郝小燕^,1, 牟春堂¹, 乔栋², 张暄梓¹, 杨文军¹, 赵俊星¹, 张春香¹, 张建新^,11山西农业大学动物科学学院,山西太谷 030801
²朔州市草牧业发展中心,山西朔州 036002

Effects of High-Concentrate Diet Supplemented with Grape Seed Proanthocyanidins on Rumen fermentation, Inflammatory and Antioxidant Indicators of Rumen and Serum in Lambs

HAO XiaoYan^,1, MU ChunTang¹, QIAO Dong², ZHANG XuanZi¹, YANG WenJun¹, ZHAO JunXing¹, ZHANG ChunXiang¹, ZHANG JianXin^{,1 1}College of Animal Science, Shanxi Agricultural University, Taigu 030801, Shanxi
²Grassland Livestock Husbandry Development Center, Shuozhou 036002, Shanxi

通讯作者: 张建新,E-mail: ypzjx@126.com

责任编辑: 林鉴非
收稿日期:2020-07-26接受日期:2020-11-27网络出版日期:2021-05-16

基金资助:

国家重点研发计划.2018YFD0502104 现代农业产业技术体系.CARS-38 山西省“1331工程”畜牧学重点学科建设计划.J201911301 山西省基础研究计划.201801D221291

Received:2020-07-26Accepted:2020-11-27Online:2021-05-16
作者简介 About authors
郝小燕,E-mail: haoxiaoyan1990@sina.com。










摘要
【目的】研究高精料饲粮条件下,添加不同水平葡萄籽原花青素（GSPs）对杜寒杂交羔羊生长性能、瘤胃发酵、瘤胃及血清炎症因子及抗氧化能力的影响。为葡萄籽原花青素在反刍动物生产中的应用提供数据参考。【方法】选择48只体重相近（22.75±1.20 kg）、月龄相近的杜泊×小尾寒羊杂交公羔,采用单因素完全随机试验设计,将48只羔羊随机分为4组,每组12只。基础饲粮精粗比为7:3,GSPs添加量分别为0（对照组,CON）、10（10GSPs）、20（20GSPs）、40（40GSPs）mg·kg^-1 BW。饲养期共60 d,前15 d为预饲期。正试期第1天晨饲前称量体重作为初始体重。正试期结束后颈静脉采集血液并分离血清,用于抗氧化指标、炎性因子和脂多糖含量测定;同时每组随机选择6只分别于饲喂后1、3、4、6、8、12 h口腔采集瘤胃液,立即测定瘤胃液pH,饲喂后3 h瘤胃液样品用于测定发酵指标及脂多糖含量。每组剩余6只进行屠宰,采集瘤胃组织测定抗氧化指标和炎症因子水平。【结果】10GSPs和20GSPs组的末体重显著高于对照组（P<0.05）,与40GSPs组差异不显著（P>0.05）。10GSPs和20GSPs组ADG和ADFI显著高于对照组和40GSPs组（P<0.05）,而40GSPs组与对照组差异不显著（P>0.05）;添加GSPs对瘤胃pH有一定的调控作用,饲喂后3、8、12 h瘤胃pH随GSPs添加水平增加而线性升高（P<0.05）,4 h瘤胃pH较对照组有线性升高趋势,但差异不显著（P=0.057）;添加GSPs后瘤胃液中乙酸、丁酸、总挥发性脂肪酸浓度有降低的趋势（P<0.1）,对丙酸、异戊酸、戊酸浓度及乙酸/丙酸值没有显著影响（P>0.05）;添加GSPs后血清脂多糖浓度较对照组显著降低（P<0.05）,但不影响瘤胃液中脂多糖浓度。20GSPs和40GSPs组瘤胃组织GSH-Px活性显著高于对照组和10GSPs组,MDA含量显著低于对照组和10GSPs组（P<0.05）。20GSPs和40GSPs组血清SOD活性显著高于对照组,GSH-Px活性显著高于对照组和10GSPs组（P<0.05）。添加GSPs对瘤胃组织炎症因子没有显著影响,但有降低IL-6和IL-10的趋势（P<0.1）。20GSPs和40GSPs组血清TNF-α水平显著低于对照组和10GSPs组（P<0.05）;40GSPs组血清IL-10含量显著低于对照组（P<0.05）,与10GSPs和20GSPs组差异不显著（P>0.05）。【结论】高精料饲粮条件下补饲适量GSPs可以提高羔羊瘤胃pH,提高血清和瘤胃组织抗氧化能力,对羔羊健康具有潜在的保护效应。在本试验条件下,GSPs的最佳饲喂量为20 mg·kg^-1BW。
关键词： 葡萄籽原花青素;羔羊;高精料;瘤胃发酵;抗氧化

Abstract
【Objective】This study was conducted to investigate the effects of dietary grape seed proanthocyanidins (GSPs) supplementation on growth performance, rumen fermentation, rumen and serum inflammatory factors and antioxidative activity in lambs under the condition of a high-concentrate diet, so as to provide a support for applying GSPs on ruminant. 【Method】A total of forty-eight 1/2 Dorper × 1/2 thin-tailed Han ram lambs with similar body weight (BW = 22.75±1.20 kg, mean ± SD) were randomly divided into four groups, with twelve lambs each group. Lambs were fed a 30:70 forage:concentrate diet with 0 (control), 10 (10GSPs), 20 (20GSPs), and 40 mg·kg^-1 BW (40GSPs) GSPs supplemented, respectively. The feeding experiment lasted for 60 days before sampling, with the first 15 days for diet transition and adaptation. On the first day of the formal experiment period, the BW before morning feeding was weighed as the initial BW. At the end of the trial period, blood samples were collected from the jugular vein, and the serum was separated for the determination of antioxidant and inflammatory indicators and lipopolysaccharide content. At the same time, six lambs in each group were randomly selected to collect rumen fluid by using an oral stomach tube at 1, 3, 4, 6, 8 and 12 hours after feeding, respectively, and the rumen pH were measured immediately. The fluid samples at 3 hours after feeding were used to measured fermentation parameters and lipopolysaccharide content. The other 6 lambs in each group were slaughtered, and the samples of rumen tissue were collected to determine the antioxidation index and inflammatory factors. 【Result】The final weight of lambs in 10GSPs and 20GSPs groups were significant greater than that in control group (P<0.05), with no difference between 40GSPs and 10GSPs or 20GSPs (P>0.05). Lambs in 10GSPs and 20GSPs groups also had higher average daily gain and average daily feed intake than control and 40GSPs (P<0.05), with no significant difference between control and 40GSPs (P>0.05). The dietary GSPs supplementation had a certain regulatory effect on rumen pH. The rumen pH increased linearly with the supplementation of GSPs (P<0.05) at 3, 8 and 12 h after feeding, and tended to increased linearly at 4 h (P=0.057). The concentrations of acetate, butyrate and total volatile fatty acid in rumen fluid tended to decrease after GSPS was added (P<0.1), but there was no significant effect on propionate, isovalerate, valerate, and the ratio of acetic acid to propionic acid (P>0.05). After GSPs was supplemented, the serum lipopolysaccharide concentration was significantly lower than that of the control group (P<0.05), but which did not affect the lipopolysaccharide concentration in the rumen fluid. The activity of GSH-Px in rumen tissue of 20GSPs and 40GSPs groups was significantly higher than that of the control group and 10GSPs group (P<0.05) , however, the content of MDA was significantly lower. The serum SOD activity of 20GSPs and 40GSPs group was significantly higher than that of the control group, and the GSH-Px activity was higher than control and 10GSPs group (P<0.05). There was no significant effect was observed in rumen inflammatory factors when GSPs was supplemented, but there was a trend of decreasing IL-6 and IL-10 (P<0.1). The level of TNF-α in 20GSPs and 40GSPs group were significantly lower than that in the control group and 10 GSPs group (P<0.05). The level of IL-10 in 40GSPs group was significantly lower than control group, with no significant difference between 40GSPs and 10GSPs or 20GSPs (P>0.05). 【Conclusion】Supplementing appropriate amount of GSPs to high-concentrate diet could improve the rumen pH, and the antioxidant capacity of serum and rumen tissue of lambs, suggesting that appropriate amount of GSPs had the potential protective effect on lamb health. The optimal feeding dose was 20 mg·kg^-1 BW under the condition of this experiment.
Keywords：grape seed proanthocyanidins;lambs;high-concentrate diet;rumen fermentation;antioxidant capacity


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本文引用格式
郝小燕, 牟春堂, 乔栋, 张暄梓, 杨文军, 赵俊星, 张春香, 张建新. 葡萄籽原花青素对羔羊瘤胃发酵、血清炎症及抗氧化指标的影响[J]. 中国农业科学, 2021, 54(10): 2239-2248 doi:10.3864/j.issn.0578-1752.2021.10.019
HAO XiaoYan, MU ChunTang, QIAO Dong, ZHANG XuanZi, YANG WenJun, ZHAO JunXing, ZHANG ChunXiang, ZHANG JianXin. Effects of High-Concentrate Diet Supplemented with Grape Seed Proanthocyanidins on Rumen fermentation, Inflammatory and Antioxidant Indicators of Rumen and Serum in Lambs[J]. Scientia Acricultura Sinica, 2021, 54(10): 2239-2248 doi:10.3864/j.issn.0578-1752.2021.10.019


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0 引言

【研究意义】在生态保护日益受到重视的背景下,传统畜牧业逐渐向先进的集约化饲养模式转变。我国优质粗饲料较为短缺,反刍动物生产中大量使用高谷物饲料以维持或提升生产性能。高精低粗的饲粮结构虽能提高育肥牛羊的生长速度,提高奶牛、奶山羊的产奶量,但长期饲喂高谷物饲粮引发的健康问题不容忽视。因此,研究高精料饲粮条件下调控瘤胃发酵、保护机体免受氧化损伤的措施对反刍动物健康、高效养殖具有重要意义。【前人研究进展】侯志高等^[1]研究发现不同精料浓度饲粮对瘤胃内环境稳定和机体氧化应激有显著影响,精料比例在55%时瘤胃内超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）活力最高,丙二醛（MDA）含量最低,清除自由基的能力最强。而随着精料比例增加,瘤胃内环境稳态被破坏,抗氧化能力显著下降,抵抗不良刺激的能力下降,故高精料饲粮下机体容易发生一系列代谢性、炎性疾病。原花青素（proanthocyanidins,PC）是自然界中广泛存在的植物多酚类化合物,由不同数量的儿茶素或表儿茶素聚合而成。有关研究表明原花青素可以增强动物机体抗氧化能力,调节机体免疫反应,进而改善动物健康状况、促进生长^[2,3]。也有研究报道原花青素可以促进机体正常细胞生长,如胃黏膜细胞^[4];可以降低脂质过氧化、DNA片段化及细胞膜微黏度,通过抗组胺作用来降低氧化应激引起的胃肠道黏膜损伤^[5];对结直肠炎、结直肠癌有一定的防护作用^[6];长期食用原花青素能够缓解内毒素对结肠屏障完整性的损伤和抑制胃肠道对内毒素的吸收^[7];可以减轻机体炎症肿胀,抑制细胞因子IL-6和TNF-α诱导的血管细胞黏附分子-1的表达水平^[8]。另外,原花青素对瘤胃发酵、微生物区系也有一定的调控作用^[9,10]。杜宇等^[11]研究发现在体外建立的亚急性瘤胃酸中毒模型中添加4.5%石榴多酚可以提高发酵液pH,降低发酵液中挥发性脂肪酸（VFA）、内毒素（LPS）和组胺浓度,同时降低牛链球菌数量和乳酸浓度。郭长征等^[12]研究表明高精料饲粮中添加另一种多羟基黄酮类化合物（槲皮素）可以提高山羊瘤胃pH,提高血清抗氧化能力,但有关植物多酚对瘤胃组织抗氧化和抗炎能力的影响及机制的研究较少。【本研究切入点】基于高精料饲粮对动物瘤胃、机体的危害及原花青素抗氧化、抗炎的生物学功能,本研究提出适宜剂量的葡萄籽原花青素可以通过调控瘤胃发酵和改善机体抗氧化能力而减轻机体氧化损伤的假设。【拟解决的关键问题】本试验研究了高精料饲粮条件下,添加不同水平葡萄籽原花青素（GSPs）对羔羊生长性能、瘤胃发酵、瘤胃及血清炎症因子及抗氧化能力的影响,为葡萄籽原花青素在反刍动物生产中的应用提供数据参考。

1 材料与方法

1.1 试验设计

葡萄籽原花青素购自南京泽朗生物科技有限公司,纯度≥96%。

试验选用48只体重相近（22.75±1.20 kg）、月龄相近的杜泊×小尾寒羊杂交公羔,采用单因素完全随机试验设计,将48只羔羊随机分为4组,每组12只。基础饲粮精粗比为70:30,GSPs添加量分别为0（对照组,CON）、10（10GSPs）、20（20GSPs）、40（40GSPs）mg·kg^-1 BW。预饲15 d,饲粮精粗比从4:6逐渐过渡到7:3,正试期45d。正试期每日晨饲前,用分析天平称取每日每只羊GSPs饲喂量,与100 g粉状饲粮混合后,逐只一次性饲喂,以保证完全摄入,GSPs的饲喂量每周根据体重调整一次。试验羔羊全部单栏（3.0 m×0.8 m）饲养,饲粮采用全混合颗粒饲料,每天饲喂2次（07:00和19:00）,自由采食,自由饮水。试验羊舍通风良好,平均温度23.7℃,平均相对湿度59%,基础饲粮组成及营养水平见表1。

Table 1
表1
表1饲粮组成及营养水平（干物质基础）
Table 1Composition and nutrient level of diets (dry matter basis, %)

项目Items	含量Content
原料 Ingredients
玉米秸秆 Corn straw	20.0
谷秸 Millet straw	10.0
玉米 Corn	46.0
豆粕 Soybean meal	7.0
棉粕 Cottonseed meal	5.0
麦麸 Wheat bran	5.0
石粉 Limestone	0.3
碳酸氢钠 Sodium bicarbonate	1.0
盐 Salt	0.7
预混料 Premix 1	5.0
营养水平 Nutrient levels
粗蛋白 CP	16.1
粗脂肪 EE	3.5
中性洗涤纤维 NDF	32.7
酸性洗涤纤维 ADF	16.9
粗灰分 Ash	10.2
总能GE; MJ·kg-1 DM	18.8

¹每千克饲粮中含有（mg·kg^-1）:Cu 15,Fe 55,Zn 25,Mn 40,Se 0.3,I 0.5,Co 0.2;饲料中维生素添加量（IU·kg^-1）:VA 20 000,VD 4 000,VE 40。饲料各营养成分均为实测值
One kilogram of diet contained the followings (mg·kg^-1): Cu 15, Fe 55, Zn 25, Mn 40, Se 0.3, I 0.5, Co 0.2; Additive vitamin premix (IU·kg^-1): VA 20 000, VD 4 000, VE 40. The compositions of diet are measured values

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1.2 样品采集

正试期第一天早6:00对所有试验羊空腹称重,作为初始体重,之后每隔15 d称重一次,记录体重数据。试验期间记录每只羊的日采食量、剩料量,正试期结束后计算平均日增重（ADG）、平均日采食量（AFDI）及料重比（F/G）。正试期第45天晨饲前颈静脉采血,常规方法分离血清后-20℃保存待测。

正试期结束当天18:00,每组随机选择6只禁食、禁水12 h,次日6:00进行屠宰。采集瘤胃背囊组织,迅速用磷酸缓冲盐溶液反复清洗,剪碎后立即置于液氮中速冻保存,采样结束后放于-80℃保存。

正试期结束后连续3 d通过口腔采集每组剩余6只羊的瘤胃液,分别于饲喂后1、3、4、6、8、12 h通过口腔采集瘤胃液（每天采2个时间点,第一天:8:00、13:00,第二天:10:00、15:00,第三天:11:00、19:00）,弃掉初始采集含有较多黏液的部分,立即测定瘤胃液pH;同时分装样品于-20℃保存待测瘤胃发酵指标。

1.3 指标测定

瘤胃组织和血清抗氧化能力测定所用试剂盒均购自南京建成生物工程研究所,具体指标包括总抗氧化能力（T-AOC）、SOD、过氧化氢酶（CAT）、GSH-Px、MDA。采用酶联免疫吸附（ELISA）法测定瘤胃组织及血清中IL-6、IL-10、TNF-α和IFN-γ含量,ELISA试剂盒购自南京建成生物工程研究所。

饲喂后3 h的瘤胃液样品用于测定VFA和LPS浓度。VFA浓度测定采用气相色谱法,具体操作参照严淑红等^[13]的方法。使用显色基质鲎试剂盒（厦门鲎试剂实验厂有限公司）测定瘤胃液和血清中的LPS浓度^[14]。

1.4 数据分析

利用Excel 2010版进行试验数据初步整理,采用SAS 9.2软件中one-way ANOVE进行分析,采用Duncan法进行多重比较,通过正交多项式分析处理的线性和二次效应。P<0.05表示为差异显著,P<0.10表示有显著的趋势。

2 结果

2.1 生长性能

羔羊生长性能数据已发表^[15],具体见表2,试验羔羊的初始体重相近,但饲粮中添加不同水平GSPs后羔羊末重（P=0.039）、ADG（P=0.005）、ADFI（P=0.001）呈二次方效应,其中10GSPs和20GSPs组的末体重显著高于对照组（P<0.05）,与40GSPs差异不显著（P>0.05）;10GSPs和20GSPs组ADG和ADFI显著高于对照组和40GSPs组（P<0.05）,而40GSPs组与对照组差异不显著（P>0.05）。饲粮中添加GSPs对料重比没有显著影响（P>0.05）。

Table 2
表2
表2高精料饲粮中添加葡萄籽原花青素对羔羊生长性能的影响
Table 2Effect of GSPs supplemented in high-concentrate diet on growth performance of lambs

项目 Items	组别 Groups				SEM	P
	CON	10GSPs	20GSPs	40GSPs		线性Linear	二次方Quadratic
初始体重Initial BW(kg)	28.39	29.10	28.64	29.32	0.82	0.532	0.985
终末体重Final BW(kg)	41.57b	43.85a	43.51a	42.64ab	0.70	0.375	0.039
平均日增重ADG(g·d-1)	280.37b	314.05a	315.90a	283.56b	10.21	0.806	0.005
平均日采食量ADFI(g·d-1)	1885.14b	2010.61a	1993.99a	1874.60b	31.74	0.737	0.001
料重比ADFI/ADG	6.76	6.42	6.31	6.65	0.210	0.639	0.123

同行数据无字母或标相同字母表示差异不显著（P>0.05）,不同小写字母表示差异显著（P<0.05）。下同
In the same row, values with no letter or the same letter mean no significant difference (P>0.05), while with different small letter mean significant difference (P<0.05). The same as below

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2.2 瘤胃pH和挥发性脂肪酸

由表3可知,与对照组相比,添加GSPs对瘤胃pH有一定的调控作用。饲喂后3、8、12 h瘤胃pH随GSPs的添加线性升高,20GSPs和40GSPs组瘤胃pH显著高于对照组和10GSPs组（P<0.05）。饲喂后4 h瘤胃液pH较对照组有线性升高趋势,但差异不显著（P=0.057）。

Table 3
表3
表3高精料饲粮中添加葡萄籽原花青素对羔羊瘤胃pH的影响
Table 3Effect of GSPs supplemented in high-concentrate diet on rumen fluid pH of lambs

项目 Items	组别 Groups				SEM	P
	CON	10GSPs	20GSPs	40GSPs		线性Linear	二次方Quadratic
1 h	6.04	6.05	6.09	6.11	0.03	0.101	0.805
3 h	5.80b	5.79b	5.93a	5.94a	0.05	0.018	0.664
4 h	5.92	5.93	5.97	6.05	0.04	0.057	0.389
6 h	6.05	6.14	6.10	6.15	0.07	0.155	0.883
8 h	6.19b	6.21b	6.34a	6.30a	0.03	0.031	0.141
12 h	6.24b	6.23b	6.33a	6.35a	0.03	0.009	0.420

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从表4可见,饲粮中添加GSPs后羔羊采食后3 h瘤胃液中乙酸（P=0.058）和丁酸（P=0.064）浓度有降低的趋势,对丙酸、异戊酸和戊酸浓度没有显著影响（P>0.05）。40GSPs组异丁酸浓度显著高于对照组和10GSPs组（P<0.05）,与20GSPs组差异不显著（P>0.05）。随着GSPs添加量的增加,总挥发性脂肪酸浓度有线性下降的趋势（P=0.062）。饲粮中添加GSPs对乙酸/丙酸值没有显著影响（P>0.05）。

Table 4
表4
表4高精料饲粮中添加葡萄籽原花青素对羔羊饲喂后3 h瘤胃挥发性脂肪酸的影响
Table 4Effect of GSPs supplemented in high-concentrate diet on rumen VFA after feeding 3 hours of lambs

项目 Items	组别 Groups				SEM	P
	CON	10GSPs	20GSPs	40GSPs		线性Linear	二次方Quadratic
乙酸 Acetate (mmol·L-1)	59.61	59.01	54.11	55.18	1.95	0.058	0.672
丙酸Propionate (mmol·L-1)	25.08	24.93	21.56	22.83	1.64	0.182	0.667
异丁酸 Isobutyrate (mmol·L-1)	0.50b	0.49b	0.55ab	0.63a	0.032	0.004	0.153
丁酸 Butyrate (mmol·L-1)	11.93	10.82	10.92	9.51	0.78	0.064	0.850
异戊酸 Isovalerate (mmol·L-1)	0.85	0.92	0.95	0.91	0.041	0.291	0.224
戊酸 Valerate (mmol·L-1)	1.21	1.21	1.10	1.19	0.069	0.584	0.464
总挥发性脂肪酸TVFA (mmol·L-1)	99.18	97.36	89.18	90.27	3.13	0.062	0.648
乙酸/丙酸Acetate/propionate	2.45	2.41	2.55	2.45	0.16	0.854	0.856

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2.3 瘤胃液和血清内毒素含量

由表5可见,高精料饲粮中添加GSPs瘤胃液LPS含量的呈线性和二次方下降趋势,其中20GSPs数值最低,但差异不显著（P=0.076）;20GSPs和40GSPs组血清LPS浓度较对照组和10GSPs组显著降低（P<0.0001）。

Table 5
表5
表5高精料饲粮中添加葡萄籽原花青素对羔羊饲喂后3 h瘤胃液脂多糖含量的影响
Table 5Effect of GSPs supplemented in high-concentrate diet on rumen LPS content after feeding 3 hours of lambs

项目 Items	组别 Groups				SEM	P
	CON	10GSPs	20GSPs	40GSPs		线性Linear	二次方Quadratic
瘤胃液LPS (EU·mL-1)	42595	34449	27116	31267	4168	0.058	0.099
血清LPS (EU·mL-1)	0.31a	0.29a	0.16b	0.17b	0.02	<0.0001	0.221

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2.4 瘤胃和血清抗氧化能力

由表6可见,高精料饲粮中添加GSPs线性提高了瘤胃GSH-Px活性,线性降低瘤胃组织MDA水平（P<0.05）。20GSPs和40GSPs组GSH-Px活性显著高于对照组和10GSPs组,MDA含量显著低于对照组和10GSPs组（P<0.05）。对于血清抗氧化指标,20GSPs和40GSPs组SOD活性显著高于对照组,GSH-Px活性显著高于对照组和10GSPs组（P<0.05）。添加GSPs有线性降低血清MDA（P=0.059）和提高T-AOC（P=0.060）的趋势。

Table 6
表6
表6高精料饲粮中添加葡萄籽原花青素对羔羊瘤胃和血清抗氧化指标的影响
Table 6Effect of GSPs supplemented in high-concentrate diet on rumen and serum antioxidant indicators of lambs

项目 Items		组别 Groups				SEM	P
		CON	10GSPs	20GSPs	40GSPs		线性Linear	二次方Quadratic
瘤胃 Rumen	T-AOC (U·mg-1)	1.13	1.22	1.28	1.23	0.08	0.282	0.370
	CAT (U·mg-1)	4.41	4.54	4.08	4.37	0.47	0.787	0.861
	SOD (U·mg-1)	6.8	6.85	7.46	7.28	0.53	0.394	0.830
	GSH-Px (U·mg-1)	66.30b	69.18b	90.55a	82.99a	3.70	0.0003	0.173
	MDA, nmol·mg-1	0.693a	0.62ab	0.52b	0.53b	0.05	0.019	0.481
血清 Serum	T-AOC (U·mL-1)	1.60	1.62	1.83	1.78	0.07	0.056	0.590
	CAT (U·mL-1)	6.6	6.66	6.52	6.79	0.52	0.855	0.839
	SOD (U·mL-1)	69.28b	72.58ab	82.9a	83.43a	4.16	0.010	0.743
	GSH-PX (U·mL-1)	85.74b	87.92b	102.60a	104.35a	4.85	0.004	0.963
	MDA (nmol·mL-1)	4.49	4.34	3.52	3.59	0.33	0.060	0.764

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2.5 瘤胃和血清炎性因子水平

由表7可见,饲粮中添加GSPs对瘤胃组织炎症因子水平没有显著影响,但有降低IL-6和IL-10的趋势（P≤0.061）。20GSPs和40GSPs组血清TNF-α水平显著低于对照组和10GSPs组（P<0.05）,IL-6水平有降低的趋势,但差异不显著（P=0.074）;40GSPs组血清IL-10含量显著低于对照组,与10GSPs和20GSPs组差异不显著（P>0.05）。

Table 7
表7
表7高精料饲粮中添加葡萄籽原花青素对羔羊瘤胃和血清炎症因子的影响
Table 7Effect of GSPs supplemented in high-concentrate diet on rumen and serum inflammatory factors of lambs

项目 Items		组别 groups				SEM	P
		CON	10GSPs	20GSPs	40GSPs		线性Linear	二次方Quadratic
瘤胃 Rumen	TNF-α (pg·mg-1)	4.48	4.45	4.25	4.30	0.23	0.497	0.871
	IL-6 (pg·mg-1)	12.05	12.06	10.16	10.73	0.66	0.061	0.676
	IFN-γ(pg·mg-1)	4.96	4.51	4.50	4.78	0.28	0.664	0.208
	IL-10 (pg·mg-1)	1.46	1.44	1.28	1.30	0.06	0.062	0.729
血清 Serum	TNF-α(pg·mL-1)	48.74a	50.52a	38.52b	39.6b	2.99	0.008	0.908
	IL-6 (pg·mL-1)	142.18	133.33	127.5	128.72	5.47	0.074	0.368
	IFN-γ (pg·mL-1)	39.13	40.44	39.86	37.09	2.21	0.506	0.365
	IL-10 (pg·mL-1)	19.86a	17.79ab	17.83ab	16.19b	0.93	0.015	0.814

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3 讨论

原花青素是植物体内重要的次生代谢产物,是一类植物多酚类化合物。葡萄籽原花青素主要存在于葡萄籽、葡萄皮和梗部,是一类重要的天然抗氧化剂。研究表明,原花青素类植物多酚在提高动物生产性能、免疫功能、抗氧化能力等方面发挥着重要作用,但高浓度的植物多酚可能会抑制动物采食量,扰乱消化生理,抑制营养物质消化和机体生长^{[16,17,18,19]}。赵家奇等^[18]研究发现在断奶仔猪饲粮中葡萄籽原花青素可以显著提高仔猪血清免疫球蛋白和补体含量,提高血清抗氧化酶活性,说明原花青素可以提高断奶仔猪免疫力和抗氧化能力。在高温热应激条件下,饲粮中添加板栗多酚可以显著提高肉鸡日增重和采食量,提高肉鸡抗氧化能力、缓解高温热应激^[20]。金亚倩等^[21]在绵羊饲料中添加葡萄渣,试验结果表明10%葡萄渣添加组较对照组日增重提高38%,可能是葡萄皮渣中的主要活性物质原花青素发挥了生物学功能,这与本试验得到的结果一致。本试验中,GSPs改善羔羊生长性能,一方面原因可能是GSPs改善了机体抗氧化能力和免疫力,进而提高生长性能;另一方面原因可能与多酚类物质降低饲粮蛋白质降解率、提高蛋白利用率有关^[17]。有关饲粮中GSPs水平对羔羊饲粮蛋白瘤胃降解率的影响还有待进一步研究。40GSPs组采食量和日增重较另外两个GSPs添加组低,可能与高剂量多酚类物质抑制瘤胃微生物（尤其是纤维分解菌）,降低饲料消化率有关^[22,23]。杨德莲等^[9]研究也表明葡萄籽原花青素能够显著降低奶牛瘤胃体外发酵液中溶纤维丁酸弧菌和产琥珀酸丝状杆菌的数量,降低体外发酵产气量。本试验中GSPs对羔羊瘤胃微生物区系的影响还需进一步深入研究。

反刍动物长期采食高精料饲粮会导致瘤胃和后肠道pH下降和VFA积累,导致微生物代谢紊乱,从而产生大量的细菌内毒素。当瘤胃pH低于5.8,且持续时间大于3 h,则被认为发生了亚急性瘤胃酸中毒。本试验中各处理组瘤胃液pH在采食后不同时间点测定结果均高于5.8（除3 h对照组为5.80,10GSPs为5.79）,提示试验羔羊尚未处于亚急性瘤胃酸中毒状态。但由于本试验中瘤胃液pH是通过口腔插管采集,可能对试验羊食道产生一定的刺激,影响了采食量,从而影响瘤胃pH。对照组和10GSPs组各时间点瘤胃pH低于6.2,添加20和40 mg·kg^-1 BW的GSPs可以在一定程度上提高瘤胃液pH,这与杨德莲等^[9,11]的研究结果相一致。瘤胃pH升高有利于提高羔羊的营养物质消化率和日增重。研究表明在体外亚急性瘤胃酸中毒模型中添加石榴皮多酚提取物可以显著抑制瘤胃液中牛链球菌的生长和繁殖^[11];在高精料饲粮中添加槲皮素可以提高山羊瘤胃pH和血清抗氧化酶活性,缓解亚急性瘤胃酸中毒^[12];在高精料饲粮中添加植物多酚和精油混合物,瘤胃pH低于5.6的时间较不添加组显著降低（18 min和199 min）^[24]。本试验中,添加GSPs有降低乙酸和总挥发性脂肪酸的趋势,可能是GSPs对纤维分解菌有一定的抑制作用所致。杜宇等^[10]的研究中石榴多酚对乙酸和丙酸均有显著的抑制作用,而本试验中丙酸浓度的降低没有达到显著水平,可能是添加剂量不同所致。前人研究也表明,单宁对瘤胃中溶纤维丁酸弧菌有显著的抑制作用^[25],而乙酸和丁酸是溶纤维丁酸弧菌的主要代谢物,因此添加GSPs后瘤胃液中丁酸浓度有所下降。GETACHEW等^[26]报道,多酚可能通过抑制微生物的氧化磷酸化而影响微生物代谢,或与微生物外源酶或底物结合而抑制底物降解,进而抑制微生物发酵产酸。

高精料引起瘤胃和肠道后段pH下降,革兰氏阴性菌崩解死亡释放出大量的LPS,LPS的积累导致瘤胃上皮和肠道黏膜屏障损伤,LPS通过损伤的屏障易位进入循环系统,从而启动机体的免疫应答,激活巨噬细胞和白细胞产生一系列细胞因子,如TNF-α、IL-6^[27]。本研究中添加GSPs后瘤胃液LPS浓度在数值上低于对照组,但统计学上没有显著差异。原因可能是GSPs对瘤胃pH的轻微调控作用尚且没有缓解不耐酸革兰氏阴性菌的死亡。但添加GSPs后血清LPS浓度显著降低,说明GSPs提高了肝脏清除LPS的能力。众多研究表明原花青素可以缓解多种因素导致的肝损伤,提高肝功能^[28,29]。本团队将进一步深入研究高精料饲粮中添加GSPs对羔羊肝功能的影响。

高精料可能导致反刍动物体内氧化和抗氧化作用失衡,使机体处于氧化应激状态^[1,30]。机体内氧化应激和炎症的发生往往是伴随发生,且促炎细胞因子的过表达正反馈促进氧化应激^[31]。植物多酚能够有效清除机体内多余的自由基,在抗氧化和抗炎方面有重要作用。DESCALZO等^[32]报道,新鲜牧草中植物多酚含量较高,因此放牧牛体内抗氧化物质含量较舍饲牛更高,机体抗氧化能力、免疫力更强。GSPs具有很强的抗氧化和抗炎能力,是一种良好的外源性抗氧化剂。宿孝奇^[33]研究表明GSPs可以显著提高泌乳早期奶牛机体抗氧化能力,降低血液中氧化代谢产物和酮体的含量。LI等^[34]研究表明GSPs可以有效缓解由三硝基苯磺酸诱导产生的小鼠急性结肠炎,主要是通过下调某些炎症介质而起抗炎作用,能够有效抑制炎性细胞浸润,抑制机体的氧化损伤,减少促炎细胞因子产生,同时通过提高氧化损伤组织的修复能力,增加抗炎细胞因子分泌而发挥作用。GSPs能够调节仔猪肠道黏膜NF-κB和Nrf2的活性,抑制仔猪肠道炎症^[35],提高母猪肠道有益菌比例^[36],抑制促炎因子TNF-α、IL-1β等的释放,有效抑制机体炎症持续发生^[37]。DE NARDI等^[24]研究发现植物多酚和精油混合物通过显著降低高谷物饲粮条件下母牛血清淀粉样蛋白A和LPS结合蛋白水平,缓解机体炎症反应,但对血清IL-6含量无显著影响。本试验中,添加20和40 mg·kg^-1 BW的GSPs显著提高了瘤胃组织和血清抗氧化酶活性,降低了MDA含量,同时降低了血清炎症因子TNF-α和IL-10的水平,对瘤胃组织也有类似的作用趋势,说明添加适量GSPs后羔羊机体抗氧化能力有所提高,对高精料所致的瘤胃及全身炎症也有缓解的潜力。

4 结论

高精料饲粮条件下,饲喂适量葡萄籽原花青素可以提高羔羊瘤胃pH,提高血清和瘤胃组织抗氧化能力,降低血清促炎因子水平,提高羔羊采食量和日增重,对羔羊健康具有潜在的保护效应。在本试验条件下,葡萄籽原花青素的最佳饲喂量为20 mg·kg^-1 BW。

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