Chinese Journal of Animal Nutrition >

2025 , Vol. 37 >Issue 9: 6254 - 6266

DOI: https://doi.org/10.12418/CJAN2025.507

RESEARCH PAPER

Metabolomics-Based Analysis of Effects of Solid Feed Feeding Level on Rumen Morphology and Function in Yak Calves

  • BIE Xinya ,
  • LI Shiguan ,
  • ZHOU Ya’nan ,
  • YUAN Jinquan ,
  • YANG Deyu ,
  • LIU Shujie ,
  • CUI Zhanhong , **
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  • Key Laboratory of Plateau Grazing Animal Nutrition and Feed Science of Qinghai Province, Yak Engineering Technology Research Center of Qinghai Province, Key Laboratory of Animal Nutrition and Forage-Feed of Grazing Yak and Tibetan Sheep in Qinghai-Tibetan Plateau, Ministry of Agriculture and Rural Affairs, Qinghai Academy of Animal Husbandry and Veterinary Sciences, Qinghai University, Xining 810016, China
** professor, E-mail:

*Contributed equally

Received date: 2025-01-06

  Online published: 2025-09-12

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Abstract

This experiment was conducted to investigate the effects of feeding levels of solid feed on rumen morphology and function of yak calves based on metabolomics analysis. Twenty-one 60-day-old weaned yak calves (male ) with similar body weight [(54.20±3.28 ) kg] were selected and divided into 3 groups with 7 calves in each group, namely high feeding level group (ad libitum feeding group, AL group), medium feeding level group (70 % feeding group, IR70 group) and low feeding level group (40 % feeding group, IR40 group). The pre-trial period was 30 days and the trial period was 90 days. The results showed as follows: 1) the thickness of mucosal epithelial, submucosal layer and muscular layer of rumen in AL group were significantly higher than those in IR70 group (P<0.05); the thickness of mucosal epithelial and muscular layer of rumen in AL group were significantly higher than those in IR40 group (P<0.05). The papillary length and muscular layer thickness of rumen in IR70 group were significantly higher than those in IR40 group (P<0.05). The rumen submucous layer thickness in IR40 group was significantly higher than that in IR70 group (P<0.05). 2) The rumen cellulase activity in IR70 group was significantly higher than that in AL group (P<0.05); the rumen cellulase activity in IR40 group was significantly higher than that in AL group (P<0.05). 3) Metabolomics analysis showed that compared with AL, the content of L-kynurenine in the metabolic pathway of African trypanosomiasis in IR40 group was significantly increased (P<0.05). Compared with AL group, the contents of differential metabolites L-asparagine and methionine in IR70 group were significantly decreased in carbon metabolism, mineral absorption and protein digestion and absorption pathways in cancer (P<0.05). In the protein digestion and absorption pathway, the content of the differential metabolite cadaverine decreased significantly (P<0.05); in the biosynthetic pathway of primary bile acids, the content of bile acids, a differential metabolite, increased significantly (P<0.05). In summary, when the yak calves are weaned at 60 days of age, and the ‘milk replacer+alfalfa hay+starter’ feeding method is used for early cultivation, increasing the level of solid feed feeding can promote the better development of rumen tissue morphology. Through metabolomics, it is found that the high feeding level improves the digestion and absorption of nutrients in yak calves, thereby improving the growth and development quality of yak calves, and providing an important basis for the scientific supplementary feeding of solid feed for lactating yak calves.

Key words： yak calves; solid feed feeding level; rumen development; metabolomics

Cite this article

BIE Xinya , LI Shiguan , ZHOU Ya’nan , YUAN Jinquan , YANG Deyu , LIU Shujie , CUI Zhanhong . Metabolomics-Based Analysis of Effects of Solid Feed Feeding Level on Rumen Morphology and Function in Yak Calves[J]. Chinese Journal of Animal Nutrition, 2025 , 37(9) : 6254 -6266 . DOI: 10.12418/CJAN2025.507

牦牛是青藏高原特有的反刍畜种资源,既是牧民群众赖以生存的生活资料来源,也是牧业稳定增收的生产资料来源。牦牛常年生活在海拔高、寒冷的青藏高原地区,以放牧饲养为主。该地区天然草地牧草枯草期长达7个月,造成了冷季放牧牦牛营养不足与缺乏的供需不平衡问题,这一实际情况对幼龄阶段牦牛犊牛生长发育的影响更加突出[1]。犊牛阶段饲粮会发生明显的改变,即以牛乳为主的液体饲料逐渐过渡到以草料为主的固体饲料,犊牛的消化方式也由化学性消化过渡到以微生物为主的消化方式,此时犊牛可塑性强,对其提供适宜的饲粮营养供给水平至关重要[2-4]。
研究表明,哺乳期在代乳粉饲喂基础上对牦牛犊牛进行固体饲料有效补饲,能够提高牦牛犊牛的生长性能[5-6]。周亚楠等[7]将苜蓿干草与燕麦干草混合饲喂牦牛犊牛,结果表明,2种干草混合优于其单独饲喂,提高了饲草料转化利用率,促进牦牛犊牛的生长发育。Stobo等[8]研究表明,随着精饲料饲喂量的增加,可增加瘤胃上皮乳头长度和密度;Nockels等[9]在试验中把高能量饲粮转变为低能量饲粮后,发现瘤胃乳头的长度变短,直至逐渐消失;而Harrison等[10]也证明,犊牛采食高能饲粮会促进瘤胃的快速生长发育。王音等[11]发现,补饲开食料后,犊牛体重得到了显著提高,也刺激了瘤胃乳头的发育。但关于哺乳期牦牛犊牛固体饲料(开食料和优质干草)适度补饲研究资料较少,缺乏对犊牛早期培育的科学指导。本课题组张颖楠等[2]研究表明,固体饲料高饲喂水平组的犊牛终末体重和平均日增重均显著高于中饲喂水平组和低饲喂水平组;低饲喂水平组瘤胃pH显著低于中饲喂水平组和高饲喂水平组,高饲喂水平组瘤胃的乙酸、丁酸、丙酸和总挥发性脂肪酸含量显著高于中饲喂水平组和低饲喂水平组。因此,本试验在此基础上,利用代谢组学技术探究固体饲料饲喂水平对牦牛犊牛瘤胃形态与功能的影响,阐释早期固体饲料补饲调控犊牛生长性能和瘤胃发育的内在机制,为进一步研究不同生长阶段牦牛犊牛精准补饲调控技术提供重要参考。

1 材料与方法

1.1 试验设计

该试验已通过青海省畜牧兽医科学院试验动物管理委员会批准,审批号:2024-QHMKY-009。
选取体重[(54.20±3.28) kg]相近、健康的60日龄断母乳牦牛犊牛(公)21头,随机分为3组(每组7头牛),即高饲喂水平组(自由采食组;AL组)、中饲喂水平组(70%采食组;IR70组)和低饲喂水平组(40%采食组;IR40组)。饲养试验于海北藏族自治州高原现代生态畜牧业科技示范园中进行,平均海拔为3 010 m,年平均温度为1.5 ℃。

1.2 试验饲粮及饲养管理

预试期30 d内,3组牦牛犊牛统一饲喂代乳粉和固体饲料。随后进入正试期,正试期90 d。试验期间各组犊牛单栏饲养,在饲喂等量代乳粉基础上,按照试验设计分3个水平饲喂由开食料和苜蓿干草组成的固体饲料。正试期开始时3组开食料饲喂量分别为0.30、0.21、0.12 kg/d,之后每10 d分别增加30、21、12 g;3组苜蓿干草饲喂量分别为0.70、0.49、0.28 kg/d,之后每10 d分别增加50、35、20 g;3组代乳粉饲喂量开始时为0.40 kg/d,之后每5 d增加10 g。代乳粉与水的比例为1∶5,分2次进行饲喂。代乳粉和基础饲粮组成及营养水平参见本课题组已经发表的文章[12]。代乳粉和饲粮的总能用氧弹量热仪(Parr 6100,美国Parr公司)进行测定;干物质(DM)、粗蛋白质(CP)、粗脂肪(EE)、中性洗涤纤维(NDF)、酸性洗涤纤维(ADF)、钙(Ca)、磷(P)、氨基酸含量分别参照GB/T 18868—2002、GB/T 6432—2018、GB/T 6433—2006、GB/T 20806—2022、NY/T 1459—2022、GB/T 6436—2018、GB/T 6437—2018、GB/T 18246—2019中方法进行测定。

1.3 样品采集

在正式试验开始的第1、45、90天分别采集代乳粉、苜蓿干草和开食料,在饲养试验完成后将3个时间点的饲粮进行混合并测定其营养成分。牦牛犊牛于前1天16:00断食断水,第2天早晨空腹称重,从每组中随机抽取5头进行屠宰。在瘤胃内部取瘤胃液,用4层纱布过滤后一部分立即测定pH,另一部分分装在5 mL离心管内立即投入液氮进行保存,取瘤胃内容物于5 mL无菌冻存管后,立即置于液氮中保存。取2 cm×2 cm瘤胃组织块样,用生理盐水清洗干净,固定于4%的多聚甲醛溶液,连续换液,直至多聚甲醛溶液澄清透明,用于后续组织形态学观察。

1.4 指标测定及方法

1.4.1 瘤胃组织形态

将 4% 多聚甲醛固定的瘤胃组织制成石蜡切片后,采用苏木精 - 伊红染色法[7]对切片进行染色。每张切片在 4 倍镜下进行观察,并用LY-WN-HP SUPPERCCD软件测量瘤胃后腹盲囊的黏膜上层厚度、黏膜下层厚度、乳头高度、乳头宽度、肌层厚度以及浆膜层厚度。

1.4.2 瘤胃消化酶活性

采用酶联免疫吸附试验法测定瘤胃中的纤维素酶、胃蛋白酶、脂肪酶、淀粉酶、木聚糖酶、内切葡聚糖酶活性。具体操作方法参考本课题组已发表文章[7]。

1.4.3 代谢组学

将15头牦牛犊牛瘤胃内容物样品送至北京诺禾致源科技股份有限公司进行代谢组学测序。取100 μL瘤胃内容物样本置于EP管中,加入400 μL的80%甲醇水溶液,涡旋振荡后冰浴静置5 min,在15 000×g、4 ℃下离心20 min;取一定量的上清液加质谱级水稀释至甲醇含量为53%;15 000×g、4 ℃离心20 min,收集上清液,进样LC-MS进行分析。色谱条件如下:色谱柱Hypesil Goldcolumn (C18),柱温为40 ℃,流速为0.2 mL/min,流动相A为0.1%甲酸,流动相B为甲醇。质谱扫描范围选择质荷比100~1 500。
将下机数据(.raw)文件导入CD 3.1搜库软件中进行处理,对每个代谢物进行保留时间、质荷比等参数的简单筛选,然后设置保留时间偏差0.2 min和质量偏差5 ppm对不同样品进行峰对齐,使鉴定更准确,随后设置质量偏差5 ppm、信号强度偏差30%、信噪比3、最小信号强度、加和离子等信息进行峰提取,同时对峰面积进行定量,再整合目标离子,然后通过分子离子峰和碎片离子进行分子式的预测并与mzCloud(https://www.mzcloud.org/)、mzVault和Masslist数据库进行比对,用blank样本去除背景离子,并对原始定量结果进行标准化处理,最后得到代谢物的鉴定和相对定量结果。数据处理部分基于Linux操作系统(CentOS版本6.6)以及软件R、Python进行。
使用KEGG数据库对鉴定到的代谢物进行注释,使用代谢组学数据处理软件meta X对数据进行转换后进行主成分分析(PCA)和偏最小二乘法判别分析(PLS-DA),采用t检验来检验各代谢物在2组间统计学显著性(P值),并计算代谢物在2组间的差异倍数(FC),差异代谢物筛选的默认标准为变量重要性投影(VIP)>1,P<0.05且FC≥2或FC≤0.5。火山图用R包ggplot 2绘制。

1.5 数据统计与分析

试验原始数据经Excel 2016初步整理后,再经SPSS 20.0软件的ANOVA程序进行单因素方差分析和Duncan氏法多重比较。结果以平均值±标准差表示,P<0.05表示差异显著,0.05≤P<0.10表示具有差异显著的趋势。

2 结果与分析

2.1 固体饲料饲喂水平对牦牛犊牛瘤胃组织形态的影响

由图1、表1可知,AL组瘤胃黏膜上皮、黏膜下层、肌层厚度均显著高于IR70组(P<0.05);AL组瘤胃黏膜上皮、肌层厚度显著高于IR40组(P<0.05);IR70组瘤胃乳头高度、肌层厚度显著高于IR40组(P<0.05);IR40组瘤胃黏膜下层厚度显著高于IR70组(P<0.05);IR70组瘤胃乳头宽度较AL组和IR40组有显著增加的趋势(0.05≤P<0.10)。
图1 牦牛犊牛的瘤胃上皮组织切片

a:AL组 AL group;b:IR70组 IR70 group;c:IR40组 IR40 group;A:乳头 papillary;B:黏膜上皮 mucosal epithelium;C:黏膜下层 submucous layer;D:肌层 muscular layer;E:浆膜层 serosa layer。

Fig.1 Section of rumen epithelium tissue of yak calves (4×)

表1 固体饲料饲喂水平对牦牛犊牛瘤胃组织形态的影响

Table 1 Effects of feeding level of solid feed on rumen histological morphology of yak calves μm

项目
Items		 组别Groups P值
P-value
AL IR70 IR40
黏膜上皮厚度Mucosal epithelium thickness 89.90±2.86a 67.76±3.74b 69.80±1.21b 0.003
黏膜下层厚度Submucous layer thickness 959.98±55.13a 549.27±10.87b 782.09±90.22a 0.009
乳头宽度Papillary width 308.37±24.20 369.77±12.55 292.28±23.12 0.081
乳头高度Papillary length 741.86±53.98ab 966.73±81.90a 602.74±81.07b 0.034
肌层厚度Muscular layer thickness 2 189.69±243.64a 1 443.14±51.31b 815.42±47.94c 0.002
浆膜层厚度Serosa layer thickness 80.61±3.34 100.35±9.39 96.41±12.84 0.354

2.2 固体饲料饲喂水平对牦牛犊牛瘤胃消化酶活性的影响

由表2可知,IR70组和IR40组瘤胃纤维素酶活性显著高于AL组(P<0.05)。
表2 固体饲料饲喂水平对牦牛犊牛瘤胃消化酶活性的影响

Table 2 Effects of feeding level of solid feed on rumen digestive enzyme activities of yak calves

项目
Items		 组别Groups P值
P-value
AL IR70 IR40
纤维素酶Cellulase/(U/mL) 461.68±7.53b 524.64±6.72a 523.79±21.41a 0.027
胃蛋白酶Pepsin/(U/mL) 19.75±1.94 22.36±1.68 24.33±0.42 0.104
脂肪酶Lipase/(U/L) 1 166.16±16.46 1 039.85±70.97 1 018.52±18.24 0.398
淀粉酶Amylase/(U/mL) 455.69±17.64 503.38±41.76 517.04±28.79 0.178
木聚糖酶Xylanase/(U/mL) 778.45±14.07 781.54±40.99 846.64±7.84 0.182
内切葡聚糖Endoglucanase/(U/L) 80.41±2.08 87.16±3.10 84.66±3.77 0.354

2.3 固体饲料饲喂水平对牦牛犊牛瘤胃代谢组学的影响

2.3.1 数据质控及PLS-DA

基于代谢物的相对定量值来计算QC样本之间的Pearson相关系数,由图2可知,测定的数据质量趋向于1,意味着测试过程是稳定的,质量较高且准确可靠。在PLS-DA模型中若R2和Q2更趋近于1,整个的数据模型更稳定可靠。如图3所示,所有的R2都比较趋近于1,R2比Q2大,且Q2回归线到Y轴的截距都小于0,说明模型能较好地描述样本,数据准确可信(图3)。
图2 数据质控

pos:正离子模式;neg:负离子模式;QC:数据质量控制。

Fig.2 Data quality control

pos: positive ion mode; neg: negative ion mode; QC: data quality control.

图3 偏最小二乘法判别分析

A、B:IR40组vs AL组在正离子和负离子的PLS-DA;C、D:IR70组vs AL组在正离子和负离子的PLS-DA。A, B: PLS-DA in positive and negative ion for IR40 group vs AL group; C, D: PLS-DA in positive and negative ion for IR70 group vs AL group.

PLS-DA得分散点图中横坐标为样本在第1主成分上的得分;纵坐标为样本在第2主成分上的得分;R2Y表示模型的解释率,Q2Y用于评价PLS-DA模型的预测能力,且R2Y大于Q2Y时表示模型建立良好。PLS-DA排序验证图中横坐标代表随机分组的Y与原始分组Y的相关性,纵坐标代表R2和Q2的得分。class为分组,R40为低饲喂水平组(IR40组),R70为中饲喂水平组(IR70组),R100为高饲喂水平组(AL组)。

Fig.3 Partial least squares discrimination analysis

The abscissa of the scatter plot obtained by PLS-DA is the score of the sample on the first principal component. The ordinate is the score of the sample on the second principal component. R2Y represents the interpretation rate of the model, and Q2Y is used to evaluate the predictive ability of the PLS-DA model. When R2Y is greater than Q2Y, the model is well established. The abscissa in the PLS-DA ranking validation plot represents the correlation between Y of the randomized group and Y of the original group, and the ordinate represents the scores of R2 and Q2. class was a group, and R40 represents the low feeding level group (IR40 group), R70 represents the medium feeding level group (IR70 group), and R100 represents the high feeding level group (AL group), respectively.

2.3.2 筛选的瘤胃差异代谢产物

对IR40组和AL组进行比较,差异代谢物显著上调47种,下调21种(图4-A、图4-B)。对IR70组和AL组进行比较,差异代谢物显著上调21种,下调52种(图4-C、图4-D)。
图4 正、负离子模式下的火山图

A、B:IR40组vs AL组在正离子和负离子下的火山图;C、D:IR70组vs AL组在正离子和负离子下的火山图。A, B: volcanic diagram in positive and negative ion for IR40 group vs AL group; C, D: volcanic diagram in positive and negative ion for IR70 group vs AL group.

R40为低饲喂水平组(IR40组),R70为中饲喂水平组(IR70组),R100为高饲喂水平组(AL组);UP代表上调;DW代表下调;NoDiff代表无差异显著性。R40 is the low feeding level group (IR40 group), R70 is the medium feeding level group (IR70 group), and R100 is the high feeding level group (AL group); “UP” stands for upward adjustment; DW stands for downward adjustment; “NoDiff” represents no significant difference.

Fig.4 Volcanic diagram in positive and negative ion mode

由图5可知,IR40组与AL组相比,共筛选出21条代谢通路,正、负离子模式下分别为10和11条。
图5 正离子(A)、负离子(B)模式下的IR40-AL通路气泡图

pos:正离子模式 positive ion mode; Tryptophan metabolism:色氨酸代谢;Taste transduction:味觉传导;Serotonergic synapse:5-羟色胺能突触;Purine metabolism:嘌呤代谢;Phenylalanine metabolism:苯丙氨酸代谢;Pantothenate and CoA biosynthesis:泛酸盐和辅酶A生物合成;Galactose metabolism:半乳糖代谢;Arginine and proline metabolism:精氨酸和脯氨酸代谢;Arachidonic acid metabolism:花生四烯酸代谢;African trypanosomiasis:非洲锥虫病;neg:负离子模式negative ion mode; Tyrosine metabolism:酪氨酸代谢;Renin secretion:肾素分泌;Regulation of lipolysis in adipocytes:脂肪细胞脂解的调控;Primary bile acid biosynthesis:初级胆汁酸生物合成;Phenylalanine, tyrosine and tryptophan biosynthesis:苯丙氨酸、酪氨酸和色氨酸生物合成;Neuroactive ligand-receptor interaction:神经活性配体-受体相互作用;Folate biosynthesis:叶酸生物合成;cAMP signaling pathway :环腺苷酸信号途径;Bile secretion:胆汁分泌;Adrenergic signaling in cardiomyocytes:心肌细胞中的肾上腺素能信号。

Fig.5 Bubble diagram of IR40-AL metabolic pathway in positive ion (A) and negative ion (B) mode

由表3可知,与AL组相比,IR40组在非洲锥虫病代谢途径中,差异代谢物L-犬尿氨酸的含量显著上升(P<0.05)。
表3 IR40-AL代谢通路以及相关差异代谢物

Table 3 Metabolic pathways of IR40-AL and related differential metabolites

序号
No.		 代谢途径
Metabolic pathways		 差异代谢物
Differential metabolites		 P值
P-value
1 初级胆汁酸的生物合成Primary bile acid biosynthesis 胆汁酸↑ 0.074
2 苯丙氨酸、酪氨酸和色氨酸的生物合成
Phenylalanine, tyrosine and tryptophan biosynthesis		 3-羟基苯甲酸↑ 0.143
3 叶酸的生物合成Folate biosynthesis 4-氨基苯甲酸↑ 0.143
4 肾上腺素能在心肌细胞中的信号传导
Adrenergic signaling in cardiomyocytes		 L-肾上腺素↑ 0.143
5 肾素的分泌Renin secretion L-肾上腺素↑ 0.208
6 酪氨酸代谢Tyrosine metabolism L-肾上腺素↑ 0.269
7 cAMP信号传导途径cAMP signaling pathway L-肾上腺素↑ 0.269
8 脂肪细胞中脂肪分解的调节Regulation of lipolysis in adipocytes L-肾上腺素↑ 0.269
9 神经活性配体与受体的相互作用Neuroactive ligand-receptor interaction L-肾上腺素↑ 0.380
10 胆汁分泌Bile secretion 胆汁酸↑ 0.477
11 非洲锥虫病African trypanosomiasis L-犬尿氨酸↑ 0.049
12 半乳糖代谢Galactose metabolism D-塔格糖↓ 0.144
13 羟色胺能突触Serotonergic synapse 前列腺素A2↑ 0.187
14 花生四烯酸的代谢Arachidonic acid metabolism 前列腺素A2↑ 0.229
15 泛酸和辅酶A的生物合成Pantothenate and CoA biosynthesis D-泛醇↑ 0.229
16 苯丙氨酸的代谢Phenylalanine metabolism D-苯丙氨酸↑ 0.269
17 味觉转导Taste transduction D-苯丙氨酸↑ 0.269
18 色氨酸的代谢Tryptophan metabolism L-犬尿喹啉↑ 0.344
19 精氨酸和脯氨酸的代谢Arginine and proline metabolism N-甲基海因↑ 0.443
20 嘌呤代谢Purine metabolism 2’-脱氧腺苷↑ 0.474

表中↑代表上调;↓代表下调。表4同。

In the table, ↑ represents upward adjustment; ↓ represents downward adjustment. The same as Table 4.

由图6可知,IR70组与AL组相比,共筛选出24条代谢通路,正、负离子模式下分别为21和3条。
图6 正离子(A)、负离子(B)模式下的IR70-AL代谢通路气泡图

pos:正离子模式positive ion mode; Protein digestion and absorption:蛋白质的消化和吸收;Vitamin digestion and absorption:维生素的消化和吸收;Mineral absorption:元素吸收;Glutathione metabolism:谷胱甘肽代谢;Central carbon metabolism in cancer:癌症中的中心碳代谢;Biosynthesis of amino acids:氨基酸的生物合成;Aminoacyl-tRNA biosynthesis:氨酰-tRNA生物合成;Vitamin B6 metabolism:维生素B6代谢;Tyrosine metabolism:酪氨酸代谢;Tryptophan metabolism:色氨酸代谢;Pyrimidine metabolism:嘧啶代谢;Purine metabolism:嘌呤代谢;Pantothenate and CoA biosynthesis:泛酸盐和CoA生物合成;Lysine degradation:赖氨酸降解;Galactose metabolism:半乳糖代谢;Cysteine and methionine metabolism:半胱氨酸和甲硫氨酸代谢;Beta-alanine metabolism:β-丙氨酸代谢;Antifolate resistance:抗叶酸抵抗;Alanine, aspartate and glutamate metabolism:丙氨酸、天冬氨酸和谷氨酸代谢;2-oxocarboxylic acid metabolism:2-氧代羧酸代谢;neg:负离子模式 negative ion mode; Primary bile acid biosynthesis:初级胆汁酸生物合成;Phenylalanine metabolism:苯丙氨酸代谢;Bile secretion:胆汁分泌。

Fig.6 Bubble diagram of IR70-AL metabolic pathway in positive ion (A) and negative ion (B) mode

由表4可知,与AL组相比,IR70组在癌症中的碳代谢、矿物质吸收和蛋白质的消化和吸收途径中,差异代谢物L-天冬酰胺和蛋氨酸的含量显著下降(P<0.05);在蛋白质的消化和吸收途径中,差异代谢物尸胺的含量显著下降(P<0.05);而在初级胆汁酸的生物合成途径中,差异代谢产物胆汁酸的含量显著上升(P<0.05)。
表4 IR70-AL代谢通路以及相关差异代谢物

Table 4 Metabolic pathways of IR70-AL and related differential metabolites

序号
No.		 代谢途径
Metabolic pathways		 差异代谢物
Differential metabolites		 P值
P-value
1 癌症中的碳代谢Central carbon metabolism in cancer L-天冬酰胺↓、蛋氨酸↓ 0.016
2 矿物质吸收Mineral absorption L-天冬酰胺↓、蛋氨酸、↓ 0.031
3 蛋白质的消化和吸收Protein digestion and absorption L-天冬酰胺↓、蛋氨酸↓、尸胺↓ 0.032
4 谷胱甘肽的代谢Glutathione metabolism 尸胺↓、L-焦谷氨酸↓ 0.121
5 氨基酸-tRNA的生物合成Aminoacyl-tRNA biosynthesis L-天冬酰胺↓、蛋氨酸↓ 0.149
6 维生素的消化和吸收Vitamin digestion and absorption 吡多胺↓、泛酸↓ 0.149
7 半乳糖代谢Galactose metabolism D-塔格糖↓ 0.219
8 半胱氨酸和蛋氨酸的代谢Cysteine and methionine metabolism 蛋氨酸↓ 0.219
9 氨基酸的生物合成Biosynthesis of amino acids L-天冬酰胺↓、蛋氨酸↓ 0.271
10 丙氨酸、天门冬氨酸和谷氨酸的代谢
Alanine, aspartate and glutamate metabolism		 L-天冬酰胺↓ 0.282
11 抗叶酸抵抗Antifolate resistance 蛋氨酸↓ 0.282
12 维生素B6代谢Vitamin B6 metabolism 吡多胺↓ 0.340
13 泛酸和辅酶A的生物合成Pantothenate and CoA biosynthesis 泛酸↓ 0.340
14 赖氨酸的降解Lysine degradation 尸胺↓ 0.394
15 酪氨酸代谢Tyrosine metabolism 3,4-二羟基苯丙酸↓ 0.443
16 β-丙氨酸的代谢Beta-alanine metabolism 泛酸↓ 0.443
17 色氨酸的代谢Tryptophan metabolism 吲哚-3-乙酸↓ 0.489
18 2-氧代羧酸的代谢2-oxocarboxylic acid metabolism 蛋氨酸↓ 0.489
19 嘧啶代谢Pyrimidine metabolism 胸苷↓ 0.571
20 初级胆汁酸的生物合成Primary bile acid biosynthesis 胆汁酸↑ 0.029
21 苯丙氨酸的代谢Phenylalanine metabolism 苯基乙酰谷氨酰胺↓ 0.170
22 胆汁分泌Bile secretion 胆汁酸↑ 0.223

3 讨论

3.1 固体饲料饲喂水平对牦牛犊牛瘤胃形态与功能的影响

乳头高度是衡量瘤胃组织形态的主要指标,较长的乳头通常与机体摄入的营养物质接触面积更大,更利于瘤胃对营养物质的吸收,影响瘤胃发挥功能的指标还有乳头宽度和肌层厚度[13]。肌层的厚度与其运动能力相关,肌层厚度越厚代表其能提供更强的机械收缩力去促进食糜蠕动吸收[14]。吕凯等[15]研究表明,饲粮蛋白质水平越高,早期断奶藏羊生长性能越高,瘤胃发育越完善。桂林生[16]研究表明,不同饲喂水平对荷斯坦公牛瘤胃组织形态影响显著。本试验结果显示,在增加固体饲料饲喂水平的情况下,高饲喂水平下瘤胃组织形态的发育趋于完善,但瘤胃乳头和浆膜层在中饲喂水平下呈现正向发育状态,这也说明中饲喂水平下犊牛瘤胃发育没有受到较大的影响。
动物生长发育的快慢与消化道酶活性的高低息息相关[17]。瘤胃中的淀粉酶和纤维素酶与瘤胃中微生物组成息息相关[18]。李瑾等[19]研究表明,饲料中蛋白质、脂肪水平对幼鳝消化道中淀粉酶活性的影响较为明显,随着饲料中蛋白质、脂肪水平的增加,其淀粉酶活性逐渐降低。侯永清等[20]报道,随着早期断奶仔猪饲粮营养水平的下降,仔猪小肠内淀粉酶活性上升。邢力[21]在降低草原红牛饲粮营养水平后发现,草原红牛血清中淀粉酶的活性反而升高。在本试验中,瘤胃中的纤维素酶和淀粉酶活性随着固体饲料饲喂水平的降低而提高,与上述研究结果一致。推测低饲喂水平下可供机体消化吸收的营养物质较少,从而会引起瘤胃消化酶活性有一定的升高。

3.2 固体饲料饲喂水平对牦牛犊牛瘤胃代谢组学的影响

与高饲喂水平相比,低饲喂水平组在非洲锥虫病代谢途径中,差异代谢物L-犬尿氨酸的含量显著上升。布氏锥虫是一种能引起非洲锥虫病的寄生虫,能通过血脑屏障,诱导细胞因子造成神经损伤。而L-犬尿氨酸正是干扰素在吲哚胺2,3-双加氧酶的作用下生成的产物,通路中表明L-犬尿氨酸随后起到了神经毒性作用,会进一步的损伤神经[22]。这表明相较于高饲喂水平,低饲喂水平犊牛的身体机能较低,这与瘤胃组织切片结果相对应。
与高饲喂水平相比,中饲喂水平下牦牛犊牛在初级胆汁酸的生物合成差异代谢物胆汁酸的含量显著上升;在癌症中的碳代谢、矿物质吸收和蛋白质的消化和吸收途径中,差异代谢物L-天冬酰胺和蛋氨酸的含量显著下降;在蛋白质的消化和吸收途径中,差异代谢物尸胺的含量显著下降。胆汁酸是肝脏生物合成的一种酸性类固醇类化合物,在脂类和脂肪消化和吸收中起重要作用,根据存在形式,胆汁酸主要分为游离型胆汁酸与结合型胆汁酸[23],胆汁酸分子内含有亲水性的羟基和羧基,同时也含有疏水性的甲基及烃基,使得胆汁酸具有界面活性分子的特征,能降低油和水两相之间的表面张力,促进脂类乳化。初级胆汁酸(如胆酸、鹅脱氧胆酸)在瘤胃微生物作用下,通过关键酶转化为次级胆汁酸(如7α-酮基胆酸、异脱氧胆酸等)[24-25]。次级胆汁酸通过血液循环进入宿主,激活核受体和膜受体,进而调控葡萄糖、氨基酸及脂质代谢通路[24]。金梦茹等[26]在癌症组织中发现其中代谢途径存在差异,还发现癌细胞消耗大量葡萄糖,能将大部分葡萄糖转化为乳酸。氨基酸在体内可用于合成新的蛋白质,支持细胞的生长和修复。在缺乏葡萄糖和脂肪时,氨基酸可以转化为葡萄糖或脂肪储存[27]。在癌症中的碳代谢通路中,天冬酰胺主要是丙酮酸通过丙氨酸、天冬氨酸和谷氨酸代谢产生,其含量下降说明机体利用葡萄糖的效率有所下降。天冬氨酸、蛋氨酸和尸胺分别参与矿物质吸收和蛋白质消化吸收途径[28-30]。矿物质通过调控微生物代谢产物影响肝脏能量代谢和营养物质利用效率[31]。饲粮蛋白质在瘤胃中被微生物降解为肽、氨基酸和氨,其中约60%的蛋白质被降解,剩余40%进入真胃和小肠进一步消化,微生物利用这些含氮物质合成菌体蛋白,后者是反刍动物小肠可消化蛋白的主要来源[32],其含量下降表明中饲喂水平下不利于机体对矿物质及蛋白质的消化吸收。

4 结论

在牦牛犊牛60日龄进行断奶,采用“代乳粉+苜蓿干草+开食料”饲喂方式进行早期培育时,提高固体饲料饲喂水平能促进瘤胃组织形态更好发育;通过代谢组学发现固体饲料高饲喂水平提高了牦牛犊牛对营养物质的消化吸收代谢能力,从而促进牦牛犊牛的生长发育。

References

Publishing order | Descend order by publishing year | Descend order by cited within
[1]
郝力壮, 王万邦, 王迅, 等. 三江源区嵩草草地枯草期牧草营养价值评定及载畜量研究[J]. 草地学报, 2013, 21(1):56-64.

HAO L Z, WANG W B, WANG X, et al. Evaluation of nutritional value and carrying capacity of Kobresia grassland during the period of withered grass in Sanjiangyuan region[J]. Acta Agrestia Sinica, 2013, 21(1):56-64. (in Chinese)

[2]
张颖楠, 李世关, 刘书杰, 等. 不同饲喂水平对断奶牦牛犊牛生长性能、瘤胃发酵及微生物区系的影响[J]. 饲料工业, 2023, 44(6):38-45.

ZHANG Y N, LI S G, LIU S J, et al. Effects of different feeding levels on growth performance,rumen fermentation and microbiota of weaned yak calves[J]. Feed Industry, 2023, 44(6):38-45. (in Chinese)

[3]
崔占鸿. 牦牛犊牛培育方式对生长和消化道发育的影响[D]. 博士学位论文. 杨凌: 西北农林科技大学, 2020.

CUI Z H. Effects of rearing patterns on growth and digstive tract development in yak calves[D]. Ph.D.Thesis. Yangling: Northwest A&F University, 2020. (in Chinese)

[4]
安乐乐, 郭文杰, 操君, 等. 哺乳期饲喂开食料对牦牛犊牛生长性能和肠道发育的影响[J]. 动物营养学报, 2023, 35(3):1716-1728.

DOI

AN L L, GUO W J, CAO J, et al. Effects of feeding starter during lactation on growth performance and intestinal development of yak calves[J]. Chinese Journal of Animal Nutrition, 2023, 35(3):1716-1728. (in Chinese)

DOI

[5]
郝文君, 薛国良, 刘书杰, 等. 补饲开食料对早期断奶牦牛犊牛胸腺和脾脏发育的转录组学分析[J]. 动物营养学报, 2023, 35(2):977-994.

DOI

HAO W J, XUE G L, LIU S J, et al. Transcriptomic analysis of starter feed supplementation on thymus and spleen development in early weaned yak calves[J]. Chinese Journal of Animal Nutrition, 2023, 35(2):977-994. (in Chinese)

DOI

[6]
焦洋. 饲粮组成及营养水平对哺乳期牦牛犊牛胰腺发育的影响[D]. 硕士学位论文. 西宁: 青海大学, 2023.

JIAO Y. Effect of diet composition and nutrient level on pancreatic development of preweaning yak calves[D]. Master’s Thesis. Xining: Qinghai University, 2023. (in Chinese)

[7]
周亚楠, 刘书杰, 杨得玉, 等. 苜蓿干草与燕麦干草及其混合饲喂对哺乳期牦牛犊牛复胃组织形态和消化酶活性的影响[J]. 动物营养学报, 2023, 35(5):3141-3153.

DOI

ZHOU Y N, LIU S J, YANG D Y, et al. Effects of alfalfa hay,oat hay and their mixed feeding on compound stomach morphology and digestive enzyme activities of yak calves during lactation period[J]. Chinese Journal of Animal Nutrition, 2023, 35(5):3141-3153. (in Chinese)

[8]
STOBO I J, ROY J H, GASTON H J. Rumen development in the calf.1.The effect of diets containing different proportions of concentrates to hay on rumen development[J]. The British Journal of Nutrition, 1966, 20(2):171-188.

[9]
NOCKELS C F, KINTNER L D, PFANDER W H. Influence of ration on morphology,histology,and trace mineral content of sheep rumen papillae[J]. Journal of Dairy Science, 1966, 49(9):1068-1074.

[10]
HARRISON H N, WARNER R G, SANDER E G, et al. Changes in the tissue and volume of the stomachs of calves following the removal of dry feed or consumption of inert bulk[J]. Journal of Dairy Science, 1960, 43(9):1301-1312.

[11]
王音, 郭文杰, 郝文君, 等. 哺乳期补饲开食料对牦牛犊牛生长性能、瘤胃发育和微生物区系的影响[J]. 动物营养学报, 2022, 34(5):3066-3076.

DOI

WANG Y, GUO W J, HAO W J, et al. Effects of supplementary starter feed on growth performance,rumen development and microbiome of yak calves during lactation[J]. Chinese Journal of Animal Nutrition, 2022, 34(5):3066-3076. (in Chinese)

DOI

[12]
李世关, 张颖楠, 杨得玉, 等. 基于代谢组学分析固体饲料饲喂水平对早期断奶牦牛犊牛皱胃形态与功能发育的影响[J]. 饲料工业, 2024, 45(9):55-64.

LI S G, ZHANG Y N, YANG D Y, et al. Metabolomics-based analysis of the effects of solid feed feeding levels on the morphology and functional development of abomasums in early-weaned yak calves[J]. Feed Industry, 2024, 45(9):55-64. (in Chinese)

[13]
徐建峰, 王燕燕, 高博, 等. 不同断奶日龄对湖羊羔羊生产性能、内脏器官发育及瘤胃形态参数的影响[J]. 西北农业学报, 2021, 30(1):18-24.

XU J F, WANG Y Y, GAO B, et al. Effects of different weaned days of age on production performance,internal organ development and morphological structure with rumen papillae of Hu lambs[J]. Acta Agriculturae Boreali-Occidentalis Sinica, 2021, 30(1):18-24. (in Chinese)

[14]
包花尔. 不同饲养条件下阿尔巴斯绒山羊前胃形态学改变的研究[D]. 硕士学位论文. 呼和浩特: 内蒙古农业大学, 2004.

BAO H E. Study of different raising conditions on variety of the morphological of Aerbasi cashmere’s proventriculus[D]. Master’s Thesis. Hohhot: Inner Mongolia Agricultural University, 2004. (in Chinese)

[15]
吕凯, 侯生珍, 王志有, 等. 蛋白水平对早期断奶藏羔羊复胃发育的影响[J]. 黑龙江畜牧兽医, 2013(10):54-56.

LV K, HOU S Z, WANG Z Y, et al. Effects of the protein levels on the development of complex stomach on early-weaning Tibetan lambs[J]. Heilongjiang Animal Science and Veterinary Medicine, 2013(10):54-56. (in Chinese)

[16]
桂林生. 日粮精粗比对荷斯坦公牛生长发育、消化道组织形态及血液生化指标影响的研究[D]. 硕士学位论文. 杨凌: 西北农林科技大学, 2009.

GUI L S. Effects of dietary concentrate to coarse ratio on growth, digestive tract morphology and blood biochemical indexes of Holstein bulls[D]. Master’s Thesis. Yangling: Northwest A&F University, 2009. (in Chinese)

[17]
孙娟, 闫素梅. 反刍动物的消化道发育及酶活性影响因素研究进展[J]. 饲料研究, 2015(2):24-28.

SUN J, YAN S M. Advances in the study of factors affecting the development of digestive tract and enzyme activity in ruminants[J]. Feed Research, 2015(2):24-28. (in Chinese)

[18]
MOHARRERY A, DAS T K. Correlation between microbial enzyme activities in the rumen fluid of sheep under different treatments[J]. Reproduction,Nutrition,Development, 2001, 41(6):513-529.

[19]
李瑾, 何瑞国, 张世萍, 等. 不同饵料对幼鳝消化系统内淀粉酶活性的影响[J]. 饲料工业, 2002, 23(12):48-50.

LI J, HE R G, ZHANG S P, et al. The effect of different feed on the amylase activity in the digestive system of young eel[J]. Feed Industry, 2002, 23(12):48-50. (in Chinese)

[20]
侯永清, 周毓平, 呙于明. 饲粮蛋白质及赖氨酸水平对早期断奶仔猪胃肠道消化酶活性、大肠蛋白质腐败作用和腹泻的影响[J]. 中国畜牧杂志, 1999, 35(3):6-7.

HOU Y Q, ZHOU Y P, GUO Y M. Effect of dietary protein levels and lysine levels on the activity of digestive enzyme in the gastro-intestinal tract,large intestinal protein putrefaction and diarrhoea incidence[J]. Chinese Journal of Animal Science, 1999, 35(3):6-7. (in Chinese)

[21]
邢力. 草原红牛与利木赞-草原红牛F1杂交牛生产性能的比较研究[D]. 硕士学位论文. 长春: 吉林农业大学, 2007.

XING L. Comparative studies on performance of grassland red cattle and crossbred F1 of grassland red cattle and Limousin[D]. Master’s Thesis. Changchun: Jilin Agricultural University, 2007. (in Chinese)

[22]
FUJIHARA T, SHEM M N. Metabolism of microbial nitrogen in ruminants with special reference to nucleic acids[J]. Animal Science Journal, 2011, 82(2):198-208.

DOI PMID

[23]
李雪菲. 低钙低锌日粮对乌冉克羊体内元素含量及代谢物的影响[D]. 硕士学位论文. 呼和浩特: 内蒙古大学, 2023.

LI X F. Effects of low calcium and zinc diets on element content and metabolites in Wu Ranke sheep[D]. Master’s Thesis. Hohhot: Inner Mongolia University, 2023. (in Chinese)

[24]
ZHANG B, JIANG X, YU Y, et al. Rumen microbiome-driven insight into bile acid metabolism and host metabolic regulation[J]. The ISME Journal, 2024, 18(1): wrae098.

[25]
CHIANG J Y L. Bile acid metabolism and signaling[J]. Comprehensive Physiology, 2013, 3(3):1191.

DOI PMID

[26]
金梦茹, 王莉, 李燕京. 乳酸对肿瘤微环境内免疫细胞的影响及相关靶点治疗的研究进展[J]. 肿瘤防治研究, 2023, 50(6):634-640.

JIN M R, WANG L, LI Y J. Effect of lactate on immune cells in tumor microenvironment and progress of related target therapy[J]. Cancer Research on Prevention and Treatment, 2023, 50(6):634-640. (in Chinese)

[27]
LOVEDAY S M. Protein digestion and absorption:the influence of food processing[J]. Nutrition Research Reviews, 2023, 36(2):544-559.

[28]
孙运菲. 轮虫摄食棕囊藻的环境因子效应及多组学水平上的差异分析[D]. 博士学位论文. 南京: 南京师范大学, 2020.

SUN Y F. Effects of environmental factors on rotifer feeding on Phaeocystis and difference analysis at multi-omics level[D]. Ph.D.Thesis. Nanjing: Nanjing Normal University, 2020. (in Chinese)

[29]
张婧. 铵硝氮素比例影响辣椒生长与果实代谢的机理研究[D]. 博士学位论文. 兰州: 甘肃农业大学, 2020.

ZHANG J. The mechanism of ammonium and nitrate nitrogen ratio on the growth and fruit metabolism of Capsicum annuum L.[D]. Ph.D.Thesis. Lanzhou: Gansu Agricultural University, 2020. (in Chinese)

[30]
王晓岩. 蒙古白丽蘑的化学成分、药理作用及其相关机制的研究[D]. 博士学位论文. 长春: 吉林农业大学, 2020.

WANG X Y. Analysis of chemical composition of Leucocalocybe ongotca and study on its pharmacological activities and mechanism[D]. Ph.D.Thesis. Changchun: Jilin Agricultural University, 2020. (in Chinese)

[31]
李胤豪, 张清月, 闫素梅. 代谢组学在反刍动物营养代谢应用中的研究进展[J]. 中国农业大学学报, 2022, 27(11):104-116.

LI Y H, ZHANG Q Y, YAN S M. Research progress of metabolomics application in nutritional metabolism of ruminants[J]. Journal of China Agricultural University, 2022, 27(11):104-116. (in Chinese)

[32]
周亚楠. 不同粗饲料来源饲粮对哺乳期牦牛犊牛生长和复胃发育的影响[D]. 硕士学位论文. 西宁: 青海大学, 2023.

ZHOU Y N. Effects of different roughage source diets on growth and compound stomach development of lactating yak calves[D]. Master’s Thesis. Xining: Qinghai University, 2023. (in Chinese)

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