Effects of Clostridium butyricum spores on growth performance, biochemical parameters in serum, intestinal flora and short-chain fatty acid content ofPaneaus vannamei
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摘要:目的
研究饲用丁酸梭菌Clostridium butyricum (CB)芽孢对凡纳滨对虾幼虾生长性能、血清生化指标、肠道菌群组成及5种短链脂肪酸含量的影响。
方法分别将质量分数为0(对照)、0.050%、0.075%和0.100%的丁酸梭菌芽孢制剂添加到基础饲料中(饲料中的活菌数分别为0、2.50×105、3.75×105、5.00×105 CFU/g)，饲喂初始体质量为(1.42±0.02) g的凡纳滨对虾幼虾30 d，然后检测生长性能、血清生化指标、肠道菌群组成以及短链脂肪酸含量。
结果与对照组相比，饲喂30 d后对虾的体质量在0.050%和0.075% CB组显著提高(P<0.05)，质量增加率则在0.050% CB组显著提高(P<0.05)；0.075% CB组对虾的血清葡萄糖、0.100% CB组的血清尿素氮浓度显著降低(P<0.05)，0.050% CB组血清磷浓度显著上升(P<0.05)。与对照组相比，0.050% CB组浮霉菌门Planctomycetes和髌骨菌门Patescibacteria丰度显著提高(P<0.05)；0.050%和0.075% CB组弧菌属Vibrio的丰度显著降低(P<0.05)。多样性分析表明，0.050%和0.075% CB组的肠道菌群组成相似度更高，且明显不同于对照组和0.100% CB组。肠道内容物中5种短链脂肪酸的含量均随丁酸梭菌添加量的升高而增加。
结论饲料中添加CB芽孢制剂可能通过抑制肠道内潜在病原菌丰度、提高肠道短链脂肪酸的含量，改善营养物质的利用，从而提高凡纳滨对虾的生长性能。本研究中CB芽孢制剂在饲料中的适宜添加量为2.50×105或3.75×105 CFU/g。
Abstract:ObjectiveTo investigate the effects of feeding Clostridium butyricum (CB) spores on the growth performance, biochemical parameters in serum, intestinal flora composition and contents of five short-chain fatty acids of Paneaus vannamei juvenile.
MethodBasic diets were supplemented with CB spore product of 0, 0.050%, 0.075% and 0.100% mass fraction (The viable counts of bacteria in the diets were 0, 2.50×105, 3.75×105 and 5.00×105 CFU/g respectively). The P. vannamei juveniles with (1.42 ± 0.02) g initial body weight of each were fed for 30 days, and growth performance, serum biochemical indicators, intestinal flora composition and short chain fatty acid contents were detected.
ResultCompared to the control group, the final body weights of the shrimps after 30 days feeding trial in the 0.050% and 0.075% CB groups significantly increased (P<0.05), and the weight gain rate significantly increased in the 0.050% CB group (P<0.05). The glucose concentration in serum of the 0.075% CB group and urea nitrogen concentration in serum of the 0.100% CB group significantly reduced (P<0.05), while the phosphorus concentration in serum of the 0.050% CB group significantly increased (P<0.05). Compared to the control group, the relative abundance of Planctomycetes and Patescibacteria significantly increased in the 0.050% CB group (P<0.05), while the relative abundance ofVibrio significantly reduced in the 0.050% and 0.075% CB groups (P<0.05). The diversity analysis indicated that the intestinal flora compositions were similar between the 0.050% and 0.075% CB groups, which were quite different from those of the control and 0.100% CB group. The intestinal contents of five short-chain fatty acids increased with dietary CB addition.
ConclusionThe supplementation of CB spore in diets helps to inhibit the proliferation of intestinal pathogens and increase the contents of intestinal short chain fatty acids, thus may enhance the nutrients utilization and growth performance of P. vannamei juvenile. In this study, the appropriate amount of Clostridium butyricum spore in diet is 2.50×105 or 3.75×105 CFU/g.
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图 1 基于5种多样性指数分析的α多样性箱形图
图中空心圆点为各样本点
Figure 1. The α diversity box plot based on analysis of five diversity indexes
The hollow dots in the figures represent individual samples
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图 2 基于主坐标分析(PCoA)的各丁酸梭菌(CB)添加组肠道菌群组成散点图
Figure 2. Scatter plot of intestinal flora composition in groups added with different amounts of Clostridium butyricum (CB) based on principal coordinates analysis (PCoA)
表 1 基础饲料配方及营养组成(风干基础)
Table 1 Feed formulation and nutrient composition of the basal diet (air-dry basis)
项目 Item 成分 Ingredient w/% 原料 Ingredient 鱼粉 Fish meal 36.0 小麦粉 Wheat flour 25.5 去皮豆粕 Soybean meal 15.0 花生粕 Peanut meal 8.0 菜籽粕 Rapeseed meal 10.0 鱼油 Fish oil 1.0 大豆卵磷脂 Soybean lecithin 1.0 磷酸二氢钙 Ca(H2PO4)2 1.5 预混料1) Premix 2.0 合计 Total 100.0 实测营养成分 Measured nutrient 粗蛋白 Crude protein 41.5 粗脂肪 Crude lipid 6.5 粗灰分 Crude ash 9.5 水分 Mositure 10.0 1)每千克预混料含有：VA 4 000 000 IU，VD3 1 500 000 IU，VE 100 000 IU，硫胺素 10 g，核黄素30 g，B6 30 g，泛酸 60 g，烟酸 80 g，B12 0.04 g，生物素 0.2 g，叶酸 6 g，肌醇120 g，VC 150 g，MgSO4·7H2O 102.45 g，KI 0.08 g，ZnSO4·7H2O 4.05 g，MnSO4·5H2O 2.19 g，CoCl2·6H2O 0.5 g，FeSO4·7H2O 16.55 g，Na2SeO4 0. 004 g，Ca(H2PO4)2 251.67 g 1)The premix per kilogram provides the following: VA 4 000 000 IU, VD3 1 500 000 IU, VE 100 000 IU, thiamine 10 g， riboflavin 30 g, B6 30 g， pantothenic acid 60 g, nicotinic acid 80 g, B12 0.04 g， biotin 0.2 g, folic acid 6 g， inositol 120 g, VC 150 g, MgSO4·7H2O 102.45 g, KI 0.08 g，ZnSO4·7H2O 4.05 g, MnSO4·5H2O 2.19 g，CoCl2·6H2O 0.5 g, FeSO4·7H2O 16.55 g，Na2SeO4 0. 004 g, Ca(H2PO4)2 251.67 g 
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表 2 饲料中添加丁酸梭菌(CB)对凡纳滨对虾生长性能的影响1)
Table 2 Effects of dietary Clostridium butyricum (CB) on growth performance of Paneaus vannamei
w(CB)/% 初质量/g Initial body weight 末质量/g Final body weight 存活率/% Survival rate 质量增加率/% Weight gain rate 饲料系数 Feed conversion rate 0(CK) 1.42±0.00a 7.03±0.14a 96.66±3.85a 394.53±9.51a 1.50±0.04a 0.050 1.41±0.00a 7.37±0.08b 97.50±1.67a 421.00±5.31b 1.44±0.04a 0.075 1.44±0.03a 7.37±0.03b 99.17±1.67a 410.43±8.65ab 1.45±0.08a 0.100 1.42±0.01a 7.31±0.07ab 100.00±0.00a 412.99±5.84ab 1.43±0.03a 1) 同列数据后的不同小写字母表示差异显著(P<0.05，Duncan’s 法) 1) Different lowercase letters of the same column indicate significant differences (P<0.05, Duncan’s method)
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表 3 饲料中添加丁酸梭菌(CB)对凡纳滨对虾营养组成的影响1)
Table 3 Effects of dietaryClostridium butyricum (CB) on nutrient compositions ofPaneaus vannamei
w/% w(CB)/% 水分 Mositure 粗蛋白 Crude protein 粗脂肪 Crude lipid 粗灰分 Crude ash 0(CK) 272.93±14.82a 70.57±0.80a 7.33±0.20a 12.87±0.99a 0.050 261.06±7.97a 70.63±0.12a 7.28±0.11a 12.73±0.23a 0.075 280.64±2.11a 70.24±0.37a 7.12±0.36a 13.10±0.60a 0.100 285.98±12.38a 70.56±0.18a 6.36±0.90a 13.60±0.67a 1)同列数据后的不同小写字母表示差异显著(P<0.05，Duncan’s 法) 1) Different lowercase letters of the same column indicate significant differences (P<0.05，Duncan’s method)
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表 4 饲料中添加丁酸梭菌(CB)对凡纳滨对虾血清生化指标的影响1)
Table 4 Effects of dietaryClostridium butyricum (CB) on biochemical parameters in serum of Paneaus vannamei
w(CB)/% c/(mmol·L−1) 总蛋白/ (U·L−1) Total protein 葡萄糖 Glucose 胆固醇 Cholesterol 甘油三酯 Triglyceride 尿素氮 Urea nitrogen 磷 Phosphorus 0(CK) 1.69±0.15b 2.21±0.23a 2.05±0.15a 1.23±0.17b 1.44±0.32a 64.38±3.13a 0.050 1.62±0.29ab 2.28±0.18a 2.09±0.16a 1.04±0.06ab 1.93±0.31b 66.43±2.94a 0.075 1.39±0.13a 1.83±0.18a 1.97±0.21a 1.11±0.14ab 1.79±0.24ab 66.53±3.83a 0.100 1.55±0.10ab 2.05±0.23a 2.03±0.19a 0.95±0.06a 1.80±0.26ab 65.47±3.72a 1) 同列数据后的不同小写字母表示差异显著(P<0.05，Duncan’s 法) 1) Different lowercase letters of the same column indicate significant differences (P<0.05, Duncan’s method)
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表 5 饲料中添加丁酸梭菌(CB) 对凡纳滨对虾肠道菌群组成(门水平)的影响1)
Table 5 Effects of dietary Clostridium butyricum (CB) on intestinal flora composition (phylum level) of Paneaus vannamei
w(CB)/% 变形菌门 Proteobacteria 拟杆菌门 Bacteroidetes 疣微菌门 Verrucomicrobia 浮霉菌门 Planctomycetes 厚壁菌门 Firmicutes 0(CK) 43.9±3.9a 41.9±3.2a 6.8±1.3a 2.8±0.4a 1.5±0.8a 0.050 36.0±2.0a 46.3±1.7a 6.7±0.7a 5.7±0.6b 0.5±0.2a 0.075 36.5±1.5a 43.0±3.5a 8.1±1.8a 7.1±0.5b 0.4±0.1a 0.100 43.2±4.3a 42.4±3.5a 5.7±1.2a 4.6±0.8b 0.7±0.3a w(CB)/% 放线菌 Actinobacteria 衣原体 Chlamydiae 髌骨菌门 Patescibacteria 埃普西隆杆菌门 Epsilonbacteraeota 柔膜菌门 Tenericutes 0(CK) 1.1±0.1a 0.8±0.4a 0.4±0.0a 0.2±0.1a 0.2±0.0a 0.050 1.9±0.7a 1.3±0.2a 1.1±0.3b 0.0±0.0a 0.0±0.0a 0.075 2.1±1.1a 1.7±0.7a 0.5±0.1a 0.0±0.0a 0.1±0.0a 0.100 0.8±0.1a 1.1±0.6a 0.3±0.0a 0.0±0.0a 0.2±0.1a 1)同列数据后的不同小写字母表示差异显著(P<0.05，Duncan’s 法) 1) Different lowercase letters of the same column indicate significant differences (P<0.05, Duncan’s method)
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表 6 饲料中添加丁酸梭菌(CB)对凡纳滨对虾肠道菌群组成(属水平)的影响1)
Table 6 Effects of dietary Clostridium butyricum (CB) on intestinal flora composition (genus level) of Paneaus vannamei
w(CB)/% 弧菌属 Vibrio Motilimonas Haloferula 黏着杆菌属 Tenacibaculum Tamlana 0(CK) 7.8±3.6b 6.4±1.9a 6.3±1.3a 5.9±0.8a 5.3±0.7a 0.050 1.2±0.3a 6.8±3.0a 6.2±0.8a 4.3±0.3a 7.6± 0.7a 0.075 1.1±0.2a 8.5±1.6a 7.7±1.8a 5.1±0.4a 6.1± 0.8a 0.100 3.8±2.5ab 6.4±1.0a 5.0±1.2a 4.1±0.7a 6.7±0.5a w(CB)% 假交替单胞菌属 Pseudoalteromonas 鲁杰氏菌属 Ruegeria Hoppeia 康氏菌属 Kangiella Halocynthiibacter 0(CK) 2.6±0.5ab 2.6±0.3a 2.5±0.8a 2.2±1.5a 1.6±0.5a 0.050 1.5±0.3a 3.4±0.5a 4.1±0.4a 2.0±0.5a 1.1±0.3a 0.075 2.3±0.4a 4.1±0.9a 3.9±0.8a 0.8±0.2a 1.6±0.3a 0.100 3.7±0.6b 2.5±0.5a 4.7±2.1a 1.3±0.6a 1.4±0.1a 1)同列数据后的不同小写字母表示差异显著(P<0.05，Duncan’s 法) 1) Different lowercase letters of the same column indicate significant differences (P<0.05, Duncan’s method)
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表 7 饲料中添加丁酸梭菌(CB)对凡纳滨对虾肠道内短链脂肪酸含量的影响1)
Table 7 Effects of dietaryClostridium butyricum (CB) on intestinal short-chain fatty acid contents ofPaneaus vannamei
w/(mg·g−1) w(CB)/% 乙酸 Acetic acid 丙酸 Propanoic acid 丁酸 Butyric acid 戊酸 Pentanoic acid 己酸 Hexanoic acid 0(CK) 5.32±0.31a 0.30±0.04a 0.20±0.00a 1.13±0.02a 0.35±0.00a 0.050 6.99±0.24b 0.32±0.03a 0.18±0.01a 1.24±0.07a 0.37±0.01a 0.075 10.54±0.61c 0.42±0.03a 0.33±0.00b 2.21±0.14b 0.59±0.01b 0.100 15.03±0.31d 1.10±0.08b 0.32±0.01b 2.72±0.11c 0.76±0.01b 1)同列数据后的不同小写字母表示差异显著(P<0.05，Duncan’s 法) 1) Different lowercase letters of the same column indicate significant differences (P<0.05, Duncan’s method)
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