Effects of Low Protein Diet Supplemented with Glutamate and Arginine on Intestinal Metabolites and Microbiota of Weaned Piglets
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摘要:
目的 探讨较大程度降低饲粮粗蛋白质（Crude protein，CP）水平后添加必需氨基酸、谷氨酸和精氨酸对断奶仔猪肠道代谢产物和微生物菌群的影响。 方法 试验选用192头、日龄（26±2） d的健康“杜长大”三元杂交断奶仔猪，平均初始体重为（6.96±0.29） kg，随机分成4个处理组，每组6个重复，每个重复8头猪，试验期16 d。组I为对照组，饲粮CP水平为21.16%；组II 为低CP饲粮组，饲粮CP水平为15.97%，补充必需氨基酸（L-赖氨酸、DL-蛋氨酸、L-苏氨酸、L-色氨酸、L-缬氨酸、L-异亮氨酸）；组III在组II 基础上添加1.50%谷氨酸（Glu）；组IV在组II 基础上添加1.50% Glu和1.00%精氨酸（Arg）。于正式试验期第11～13 d测定各处理断奶仔猪肠道代谢产物成分及微生物群落组成。 结果 组II 、组III和组IV断奶仔猪粪样腐胺和亚精胺含量均显著低于组I（P＜0.05），组II、组III和组IV间差异不显著（P＞0.05），尸胺含量组II 和组III显著低于组I和组IV（P＜0.05），组I显著低于组IV（P＜0.05），组II 和组III间差异不显著（P＞0.05）。粪样乙酸和丙酸含量各组间差异不显著（P＞0.05），丁酸含量组II 、组III和组IV显著高于组I（P＜0.05），组II 、组III和组IV间差异不显著（P＞0.05）。仔猪肠道菌群丰富度和多样性组II 、组III和组IV显著高于组I（P＜0.05）。仔猪粪样菌群组成在门分类水平上，组II 、组III和组IV厚壁菌门（Firmicutes）相对丰度显著高于组I（P＜0.05），拟杆菌门（Bacteroidetes）和变形菌门（Proteobacteria）相对丰度显著低于组I（P＜0.05）。在科属分类水平上，各组菌群组成亦有较大差异，组II 、组III和组IV梭菌目下未明确的科和梭菌目下未明确的属相对丰度显著高于组I（P＜0.05），普雷沃氏菌科（Prevotellaceae）、肠杆菌科（Enterobacteriaceae）和丁酸球菌属（Lachnospiraceae）相对丰度显著低于组I（P＜0.05），组IV毛螺菌科（Lachnospiraceae）、乳杆菌科（Lactobacillaceae）和布劳特氏菌属（Blautia）、乳杆菌属（Lactobacillus）相对丰度显著高于组I（P＜0.05），组IV消化链球菌科（Peptostreptococcaceae）和土孢杆菌属（Terrisporobacter）相对丰度显著低于组I（P＜0.05）。 结论 将饲粮CP水平从21.16%降低至15.97%，平衡重要必需氨基酸并补充Glu+Arg，能降低断奶仔猪肠道生物胺含量，提高丁酸含量，增加肠道菌群多样性和丰富度，影响肠道菌群组成。 Abstract:Objective Effects of a highly reduced crude protein diet supplemented with glutamate (Glu) and arginine (Arg) on the intestinal metabolites and microbiota of weaned piglets were examined for aquaculture feed formulation. Method One-hundred-ninety-two healthy (26±2)-day-old crossbred (Duroc × Landrace × Large White) piglets with an average body weight of (6.96±0.29) kg were randomly divided into 4 groups. Each group contained 6 replicates of 8 piglets each. Group I (CK) was fed on the control diet that provided 21.16% crude protein (CP). Group II was on a low-protein diet containing 15.97% CP supplemented with some limiting amino acids. Added to the Group II diet, 1.50% Glu was included for feeding Group III and 1.50% Glu plus 1.00% Arg for Group IV. From 11th to 13th day in the 16 d feeding program, metabolites and microbial community in the piglet intestines were monitored. Result The fecal putrescine and spermidine of the treatment piglets were similar but significantly lower than those of CK (P＜0.05). The cadaverine in the Groups II and III piglets showed no significant differences but were significantly lower than that of CK or Group IV (P＜0.05), and that of Group I significantly lower than that of Group IV (P＜0.05). No significant differences were observed on the fecal acetic acid and propionic acid among all groups (P＞0.05), but the butyric acid in the piglets of all treatment groups was significantly higher than those of CK (P＜0.05). The intestinal microbiota richness and diversity of the animals under treatments were significantly higher than those of CK (P＜0.05). At phylum level, the relative abundance of Firmicutes in the fecal microbiota of the piglets were significantly higher under the treatments than CK (P＜0.05), but those of Bacteroidetes and Proteobacteria significantly lower than CK (P＜0.05). Significant differences were also observed among the groups at family and genus levels. The relative abundances of unspecified families and unspecified genera under clostridiales were significantly higher under the treatments than CK (P＜0.05), those of prevotellaceae, enterobacteriaceae, and lachnospiraceae were significantly lower under the treatments than CK (P＜0.05), those of lachnospiraceae, lactobacillaceae, blautia, and lactobacillus of Group IV significantly higher than those of CK (P＜0.05), and those of peptostreptococcaceae and terrisporobacter of Group IV were significantly lower than those of CK (P＜0.05). Conclusion A forage of CP being reduced from 21.16% to 15.97% and supplemented with Glu and Arg lowered the intestinal biogenic amines, increased the butyric acid, and enhanced the diversity and richness of microbiota in the weaned piglets. -
Key words:
- low-protein diet /
- glutamate /
- arginine /
- intestinal metabolites /
- microbiota /
- weaned piglets
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图 1 粪样稀释性曲线和丰度等级曲线
a、b分别为稀释性曲线和OTU丰度等级曲线。
Figure 1. Dilution and OTU rank-abundance curves of fecal samples
a and b indicate dilution curve and OTU rank-abundance curve, respectively.
图 2 粪样中优势菌群结构分布情况
A、B、C分别表示肠道菌群在门水平、科水平、属水平上的结构组成。
Figure 2. Distribution of dominant fecal microbes
A, B, C represented the distruction of fecal dominant microbiota at phylum level, family level, and genus level, respectively.
表 1 饲粮组成与营养水平（风干基础）
Table 1. Nutritional composition of basal diets (on air-dry basis)
项目
Items组I（CK）
Group I组II
Group II组III
Group III组IV
Group IV原料 Ingredient 玉米 Corn/% 35.12 48.00 47.00 45.00 豆粕 Soybean meal/% 15.00 6.30 6.20 6.60 膨化大豆
Extruded soybean/%18.00 11.00 12.00 12.00 麦麸 Wheat bran/% 1.70 鱼粉 Fish meal/% 7.00 7.00 7.00 7.00 乳清粉 Whey powder/% 16.00 16.00 16.00 16.00 蔗糖 Sucrose/% 2.00 2.00 2.00 2.00 柠檬酸 Citric acid/% 2.00 2.00 2.00 2.00 豆油 Soybean oil/% 2.50 2.26 2.56 3.15 石粉 Limestone/% 0.30 0.32 0.23 0.47 磷酸氢钙 CaHPO4/% 0.54 0.72 0.82 0.60 食盐 NaCl/% 0.30 0.30 0.30 0.30 氯化胆碱
Choline chloride （50%）/%0.08 0.08 0.08 0.08 L-赖氨酸盐酸盐
L-lysine•HCL/%0.03 0.42 0.41 0.40 DL-蛋氨酸
DL-methionine/%0.09 0.15 0.15 0.15 L-苏氨酸 L-threonine/% 0.03 0.20 0.20 0.20 L-色氨酸 L-tryptophan/% 0.01 0.07 0.07 0.07 L-缬氨酸 L-Valine/% 0.20 0.20 0.20 L-异亮氨酸 L-isoleucine/% 0.21 0.20 0.20 L-谷氨酸 L-glutamate/% 1.50 1.50 L-精氨酸 L-arginine/% 1.00 ①预混料 Premix/% 1.00 1.00 1.00 1.00 合计 Total/% 100.00 100.00 100.00 100.00 ②营养水平 Nutrient levels 消化能
Digestible energy/（MJ·kg−1）14.99 14.42 14.39 14.39 净能
Net energy/（MJ·kg−1）10.55 10.54 10.53 10.54 粗蛋白质 Crude protein/% 21.16 15.97 15.93 15.95 粗脂肪 EE/% 8.03 6.85 7.23 7.74 中性洗涤纤维 NDF/% 7.50 7.45 6.82 6.67 酸性洗涤纤维 ADF/% 5.40 5.37 5.12 5.00 淀粉 Starch/% 24.89 32.01 31.20 30.98 标准回肠可消化氨基酸 Standardized ileal digestible amino acids 赖氨酸 Lys/% 1.09 1.09 1.09 1.09 蛋氨酸 Met/% 0.41 0.41 0.41 0.41 蛋氨酸+胱氨酸 Met+Cys/% 0.65 0.65 0.65 0.65 苏氨酸 Thr/% 0.69 0.69 0.69 0.69 色氨酸 Trp/% 0.23 0.23 0.23 0.23 缬氨酸 Val/% 0.80 0.80 0.80 0.80 异亮氨酸 Ile/% 0.74 0.74 0.74 0.74 亮氨酸 Leu/% 1.41 1.14 1.14 1.13 总谷氨酸 Total Glu/% 3.60 2.67 4.15 4.16 总精氨酸 Total Arg/% 1.36 0.95 0.95 1.96 ①预混料为每千克饲粮提供：维生素A 12 500.00 IU，维生素D3 2 500.00 IU，维生素E 80.00 mg，维生素K3 3.00 mg，维生素B1 2.50 mg，维生素B2 10.00 mg，维生素B6 3.00 mg，维生素B12 0.035 mg，烟酸 30.00 mg，泛酸 15.00 mg，叶酸 0.45 mg，生物素 0.50 mg，铁 140.00 mg，铜 15.00 mg，锌 140.00 mg，锰 30.00 mg， 碘 0.50 mg，硒 0.25 mg；②粗蛋白质为实测值，其他营养指标为计算值。
① Premix provided following nutrients in per kg of diet：VA 12 500.00 IU, VD3 2 500.00 IU, VE 80.00 mg, VK3 3.00 mg, VB1 2.50 mg; VB2 10.00 mg, VB6 3.00 mg, VB12 0.035 mg, nicotinic acid 30.00 mg, pantothenic acid 15.00 mg, folic acid 0.45 mg; biotin 0.50 mg, Fe 140.00 mg, Cu 15.00 mg, Zn 140.00 mg, Mn 30.00 mg, I 0.50 mg, Se 0.25 mg. ② CP is presented as measured; others, calculated values.
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表 2 低蛋白质饲粮添加谷氨酸和精氨酸对断奶仔猪粪样生物胺的影响
Table 2. Effects of low-protein diet supplemented with Glu + Arg on fecal biogenic amines of weaned piglets （单位：µg·g−1）
项目
Items组I
Group I组II
Group II组III
Group III组IV
Group IV腐胺
Putrescine36.10±1.45 a 23.31±0.99 b 25.63±0.61 b 27.76±1.67 b 尸胺
Cadaverine8.66±0.48 b 5.65±0.56 c 5.44±0.22 c 13.14±0.65 a 亚精胺
Spermidine57.86±2.21 a 25.44±1.37 b 31.46±1.39 b 27.16±1.31 b 同行数据后不同小写字母表示不同处理间差异显著（P ＜0.05），表3、4同。
Data with different lowercase letters on same row indicate significant difference at P＜0.05; those with same or no letter, no significant difference at P＞0.05. Same for below.
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表 3 低蛋白质饲粮添加谷氨酸和精氨酸对断奶仔猪粪样短链脂肪酸的影响
Table 3. Effects of low-protein diet supplemented with Glu + Arg on fecal short-chain fatty acids of weaned piglets （单位：mg·g−1）
项目
Items组I
Group I组II
Group II组III
Group III组IV
Group IV乙酸
Acetic acid1.27±0.09 1.43±0.03 1.280±0.02 1.39±0.03 丙酸
Propionic acid0.78±0.01 0.81±0.03 0.88±0.02 0.77±0.01 丁酸
Butyric acid0.49±0.03 b 0.56±0.04 a 0.61±0.03 a 0.64±0.02 a
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表 4 低蛋白质饲粮添加谷氨酸和精氨酸对断奶仔猪肠道菌群多样性的影响
Table 4. Effects of low-protein diet supplemented with Glu + Arg on intestinal microflora diversity of weaned piglets
项目
Items组I
Group I组II
Group II组III
Group III组IV
Group IV物种数目 observed species 568.00±36.27 b 632.33±48.56 a 648.00±62.78 a 680.00±21.23 a 测序深度 Coverage/% 99.99 99.99 99.99 99.98 Shannon指数 Shannon index 4.68±0.18 b 4.92±0.04 ab 4.81±0.11 ab 5.05±0.13 a Simpson指数 Simpson index 0.93±0.01 a 0.86±0.02 b 0.85±0.02 b 0.86±0.05 b Chao1指数 Chao1 index 574.81±74.76 b 631.53±13.40 ab 629.60±17.09 ab 658.02±46.58 a ACE指数 ACE index 590.86±33.17 b 641.19±17.34 ab 646.50±19.77 ab 673.04±47.19 a
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