Chinese Journal of Animal Nutrition >

2023 , Vol. 35 >Issue 10: 6516 - 6525

DOI: https://doi.org/10.12418/CJAN2023.596

RESEARCH PAPER

Feasibility Evaluation on Replacing Dietary Alfalfa Protein with Slow-Release Urea Using in Vitro Gas Production Technique for Goats

  • AN Jing , 1 ,
  • YANG Yi 1 ,
  • WU Wenxuan , 1, 2, * ,
  • ZHOU Chuanshe , 3, * ,
  • CHEN Xinzhu 4 ,
  • WANG Chong 5 ,
  • MAO Huiling 5 ,
  • ZHU Wen 6
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  • 1 Key Lab of Animal Genetics, Breeding and Reproduction in the Plateau Mountainous Region of Education Ministry, Institute of Animal Nutrition and Feed Science, College of Animal Science, Guizhou University, Guiyang 550025, China
  • 2 Key Lab of Plateau Mountain Livestock Pollution Control and Resource Reuse Technology of Guizhou Province, Institute of New Rural Development,Guizhou University, Guiyang 550025, China
  • 3 Key Lab of Agro-Ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Science, Changsha 410125, China
  • 4 Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou 350013, China
  • 5 College of Animal Science and Technology & College of Veterinary Medicine, Zhejiang A&F University, Hangzhou 311300, China
  • 6 College of Animal Science, Anhui Agricultural University, Hefei 230036, China
*WU Wenxuan, professor, E-mail: ;
ZHOU Chuanshe, professor, E-mail:

Received date: 2023-02-20

  Online published: 2023-10-12

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Abstract

This study was conducted to evaluate the feasibility of replacing dietary alfalfa protein with slow-release urea using in vitro gas production technique for goats, and then to provide an understanding of exploitation of slow-release urea used as protein feed for ruminants. Four goats were used as rumen fluid donors. The ANKOM RFS gas production measurement system was used to gas production collection. Fermentation substrates were 3 diets: control group, the diet contained 48% alfalfa without slow-release urea; test group 1, the diet contained 0.5% slow-release urea and 24% alfalfa; test group 2, the diet contained 1.0% slow-release urea without alfalfa. The in vitro fermentation was terminated after 72 h, and the gas production, fermentation parameters, cellulase activities, in vitro dry matter degradation rate, and volatile fatty acid concentrations and methane production were measured. The results are showed as follows: 1) test group 1 and test group 2 had significantly higher cumulative gas production of 24, 48 and 72 h (GP24, GP48 and GP72), gas production from slowly degradable fraction (b), potential gas production (a+b), digestible organic matter content, and metabolizable energy than those of control group (P<0.05). Test group 1 showed significantly lower gas production from rapidly degradable fraction (a) than that of control group and test group 2 (P<0.05). The gas production rate (c) of test group 1, test group 2 and control decreased successively, and the difference was significant among all groups (P<0.05). 2) The pH of control group, test group 2 and test group 1 decreased successively, and was significantly different from each other (P<0.05), but the difference of values was quite small. The concentration of ammonia nitrogen (NH3-N) of test group 1 was significantly lower than that of control group and test group 2 (P<0.05). There were no differences in microprotein concentration, cellulase activities, and in vitro dry matter degradation rate among 3 groups (P>0.05). 3) The propionate concentration of test group 1 and test group 2 was significantly higher than that of control group (P<0.05). Acetate/propionate of control group, test group 1 and test group 2 decreased successively and was significantly different from each other (P<0.05). The concentrations of total volatile fatty acids, acetate, propionate, and methane production were not significantly affected among 3 groups (P>0.05). In conclusion, it is found that an appropriate level of slow-release urea is able to replace dietary alfalfa protein, which is beneficial to in vitro rumen fermentation. Under the experimental conditions, it is recommended to replace 50% alfalfa protein with 0.5% slow-release urea in the diet, and the proportion of alfalfa in the diet decreased from 48% to 24%.[Chinese Journal of Animal Nutrition, 2023, 35(10):6516-6525]

Key words： slow-release urea; alfalfa protein; in vitro gas production technique; rumen fermentation parameters; goats

Cite this article

AN Jing , YANG Yi , WU Wenxuan , ZHOU Chuanshe , CHEN Xinzhu , WANG Chong , MAO Huiling , ZHU Wen . Feasibility Evaluation on Replacing Dietary Alfalfa Protein with Slow-Release Urea Using in Vitro Gas Production Technique for Goats[J]. Chinese Journal of Animal Nutrition, 2023 , 35(10) : 6516 -6525 . DOI: 10.12418/CJAN2023.596

近年来,我国草食畜牧业快速发展,饲养方式逐渐规模化、集约化,对饲草尤其是苜蓿等优质饲草需求不断扩大。但贵州等南方山区受气候、环境、地形、土壤等影响,种植和生产苜蓿存在很大的困难,苜蓿资源长期短缺,且难以改变,成为制约当地牛、羊产业高质量发展的主要瓶颈。反刍动物具有区别于单胃动物的特殊器官瘤胃,瘤胃微生物可利用非蛋白氮降解产生的氨(NH3)合成微生物蛋白满足机体大部分蛋白质需要。在当前蛋白质饲料严重不足的形势下,开发利用尿素等非蛋白氮替代优质蛋白质饲料具有重要意义,已得到研究者的重视。张苏江等[1]发现,添加20和30 mg/kg未包被尿素能促进肉羊瘤胃体外发酵,但添加量超过40 mg/kg则会抑制产气量和挥发性脂肪酸(VFA)产量。孙美杰等[2]以玉米秸秆为湖羊瘤胃体外发酵底物,发现添加20 mg/dL的未包被尿素可显著提高总挥发性脂肪酸(TVFA)产量,过量则抑制瘤胃发酵。
根据反刍动物学原理,瘤胃微生物分解纯尿素产生NH3的速度远超其将NH3合成微生物蛋白的速度,容易导致瘤胃NH3中毒,损害机体健康,所以需要对尿素进行包被,使瘤胃微生物降解NH3和合成微生物蛋白的速度相适应,提高非蛋白氮的利用率,同时降低氮排泄量[3]。缓释尿素是用1层或多层特殊的包被材料(天然或人工合成的聚合物、生物复合材料以及黏土矿物的纳米复合材料等)包裹尿素制成[4],以替代豆粕最常见。赵二龙等[5]研究发现,在饲粮中添加1.41%的缓释尿素替代75%的豆粕,肉羊可获得最佳的生长性能与饲料效率。Dávila-Ramos等[6]发现,在羔羊饲粮中添加0.8%的缓释尿素改善了其生长性能。Estrada-Angulo等[7]研究显示,在淀粉与酸性洗涤纤维为4∶1的饲粮中添加0.8%的缓释尿素时羔羊的生长性能最佳。Liang等[8]在肉牛饲粮中添加1.41%的缓释尿素替代75%的豆粕,结果发现,该组肉牛的生长性能与不含缓释尿素,豆粕占饲粮的11.12%的对照组相比差异不显著。
瘤胃体外发酵培养能较好模拟体内瘤胃发酵过程,具有操作简单、可批次培养、重复性好等优点,是研究反刍动物饲料营养价值的重要方法;同时,直接将动物采食的饲粮用作体外发酵底物获取的发酵参数,更加客观,也更加接近瘤胃原真数值,已得到学术界的认可[9]。据此,本试验应用体外产气法研究尿素替代山羊饲粮中不同水平苜蓿蛋白对山羊瘤胃体外发酵参数的影响,为验证缓释尿素替代苜蓿蛋白的可行性提供数据参考。

1 材料与方法

1.1 试验设计

选取4只体重相近[(32.13±2.18) kg]的贵州黑山羊(羯羊)作为瘤胃液供体羊,按满足山羊的维持需要提供饲粮饲喂量。山羊饲粮参照《肉羊营养需要量》(NY/T 816—2021)配制,由精补颗粒料和苜蓿干草组成,精粗比为40∶60,饲粮的干物质(DM)含量为90.45%,干物质中含总能(GE)16.73 MJ/kg、粗蛋白质(CP)18.02%、中性洗涤纤维(NDF)33.78%和酸性洗涤纤维(ADF)18.48%。其中,精补颗粒料长6 mm、粒径3.5 mm,含玉米37.00%、小麦15.00%、豆粕12.50%、干酒糟及其可溶物(DDGS)10.00%、麦麸10.00%、玉米胚芽粕10.00%、石粉2.00%、磷酸氢钙0.60%、食盐0.83%、小苏打1.20%、一水合硫酸镁0.50%、甜味剂0.02%、香味剂0.02%、抗氧化剂0.02%、防霉剂0.10%、预混料0.21%。
山羊持续饲喂14 d后于第15天晨饲前使用负压真空泵(VP30,北京莱伯科泰有限公司)连接胃管式采样器从供体羊口腔中抽取瘤胃液,去掉前面的唾液并用少量瘤胃液润洗接收瓶防备污染,经4层纱布过滤后迅速装入已经通有二氧化碳(CO2)且将温度设为39 ℃的密闭保温瓶中用于配制人工瘤胃液。人工唾液参照Benedeti等[10]给出的方法配制。

1.2 瘤胃体外培养

利用ANKOM RFS产气测量系统进行体外产气,将21套发酵模块随机分为3组:对照组、试验1组、试验2组,每组7个重复,每个重复1套模块。每个发酵瓶中加入4.000 0 g发酵底物和100 mL人工瘤胃液(20 mL瘤胃原液+80 mL人工唾液),迅速通入CO2,在温度为39 ℃、转速为60 r/min的气浴恒温箱(HNY-211B,天津欧诺仪器股份有限公司)中持续发酵72 h后终止发酵,分离固液,测定相关指标。体外发酵底物为本课题组前期完成的动物饲养试验实际采食的3种饲粮,其组成及营养水平见表1。
表1 发酵底物组成及营养水平(干物质基础)

Table 1 Composition and nutrient levels of fermented substrates (DM basis)%

项目 Items 对照组 Control group 试验1组 Test group 1 试验2组Test group 2
原料 Ingredients
缓释尿素 Slow-release urea 0.50 1.00
苜蓿 Alfalfa 48.00 24.00
花生秧 Peanut vine 10.00 30.00 50.40
稻壳粉 Rice hull powder 0.00 3.70 6.10
玉米 Corn 27.00 25.00 25.00
豆粕 Soybean meal 7.00 8.80 11.00
麦麸 Wheat bran 2.50 2.50 1.00
柠檬渣 Lemon residue 2.30 2.30 2.30
稻米油 Rice bran oil 2.00 2.00 2.00
石粉 Limestone 0.50 0.50 0.50
食盐 NaCl 0.50 0.50 0.50
多矿 Multi-minerals1) 0.06 0.06 0.06
多维 Multi-vitamins2) 0.14 0.14 0.14
合计 Total 100.00 100.00 100.00
营养水平 Nutrient levels3)
干物质 DM 91.22 90.38 90.77
有机物 OM 89.25 90.27 89.58
代谢能 ME/(MJ/kg) 9.62 9.58 9.38
粗蛋白质 CP 17.37 17.10 17.27
中性洗涤纤维 NDF 40.11 35.95 32.00
酸性洗涤纤维 ADF 25.60 22.94 20.94

1)每千克多矿中含有 One kg of multi-minerals contained the following:Cu 10 000 mg,Fe 71 000 mg,Zn 63 000 mg,Mn 62 000 mg,Se 300 mg,I 800 mg,Co 1 000 mg。

2)每千克多维中含有 One kg of multi-vitamins contained the following:VA 25 000 000 IU,VD3 5 000 000 IU,VE 175 000 mg,VK3 5 000 mg,VB1 7 840 mg,VB2 17 600 mg,VB6 7 840 mg,VB12 100 mg,生物素 biotin 1 600 mg,叶酸 folic acid 4 800 mg,烟酸 niacin 79 200 mg,泛酸 pantothenic acid 48 950 mg。

3)代谢能为计算值,其他营养水平均为实测值。ME was a calculated value, while the other nutrient levels were measured values.

1.3 指标检测

1.3.1 常规营养成分检测

干物质含量参照GB/T 6435—2014测定,粗灰分(Ash)含量参照GB/T 6438—2007测定,干物质减去粗灰分便得到有机物(OM)的含量,总能使用氧弹式热量计(Parr-6400,美国)测定,粗蛋白质含量参照GB/T 6432—2008使用全自动凯氏定氮仪(KF1100,济南海能仪器股份有限公司)测定,中性洗涤纤维与酸性洗涤纤维含量参照Van Soest[11]的方法使用全自动纤维分析仪(FT12,格哈特,德国)测定。

1.3.2 体外发酵产气量、产气参数、可消化有机物(DOM)含量、有机物降解率(OMD)及代谢能(ME)

(1)产气量
利用ANKOM RFS产气测量系统,每间隔1 h记录1次产气压力,通过公式计算产气量。计算公式如下:
GP=Vj×Ppsi×0.068 。
式中:GP为39 ℃时的总产气量(mL);Vj为发酵瓶中气体体积(150 mL);Ppsi为ANKOM RFS产气测量系统记录的累积产气压力(kPa)。
(2)产气参数
利用SAS 9.2 NINE程序将样品各时间点的产气量带入Ørskov等[12]提出的模型中计算产气参数,计算公式为:
GPt=a+b×(1-e-ct)
式中:GPt为t时刻的累积产气量(mL);a为快速降解部分的产气量(mL);b为慢速降解部分的产气量(mL);a+b为潜在产气量(mL);c为产气速率(%/h);t为时间(h)。
(3)可消化有机物含量、有机物降解率和代谢能
参照Menke等[13]提出的方法,以200 mg样品(干物质基础)为每单位体外发酵底物时,根据样品体外培养24 h产气量计算可消化有机物含量、有机物降解率和代谢能,计算公式如下:
DOM(%)=7.65×GP24+353;
OMD(%)=17.04+1.108 5×GP24;
ME=-0.20+0.141 0×OMD。
式中:GP24表示24 h的累积产气量(mL);DOM为可消化有机物含量(g/kg);OMD为有机物降解率(%);ME为代谢能(MJ/kg)。

1.3.3 体外发酵参数

1.3.3.1 pH、氨态氮与微生物蛋白浓度、纤维素酶活性及体外干物质降解率

用Seven2GoTM pH计(Mettler Toledo,瑞士)测定终止发酵液的pH。将发酵液倒入500 mL离心管中,4 ℃、10 000 r/min离心10 min,取上清液测定氨态氮、微生物蛋白浓度及纤维素酶活性。氨态氮浓度参照冯宗慈等[14]提出的比色法测定。微生物蛋白含量参照Zinn等[15]报道的考马斯亮蓝染色法测定。纤维素二糖酶、羧甲基纤维素酶、木聚糖酶活性采用3,5-二硝基水杨酸比色法(DNS法)[16]测定,纤维素酶活性计算公式如下:
纤维素酶活性[μmol/(min·mL)]=[还原糖生成量(μg)×稀释倍数]/[试液量(mL)×还原糖相对分子质量×反应时间(min)]。
将500 mL离心管内剩余的固形物经105 ℃烘至恒重后,计算体外干物质降解率,计算公式如下:
体外干物质降解率(%)={[底物质量(g)-105 ℃烘至恒重后固形物质量(g)]/底物质量(g)}×100。

1.3.3.2 挥发性脂肪酸浓度及甲烷(CH4)产量

参照文献[16]给出的方法采用气相色谱仪(GC-2010 Plus,岛津,日本)测定发酵液中乙酸、丙酸、丁酸等挥发性脂肪酸组分的浓度,并计算乙酸/丙酸和总挥发性脂肪酸浓度。
总挥发性脂肪酸浓度(mmol/L)=乙酸浓度(mmol/L)+丙酸浓度(mmol/L)+丁酸浓度(mmol/L)。
根据乙酸、丙酸、丁酸浓度,参照Moss等[17]的方法计算甲烷产量,计算公式如下:
甲烷产量(mmol/L)=0.45×乙酸浓度(mmol/L)-0.275×丙酸浓度(mmol/L)+0.4×丁酸浓度(mmol/L)。

1.4 数据处理

数据均以“平均值±标准差(mean±SD)”表示。用Excel 2019对数据进行初步处理,再用SAS 9.4统计软件的ANOVA程序对3组试验数据进行统计分析,运用Duncan氏法多重比较检验差异的显著性,P<0.05表示差异显著。

2 结果与分析

2.1 体外发酵产气量、产气参数、可消化有机物含量、有机物降解率及代谢能

由表2可知,试验1组和试验2组的24 h累积产气量(GP24)、48 h累积产气量(GP48)、72 h累积产气量(GP72)、慢速降解部分产气量(b)、潜在产气量(a+b)、可消化有机物含量、代谢能均显著高于对照组(P<0.05)。试验1组的快速降解部分产气量(a)显著低于对照组、试验2组(P<0.05),后2组之间差异不显著(P>0.05)。试验1组、试验2组、对照组的产气速率(c)数值依次降低,且各组间差异显著(P<0.05)。各组间有机物降解率差异不显著(P>0.05)。
表2 缓释尿素替代苜蓿蛋白对山羊体外发酵产气量、产气参数、可消化有机物含量、有机物降解率及代谢能的影响

Table 2 Effects of replacing alfalfa protein with slow-release urea on in vitro rumen fermentation gas production, gas production parameters, digestible organic matter content, organic matter degradation rate and ME of goats

项目
Items		 对照组
Control		 试验1组
Test group 1		 试验2组
Test group 2		 P值
P-value
24 h累积产气量 GP24/mL 223.82±8.29b 252.59±8.07a 247.11±13.33a <0.01
48 h累积产气量 GP48/mL 258.44±11.84b 290.48±8.44a 284.16±12.97a <0.01
72 h累积产气量 GP72/mL 267.02±13.66b 298.89±8.87a 292.97±10.55a <0.01
快速降解部分产气量
GP from rapidly degradable fraction (a)/mL		 11.40±2.29a 6.39±1.22b 9.10±2.69a <0.01
慢速降解部分产气量
GP from slowly degradable fraction (b)/mL		 224.87±6.07b 259.84±7.09a 252.16±12.40a <0.01
潜在产气量 Potential GP (a+b)/mL 236.27±6.31b 266.23±5.97a 261.25±11.30a <0.01
产气速率 GP rate (c)/(%/h) 5.42±0.20c 6.28±0.16a 6.06±0.11b <0.01
可消化有机物含量
Digestible organic matter content/(g/kg)		 438.61±2.08b 449.61±2.33a 447.52±4.72a <0.01
有机物降解率 Organic matter degradation rate/% 29.45±0.30 31.04±0.34 30.74±0.68 0.40
代谢能 ME/(MJ/kg) 3.95±0.04b 4.18±0.05a 4.13±0.10a <0.01

同行数据肩标不同字母表示差异显著(P<0.05),无字母或相同字母表示差异不显著(P>0.05)。下表同。

In the same row, values with different letter superscripts mean significant difference (P<0.05), while with the same or no letter superscripts mean no significant difference (P>0.05). The same as below.

2.2 体外发酵参数

2.2.1 pH、氨态氮与微生物蛋白浓度、纤维素酶活及体外干物质降解率

由表3可知,对照组、试验2组、试验1组体外发酵pH依次降低且互相之间差异显著(P<0.05);试验1组氨态氮浓度显著低于对照组和试验2组(P<0.05);各组间微生物蛋白浓度与羧甲基纤维素酶、木聚糖酶、纤维素二糖酶活性及体外干物质降解率差异不显著(P>0.05)。
表3 缓释尿素替代苜蓿蛋白对山羊体外发酵pH、氨态氮与微生物蛋白浓度、纤维素酶活性及体外干物质降解率的影响

Table 3 Effects of replacing alfalfa protein with slow-release urea on in vitro fermentation pH, NH3-N, MCP concentrations, cellulase activities and IVDMD of goats

项目
Items		 对照组
Control		 试验1组
Test group 1		 试验2组
Test group 2		 P值
P-value
pH 5.64±0.01a 5.54±0.01c 5.59±0.01b <0.01
氨态氮 NH3-N/(mg/dL) 27.36±0.45a 17.47±1.80b 24.36±2.22a <0.01
微生物蛋白 MCP/(g/L) 0.23±0.01 0.23±0.01 0.23±0.01 0.92
羧甲基纤维素酶 Carboxymethyl cellulose/[μmol/(min·mL) ] 0.15±0.03 0.17±0.01 0.16±0.02 0.29
木聚糖酶 Xylanase/[μmol/(min·mL) ] 0.22±0.05 0.30±010 0.25±0.05 0.73
纤维素二糖酶 Cellobiase/[μmol/(min·mL) ] 0.15±0.01 0.15±0.02 0.15±0.01 0.58
体外干物质降解率 IVDMD/% 54.09±4.89 54.90±2.65 56.40±0.62 0.48

2.2.2 挥发性脂肪酸浓度及甲烷产量

由表4可知,试验1组和试验2组丙酸浓度显著高于对照组(P<0.05);对照组、试验1组、试验2组乙酸/丙酸依次降低且互相之间差异显著(P<0.05);各组间总挥发性脂肪酸、乙酸、丁酸浓度与甲烷产量差异不显著(P>0.05)。
表4 缓释尿素替代苜蓿对山羊体外发酵挥发性脂肪酸浓度及甲烷产量的影响

Table 4 Effects of replacing alfalfa protein with slow-release urea on in vitro fermentation volatile fatty acid concentrations and CH4 production of goats

项目
Items		 对照组
Control		 试验1组
Test group 1		 试验2组
Test group 2		 P值
P-value
总挥发性脂肪酸 TVFA/(mmol/L) 168.45±6.23 174.31±2.64 177.18±13.35 0.22
乙酸 Acetate/(mmol/L) 97.07±2.79 98.29±1.35 99.01±7.44 0.63
丙酸 Propionate/(mmol/L) 57.60±2.59b 62.41±0.80a 64.34±4.89a 0.01
丁酸 Butyrate/(mmol/L) 13.76±1.12 13.61±0.69 13.83±1.06 0.75
乙酸/丙酸 Acetate/propionate 1.69±0.04a 1.57±0.02b 1.54±0.02c <0.01
甲烷产量 CH4 production/(mmol/L) 33.35±0.94 32.51±0.67 32.39±2.45 0.58

3 讨论

3.1 缓释尿素替代苜蓿蛋白对山羊体外发酵产气量、产气参数、可消化有机物含量、有机物降解率及代谢能的影响

瘤胃微生物可将饲粮中的碳水化合物和蛋白质降解,产生大量的甲烷、CO2气体以及少量的氢气(H2)、氧气(O2)、氮气(N2)。产气量是评价瘤胃发酵性能的重要指标,与发酵底物可利用程度正相关[13,18]。根据康奈尔净碳水化合物-蛋白质体系(CNCPS体系),瘤胃体外发酵的快速降解部分产气量由瘤胃微生物降解葡萄糖和淀粉等容易利用的有机物产生,慢速降解部分产气量由瘤胃微生物降解纤维素和半纤维素等难以利用的有机物产生。本试验中,试验1组和试验2组GP24、GP48、GP72、慢速降解部分产气量、潜在产气量、可消化有机物含量、代谢能均显著高于对照组,表明适量的缓释尿素替代苜蓿蛋白可以促进瘤胃微生物的定植,提高饲粮中碳水化合物和蛋白质在瘤胃的降解。这可能与这2组发酵液中细菌、真菌和原虫数量增加有关,有待于下一步研究验证。
同时,试验1组、试验2组、对照组的产气速率依次降低,这提示缓释尿素对产气量的影响存在剂量效应,即适宜的缓释尿素(0.5%)替代苜蓿蛋白可提高慢速降解部分产气量、潜在产气量、产气速率、可消化有机物含量、代谢能,而高比例缓释尿素(1.0%)替代苜蓿蛋白对产气速率的提高则不如比例替代理想。张娇娇[19]也得到类似的结论,其在基础饲粮中添加2.0%的缓释尿素时产气速率显著低于对照组和低剂量(0.5%、1.0%)组。本试验中,试验1组快速降解部分产气量显著低于对照组、试验2组,与Ahmed等[20]所得类似,说明本研究中的快速降解部分的降解存在滞后现象。这可能与人工瘤胃液(瘤胃原液和人工唾液)预热时间较短,导致体外发酵液的温度与发酵瓶的温度未能在短时间内达到肉羊瘤胃的温度(39 ℃),使发酵液中的微生物活化不充分有关。

3.2 缓释尿素替代苜蓿蛋白对山羊体外发酵参数的影响

3.2.1 pH、氨态氮与微生物蛋白浓度、纤维素酶活性及体外干物质降解率

与传统尿素相比,缓释尿素经特殊材料包被后,可降低尿素分解成氨的速率,延长氨的生成与利用(合成微生物蛋白)时间,其缓释作用不会造成瘤胃发酵内环境出现剧烈失衡,进而提高尿素的利用效率[3]。
瘤胃液pH是衡量瘤胃发酵环境的重要指标,其正常范围为5.5~7.5,pH处于正常范围内才能保证正常发酵[21-22]。研究表明,用羟甲基尿素代替饲粮精料18.96%的粗蛋白质,试验羊瘤胃液pH显著低于未添加羟甲基尿素组[23]。本试验中,试验1组和试验2组发酵液pH虽然显著低于对照组,但各组的pH在数值上差异很小,表明缓释尿素对瘤胃内环境pH的影响有限。
氨态氮是瘤胃微生物生长的重要氮源,反映了瘤胃微生物分解饲粮蛋白质产生氨态氮,然后利用氨态氮合成微生物蛋白的动态平衡能力,是评价瘤胃内环境稳态的关键指标[24]。瘤胃中的氨态氮浓度保持动态平衡,适宜浓度为5~30 mg/d[25],氨态氮浓度在合理范围内才利于瘤胃微生物的生长繁殖和瘤胃微生物区系的完善。有研究表明,饲粮中添加1%的缓释尿素能够促进瘤胃微生物利用氨态氮合成微生物蛋白的效率,降低瘤胃微生物降解饲粮中的蛋白质产生氨态氮,使其浓度保持在合理范围,维持瘤胃内环境稳态[26]。本研究结果显示,各组氨态氮浓度均在合理范围内,表明缓释尿素替代苜蓿蛋白不会造成反刍动物瘤胃中氨态氮浓度失衡;试验1组氨态氮浓度显著低于对照组和试验2组,说明0.5%的缓释尿素替代苜蓿蛋白会降低氨态氮浓度,更利于微生物利用氨态氮合成微生物蛋白,为反刍动物机体生长发育提供更多的氮源。
微生物蛋白含量能够反映瘤胃微生物对氨态氮的利用效率,微生物蛋白能够满足反刍动物所需蛋白质的40%~80%,是反刍动物机体氮源的主要提供者[27]。本试验结果表明,各组间微生物蛋白浓度差异不显著。这提示,缓释尿素替代苜蓿蛋白不影响反刍动物机体生长发育氮源正常供应。但也有研究表明,缓释尿素可提高瘤胃微生物蛋白合成。Norrapoke等[28]报道,向发酵木薯粕中加入4%的缓释尿素能提高肉牛瘤胃微生物蛋白的合成效率。此外,有研究显示,1%的缓释尿素较等比例的未包被尿素更能增强肉牛瘤胃合成微生物蛋白[29]。产生结果差异的原因可能与尿素替代对象(本试验是用缓释尿素替代优质苜蓿蛋白,而上述文献均为替代精饲料)、基础饲粮组成等不同有关。
纤维素酶活性是衡量反刍动物瘤胃微生物降解饲粮中难以降解的纤维素、木质素等能力的重要指标,是纤维分解菌分泌的多酶复合体的统称[30]。反刍动物瘤胃内分解纤维素的酶主要分为3类:木聚糖酶、羧甲基纤维素酶、纤维素二糖酶,三者联合作用于饲粮中难以被降解吸收的纤维素、木质素、半纤维素等物质[31]。本试验结果显示,缓释尿素替代苜蓿蛋白对山羊体外发酵木聚糖酶、羧甲基纤维素酶、纤维素二糖酶活性无显著影响,表明适量缓释尿素替代苜蓿蛋白不会对瘤胃内环境的纤维素酶活性产生负面影响。
体外干物质降解率与饲粮被瘤胃降解利用程度正相关。王里彦等[32]利用缓释尿素替代不同比例豆粕(0、37%、71%、100%)进行肉羊瘤胃体外发酵发现,低比例(37%)替代不影响所有养分的体外降解率,而高比例(71%、100%)替代虽然能提高体外干物质降解率,但显著降低了其他养分的体外降解率。本试验中,3种饲粮的体外干物质降解率差异不显著。这与上述结果存在差异,可能与试验设计有关,本试验是用缓释尿素替代优质苜蓿蛋白,而上述文献则是用缓释尿素替代豆粕。

3.2.2 挥发性脂肪酸浓度及甲烷产量

反刍动物由于特殊的消化系统,可将饲粮中营养物质在瘤胃微生物降解作用下分解为挥发性脂肪酸、NH3等物质,供给动物机体所利用[33]。挥发性脂肪酸由瘤胃微生物分解碳水化合物所产生,主要包括乙酸、丙酸、丁酸,分别占总挥发性脂肪酸的50%~65%、18%~25%、12%~20%,可提供机体70%~80%的能源。乙酸/丙酸是评价反刍动物瘤胃发酵类型的重要指数。当乙酸/丙酸大于2.5时为乙酸型发酵,有利于体脂和乳脂的合成;当乙酸/丙酸小于2.5时为丙酸型发酵,机体利用丙酸异生为葡萄糖供能[34],也可促进机体氨态氮合成微生物蛋白[35]。
本试验结果显示,试验1组和试验2组丙酸浓度显著高于对照组,表明适量缓释尿素替代苜蓿蛋白有利于提高肉羊对饲粮中营养物质的消化代谢、吸收利用和生产性能。丙酸浓度高,意味着糖异生效率高,可以为机体提供更多的能量促进动物生长,但这在我们已完成的肉羊饲养试验中并未体现出来,这可能与体外发酵试验程序化、可控性强、误差较小,而动物生长试验受的影响因素较多有关,我们下一步将测定肉羊瘤胃液内的丙酸浓度来相互验证。对照组、试验1组、试验2组乙酸/丙酸依次降低且互相之间差异显著,这可能与饲粮的纤维水平(对照组、试验1组、试验2组依次递减)有关,粗饲料中纤维类物质含量较高,瘤胃发酵主要以产乙酸为主,Sutton等[36]的研究也得到类似结果。此外,本试验结果显著各组间总挥发性脂肪酸、乙酸、丁酸浓度与甲烷产量差异不显著。
总之,开发利用缓释尿素等苜蓿蛋白替代物,是缓解我国优质牧草饲料资源缺乏的必然道路。通过体外发酵试验发现缓释尿素替代苜蓿蛋白具有可行性,可为今后将其作为蛋白质饲料资源开发利用提供数据积累,有关缓释尿素在动物生产上替代苜蓿蛋白的效果,有待于后续深入研究。

4 结论

在体外发酵条件下,适宜水平的缓释尿素替代苜蓿蛋白有利于瘤胃发酵。在本试验条件下,以0.5%的缓释尿素替代50%的苜蓿蛋白(饲粮中苜蓿占比由48%降至24%)为宜。

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