1 材料与方法
1.1 试验材料
1.2 试验动物与试验设计
表1 基础饲粮组成及营养水平(风干基础)Table 1 Composition and nutrient levels of the basal diet (air-dry basis) |
| 原料Ingredients | 含量Content | 营养水平Nutrient levels2) | 含量Content | |
|---|---|---|---|---|
| 玉米Corn | 55.00 | 代谢能ME/(MJ/kg) | 11.79 | |
| 豆粕Soybean meal | 15.00 | 粗蛋白质CP | 16.93 | |
| 麸皮Bran | 12.00 | 粗纤维CF | 2.02 | |
| 大豆油Soybean oil | 2.00 | 粗灰分Ash | 11.82 | |
| 石粒Stone particles | 7.50 | 钙Ca | 3.49 | |
| 石粉Limestone | 5.00 | 有效磷AP | 0.53 | |
| 食盐NaCl | 0.50 | 蛋氨酸Met | 0.49 | |
| 预混料Premix1) | 3.00 | 赖氨酸Lys | 0.81 | |
| 合计Total | 100.00 | 蛋氨酸+胱氨酸Met+Cys | 0.51 |
1)每千克预混料包含 One kg premix contained the following:VA 200 000 IU,VD3 100 000 IU,VE 500 IU,VK3 60 mg,VB2 100 mg,VB6 70 mg,烟酰胺 niacinamide 700 mg,泛酸 pantothenic acid 300 mg,生物素 biotin 2 mg,叶酸 folic acid 20 mg,Cu (as copper sulfate) 500 mg,Fe (as ferrous sulfate) 13 000 mg,Mn (as manganese sulfate) 500 mg,Zn (as zinc sulfate) 2 400 mg。 2)代谢能、有效磷和氨基酸为计算值计算值,其余为实测值。ME, AP and amino acids were calculated values, while the others were measured values. |
1.3 饲养管理
1.4 样品采集与指标测定
1.4.1 饲粮营养水平
1.4.2 生产性能和粪便评分
表2 粪便评分标准Table 2 Fecal scoring criteria |
| 项目Items | 评分标准Scoring criteria |
|---|---|
| 1分1 point | 正常的干粪便,呈圆锥形 |
| 2分2 point | 轻微疏松粪便,一些呈圆锥形,无游离水 |
| 3分3 point | 疏松粪便,有少量呈圆锥形,一些游离水 |
| 4分4 point | 粪便极其疏松,无圆锥形粪便,大量游离水 |
1.4.3 器官指数和肠道长度
1.4.4 肠道抗氧化指标测定
1.4.5 肠道形态和回肠消化酶活性
1.4.6 盲肠菌群
1.4.7 回肠消化酶相关基因表达
表3 PCR反应条件Table 3 PCR reaction conditions |
| 项目 Items | 温度 Temperature/℃ | 时间 Time | 循环次数 Number of cycles |
|---|---|---|---|
预变性Pre-denaturation | 52 | 2 min | 1 |
| 95 | 10 min | ||
循环反应Cyclic reaction | 95 | 15 s | 40 |
| 60 | 1 min | ||
熔解曲线Melting curve | 95 | 15 s | |
| 60 | 1 min | ||
| 95 | 15 s |
表4 引物序列Table 4 Primer sequence |
| 基因 Genes | GenBank登录号 GenBank accession number | 引物序列 Primer sequence (5'—3') | 产物长度 Product size/bp |
|---|---|---|---|
| β-肌动蛋白 β-actin | NM_205518.1 | F:AACACCCACACCCCTGTGAT R:TGAGTCAAGCGCCAAAAGAA | 116 |
| α-淀粉酶 AMS | NM_001001473.2 | F:AAGTGGAATGGAGAGAAGATG R:CCAGAAAGTAAGAATGGAAGC | 147 |
| 脂肪酶 LPS | XM_015288675.2 | F:CCCTGCCCAACCTTATTT R:GCATTTCCACTCCTCCGT | 176 |
| 胰蛋白酶 TPS | XM_025143180.1 | F:AGCGAGCAGACCATTAGTTC R:AGGAGAGTACAGGGGCATTC | 253 |
| 脂肪酸转运蛋白4 FATP4 | XM_046929199.1 | F:ATACCTCTGGCACTACGGGAAT R:CATACATCACATCATCGGGTCT | 117 |
| 乙酰辅酶A羧化酶 ACC | NM_205505 | F:TTGTGGCACAGAAGAGGGAA R:GTTGGCACATGGAATGGCAG | 161 |
1.5 数据统计与分析
2 结果
2.1 饲粮中添加木纤维对70~77周龄蛋鸡生产性能的影响
表5 饲粮中添加木纤维对70~77周龄蛋鸡生产性能的影响Table 5 Effects of dietary wood fiber supplementation on performance of laying hens aged from 70 to 77 weeks |
| 项目 Items | 组别Groups | P值 P-value | |||
|---|---|---|---|---|---|
| CON | D | Z | G | ||
| 产蛋率Laying rate/% | |||||
| 70~71周龄70 to 71 weeks of age | 89.92±0.95c | 91.26±0.78b | 91.56±0.78b | 92.56±0.34a | 0.001 |
| 72~73周龄72 to 73 weeks of age | 89.36±1.23c | 91.19±0.65b | 90.93±0.63b | 91.88±0.54a | 0.001 |
| 74~75周龄74 to 75 weeks of age | 88.73±1.36c | 90.60±0.62b | 90.16±0.75b | 91.34±0.80a | 0.010 |
| 76~77周龄76 to 77 weeks of age | 87.08±1.60b | 88.82±1.71a | 88.20±1.17ab | 89.05±1.77a | 0.033 |
| 料蛋比Feed to egg ratio | |||||
| 70~71周龄70 to 71 weeks of age | 2.13±0.03a | 2.04±0.02c | 2.07±0.02b | 2.04±0.02c | 0.010 |
| 72~73周龄72 to 73 weeks of age | 2.13±0.03a | 2.03±0.03c | 2.07±0.02b | 2.06±0.02b | 0.001 |
| 74~75周龄74 to 75 weeks of age | 2.15±0.04a | 2.02±0.02c | 2.10±0.02b | 2.08±0.03b | 0.001 |
| 76~77周龄76 to 77 weeks of age | 2.26±0.11a | 2.09±0.05c | 2.17±0.04b | 2.16±0.06b | 0.010 |
| 平均蛋重Average egg weight/g | |||||
| 70~71周龄70 to 71 weeks of age | 57.56±0.34c | 59.36±0.56a | 58.21±0.39b | 58.18±0.28b | 0.001 |
| 72~73周龄72 to 73 weeks of age | 58.52±0.78b | 59.85±0.71a | 58.52±0.57b | 58.19±0.27b | 0.010 |
| 74~75周龄74 to 75 weeks of age | 58.13±0.35ab | 60.27±0.56a | 58.33±0.27b | 57.99±0.56c | 0.001 |
| 76~77周龄76 to 77 weeks of age | 57.96±0.97b | 59.67±0.72a | 58.29±0.73b | 58.21±0.77b | 0.010 |
同行数据肩标不同小写字母表示差异显著(P<0.05),相同或无字母表示差异不显著(P>0.05)。下表同。 | |
In the same row, values with different small letter superscripts mean significant difference (P<0.05), while with the same or no letter superscripts mean no significant difference (P>0.05). The same as below. |
2.2 饲粮中添加木纤维对70~77周龄蛋鸡粪便评分和体重的影响
表6 饲粮中添加木纤维对70~77周龄蛋鸡粪便评分的影响Table 6 Effects of dietary wood fiber supplementation on fecal scores of laying hens aged from 70 to 77 weeks 分 |
| 项目 Items | 组别Groups | P值 P-value | |||
|---|---|---|---|---|---|
| CON | D | Z | G | ||
| 70周龄70 weeks of age | 3.50±0.55 | 3.17±0.41 | 3.00±0.63 | 2.84±0.75 | 0.285 |
| 71周龄71 weeks of age | 3.50±0.55 | 3.33±0.52 | 3.17±0.41 | 3.98±2.66 | 0.774 |
| 72周龄72 weeks of age | 3.67±0.52a | 3.00±0.63b | 3.00±0.00b | 2.83±0.41b | 0.023 |
| 73周龄73 weeks of age | 3.33±0.52 | 3.00±0.63 | 2.83±0.41 | 2.67±0.52 | 0.186 |
| 74周龄74 weeks of age | 3.33±0.52 | 2.67±0.52 | 2.67±0.52 | 2.83±0.41 | 0.091 |
| 75周龄75 weeks of age | 3.33±0.52a | 2.83±0.41ab | 2.67±0.52b | 2.50±0.55b | 0.049 |
| 76周龄76 weeks of age | 3.33±0.52 | 2.67±0.52 | 2.67±0.52 | 2.67±0.52 | 0.089 |
| 77周龄77 weeks of age | 3.33±0.52 | 2.67±0.52 | 2.50±0.55 | 2.67±0.52 | 0.056 |
| 70~77周龄70 to 77 weeks of age | 3.42±0.13a | 2.92±0.25b | 2.81±0.23b | 3.00±0.57b | 0.010 |
表7 饲粮中添加木纤维对70~77周龄蛋鸡平均体重的影响Table 7 Effects of dietary wood fiber supplementation on average body weights of laying hens aged from 70 to 77 weeks kg |
| 项目 Items | 组别Groups | P值 P-value | |||
|---|---|---|---|---|---|
| CON | D | Z | G | ||
| 70周龄70 weeks of age | 1.93±0.06 | 1.94±0.03 | 1.92±0.07 | 1.89±0.07 | 0.648 |
| 71周龄71 weeks of age | 1.95±0.05 | 1.94±0.03 | 1.93±0.08 | 1.93±0.03 | 0.964 |
| 72周龄72 weeks of age | 1.94±0.05 | 1.96±0.03 | 1.93±0.06 | 1.95±0.04 | 0.836 |
| 73周龄73 weeks of age | 1.99±0.08 | 2.07±0.03 | 2.01±0.14 | 1.98±0.03 | 0.497 |
| 74周龄74 weeks of age | 2.00±0.07 | 2.05±0.06 | 2.08±0.17 | 2.00±0.05 | 0.654 |
| 75周龄75 weeks of age | 2.03±0.07 | 2.08±0.04 | 1.97±0.03 | 2.02±0.04 | 0.056 |
| 76周龄76 weeks of age | 2.05±0.06 | 2.01±0.02 | 1.98±0.02 | 2.67±0.52 | 0.118 |
| 77周龄77 weeks of age | 1.95±0.03 | 2.00±0.05 | 1.99±0.04 | 1.99±0.01 | 0.327 |
| 70~77周龄70 to 77 weeks of age | 1.98±0.07 | 2.01±0.06 | 1.98±0.09 | 1.98±0.06 | 0.316 |
2.3 饲粮中添加木纤维对蛋鸡消化器官指数和肠道长度的影响
表8 饲粮中添加木纤维对蛋鸡器官指数和肠道长度的影响Table 8 Effects of dietary wood fiber supplementation on organ indexes and intestinal lengths of laying hens |
| 项目 Items | 组别Groups | P值 P-value | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| CON | D | Z | G | ||||||||
| 器官指数Organ indexes/% | |||||||||||
| 腺胃Glandular stomach | 0.47±0.04 | 0.46±0.04 | 0.50±0.08 | 0.49±0.10 | 0.113 | ||||||
| 肌胃Gizzard | 1.64±0.42 | 1.62±0.42 | 1.60±0.27 | 1.58±0.23 | 0.992 | ||||||
| 十二指肠Duodenum | 0.62±0.09 | 0.65±0.08 | 0.62±0.10 | 0.61±0.07 | 0.796 | ||||||
| 空肠Jejunum | 1.03±0.14b | 1.46±0.13a | 1.45±0.13a | 1.31±0.29a | 0.003 | ||||||
| 回肠Ileum | 1.09±0.24b | 1.61±0.19a | 1.30±0.15b | 1.15±0.19b | 0.003 | ||||||
| 肠道长度Intestinal lengths/cm | |||||||||||
| 回肠Ileum | 53.42±10.38 | 55.37±3.28 | 53.22±3.30 | 57.40±2.79 | 0.635 | ||||||
| 十二指肠Duodenum | 27.63±1.45 | 27.65±1.90 | 26.45±1.54 | 27.38±1.60 | 0.839 | ||||||
| 空肠Jejunum | 52.05±8.10 | 54.50±2.35 | 56.25±4.55 | 56.25±0.50 | 0.497 | ||||||
2.4 饲粮中添加木纤维对蛋鸡肠道抗氧化指标的影响
表9 饲粮中添加木纤维对蛋鸡肠道抗氧化指标的影响Table 9 Effects of dietary wood fiber supplementation on intestinal antioxidant indices of laying hens |
| 项目 Items | 组别Groups | P值 P-value | |||
|---|---|---|---|---|---|
| CON | D | Z | G | ||
| 空肠Jejunum | |||||
| 总抗氧化能力T-AOC/(U/mg) | 0.34±0.10b | 0.38±0.05ab | 0.54±0.17a | 0.47±0.11a | 0.033 |
| 谷胱甘肽过氧化物酶GSH-Px/(U/mg) | 154.80±47.03c | 172.90±22.86b | 248.18±76.82a | 217.33±51.86a | 0.033 |
| 超氧化物歧化酶SOD/(U/mg) | 19.12±5.81b | 21.36±2.82ab | 30.66±9.49a | 26.85±6.41a | 0.033 |
| 丙二醛MDA/(nmol/mg) | 5.45±1.30 | 4.79±0.63 | 5.16±1.11 | 5.37±0.89 | 0.725 |
| 回肠Ileum | |||||
| 总抗氧化能力T-AOC/(U/mg) | 0.21±0.02b | 0.32±0.07a | 0.33±0.09a | 0.24±0.03b | 0.001 |
| 谷胱甘肽过氧化物酶GSH-Px/(U/mg) | 94.94±10.02b | 135.37±21.20a | 150.65±39.21a | 110.64±11.42b | 0.010 |
| 超氧化物歧化酶SOD/(U/mg) | 11.73±1.24b | 18.13±4.16a | 18.61±4.84a | 13.38±1.45b | 0.010 |
| 丙二醛MDA/(nmol/mg) | 2.63±0.28b | 3.75±0.59a | 3.62±0.51a | 3.02±0.36a | 0.010 |
| 十二指肠Duodenum | |||||
| 总抗氧化能力T-AOC/(U/mg) | 0.25±0.03 | 0.24±0.01 | 0.25±0.02 | 0.25±0.01 | 0.618 |
| 谷胱甘肽过氧化物酶GSH-Px/(U/mg) | 116.62±11.55 | 111.03±4.67 | 115.05±8.69 | 116.05±4.62 | 0.618 |
| 超氧化物歧化酶SOD/(U/mg) | 14.41±1.43 | 13.72±0.58 | 14.21±1.07 | 14.34±0.57 | 0.618 |
| 丙二醛MDA/(nmol/mg) | 3.23±0.32 | 3.08±0.13 | 3.19±0.24 | 3.21±0.13 | 0.618 |
2.5 饲粮中添加木纤维对蛋鸡肠道形态的影响
表10 饲粮中添加木纤维对蛋鸡肠道形态的影响Table 10 Effects of dietary wood fiber supplementation on intestinal morphology of laying hens |
| 项目 Items | 组别Groups | P值 P-value | |||
|---|---|---|---|---|---|
| CON | D | Z | G | ||
| 十二指肠Duodenum | |||||
| 绒毛高度Villi heigh/μm | 1 414.84±18.82 | 1 326.68±37.15 | 1 352.52±182.33 | 1 360.05±41.00 | 0.724 |
| 隐窝深度Crypt depth/μm | 95.50±2.35 | 96.12±1.89 | 98.70±9.50 | 106.36±7.84 | 0.215 |
| 绒隐比V/C | 13.70±0.75 | 14.87±0.27 | 13.89±3.02 | 12.85±1.09 | 0.561 |
| 空肠Jejunum | |||||
| 绒毛高度Villi heigh/μm | 803.34±5.44 | 786.33±13.07 | 783.99±28.42 | 814.70±7.80 | 0.145 |
| 隐窝深度Crypt depth/μm | 70.53±4.65b | 80.45±0.86a | 78.08±6.12a | 82.25±0.83a | 0.027 |
| 绒隐比V/C | 10.49±0.71 | 9.91±0.30 | 9.91±0.69 | 10.10±0.15 | 0.394 |
| 回肠Ileum | |||||
| 绒毛高度Villi heigh/μm | 678.70±4.51 | 669.24±8.07 | 679.60±1.25 | 671.91±2.51 | 0.077 |
| 隐窝深度Crypt depth/μm | 76.37±3.18 | 73.23±3.16 | 71.63±1.82 | 72.98±1.33 | 0.126 |
| 绒隐比V/C | 8.69±0.49 | 8.72±0.51 | 8.64±0.56 | 8.41±0.38 | 0.790 |
2.6 饲粮中添加木纤维对蛋鸡回肠消化酶活性和相关基因表达的影响
表11 饲粮中添加木纤维对蛋鸡回肠消化酶活性的影响Table 11 Effects of dietary wood fiber supplementation on ileal digestive enzyme activities of laying hens |
| 项目 Items | 组别Groups | P值 P-value | |||
|---|---|---|---|---|---|
| CON | D | Z | G | ||
| α-淀粉酶 AMS/(U/mg prot) | 4.69±0.40b | 5.78±0.32a | 5.12±0.58ab | 5.41±1.04a | 0.042 |
| 胰蛋白酶 TPS/(U/mg prot) | 2 562.41±239.82 | 2 674.63±844.03 | 2 936.48±1 109.62 | 2 468.89±250.94 | 0.733 |
| 脂肪酶 LPS/(U/g prot) | 10.62±4.18 | 10.69±1.14 | 8.19±8.00 | 7.89±4.07 | 0.862 |
表12 饲粮中添加木纤维对蛋鸡回肠消化酶相关基因表达的影响Table 12 Effects of dietary wood fiber supplementation on expression of ileal digestive enzyme-related genes of laying hens |
| 项目 Items | 组别Groups | P值 P-value | |||
|---|---|---|---|---|---|
| CON | D | Z | G | ||
| 脂肪酸转运蛋白4 FATP4 | 1.00±0.03b | 0.74±0.13b | 1.05±0.70b | 3.15±0.38a | 0.017 |
| 乙酰辅酶A羧化酶ACC | 1.01±0.14b | 0.61±0.11c | 2.56±0.20a | 1.35±0.48b | 0.010 |
| α-淀粉酶AMS | 1.02±0.22b | 0.73±0.05b | 0.70±0.13b | 2.26±0.45a | 0.001 |
| 脂肪酶LPS | 1.01±0.13c | 1.68±0.15b | 1.25±0.10b | 5.74±0.08a | 0.001 |
| 胰蛋白酶TPS | 1.02±0.23b | 2.83±0.94a | 2.25±0.53a | 1.79±0.36b | 0.027 |
2.7 饲粮中添加木纤维对蛋鸡盲肠菌群的影响
2.7.1 OTU聚类分析
2.7.2 饲粮中添加木纤维对蛋鸡盲肠菌群Alpha多样性的影响
表13 饲粮中添加木纤维对蛋鸡盲肠菌群Alpha多样性的影响Table 13 Effects of dietary wood fiber supplementation on cecal microbiota Alpha diversity of laying hens |
| 项目 Items | 组别Groups | P值 P-value | |||
|---|---|---|---|---|---|
| CON | D | Z | G | ||
| Observed_OTUs指数 Observed_OTUs index | 1 607.83±180.71 | 1 557.00±249.33 | 1 538.00±96.73 | 1 785.83±266.00 | 0.216 |
| Shannon指数Shannon index | 8.17±0.29 | 8.17±0.39 | 8.23±0.12 | 8.53±0.21 | 0.142 |
| Simpson指数Simpson index | 0.99±0.00 | 0.99±0.00 | 0.99±0.00 | 0.99±0.00 | 0.317 |
| Chao1指数Chao1 index | 1 516.57±54.56c | 1 612.99±262.39b | 1 656.69±172.03b | 1 897.08±231.61a | 0.030 |
2.7.3 饲粮中添加木纤维对蛋鸡盲肠菌群Beta多样性的影响
图2 Beta多样性指数热图矩阵中的数值表示样品间的相异性指数,该指数与微生物群落差异程度呈负相关。每个单元格内自上而下依次为Bray-Curtis距离、非加权UniFrac距离和加权UniFrac距离的定量结果。 Fig.2 Heat map of Beta diversity index Values in the matrix represent the dissimilarity index between samples, which is negatively correlated with microbial community degree of deviation. Within each cell, the quantitative results are presented from top to bottom as Bray-Curtis distance, unweighted UniFrac distance and weighted UniFrac distance. |
2.7.4 饲粮中添加木纤维对蛋鸡盲肠菌群结构组成的影响
图4 门水平上的盲肠菌群组成柱状图p_Bacteroidota:拟杆菌门;p_Firmicutes:厚壁菌门;p_Campilobacterota:弯曲杆菌门;p_Actinobacteriota:放线菌门;p_Proteobacteria:变形菌门;p_Desulfobacterota:脱硫杆菌门;p_Spirochaetota:螺旋体菌门;p_Fusobacteriota:梭杆菌门;p_Synergistota:互养菌门;p_WPS-2:WPS-2菌门;p_Cyanobacteria:蓝藻菌门;p_Eusimicrobiota:真微生物菌门;p_Patescibacteria:候选菌辐射门;p_Deferribacterota:脱铁杆菌门;p_Verrucomicrobiota:疣微菌门;p_Chloroflexi:绿弯菌门;p_Acidobacteriota:酸杆菌门;p_Planctomycetota:浮霉菌门;p_Gemmatimonadota:芽单胞菌门;p_Myxococcota:黏液球菌门;Other:其他。 Fig.4 Bar chart of cecal microbiota composition at phylum level |
表14 优势盲肠菌群门水平相对丰度Table 14 Relative abundances of dominant cecal microbiota at phylum level |
| 项目 Items | 组别Groups | P值 P-value | |||
|---|---|---|---|---|---|
| CON | D | Z | G | ||
| 拟杆菌门Bacteroidota | 42.77±3.72b | 51.38±2.84a | 46.70±3.09ab | 46.15±1.84ab | 0.010 |
| 厚壁菌门Firmicutes | 42.37±3.84 | 38.03±4.73 | 41.99±3.10 | 43.86±1.23 | 0.068 |
| 弯曲杆菌门Campilobacterota | 2.77±1.47 | 1.94±1.21 | 2.47±1.83 | 1.14±0.76 | 0.278 |
图5 属水平上的盲肠菌群组成柱状图g_Bacteroides:拟杆菌属;g_Megamonas:巨单胞菌属;g_Phascolarctobacterium:考拉杆菌属;g_Rikenellaceae_RC9_gut_group:理研菌科RC9肠道群;g_Faecalibacterium:粪杆菌属;g_Helicobacter:螺杆菌属;g_[Ruminococcus]_torques_group:瘤胃球菌属torques群;g_F082:F082菌属;g_Megasphaera:巨球形菌属;g_Subdoligranulum:罕见小球菌属;g_Lactobacillus:乳杆菌属;g_Clostridia_vadinBB60_group:梭菌纲vadinBB60群;g_Parabacteroides:副拟杆菌属;g_Prevotellaceae_UCG-001:普雷沃氏菌科UCG-001;g_Desulfovibrio:脱硫弧菌属;g_Olsenella:奥尔森菌属;g_Muribaculaceae:鼠杆菌科(属级);g_Fusobacterium:梭杆菌属;g_Synergistes:互养菌属;g_Alloprevotella:拟普雷沃氏菌属;Other:其他。 Fig.5 Bar chart of cecal microbiota composition at genus level |
表15 优势盲肠菌群属水平相对丰度Table 15 Relative abundances of dominant cecal microbiota at genus level |
| 项目 Items | 组别Groups | P值 P-value | |||
|---|---|---|---|---|---|
| CON | D | Z | G | ||
| 拟杆菌属Bacteroides | 19.72±4.86c | 27.18±4.89a | 22.05±2.88b | 18.97±1.99c | 0.001 |
| 巨单胞菌属Megamonas | 7.83±4.85 | 4.81±3.65 | 6.50±4.17 | 5.86±3.35 | 0.632 |
| 理研菌科RC9肠道群 Rikenellaceae_RC9_gut_group | 5.17±1.78 | 5.56±2.17 | 5.88±0.33 | 6.78±1.10 | 0.346 |
| 考拉杆菌属Phascolarctobacterium | 5.03±1.17 | 5.55±1.76 | 6.02±2.16 | 6.36±0.65 | 0.577 |

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