Chinese Journal of Animal Nutrition >

2025 , Vol. 37 >Issue 9: 6328 - 6338

DOI: https://doi.org/10.12418/CJAN2025.513

RESEARCH PAPER

Effects of Compound Microecological Preparations on Rumen Fermentation Gas Production, Fermentation Parameters and Nutrient Degradation Rates of Dairy Cows in Vitro

  • LIU Suran , 1 ,
  • ZENG Yichen 1 ,
  • DENG Ming 1 ,
  • SUN Baoli 1 ,
  • HU Wenfeng 2 ,
  • GUO Yongqing , 1, **
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  • 1 College of Animal Science, South China Agricultural University, Guangzhou 510642, China
  • 2 College of Food Science, South China Agricultural University, Guangzhou 510642, China
** associate professor, E-mail:

*Contributed equally

Received date: 2025-01-10

  Online published: 2025-09-12

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Abstract

This experiment was conducted to investigate the effects of compound microecological preparations on rumen fermentation gas production, fermentation parameters and nutrient degradation rates of dairy cows in vitro. A single-factor experimental design was adopted, and it was divided into 4 groups, which were supplemented with 0 (control group, CK group), 0.4 (T1 group), 0.8 (T2 group) and 1.2 g/kg (T3 group) compound microecological preparations [inactivated Lactobacillus acidophilus≥107 CFU/g, Bacillus subtilis≥107 CFU/g, and Aspergillus oryzae (counted by spores)≥107 CFU/g] in the total mixed ration (TMR) substrate, respectively. Each group had 6 replicates and was cultured in vitro for 48 h. The results showed as follows: 1) the gas production in T2 group was the highest among all groups after 2 h, and it was significantly higher than that in CK group from 12 to 48 h (P<0.05). With the increase of the supplemental level of compound microecological preparations, the rumen gas production showed a quadratic change of first increasing and then decreasing from 4 to 48 h (P<0.05). 2) Compared with CK group, the ammonia nitrogen concentration in the compound microecological preparation supplemental groups was significantly decreased (P<0.05), with the lowest in T2 group; the concentrations of acetic acid, valeric acid and total volatile fatty acid in T2 group were significantly increased (P<0.05). 3) The dry matter degradation rate in T2 group was significantly higher than that in T1 group (P<0.05), and the starch degradation rate in T3 group was significantly higher than that in T1 group (P<0.05). There was no significant difference in the degradation rates of neutral detergent fiber and acid detergent fiber among all groups (P>0.05). 4) The amylase activity in T2 group and T3 group was significantly higher than that in T1 group (P<0.05), and the microbial protein concentration was significantly higher than that in CK group and T1 group (P<0.05). In conclusion, the compound microecological preparations can improve the rumen fermentation of dairy cows in vitro, with an appropriate supplemental level of 0.8 to 1.2 g/kg.

Key words： compound microecological preparations; rumen fermentation; gas production; fermentation parameters; nutrient degradation rates; in vitro

Cite this article

LIU Suran , ZENG Yichen , DENG Ming , SUN Baoli , HU Wenfeng , GUO Yongqing . Effects of Compound Microecological Preparations on Rumen Fermentation Gas Production, Fermentation Parameters and Nutrient Degradation Rates of Dairy Cows in Vitro[J]. Chinese Journal of Animal Nutrition, 2025 , 37(9) : 6328 -6338 . DOI: 10.12418/CJAN2025.513

广东省粗饲料资源相对短缺,为满足奶牛的能量需要不得不提高饲粮中的精料水平。精料中玉米等谷物饲料含有大量淀粉,易发酵产生有机酸,致使瘤胃及后肠道pH快速下降,进一步诱发急性或亚急性瘤胃酸中毒、乳脂抑制综合征等代谢病[1-2]。生产中,除了调整饲粮组成外,可以采用饲料添加剂来降低高产牛瘤胃代谢异常的多发。其中,微生态制剂具有改善奶牛胃肠道菌群结构、降低胃肠道异常代谢和提高生产水平的潜力,逐渐受到行业关注。
微生态制剂主要包括乳酸菌、芽孢杆菌以及米曲霉等益生菌及其代谢产物。近年来,微生态制剂在奶牛饲粮中的应用取得了一定进展。研究表明,以占饲粮干物质(DM)0.05%或0.10%的比例向高产奶牛直接饲喂1.35×109 CFU/g的固态植物乳杆菌,能够降低瘤胃中与氨态氮(NH3-N)产生相关的细菌相对丰度[3]。Chang等[4]体外试验结果表明,在30 mL人工瘤胃液中添加0.2 g饲粮和0.01 g嗜酸乳杆菌粉剂(含菌量为2.0×1011 CFU/g)能够提升产气量和DM降解率,同时提高牛链球菌和嗜酸乳杆菌等菌的相对丰度。Jia等[5]在奶牛饲粮中以50 g/(头·d)的剂量添加枯草芽孢杆菌,发现可以降低瘤胃中乙酸摩尔比例,并提高丙酸摩尔比例,同时降低Methanosphaera sp.WGK6和Methanosphaera stadtmanae相对丰度,显著提高奶牛产奶量,降低甲烷(CH4)排放量。Sun等[6]研究发现,在奶牛饲粮中添加1×1011 CFU/(头·d)的枯草芽孢杆菌,能够提高产奶量和乳成分产量,降低乳中体细胞数,促进瘤胃总菌、蛋白质降解菌和淀粉降解菌的生长。研究显示,在泌乳中期奶牛饲粮中以5 g/(头·d)的剂量添加米曲霉培养物,促进了瘤胃中总挥发性脂肪酸(TVFA)的合成,降低了血浆中游离脂肪酸浓度,改善了奶牛的能量代谢[7]。Sosa等[8]研究发现,在奶牛饲粮中添加2 g/(头·d)米曲霉培养物,提高了瘤胃TVFA、乙酸、异戊酸和戊酸浓度,同时也提高了奶牛的干物质采食量和DM表观消化率。
不过,以往的研究多以单一微生态制剂为主,复合微生态制剂在奶牛上的应用研究相对缺乏。因此,本研究采用人工瘤胃的方法,探究了由灭活嗜酸乳杆菌、枯草芽孢杆菌等组成的复合微生态制剂对奶牛瘤胃发酵和营养物质降解率等参数的影响,旨在为提高奶牛瘤胃健康水平和复合微生态制剂的科学应用提供参考。

1 材料与方法

1.1 试验材料和试验设计

本试验所用复合微生态添加剂主要成分为灭活嗜酸乳杆菌≥107 CFU/g、枯草芽孢杆菌≥107 CFU/g和米曲霉(以孢子计)≥107 CFU/g。
采用单因素试验设计,共分为4组,分别在全混合日粮(TMR)底物中添加0(对照组,CK组)、0.4(T1组)、0.8(T2组)和1.2 g/kg(T3组)复合微生态制剂,每组6个重复,体外培养48 h。依据NRC(2001)泌乳中期奶牛营养需要水平配制饲粮,其组成及营养水平见表1。
表1 饲粮组成及营养水平(干物质基础)

Table 1 Composition and nutrient levels of the diet (DM basis) %

项目Items 含量Content
原料Ingredients
苜蓿干草Alfalfa hay 16.95
燕麦草Oat hay 27.51
全株玉米青贮Whole corn silage 14.25
玉米Corn 16.22
豆粕Soybean meal 25.07
合计Total 100.00
营养水平Nutrient levels
粗蛋白质CP 17.01
中性洗涤纤维NDF 32.10
酸性洗涤纤维ADF 20.94
淀粉Starch 24.96
泌乳净能NEL/(MJ/kg) 6.93

泌乳净能依据中国饲料数据库(www.chinafeeddata.org.cn)计算得出,其余营养水平均为实测值。

NEL was calculated according to China Feed Database (www.chinafeeddata.org.cn), while the other nutrient levels were measured values.

1.2 饲养管理

试验地点为华南农业大学奶牛试验基地(广东肇庆),试验中所有程序均得到了华南农业大学动物实验委员会的批准(批准编号:2020G012)。选择3头泌乳天数[(91±16) d]和产奶量[(28.58±2.16) kg/d]相近,安装有瘤胃瘘管的荷斯坦奶牛作为瘤胃液供体动物。牛只单栏饲养,舍内铺有橡胶垫,于每日08:30和17:30进行TMR饲喂,自由饮水,每日清粪2次,机械挤奶,挤奶时间为08:00和17:00。

1.3 样品采集与处理

在晨饲前采集3头奶牛瘤胃液,并用4层医用无菌纱布进行过滤,共收集约1.5 L后放入保温瓶中迅速带回实验室。在实验室内,将瘤胃液与预先配制好的瘤胃缓冲液以1∶2比例混合(期间不断通入二氧化碳),得到人工瘤胃液。
参照Menke等[9]的方法配制瘤胃缓冲液,并参考李金辉等[10]的方法进行瘤胃体外发酵。准确称取1 g(±0.005 g)饲粮底物与相应剂量的微生态制剂于纤维袋中,并放入提前预热至39 ℃的500 mL发酵袋中,然后向每个发酵袋加入混合人工瘤胃液150 mL,盖紧橡胶塞并放至39 ℃水浴锅中振荡培养48 h,振荡频率为45 r/min,每组6个重复;同时,设置3个空白对照。分别在2、4、6、8、12、24、36和48 h时间点对试验数据进行记录;48 h培养结束后,将发酵袋放入冰水中水浴5 min使其终止发酵,并对发酵液进行收集和处理。

1.4 测定指标及方法

1.4.1 产气量

分别于发酵2、4、6、8、12、24、36和48 h,采用50 mL医用注射针管对发酵袋中的气体进行收集记录,并计算产气量,计算公式如下:
产气量=某时间点产气量-对应时间点3个空白对照平均产气量。

1.4.2 瘤胃发酵参数

终止发酵后,将发酵袋中的瘤胃液分装至4管10 mL离心管中,一管用于测定pH;另一管加入溶液体积1/4的25%偏磷酸放入-20 ℃冰箱中保存,用于后续挥发性脂肪酸浓度的测定;其余2管则直接放入-20 ℃冰箱中,用于测定NH3-N和微生物蛋白(MCP)浓度以及消化酶活性指标。采用pH仪(FE28-Standard,Mettler Toledo,瑞士)测定样品pH;NH3-N浓度参考Broderick等[11]的方法测定,采用酶标仪(Synergy H1,BioTek,美国)进行比色,测定标准品吸光度(OD)值,以求出标准曲线,将处理好的样品放在630 nm波长下比色并计算OD值;挥发性脂肪酸测定则依据Erwin等[12]所描述的方法进行,利用装有HP-INNOwax(30.0 m×320 μm×0.5 μm,Catalog No:19091N-213)毛细管色谱柱的Agilent 6890B气相色谱仪测定,具体参数参考Liu等[13]。

1.4.3 营养物质降解率

终止发酵后,将纤维袋从发酵袋中取出,并用冰蒸馏水冲洗其表面杂质,放入烘箱中65 ℃烘干48 h。DM和粗蛋白质(CP)含量参照AOAC(1990)[14]的方法测定,中性洗涤纤维(NDF)和酸性洗涤纤维(ADF)含量参考Van Soest等[15]的方法测定;淀粉含量参考Bal等[16]的方法,采用高氯酸水解-蒽酮比色法测定。营养物质降解率计算公式如下:
某营养物质降解率=100×(发酵前该营养物质含量-发酵后该营养物质含量)/发酵前该营养物质含量。

1.4.4 消化酶活性和MCP浓度

淀粉酶和蛋白酶活性分别采用淀粉-碘比色法和比色法测定;MCP浓度采用嘌呤法测定,将处理过的样品放至波长260 mm下比色,并代入标准曲线中进行计算。测定步骤按照试剂盒说明书标明的方法进行,试剂盒均购自南京建成生物工程研究所。

1.5 数据处理与统计分析

试验所有数据均使用Excel 2016进行初步整理,然后采用SAS 9.4软件的一般线性模型(GLM)程序对试验数据进行分析,模型如下:
Yi=μ+Ti+εi。
式中:Yi为不同处理的因变量值;μ为总体平均值;Ti为微生态制剂处理效应(i=1,2,3,4);εi为随机误差。
采用Tukey法进行多重比较,并采用Spearman秩进行相关性分析,应用正交多项式分析提高微生态制剂水平与各对应指标的关系(处理、线性和二次),结果数据以平均值和均值标准误(SEM)表示,P<0.05表示差异显著,P>0.05表示差异不显著。

2 结果

2.1 复合微生态制剂对奶牛瘤胃体外发酵产气量的影响

由表2可知,T2组奶牛瘤胃体外发酵产气量在2 h后为各组最高,且在12~48 h时显著高于CK组(P<0.05),4和36 h时显著高于T1组(P<0.05),8~48 h时显著高于T3组(P<0.05),其余各组间无显著差异(P>0.05)。随着复合微生态制剂添加水平的提高,瘤胃产气量在4~48 h时呈先升高后降低的二次变化(P<0.05)。
表2 复合微生态制剂对奶牛瘤胃体外发酵产气量的影响

Table 2 Effects of compound microecological preparations on rumen fermentation gas production of dairy cows in vitro mL

时间
Time/h		 组别Groups 均值
标准误
SEM		 P值P-value
CK T1 T2 T3 处理
Treatment		 线性
Linear		 二次
Quadratic
2 39.00 38.70 40.60 41.20 0.569 0.357 0.136 0.646
4 73.00ab 72.37a 76.50b 73.53ab 0.511 0.046 0.430 0.010
6 106.50 106.70 110.70 104.20 0.792 0.060 0.278 0.014
8 134.40ab 135.62ab 140.20b 133.20a 0.785 0.026 0.289 0.007
12 184.73a 185.20ab 189.70b 181.03a 0.617 0.001 0.027 <0.001
24 258.57a 260.30ab 267.10b 254.78a 0.942 0.002 0.052 0.001
36 325.73a 327.30a 333.77b 323.62a 0.610 <0.001 0.045 <0.001
48 365.23a 369.30ab 374.85b 364.53a 1.080 0.011 0.134 0.007

同行数据肩标相同字母或无字母表示差异不显著(P>0.05),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 复合微生态制剂对奶牛瘤胃体外发酵参数的影响

由表3可知,各组间奶牛瘤胃体外发酵pH无显著差异(P>0.05);CK组NH3-N浓度显著高于其余各组(P<0.05),丙酸浓度显著高于T1组(P<0.05);T2组和T3组异戊酸浓度显著高于T1组(P<0.05);T2组乙酸浓度显著高于其余各组(P<0.05),戊酸和TVFA浓度显著高于CK组和T1组(P<0.05),NH3-N浓度显著低于其余各组(P<0.05);T3组戊酸浓度显著高于CK组和T1组(P<0.05);T1组和T2组乙酸/丙酸值显著高于CK组和T3组(P<0.05)。随着复合微生态制剂添加水平的提高,瘤胃NH3-N、戊酸、异戊酸和TVFA浓度呈线性和二次变化(P<0.05)。
表3 复合微生态制剂对奶牛瘤胃体外发酵参数的影响

Table 3 Effects of compound microecological preparations on rumen fermentation parameters of dairy cows in vitro

项目
Items		 组别Groups 均值
标准误
SEM		 P值P-value
CK T1 T2 T3 处理
Treatment		 线性
Linear		 二次
Quadratic
pH 6.28 6.29 6.28 6.29 0.004 0.719 0.887 0.293
氨态氮
NH3-N/(mg/dL)		 26.37d 21.17c 9.95a 14.42b 0.344 <0.001 <0.001 <0.001
挥发性脂肪酸VFA/(mmol/L)
乙酸Acetic acid 38.67a 40.15a 47.36b 41.68a 0.660 0.001 0.394 0.001
丙酸Propanoic acid 12.38b 9.83a 10.89ab 11.22ab 0.283 0.047 0.077 0.598
丁酸Butyric acid 8.27 8.12 8.74 8.58 0.087 0.068 0.062 0.072
戊酸Valeric acid 2.13a 1.81a 2.75b 2.67b 0.056 <0.001 <0.001 0.001
异丁酸
Isobutyric acid		 1.56 1.38 1.31 1.54 0.080 0.647 0.479 0.447
异戊酸
Isovaleric acid		 2.13ab 1.80a 2.64b 2.36b 0.065 0.001 0.005 0.002
总挥发性脂肪酸
TVFA		 65.14a 63.09a 74.49b 68.66ab 0.902 0.002 0.035 0.001
乙酸/丙酸
Acetic acid/
propanoic acid		 3.13a 4.12b 4.43b 3.77a 0.093 0.002 0.170 0.045
摩尔比例Molar ratio/%
乙酸Acetic acid 59.30a 63.63bc 64.57c 61.51ab 0.371 0.001 0.050 0.038
丙酸Propanoic acid 18.99b 15.57a 14.59a 16.66ab 0.309 0.001 0.205 0.056
丁酸Butyric acid 12.73 12.90 11.94 12.58 0.145 0.127 0.424 0.035
戊酸Valeric acid 3.29ab 2.88a 3.67b 3.88b 0.091 0.004 0.001 0.197
异丁酸
Isobutyric acid		 2.44 2.18 1.69 2.08 0.128 0.291 0.791 0.172
异戊酸
Isovaleric acid		 3.27 2.86 3.54 3.29 0.089 0.072 0.090 0.045
瘤胃挥发性脂肪酸摩尔比例方面,T2组乙酸摩尔比例显著高于CK组和T3组(P<0.05),丙酸摩尔比例显著低于CK组(P<0.05);T1组乙酸摩尔比例显著高于CK组(P<0.05),丙酸摩尔比例显著低于CK组(P<0.05);T2组和T3组戊酸摩尔比例显著高于T1组(P<0.05)。随着复合微生态制剂添加水平的提高,瘤胃戊酸摩尔比例呈线性提高(P<0.05),瘤胃乙酸、丁酸和异戊酸摩尔比例呈二次变化(P<0.05)。

2.3 复合微生态制剂对奶牛瘤胃体外发酵营养物质降解率的影响

由表4可知,T2组奶牛瘤胃体外发酵DM降解率显著高于T1组(P<0.05),CP降解率显著低于CK组和T1组(P<0.05);CK组和T3组淀粉降解率显著高于T1组(P<0.05);各组间NDF和ADF降解率无显著差异(P>0.05)。随着复合微生态制剂添加水平的提高,瘤胃DM降解率呈二次变化(P<0.05),CP和淀粉降解率呈线性和二次变化(P<0.05)。
表4 复合微生态制剂对奶牛瘤胃体外发酵营养物质降解率的影响

Table 4 Effects of compound microecological preparations on rumen fermentation nutrient degradation rates of dairy cows in vitro %

项目
Items		 组别Groups 均值
标准误
SEM		 P值P-value
CK T1 T2 T3 处理
Treatment		 线性
Linear		 二次
Quadratic
干物质DM 44.78ab 44.15a 48.35b 44.83ab 0.469 0.028 0.605 0.004
粗蛋白质CP 72.65b 72.12b 62.42a 66.41ab 0.812 0.006 0.038 0.009
淀粉Starch 82.03b 74.12a 79.99ab 85.66b 0.693 0.006 0.038 0.009
中性洗涤纤维
NDF		 40.94 41.15 42.92 41.84 0.483 0.504 0.624 0.263
酸性洗涤纤维
ADF		 27.25 26.63 27.43 27.05 0.439 0.924 0.742 0.595

2.4 复合微生态制剂对奶牛瘤胃体外发酵消化酶活性和MCP浓度的影响

由表5可知,T2组和T3组奶牛瘤胃体外发酵淀粉酶活性显著高于T1组(P<0.05),MCP浓度显著高于CK组和T1组(P<0.05);T2组蛋白酶活性显著低于T1组(P<0.05);T2组淀粉酶活性显著低于T3组(P<0.05)。随着复合微生态制剂添加水平的提高,瘤胃淀粉酶活性呈线性提高(P<0.05),蛋白酶活性呈二次变化(P<0.05),MCP浓度呈线性和二次变化(P<0.05)。
表5 复合微生态制剂对奶牛瘤胃体外发酵消化酶活性和MCP浓度的影响

Table 5 Effects of compound microecological preparations on rumen fermentation digestive enzyme activity and MCP concentration of dairy cows in vitro

项目
Items		 组别Groups 均值
标准误
SEM		 P值P-value
CK T1 T2 T3 处理
Treatment		 线性
Linear		 二次
Quadratic
淀粉酶
Amylase/(U/dL)		 32.74bc 25.20a 30.23b 35.37c 0.601 <0.001 <0.001 0.863
蛋白酶
Protease/(U/mL)		 17.05ab 18.13b 13.31a 15.69ab 0.525 0.015 0.153 0.010
微生物蛋白
MCP/(mg/dL)		 35.99a 37.02a 53.88b 49.34b 1.404 <0.001 0.006 0.006

3 讨论

3.1 复合微生态制剂对奶牛瘤胃体外发酵产气量的影响

瘤胃中有大量微生物栖息,如原虫、细菌和真菌等,其在发酵饲粮的过程中会释放大量气体,如甲烷、氢气和二氧化碳等。因此,瘤胃产气量与底物降解率和瘤胃微生物活性之间呈正相关关系,是评估饲粮体外发酵程度的一项重要指标[17]。本研究中,奶牛瘤胃体外发酵产气量在4~48 h时随复合微生态制剂添加水平的提高呈先升高后降低的二次变化。赵鹏等[18]研究结果也显示,饲粮中添加复合微生态制剂可以显著提高瘤胃体外发酵产气量。微生态制剂中的益生菌可利用饲粮底物中的氮源和碳源代谢产生各类消化酶,如芽孢杆菌可产生淀粉酶、纤维酶和脂肪酶等,促进营养物质降解并产生乙酸、丙酸和甲烷等,提高产气量[19]。有研究结果显示,复合微生态制剂能显著提高瘤胃体外发酵总产气量,并降低甲烷和二氧化碳的产生量[20]。由此可知,复合微生态制剂可以提高反刍动物瘤胃对营养物质的降解能力和利用率。

3.2 复合微生态制剂对奶牛瘤胃体外发酵参数的影响

pH是衡量瘤胃内环境的重要指标之一,可反映瘤胃内微生物的代谢状况与活动水平,正常范围在6.0~7.0[21]。本试验中,各组瘤胃体外发酵pH均在正常范围内,表明复合微生态制剂并未对瘤胃内环境稳态造成不良影响。NH3-N作为瘤胃内蛋白质降解的中间产物,反映了机体对蛋白质的利用效率[22]。同时,NH3-N是瘤胃内众多微生物的唯一氮源,也是MCP合成的重要前体物质之一,其浓度受采食量、饲粮蛋白质水平以及微生物利用效率等多种因素的影响。Chen等[23]研究发现,微生态制剂能够显著降低瘤胃体外发酵的NH3-N浓度。本研究也得到相似的结果,复合微生态制剂添加组NH3-N浓度较CK组显著降低,并以T2组浓度最低,表明复合微生态制剂中的益生菌能通过协同作用促进瘤胃内微生物的代谢,加速MCP的合成。
乙酸、丙酸和丁酸等挥发性脂肪酸主要由瘤胃内微生物发酵代谢产生,为反刍动物提供了70%~80%的能量来源,是反刍动物赖以生存的必需物质之一[24]。乙酸除供能外也是乳脂合成的前体物质之一,丙酸则是机体糖异生作用的重要原料。本试验中,T2组瘤胃体外发酵乙酸和TVFA浓度显著提高,表明复合微生态制剂具有改善瘤胃发酵的作用。研究发现,益生菌能促进瘤胃原生菌对抗性碳水化合物的降解,从而提高瘤胃发挥性脂肪酸浓度[25]。本试验中,T1组和T2组瘤胃较低的丙酸浓度和摩尔比例与较低的淀粉降解率和淀粉酶活性有关,研究表明非结构性碳水化合物降解发酵能提高奶牛胃肠道中丙酸浓度[26]。
戊酸、异丁酸和异戊酸等挥发性脂肪酸都属于异位酸,由支链氨基酸通过氧化脱氨基而来,具有调节瘤胃发酵、MCP合成、瘤胃纤维菌生长以及促进瘤胃和小肠绒毛生长发育等作用[27]。本研究中,随着复合微生态制剂添加水平的提高,瘤胃异位酸浓度和摩尔比例整体呈线性提高。Lamontagne等[28]在泌乳期奶牛饲粮中添加200 g/d的微生态制剂,显著提高了瘤胃中戊酸和异戊酸的水平,表明复合微生态制剂具有改善瘤胃菌群平衡和促进瘤胃发酵的作用。依据乙酸/丙酸值的大小,可将瘤胃发酵类型分为乙酸型、丙酸型、丁酸型以及混合型等多种发酵模式。本试验中,奶牛瘤胃体外发酵乙酸/丙酸值随复合微生态制剂添加水平的提高呈二次变化,并属于乙酸型发酵模式。研究表明,瘤胃内脂质代谢通路的改变会影响乳脂率,乙酸/丙酸值与乳脂率呈显著正相关[29-30]。Cappellozza等[31]研究表明,在泌乳奶牛饲粮中添加微生态制剂,可以显著提高乙酸摩尔比例,显著降低丙酸摩尔比例,从而提高乙酸/丙酸值。

3.3 复合微生态制剂对奶牛瘤胃体外发酵营养物质降解率的影响

营养物质降解率通常用来评判饲粮营养价值和瘤胃微生物状态,而瘤胃微生物状态又反映了瘤胃内微生物对饲粮中营养物质的降解和利用能力。瘤胃微生物生长发育所需要的氮源和碳源主要由饲粮中的蛋白质和淀粉提供[32]。本研究得出,随着复合微生态制剂添加水平的提高,奶牛瘤胃体外发酵DM降解率呈二次变化。王志远[33]研究也显示,微生态制剂可以显著提高全株谷草青贮瘤胃体外发酵全期的DM降解率。DM降解率与瘤胃微生物活性和体外产气量之间呈正相关[34],表明复合微生态制剂加速了瘤胃菌群对营养物质的降解和利用。
饲料CP降解率取决于多种因素,如蛋白质品质、瘤胃停留时间和发酵添加剂等。本研究中,T2组奶牛瘤胃体外发酵CP降解率显著低于CK组。Suprayogi等[35]研究表明,米曲霉在发酵过程中会降低豆粕中组氨酸和苯丙氨酸含量;采用5 mL/100 g的米曲霉对豆粕进行发酵,结果表明,发酵豆粕中真蛋白质、组氨酸和赖氨酸含量均有所降低[36]。米曲霉在发酵过程中直接或间接形成初级代谢物和次级挥发性代谢物,具有抑制蛋白质降解的作用,并能与蛋白质结合,形成复合物,从而降低蛋白质的降解速率[37-38]。此外,温度是影响米曲霉降解蛋白质的重要因素之一,当温度超过30 ℃时,米曲霉分泌的蛋白酶活性显著降低,从而影响CP降解率[39-40],人工瘤胃试验所模拟瘤胃温度一般为37 ℃,这可能会导致米曲霉活性降低。本试验中,淀粉降解率随微生态制剂添加水平的提高呈线性提高。乳杆菌和芽孢类杆菌在适宜的胃肠内环境中能分泌多种功能性酶类如淀粉酶和纤维素酶,同时也能破坏饲粮纤维结构,促进饲粮中营养物质的降解[41-42]。本试验中,复合微生态制剂未能显著降低NDF和ADF降解率,这可能是因为发酵底物中半纤维素含量较高,而本试验添加水平下的复合微生态制剂无法分泌足够的纤维素酶以提高NDF和ADF降解率。

3.4 复合微生态制剂对奶牛瘤胃体外发酵消化酶活性和MCP浓度的影响

反刍动物最大的生理特点之一是可借助瘤胃内的微生物,将饲粮中的各种营养物质进行降解,并利用其中的小肽、氨基酸和挥发性脂肪酸等氮源和碳源合成MCP。MCP是反刍动物小肠吸收利用蛋白质的主要来源,为机体提供50%左右所需蛋白质[43]。本研究中,T2组和T3组瘤胃体外发酵MCP浓度显著高于CK组,这可能与其较低的瘤胃NH3-N浓度有关。NH3-N是MCP合成的重要前体物质[44]。王诚等[45]在奶牛饲粮中添加15 mL/(头·d)复合微生态制剂,显著降低了瘤胃NH3-N和丁酸浓度,表明微生态制剂能促进瘤胃微生物对NH3-N的吸收,从而提高MCP的合成量。瘤胃微生物在降解饲粮纤维素、半纤维素等多种难以消化的物质时,会产生各种酶以助分解,因此瘤胃消化酶活性与营养物质消化之间的关联非常密切。本研究中,奶牛瘤胃体外发酵MCP浓度和淀粉酶活性随复合微生态制剂添加水平的提高呈线性提高,表明复合微生态制剂促进了瘤胃微生物的生长及消化酶活性的提高。

4 结论

在本试验条件下,向TMR底物添加复合微生态制剂能够降低奶牛瘤胃体外发酵NH3-N浓度,但对NDF和ADF降解率无显著影响。添加0.8 g/kg复合微生态制剂可以提高瘤胃体外发酵产气量以及乙酸、戊酸和TVFA浓度,添加0.8和1.2 g/kg复合微生态制剂均可提高MCP浓度。因此,复合微生态制剂可以改善奶牛瘤胃体外发酵,以添加0.8~1.2 g/kg为宜。

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