Chinese Journal of Animal Nutrition >

2023 , Vol. 35 >Issue 1: 579 - 592

DOI: https://doi.org/10.3969/j.issn.1006-267x.2023.01.055

RESEARCH PAPER

Effects of Apparent Digestible Threonine on Apparent Digestibility of Nutrients, Intestinal Morphology and Antioxidant Capacity of Meat Rabbits Challenged by Lipopolysaccharide

  • JIAO Yanna , 1 ,
  • YU Bing 1, 2 ,
  • LI Zimei 1 ,
  • ZENG Tingxuan 1 ,
  • CHEN Xiang 1 ,
  • JING Jieying 1 ,
  • SU Zexin 1 ,
  • CAI Jingyi 1, 2 ,
  • TIAN Gang , 1, 2, *
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  • 1 Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, China
  • 2 Key Laboratory of Animal Disease-Resistant Nutrition, Ministry of Education, Chengdu 611130, China
* professor, E-mail:

Received date: 2022-06-21

  Online published: 2023-01-11

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Abstract

This study was conducted to investigate the effects of apparent digestible threonine (AFDT) on apparent digestibility of nutrients, intestinal morphology and antioxidant capacity of meat rabbits with intestinal injury induced by lipopolysaccharide (LPS). A 2×3 two factors design was used in this experiment. Two kinds of challenge treatments were intraperitoneal injection of 100 μg/kg BW LPS or equivalent physiological saline; AFDT levels were 0.43%, 0.54% and 0.64%, respectively. A total of 180 healthy 35-day-old weaned New Zealand white rabbits with similar initial weight [(808.11±12.17) g] were selected and randomly divided into 6 groups, with 30 replicates in each group and 1 rabbit in each replicate. The experiment lasted for 35 days, and was injected on the morning of days 17 and 20. On day 22 of experiment, weighed, slaughtered and collected intestinal tissue and mucosa samples. Fecal samples were collected from days 4 to 7 after the experiment (intraperitoneal injection of 100 μg/kg BW LPS and the same amount of sterile normal saline were given on days 3 and 6). The results showed as follows: 1) dietary AFDT significantly increased the apparent digestibility of threonine (P<0.05), significantly decreased the apparent digestibility of dry matter (DM), nitrogen-free extract (NFE) and alanine (Ala) (P<0.05), and significantly decreased the total antioxidant capacity (T-AOC) of duodenum (P<0.05). 2) LPS challenge significantly decreased the apparent digestibility of DM, organic matter (OM), gross energy (GE), NFE, acid detergent fiber (ADF) and cysteine (Cys) of meat rabbits (P<0.05), significantly reduced the villus height (VH) of jejunum and the malondialdehyde (MDA) content in duodenum (P<0.05), and significantly increased the MDA content and T-AOC of jejunum (P<0.05). 3) There was significant interaction between dietary AFDT level and LPS challenge on VH in duodenum, and T-AOC, MDA content, xanthine oxidase from buttermilk (XOD) and glutathione peroxidase (GSH-Px) activities in ileum in meat rabbits (P<0.05). It was showed that under the conditions of LPS challenge, AFDT significantly increased VH in duodenum and GSH-Px activity in ileum (P<0.05), and decreased XOD activity in ileum (P<0.05), which had a tendency to decrease MDA content in ileum (P=0.07). In conclusion, the supplementation of dietary AFDT alleviates intestinal morphology damage and oxidative stress which induced by LPS challenge to a certain extent, and alleviates negative impact of meat rabbits challenged by LPS.

Key words： apparent digestible threonine; lipopolysaccharide; meat rabbit; apparent digestibility of nutrients; intestinal morphology; antioxidant capacity

Cite this article

JIAO Yanna , YU Bing , LI Zimei , ZENG Tingxuan , CHEN Xiang , JING Jieying , SU Zexin , CAI Jingyi , TIAN Gang . Effects of Apparent Digestible Threonine on Apparent Digestibility of Nutrients, Intestinal Morphology and Antioxidant Capacity of Meat Rabbits Challenged by Lipopolysaccharide[J]. Chinese Journal of Animal Nutrition, 2023 , 35(1) : 579 -592 . DOI: 10.3969/j.issn.1006-267x.2023.01.055

肠道作为营养物质吸收和代谢的主要场所,对动物的健康生长发挥着重要作用[1]。在家兔养殖过程中,肠道健康问题一直备受关注,断奶前后经常发生肠道疾病,引起肠道黏膜受损、免疫功能和消化吸收能力降低等问题,导致腹泻率增加,生长性能下降,给家兔养殖业造成较大的经济损失[2]。家兔的肠道健康与营养密切相关,为了保障家兔的肠道健康,通过营养调控的方法开展疾病防治是家兔生产过程中的重要环节,对优化家兔营养方案具有重要意义。苏氨酸作为机体的必需氨基酸,被广泛应用于畜禽生产中。苏氨酸具有改善肠道形态发育和营养物质的消化吸收[3]、参与黏蛋白和免疫球蛋白的合成[4⇓-6]、维护肠道屏障等功能[7],对改善动物肠道健康具有重要意义。脂多糖(lipopolysaccharides,LPS)在动物营养学中是常见的诱导肠道损伤的化学方法,其可诱导机体氧化应激,阻碍肠道形态发育与消化吸收,从而降低生长性能[8]。在其他动物的研究中表明,饲粮添加苏氨酸可调节肠道屏障功能,缓解应激和攻毒造成的肠道炎症反应[3-4]。但苏氨酸能否缓解LPS应激对肉兔肠道健康的影响还鲜见报道。因此,本试验通过研究饲粮中添加表观可消化苏氨酸(AFDT)对LPS应激肉兔营养物质表观消化率、肠道形态和氧化还原状态的影响,以阐明苏氨酸对生长肉兔肠道健康的调节作用。

1 材料与方法

1.1 试验材料

苏氨酸含量为98.5%。LPS来源于大肠杆菌(Escherichia coli),血清型O127∶B8,购自美国Sigma试剂公司。

1.2 试验设计及饲养管理

选取180只平均体重为(808.11±12.17) g、健康的35日龄断奶新西兰白兔,采用2×3双因子试验设计,随机分为6组,每组30个重复,每个重复1只兔。试验饲粮参照De Blas等[9]育肥兔营养需要标准进行配制,3种饲粮组成及营养水平见表1,饲粮中AFDT水平分别为0.43%、0.54%和0.65%(实测值分别为0.43%、0.54%和0.64%),分别为推荐量的100%、125%和150%,试验饲粮以苏氨酸替代基础饲粮中的玉米和丙氨酸,达到等能等氮处理。于试验第17天和第20天腹腔注射100 μg/kg BW LPS或等量灭菌生理盐水,注射前12 h进行结料,正常饮水,LPS血清型和注射剂量参照Grasa等[10]标准进行。试验期为35 d,在试验第22天从各组中随机选取6只接近平均体重的肉兔进行屠宰,取肠道组织和黏膜样。试验期间自由采食和饮水,自然通风和光照。
表1 饲粮组成及营养水平(风干基础)

Table 1 Composition and nutrient levels of diets (air-dry basis) %

项目
Items		 饲粮Diets
1 2 3
原料Ingredients
苜蓿草粉Alfalfa meal (CP 14%) 25.65 25.65 25.65
豆粕Soybean meal (CP 44%) 7.26 7.26 7.26
玉米Corn 11.50 11.47 11.44
麦麸Wheat bran 13.86 13.86 13.86
玉米胚芽粕Corn germ meal 16.14 16.14 16.14
甜菜粕Beet pulp 14.50 14.50 14.50
统糠Rice bran and hulls 8.00 8.00 8.00
大豆油Soybean oil 1.18 1.18 1.18
碳酸钙CaCO3 0.08 0.08 0.08
二水磷酸氢钙CaHPO4·2H2O 0.03 0.03 0.03
氯化钠NaCl 0.34 0.34 0.34
L-赖氨酸盐酸盐L-Lys HCl (≥98.5%) 0.14 0.14 0.14
L-蛋氨酸L-Met (≥99%) 0.06 0.06 0.06
L-丙氨酸L-Ala (≥99%) 0.16 0.08
L-苏氨酸L-Thr (≥98.5%) 0.10 0.21 0.32
预混料Premix1) 1.00 1.00 1.00
合计Total 100.00 100.00 100.00
营养水平Nutrient levels2)
消化能DE/(MJ/kg) 10.20(10.66) 10.20(10.53) 10.20(10.60)
粗蛋白质CP 15.41(16.05) 15.41(16.48) 15.41(16.22)
粗纤维CF 15.55(21.67) 15.55(21.54) 15.55(22.09)
项目
Items		 饲粮Diets
1 2 3
中性洗涤纤维NDF 35.08(44.78) 35.08(45.85) 35.07(45.43)
酸性洗涤纤维ADF 19.34(23.90) 19.34(25.20) 19.34(24.93)
酸性木质素ADL 4.32(4.61) 4.32(4.61) 4.32(4.18)
粗脂肪EE 2.41(2.98) 2.41(2.95) 2.41(2.91)
粗灰分Ash 6.42(5.79) 6.42(6.44) 6.42(6.15)
钙Ca 0.60 0.60 0.60
总磷TP 0.40 0.40 0.40
可消化赖氨酸AFDL 0.57 0.57 0.57
可消化苏氨酸AFDT 0.43(0.43) 0.54(0.54) 0.65(0.64)

1)预混料为每千克饲粮提供The premix provided the following per kg of diets:Zn(ZnSO4·H2O)35 mg,Fe(FeSO4·7H2O)30 mg,Mn(MnSO4·H2O)8 mg,Cu(CuSO4·5H2O)6 mg,I(KI)0.4 mg,Se(Na2SeO3)0.05 mg,Co(CoCl2·6H2O)0.3 mg,胆碱 choline 100 mg,烟酸 niacin 20 mg,泛酸 pantothenic acid 12.5 mg,VB1 1 mg,VB2 3.6 mg,VB6 1.8 mg,VB12 0.125 mg,生物素 biotin 2.4 mg,VA 6 000 IU,VD 1 200 IU,VE 50 IU,VK3 2.4 mg。

2)营养水平为计算值,括号内为实测值。可消化赖氨酸与苏氨酸通过未攻毒组的消化率与饲粮中赖氨酸与苏氨酸水平计算而得。Nutrient levels were calculated values, the measured values were in parentheses. Digestible lysine and threonine were calculated from the digestibility of the non-challenged group and the levels of lysine and threonine in the diet.

1.3 指标测定及方法

1.3.1 营养物质表观消化率的测定

采用四分法采集3种饲粮各1 000 g。饲养试验结束后的第3天和第6天,每组选取15只健康且接近平均体重的肉兔,腹腔注射100 μg/kg BW LPS稀释液和等量无菌生理盐水。采用酸不溶灰分(AIA)指示剂法,待试验结束后的第4天至第7天每组收集10只肉兔的粪便,连续收集4 d[11]。每日收集到的粪便喷洒10%的盐酸进行固氮,将4 d收集到的同一只肉兔的粪样进行充分混匀,放入烘箱65 ℃烘干至恒重,粉碎过筛,-20 ℃保存。参照Papadomichelakis等[11]的方法对饲粮和粪便样品中AIA含量进行测定。饲粮和粪便中干物质(DM)、有机物(OM)、总能(GE)、粗蛋白质(CP)、粗脂肪(EE)、无氮浸出物(NFE)、粗纤维(CF)、中性洗涤纤维(NDF)、酸性洗涤纤维(ADF)、酸性木质素(ADL)和氨基酸含量参照张丽英[12]的方法进行测定和计算。采用下列公式计算营养物质表观消化率:
营养物质表观消化率(%)=100-100×(饲粮中指示剂含量/粪样中指示剂含量)×(粪样中营养物质含量/饲粮中营养物质含量)[13]。

1.3.2 肠道形态的测定

在试验第22天空腹称重后,从每组随机选取6只接近平均体重的肉兔进行屠宰,取肠道组织样后保存于4%多聚甲醛溶液瓶中,取肠道黏膜样-80 ℃保存备用。将保存于多聚甲醛溶液中的十二指肠、空肠和回肠样品组织经脱水、修剪、浸蜡、包埋和切片等处理后,进行苏木精-伊红(HE)染色。用光学显微镜(40倍成像)对肠道组织进行形态的观察,随机选取形态完整的目的区域进行拍照,每个样品选取10个绒毛形态完整和延展均匀的视野进行绒毛高度(VH)和隐窝深度(CD)的测定,并计算绒隐比(V/C)。肠道组织样品送至四川农业大学动物医学院病理研究室测定。

1.3.3 肠道抗氧化能力的测定

采用南京建成生物工程研究所试剂盒测定肉兔肠道黏膜中的谷胱甘肽过氧化物酶(GSH-Px)、总超氧化物歧化酶(T-SOD)和黄嘌呤氧化酶(XOD)活性、总抗氧化能力(T-AOC)和丙二醛(MDA)含量。具体操作严格按照试剂盒中的说明书进行。

1.4 数据处理与统计分析

采用Excel 2013对数据进行初步处理,用SAS 9.2的GLM模型进行2×3双因子方差分析。主效应包括饲粮AFDT水平和LPS应激以及两者互作。在交互作用显著时,用Duncan氏法进行多重比较。结果用平均值及均值标准误(SEM)表示,并以P<0.05表示差异显著,P<0.01表示差异极显著,0.05≤P<0.10表示差异有显著趋势。

2 结果

2.1 AFDT对LPS应激肉兔营养物质和氨基酸表观消化率的影响

2.1.1 AFDT对LPS应激肉兔营养物质表观消化率的影响

由表2可知,LPS应激对肉兔GE、CP、CF、NDF和EE的表观消化率均无显著影响(P>0.05),但LPS应激显著降低了DM、OM、GE、NFE和ADF的表观消化率(P<0.05)。AFDT对肉兔GE、CP、CF、ADF、NDF、EE和OM的表观消化率均无显著影响(P>0.05),且与AFDT 0.54%和0.64%组相比,AFDT 0.43%组显著增加了DM和NFE的表观消化率(P<0.05)。饲粮AFDT水平与LPS应激对肉兔营养物质表观消化率无显著交互效应(P>0.05)。
表2 LPS应激和不同AFDT水平对生长肉兔营养物质表观消化率的影响

Table 2 Effects of LPS challenge and different levels of AFDT on apparent digestibility of nutrients of meat rabbits (n=10)%

项目
Items		 总能
GE		 粗蛋白质
CP		 粗纤维
CF		 酸性洗涤纤维
ADF		 中性洗涤纤维
NDF		 粗脂肪
EE		 有机物
OM		 无氮浸出物
NFE		 干物质
DM
表观可消化苏氨酸水平
AFDT level/%		 应激Challenge
0.43 NS 62.96 76.97 26.39 31.84 42.28 83.57 63.75 75.35 63.08
0.54 NS 61.79 76.37 25.84 33.77 41.09 81.46 62.19 73.64 61.64
0.64 NS 62.38 77.34 26.42 32.72 40.38 84.71 62.84 74.42 62.34
0.43 LPS 61.99 75.90 26.29 32.08 40.44 82.16 62.55 74.07 62.07
0.54 LPS 61.58 76.86 24.35 31.34 40.54 85.48 61.85 73.02 61.43
0.64 LPS 60.54 75.51 25.82 30.74 39.88 83.84 60.64 72.39 60.58
SEM 0.89 0.73 1.01 1.16 1.43 1.53 0.58 0.48 0.48
主效应Main effect
表观可消化苏氨酸水平
AFDT level/%		 0.43 62.42 76.97 26.22 30.38 41.72 82.51 63.07 74.65a 62.50a
0.54 61.69 76.37 26.12 32.60 40.92 83.81 62.02 73.33b 61.53b
0.64 61.46 76.43 25.10 32.11 40.30 84.21 61.74 73.40b 61.46b
应激 NS 62.33a 77.10 26.13 33.03a 41.42 83.25 62.86a 74.41a 62.29a
Challenge LPS 61.37b 76.09 25.49 30.56b 40.52 83.82 61.68b 73.16b 61.36b
P值
P-value		 表观可消化
苏氨酸水平
AFDT level		 0.11 0.72 0.39 0.31 0.43 0.51 0.14 0.03 0.04
应激Challenge 0.02 0.18 0.37 0.04 0.30 0.60 0.04 0.01 0.01
表观可消化苏氨
酸水平×应激
AFDT level×challenge		 0.24 0.19 0.67 0.96 0.97 0.09 0.42 0.45 0.25

NS表示注射生理盐水;LPS表示注射脂多糖。同列数据肩标不同小写字母表示差异显著(P<0.05)。下表同。

NS mean the injection of normal saline; LPS mean the injection of lipopolysaccharide. In the same column, values with different small letter superscripts mean significant difference (P<0.05). The same as below.

2.1.2 AFDT对LPS应激肉兔氨基酸表观消化率的影响

由表3和表4可知,LPS应激显著降低了肉兔半胱氨酸(Cys)的表观消化率(P<0.05),显著提高了蛋氨酸(Met)的表观消化率(P<0.05),并有提高组氨酸(His)表观消化率的趋势(P=0.05),而其他氨基酸表观消化率无规律性变化。与AFDT 0.43%组相比,AFDT 0.54%组显著降低了亮氨酸(Leu)、His和丙氨酸(Ala)的表观消化率(P<0.05)。随着饲粮AFDT水平的提高,苏氨酸表观消化率显著提高(P<0.05),而Ala表观消化率显著降低(P<0.05)。饲粮AFDT水平与LPS应激互作对肉兔氨基酸表观消化率无显著影响(P>0.05)。
表3 LPS应激和不同AFDT水平对肉兔必需氨基酸表观消化率的影响

Table 3 Effects of LPS challenge and different levels of AFDT on apparent digestibility of essential amino acids of meat rabbits (n=4)%

项目
Items		 赖氨酸
Lys		 蛋氨酸
Met		 苏氨酸
Thr		 缬氨酸
Val		 异亮氨酸
Ile		 亮氨酸
Leu		 苯丙氨酸
Phe		 组氨酸
His
表观可消化苏氨酸水平
AFDT level/%		 应激Challenge
0.43 NS 84.19 81.95 73.95 79.03 78.46 82.29 81.52 84.19
0.54 NS 81.43 81.12 74.63 75.62 74.63 79.29 78.72 82.06
0.64 NS 83.79 87.65 81.00 79.32 78.56 82.34 81.41 83.98
0.43 LPS 83.08 89.16 73.87 78.32 78.01 82.36 81.39 84.82
0.54 LPS 82.12 88.31 76.20 76.58 76.03 80.56 79.82 83.68
0.64 LPS 83.18 88.67 80.08 77.73 77.33 81.88 80.81 84.67
SEM 0.77 1.97 1.24 1.08 1.15 0.82 0.86 0.56
主效应Main effect
表观可消化苏氨酸水平
AFDT level/%		 0.43 83.64 85.55 73.91b 78.68 83.57a 82.33a 81.46 84.50a
0.54 81.78 84.72 75.42b 76.10 88.71a 79.92b 79.27 82.87b
0.64 83.49 88.16 80.54a 78.52 75.33b 82.11a 81.11 84.32a
应激 NS 83.14 83.57b 76.53 77.99 77.22 81.31 80.55 83.41
Challenge LPS 82.79 88.71a 76.72 77.55 77.09 81.60 80.67 84.39
P值
P-value		 表观可消化苏氨酸水平
AFDT level		 0.05 0.22 <0.01 0.05 0.04 0.02 0.05 0.02
应激Challenge 0.59 <0.01 0.85 0.62 0.89 0.67 0.80 0.05
表观可消化苏
氨酸水平×应激
AFDT level×challenge		 0.50 0.22 0.60 0.50 0.49 0.57 0.60 0.62
表4 LPS应激和不同AFDT水平对肉兔非必需氨基酸表观消化率的影响

Table 4 Effects of LPS challenge and different levels of AFDT on apparent digestibility of non-essential amino acids of meat rabbits (n=4)%

项目
Items		 天冬氨酸
Asp		 丝氨酸
Ser		 谷氨酸
Glu		 甘氨酸
Gly		 丙氨酸
Ala		 半胱氨酸
Cys		 酪氨酸
Tyr		 精氨酸
Arg		 脯氨酸
Pro
表观可消化苏氨酸水平
AFDT level/%		 应激Challenge
0.43 NS 80.66 79.19 85.80 74.26 80.86 83.33 77.99 89.60 80.49
0.54 NS 77.48 76.51 83.40 71.33 75.82 82.75 75.32 87.96 79.27
0.64 NS 80.81 79.01 85.53 74.33 77.04 82.50 78.43 89.50 81.12
0.43 LPS 80.42 79.32 85.51 74.46 80.39 74.98 80.14 89.43 82.35
0.54 LPS 79.18 77.76 84.14 72.80 76.88 71.14 77.39 88.77 80.42
0.64 LPS 79.97 78.77 84.91 73.96 75.51 72.36 78.36 89.45 80.56
SEM 1.07 0.99 0.78 1.15 1.15 2.44 1.10 0.54 1.20
主效应Main effect
表观可消化苏氨酸水平
AFDT level/%		 0.43 80.54 79.26 85.66 74.36 80.62a 79.16 79.06 89.52 81.42
0.54 78.33 77.14 83.77 72.06 76.35b 76.94 76.35 88.36 79.84
0.64 80.39 78.89 85.22 74.15 76.27b 77.43 78.39 89.48 80.86
应激 NS 79.65 78.24 84.91 73.31 77.91 82.60a 77.24 89.02 80.30
Challenge LPS 79.85 78.61 84.85 73.74 77.59 72.83b 78.63 89.22 81.10
P值
P-value		 表观可消化苏氨酸水平
AFDT level		 0.09 0.10 0.07 0.12 <0.01 0.64 0.06 0.08 0.43
应激Challenge 0.82 0.64 0.93 0.65 0.74 <0.01 0.14 0.66 0.42
表观可消化苏氨酸水平×应激
AFDT level×challenge		 0.48 0.74 0.67 0.72 0.54 0.80 0.53 0.62 0.58

2.2 AFDT对LPS应激肉兔肠道形态的影响

由表5可知,LPS应激对肉兔十二指肠和空肠CD以及回肠的3个肠道形态指标均无显著影响(P>0.05),但LPS应激显著降低了空肠的VH(P<0.05),显著增加了十二指肠的V/C(P<0.05),有降低十二指肠VH(P=0.06)和空肠V/C(P=0.09)的趋势。饲粮AFDT水平对肉兔的肠道形态无显著影响(P>0.05)。饲粮AFDT水平与LPS应激对肉兔十二指肠VH存在显著交互效应(P<0.05)。在生理盐水组中,与AFDT 0.64%组相比,AFDT 0.54%组显著增加了十二指肠VH(P<0.05);在LPS应激组中,与AFDT 0.54%组相比,AFDT 0.64%组有提高十二指肠VH的趋势(P=0.07)。
表5 LPS应激和不同AFDT水平对肉兔肠道形态的影响

Table 5 Effects of LPS challenge and different levels of AFDT on intestinal morphology of meat rabbits (n=4)

项目
Items		 十二指肠Duodenum 空肠Jejunum 回肠Ileum
绒毛高度
VH/μm		 隐窝深度
CD/μm		 绒隐比
V/C		 绒毛高度
VH/μm		 隐窝深度
CD/μm		 绒隐比
V/C		 绒毛高度
VH/μm		 隐窝深度
CD/μm		 绒隐比
V/C
表观可消化苏氨酸水平
AFDT level/%		 应激Challenge
0.43 NS 790.12ab 200.58 4.17 786.55 221.68 4.10 517.38 219.58 3.15
0.54 NS 864.26a 228.17 4.14 746.56 196.17 4.10 424.34 101.69 4.65
0.64 NS 645.12b 171.01 3.92 762.93 198.16 4.54 455.91 144.19 4.12
0.43 LPS 666.79b 148.44 6.31 543.41 195.32 3.03 525.15 112.69 5.79
0.54 LPS 644.13b 212.23 4.25 553.56 231.48 2.75 508.86 175.88 4.11
0.64 LPS 770.79ab 156.61 6.00 616.30 169.04 4.35 453.92 160.90 4.11
SEM 50.27 22.98 0.64 56.88 21.86 0.59 29.38 40.21 0.66
主效应Main effect
表观可消化苏氨酸水平
AFDT level/%		 0.43 728.45 173.51 5.24 664.98 204.00 3.56 521.26 166.14 4.65
0.54 754.20 220.19 4.19 650.06 213.82 3.42 466.60 138.50 4.38
0.64 707.96 163.81 4.96 689.62 183.60 4.46 454.90 152.55 4.12
应激 NS 777.53 199.92 4.08b 765.35a 202.34 4.25 495.98 154.97 4.10
Challenge LPS 686.91 172.42 5.52a 571.09b 198.61 3.38 465.88 149.82 4.67
P值
P-value		 表观可消化苏氨酸水平
AFDT level		 0.62 0.06 0.26 0.78 0.39 0.20 0.09 0.75 0.72
应激Challenge 0.06 0.16 0.01 <0.01 0.84 0.09 0.23 0.86 0.30
表观可消化
苏氨酸水平×应激
AFDT level×challenge		 <0.01 0.66 0.22 0.70 0.31 0.60 0.30 0.06 0.10

2.3 AFDT对LPS应激肉兔肠道抗氧化能力的影响

由表6可知,LPS应激对肉兔十二指肠、空肠和回肠SOD、XOD和GSH-Px活性以及回肠的T-AOC和MDA含量均无显著影响(P>0.05),但LPS应激显著增加了空肠MDA含量和T-AOC(P<0.05),显著降低了十二指肠MDA含量(P<0.05)。饲粮添加AFDT对肉兔肠道抗氧化能力无显著改善作用,且与AFDT 0.54%和0.64%组相比,AFDT 0.43%组显著提高了十二指肠T-AOC(P<0.05)。饲粮AFDT水平与LPS应激互作显著影响肉兔回肠的MDA含量、T-AOC以及XOD和GSH-Px活性(P<0.05)。在生理盐水组中,与AFDT 0.43%组相比,AFDT 0.54%组回肠MDA含量显著降低(P<0.05),AFDT 0.64%组回肠的T-AOC显著提高(P<0.05);在LPS应激组中,与AFDT 0.43%组相比,AFDT 0.54%组回肠GSH-Px活性显著增加(P<0.05),AFDT 0.64%组回肠XOD活性显著降低(P<0.05),且有增加回肠GSH-Px活性(P=0.09)以及降低回肠MDA含量的趋势(P=0.07)。
表6 LPS应激和不同AFDT水平对肉兔肠道抗氧化的影响

Table 6 Effects of LPS challenge and different levels of AFDT on intestinal antioxidant of meat rabbits (n=4)

项目
Items		 十二指肠Duodenum 空肠Jejunum 回肠Ileum
超氧化物
歧化酶
SOD/
(mg
prot)		 丙二醛
MDA/
(nmol/mg
prot)		 总抗氧
化能力
T-AOC/
(U/mg
prot)		 黄嘌呤
氧化酶
XOD/
(U/mg
prot)		 谷胱甘
肽过氧
化物酶
GSH-Px/
(U/mg
prot)		 超氧化物
歧化酶
SOD/
(mg
prot)		 丙二醛
MDA/
(nmol/mg
prot)		 总抗氧
化能力
T-AOC/
(U/mg
prot)		 黄嘌呤
氧化酶
XOD/
(U/mg
prot)		 谷胱甘
肽过氧
化物酶
GSH-Px/
(U/mg
prot)		 超氧化物
歧化酶
SOD/
(mg
prot)		 丙二醛
MDA/
(nmol/mg
prot)		 总抗氧
化能力
T-AOC/
(U/mg
prot)		 黄嘌呤
氧化酶
XOD/
(U/mg
prot)		 谷胱甘
肽过氧
化物酶
GSH-Px/
(U/mg
prot)
表观可消化
苏氨酸水平
AFDT level/%		 应激
Challenge
0.43 NS 35.47 12.84 21.79 47.81 20.64 26.18 2.49 14.61 72.82 14.67 24.76 10.87a 12.02b 42.64d 13.86ab
0.54 NS 29.39 9.04 13.80 56.92 14.16 22.31 2.09 12.97 74.22 13.14 21.55 4.90b 11.03b 67.16abc 9.11b
0.64 NS 31.29 12.61 14.60 49.90 21.35 26.52 3.53 16.12 60.86 14.13 26.39 7.80ab 15.64a 74.80ab 13.97a
0.43 LPS 29.24 5.71 17.20 56.40 20.53 26.15 7.60 17.43 60.05 14.2 25.36 6.39ab 12.10ab 80.96a 9.14b
0.54 LPS 31.37 6.61 16.11 54.28 19.67 28.46 1.88 18.61 56.43 15.98 29.34 7.81ab 14.75ab 57.41bcd 15.78a
0.64 LPS 30.73 5.96 15.17 49.85 19.36 24.70 2.91 17.29 69.72 16.59 26.57 3.99b 12.24ab 51.42cd 12.63ab
SEM 1.98 2.55 1.77 5.73 2.73 1.63 1.65 1.58 8.27 1.03 2.47 1.45 1.31 5.78 1.44
主效应Main effect
表观可消化苏氨酸水平
AFDT level/%		 0.43 32.07 8.96 19.29a 52.49 20.58 26.16 3.84 16.02 66.43 14.43 25.06 8.63 12.79 61.80 11.50
0.54 30.38 7.83 14.95b 55.60 16.92 25.39 4.39 15.79 65.32 14.55 25.44 6.35 13.24 62.29 12.44
0.64 31.01 9.28 14.88b 49.87 20.35 25.61 3.77 16.71 65.29 15.36 26.48 6.00 14.58 63.11 13.30
应激 NS 31.85 11.42a 16.43 51.51 18.61 25.00 2.31b 14.57b 69.30 13.98 24.23 7.92 13.97 61.53 12.31
Challenge LPS 30.45 6.10b 16.16 51.76 19.85 26.44 5.72a 17.78a 62.07 15.59 27.09 6.07 13.10 63.26 12.52
P值
P-value		 表观可消化
苏氨酸水平
AFDT level		 0.62 0.81 0.03 0.61 0.34 0.89 0.91 0.83 0.99 0.62 0.84 0.18 0.60 0.98 0.49
应激
Challenge		 0.34 0.02 0.70 0.68 0.62 0.30 0.02 0.02 0.30 0.07 0.17 0.13 0.56 0.72 0.86
表观可消化苏氨
酸水平×应激
AFDT level×
challenge		 0.14 0.61 0.16 0.62 0.37 0.05 0.33 0.37 0.25 0.25 0.24 0.03 0.02 <0.01 <0.01

3 讨论

3.1 LPS应激模型建立

肠道黏膜的完整性是肠道功能正常运行的结构基础,可抵抗细菌和内毒素移位。而LPS诱导的免疫应激会导致肠道屏障受损,细菌和毒素移位进入体循环,从而引发一系列的全身性炎症反应[14]。如LPS会导致家兔的体温升高、采食量下降、精神萎靡和腹泻等症状,影响肠道的收缩与分泌,诱导肠道黏膜损伤以及肠道功能的改变,从而影响肠道对营养物质的转运和吸收[15-16]。本研究前期结果发现,LPS应激诱导肉兔体温升高、嗜睡和生长性能下降等症状。在本试验中,LPS导致肉兔肠道发生氧化应激、肠道形态受损以及营养物质消化吸收受阻,这说明LPS刺激可导致肉兔发生免疫应激反应。

3.2 AFDT对LPS应激肉兔营养物质表观消化率的影响

家兔对营养物质的消化吸收可反映它对营养物质的利用情况,进而反映生长性能和肠道健康状况。研究表明,LPS应激会损害动物机体的消化吸收功能,显著降低机体对DM、OM、CP和EE的表观消化率[17]。在本试验中,LPS应激显著降低了DM、OM、NFE、GE、ADF和Cys的表观消化率,可能是应激损害了肉兔胃肠道的消化功能,降低了蠕动速度,从而抑制了生长且影响采食量,这与本试验前期的生长性能下降结果相对应,同时食糜在肠道中的停留时间延长,这可能是Met表观消化率增加的原因[18]。前人研究结果发现,低蛋白质饲粮中额外添加0.15%苏氨酸改善了离乳前期梅花鹿部分氨基酸的表观消化率[19]。此外,饲粮中适宜水平的苏氨酸可改善幼鱼对脂类的消化与吸收[20]。本试验中,饲粮额外添加AFDT对营养物质表观消化率无显著改善作用,且降低了NFE、DM和Ala的表观消化率。营养物质表观消化率受饲粮中营养水平的影响,同时机体的代谢反馈也会影响其消化吸收[21]。苏氨酸用于肠道维持的需要量较高,因此其在肠道的周转率更快,苏氨酸消化率的增加有利于肠道黏蛋白和免疫球蛋白的合成,便于更好地维护肠道健康[22]。此外,苏氨酸属于中性氨基酸,和肠道内其他中性氨基酸共用相同的氨基酸转运系统,额外添加苏氨酸会导致肠道中游离的苏氨酸含量增加,与其他氨基酸竞争转运载体,从而影响这些氨基酸在肠道中的消化吸收[23],也正好解释了额外添加AFDT在提高苏氨酸表观消化率的同时降低了部分氨基酸的表观消化率。而苏氨酸表观消化率的增加便于更好地调节肠道免疫功能。

3.3 AFDT对LPS应激肉兔肠道形态的影响

肠道是动物机体消化和吸收营养物质的主要场所,肠道的形态结构可反映肠道的健康状况以及对营养物质的消化吸收能力[24]。VH和CD是反映肠道的吸收能力和上皮细胞成熟率的指标,VH增高可增加营养物质与肠道的接触面积,促进营养物质的吸收,CD降低时表明上皮细胞成熟周期缩短,分泌增多,消化能力增强,两者可提高对肠道营养物质的消化吸收,两者的比值V/C在一定程度上也可反映肠道的发育和消化吸收能力[25]。有研究表明,LPS应激会破坏肠道黏膜的形态结构,影响机体对营养物质的消化吸收[26-27]。在本试验LPS应激结束后,LPS应激降低了肉兔十二指肠和空肠的VH,破坏了肠道形态结构,这与上述研究结果相似。研究表明,饲粮中苏氨酸缺乏或过量在一定程度上会对动物的肠道形态结构产生不利影响[28-29]。Ahmed等[22]通过给肉鸡饲喂NRC(1994)推荐量的110%和120%的AFDT可显著改善35日龄肉鸡的肠道形态,提高十二指肠VH和V/C,降低CD。本试验与前人研究结果不完全一致,饲粮添加AFDT对肉兔肠道形态没有显著改善作用,这与Wellington等[30]和Zarrin-Kavyani等[31]在仔猪、肉鸡上的研究结果相似,这可能与动物的种属、日龄、基因型和饲养管理等因素有关。本试验还发现,在LPS应激条件下,AFDT 0.64%组对肉兔肠道形态的缓解效果最佳。陈跃平等[7]研究表明,在LPS应激条件下,饲粮中额外添加0.30%苏氨酸可通过增加21日龄肉鸡空肠的VH、V/C和回肠的V/C来保护肠道形态结构,维护肠道健康。这与本试验结果相似,可能原因是试验前期高水平AFDT提高了肉兔的采食量,使营养物质的摄入量增多,促进肉兔肠道发育,从而在一定程度上有利于缓解肠道损伤。由此可以推测,饲粮添加AFDT对肠道损伤条件下肉兔肠道形态的完整性发挥了一定的调节作用。

3.4 AFDT对LPS应激肉兔肠道抗氧化能力的影响

肠道黏膜可为机体提供屏障保护作用,但机体摄入的外源物质和病原微生物等可诱导氧化损伤和肠道的炎症反应,而胃肠道是活性氧(ROS)的关键来源,其内部的抗氧化酶可保护肠道免受ROS的损伤[32]。SOD、GSH-Px和T-AOC是机体抗氧化系统中的主要抗氧化酶[33]。MDA是脂质代谢的终产物,其含量可间接反映机体的活性氧自由基水平,其在体内积累过多会导致机体产生过氧化反应[34]。XOD是肠道氧自由基产生的关键酶,对肠道的损伤起着直接作用,是反映组织氧化损伤的灵敏指标[35]。LPS可诱导机体产生活性氧,造成氧化应激[36]。Li等[37]研究表明,腹腔注射LPS会诱导肉鸡肠道氧化应激,肠道中MDA含量增加,而SOD活性降低。本试验中,LPS应激增加了肉兔空肠MDA含量,造成空肠氧化应激,而应激引起的十二指肠MDA含量降低以及空肠T-AOC的升高可能是不同部位其氧化酶活性存在差异以及机体抗氧化能力的代偿性升高所致[38]。已有研究报道,饲粮添加苏氨酸可缓解LPS对机体造成的氧化应激,提高肠道抗氧化酶活性,降低MDA含量,从而提高机体肠道的抗氧化能力[7]。本试验在LPS应激条件下,AFDT 0.64%组降低了肉兔回肠XOD活性和MDA含量,AFDT 0.54%组提高回肠GSH-Px活性。由此推测,在LPS应激条件下,饲粮添加AFDT可能通过抑制机体XOD活性和降低MDA含量,减少ROS的释放从而缓解LPS应激对肉兔肠道的损伤,同时通过提高机体GSH-Px活性来清除体内的自由基,提高肉兔肠道的抗氧化能力。原因可能是苏氨酸能与机体的金属元素螯合,金属元素参与抗氧化酶的组成,饲粮添加苏氨酸可参与这些金属元素的储存和运输过程,提供抗氧化酶所需的金属元素,从而增强机体的抗氧化能力,缓解氧化应激对肉兔肠道的损伤[33,39]。

4 结论

在本试验中,LPS应激诱导肉兔肠道氧化应激、肠道形态受损,从而导致营养物质消化吸收受阻。饲粮AFDT水平为0.64%时,可以通过提高肉兔的肠道抗氧化能力以及改善肠道形态,从而在一定程度上缓解LPS应激对肉兔肠道健康的影响。

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