动物营养学报    2020, Vol. 32 Issue (9): 4177-4184    PDF    
丁酸钠与丝兰对大肠杆菌K99攻毒哺乳犊牛生长性能及血清抗氧化指标的影响
刘文慧1 , 阿拉腾珠拉1 , 马露1,2 , 任丽娟1 , 高胜涛1 , 卜登攀1,2     
1. 中国农业科学院北京畜牧兽医研究所, 动物营养学国家重点实验室, 北京 100193;
2. 中国农业科学院与世界农用林业中心农用林业与可持续畜牧业联合实验室, 北京 100193
摘要: 本试验旨在研究丁酸钠与丝兰对大肠杆菌K99攻毒哺乳犊牛生长性能及血清抗氧化指标的影响。试验选用40头健康、体重[(40±5)kg]相近的1日龄荷斯坦公犊牛,随机分为4组,每组10头。对照组(C组)饲喂基础饲粮,丁酸钠组(SB组)饲喂基础饲粮+30 g/d丁酸钠;丝兰组(Y组)饲喂基础饲粮+9 g/d丝兰,丁酸钠+丝兰组(SB+Y组)饲喂基础饲粮+30 g/d丁酸钠+9 g/d丝兰。在犊牛7日龄时进行大肠杆菌K99攻毒,试验进行至14日龄结束。结果表明:1)1~7日龄时,与SB组相比,SB+Y组犊牛平均日采食量显著降低(P < 0.05)。2)11日龄时,SB组犊牛血清中过氧化氢酶(CAT)活性显著高于C组(P < 0.05);14日龄时,SB组和Y组犊牛血清中CAT活性显著高于C组和SB+Y组(P < 0.05)。11日龄时,与C组相比,Y组犊牛血清中谷胱甘肽过氧化物酶(GSH-Px)活性有升高的趋势(P=0.088),SB+Y组犊牛血清中GSH-Px活性显著提高(P < 0.05)。7日龄时,与C组相比,Y组犊牛血清中丙二醛(MDA)含量有降低的趋势(P=0.050);14日龄时,与C组相比,SB+Y组犊牛血清中MDA含量有降低的趋势(P=0.089)。综上所述,哺乳犊牛处于大肠杆菌K99攻毒的应激模式下,丁酸钠与丝兰单独添加均可提高犊牛的抗氧化能力,但对犊牛的生长性能没有影响。丁酸钠与丝兰组合添加可提高犊牛的抗氧化能力,但降低了犊牛的平均日采食量。
关键词: 哺乳犊牛    丁酸钠    丝兰    生长性能    抗氧化    
Effects of Sodium Butyrate and Yucca on Growth Performance and Serum Antioxidant Indices of Sucking Calves Challenged with Escherichia coli K99
LIU Wenhui1 , Alatengzhula1 , MA Lu1,2 , REN Lijuan1 , GAO Shengtao1 , BU Dengpan1,2     
1. Institute of Animal Science, State Key Laboratory of Animal Nutrition, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
2. Chinese Academy of Agricultural Sciences and World Agroforestry Center Joint Lab on Agroforestry and Sustainable Animal Husbandry, Beijing 100193, China
Abstract: This experiment was conducted to evaluate effects of sodium butyrate and Yucca on growth performance and serum antioxidant indices of sucking calves challenged with Escherichia coli K99. A total of 40 healthy Holstein calves with similar body weight of (40±5) kg were randomly allocated to 4 groups with 10 calves per group. The control group (C group) was fed a basic diet, the sodium butyrate group (SB group) was fed the basic diet+30 g/d sodium butyrate, the Yucca group (Y group) was fed the basic diet+9 g/d Yucca, and the sodium butyrate+Yucca group (SB+Y group) was fed the basic diet+30 g/d sodium butyrate+9 g/d Yucca. The calves were challenged with Escherichia coli K99 at 7 days of age, and the experiment ended at 14 days of age. The results showed as follows:1) during 1 to 7 days of age, compared with the SB group, the average daily feed intake of calves of SB+Y group was significantly decreased (P < 0.05). 2) At 11 days of age, the serum catalase (CAT) activity of calves of SB group was significantly higher than that of C group (P < 0.05); at 14 days of age, the serum CAT activity of calves of SB group and Y group was significantly higher than that of C group and SB+Y group (P < 0.05). At 11 days of age, compared with C group, the serum glutathione peroxidase (GSH-Px) activity of calves of Y group had a tendency to increase (P=0.088), and the serum GSH-Px activity of calves of SB+Y group was significantly increased (P < 0.05). At 7 days of age, compared with C group, the serum malondialdehyde (MDA) content of calves of Y group had a tendency to decrease (P=0.050); at 7 days of age, compared with C group, the serum MDA content of calves of SB+Y group had a tendency to decrease (P=0.089). In conclusion, when sucking calves are under stress condition challenged with Escherichia coli K99, the separately supplementation of sodium butyrate and Yucca can improve the antioxidant capacity of calves, but it has no effect on the growth performance of calves. The combined supplementation of sodium butyrate and Yucca can improve the antioxidant capacity of calves, but reduce the average daily feed intake of calves.
Key words: sucking calves    sodium butyrate    Yucca    growth performance    antioxidant    

犊牛在刚出生时,肠道发育尚未完全,此阶段犊牛极易受到外界病原体的侵害,造成腹泻的发生,严重时导致死亡,给养殖业造成了巨大的损失[1]。大肠杆菌K99是造成新生犊牛腹泻的主要细菌之一[2-4]。在实际生产中,对于细菌导致的犊牛腹泻,养殖户通常使用抗生素来治疗,但因抗生素药品残留问题,对牧场用药治疗造成了巨大的局限性。因此,研究如何利用绿色有机的方法预防及治疗哺乳犊牛腹泻成为广大学者研究的焦点。

丁酸钠(sodium butyrate)以无耐药性、无残留和无污染等特点被作为一种饲料添加剂,在动物饲料行业以及促进动物健康方面受到越来越多的关注[5-6]。丁酸钠作为一种生物调节剂,其有效成分丁酸是一种短链挥发性性脂肪酸,由厌氧细菌发酵未消化吸收的碳水化合物和纤维多糖产生[7]。已有研究表明,丁酸钠可促进犊牛生长,提高消化酶活性,促进小肠对营养物质的消化吸收[8-9],可为肠上皮细胞提供能量[10],调节炎症反应,提高机体抗氧化及免疫性能[11],维持肠道屏障完整性[12]。Gálfi等[13]研究表明,仔猪饲粮中添加0.17%的丁酸钠可增加仔猪采食量、提高日增重以及降低仔猪回肠大肠杆菌数量。Hill等[14]研究表明,犊牛饲粮中添加3%的丁酸钠可显著提高犊牛的体重,并降低腹泻率。Xiong等[15]研究表明,仔猪饲粮中添加0.2%的丁酸钠可缓解大肠杆菌O157:H7引起的肠道炎症反应。

丝兰(Yucca)作为一种纯天然多功能植物添加剂,已在动物生产中得到广泛应用,为生产者带来了良好的经济效益,其主要活性成分有甾类皂苷、多酚及多糖[16]。丝兰的成分丝兰皂苷具有广谱抗菌的作用,对大肠杆菌、痢疾杆菌及伤寒杆菌等具有较强的抑制作用[17]。研究表明,多酚具有抗炎、抗氧化和提高机体免疫力等生理功能,并能够促进动物生长发育[18]。田丽新等[19]研究发现,饲粮中添加丝兰提取物可显著提高奶牛免疫性能及抗氧化能力。De Sousa等[20]研究表明,断奶犊牛饲粮中添加2 g/d的丝兰提取物可提高犊牛的生长性能。Abdel-Raheem等[21]在断奶犊牛饲粮中添加2 g/kg的丝兰提取物,可改善犊牛的瘤胃发酵功能,提高生长性能。目前,丁酸钠在犊牛上的研究多集中在生长性能方面,对于犊牛的抗菌效果及抗氧化能力的影响研究较少,且丝兰对哺乳犊牛抗氧化功能的影响以及丁酸钠与丝兰的组合对犊牛的应用效果尚未见报道。因此,本研究利用大肠杆菌K99对哺乳犊牛进行攻毒,探究饲粮中添加丁酸钠、丝兰及二者组合对哺乳犊牛生长性能及血清抗氧化指标的影响。

1 材料与方法 1.1 试验材料

丁酸钠,白色结晶粉末,纯度为98.5%,易溶于水。丝兰,取自生长于美国西南部及墨西哥北部半沙漠地区的丝兰类植物麟凤兰,纯植物粉末状,食品级。

1.2 试验动物管理

犊牛出生后2 h灌服初乳4 L,出生后6 h灌服初乳2 L。从2日龄开始饲喂常乳,每天饲喂2次(07:00和15:00)。饲喂量:2~7日龄,2.5 L/次;8~14日龄,4.5 L/次。犊牛开食料从3日龄时开始饲喂,自由采食。每头犊牛单独饲养于1.5 m×3.0 m犊牛笼内。初乳、常乳的密度及营养水平见表 1,开食料的营养水平见表 2

表 1 初乳、常乳的密度及营养水平 Table 1 Density and nutrient levels of colostrum and milk
表 2 开食料的营养水平(干物质基础) Table 2 Nutrient levels of starter (DM basis) 
1.3 试验设计

试验采用完全随机试验设计,共选取40头出生健康、体重[(40±5) kg]相近的1日龄中国荷斯坦公犊牛,随机分为4组,每组10头。对照组(C组)饲喂基础饲粮,无添加剂;丁酸钠组(SB组)饲喂基础饲粮+30 g/d丁酸钠;丝兰组(Y组)饲喂基础饲粮+9 g/d丝兰;丁酸钠+丝兰组(SB+Y组)饲喂基础饲粮+30 g/d丁酸钠+9 g/d丝兰。于犊牛2日龄时将丁酸钠和丝兰混合在牛奶中进行饲喂。试验进行至14日龄时结束。在犊牛7日龄晨饲前,给每头犊牛经口灌服大肠杆菌K99(菌株号:CVCC 237;血清型:O101:K99),大肠杆菌K99由中国兽医药品监察所提供。参照Pal等[23]和Besser等[24]的研究,本试验攻毒大肠杆菌的剂量是取1 g含量为1×1011 CFU/g的大肠杆菌K99冻干粉溶解于10 mL生理盐水中,每头犊牛灌服10 mL。

1.4 样品采集与指标测定 1.4.1 初乳、常乳及开食料

给犊牛第1次饲喂初乳时采集初乳样品,每周采集常乳和开食料样品,于-20 ℃保存。采用乳成分分析仪(FOSS FT120)测定初乳及常乳的干物质(DM)、乳蛋白、乳脂肪、乳糖、总固形物含量;并参照AOAC(2000)[25]的方法测定开食料干物质、粗蛋白质(CP)、粗脂肪(EE)和粗灰分(Ash)含量;参照Van Soest等[26]的方法测定开食料中性洗涤纤维(NDF)和酸性洗涤纤维(ADF)含量,钙(Ca)和磷(P)含量采用电感耦合等离子体发射光谱仪(ICP-OES 9000,Shimadzu公司,日本)测定。其中,CP含量采用K1100全自动凯氏定氮仪测定,NDF和ADF含量采用Y078自动纤维测定仪(A2000i)测定,EE含量采用SoxtecTM 2050全自动脂肪分析仪测定。

1.4.2 生长性能

于7、11及14日龄晨饲前使用精确度0.5 kg、量程200 kg的电子秤对每头犊牛进行称重。每天记录每头犊牛当日的开食料投喂量和次日同一时间投喂时剩料量,以此计算犊牛的平均日采食量及料重比,计算方法如下:

1.4.3 血清抗氧化指标

于7、11和14日龄午饲后2 h,通过颈静脉法采集血液20 mL,静置至分层(约30 min),于4 ℃、1 240×g离心15 min,取上清液(血清)分装至5个2 mL离心管中,-20 ℃保存待测。血清中超氧化物歧化酶(SOD)活性采用黄嘌呤氧化酶法测定,谷胱甘肽过氧化物酶(GSH-Px)活性采用比色法测定,丙二醛(MDA)含量采用硫代巴比妥(TBA)法测定,过氧化氢酶(CAT)活性采用钼酸铵法测定,所采用的测定仪器为Y040多功能酶标仪(Tecan Infinite 200 Pro,Tecan国际有限公司)。试剂盒均购自于南京建成生物工程研究所,并严格按照试剂盒说明书上的操作步骤进行操作。

1.4.4 粪便评分

于每日07:00观察试验犊牛粪便进行评分,并记录。粪便评分标准按表 3执行[14],采用4分制粪便评分法,以评分≥3记为1次腹泻。

表 3 粪便评分标准 Table 3 Fecal consistency score standard
1.5 数据统计分析

试验数据采用SAS 9.4软件MIXED模型进行统计分析,统计模型中包括试验犊牛的随机因素以及试验处理及交互作用的固定因素。采用最小二乘均值法(least squares means,LSMEANS)进行显著性比较,P < .05为差异显著,0.05≤P < 0.10为有差异的趋势。

2 结果 2.1 丁酸钠与丝兰对哺乳犊牛生长性能及粪便评分的影响

表 4可知,饲粮添加丁酸钠与丝兰对大肠杆菌K99攻毒前(1~7日龄)、攻毒后(8~14日龄)犊牛日增重、料重比及粪便评分无显著影响(P>0.05)。1~7日龄时,SB+Y组犊牛平均日采食量显著降低(P < 0.05),且SB+Y组犊牛粪便评分有增加的趋势(P=0.055)。

表 4 丁酸钠与丝兰对哺乳犊牛生长性能及粪便评分的影响 Table 4 Effects of sodium butyrate and Yucca on growth performance and fecal score of sucking calves
2.2 丁酸钠与丝兰对哺乳犊牛血清抗氧化指标的影响

表 5可知,7日龄时,与C组相比,Y组犊牛血清中CAT活性有升高的趋势(P=0.062);11日龄时,SB组犊牛血清中CAT活性显著高于C组(P < 0.05);14日龄时,SB组和Y组犊牛血清中CAT活性显著高于C组和SB+Y组(P < 0.05)。11日龄时,与C组相比,Y组犊牛血清中GSH-Px活性有升高的趋势(P=0.088),SB+Y组犊牛血清中GSH-Px活性显著提高(P < 0.05)。7日龄时,与C组相比,Y组犊牛血清中MDA含量有降低的趋势(P=0.050);14日龄时,与C组相比,SB+Y组犊牛血清中MDA含量有降低的趋势(P=0.089)。

表 5 丁酸钠与丝兰对哺乳犊牛血清抗氧化指标的影响 Table 5 Effects of sodium butyrate and Yucca on serum antioxidant indices of sucking calves
3 讨论 3.1 丁酸钠与丝兰对哺乳犊牛生长性能及健康状况的影响

犊牛幼龄时期的生长状况与其后期的生产水平直接相关,可通过营养性添加剂改善犊牛的生长状况,提高其成年时期的生产性能[27]。已有研究表明,丁酸钠对犊牛的生长性能具有积极的影响[28-30],而本研究结果表明,丁酸钠对攻毒前、攻毒后犊牛的平均日增重、平均日采食量及料重比没有显著影响,这与Gorka等[31]和Koch等[32]的研究结果一致;丝兰对攻毒前、攻毒后犊牛的生长性能也没有显著影响。这可能由于试验期较短,犊牛哺乳时期主要的营养来源是牛奶,此阶段给犊牛提供的牛奶是定量的,且开食料采食量较低,因此,短时间内饲喂丁酸钠与丝兰并不能影响犊牛的采食量及犊牛的生长。然而丁酸钠与丝兰的组合显著影响了攻毒前犊牛的平均日采食量,这可能是由于丁酸钠与丝兰的组合使犊牛腹泻增加,导致犊牛食欲不振,进而降低犊牛采食量。

腹泻是犊牛时期最常见的健康问题之一,不仅影响其生长性能,严重时将造成死亡。肠道分泌增加或液体和电解质的损失可被视为腹泻产生的最终途径,并且许多因素在腹泻的发展中以微妙的方式相互作用[33]。本试验中粪便评分结果显示,攻毒大肠杆菌K99后犊牛腹泻率从30%增加到70%,且其他组的腹泻率也有增加的现象,这表明利用大肠杆菌K99攻毒使哺乳犊牛腹泻是有效的,这与前人的研究结果[34]相似。本试验结果表示,丁酸钠与丝兰的单独添加并没有显著降低犊牛粪便评分,但有缓解的作用。这可能是由于丁酸根阴离子能抑制肠道巨噬细胞组蛋白去乙酰化酶活性,上调巨噬细胞内源性抗菌肽的活性表达,增强巨噬细胞对大肠杆菌的杀菌能力[35]。有研究表明,在仔猪饲粮中添加丁酸可明显降低回肠大肠菌群的数量,同时提高乳酸杆菌的数量,提高乳酸杆菌与大肠杆菌数量的比值[13]。丝兰的活性成分皂苷对大肠杆菌具有抑制作用[36],因此,丁酸钠与丝兰的单独添加可降低腹泻的发生。而丁酸钠与丝兰的组合添加增加了犊牛腹泻的发生,这可能是由于丁酸钠与丝兰组合后产生某种物质致使腹泻增加,具体机制需要进一步的研究。

3.2 丁酸钠与丝兰对哺乳犊牛血清抗氧化指标的影响

抗氧化酶是动物抗氧化防御系统中必不可少的关键因子,主要的抗氧化酶包括SOD、GSH-Px及CAT等[37]。本试验结果表明,丁酸钠的添加显著提高了血清中CAT活性,CAT可参与过氧化氢(H2O2)的降解,最终转化为水[38]。因此,这可能是由于丁酸钠降低H2O2诱导的DNA损伤,增加CAT基因的表达[39],进而提高了CAT活性。GSH-Px可防止磷脂膜、酶和自由基的损伤[40]。MDA是脂质过氧化产物,其含量可间接反映氧自由基对细胞的损伤程度,氧自由基和CAT的稳态水平对于机体的健康至关重要[41]。Cigerci等[42]在模拟瘤胃发酵液中添加丝兰提取物降低了小鼠血清MDA含量,同时增加了血清GSH-Px活性,提高了小鼠的抗氧化能力,与本研究结果相似。这可能是丝兰的主要活性成分中多酚的作用,多酚的结构特点使其具有较强的抗氧化及清除自由基的能力[18]。丁酸钠与丝兰的组合显著影响了11日龄犊牛血清中GSH-Px活性,表明丁酸钠与丝兰的组合对哺乳犊牛抗氧化能力具有积极的作用。本试验中,与7和14日龄相比,11日龄时犊牛血清中GSH-Px活性均下降,这可能由于攻毒大肠杆菌K99后,11日龄犊牛腹泻增加,腹泻导致动物机体血清中GSH-Px活性显著降低[43-44]

4 结论

哺乳犊牛处于大肠杆菌K99攻毒的应激模式下,丁酸钠与丝兰单独添加均可提高犊牛的抗氧化能力,降低腹泻,但对生长性能没有影响;丁酸钠与丝兰的组合添加可提高犊牛的抗氧化能力,但降低了平均日采食量,增加了犊牛腹泻。

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