CFU/g);在基础饲料中添加蜡样芽孢杆菌活菌生物膜作为生物膜组(活菌含量为108 CFU/g);每组8个平行.在养殖大棚暂养7d后,对凡纳滨对虾进行40 d的养殖实验.在第17天进行白斑综合征(White Spot Syndrome Virus,WSSV)攻毒实验;第22天进行副溶血弧菌(Vibrio parahaemolyticus)攻毒实验.期间在第1、5、10、15天采样,称重并测量体长计算生长速率.在第1、5、10、15、20、25天采样,并取其肠道内容物提取DNA,用16S rDNA序列V3+V4区高通量测序方法检测对虾肠道内微生物群落的结构及变化情况.结果显示,生物膜组和游离态组对虾体重、体长增长速率高于空白组,生物膜组与游离态组对虾体重、体长差异性不显著,生物膜组和游离态组对虾体重、体长与空白组相比差异性显著.实验中凡纳滨对虾肠道微生物由变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、放线菌门(Actinobacteria)、厚壁菌门(Firmicutes)等组成,其中,变形菌门平均占到总量的94.0％.变形菌门中主要以弧菌属(Vibrio)、发光杆菌属(Photobacterium)、Octadecabacter等为主,生物膜组、游离态组、空白对照组弧菌属平均含量分别为34.65％、39.27％、58.00％.在WSSV攻毒实验中,生物膜组、游离态组、空白对照组平均累积死亡率分别为80.0％、77.0％、92.0％,各组差异性不显著(p＞0.05).在副溶血弧菌攻毒实验中,生物膜组、游离态组、空白对照组平均累积死亡率分别为61.3％、75.0％、77.3％,生物膜组与游离态组和空白对照组相比差异性显著(P＜0.05).研究表明,饲料中添加蜡样芽孢杆菌生物膜和游离态蜡样芽孢杆菌投喂凡纳滨对虾后,可改变对虾肠道的微生物组成,提高凡纳滨对虾生长速度、增强抗病能力,而且添加蜡样芽孢杆菌生物膜效果最明显.     

Effects of Pediococcus pentosaceus supplementation on growth performance,intestinal microflora and disease resistance of white shrimp,Litopenaeus vannamei

Litopenaeus vannamei is economically important shrimp species in worldwide aquaculture. This study was conducted to assess the effect of different levels of probiotic Pediococcus pentosaceus (PP) on growth performance, feed utilization, digestive enzyme activity, intestinal microflora count and body composition of L. vannamei. Four diets containing different concentrations [0 (PP0), 10⁶ (PPI), 10⁷ (PPII) and 10⁸ (PPIII) CFU/g] of PP were formulated. After 8 weeks feeding trial, the experimental shrimps were challenged with Vibrio anguillarum and noted the surveillance. At the end of the feeding trial, the obtained results revealed a significant increase (p < .05) in final body weight, final length, weight gain (WG), survival rate, protease and amylase activities, lactobacillus sp. and Bacillus sp. intestinal count, total haemocyte counts (THC) and lysozyme activity in PPIII group when compared with the other groups. Similarly, WG, amylase activity, Bacillus sp. count, THC and lysozyme activity were significantly enhanced in case of PPII compared to the control group (p < .05). Interestingly, FCR and Vibrio sp. counts were significantly decreased in case of PPIII group when compared to the other groups (p < .05). Also, significant level of surveillance was noted in the challenging test with V. anguillarum. These results suggested that the P. pentosaceus improved the growth performance, digestive enzyme activity, immunity and tolerance against V. anguillarum of L. vannamei.     

水解单宁对凡纳滨对虾生长性能和肠道微生物的影响

为分析水解单宁对凡纳滨对虾生长及其肠道微生物菌群结构的影响,以基础饲料为空白组,在基础饲料中分别添加0.1%、0.2%和0.3%的水解单宁作为实验组,进行为期60 d的养殖实验后,统计生长性能,并取其肠道内容物提取DNA,用Illumina MiSeq测序平台进行16S rDNA基因V3～V4区高通量测序,检测对虾肠道内菌群结构及变化情况。结果表明:(1) 3个水解单宁添加组的增重率、特定生长率和肥满度与对照组相比均显著升高(P0.05),肝体比均显著降低(P0.05)。(2)4组样品中共获得206192条优化序列,操作分类单元(OUT)总数达542个。对照组凡纳滨对虾肠道微生物以变形菌门和蓝细菌门为主,其次是放线菌门和拟杆菌门;试验组肠道主要菌群为变形菌门、软壁菌门。试验组与对照组相比,蓝细菌门、放线菌门和拟杆菌门的比例降低,变形菌门、软壁菌门和厚壁菌门比例增加。(3)Rank-Abundance曲线和多样性指数结果可见,实验组的物种丰度和均匀度大体上均高于对照组。PCoA分析发现, 0.1%和0.3%添加组的微生物群落较为接近,而与0.2%添加组差别较大,结合生长性能指标可知,饲料中水解单宁的最适添加量为0.1%。以上研究表明,饲料添加水解单宁可显著改变对虾肠道的微生物组成,提高对虾生长速度,影响凡纳滨对虾的生长性能。     

Effect of dietary supplementation with Bacillus subtilis on the growth,performance, immune response and antioxidant activities of the shrimp (Litopenaeus vannamei)

The objective of this study was to evaluate the effect of dietary supplementation of a probiotic bacterium, Bacillus subtilis, on the growth, immune response and antioxidant activities of shrimp (Litopenaeus vannamei). Shrimps with an average initial weight of 2.11±0.17 g were randomly assigned to four groups with three replicates. The control group was fed a basal diet, and three treated groups were fed diets supplemented with B. subtilis at doses of 1 × 10⁴, 5 × 10⁴ and 10 × 10⁴ colony‐forming unit (CFU) g^?1 feed respectively. After 40 days of culture, 10 shrimps from each replicate were taken randomly for the determination of immune response and oxidization resistance indices. The results showed that the shrimps fed with B. subtilis at a dose of 1 × 10⁴, 5 × 10⁴ CFU g^?1 feed showed significantly better growth than that of the control diet. The phenoloxidase activities showed a tendency to increase with an increased dose of B. subtilis in diets but there was no significant difference among the three treated groups. In addition, phenoloxidase activities were found to be significantly higher (P<0.05) in the groups treated with B. subtilis than that of the control group. Shrimps treated with 5 × 10⁴ CFU g^?1 feed probiotic bacterium showed the highest lysozyme activity and it was significantly higher (P<0.05) than the other groups. However, there was no significant difference in acid phosphatase and alkaline phosphatase activity across all the groups. The total antioxidant capacity, superoxide dismutase and glutathione peroxidase activities in the probiotic‐treated groups were significantly increased (P<0.05) as compared with the control groups. Both maleic dialdehyde concentration and superoxide anion activities in the probiotic‐treated groups were significantly lower (P<0.05) than those of the control. The probiotic did not affect the nitric oxide synthase and the catalase activity in any of the control and treated groups. These results indicated that the probiotic B. subtilis could significantly promote the growth rate of shrimp by increasing the immune function and antioxidant capacity. The most effective dose of B. subtilis in the diet was 5 × 10⁴ CFU g^?1 feed.