动物营养学报    2021, Vol. 33 Issue (2): 760-768    PDF    
菊粉对断奶仔猪生长性能、养分表观消化率及抗氧化能力的影响
王维康 , 陈代文 , 余冰 , 黄志清 , 毛湘冰 , 郑萍 , 虞洁 , 罗钧秋 , 罗玉衡 , 阎辉 , 何军     
四川农业大学动物营养研究所, 动物抗病营养教育部重点实验室, 动物抗病营养四川省重点实验室, 成都 611130
摘要: 本试验旨在研究饲粮添加不同水平菊粉对断奶仔猪生长性能、养分表观消化率及抗氧化能力的影响。选取32头平均体重为(7.10±0.20) kg的杜长大(DLY)断奶仔猪,随机分为4组,每组8个重复,每个重复1头猪。对照组饲喂基础饲粮,试验组分别在基础饲粮中添加2.5、5.0和10.0 g/kg的菊粉(等量替代基础饲粮中的玉米)。试验期21 d。结果表明,与对照组相比:1)饲粮添加2.5、5.0和10.0 g/kg菊粉对断奶仔猪平均日采食量和平均日增重无显著影响(P>0.05),饲粮添加2.5、5.0和10.0 g/kg菊粉可显著提高断奶仔猪粗灰分表观消化率(P < 0.05),饲粮添加2.5和5.0 g/kg菊粉可显著提高断奶仔猪粗蛋白质表观消化率(P < 0.05)。2)饲粮添加2.5 g/kg菊粉可显著提高血清中总超氧化物歧化酶(T-SOD)活性(P < 0.05);饲粮添加2.5、5.0和10.0 g/kg菊粉可显著降低肝脏中丙二醛(MDA)含量(P < 0.05);饲粮添加2.5和5.0 g/kg菊粉可显著提高空肠中过氧化氢酶(CAT)活性(P < 0.05),饲粮添加2.5和5.0 g/kg菊粉可显著降低回肠中MDA含量(P < 0.05),饲粮添加2.5 g/kg菊粉可显著提高十二指肠中CAT活性(P < 0.05)。3)饲粮添加2.5、5.0和10.0 g/kg菊粉可显著提高肝脏中核因子E2相关因子2(Nrf2)的mRNA相对表达量(P < 0.05),饲粮添加5.0和10.0 g/kg菊粉可显著提高肝脏中血红素氧合酶-1(HO-1)的mRNA相对表达量(P < 0.05)。4)饲粮添加2.5、5.0和10.0 g/kg菊粉可显著提高十二指肠中HO-1的mRNA相对表达量和空肠中Nrf2的mRNA相对表达量(P < 0.05),饲粮添加2.5和5.0 g/kg菊粉可显著提高回肠中HO-1和Nrf2的mRNA相对表达量(P < 0.05),饲粮添加2.5 g/kg菊粉可显著提高空肠中Kelch样环氧氯丙烷相关蛋白1(Keap1)的mRNA相对表达量(P < 0.05),饲粮添加10.0 g/kg菊粉可显著提高空肠中HO-1和Keap1的mRNA相对表达量(P < 0.05)。综上所述,饲粮添加菊粉对断奶仔猪生长性能无显著影响,但可改善养分表观消化率,提高抗氧化能力。本试验条件下,断奶仔猪饲粮中菊粉适宜添加水平为2.5 g/kg。
关键词: 菊粉    断奶仔猪    养分表观消化率    抗氧化    
Effects of Inulin on Growth Performance, Nutrient Apparent Digestibilities and Antioxidant Capacity of Weaned Piglets
WANG Weikang , CHEN Daiwen , YU Bing , HUANG Zhiqing , MAO Xiangbing , ZHENG Ping , YU Jie , LUO Junqiu , LUO Yuheng , YAN Hui , HE Jun     
Key Laboratory of Animal Disease-Resistance and Nutrition of Sichuan Province, Key Laboratory of Animal Disease-Resistance and Nutrition of Ministry of Education, College of Institute of Animal Nutrition, Sichuan Agricultural University, Chengdu 611130, China
Abstract: This experiment was conducted to investigate the effects of inulin on growth performance, nutrient apparent digestibilities and antioxidant capacity of weaned piglets. Thirty-two Duroc×Landrace×Yorkshire (DLY) weaned piglets with an average weight of (7.10±0.20) kg were randomly divided into 4 groups with 8 replicates in each group and 1 pig in each replicate. Pigs in the control group were fed a basal diet, and others in experimental groups were fed basal diets supplemented with 2.5, 5.0 and 10.0 g/kg inulin (equal replaced the corn in basal diet), respectively. The experiment lasted for 21 days. The results showed that compared with the control group: 1) dietary 2.5, 5.0 and 10.0 g/kg inulin had no significant effects on the average daily feed intake and average daily gain of weaned piglets (P>0.05), dietary 2.5, 5.0 and 10.0 g/kg inulin significantly increased the ash apparent digestibility of weaned piglets (P < 0.05), and dietary 2.5 and 5.0 g/kg inulin significantly increased the crude protein apparent digestibility of weaned piglets (P < 0.05). 2) Dietary 2.5 g/kg inulin significantly increased the serum total superoxide dismutase (T-SOD) activity (P < 0.05); dietary 2.5, 5.0 and 10.0 g/kg inulin significantly decreased the liver malondialdehyde (MDA) content (P < 0.05); dietary 2.5 and 5.0 g/kg inulin significantly increased the jejunum catalase (CAT) activity (P < 0.05), dietary 2.5 and 5.0 g/kg inulin significantly decreased the ileum MDA content (P < 0.05), and dietary 2.5 g/kg inulin significantly increased the duodenum CAT activity (P < 0.05). 3) Dietary 2.5, 5.0 and 10.0 g/kg inulin significantly increased the mRNA relative expression level of nuclear factor-E2-related factor 2 (Nrf2) in liver (P < 0.05), and dietary 5.0 and 10.0 g/kg inulin significantly increased the mRNA relative expression level of heme oxygenase-1 (HO-1) in liver (P < 0.05). 4) Dietary 2.5, 5.0 and 10.0 g/kg inulin significantly increased the mRNA relative expression level of HO-1 in duodenum and mRNA relative expression level of Nrf2 in jejunum (P < 0.05), dietary 2.5 and 5.0 g/kg inulin significantly increased the mRNA relative expression level of HO-1 and Nrf2 in ileum (P < 0.05), dietary 2.5 g/kg inulin significantly increased the mRNA relative expression level of Kelch-like epichlorohydrin-associated protein 1 (Keap1) in jejunum (P < 0.05), and dietary 10.0 g/kg inulin significantly increased the mRNA relative expression level of HO-1 and Keap1 in jejunum (P < 0.05). In summary, dietary inulin has no significant effect on growth performance, but can improve nutrient apparent digestibilities and antioxidant capacity of weaned piglets. Under the condition of this experiment, the dietary suitable supplemental level of inulin for weaned piglets is 2.5 g/kg.
Key words: inulin    weaned piglets    nutrient apparent digestibilities    antioxidant    

仔猪断奶后消化系统和免疫器官尚没有发育完全,加之断奶过程中环境改变和饲粮摄入改变所带来的应激,断奶导致仔猪对养分消化吸收下降、抗病能力下降、腹泻率增加,最终导致仔猪产生“断奶仔猪应激综合征”[1-2]。饲粮纤维在小肠内不被消化吸收,但可在大肠被发酵,可降低血糖、血脂,预防高血压、高血脂、高血糖及结肠癌等疾病。随着对饲粮纤维结构和功能的深入研究,发现饲粮纤维可改善肠道微生物群落结构及其代谢产物,维持肠道内环境稳态,从而促进营养物质的吸收和改善肠道健康[3-4]。菊粉是一种绿色天然的膳食纤维。菊粉的聚合度一般在2~60,平均聚合度为10[5]。大量水果蔬菜中可以分离出菊粉,如香蕉、芦笋、韭菜和洋葱。目前工业生产菊粉大多数是从菊苣和菊芋中加工提取[6]。作为一种膳食纤维,菊粉在哺乳动物小肠内不能被消化吸收,但在后肠可以被微生物发酵分解[7]。研究表明,肠道细菌发酵膳食纤维会产生大量挥发性脂肪酸(VFA),如乙酸、丙酸和丁酸,可作为肠上皮细胞的能量来源,并可预防各种炎症[8-9]。此外,菊粉可通过增加有益菌群的数量和代谢活性来改善断奶仔猪肠道功能和胃肠道环境[10]。然而到目前为止,菊粉对畜禽抗氧化的研究应用效果还鲜有报道。本试验通过在断奶仔猪基础饲粮中添加不同水平的菊粉,研究菊粉对断奶仔猪生长性能、养分消化率和抗氧化能力的影响,以期为断奶仔猪中菊粉的应用提供理论依据。

1 材料与方法 1.1 试验设计

试验采用单因子设计,选取32头平均体重为(7.10±0.20) kg的杜长大(DLY)断奶仔猪,随机分为4组,每组8个重复,每个重复1头猪。对照组饲喂基础饲粮,试验组分别在基础饲粮中添加2.5、5.0和10.0 g/kg的菊粉(等量替代基础饲粮中的玉米)。试验期21 d。

1.2 基础饲粮

玉米-豆粕型基础饲粮参照NRC(2012)中7~11 kg断奶仔猪的营养需要进行配制,基础饲粮组成及营养水平见表 1

表 1 基础饲粮组成及营养水平(风干基础) Table 1 Composition and nutrient levels of the basal diet (air-dry basis) 
1.3 饲养管理

本试验在四川农业大学动物营养研究所试验场进行。试验使用的圈舍和代谢笼多次冲洗后,用消毒剂消毒,24 h后再次冲洗,然后用2 : 1比例的甲醛与高锰酸钾熏蒸圈室,熏蒸持续24 h,全程门窗密闭,结束后通风3 d。试验期间严格按照断奶仔猪饲养管理饲养,每日饲喂3次(08:00、14:00和20:00),每次喂食遵循多次少量原则,自由饮水。圈舍卫生每日清扫,每周消毒。试验期间早晚观察仔猪精神状态,并记录每日采食量与腹泻情况。

1.4 样品采集 1.4.1 饲粮与粪便样品收集

采用四分法收集饲粮样品,每个组取500 g左右,装入样品袋,记号,放入-20 ℃冰箱保存,用于常规养分的分析。在试验第18~21天对所有仔猪采用内源指示剂法进行消化试验,采用部分收粪法收集粪便。

1.4.2 血清样品收集

于试验开始后第21天早上,对空腹仔猪前腔静脉采血15 mL,置于普通离心管,室温下放置30 min后,4 ℃、3 000×g离心10 min,分离血清,置于-20 ℃保存,待测血清抗氧化指标。

1.4.3 组织样品收集

于试验第21天早上仔猪采血后,立即进行屠宰,取出肝脏、小肠等内脏。采集多份肝脏样品于冻存管中,用锡箔纸包严放入液氮,然后转至-80 ℃冻存。使用生理盐水对小肠段进行冲洗,待洗净残留在肠段上的食糜后,用载玻片将十二肠、空肠、回肠黏膜刮下来,然后置于无菌EP管中,迅速用锡箔纸包严放入液氮,然后转至-80 ℃冻存。

1.5 指标测定 1.5.1 生长性能测定

在试验第1天和第21天08:00对仔猪进行空腹称重,统计仔猪每日采食量,分别计算平均日采食量(ADFI)和平均日增重(ADG)。

1.5.2 养分表观消化率测定

消化试验采用内源指示剂法,利用盐酸不溶灰分(AIA)为指示剂。饲粮养分表观消化率计算公式为:

式中:F1是饲粮中该养分含量(%);F2是粪中该养分含量(%);A1是饲粮中AIA含量(%);A2是粪中AIA含量(%)。

1.5.3 血清、肝脏和肠道抗氧化指标测定

使用南京建成生物工程研究所试剂盒测定仔猪血清、肝脏和肠道中总抗氧化能力(T-AOC)和谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)、总超氧化物歧化酶(T-SOD)活性及丙二醛(MDA)含量,严格按照说明书操作。

1.5.4 肝脏和肠道抗氧化相关基因表达测定

总RNA的提取:按照RNA提取试剂盒(Trizol Reagent TaKaRa,日本)严格操作,得到总RNA后,在核酸蛋白仪上取RNA样品测出相应的吸光度(OD)260 nm和OD280 nm,样品检测数值处在1.8≤OD260 nm/OD280 nm≤2.0,用1%琼脂糖凝胶电泳测定RNA的质量和完整性。

反转录:利用反转录得到cDNA,所有步骤严格按照反转录试剂盒[TaKaRa,宝生物工程(大连)有限公司]说明书进行。反转录所用仪器是PCR仪(Bio-Rad DNA Engine,美国),反应共有2步,分别为:反转录反应37 ℃,15 min;反转录酶失活反应85 ℃,5 s。

引物设计:肝脏和肠道抗氧化相关基因[核因子E2相关因子2(nuclear factor-E2-related factor 2,Nrf2)、Kelch样环氧氯丙烷相关蛋白1(Kelch-like epichlorohydrin-associated protein 1,Keap1)和血红素氧合酶-1(heme oxygenase-1,HO-1)]引物序列见表 2

表 2 肝脏和肠道抗氧化相关基因引物序列 Table 2 Primer sequences of antioxidant related genes in liver and intestine

实时荧光定量PCR:使用染料为SYBR Green PCR Mix[TaKaRa,宝生物工程(大连)有限公司],使用仪器是实时荧光定量PCR仪(CFX-96 Real-Time PCR System,Bio-Rad公司,美国)。以β-肌动蛋白(β-actin)为内参基因。用2-ΔΔCt[11]计算目的基因的mRNA相对表达量。

1.6 数据统计及分析

所有试验数据经Excel 2016初步分析后,使用SPSS 22.0统计软件对4组的试验数据进行单因素方差分析,差异显著时进行Duncan氏多重比较,所有试验数据均以“平均值±标准误”表示,P<0.05为差异显著,0.05≤P≤0.10为有趋势。

2 结果与分析 2.1 菊粉对断奶仔猪生长性能的影响

表 3可见,与对照组相比,饲粮添加2.5、5.0和10.0 g/kg菊粉对断奶仔猪ADFI和ADG无显著影响(P>0.05),但饲粮添加2.5 g/kg菊粉后断奶仔猪ADFI和ADG分别提高了12.2%和20.1%。

表 3 菊粉对断奶仔猪生长性能的影响 Table 3 Effects of inulin on growth performance of weaned piglets  
2.2 菊粉对断奶仔猪养分表观消化率的影响

表 4可见,与对照组相比,饲粮添加2.5、5.0和10.0 g/kg菊粉可显著提高断奶仔猪粗灰分表观消化率(P < 0.05),饲粮添加2.5和5.0 g/kg菊粉可显著提高断奶仔猪粗蛋白质表观消化率(P < 0.05),饲粮添加2.5、5.0和10.0 g/kg菊粉对断奶仔猪干物质、粗脂肪和能量表观消化率无显著影响(P>0.05)。

表 4 菊粉对断奶仔猪养分表观消化率的影响 Table 4 Effects of inulin on nutrient apparent digestibilities of weaned piglets 
2.3 菊粉对断奶仔猪血清、肝脏和肠道抗氧化指标的影响

表 5可见,与对照组相比,饲粮添加2.5 g/kg菊粉可显著提高血清中T-SOD活性(P < 0.05),但饲粮添加2.5、5.0和10.0 g/kg菊粉对血清中CAT和GSH-Px活性、T-AOC及MDA含量无显著影响(P>0.05);饲粮添加2.5、5.0和10.0 g/kg菊粉可显著降低肝脏中MDA含量(P < 0.05),但饲粮添加2.5、5.0和10.0 g/kg菊粉对肝脏中T-AOC及CAT、T-SOD和GSH-Px活性无显著影响(P>0.05);饲粮添加2.5和5.0 g/kg菊粉可显著提高空肠中CAT活性(P < 0.05),饲粮添加2.5和5.0 g/kg菊粉可显著降低回肠中MDA含量(P < 0.05),饲粮添加2.5 g/kg菊粉可显著提高十二指肠中CAT活性(P < 0.05),但饲粮添加2.5、5.0和10.0 g/kg菊粉对十二指肠、空肠和回肠中T-AOC及T-SOD和GSH-Px活性无显著影响(P>0.05)。

表 5 菊粉对断奶仔猪血清、肝脏和肠道抗氧化指标的影响 Table 5 Effects of inulin on serum, liver and intestine antioxidant indexes of weaned piglets
2.4 菊粉对断奶仔猪肝脏抗氧化相关基因表达的影响

表 6可见,与对照组相比,饲粮添加2.5、5.0和10.0 g/kg菊粉可显著提高肝脏中Nrf2的mRNA相对表达量(P < 0.05),饲粮添加5.0和10.0 g/kg菊粉可显著提高肝脏中HO-1的mRNA相对表达量(P < 0.05),饲粮添加2.5、5.0和10.0 g/kg菊粉对肝脏中Keap1的mRNA相对表达量无显著影响(P>0.05)。

表 6 菊粉对断奶仔猪肝脏抗氧化相关基因表达的影响 Table 6 Effects of inulin on expression of liver antioxidant related genes of weaned piglets
2.5 菊粉对断奶仔猪肠道抗氧化相关基因表达的影响

表 7可见,与对照组相比,饲粮添加2.5、5.0和10.0 g/kg菊粉可显著提高十二指肠中HO-1的mRNA相对表达量和空肠中Nrf2的mRNA相对表达量(P < 0.05),饲粮添加2.5和5.0 g/kg菊粉可显著提高回肠中HO-1和Nrf2的mRNA相对表达量(P < 0.05),饲粮添加2.5 g/kg菊粉可显著提高空肠中Keap1的mRNA相对表达量(P < 0.05),饲粮添加10.0 g/kg菊粉可显著提高空肠中HO-1和Keap1的mRNA相对表达量(P < 0.05)。

表 7 菊粉对断奶仔猪小肠抗氧化相关基因表达的影响 Table 7 Effects of inulin on expression of intestinal antioxidant-related genes of weaned piglets
3 讨论

菊粉是一种从菊苣和菊芋等天然植物中提取的可溶性膳食纤维,是一种潜在的绿色饲料添加剂,可用来提高动物的生长性能肠道健康。研究发现,饲粮添加10.0、15.0和20.0 g/kg菊粉均可显著断奶仔猪ADG[12]。周锡红等[13]将含有5%菊粉的饲粮喂给仔猪,结果发现,菊粉可显著提高ADG,并显著降低料重比。在本试验中,饲粮添加不同水平菊粉对断奶仔猪ADFI和ADG无显著影响。与前人试验相比,本试验饲粮中添加菊粉水平较低可能是造成断奶仔猪ADFI和ADG无显著变化的原因。本试验中,饲粮添加菊粉可提高粗蛋白质和粗灰分表观消化率,与前人研究结果[14-16]一致。

通常情况下机体内自身产生的活性氧(ROS)可以被自身酶类清除,因此对机体并无太大影响。但机体受到刺激后产生的活性氧数量会远远超过机体酶类清除能力,机体会出现应激状态[17-19],最终对机体造成不可逆的伤害[20]。断奶仔猪早期断奶破坏了仔猪的机体抗氧化防御系统,导致机体进入应激状态[21]。机体产生的超氧化物歧化酶(SOD)、GSH-Px、CAT等抗氧化酶能够有效清除机体内的自由基[22]。机体自身产生的这些抗氧化酶的活性高低直接反映出机体的抗氧化能力,也关系到机体的健康状况[23]。研究发现,在断奶仔猪饲粮中添加菊粉可以提高血清SOD活性[24]。尚红梅[25]研究发现,饲粮添加菊粉可显著提高血清中SOD活性,显著降低血清中MDA含量。魏轶男等[26]研究发现,肉仔鸡饲粮中添加菊粉可以显著提高肉仔鸡肝脏和血清中SOD、GSH-Px活性,并显著降低肉仔鸡肝脏和血清中MDA含量。本试验中,饲粮添加2.5 g/kg菊粉可显著提高血清中T-SOD活性和十二指肠CAT活性;饲粮添加2.5、5.0和10.0 g/kg菊粉可显著降低肝脏中MDA含量;饲粮添加2.5和5.0 g/kg菊粉可显著提高空肠中CAT活性,降低回肠中MDA含量。这表明饲粮添加菊粉可增加断奶仔猪血清、肝脏和肠道黏膜系统抗氧化酶活性,从而提高断奶仔猪的抗氧化能力。

Nrf2/Keap1是机体细胞内维持氧化还原平衡的重要信号通路之一,同时Nrf2作为一种应激基本表达的关键转录因子,存在于机体多个器官,Nrf2的缺失或激活障碍直接导致细胞对应激源的敏感性变化[27-28]。应激可以损坏细胞内氧化还原平衡,进而激活或抑制一些信号通路和许多信号介导分子,例如Nrf2/Keap1信号通路[29]。HO-1作为Nrf2信号通路下游的重要因子[30],是反映细胞应激状态敏感性的关键指标[31]。前人的研究发现,Nrf2的mRNA表达水平上升,可以提高机体抗氧化酶的活性[32]。本试验研究发现,饲粮添加菊粉可提高肝脏HO-1和Nrf2、十二指肠HO-1和空肠Nrf2的mRNA相对表达量。这表明饲粮添加菊粉可以通过活化Nrf2通路增强断奶仔猪抗氧化酶活性,进而提高断奶仔猪的抗氧化能力。

4 结论

饲粮添加菊粉对断奶仔猪生长性能无显著影响,但可改善养分表观消化率,提高抗氧化能力。本试验条件下,断奶仔猪饲粮中菊粉适宜添加水平为2.5 g/kg。

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