Effects of Navel Orange Residue and Stevia Residue Microbial Silage Feed on Growth Performance, Plasma Biochemical Indices and Fecal Microbial Diversity of Beef Cattle

CAO Xianhong; QIU Jingyun; LIAN Hai; OU Xiang; WU Lijuan; ZHANG Qiang; CHEN Rongqiang; SHI Xiaosheng; LEI Xiaowen

doi:10.12418/CJAN2024.264

2024 , Vol. 36 >Issue 5: 3070 - 3081

DOI: https://doi.org/10.12418/CJAN2024.264

RESEARCH PAPER

Effects of Navel Orange Residue and Stevia Residue Microbial Silage Feed on Growth Performance, Plasma Biochemical Indices and Fecal Microbial Diversity of Beef Cattle

CAO Xianhong ^,¹ ,
QIU Jingyun ¹ ,
LIAN Hai ¹ ,
OU Xiang ¹ ,
WU Lijuan ¹ ,
ZHANG Qiang ¹ ,
CHEN Rongqiang ¹ ,
SHI Xiaosheng ² ,
LEI Xiaowen ^,¹^,^*

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¹ Ganzhou Animal Husbandry and Fisheries Research Institute, Gannan Academy of Sciences, Ganzhou 341401, China
² Xinfeng County Agriculture and Rural Bureau, Ganzhou 341699, China

^* associate professor, E-mail: 343224896@qq.com

Received date: 2023-12-11

Online published: 2024-05-15

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Abstract

This study was conducted to explore the effects of navel orange residue and stevia residue microbial silage feed on growth performance, plasma biochemical indices and fecal microbial diversity of beef cattle. A total of 24 Simmental crossbred beef cattle at 22 months of age in good health and similar weight were randomly divided into 4 groups with 6 cattle per group. The control group was fed the experimental diet contained 30% microbial silage rice straw, the experiment group Ⅰ was fed the experimental diet contained 6.0% microbial silage rice straw and 24% microbial silage stevia residue, the experiment group Ⅱ was fed the experimental diet contained 6.0% microbial silage rice straw and 24% microbial silage navel orange residue, and the experiment group Ⅲ was fed the experimental diet contained 6.0% microbial silage rice straw, 12% microbial silage stevia residue and 12% microbial silage navel orange residue. The pre-trial period was 1 week and the trial period was 11 weeks. The results showed as follows: 1) the average daily gain of experiment group Ⅲ was significantly higher than that of control group and experiment group Ⅱ (P<0.05), and the feed to gain ratio was significantly lower than that of control group and experiment group Ⅱ (P<0.05). 2) The contents of glutamate (Glu), phosphofructokinase (PFK) and glucose-6-phosphate dehydrogenase (G6PDH) in plasma of experiment group Ⅲ were significantly higher than those of control group and experiment group II (P<0.05), and the contents of urea nitrogen (UN) and pyruvate kinase (PK) in plasma were significantly low than those of control group and experiment group I (P<0.05). 3) At the phylum level, the relative abundance of Proteobacteria in feces of experiment group Ⅲ was significantly higher than that of other three groups (P<0.05), and the relative abundance of Firmicutes in feces was significantly lower than that of other three groups (P<0.05). At the genus level, the relative abundance of Acinetobacter in feces of experiment group Ⅲ were significantly higher than that of other three groups (P<0.05), the relative abundances of UCG-005 and Alistipes in feces were significantly lower than those of other three groups (P<0.05). and the relative abundance of Rikenellaceae_ RC9_ gut_ group and UCG-010 in feces were significantly lower than those of control group and experiment group Ⅰ (P<0.05). 4) The Faith_pd index of experiment group Ⅲ was significantly higher than that of other three groups (P<0.05), and the Shannon index and Simpson index were significantly lower than those of the control group (P<0.05). Furthermore, there was a significant difference in Beta diversity between experiment group Ⅲ and control group, and there were significant differences between the control group and other experiment groups in amino acid metabolism, energy metabolism and other functions (P<0.05). In summary, the addition of navel orange residue and stevia residue mixed microbial silage feed can improve the growth performance of beef cattle, improve the plasma biochemical indices, change the Alpha diversity and Beta diversity of fecal microbial community of beef cattle, and regulate digestive and absorptive functions of intestinal microorganisms in beef cattle.

Key words： navel orange residue; stevia residue; beef cattle; growth performance; plasma biochemical indices; microbial diversity

Cite this article

CAO Xianhong , QIU Jingyun , LIAN Hai , OU Xiang , WU Lijuan , ZHANG Qiang , CHEN Rongqiang , SHI Xiaosheng , LEI Xiaowen . Effects of Navel Orange Residue and Stevia Residue Microbial Silage Feed on Growth Performance, Plasma Biochemical Indices and Fecal Microbial Diversity of Beef Cattle[J]. Chinese Journal of Animal Nutrition, 2024 , 36(5) : 3070 -3081 . DOI: 10.12418/CJAN2024.264

随着日常饮食结构变化和生活水平的提高,肉牛养殖业在我国南方畜牧业的结构占比越来越高,但是养殖规模的扩大与粗饲料资源短缺之间的矛盾也越来越深^[1]。开发农业副产物替代常规饲料中的粗饲料是发展新型粗饲料的重要途径,包括常见的脐橙渣和甜叶菊渣等^[2-3]。其中,脐橙渣是脐橙加工后的副产品,是一种富含膳食纤维的饲料原料。据统计,脐橙在赣南地区种植面积高达10.8万hm²,每年总产量122万t,每年因加工产生的废渣约50万t,但其除少部分用于饲料原料,大部分得不到有效的利用^[4-5]。甜叶菊渣是甜叶菊加工提取甜菊糖苷后的副产物,富含粗蛋白质和氨基酸等营养成分。在我国南方地区,甜叶菊是种植面积较广的一种优质农作物,但其加工后产生的甜叶菊渣利用率很低,大部分被当成废弃物丢弃或掩埋^[6]。因此,将脐橙渣和甜叶菊渣饲料化开发,对缓解肉牛粗饲料资源短缺问题和减少农业副产物资源浪费具有重要意义。

由于脐橙渣和甜叶菊渣的含水量高,易腐败,导致其不能长期储存,因而饲料资源化利用率低。研究表明,通过微生物微贮技术将农业副产物制成非常规饲料,不仅能长期提供优质新型粗饲料,还能提高动物饲料转化率和营养吸收水平^[7-8]。在微贮脐橙渣替代部分基础饲粮饲喂番鸭的试验中,表明脐橙渣的利用率有所提高,番鸭的消化吸收能力和生长性能得到提高^[9]。卢占军等^[10]以甜叶菊渣为主要原料生产微贮饲料,表明处理过的甜叶菊渣能显著提高畜禽的饲料利用效率,降低饲养成本。目前尚无关于脐橙渣和甜叶菊渣微贮饲料在肉牛体内的应用研究报道,为了更好地利用脐橙渣和甜叶菊渣资源,开发肉牛新型粗饲料,本试验利用微生物微贮技术对脐橙渣和甜叶菊渣进行微贮,在肉牛育肥期间用其替代部分粗饲料中的微贮稻草,通过研究脐橙渣和甜叶菊渣微贮饲料对肉牛生长性能、血浆生化指标和粪便微生物多样性的影响,进一步为脐橙渣和甜叶菊渣微贮饲料产品的研发与应用价值提供科学依据。

1 材料与方法

1.1 微贮饲料的制备

脐橙渣由信丰农夫山泉饮料有限公司提供,甜叶菊渣由赣州菊隆高科技实业有限公司提供,稻草由赣州大圣牧业发展有限公司提供。上述3种原料的营养成分见表1,按照《饲料分析及饲料质量检测技术》^[11]中的方法进行测定。微贮用酵母菌(yeast)活菌数≥10⁶ CFU/g,乳酸菌(Lactobacillus)活菌数≥10¹¹ CFU/g。根据试验需求,将上述3种原料按照干物质含量计算,制备微贮稻草、微贮甜叶菊渣+稻草(甜叶菊渣∶稻草=4∶1)、微贮脐橙渣+稻草(脐橙渣∶稻草=4∶1)和微贮脐橙渣+甜叶菊渣+稻草(脐橙渣∶甜叶菊渣∶稻草=2∶2∶1),控制整体水分含量为65%~70%,再按各组原料的质量比例添加蔗糖(0.15%)、酵母菌(0.005%)和乳酸菌(0.01%)等辅料,制备过程中控制温度为15~25 ℃,搅拌混匀后密封微贮8周。

表1 脐橙渣、甜叶菊渣和稻草的营养成分

Table 1 Nutrient composition of navel orange residue, stevia residue and rice straw %

项目 Items	含量Content
项目 Items	脐橙渣Navel orange residue	甜叶菊渣Stevia residue	稻草Rice straw
干物质DM	30	35	88
粗脂肪EE	0.71	4.75	1.44
粗蛋白质CP	7.00	13.31	5.53
粗灰分Ash	2.96	8.64	11.31
水溶性碳水化合物WSC	48.05	0.65	8.99
中性洗涤纤维NDF	12.12	60.54	59.51
酸性洗涤纤维ADF	11.72	43.89	39.10

	干物质含量以鲜样为基础,其余指标以干物质为基础。
	DM content was based on the fresh sample, and the other indexes were based on DM.

1.2 试验设计和饲养管理

选取24头健康状况良好、体重无显著差异(P>0.05)的22月龄西门塔尔杂交牛(简称西杂牛)为试验动物,随机分为4组,每组6头牛。对照组饲喂含30%微贮稻草的试验饲粮,试验Ⅰ组饲喂含6.0%微贮稻草和24%微贮甜叶菊渣的试验饲粮,试验Ⅱ组饲喂含6.0%微贮稻草和24%微贮脐橙渣的试验饲粮,试验Ⅲ组饲喂含6.0%微贮稻草、12%微贮甜叶菊渣和12%微贮脐橙渣的试验饲粮。试验饲粮组成及营养水平见表2。试验期12周,预试期1周,正试期11周。每天08:00和17:00定时饲喂。按照江西省赣州市会昌县庄埠乡华弘种养专业合作社日常饲养程序进行管理。

表2 试验饲粮组成及营养水平(风干基础)

Table 2 Composition and nutrient levels of experimental diets (air-dry basis) %

项目 Items	含量Content
项目 Items	对照组 Control group	试验Ⅰ组 Experiment group Ⅰ	试验Ⅱ组 Experiment group Ⅱ	试验Ⅲ组 Experiment group Ⅲ
原料Ingredients
微贮稻草Microbial silage rice straw	30.0	6.0	6.0	6.0
微贮脐橙渣 Microbial silage navel orange residue			24.0	12.0
微贮甜叶菊渣 Microbial silage stevia residue		24.0		12.0
豆渣Soybean residue	20.0	20.0	20.0	20.0
麦秆Wheat straw	6.0	6.0	6.0	6.0
麦壳Wheat hulls	4.7	5.8	5.5	5.4
玉米Corn	26.2	26.2	26.2	26.2
豆粕Soybean meal	7.5	6.4	6.7	6.8
麦麸Wheat bran	3.0	3.0	3.0	3.0
预混料Premix¹⁾	1.6	1.6	1.6	1.6
小苏打NaHCO₃	1.0	1.0	1.0	1.0
合计Total	100.0	100.0	100.0	100.0
营养水平Nutrient levels²⁾
总能GE/(MJ/kg)	15.32	15.08	14.91	14.97
粗脂肪EE	2.13	2.81	2.03	2.37
粗蛋白质CP	12.28	13.34	12.55	13.01
中性洗涤纤维NDF	36.65	37.47	31.69	33.45
酸性洗涤纤维ADF	24.48	25.84	20.83	22.91
钙Ca	0.42	0.37	0.34	0.37
磷P	0.25	0.22	0.21	0.22

	1)预混料为每千克饲粮提供 Premix provided the following per kg of diets:VA 3 300 IU,VD 880 IU,VE 60 mg,Cu 10 mg,Fe 50 mg,Mn 20 mg,I 0.5 mg,Se 0.1 mg,Co 0.1 mg。
	2)总能为计算值,其余均为实测值。GE was a calculated value, while the others were measured values.

1.3 样品采集和指标测定

1.3.1 饲粮营养水平

饲粮总能为计算值,参考Caton等^[12]计算得出;其余营养水平均为实测值,参考《饲料分析及饲料质量检测技术》测定。

1.3.2 生长性能

正式期第1天和最后1天分别称量肉牛晨饲前的空腹体重,计算出各组平均日增重(ADG);每周记录1次肉牛的饲喂量和剩料量,统计整个正试期各组的饲料消耗量,计算出平日采食量(ADFI);根据统计出的ADG和ADFI计算各组肉牛料重比(F/G)。

1.3.3 血浆生化指标检测

正试期最后1天晨饲前限位保定肉牛,通过颈静脉采血收集血液样本到抗凝真空采血管中。将血液静置20 min后,3 500 r/min离心10 min,取上层血浆样品于离心管中。采用酶联免疫吸附试验(ELISA)试剂盒检测血浆中谷草转氨酶(AST)、谷丙转氨酶(ALT)、肌酐(CRE)、尿素氮(UN)、尿酸(UA)、丙酮酸激酶(PK)、谷氨酸(Glu)、磷酸果糖激酶(PFK)和葡萄糖-6-磷酸脱氢酶(G6PDH)含量。所有操作步骤严格按照相应试剂盒说明书进行。

1.3.4 粪便微生物多样性

穿戴好一次性无菌手套,将手伸入肉牛肛门内,收集正式期最后1天晨饲前肉牛的新鲜粪便样品。每份样品取100 g,置于对应编号的50 mL离心管中,立即干冰保存运输。回到实验室后立即将样品放于-80 ℃保存,用于提取DNA,以备后期16S rDNA扩增子测序测定。粪便微生物多样性由南京集思慧远生物科技有限公司测定。

1.4 数据统计与分析

数据经Excel 2019整理后,采用SPSS 25.0统计软件中的LSD多重比较法进行组间差异比较。结果以平均值±标准误(SE)表示,P<0.05表示差异显著。

2 结果与分析

2.1 不同微贮饲料对肉牛生长性能的影响

由表3可知,各组之间肉牛的初重、末重和ADFI无显著差异(P>0.05)。与对照组相比,试验Ⅰ组和试验Ⅲ组的ADG显著升高(P<0.05),F/G显著降低(P<0.05)。与试验Ⅲ组相比,试验Ⅱ组的ADG显著降低(P<0.05),F/G显著升高(P<0.05),且试验Ⅲ组和试验Ⅰ组之间无显著差异(P>0.05)。

表3 不同微贮饲料对肉牛生长性能的影响

Table 3 Effects of different microbial silage feed on growth performance of beef cattle

项目 Items	对照组 Control group	试验Ⅰ组 Experiment group Ⅰ	试验Ⅱ组 Experiment group Ⅱ	试验Ⅲ组 Experiment group Ⅲ
初重IBW/kg	617.25±32.35	648.56±56.81	607.33±60.96	614.00±76.75
末重FBW/kg	716.50±38.15	773.50±60.75	709.28±59.90	741.63±88.13
平均日增重ADG/kg	1.27±0.08^b	1.60±0.05^a	1.31±0.04^b	1.64±0.15^a
平均日采食量ADFI/kg	8.75±0.20	9.01±0.14	8.51±0.37	9.19±0.10
料重比F/G	6.91±0.29^a	5.71±0.23^b	6.51±0.10^a	5.49±0.26^b

	同行数据肩标无字母或相同字母表示差异不显著(P>0.05),肩标不同字母表示差异显著(P<0.05)。下表同。
	In the same row, values with no letter or the same letter superscripts mean no significant difference (P>0.05), while with different letter superscripts mean significant difference (P<0.05). The same as below.

2.2 不同微贮饲料对肉牛血浆生化指标的影响

由表4可知,与对照组相比,试验Ⅰ组的血浆Glu和G6PDH含量均显著升高(P<0.05);试验Ⅱ组的血浆Glu含量显著升高(P<0.05);试验Ⅲ组的血浆Glu、PFK和G6PDH含量均显著升高(P<0.05),UN和PK含量均显著下降(P<0.05)。与试验Ⅲ组相比,试验Ⅰ组的血浆AST/ALT及UN、PK和Glu含量均显著升高(P<0.05),PFK含量显著下降(P<0.05);试验Ⅱ组的血浆UN含量显著升高(P<0.05),Glu、PFK和G6PDH含量均显著下降(P<0.05)。

表4 不同微贮饲料对肉牛血浆生化指标的影响

Table 4 Effects of different microbial silage feed on blood biochemical indicators of beef cattle

项目 Items	对照组 Control group	试验Ⅰ组 Experiment group Ⅰ	试验Ⅱ组 Experiment group Ⅱ	试验Ⅲ组 Experiment group Ⅲ
谷草转氨酶AST/(ng/L)	341.10±31.71	348.99±3.03	346.91±0.29	321.05±35.71
谷丙转氨酶ALT/(ng/L)	209.01±1.43	167.56±23.29	199.95±15.41	210.89±10.48
谷草转氨酶/谷丙转氨酶 AST/ALT	1.74±0.03^ab	1.86±0.03^a	1.51±0.10^b	1.45±0.16^b
肌酐CRE/(μmol/L)	99.72±19.62	87.24±3.07	90.58±4.89	88.08±13.83
尿素氮UN/(μmol/L)	1 692.48±122.01^a	1 778.65±173.32^a	1 859.42±23.81^a	1 087.66±31.02^b
尿酸UA/(μmol/L)	39.10±5.78	36.49±1.88	41.72±0.85	35.73±1.24
丙酮酸激酶PK/(ng/L)	607.69±3.78^a	592.48±12.18^a	549.51±11.85^ab	476.56±37.41^b
谷氨酸Glu/(μmol/L)	16.35±0.14^d	33.13±0.41^a	26.89±0.38^c	31.29±0.38^b
磷酸果糖激酶PFK/(ng/L)	503.28±3.45^b	604.56±32.86^b	618.71±17.97^b	698.93±77.51^a
葡萄糖-6-磷酸脱氢 G6PDH/(ng/L)	404.56±44.43^bc	585.61±17.59^a	444.61±5.38^b	588.32±4.88^a

2.3 不同微贮饲料对肉牛粪便微生物多样性的影响

2.3.1 肉牛粪便微生物门水平结构分析

由表5可知,各组肉牛粪便相对丰度较高的优势细菌门分别为厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidota)、变形菌门(Proteobacteria)和螺旋体门(Spirochaetota)。试验Ⅲ组的Firmicutes相对丰度显著低于其他3组(P<0.05),Proteobacteria相对丰度显著高于其他3组(P<0.05);对照组的Bacteroidota相对丰度均显著高于其他3组(P<0.05),试验Ⅰ组和试验Ⅲ组的Bacteroidota相对丰度均显著高于试验Ⅱ组(P<0.05);试验Ⅰ组的Spirochaetota相对丰度显著高于其他3组(P<0.05),试验Ⅱ组的Spirochaetota相对丰度显著低于对照组(P<0.05)。

表5 肉牛粪便微生物优势细菌门水平的相对丰度

Table 5 Relative abundance of dominant bacteria in feces of beef cattle at phylum level %

项目 Items	对照组 Control group	试验Ⅰ组 Experiment group Ⅰ	试验Ⅱ组 Experiment group Ⅱ	试验Ⅲ组 Experiment group Ⅲ
厚壁菌门Firmicutes	60.39±0.16^a	63.25±0.09^a	66.05±1.24^a	47.54±5.09^b
拟杆菌门Bacteroidota	36.88±0.19^a	28.43±0.71^b	25.62±0.23^c	28.51±0.20^b
变形菌门Proteobacteria	0.24±0.02^b	4.06±0.72^b	5.62±1.28^b	20.60±5.13^a
螺旋体门Spirochaetota	1.37±0.10^b	3.01±0.20^a	0.79±0.11^c	1.04±0.10^bc

2.3.2 属水平肉牛粪便微生物结构分析

由表6可知,在属水平下,各组相对丰度较高的前5种优势细菌属分别为UCG-005、理研菌科RC9肠道群(Rikenellaceae_RC9_gut_group)、UCG-010、不动菌属(Acinetobacter)和另枝菌属(Alistipes)。与对照组相比,试验Ⅰ组和试验Ⅱ组的UCG-005和Acinetobacter相对丰度显著升高(P<0.05),Rikenellaceae_RC9_gut_group和UCG-010相对丰度显著下降(P<0.05),试验Ⅲ组的UCG-005、Rikenellaceae_RC9_gut_group、UCG-010和Alistipes相对丰度显著下降(P<0.05),Acinetobacter相对丰度显著升高(P<0.05)。与试验Ⅰ组相比,试验Ⅲ组的UCG-005、Rikenellaceae_RC9_gut_group、UCG-010和Alistipes相对丰度显著下降(P<0.05),Acinetobacter相对丰度显著升高(P<0.05)。与试验Ⅱ组相比,试验Ⅲ组的UCG-005和Alistipes相对丰度显著下降(P<0.05),Rikenellaceae_RC9_gut_group和Acinetobacter相对丰度显著升高(P<0.05),UCG-010相对丰度无显著差异(P>0.05)。

表6 肉牛粪便微生物优势细菌属水平的相对丰度

Table 6 Relative abundance of dominant bacteria in feces of beef cattle at genus level %

项目 Items	对照组 Control group	试验Ⅰ组 Experiment group Ⅰ	试验Ⅱ组 Experiment group Ⅱ	试验Ⅲ组 Experiment group Ⅲ
UCG-005	11.76±0.30^b	14.06±0.23^a	14.19±0.44^a	8.78±0.82^c
理研菌科RC9肠道群 Rikenellaceae_RC9_gut_group	11.32±0.09^a	8.83±0.20^b	5.01±0.10^d	7.61±0.77^c
UCG-010	8.59±0.20^a	5.09±0.07^b	4.39±0.21^c	3.79±0.87^c
不动菌属Acinetobacter	0.00±0.00^c	2.91±0.51^b	2.65±1.08^b	14.44±4.05^a
另枝菌属Alistipes	4.57±0.12^b	2.45±0.05^c	6.58±0.26^a	2.02±0.13^d

2.3.3 肉牛粪便微生物Alpha多样性分析

由表7可知,各组的覆盖度指数均高于0.999,说明样品测序结果基本覆盖到了样品中所有的物种。试验Ⅲ组的Faith_pd指数显著高于其他3组(P<0.05),说明试验Ⅲ组粪便微生物多样性最复杂;试验Ⅲ组的ACE指数和Chao1指数最高,且试验Ⅱ组的ACE指数和Chao1指数显著低于其他3组(P<0.05),说明试验Ⅲ组粪便微生物群落的丰富度最高;对照组Shannon指数和Simpson指数数值最高,显著高于试验Ⅲ组(P<0.05),说明试验Ⅲ组粪便微生物的物种丰富度低于对照组。这表明添加脐橙渣和甜叶菊渣混合微贮饲料对肉牛粪便微生物菌群Alpha多样性有显著影响。

表7 不同微贮饲料对肉牛粪便微生物Alpha多样性指数的影响

Table 7 Effects of different microbial silage on fecal microbial Alpha diversity indexes of beef cattle

项目 Items	对照组 Control group	试验Ⅰ组 Experiment group Ⅰ	试验Ⅱ组 Experiment group Ⅱ	试验Ⅲ组 Experiment group Ⅲ
覆盖度指数Coverage index	0.999 4±0.000 2	0.999 6±0.000 2	0.999 1±0.000 2	0.999 2±0.000 4
Faith_pd指数Faith_pd index	227.38±1.68^c	250.55±5.28^b	228.53±6.32^c	274.67±8.66^a
ACE指数ACE index	3 339.33±46.68^a	3 374.86±56.45^a	3 153.96±56.73^b	3 557.53±197.84^a
Chao1指数Chao1 index	3 333.61±47.78^a	3 369.49±53.43^a	3 144.80±57.38^b	3 548.52±194.82^a
Shannon指数Shannon index	10.010±0.035^a	9.919±0.002^a	9.592±0.103^b	9.604±0.271^b
Simpson指数Simpson index	0.997±0.000^a	0.996±0.000^a	0.994±0.001^ab	0.989±0.004^b

2.3.4 肉牛粪便微生物Beta多样性分析

由图1可知,各试验组粪便微生物Beta多样性主坐标分析(principal co-ordinates analysis,PCoA)的第1主成分与对照组良好区分开来,并且试验Ⅲ组距离最远,说明添加脐橙渣和甜叶菊渣混合微贮饲料可引起肉牛粪便微生物菌群Beta多样性改变。另外,第2主成分上各试验组均与对照组具有明显的Bray-curtis距离和Weighted-Unifrac距离,但在Unweighted-Unifrac距离上试验Ⅰ组与对照组较近。

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图1 粪便微生物Bray-curtis距离(A)、Weighted-Unifrac距离(B)和Unweighted-Unifrac距离(C)PCoA结果

图中圆点分别表示各个样品,不同颜色表示样品属于不同的分组。

F1、F2、F3、F4分别表示对照组、试验Ⅰ组、试验Ⅱ组、试验Ⅲ组。图2同。

Fig.1 PCoA results of Bray-curtis distance (A), Weighted-Unifrac distance (B) and Unweighted-Unifrac distance (C) of fecal microorganisms

The dots in the figure indicated the samples, and different colors indicated that the samples belong to different groups

F1, F2, F3 and F4 indicated the control group, experiment group Ⅰ, experiment group Ⅱ, and experiment group Ⅲ, respectively. The same as Fig.2.

2.4 肉牛粪便微生物基因功能预测分析

由图2-A可知,对照组与试验Ⅰ组粪便微生物KEGG代谢途径二级通路注释丰度差异分析主要表现为氨基酸代谢、能量代谢、聚糖的生物合成和代谢、信号转导、核苷酸代谢、辅助因子和维生素的代谢、细胞生长与死亡等功能性显著上升(P<0.05),传染病:细菌性、萜类和多酮类的代谢、细胞活性、膜运输等功能性显著下降(P<0.05)。

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图2 各组肉牛粪便的差异微生物功能预测

Amino acid metabolism:氨基酸代谢;Energy metabolism:能量代谢;Glycan biosynthesis and metabolism:聚糖的生物合成和代谢;Signal transduction:信号转导;Nucleotide metabolism:核苷酸代谢;Metabolism of cofactors and vitamins:辅助因子和维生素的代谢;Cell growth and death:细胞生长与死亡;Infectious diseases: Bacterial:传染病:细菌性;Metabolism of terpenoids and polyketides:萜类和多酮类的代谢;Cell motility:细胞活性;Membrane transport:膜运输;Folding, sorting and degradation:折叠、分类和降解;Lipid metabolism:脂质代谢;Carbohydrate metabolism:碳水化合物代谢;Translation:翻译;Replication and repair:复制和修复;Metabolism of other amino acids:其他氨基酸的代谢;Xenobiotics biodegradation and metabolism:异种生物降解和代谢。

图为第2层级下KEGG代谢途径的差异分析,图中不同颜色代表不同的分组。图片中左图所示为不同功能在2组样品中的相对丰度比例,中间所示为95%置信区间内功能相对丰度的差异比例。

Fig.2 Prediction of differential bacterial function in the feces of beef cattle in different groups

The figure presents the differential analysis of KEGG pathways at the second level, where different groups are represented by different colors. The left image displays the abundance ratio of different functions in two groups of samples, while the middle image shows the difference ratio of functional abundance within the 95% confidence interval.

由图2-B可知,对照组与试验Ⅱ组主要表现为氨基酸代谢、能量代谢、信号转导、核苷酸代谢等功能性显著上升(P<0.05),折叠、分类和降解,聚糖的生物合成和代谢,传染病:细菌性,萜类和多酮类的代谢,脂质代谢,膜运输,碳水化合物代谢(Carbohydrate metabolism)等功能性显著下降(P<0.05)。

由图2-C可知,对照组与试验Ⅲ组主要表现为翻译,复制和修复,能量代谢,碳水化合物代谢,聚糖的生物合成和代谢,细胞生长与死亡,折叠、分类和降解等功能性显著上升(P<0.05),其他氨基酸的代谢、萜类和多酮类的代谢、异种生物降解和代谢、膜运输等功能性显著下降(P<0.05)。

3 讨论

3.1 不同微贮饲料对肉牛生长性能的影响

肉牛的ADG、ADFI和F/G是衡量其生长性能的重要指标,同时也是最直接和最有价值反映动物营养水平的指标^[13]。本试验中,与对照组相比,各试验组ADFI无显著差异,添加脐橙渣和甜叶菊渣混合微贮饲料能显著提高肉牛ADG,降低F/G。研究表明,脐橙渣在微生物作用下营养成分会迅速转化,粗蛋白质含量显著增加32.69%,从而进一步提高其营养价值^[14]。雷小文等^[9]利用脐橙渣替代基础饲粮,发现番鸭的消化功能和营养吸收水平得到提高,番鸭ADG显著提高,F/G显著下降。甜叶菊渣对肉兔生长性能的影响中发现,饲粮中适当添加甜叶菊渣不会影响肉兔生长速度和饲料利用率^[15]。然而,在本试验中,添加甜叶菊渣微贮饲料能显著提高肉牛ADG,降低F/G,可能因为肉牛对甜叶菊渣的粗纤维具有更好的消化吸收能力。另外,与单一脐橙渣微贮饲料饲喂效果相比,脐橙渣和甜叶菊渣混合微贮饲料更能显著增加肉牛ADG,降低F/G,这是由于单一脐橙渣微贮饲料会产生过多的微生物活菌,增加动物肠道对营养物质的额外消耗,进而影响机体对营养物质的吸收利用^[16]。在本试验条件下,通过脐橙渣和甜叶菊渣混合微贮饲料替换部分肉牛微贮稻草可提高肉牛的增重性能和饲料利用率,达到开发利用新型粗饲料资源的目的。

3.2 不同微贮饲料对肉牛血浆生化指标的影响

血浆中AST、ALT和AST/ALT指标可以反映肝脏功能和代谢情况,间接参与机体糖类、脂类和蛋白质的代谢^[17];CRE、UN和UA指标可以评价肾功能和代谢情况,反映出机体蛋白质和氨基酸代谢的平衡^[18];PK、Glu、PFK和G6PDH是肝脏糖代谢过程中的重要合成酶,对机体糖代谢的调控具有重要作用^[19]。本试验中,与对照组相比,试验组肉牛血浆中AST、ALT、CRE和UA含量无显著差异,而试验Ⅲ组Glu、PFK和G6PDH含量显著升高,其UN和PK含量显著下降,说明添加脐橙渣和甜叶菊渣混合微贮饲料对肉牛肝脏和肾脏没有产生不利影响,同时能维持机体对蛋白质和氨基酸代谢的平衡。研究表明,饲喂微贮后的甜叶菊渣能影响妊娠母猪脐带血浆中的生化指标,显著降低UN含量,可提高肾脏的代谢能力^[20]。Caputo等^[21]研究表明,G6PDH的表达增加能使葡萄糖前体的供应得到有效保障,揭示肝脏糖代谢作用有所提高。然而,本试验中试验Ⅰ组血浆UN含量与试验Ⅲ组相比显著升高,血浆G6PDH含量与对照组相比显著升高,表明脐橙渣和甜叶菊渣混合微贮饲料比单一甜叶菊渣微贮饲料更能提高肉牛肾脏对蛋白质和氨基酸的代谢。该试验中关于脐橙渣和甜叶菊渣混合微贮饲料对肉牛血浆生化指标的影响机制仍需进一步深入研究。

3.3 不同微贮饲料对肉牛粪便微生物多样性的影响

反刍动物因拥有特殊的消化系统结构,导致肠道微生物区系较为复杂,因此粗饲料的营养成分直接影响反刍动物的消化和吸收^[22]。研究发现,微生物微贮工艺能够改善粗饲料营养成分,并产生大量的有机酸限制病原菌的生长繁殖,从而维持肠道微生态的平衡^[23-24]。在本试验中,与其他3组相比,试验Ⅲ组的Proteobacteria和Acinetobacter相对丰度显著升高,Firmicutes、UCG-005和Alistipes相对丰度显著下降,且试验Ⅲ组的Rikenellaceae_RC9_gut_group和UCG-010相对丰度显著低于对照组和试验Ⅰ组。其中,Proteobacteria是促进新陈代谢的主要菌群;Acinetobacter能够促进免疫细胞的成熟并帮助机体抵御肠道感染;Firmicutes有利于宿主对纤维素的消化吸收;UCG-005和UCG-010有利于宿主对纤维素的降解和淀粉消化;Alistipes对肝纤维化、心血管疾病等具有保护作用;Rikenellaceae_RC9_gut_group能够调节脂质代谢和蛋白质代谢^[25⇓-27]。马艳等^[25]通过对放牧耗牛肠道菌群多样性进行分析,发现Firmicutes、UCG-005和Rikenellaceae_RC9_gut_group在试验的不同组别中形成显著差异,且该菌群的变化能显著提高耗牛新陈代谢能力和增强纤维降解能力。因此,本试验结果表明脐橙渣和甜叶菊渣混合微贮饲料能调节肉牛肠道菌群相对丰度,促进肉牛新陈代谢,提高肠道抵御感染能力。

Alpha多样性中Faith_pd指数越高,表明微生物组成多样性越复杂;ACE指数和Chao1指数越高,表明微生物群落丰富度越高;Shannon指数和Simpson指数越高,表明微生物物种越丰富^[28]。本试验中,试验Ⅲ组Faith_pd指数与其他3组相比显著升高,Shannon指数和Simpson指数与对照组相比显著降低,表明饲料中添加脐橙渣和甜叶菊渣混合微贮饲料能显著增加肉牛肠道微生物组成的多样性,降低物种丰富度。另外,Beta多样性PCoA结果表明,试验Ⅲ组与对照组之间的第1主成分距离最远,且第2主成分上具有明显的Bray-curtis距离和Weighted-Unifrac距离。吴占月等^[29]在欧拉羊粪便微生物多样性的研究中表明,添加发酵菌的试验组肠道微生物第1主成分上与对照组良好区分开来,第2主成分上Bray-curtis距离与对照组明显,试验组欧拉羊引起了肠道Beta多样性改变。因此,添加脐橙渣和甜叶菊渣混合微贮饲料会引起肉牛肠道菌群Alpha多样性和Beta多样性的改变,从而肠道微生物区系发生变化。

3.4 不同微贮饲料对肉牛粪便微生物功能的影响

通过KEGG功能注释发现,添加脐橙渣和甜叶菊渣混合微贮饲料会对肉牛肠道微生物功能产生显著影响。本试验中,以试验Ⅲ组和对照组为例,在二级代谢分类中,试验Ⅲ组其他氨基酸的代谢、萜类和多酮类的代谢、异种生物降解和代谢以及膜运输功能显著高于对照组,表明脐橙渣和甜叶菊渣混合微贮饲料对肉牛肠道其他氨基酸的代谢以及萜类和多酮类的代谢等有所提高。研究表明,肠道菌群中的Proteobacteria和Acinetobacter不仅有利于宿主进行新陈代谢和促进免疫细胞的成熟,同时还有利于提高宿主的营养物质利用率,调节能量和营养物质代谢平衡^[30]。其他氨基酸的代谢以及萜类和多酮类的代谢会促进动物机体肝脏和肾脏对糖类、脂类、蛋白质等营养物质的代谢,有利于动物生长性能的提高^[31-32]。因此,添加脐橙渣和甜叶菊渣混合微贮饲料可以显著影响肉牛肠道微生物功能,提高肉牛营养物质的代谢效率,从而有利于改善肉牛生长性能。

4 结论

① 添加脐橙渣和甜叶菊渣混合微贮饲料替代部分微贮稻草能显著提高肉牛ADG,降低F/G,改善血浆生化指标,提高肉牛的机体健康状况,并且与脐橙渣和甜叶菊渣的单一微贮饲料的饲喂效果相比,两者的混合微贮饲料对肉牛饲喂效果更佳。

② 添加脐橙渣和甜叶菊渣混合微贮饲料对肉牛肠道菌群Alpha多样性、Beta多样性和微生物功能有调节作用,并能促进肉牛肠道对营养物质的消化和吸收,从而提高其生长性能。

References

Publishing order | Descend order by publishing year | Descend order by cited within

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