动物营养学报 >

2023 , Vol. 35 >Issue 11: 7270 - 7283

DOI: https://doi.org/10.12418/CJAN2023.662

研究论文

不同粗饲料组合添加支链挥发性脂肪酸对牦牛体外瘤胃发酵特性的影响

  • 马秀莲 , 1 ,
  • 姜菲 1 ,
  • 高彦华 , 1, * ,
  • 彭忠利 1 ,
  • 黄艳玲 1 ,
  • 黎小银 1 ,
  • 胡志斌 1 ,
  • 李博为 1 ,
  • 张康林 2
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  • 1 西南民族大学畜牧兽医学院,青藏高原动物遗传资源保护与利用教育部重点实验室,动物科学国家民委重点实验室,成都 610041
  • 2 甘孜藏族自治州畜牧业科学研究所,康定 626000
* 高彦华,讲师,硕士生导师,E-mail:

马秀莲(1997—),女,甘肃康乐人,硕士研究生,从事反刍动物营养研究。E-mail:

Copy editor: 田艳明

收稿日期: 2023-06-05

  网络出版日期: 2023-11-12

基金资助

四川省科技计划项目(2023NSFSC1148)

国家现代农业技术体系四川肉牛创新团队建设项目(sccxtd-2020-13)

西南民族大学双一流项目(XM2023010)

甘孜州科技计划(23Kjjh00017)

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Effects of Different Roughage Combinations Supplemented with Branched-Chain Volatile Fatty Acids on Rumen Fermentation Characteristics of Yaks in Vitro

  • MA Xiulian , 1 ,
  • JIANG Fei 1 ,
  • GAO Yanhua , 1, * ,
  • PENG Zhongli 1 ,
  • HUANG Yanling 1 ,
  • LI Xiaoyin 1 ,
  • HU Zhibin 1 ,
  • LI Bowei 1 ,
  • ZHANG Kanglin 2
Expand
  • 1 Key Laboratory of Animal Science of National Ethnic Affairs Commission of China, Ministry of Education Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resources Reservation and Utilization, College of Animal and Veterinary Sciences, Southwest Minzu University, Chengdu 610041, China
  • 2 Institute of Animal Husbandry Science, Ganzi Tibetan Autonomous Prefecture, Kangding 626000, China
* lecturer, E-mail:

Received date: 2023-06-05

  Online published: 2023-11-12

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摘要

本研究旨在探讨以不同比例油菜秸秆(RAS)和稻草(RIS)替代全株玉米青贮(WCS)对牦牛体外瘤胃发酵特性的影响,筛选出1种适宜组合后,继续研究添加不同种类支链挥发性脂肪酸(BCVFA)对牦牛体外瘤胃发酵特性的影响。试验1设置12个组,每组4个重复,各组底物组成分别为100%WCS、100%RAS、100%RIS、90%WCS+10%RAS、80%WCS+20%RAS、70%WCS+30%RAS、90%WCS+10%RIS、80%WCS+20%RIS、70%WCS+30%RIS、80%WCS+10%RAS+10%RIS、70%WCS+10%RAS+20%RIS及70%WCS+20%RAS+10%RIS,经体外发酵72 h后测定瘤胃发酵参数,确定1种适宜组合。试验2选择1种适宜组合(70%WCS+20%RAS+10%RIS)为底物,添加0.3%BCVFA(干物质基础),设置对照组(不添加BCVFA)及7个试验组,每组4个重复,各试验组分别添加异丁酸(IB)、异戊酸(IV)、2-甲基丁酸(ME)、IB∶IV=1∶1、IB∶ME=1∶1、IV∶ME=1∶1及IB∶IV∶ME=1∶1∶1,经体外发酵72 h后测定瘤胃发酵参数及纤维素酶活性。结果表明:1)与RAS组和RIS组相比,70%WCS+30%RAS组pH显著降低(P<0.05),微生物蛋白(MCP)和丁酸含量以及干物质降解率(DMD)均显著提高(P<0.05),且中性洗涤纤维降解率(NDFD)显著高于RAS组(P<0.05);80%WCS+20%RIS组pH显著降低(P<0.05),MCP、乙酸、丙酸、丁酸和总挥发性脂肪酸含量以及DMD均显著提高(P<0.05),且NDFD显著高于RAS组(P<0.05);70%WCS+10%RAS+20%RIS组pH显著降低(P<0.05),MCP含量和DMD均显著提高(P<0.05);70%WCS+20%RAS+10%RIS组pH显著降低(P<0.05),MCP含量和DMD均显著提高(P<0.05)。综合比较,70%WCS+20%RAS+10%RIS组为适宜组合。2)与对照组相比,各试验组MCP含量均显著提高(P<0.05),其中IV∶ME组最高;挥发性脂肪酸方面,IB∶IV组和IB∶ME组丙酸和戊酸含量均显著高于对照组(P<0.05);养分降解率方面,对照组和IB∶IV∶ME组显著高于其余各组(P<0.05);纤维素酶活性方面,IB∶ME组、IV∶ME组及IB∶IV∶ME组羧甲基纤维素酶和木聚糖酶活性均显著高于对照组(P<0.05),其中IV∶ME组最高。综上所述,以70%WCS+20%RAS+10%RIS为底物添加0.3%BCVFA(IV∶IB∶ME=1∶1∶1)能有效改善牦牛体外瘤胃发酵特性。

关键词： 油菜秸秆; 稻草; 支链挥发性脂肪酸; 体外瘤胃发酵; 牦牛

本文引用格式

马秀莲 , 姜菲 , 高彦华 , 彭忠利 , 黄艳玲 , 黎小银 , 胡志斌 , 李博为 , 张康林 . 不同粗饲料组合添加支链挥发性脂肪酸对牦牛体外瘤胃发酵特性的影响[J]. 动物营养学报, 2023 , 35(11) : 7270 -7283 . DOI: 10.12418/CJAN2023.662

Abstract

The aim of this study was to investigate the effects of replacing whole corn silage (WCS) with different proportions of rape straw (RAS) and rice straw (RIS) on rumen fermentation characteristics of yaks in vitro. After selecting a suitable combination, the effects of supplementation of different types of branch chain volatile fatty acid (BCVFA) on rumen fermentation characteristics of yaks in vitro were further studied. The experiment 1 consisted of 12 groups with 4 replicates per group. The substrate composition of each group was 100%WCS, 100%RAS, 100%RIS, 90%WCS+10%RAS, 80%WCS+20%RAS, 70%WCS+30%RAS, 90%WCS+10%RIS, 80%WCS+20%RIS, 70%WCS+30%RIS, 80%WCS+10%RAS+10%RIS, 70%WCS+10%RAS+20%RIS and 70%WCS+20%RAS+10%RIS, respectively. The rumen fermentation parameters were determined after 72 h in vitro fermentation, and an optimal combination was determined. In experiment 2, an optimal combination (70%WCS+20%RAS+10%RIS) was selected as the substrate, 0.3% BCVFA (dry matter basis) was added, and a control group (without BCVFA) and 7 experimental groups were set up, with 4 replicates in each group. The experimental groups were supplemented with isobutyrate (IB), isovalerate (IV), 2-methyl butyrate (ME), IB∶IV=1∶1, IB∶ME=1∶1, IV∶ME=1∶1 and IB∶IV∶ME=1∶1∶1, respectively. Then the rumen fermentation parameters and cellulase activity were determined after 72 h in vitro fermentation. The results showed as follows: 1) compared with RAS and RIS groups, pH in 70%WCS+30%RAS group was significantly decreased (P<0.05), the contents of microbial protein (MCP) and butyrate as well as dry matter degradation rate (DMD) were significantly increased (P<0.05), and the neutral detergent fiber degradation rate (NDFD) was significantly higher than that in RAS group (P<0.05); pH in 80%WCS+20%RIS group was significantly decreased (P<0.05), the contents of MCP, acetate, propionate, butyrate and total volatile fatty acid as well as DMD were significantly increased (P<0.05), and the NDFD was significantly higher than that in RAS group (P<0.05); pH in 70%WCS+10%RAS+20%RIS group was significantly decreased (P<0.05), and the MCP content and DMD were significantly increased (P<0.05); pH in 70%WCS+20%RAS+10%RIS group was significantly decreased (P<0.05), and the MCP content and DMD were significantly increased (P<0.05). By comprehensive comparison, 70%WCS+20%RAS+10%RIS group was the appropriate combination. 2) Compared with control group, the MCP content in experimental groups was significantly increased (P<0.05), and the highest value was found in IV∶ME group; in terms of volatile fatty acid, the contents of propionate and valerate in IB∶IV and IB∶ME groups were significantly higher than those in the control group (P<0.05); the nutrient degradation rate in the control group and IB∶IV∶ME group was significantly higher than that in the other groups (P<0.05); in terms of cellulase activity, the activities of carboxymethyl cellulase and xylanase in IB∶ME, IV∶ME and IB∶IV∶ME groups were significantly higher than those in the control group (P<0.05), and the highest values were found in IV∶ME group. In conclusion, supplementation of 0.3% BCVFA (IV∶IB∶ME=1∶1∶1) with 70%WCS+20%RAS+10%RIS as substrate can effectively improve rumen fermentation characteristics of yaks in vitro.

Key words： rape straw; rice straw; branched-chain volatile fatty acid; in vitro rumen fermentation; yaks

牦牛是青藏高原地区特有的畜种,对当地的经济发展尤为重要,但高原地区气候寒冷,枯草期长,导致牦牛粗饲料供应不足,一定程度限制了牦牛养殖业的发展。高海拔地区放牧牦牛瘤胃中存在大量螺旋体和密螺旋体,且瘤胃球菌科和脱硫弧菌科的相对丰度高于舍饲牦牛,这使牦牛对低质粗饲料具有更强的消化能力[1-2]。因此,可考虑将低质粗饲料如油菜秸秆(rapeseed straw,RAS)、小麦秸秆和稻草(rice straw,RIS)等用于饲喂牦牛,以缓解粗饲料供应不足的问题。
油菜秸秆和水稻是四川平原地区的主要农作物副产物,具有产量大及纤维含量高[中性洗涤纤维(neutral detergent fiber,NDF)含量为55.75%~79.70%,粗蛋白质(crude protein,CP)含量为3.10%~12.33%]的特点,但其营养价值单一,消化率低,且直接饲喂会降低牦牛的采食量[3-4]。目前,关于提高低品质粗饲料的研究其主要处理方法为氨化法、碱化法以及好氧发酵和厌氧发酵等微贮法[5⇓⇓-8],但这些方法存在环境污染、易腐化和时间久等的缺点[9-10]。研究发现,将低质粗饲料与优质粗饲料混合使用后,其利用率可得到提高,如程景等[11]将全株玉米青贮(whole corn silage,WSC)、小麦秸秆和苜蓿干草进行不同组合的体外发酵,结果显示三者的比例为7∶2∶1时,发酵液中乙酸、丙酸、丁酸及总挥发性脂肪酸(total volatile fatty acid,TVFA)含量显著提高。另有研究发现,不同粗饲料饲喂反刍动物对瘤胃微生物合成微生物蛋白(microbial protein,MCP)的影响不同,如冶文兴[12]使用稻草替代部分全株玉米青贮饲喂奶牛后发现,试验组奶牛瘤胃内MCP含量显著高于对照组。
支链挥发性脂肪酸(branched-chain volatile fatty acid,BCFVA)由4~5个碳原子构成,主要包括异丁酸(isobutyrate,IB)、异戊酸(isovalerate,IV)和2-甲基丁酸(2-methyl butyrate,ME)等,并主要由支链氨基酸(亮氨酸、缬氨酸和异亮氨酸)通过氧化脱羧基和脱氨基生成[13-14]。研究发现,在不同品质粗饲料中添加BCVFA可以提高南江黄羊体外瘤胃微生物利用氨合成MCP的能力[15]。饲粮添加BCVFA也可以提高奶牛养分降解率和乙酸、丙酸含量,并提高动物产奶量和日增重[16]。饲粮添加BCVFA还能显著提高反刍动物的瘤胃挥发性脂肪酸(volatile fatty acid,VFA)含量,如最近的研究发现在牧草组合(箭荞豌豆与燕麦比例为5∶5)中添加0.3% ME,可以促进TVFA合成[17]。此外,研究还发现,不同种类的BCVFA对反刍动物的瘤胃发酵特性影响不同,如提高瘤胃纤维素酶活性[18]。目前,关于添加BCVFA对改善低质粗饲料如油菜秸秆和稻草等的研究尚未见报道。因此,本研究首先通过体外发酵技术评价油菜秸秆和稻草部分替代全株玉米青贮的组合效应,以筛选出1种适宜组合为底物,并探究粗饲料中添加不同种类BCVFA对牦牛体外瘤胃发酵特性的影响,为牦牛养殖业中提高低品质粗饲料利用率以及合理利用BCVFA提供参考。

1 材料与方法

1.1 试验材料

油菜秸秆、稻草及全株玉米青贮取自罗江奶牛养殖场,按照张丽英[19]的方法将采集的粗饲料切碎至3~4 cm,于65 ℃烘箱烘48 h后室温回潮5~6 h,粉碎,过40目网筛制备成风干样,室温保存。
牦牛瘤胃液于广汉市江南屠宰场采集,取4头刚屠宰后的牦牛瘤胃液经4层纱布过滤,迅速装入提前用热水预热的保温瓶中,并持续通入二氧化碳(CO2),使其处于完全厌氧状态。
IB(纯度>99.0%;CAS:79-31-2)、ME(纯度>98.0%;CAS:116-53-0)和IV(纯度>99.9%;CAS:503-74-2)由上海阿拉丁生化科技股份有限公司提供。

1.2 试验设计

试验分为试验1和试验2,均采用单因素设计。试验1共设置12个组,每组4个重复,分为单一粗饲料组及混合粗饲料组(表1),经体外发酵后确定适宜组合。试验2在试验1的研究结果上,选择1种适宜组合为底物,设置对照组(不添加BCVFA)和7个试验组,每组4个重复,添加不同种类的BCVFA,各试验组的BCVFA添加量均为底物干物质基础的0.3%(表2)。
表1 试验1粗饲料组成(干物质基础)

Table 1 Composition of roughage in experiment 1 (DM basis)

组别Groups 底物组成Substrate composition
WCS 100%全株玉米青贮
RAS 100%油菜秸秆
RIS 100%稻草
90%WCS+10%RAS 90%全株玉米青贮+10%油菜秸秆
80%WCS+20%RAS 80%全株玉米青贮+20%油菜秸秆
70%WCS+30%RAS 70%全株玉米青贮+30%油菜秸秆
90%WCS+10%RIS 90%全株玉米青贮+10%稻草
80%WCS+20%RIS 80%全株玉米青贮+20%稻草
70%WCS+30%RIS 70%全株玉米青贮+30%稻草
80%WCS+10%RAS+10%RIS 80%全株玉米青贮+10%油菜秸秆+10%稻草
70%WCS+10%RAS+20%RIS 70%全株玉米青贮+10%油菜秸秆+20%稻草
70%WCS+20%RAS+10%RIS 70%全株玉米青贮+20%油菜秸秆+10%稻草

WCS:全株玉米青贮 whole corn silage;RAS:油菜秸秆 rapeseed straw;RIS:稻草 rice straw。表3~表6同 the same as Table 3 to Table 6。

表2 试验2添加的BCVFA(干物质基础)

Table 2 BCVFA supplementation in experiment 2 (DM basis)

组别
Groups		 BCVFA组成
BCVFA composition
CK 无
IB 异丁酸
IV 异戊酸
ME 2-甲基丁酸
IB∶IV 异丁酸∶异戊酸=1∶1
IB∶ME 异丁酸∶2-甲基丁酸=1∶1
IV∶ME 异戊酸∶2-甲基丁酸=1∶1
IB∶IV∶ME 异丁酸∶异戊酸∶2-甲基丁酸=1∶1∶1

BCVFA添加量为底物干物质基础的0.3%。CK:对照;IB:异丁酸;IV:异戊酸组;ME:2-甲基丁酸。

BCVFA was added at a dosage of 0.3% on substrate dry matter basis. CK: control; IB: isobutyrate; IV: isovalerate; ME: 2-methylbutyrate.

1.3 试验方法

称取1 g底物置于一次性注射器(100 mL)中,参照Menke等[20]的方法配制缓冲液,取经4层纱布过滤后的瘤胃液与缓冲液以1∶2(体积比)比例均匀混合成人工瘤胃液,取50 mL混合液加入到一次性注射器中(试验2在加入底物后加入BCVFA,再加入人工瘤胃液),置于39 ℃恒温摇床发酵72 h,记录0、8、16、24、36、48及72 h累积产气量。发酵结束后将注射器置于冰上停止发酵,立即测定发酵液pH,收集发酵液分别于-20和-80 ℃保存,用于测定MCP、氨态氮(ammoniacal nitrogen,NH3-N)、VFA含量以及纤维素酶活性;发酵残渣于65 ℃烘箱烘干,用于测定养分降解率,包括干物质降解率(dry matter degradation rate,DMD)、中性洗涤纤维降解率(neutral detergent fiber degradation rate,NDFD)和酸性洗涤纤维降解率(acid detergent fiber degradation rate,ADFD)。

1.4 测定指标及方法

1.4.1 常规营养成分含量测定

CP含量采用杜马斯燃烧法测定,粗脂肪(ether extract,EE)含量采用全自动脂肪仪索氏提取法测定,粗灰分(Ash)含量采用马福炉灰化法测定,钙(Ca)和磷(P)含量分别按照GB/T 6436—2018和GB/T 6437—2018测定,NDF和酸性洗涤纤维(acid detergent fiber,ADF)含量参照Van Soest等[21]报道的纤维分析法利用纤维测定分析仪(Gerhardt F12)测定。各养分降解率计算方法如下:
DMD(%)=100×(DM1-DM2)/DM1;
NDFD(%)=100×(DM1×前NDF-DM2×后NDF)/(DM1×前NDF);
ADFD(%)=100×(DM1×前ADF-DM2×后ADF)/(DM1×前ADF)。
式中:DM1为发酵前底物重量(g);DM2为发酵后残渣重量(g);前NDF为发酵前底物NDF含量(%);后NDF为发酵后残渣NDF含量(%);前ADF为发酵前底物ADF含量(%);后ADF为发酵后残渣ADF含量(%)。
各种粗饲料营养成分含量见表3。
表3 各种粗饲料营养成分含量(干物质基础)

Table 3 Nutrient contents of various roughage (DM basis) %

组别
Groups		 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		 粗灰分
Ash		 钙
Ca		 磷
P
WCS 94.15 7.89 2.58 49.23 26.55 6.84 0.54 0.36
RAS 95.80 5.00 2.51 70.39 49.64 8.56 0.78 0.48
RIS 95.58 3.49 1.17 63.30 34.45 15.98 0.36 0.21
90%WCS+10%RAS 93.66 7.06 2.57 44.66 26.12 6.79 0.54 0.27
80%WCS+20%RAS 93.91 6.78 2.57 53.58 32.33 7.06 0.59 0.44
70%WCS+30%RAS 94.15 7.00 2.56 53.31 29.13 7.29 0.63 0.38
90%WCS+10%RIS 93.53 7.15 2.44 43.63 23.95 7.60 0.61 0.38
80%WCS+20%RIS 93.80 7.28 2.30 46.59 25.90 7.83 0.54 0.34
70%WCS+30%RIS 93.93 6.84 2.16 51.53 27.99 9.51 0.54 0.11
80%WCS+10%RAS+10%RIS 93.16 7.16 2.43 53.50 31.40 7.94 0.58 0.32
70%WCS+10%RAS+20%RIS 93.96 6.83 2.29 53.45 29.73 8.76 0.54 0.14
70%WCS+20%RAS+10%RIS 93.92 6.53 2.43 52.99 31.36 8.09 0.57 0.36

1.4.2 瘤胃发酵参数

pH通过便携pH计(PS-101)测定;NH3-N含量参照冯宗慈等[22]改进的比色法测定,将解冻后发酵液于1 972×g、4 ℃离心10 min,取上清液测定;MCP含量参照姜菲等[17]的方法测定,样品经冰浴超声波破碎后(350 W,总时长2 min,超声开时间5 s,超声关时间5 s),离心(16 200×g、4 ℃离心20 min)2次,取沉淀,经1 mL生理盐水重悬后作为待测液,使用索莱宝二喹啉甲酸(BCA)蛋白试剂盒测定;VFA含量参照曹庆云等[23]的方法测定,将样品解冻后摇匀,取2 mL于9 600×g、4 ℃离心10 min,取1 mL上清液,加入0.2 mL含有内标(2-乙基丁酸)的偏磷酸溶液(25%),混匀,4 ℃静置过夜,经9 600×g、4 ℃离心10 min,取1 μL上清液作为待测液,使用安捷伦7890B GC气相色谱仪测定。

1.4.3 纤维素酶活性

参照Agarwal等[24]的方法对样品进行前处理:以20 s的脉速率超声处理10 min,然后在4 ℃、25 000×g离心15 min分离上清液。采用二硝基水杨酸(DNS)法测定羧甲基纤维素酶、木聚糖酶和β-葡萄糖苷酶活性。所有酶活性均定义为每分钟每毫升发酵液产生1 μmol还原糖的酶活量。

1.5 体外发酵组合效应指数计算

参照张吉鹍[25]的方法计算单项组合效应指数(single-factor associative effect index,SFAEI)和多项组合效应指数(multiple-factor associative effect index,MFAEI),计算公式如下:
SFAEI=(组合实测值-加权估算值)/加权估算值;
加权估算值=A饲料×A所占比例(%)+B饲料×B所占比例(%)+C饲料×C所占比例(%);
MFAEI=􀰑SFAEI。

1.6 数据统计分析

原始数据使用Excel 2019整理后,采用SPSS 26.0软件进行数据分析,其中,产气量采用一般线性模型的重复测量数据分析进行莫莱奇球形检验,并通过GraphPad Prism 9.1作图;发酵参数和养分降解率采用单因素方差分析(one-way ANVOA)及Duncan氏法进行多重比较。结果以“平均值±标准误”表示,P<0.05表示差异显著。

2 结果与分析

2.1 不同粗饲料组合对牦牛体外瘤胃发酵特性的影响

2.1.1 不同粗饲料组合对牦牛体外瘤胃发酵产气量的影响

由图1可知,不同粗饲料组合和不同发酵时间对牦牛体外瘤胃发酵产气量有显著影响(P<0.05),且粗饲料组合与发酵时间对牦牛体外瘤胃发酵产气量有显著交互作用(P<0.05)。其中,与RAS组和RIS组相比,各混合组产气量均显著升高(P<0.05)。
图1 不同粗饲料组合对牦牛体外瘤胃发酵产气量的影响

WCS:全株玉米青贮 whole corn silage;RAS:油菜秸秆 rapeseed straw;RIS:稻草 rice straw。

Fig.1 Effects of different roughage combinations on gas production in rumen fermentation of yaks in vitro

2.1.2 不同粗饲料组合对牦牛体外瘤胃发酵参数的影响

由表4可知,与WCS组相比,各混合组pH无显著差异(P>0.05);与RAS组和RIS组相比,各混合组pH均显著降低(P<0.05)。与WCS组相比,各混合组MCP含量无显著差异(P>0.05);与RAS组和RIS组相比,各混合组MCP含量均显著提高(P<0.05)。各组间NH3-N含量无显著差异(P>0.05)。在VFA含量上,与WCS组相比,80%WCS+20%RIS组TVFA含量显著提高(P<0.05);而与RAS组相比,80%WCS+20%RIS组的乙酸、丙酸、丁酸和TVFA含量均显著提高(P<0.05),90%WCS+10%RAS组、80%WCS+20%RAS组及80%WCS+10%RAS+10%RIS组乙酸、丁酸和TVFA含量均显著提高(P<0.05);与RIS组相比,各混合组丁酸含量均显著提高(P<0.05),且80%WCS+20%RIS组乙酸、丙酸和TVFA含量均显著提高(P<0.05)。
表4 不同粗饲料组合对牦牛体外瘤胃发酵参数的影响

Table 4 Effects of different roughage combinations on rumen fermentation parameters of yaks in vitro

项目
Items		 pH 氨态氮
NH3-N/
(mg/dL)		 微生物
蛋白
MCP/
(mg/dL)		 乙酸
Acetate/
(mmol/L)		 丙酸
Propionate/
(mmol/L)		 丁酸
Butyrate/
(mmol/L)		 戊酸
Valerate/
(mmol/L)		 总挥发性
脂肪酸
TVFA/
(mmol/L)
组别Groups
WCS 5.70
±0.07bcd		 11.44
±0.93		 19.74
±0.72ab		 80.25
±2.63abc		 31.08
±1.74ab		 13.76
±0.90bc		 4.68
±0.62		 133.63
±5.21bcd
RAS 6.20
±0.07a		 11.16
±0.92		 13.45
±0.43c		 65.84
±4.87cd		 30.46
±2.30b		 8.19
±0.61d		 3.89
±0.20		 114.33
±7.64cd
RIS 6.20
±0.15a		 12.16
±0.69		 14.24
±0.88c		 68.23
±7.45bcd		 28.96
±2.95bc		 8.42
±1.97d		 4.32
±0.25		 116.00
±11.06cd
90%WCS+
10%RAS		 5.70
±0.04cd		 10.91
±0.40		 20.95
±0.60a		 82.55
±6.89ab		 34.95
±2.71ab		 17.80
±1.36ab		 5.56
±0.37		 145.51
±10.81ab
80%WCS+
20%RAS		 5.73
±0.03bcd		 12.50
±0.61		 19.90
±0.47ab		 83.66
±6.03ab		 32.63
±0.64ab		 17.03
±1.42abc		 4.64
±0.30		 142.97
±7.52ab
70%WCS+
30%RAS		 5.98
±0.05b		 10.55
±0.34		 19.47
±0.37ab		 75.87
±3.85abc		 30.70
±1.96b		 15.48
±0.95abc		 5.49
±0.48		 132.26
±7.00bc
90%WCS+
10%RIS		 5.65
±0.09cd		 10.65
±0.60		 19.62
±1.02ab		 59.07
±2.07d		 23.41
±0.78c		 12.96
±1.53c		 4.10
±0.31		 103.13
±4.26cd
80%WCS+
20%RIS		 5.58
±0.16d		 11.34
±0.68		 18.18
±0.49b		 89.67
±1.94a		 37.69
±1.39a		 19.67
±0.87a		 6.26
±0.18		 158.45
±3.51a
70%WCS+
30%RIS		 5.90
±0.04bc		 11.23
±0.89		 18.24
±1.49b		 78.29
±3.46abc		 33.13
±2.16ab		 15.78
±1.21abc		 6.09
±0.85		 138.13
±6.80bcd
80%WCS+10%
RAS+10%RIS		 5.80
±0.07bcd		 10.95
±0.96		 18.17
±0.35b		 81.74
±3.04ab		 35.76
±2.52ab		 14.59
±0.85bc		 5.59
±1.23		 144.14
±6.14ab
70%WCS+10%
RAS+20%RIS		 5.93
±0.13bcd		 10.48
±0.88		 20.67
±0.67a		 75.70
±6.88abc		 31.83
±2.00ab		 14.40
±2.45bc		 4.67
±0.40		 131.66
±11.51bc
70%WCS+20%
RAS+10%RIS		 5.88
±0.10bc		 10.85
±0.30		 19.57
±0.43b		 78.38
±2.69abc		 32.04
±2.54ab		 14.14
±1.31bc		 4.77
±0.68		 133.88
±7.09bcd
P值P-value <0.001 0.257 <0.001 0.004 0.006 <0.001 0.085 0.001

同列数据肩标不同字母表示差异显著(P<0.05),相同字母或无字母表示差异不显著(P>0.05)。表5、表7、表8和表9同。

In the same column, values with different letter superscripts mean significant difference (P<0.05), while with the same letter or no letter superscripts mean no significant difference (P>0.05). The same as Table 5, Table 7, Table 8 and Table 9.

2.1.3 不同粗饲料组合对牦牛体外瘤胃发酵养分降解率的影响

由表5可知,与WCS组相比,除70%WCS+30%RAS组外,其余各组DMD均显著降低(P<0.05);与RAS组相比,各混合组DMD均显著提高(P<0.05),70%WCS+30%RAS组、80%WCS +20%RIS组、70%WCS+30%RIS组及80%WCS+10%RAS组+10%RIS组NDFD均显著提高(P<0.05);与RIS组相比,各混合组DMD均显著提高(P<0.05),NDFD和ADFD则无显著差异(P>0.05)。
表5 不同粗饲料组合对牦牛体外瘤胃发酵养分降解率的影响

Table 5 Effects of different roughage combinations on nutrient degradation rates in rumen fermentation of yaks in vitro %

项目
Items		 干物质降解率
DMD		 中性洗涤纤维降解率
NDFD		 酸性洗涤纤维降解率
ADFD
组别Groups
WCS 67.51±0.00a 50.78±0.01a 46.66±0.01
RAS 38.04±0.00e 32.84±0.02c 31.90±0.02
RIS 48.36±0.03d 46.39±0.06ab 44.27±0.07
90%WCS+10%RAS 61.95±0.02bc 41.77±0.03abc 38.59±0.03
80%WCS+20%RAS 61.10±0.01bc 41.54±0.03abc 40.34±0.02
70%WCS+30%RAS 64.23±0.00ab 52.44±0.02a 48.38±0.01
90%WCS+10%RIS 61.97±0.01bc 36.65±0.05bc 32.53±0.10
80%WCS+20%RIS 62.47±0.01bc 46.17±0.03ab 41.65±0.03
70%WCS+30%RIS 58.39±0.02c 49.35±0.05a 39.45±0.09
80%WCS+10%RAS+10%RIS 58.33±0.01c 48.14±0.04ab 44.46±0.02
70%WCS+10%RAS+20%RIS 60.53±0.01bc 44.05±0.05abc 41.34±0.03
70%WCS+20%RAS+10%RIS 60.26±0.01bc 42.94±0.01abc 39.82±0.02
P值P-value <0.001 0.024 0.314

2.1.4 不同粗饲料组合对牦牛体外瘤胃发酵组合效应指数的影响

由表6可知,MFAEI计算结果显示:70%WCS+30%RAS组、80%WCS+20%RIS组、70%WCS+10%RAS+20%RIS组及70%WCS+20%RAS+10%RIS组均有正向组合效应,结合粗饲料组成及营养成分考虑,确定70%WCS+20%RAS+10%RIS组为适宜组合,选择该组合进行后续试验。
表6 不同粗饲料组合对牦牛体外瘤胃发酵组合效应指数的影响

Table 6 Effects of different roughage combinations on associative effect index of rumen fermentation in yaks in vitro

组别
Groups		 单项组合效应指数SFAEI 多项组合
效应指数
MFAEI
pH 干物质
降解率
DMD		 微生物
蛋白
MCP		 中性洗涤纤
维降解率
NDFD		 酸性洗涤纤
维降解率
ADFD
90%WCS+10%RAS -0.016 4 -0.040 5 0.096 3 -0.146 3 0.069 6 -0.037 3
80%WCS+20%RAS -0.019 7 -0.008 4 0.076 7 -0.113 9 0.063 1 -0.002 2
70%WCS+30%RAS 0.015 3 0.039 9 0.076 2 0.050 5 0.030 5 0.212 4
90%WCS+10%RIS -0.025 0 -0.055 3 0.022 7 -0.267 3 -0.245 1 -0.570 0
80%WCS+20%RIS -0.045 4 -0.019 0 -0.024 8 -0.080 0 0.178 2 0.009 0
70%WCS+30%RIS 0.002 5 -0.054 6 0.008 3 -0.010 1 0.038 6 -0.015 3
80%WCS+10%RAS+10%RIS -0.006 8 -0.068 9 -0.021 1 -0.014 4 0.059 6 -0.051 6
70%WCS+10%RAS+20%RIS 0.006 8 -0.003 3 0.120 0 -0.087 1 -0.012 3 0.024 1
70%WCS+20%RAS+10%RIS -0.001 7 0.009 4 0.091 2 -0.083 2 0.017 6 0.033 3

2.2 粗饲料组合添加BCVFA对牦牛体外瘤胃发酵特性和纤维素酶活性的影响

2.2.1 粗饲料组合添加BCVFA对牦牛体外瘤胃发酵产气量的影响

由图2可知,不同BCVFA种类和不同发酵时间对牦牛体外瘤胃发酵产气量有显著影响(P<0.05),且BCVFA种类与发酵时间对牦牛体外瘤胃发酵产气量有显著交互作用(P<0.05)。与对照组相比,除IB组和IB∶IV∶ME组外,各试验组产气量均显著降低(P<0.05)。
图2 粗饲料组合添加BCVFA对牦牛体外瘤胃发酵产气量的影响

CK:对照 control;IB:异丁酸 isobutyrate;IV:异戊酸组 isovalerate;ME:2-甲基丁酸 2-methylbutyrate。

Fig.2 Effects of BCVFA supplementation in roughage combination on gas production in rumen fermentation of yaks in vitro

2.2.2 粗饲料组合添加BCVFA对牦牛体外瘤胃发酵参数的影响

由表7可知,与对照组相比,IB组pH显著降低(P<0.05),ME组、IB∶ME组、IV∶ME组及IB∶ME组pH显著提高(P<0.05);各试验组MCP含量均显著提高(P<0.05);IB组NH3-N含量显著降低(P<0.05);IV组和IV∶ME组乙酸含量显著降低(P<0.05),ME组、IB∶IV组、IB∶ME组及IV∶ME组丙酸含量均显著提高(P<0.05),IB∶IV组和IB∶ME组戊酸含量显著提高(P<0.05)。
表7 粗饲料组合添加BCVFA对牦牛体外瘤胃发酵参数的影响

Table 7 Effects of BCVFA supplementation in roughage combination on rumen fermentation parameters of yaks in vitro

项目
Items		 pH 氨态氮
NH3-N/
(mg/dL)		 微生物蛋白
MCP/
(mg/dL)		 乙酸
Acetate/
(mmol/L)		 丙酸
Propionate/
(mmol/L)		 丁酸
Butyrate/
(mmol/L)		 戊酸
Valerate/
(mmol/L)		 总挥发性
脂肪酸
TVFA/
(mmol/L)
组别Groups
CK 5.98
±0.02b		 4.42
±0.28a		 14.97
±0.77f		 58.04
±3.62a		 17.96
±1.46d		 6.15
±1.57		 1.18
±0.15c		 82.73
±6.58
IB 5.87
±0.02c		 3.45
±0.12b		 23.67
±0.66bc		 53.77
±1.62ab		 21.04
±1.29bcd		 4.72
±0.54		 1.36
±0.19c		 80.89
±3.04
IV 6.02
±0.03ab		 4.48
±0.24a		 23.34
±1.21cd		 45.08
±4.56b		 23.43
±2.54abcd		 4.71
±0.78		 1.80
±0.37bc		 75.02
±7.15
ME 6.10
±0.03a		 4.48
±0.28a		 22.64
±0.87cde		 49.70
±0.93ab		 25.18
±1.66abc		 4.45
±0.49		 2.14
±0.38abc		 81.47
±3.03
IB∶IV 6.07
±0.02a		 4.33
±0.24a		 20.76
±0.42e		 52.50
±3.08ab		 28.49
±2.60a		 4.84
±0.62		 3.06
±0.36ab		 88.88
±6.24
IB∶ME 6.08
±0.03a		 4.70
±0.30a		 21.19
±0.36de		 50.95
±3.13ab		 27.42
±3.40ab		 4.24
±0.70		 3.22
±0.83a		 85.84
±7.90
IV∶ME 6.06
±0.03a		 4.32
±0.22a		 26.19
±0.43a		 45.81
±1.42b		 24.69
±1.49abc		 3.50
±0.32		 2.08
±0.24abc		 76.08
±3.33
IB∶IV∶ME 5.99
±0.01b		 4.00
±0.15ab		 25.68
±0.60ab		 55.63
±2.73ab		 19.58
±1.27cd		 4.69
±0.41		 1.18
±0.10c		 81.08
±4.21
P值P-value <0.001 0.035 <0.001 0.033 0.010 0.504 0.005 0.680

CK:对照 control;IB:异丁酸 isobutyrate;IV:异戊酸组 isovalerate;ME:2-甲基丁酸 2-methylbutyrate。下表同 the same as below.

2.2.3 粗饲料组合添加BCVFA对牦牛体外瘤胃发酵养分降解率的影响

由表8可知,与对照组相比,除IB∶IV∶ME组外,其他试验组DMD、NDFD和ADFD均显著降低(P<0.05)。
表8 粗饲料组合添加BCVFA对牦牛体外瘤胃发酵养分降解率的影响

Table 8 Effects of BCVFA supplementation in roughage combination on nutrient degradation rates in rumen fermentation of yaks in vitro %

项目
Items		 干物质降解率
DMD		 中性洗涤纤维降解率
NDFD		 酸性洗涤纤维降解率
ADFD
组别Groups
CK 70.78±0.49a 55.59±0.57a 51.59±0.54a
IB 67.30±0.68bc 49.65±0.70bc 44.52±0.77bc
IV 65.85±0.62c 47.84±1.13c 43.16±0.84bc
ME 65.74±0.67c 47.61±1.30c 43.78±1.18bc
IB∶IV 66.48±0.60bc 47.60±1.21c 41.75±1.92c
IB∶ME 66.55±1.09bc 49.62±1.66bc 45.82±2.90bc
IV∶ME 67.99±0.45b 51.39±0.73b 46.47±0.64b
IB∶IV∶ME 70.83±0.31a 55.28±0.43a 51.15±0.34a
P值P-value <0.001 <0.001 <0.001

2.2.4 粗饲料组合添加BCVFA对牦牛体外瘤胃发酵纤维素酶活性的影响

由表9可知,与对照组相比,IB∶ME组、IV∶ME组及IB∶IV∶ME组羧甲基纤维素酶和木聚糖酶活性均显著提高(P<0.05),IB组、IV组、ME组、IB∶IV组及IB∶ME组β-葡萄糖苷酶活性均显著降低(P<0.05)
表9 粗饲料组合添加BCVFA对牦牛体外瘤胃发酵纤维素酶活性的影响

Table 9 Effects of BCVFA supplementation in roughage combination on cellulase activity in rumen fermentation of yaks in vitro

项目
Items		 羧甲基纤维素酶
Carboxymethyl cellulase/
[nmol glucose/(min·mL)]		 木聚糖酶
Xylanase/[nmol xylose/
(min·mL)]		 β-葡萄糖苷酶
β-glucoside enzyme/
[(nmol p-nitrophenol/(min·mg prot)]
组别Groups
CK 2.10±0.13c 34.11±1.35d 16.68±0.68a
IB 3.09±0.25abc 33.26±1.04d 8.64±0.73c
IV 2.40±0.36bc 33.08±1.36d 9.38±0.88c
ME 3.05±0.16abc 37.99±0.75bcd 10.54±1.15c
IB∶IV 3.01±0.57abc 36.62±2.76cd 10.20±1.34c
IB∶ME 3.36±0.40ab 40.75±2.52abc 13.64±1.12b
IV∶ME 3.84±0.35a 43.86±1.09a 14.11±0.92ab
IB∶IV∶ME 3.66±0.49a 43.13±2.84ab 15.48±0.42ab
P值P-value 0.018 <0.001 <0.001

3 讨论

瘤胃微生物通过分解饲料蛋白质和碳水化合物,产生CO2、甲烷(CH4)及氢气(H2)等气体,因此,体外发酵产气量可在一定程度上反映饲料的养分降解率。本研究试验1中,与WCS组相比,油菜秸秆和稻草替代全株玉米青贮比例在20%以内时各组产气量差异不显著,但与RAS组和RIS组相比显著提高,说明低品质粗饲料中加入适宜比例的优质粗饲料能一定程度提高低品质粗饲料的利用率,这与Ouda等[26]的研究结果相似。试验2中,与对照组相比,IB组和IB∶IV∶ME组产气量无显著变化,这与任莹等[27]在山羊饲粮中添加异戊酸的研究结果部分相似,但其他各组有降低的趋势,可能与BCVFA的组成有关。
pH是体现瘤胃状态的重要指标,正常范围在5.5~7.5[28]。在本研究试验1中,各组pH均在此范围,表明几种粗饲料作为底物进行发酵未对牦牛瘤胃内环境产生不利影响;在本研究试验2中,IB组pH有降低的趋势,这与Liu等[29]的研究结果相似。NH3-N可反映瘤胃发酵过程中瘤胃微生物对饲料蛋白质的降解情况和氨的利用情况[30]。Murphy等[31]和Satter等[32]研究发现,NH3-N含量在6.30~27.50 mg/dL适合微生物生长。在本试验1中,NH3-N含量在10.48~12.50 mg/dL,这与Rivero等[33]的研究结果一致。不同粗饲料替代全株玉米青贮后,粗饲料CP含量并未出现明显变化(6.52%~7.28%),这可能是各组NH3-N含量无显著变化的原因;在试验2中,IB组NH3-N含量显著降低,这与张慧玲等[34]的研究结果一致,表明粗饲料中添加异丁酸可能具有提高瘤胃微生物利用NH3-N能力的作用。
瘤胃微生物利用饲料能量、蛋白质及其他养分可合成MCP[35],有60%~80%的MCP可进入小肠被动物机体吸收利用,从而促进动物机体生长[36]。在本研究试验1中,油菜秸秆或稻草部分替代全株玉米青贮后,与RAS组或RIS组相比,MCP含量均显著提高,这与马艳艳等[37]的研究结果部分一致,说明在低品质粗饲料中加入部分优质粗饲料可提高牦牛瘤胃微生物合成MCP的能力。在本研究试验2中,各试验组MCP含量显著高于对照组,这与沈冰蕾等[38]的研究结果一致。此外,有研究表明,瘤胃微生物对饲料蛋白质降解得越快,所产生的MCP也就越多[39]。VFA是反刍动物体内主要的能源[40]。在本研究试验1中,利用油菜秸秆替代10%或20%全株玉米青贮后,与RAS组相比,牦牛瘤胃乙酸、丙酸、丁酸及TVFA含量均显著提高,这与pH的变化相符合,表明瘤胃微生物对不同组成的粗饲料的利用情况不同。在本研究试验2中,与对照组相比,IB∶IV组和IB∶ME组丙酸含量均显著提高,这与Zhang等[41]的研究结果一致,而VFA主要由瘤胃微生物通过降解粗饲料中的碳水化合物产生,结合MCP含量的变化,推测BCVFA可能具有促进瘤胃微生物生长的作用,从而影响MCP和VFA合成[42]。
体外发酵DMD与累积产气量具有相关性,DMD越高产气量越高[43],且两者都可反映饲料在反刍动物瘤胃内的实际消化情况。在本研究试验1中,DMD和产气量均随着油菜秸秆或稻草替代全株玉米青贮比例的升高而升高,这与杜瑞平等[44]和冉生斌等[45]的研究结果过一致,表明在低品质粗饲料中添加优质粗饲料具有提高低品质粗饲料利用率的作用;而试验2中,与对照组相比,添加单独或2种BCVFA显著降低DMD,这与Mitchell等[46]的研究结果不一致,可能与添加比例不同有关。纤维素酶是一种复合酶系,主要包括内切葡聚糖酶、外切葡聚糖酶和β-葡萄糖苷酶3种酶,在这3种酶的协同作用下,可将纤维素水解为纤维二糖和寡糖,最终水解为葡萄糖[47⇓-49]。饲料纤维降解率的变化主要是细胞壁碳水化合物(主要为NDF)的类型及其降解的差异[50],Wang等[51]研究表明,BCVFA有提高纤维素酶活性的作用。在本研究试验2中,添加2-甲基丁酸和异丁酸或异戊酸及其三者组合能显著提高羧甲基纤维素酶和木聚糖酶活性,这与Wang等[52]及Roman-Garcia等[53]的研究结果相似,表明粗饲料中添加BCVFA具有提高牦牛体外瘤胃纤维素酶活性的作用。

4 结论

① 与单独使用油菜秸秆或稻草相比,将不同比例的油菜秸秆或稻草与全株玉米青贮组合能显著改善牦牛瘤胃发酵特性,其中以70%全株玉米青贮+20%油菜秸秆+10%稻草为适宜组合。
② 粗饲料组合中添加BCVFA能显著提高牦牛体外瘤胃发酵MCP含量以及羧甲基纤维素酶和木聚糖酶活性,且以异丁酸∶异戊酸∶2-甲基丁酸=1∶1∶1的组合添加效果较好。

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