Scientia Agricultura Sinica ›› 2026, Vol. 59 ›› Issue (20): 4655-4672.doi: 10.3864/j.issn.0578-1752.2026.20.017

• SPECIAL FOCUS: THEORY, TECHNOLOGY AND PRACTICE OF LOW-CARBON TRANSITION IN AGRIFOOD SYSTEMS AND ENTERIC METHANE MITIGATION IN RUMINANTS • Previous Articles     Next Articles

Effects of Dietary Soluble Protein Levels on Production Performance, Methane Emission and Rumen Microbial Flora of Reserve Cattle

ZHANG MengMeng(), ZHANG ChuanKai, SUN ShiQin, YIN XiangHui, LI WenJuan, ZHANG LiYang, FU Tong, GAO TengYun(), LIU KaiZhen()   

  1. Henan Agricultural University of Animal Science and Technology/Henan Provincial Livestock Nutrition Regulation and Ecological Breeding International Joint Laboratory, Zhengzhou 450046
  • Received:2025-09-09 Accepted:2026-01-22 Online:2026-10-16 Published:2026-10-21
  • Contact: GAO TengYun, LIU KaiZhen

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Abstract:

【Objective】Soluble protein ( SP ) can be rapidly degraded and utilized by microorganisms in the rumen. Through the analysis of the changes of soluble protein in the diet, the rumen microorganisms and their metabolites were analyzed to explore the effect of soluble protein on methane emission. 【Method】A total of thirty healthy, five-month-old Holstein heifers (94.55 ± 6.27 kg body weight) were randomly allocated to three dietary treatments (n=10 per group). All groups were fed a diet with a 60:40 concentrate-to-roughage ratio. The three dietary treatments differed only in their soluble protein (SP) content, expressed as a percentage of total dietary dry matter: Diet 1 (6.00% SP), Diet 2 (6.48% SP), and Diet 3 (6.96% SP). Following an eight-week feeding period, methane production was measured over three consecutive days, and rumen fluid samples were collected for subsequent analysis.【Result】(1)The daily gain of Diet 3 was significantly higher than that of Diet 2 and Diet 1 during 29-56 days in the whole experimental period (P<0.05 ),There was no significant difference in dry matter intake during the whole experimental period (P>0.05).No significant differences in methane emissions were observed among the treatment groups(P>0.05), while methane to body weight ratio, Diet 2 was significantly higher than Diet 3 and Diet 1 ( <0.05).In the rumen fermentation parameters, the ammonia nitrogen content increased first and then decreased with the soluble protein content. The content of volatile fatty acids was the highest in Diet 3. (2) In microbial diversity, there was no significant difference in ACE, Chao1, Shannon and index among the three groups (P>0.05); at the phylum level, Bacteroidetes in Diet 3 was lower than that in Diet 2 and Diet 1. At the genus level, Prevotella in Diet 2 was slightly higher than the other two groups. LEfSe analysis identified microbial taxa with LDA scores greater than 4.5 in the Diet 3 group, which included Pseudoruminococcus、Streptococcaceae、Veillonellales、Streptococcus_mutans、Ruminiclostridium_josui、Prevotella_histicola、Prevotella_veroralis.(3) There were 240 significantly different metabolites between Diet 1 and Diet 3, 109 significantly different metabolites between Diet 2 and Diet 1, and 60 significantly different metabolites between Diet 3 and Diet 2. Among them, the differences in metabolites were mainly in organic acids and their derivatives, lipids and lipid molecules. The metabolites of succinic acid, 23-demethoxycholic acid, spermidine, histidine, threonine and glutamine increased significantly with the increase of SP content. D-erythritol-4-phosphate, isocitrate, leucine and other metabolites were enriched in Diet 1. (4)There was a significant positive correlation between Veillonella and propionic acid. Ruminiclostridium _ josui, Prevotella _ veroralis and Streptococcus _ mutans were significantly positively correlated with Histidine, threonine and glutamine.【Conclusion】Increasing the level of soluble protein (SP) in the diet improved the average daily gain of heifers and concomitantly exerted a moderate inhibitory effect on methane emissions per unit of body weight. Furthermore, the higher SP level promoted the colonization of beneficial bacteria such as Prevotella, which facilitates volatile fatty acid (VFA) production. These shifts in the rumen enhanced the synthesis of energy and amino acids through ruminal metabolism, thereby potentially supporting immune function.

Key words: soluble protein, methane emissions, reserve cattle, growth performance, rumen microorganisms

Table 1

Basic diet composition and nutrient level (air-dried basis %)"

项目
Item		 含量 Content (%)
Diet 1 Diet 2 Diet 3
原料组成Ingredients
燕麦干草Dried oat hay 20.00 20.00 20.00
苜蓿干草Alfalfa hay 13.34 6.67 0.00
苜蓿青贮Alfalfa silage 0.00 6.67 13.34
小麦秸秆Wheat straw 6.33 6.33 6.33
颗粒料Granular compound1) 60.33 60.33 60.33
营养水平Nutritional level2)
粗蛋白CP (% of DM) 16.88 16.82 16.75
代谢能ME (Mcal/kg) 2.99 2.98 2.98
可溶性蛋白SP3） (% of DM) 6.00 6.48 6.96
SP/ME 2.01 2.17 2.34
SP/CP 0.36 0.39 0.42
中性洗涤纤维NDF (% of DM) 33.03 33.56 34.09
酸性洗涤纤维ADF (% of DM) 17.89 18.29 18.69

Table 2

Regression equation of penetration tube"

渗透管号
Permeation tube number		 牛号
Cattle number		 回归方程
Regression equation		 R2 渗透速率
Permeation rate (mg·d-1)
73 24170 y = -0.0015x + 101.71 0.9992 1.5
64 2948 y = -0.0031x + 177.33 0.9996 3.1
138 2465 y = -0.0041x + 35.625 0.9998 4.1
1 2963 y = -0.0029x + 165.90 0.9998 2.9
39 2464 y = -0.0026x + 155.26 0.9993 2.6
74 2979 y = -0.0014x + 98.074 0.9990 1.4
148 24174 y = -0.0019x + 119.67 0.9998 1.9
86 3000 y = -0.0012x + 89.577 0.9996 1.2
11 3009 y = -0.0073x + 365.99 0.9997 7.3
19 3019 y = -0.0046x + 243.39 0.9998 4.6
90 3012 y = -0.0029x + 35.671 0.9996 2.9
20 2468 y = -0.0014x + 101.07 0.9998 1.4
92 3008 y = -0.0067x + 343.84 0.9996 6.7
63 2938 y = -0.0028x + 161.05 0.9999 2.8
95 24155 y = -0.0096x + 472.28 0.9998 9.6

Table 3

Effects of soluble protein on growth performance of heifers"

项目
Item		 组别Group P值
P value
Diet 1 Diet 2 Diet 3
第1天体重 (kg) 96.60±11.77 98.30±7.01 95.60±6.20 0.885
第28天体重 (kg) 120.40±12.27 126.90±9.93 119.40±3.47 0.413
第56天体重 (kg) 144.70±13.48 151.20±10.52 150.00±4.32 0.576
1-28 d
干物质采食量 (DMI, kg) 3.10±0.36 3.08±0.17 2.91±0.16 0.445
平均日增重 (ADG, kg) 0.85±0.17 1.02±0.28 0.85±0.14 0.342
料重比F/G 3.74±0.70 3.17±0.72 3.50±0.62 0.431
29-56 d
干物质采食量 (DMI, kg) 3.86±0.29 4.02±0.21 3.95±0.08 0.497
平均日增重 (ADG, kg) 0.87b±0.09 0.87b±0.11 1.09a±0.17 0.025
料重比F/G 4.48a±0.49 4.68a±0.55 3.69b±0.55 0.028
1-56 d
干物质采食量 (DMI, kg) 3.48±0.31 3.55±0.19 3.43±0.09 0.694
平均日增重 (ADG, kg) 0.86±0.09 0.94±0.18 0.97±0.14 0.433
料重比F/G 4.06±0.30 3.83±0.50 3.59±0.52 0.290

Table 4

Effects of soluble protein on methane emission intensity of heifers"

项目
Item		 组别Group P值
P value
Diet 1 Diet 2 Diet 3
平均甲烷排放量Average methane emission(g·d-1) 101.47±26.00 69.82±19.48 92.83±26.37 0.143
CH4/BW 0.70a±0.12 0.45b±0.13 0.62ab±0.17 0.049
CH4/DMI 23.81±5.72 16.92±5.57 17.48±8.93 0.256

Table 5

Effects of soluble protein on rumen fermentation parameters of heifers"

项目
Item		 组别Group P值
P value
Diet 1 Diet 2 Diet 3
pH 6.70±0.39 6.65±0.10 6.63±0.18 0.913
NH3 -N/(mg·dL-1) 5.74±1.33 6.90±1.40 6.31±1.31 0.072
MCP ( mg·mL-1) 1.33±0.10 1.26±0.18 1.25±0.13 0.516
乙酸 Acetic acid (AA, mmol·L-1) 45.01±5.70 43.69±4.58 50.07±5.54 0.175
丙酸 Propionic acid (PA, mmol·L-1) 13.39±1.96 12.53±0.74 14.08±1.44 0.286
异丁酸 Isobutyric acid (mmol·L-1) 0.96±0.11 0.97±0.10 1.05±0.24 0.658
丁酸 Butyric acid (mmol·L-1) 4.28±0.93 4.49±0.37 5.15±1.23 0.325
异戊酸 Isovaleric acid (mmol·L-1) 1.24±0.06 1.24±0.05 1.49±0.65 0.488
戊酸 Valeric acid (mmol·L-1) 1.54±0.15 1.53±0.08 1.85±0.56 0.281
乙/丙 (AA/PA) 3.39±0.36 3.48±0.20 3.56±0.22 0.147
总挥发性脂肪酸 Total VFAs (mmol·L-1) 66.42±8.04 64.46±5.29 73.69±8.04 0.603

Fig. 1

Rumen microbial α diversity index of replacement cattle fed with different soluble protein levels"

Fig. 2

PCoA results of rumen microorganisms fed with different soluble protein levels of reserve cattle"

Fig. 3

Differences in the composition of rumen microorganisms in reserve cattle (phylum level and genus level)"

Fig. 4

Screening of differential microorganisms in rumen of reserve cattle A: OTUs Venn diagram between groups; B: LEfSe analysis was used to screen microorganisms with significant differences between groups"

Fig. 5

Pairwise comparison of PLS-DA principal component score plot"

Fig. 6

Volcano diagram of differential metabolites by pairwise comparison"

Fig. 7

Thermograms of three groups of related differential metabolites"

Fig. 8

KEGG enrichment map of differential metabolites by pairwise comparison"

Fig. 9

Heatmap of correlation between rumen microorganisms and rumen fermentation and methane emission"

Fig. 10

Heatmap of correlation between rumen microorganisms and rumen metabolites"

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