Chinese Journal of Animal Nutrition >

2024 , Vol. 36 >Issue 10: 6459 - 6467

DOI: https://doi.org/10.12418/CJAN2024.549

RESEARCH PAPER

Effects of Noni Fruit Flavonoids on in Vitro Rumen Fermentation of Cashmere Goats with High-Concentrate Substrate

  • ZHANG Qingyue , 1 ,
  • DONG Shuhui 1 ,
  • LI Yinhao 1 ,
  • LIU Jintao 1 ,
  • ZHANG Shangxiong 2 ,
  • YU Hao 1 ,
  • ZHAO Yanli 1 ,
  • GUO Xiaoyu 1 ,
  • GUO Yongmei 1 ,
  • SHI Binlin 1 ,
  • YAN Sumei , 1, *
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  • 1 Key Laboratory of Animal Nutrition and Feed Science at Universities of Inner Mongolia Autonomous Region, College of Animal Science, Inner Mongolia Agricultural University, Hohhot 010018, China
  • 2 National Center of Pratacultural Technology Innovation (Under Preparation), Hohhot 010070, China
*professor, E-mail:

Received date: 2024-03-26

  Online published: 2024-10-14

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Abstract

This experiment aimed to screen one extract of noni fruit from noni fruit polyphenols (NFP) and noni fruit flavonoids (NFF), which can regulate the in vitro rumen fermentation of cashmere goats under the condition of high-concentrate substrate, and to screen the appropriate amount of its additives to provide theoretical basis for the development of green feed additives for cashmere goats and the rational utilization of noni fruit resources. The experiment was conducted in two parts using Albas white cashmere goats as rumen fluid donor animals. Part 1: resveratrol (RSV) was used as a positive control. A single-factor completely randomized trial design was used to divide the fermentation bottles into four treatments with high-concentrate diet (the ratio of concentrate to roughage=65∶35) and the high-concentrate diet supplemented with 0.25% NFP, 0.25% NFF and 0.02% RSV as substrates, respectively, to conducted the in vitro rumen fermentation experiment. Each treatment was performed in six replicates. Part 2: a single-factor completely randomized trial design was used to divide the fermentation bottles into five treatments with the high-concentrate diet supplemented with 0, 0.025%, 0.050%, 0.100% and 0.250% NFF as substrates, respectively, to conducted the in vitro rumen fermentation experiment. The results of the part 1 showed that NFF promoted rumen fermentation with the highest of multiple forages associative effect indicator (MFAEI), and its regulatory effect was better than that of NFP in alleviating pH decline, reducing concentrations of lactic acid and histamine (HIS), and improving nitrogen utilization efficiency in the fermentation broth. Therefore, NFF was screened as a noni fruit extract with better modulation of rumen fermentation in vitro in cashmere goats for subsequent experiment. The results of the part 2 showed that the highest MEAEI to promote rumen fermentation was observed when 0.100% NFE was added to the high-concentrate substrate, while it significantly reduced the concentrations of abnormal metabolites HIS and lipopolysaccharide (LPS) in the fermentation broth in vitro (P<0.05), and it also significantly alleviated the pH decrease under the condition of high-concentrate substrate (P<0.05). In conclusion, NFF has a good effect of promoting rumen fermentation in vitro in cashmere goats, and its suitable additive amount in high concentrate substrate is 0.100%.

Key words： noni fruit; flavonoids; polyphenol; high-concentrate; cashmere goat; rumen fermentation in vitro

Cite this article

ZHANG Qingyue , DONG Shuhui , LI Yinhao , LIU Jintao , ZHANG Shangxiong , YU Hao , ZHAO Yanli , GUO Xiaoyu , GUO Yongmei , SHI Binlin , YAN Sumei . Effects of Noni Fruit Flavonoids on in Vitro Rumen Fermentation of Cashmere Goats with High-Concentrate Substrate[J]. Chinese Journal of Animal Nutrition, 2024 , 36(10) : 6459 -6467 . DOI: 10.12418/CJAN2024.549

近年来,由于受到天然草场资源的限制,绒山羊的饲养方式由传统放牧转为舍饲圈养,集约化的舍饲圈养已经成为了绒山羊主要的饲养方式,而为了提高生产效益,集约化养殖通常会采用精料占比较高的饲粮。反刍动物长期采食高精料饲粮会危害瘤胃等器官的健康[1],甚至引起肝损伤等一系列代谢性疾病[2]。因此,研发可缓解高精料饲粮所致损伤的新型绿色饲料添加剂成为当前研究的一大热点。诺丽在我国主要分布于华南地区,其果实药食两用的习俗历史悠久。诺丽果中含有丰富的营养物质和生物活性物质,具有抗氧化[3]、抗癌[4]、提高免疫力[5]和调节消化道微生物[6]等多种功效。诺丽果中的多酚类和黄酮类物质还具有消炎功效,富含多酚的诺丽果提取物可治疗家兔口腔溃疡[7],并可通过调节肠道菌群改善高脂引起的小鼠肠道损伤[6]。从诺丽果汁中分离出的芦丁和五羟黄酮可抑制脂多糖(LPS)诱导的巨噬细胞中一氧化氮(NO)的产生,并下调炎症因子的蛋白表达[8];诺丽果渣黄酮则可以通过减少乳酸的积累以及肝糖原的储备缓解小鼠的运动后疲劳[2]。本课题组前期研究也表明,多酚类和黄酮类物质作为诺丽果的主要功效成分具有较好的体外抗氧化活性,且优于诺丽果多糖[9];诺丽果及其多糖提取物具有促进绒山羊体内外瘤胃发酵的功效[10-11]。这些研究结果提示诺丽果黄酮类和多酚类提取物可能均具备一定的瘤胃发酵调控活性,并具备缓解高精料饲粮引起的潜在危害机体健康的能力。
然而,目前国内外对于诺丽果的研究仍集中在其活性成分的提取及纯化工艺上,研究对象多以人和小鼠为主;而对于诺丽果提取物及其分离得到的单体化合物活性的比较研究还不够充分,尤其是对诺丽果中多酚和黄酮等主功效成分调控瘤胃发酵水平的比较研究仍缺乏相关资料报道。白藜芦醇(RSV)作为研究的较为成熟的非类黄酮多酚,具有抗氧化[12]、代谢调节[13]、提高绵羊营养物质消化率[14]和缓解LPS引起的氧化应激[15]等功能。鉴于此,本研究以RSV为阳性对照,利用体外试验比较研究和综合评价高精料底物中添加诺丽果多酚(NFP)和诺丽果黄酮(NFF)对绒山羊体外瘤胃发酵的影响,筛选出可调控体外瘤胃发酵的诺丽果提取物,并探究其适宜添加剂量。该研究结果对诺丽果资源的活性成分挖掘与综合利用及高精料集约化养殖中新型饲料添加剂的开发具有重要意义。

1 材料与方法

1.1 试验材料

NFP和NFF采用超声波辅助浸提法制备[9],制备方法简述如下:将诺丽果粉与53%的乙醇溶液以1∶32的比例混匀,于50 ℃超声浸提33 min后,离心得到NFP溶液,旋蒸浓缩后,冻干得到NFP(总酚含量为423.0 mg/g);将诺丽果粉与90%的乙醇溶液以1∶35的比例混匀,于70 ℃超声浸提15 min后,得NFF溶液,旋蒸浓缩后,冻干得到NFF(总黄酮含量为23.14 mg/g)。RSV(纯度为98%)由长沙世唯生物科技有限公司提供。

1.2 试验设计

试验选取3只体况相近的内蒙古阿尔巴斯白绒山羊羯羊作为瘤胃液供体动物。每天于07:00和15:00对供体羊进行2次饲喂,供体羊自由采食并自由饮水,供体羊所喂饲粮和体外发酵底物均为同一种高精料饲粮(精料∶粗料=65∶35),参照《肉羊营养需要量》(NY/T 816—2021)配制,其组成及营养水平见表1。试验分两部分进行,第1部分是比较研究NFP和NFF对绒山羊体外瘤胃发酵影响的差异,筛选出对体外瘤胃发酵具有较好调控作用的诺丽果提取物;第2部分是在第1部分得出对体外瘤胃发酵具有较佳调控作用的诺丽果提取物为NFF后,探究该诺丽果提取物的适宜添加剂量。第1部分:采用单因素完全随机试验设计,分别以高精料饲粮(CON组)以及在高精料饲粮中添加0.25% NFP(NFP组)、0.25% NFF(NFF组)、0.02%RSV(RSV组,作为阳性对照)为底物进行体外瘤胃发酵,NFP和NFF的添加剂量参考本课题组前期研究结果[6,11],RSV的添加剂量参考Ma等[14]的研究结果,每组6个重复。第2部分:采用单因素完全随机试验设计,分别在高精料饲粮中添加0(CON组)、0.025%(F1组)、0.050%(F2组)、0.100%(F3组)和0.250%(F4组)的NFF为底物进行体外瘤胃发酵,每组6个重复。
表1 高精料饲粮组成及营养水平(风干基础)

Table 1 Composition and nutrient levels of the high-concentrate diet (air-dry basis)%

项目 Items 含量 Content
原料 Ingredients
苜蓿草 Alfalfa hay 11.90
玉米秸秆 Corn stalk 23.10
玉米 Corn grain 32.03
豆粕 Soybean meal 3.07
麸皮 Bran 14.20
玉米胚芽粕 Corn germ meal 11.00
玉米干酒糟及其可溶物
Corn DDGS		 2.50
预混料 Premix1) 0.30
石粉 Limestone 1.10
磷酸氢钙 CaHPO4 0.50
食盐 NaCl 0.30
合计 Total 100.00
营养水平 Nutrient levels2)
消化能 DE/(MJ/kg) 11.63
干物质 DM 93.95
粗蛋白质 CP 11.50
粗脂肪 EE 3.10
中性洗涤纤维 NDF 30.71
酸性洗涤纤维 ADF 13.57
钙 Ca 0.81
磷 P 0.41

1)预混料为每千克饲粮提供The premix provided the following per kg of the diet:Fe 0.024 g,Cu 0.004 8 g,Zn 0.03 g,Mn 0.018 g,I 0.18 mg,Se 0.18 mg,Co 0.15 mg,VA 3 600 IU,VD3 1 500 IU,VE 7.5 IU,VK3 1.08 mg,VB1 0.021 mg,VB2 5.1 mg,VB6 0.54 mg,烟酸 nicotinic acid 13.2 mg,D-泛酸 D-pantothenic acid 10.2 mg,VB12 0.018 mg,生物素 biotin 0.084 mg,叶酸 folic acid 0.9 mg。

2)消化能为参考《肉羊饲养标准》(NY/T 816—2021)计算得出,其余为实测值。DE was calculated with reference to Meat Sheep Breeding Standards of China (NY/T 816—2021), while the rest were measured values.

1.3 体外瘤胃发酵方法

参考Menke等[16]的方法配制缓冲液,用缓冲液分别配制5 g/L的NFP溶液和NFF溶液,以及0.4 g/L的RSV溶液。准确称取1.000 g高精料饲粮作为底物(高精料底物)放入厌氧发酵瓶中,于39 ℃预热。第1部分试验具体操作:NFP组、NFF组、RSV组厌氧发酵瓶中分别加入500 μL NFP溶液、NFF溶液和RSV溶液(即NFP和NFF的添加剂量为0.25%,RSV的添加剂量为0.02%),CON组加入等体积的缓冲液。于晨饲前经口腔采集3只绒山羊的瘤胃液,混合后经2层纱布过滤至已预热达39 ℃并通有CO2的保温瓶中。将瘤胃液与缓冲液按1∶2的比例混合,并分装至上述厌氧发酵瓶中,培养体系为60 mL,该步骤全程通CO2进行。将厌氧发酵瓶密封后于39 ℃培养箱培养,每隔3 h摇动1次发酵瓶。培养24 h后迅速将发酵瓶取出,冰浴终止发酵,并取样测定各项指标。第2部分试验具体操作:在CON组、F1组、F2组、F3组和F4组的厌氧发酵瓶中分别加入0、50、100、200和500 μL的NFF溶液,添加剂量不足500 μL的组用缓冲液补至500 μL(即CON组、F1组、F2组、F3组和F4组NFF的添加剂量分别为0、0.025%、0.050%、0.100%和0.250%),其余步骤同第1部分试验。

1.4 测定指标与方法

1.4.1 饲粮营养水平

参照《饲料中水分的测定》(GB/T 6435—2014)测定吸附水含量并据此计算干物质含量(干物质含量=100-吸附水含量);粗蛋白质含量参照《饲料中粗蛋白的测定 凯氏定氮法》(GB/T 6432—2018)进行测定;粗脂肪含量参照《饲料中粗脂肪的测定》(GB/T 6433—2006)进行测定;参照《饲料中钙的测定》(GB/T 6436—2018),采用高锰酸钾法测定钙含量;磷含量参照《饲料中总磷的测定 分光光度法》(GB/T 6437—2018)进行测定;参考Van Soest等[17]的方法,采用ANKOM纤维分析仪测定中性洗涤纤维和酸性洗涤纤维含量。

1.4.2 体外瘤胃发酵参数

体外发酵产气量采用AGRS-Ⅲ微生物发酵微量产气记录系统[18]进行测定。发酵液pH采用台式pH计测定;氨态氮(NH3-N)浓度采用比色法[19]测定;微生物蛋白(MCP)浓度采用考马斯亮蓝法[20]测定;原虫数量使用血细胞计数板进行镜检[21]。采用内标(二乙基丁酸)法使用气相色谱仪测定发酵液中各挥发性脂肪酸(VFA)浓度[11],并计算总挥发性脂肪酸(TVFA)浓度和乙酸/丙酸比值;采用比色法测定发酵液中乳酸浓度,试剂盒购自南京建成生物工程研究所;计算体外瘤胃发酵参数的单项组合效应值(SFAEI)和多项组合效应值[MFAEI,不同组合(处理)各指标SFAEI之和][22]。SFAEI计算公式如下:

SFAEI= A 2 - A 1 A 2。

式中:A1为组合前(CON组)各单一指标的平均值;A2为组合后(试验组)各单一指标的平均值。

1.4.3 发酵液中异常代谢物LPS和组胺(HIS)浓度

发酵液中HIS和LPS浓度采用酶联免疫吸附法进行测定,试剂盒购自泉州市睿信生物科技有限公司。

1.5 统计与分析

试验第1部分数据使用SAS 8.1软件中的MIXED模型进行方差分析,混合模型包括饲粮处理效应和发酵瓶的随机效应。试验第2部分数据使用SAS 8.1软件中的MIXED模型进行方差分析和回归分析,混合模型包括处理效应和发酵瓶的随机效应。P<0.05表示组间差异显著,0.05≤P<0.10表示存在显著差异的趋势。试验数据表示为平均值和均值标准误(SEM)。

2 结果

2.1 NFF和NFP对绒山羊体外瘤胃发酵参数的影响

如表2所示,与CON组和NFP组相比,NFF组和RSV组的pH显著升高(P<0.05),但原虫数量显著降低(P<0.05)。与NFF组相比,其他3组的NH3-N浓度显著降低(P<0.05),CON组和NFP组的MCP浓度也显著降低(<0.05)。与CON组相比,NFP组、NFF组和RSV组的产气量、乙酸/丙酸比值和丙酸浓度均显著升高(P<0.05),NFF组和RSV组的乳酸浓度显著下降(P<0.05)。与RSV组相比,另3个组的乙酸浓度显著升高(P<0.05),且这3组间无显著差异(P>0.05)。与NFP组相比,其他3组的丁酸和异戊酸浓度显著降低(P<0.05),CON组和RSV组的异丁酸和戊酸浓度显著降低(P<0.05),RSV组的TVFA浓度显著降低(P<0.05)。
表2 高精料底物中添加NFF或NFP对绒山羊体外瘤胃发酵参数的影响

Table 2 Effects of adding NFF or NFP to high-concentrate substrate on rumen fermentation parameters of cashmere goats in vitro

项目
Items		 组别 Groups SEM P值
P-value
CON NFP NFF RSV
pH 5.84b 5.98b 6.22a 6.29a 0.05 <0.001
氨态氮 NH3-N/(mg/dL) 35.3b 39.0b 53.2a 35.3b 2.02 <0.001
微生物蛋白 MCP/(mg/dL) 10.0b 11.7b 15.3a 12.9ab 0.79 0.002
原虫 Protozoa/(×104个/mL) 66.7a 64.4a 55.8b 54.3b 1.78 <0.001
产气量 Gas production/mL 99.2b 120.1a 128.6a 124.5a 4.52 0.002
乳酸 Lactic acid/(mmol/L) 0.092a 0.041ab 0.033b 0.027b 0.01 0.021
挥发性脂肪酸 VFA/(mmol/L)
乙酸 Acetate 57.3a 61.4a 59.8a 54.0b 1.48 0.001
丙酸 Propionate 27.3a 24.2b 24.3b 22.3b 0.82 0.004
丁酸 Butyrate 13.3b 17.2a 13.9b 11.8b 0.94 0.006
异丁酸 Iso-butyrate 0.97b 1.22a 1.04ab 0.96b 0.06 0.020
戊酸 Valerate 1.45b 1.61a 1.48ab 1.38b 0.04 0.008
异戊酸 Iso-valerate 2.05b 2.75a 2.13b 2.02b 0.18 0.030
总挥发性脂肪酸 TVFA 102.3ab 111.5a 101.2ab 92.4b 3.54 0.014
乙酸/丙酸 Acetate/propionate 2.11b 2.63a 2.41a 2.45a 0.09 0.006

同行数据肩标不同字母表示差异显著(P<0.05)。下表同。

Values with different letter superscripts in the same row are significantly different (P<0.05). The below tables are the same.

2.2 NFF和NFP对绒山羊体外瘤胃发酵参数影响的综合评定

由表3可知,添加NFF、NFP、RSV均产生了正组合效应(各试验组的MFAEI均大于0),MFAEI由大到小依次为NFF>NFP>RSV。
表3 高精料底物中添加NFF或NFP对绒山羊体外瘤胃发酵影响的综合评定

Table 3 Comprehensive evaluation of effects of adding NFF or NFP to high-concentrate substrate on rumen fermentation of cashmere goats in vitro

项目
Items		 组别 Groups
CON NFP NFF RSV
单项组合效应值 SFAEI(×10-2)
pH 0 2.34 6.11 7.15
氨态氮 NH3-N 0 9.49 33.65 0.00
微生物蛋白 MCP 0 14.53 34.64 22.48
产气量 Gas production 0 17.33 23.10 20.64
总挥发性脂肪酸 TVFA 0 8.11 -0.99 -10.39
多项组合效应值 MFAEI 0 0.518 0.965 0.399

2.3 NFF和NFP对绒山羊体外瘤胃发酵液中异常代谢物LPS和HIS浓度的影响

由表4可知,与CON组相比,NFP组、NFF组和RSV组的LPS浓度显著下降(P<0.05),NFF组的HIS浓度显著下降(P<0.05)。
表4 高精料底物中添加NFF或NFP对绒山羊体外瘤胃发酵液中异常代谢物LPS和HIS浓度的影响

Table 4 Effects of adding NFF or NFP to high-concentrate substrate on concentrations of abnormal metabolites LPS and HIS in rumen fermentation fluid of cashmere goats in vitrong/mL

项目
Items		 组别 Groups SEM P值
P-value
CON NFP NFF RSV
脂多糖 LPS 117.0a 99.2b 104.0b 95.4b 3.01 <0.001
组胺 HIS 8.79a 8.60ab 8.25b 8.49ab 0.12 0.040

2.4 不同添加剂量的NFF对绒山羊体外瘤胃发酵参数的影响

由表5可知,与CON组相比,F2组、F3组、F4组的pH和产气量均显著升高(P<0.05),且产气量以F2组和F3组较高,并显著高于F1组(P<0.05);F1组、F2组、F3组的MCP浓度显著升高(P<0.05);F3组、F4组的NH3-N浓度显著升高(P<0.05),同时原虫数量显著下降(P<0.05),且NH3-N浓度以F4组最高,显著高于F1组和F2组(P<0.05)。添加NFF趋于显著地降低了丙酸(P=0.068)和乳酸浓度(P=0.054)。随NFF添加剂量的增加,pH和产气量呈显著的一次线性(P=0.001,P=0.022)和二次曲线(P=0.001,P<0.001)升高;原虫数量则呈显著的一次线性(P=0.001)和二次曲线(P=0.021)下降;MCP浓度呈显著的二次曲线(P=0.010)升高;丙酸浓度呈显著的二次曲线(P=0.005)下降,并以F3组的丙酸浓度最低;NH3-N浓度呈显著的一次线性(P<0.001)升高;乳酸浓度则呈显著的一次线性(P=0.014)下降。
表5 高精料底物中添加不同剂量的NFF对绒山羊体外瘤胃发酵参数的影响

Table 5 Effects of adding different doses of NFF to high-concentrate substrate on rumen fermentation parameters of cashmere goats in vitro

项目
Items		 组别 Groups SEM P值 P-value
CON F1 F2 F3 F4 处理
Treatment		 线性
Linear		 二次
Quadratic
pH 6.04b 6.12ab 6.14a 6.18a 6.17a 0.02 <0.001 0.001 0.001
氨态氮 NH3-N/(mg/dL) 48.2c 50.9bc 50.2bc 51.9ab 54.8a 0.81 <0.001 <0.001 0.393
微生物蛋白 MCP/(mg/dL) 12.0b 17.3a 17.4a 17.7a 15.9ab 1.30 0.032 0.411 0.010
原虫 Protozoa/(×104个/mL) 63.5a 59.4ab 59.5ab 56.6b 55.9b 1.28 0.002 0.001 0.021
产气量 Gas production/mL 103c 114bc 135a 135a 130ab 5.09 <0.001 0.022 <0.001
乳酸 Lactic acid/(mmol/L) 0.067 0.056 0.054 0.045 0.044 0.01 0.054 0.014 0.073
挥发性脂肪酸 VFA/(mmol/L)
乙酸 Acetate 55.1 56.0 56.4 57.1 55.6 1.00 0.688 0.978 0.147
丙酸 Propionate 20.9 20.6 20.4 20.1 20.7 0.21 0.068 0.787 0.005
丁酸 Butyrate 14.4 13.9 13.7 13.7 14.0 0.57 0.887 0.913 0.340
异丁酸 Iso-butyrate 1.14 1.14 1.16 1.15 1.18 0.03 0.889 0.348 0.899
戊酸 Valerate 1.55 1.56 1.61 1.59 1.50 0.05 0.632 0.346 0.269
异戊酸 Iso-valerate 2.22 2.26 2.22 2.28 2.29 0.09 0.972 0.574 0.875
总挥发性脂肪酸 TVFA 95.3 95.4 95.4 95.8 95.7 1.35 0.998 0.805 0.851
乙酸/丙酸 Acetate/propionate 2.71 2.79 2.71 2.78 2.74 0.05 0.732 0.900 0.610

2.5 不同添加剂量的NFF对绒山羊体外瘤胃发酵影响的综合评定

由表6可知,NFF的各添加剂量均产生了正组合效应(4个添加NFF组的MFAEI均大于0),MFAEI由大到小依次是F3组>F2组>F4组>F1组。
表6 高精料底物中添加不同剂量的NFF对绒山羊体外瘤胃发酵影响的综合评定

Table 6 Comprehensive evaluation of effects of adding different doses of NFF to high-concentrate substrate on rumen fermentation of cashmere goats in vitro

项目
Items		 组别 Groups
CON F1 F2 F3 F4
单项组合效应值 SFAEI(×10-2)
pH 0 1.31 1.63 2.27 2.11
氨态氮 NH3-N 0 5.30 3.98 7.13 12.04
微生物蛋白 MCP 0 30.6 31.0 32.2 24.5
产气量 Gas production 0 9.65 23.70 23.70 20.77
总挥发性脂肪酸 TVFA 0 0.105 0.105 0.522 0.418
多项组合效应值 MFAEI 0 0.470 0.605 0.658 0.599

2.6 不同添加剂量的NFF对绒山羊体外瘤胃发酵液中异常代谢物LPS和HIS浓度的影响

由表7可知,与CON组相比,4个添加NFF组的HIS浓度均显著下降(P<0.05),并以F2组、F3组和F4组的浓度较低,显著低于F1组(P<0.05)。与CON组相比,F3组和F4组的LPS浓度显著降低(P<0.05),并以F3组的浓度最低,显著低于F1组和F2组(P<0.05)。随着NFF添加剂量的增加,LPS和HIS浓度呈显著的一次线性(P=0.003,P<0.001)和二次曲线(P=0.002,P<0.001)下降。
表7 高精料底物中添加不同剂量的NFF对绒山羊体外瘤胃发酵液中异常代谢物LPS和HIS浓度的影响

Table 7 Effects of adding different does of NFF to high-concentrate substrate on concentrations of abnormal metabolites LPS and HIS in rumen fermentation fluid of cashmere goats in vitrong/mL

项目
Items		 组别 Groups SEM P值 P-value
CON F1 F2 F3 F4 处理
Treatment		 线性
Linear		 二次
Quadratic
脂多糖 LPS 131.1a 119.7ab 121.2ab 96.5c 107.1bc 5.26 0.001 0.003 0.002
组胺 HIS 8.52a 6.82b 5.12c 4.13c 4.26c 0.43 <0.001 <0.001 <0.001

3 讨论

RSV是一种具有较强生物活性的非类黄酮多酚,可以提高绵羊对营养物质的消化率[14],缓解LPS引起的氧化应激[15],因而选择其作为本试验的阳性对照。本试验中2种诺丽果提取物NFF和NFP的MFAEI均优于RSV,并以NFF最高,说明NFF具有较好的促进绒山羊体外瘤胃发酵的效果,且F3组的MFAEI优于其他添加剂量组,即0.100% NFF对高精料底物条件下绒山羊体外瘤胃发酵的促进效果最好。究其原因,可能与NFF调控了瘤胃氮利用有关。原虫、MCP及其前体物NH3-N均是反映反刍动物消化利用蛋白质的重要物质。本试验中仅NFF组显著提高了发酵液中NH3-N和MCP的浓度,并且降原虫的效果达到了RSV的水平。这表明NFF可促进蛋白质的降解并减少氮周转,进而提高瘤胃氮利用效率,促进MCP的合成。本课题组前期研究表明,NFF中含有大量的芦丁和槲皮素[11],槲皮素可以减少发酵液中的原虫及其共生菌产甲烷菌[23]。因此,本试验中NFF可能通过降原虫作用来减少原虫对细菌的吞噬,从而间接促进细菌对营养物质的消化降解,提高了体外发酵的产气量。此外,有研究表明,芦丁[24]、富含黄酮的假蒟提取物[25]对瘤胃发酵的促进效果均存在剂量依赖效应,并在高剂量时效果较佳,这与本研究结果一致。
高精料底物的快速降解会使VFA迅速积累、乳酸浓度升高,进而导致pH快速下降。pH过低时大量革兰氏阴性菌死亡,从而释放异常代谢产物LPS和HIS等[26],甚至引起亚急性瘤胃酸中毒(SARA)。有研究表明,瘤胃液pH低于5.8是判定SARA发生的重要指标[27]。本试验中CON组的pH(5.84)接近5.8,在2种诺丽果提取物中仅NFF显著提高了发酵液pH并降低了乳酸和HIS浓度。因此,NFF对于发酵液的酸度具有更好的缓冲作用,具有缓解SARA的潜力。乳酸作为瘤胃发酵的产物在瘤胃中的浓度较低,但酸度却是VFA的10倍。研究表明,柑橘类黄酮提取物通过增加乳酸消耗菌(反刍月形单胞菌和埃氏巨球型菌)与减少乳酸利用菌(牛链球菌)的相对丰度,降低了高精料饲粮条件下瘤胃的乳酸浓度,提高了pH[28];芦丁可以抑制奶牛体外瘤胃发酵乳酸产生菌的生长,从而降低乳酸浓度[24]。这提示NFF可能通过调控乳酸生成菌和乳酸消耗菌的相对丰度,降低乳酸浓度,升高pH,进而减少LPS和HIS生成,但还需要后续试验进行验证。

4 结论

在2种诺丽果提取物(NFF和NFP)中,NFF具备调控高精料底物条件下绒山羊体外瘤胃发酵的作用,其在高精料底物中的适宜添加剂量为0.100%。

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