Chinese Journal of Animal Nutrition >

2025 , Vol. 37 >Issue 7: 4494 - 4506

DOI: https://doi.org/10.12418/CJAN2025.368

RESEARCH PAPER

Effects of Dietary Oleanolic Acid on Performance, Serum Biochemical Indices and Antioxidant Function of Laying Hens with Fatty Liver Hemorrhagic Syndrome Induced by High-Energy and Low-Protein Diet

  • WU Liyang , 1 ,
  • ZENG Fajiao 1, 2 ,
  • ZHOU Xiaoling 1, 2 ,
  • WANG Bin 1 ,
  • GAO Shufeng 1, 2 ,
  • ZHOU Yinghua 1, 2 ,
  • ZENG Ao 1, 2 ,
  • SHU Yan , 1, 2, *
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  • 1 Hunan Institute for Microbiology, Changsha 410009, China
  • 2 Hunan Engineering Laboratory of Feeding Microbioecologics, Changsha 410009, China
* senior veterinarian, E-mail:

Received date: 2024-12-13

  Online published: 2025-07-12

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Abstract

This experiment was conducted to investigate the effects of dietary oleanolic acid on performance, serum biochemical indices and antioxidant function of laying hens with fatty liver hemorrhagic syndrome (FLHS) induced by high-energy and low-protein diet. Two hundred healthy Hy-Line brown laying hens of 26 weeks of age with similar body weight and laying rate were selected and randomly divided into 4 groups with 5 replicates in each group and 10 hens in each replicate. Hens in the control group were fed a corn-soybean meal type basal diet, those in FLHS model group were fed a high-energy and low-protein diet, and those in experimental groups Ⅰ and Ⅱ were fed the high-energy and low-protein diet supplemented with 0.1% and 0.2% oleanolic acid, respectively. The pre-experiment was 10 days and the formal experiment was 90 days. The results showed as follows: 1) compared with the control group, the laying rate and daily egg production of laying hens in FLHS model group were significantly decreased (P<0.05), and the feed to egg ratio was significantly increased (P<0.05); compared with FLHS model group, the laying rate and daily egg production in experimental groups Ⅰ and Ⅱ were significantly increased (P<0.05), and the feed to egg ratio was significantly decreased (P<0.05). 2) The observation of liver tissue pathological sections showed that the hepatocytic cords in the control group were clearly visible, and there were no lipid droplets; in FLHS model group, the volume and number of lipid vacuoles in hepatocytes were large; and the volume of lipid vacuoles in hepatocytes in experimental groups Ⅰ and Ⅱ was decreased, showing obvious signs of recovery compared with FLHS model group. 3) Compared with FLHS model group, the serum alanine aminotransferase activity, albumin to globulin ratio, and contents of creatinine, triglyceride and total cholesterol of laying hens in experimental groups Ⅰ and Ⅱ were significantly decreased (P<0.05), while the serum contents of total protein, albumin and globulin were significantly increased (P<0.05); the serum high-density lipoprotein cholesterol content in experimental group Ⅰ was significantly increased (P<0.05). 4) Compared with FLHS model group, the serum contents of interleukin-6, interleukin-8, interleukin-18 and tumor necrosis factor-α of laying hens in experimental groups Ⅰ and Ⅱ were significantly decreased (P<0.05), and the serum interleukin-10 content was significantly increased (P<0.05). 5) Compared with the control group, the mRNA relative expression levels of nuclear factor-κB (NF-κB), inducible nitric oxide synthase (iNOS), Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88) in liver of laying hens in FLHS model group were significantly increased (P<0.05); compared with FLHS model group, the mRNA relative expression levels of NF-κB, iNOS, TLR4 and MyD88 in liver in experimental groups Ⅰ and Ⅱ were significantly decreased (P<0.05). 6) Compared with the control group, the activities of total superoxide dismutase (T-SOD) and catalase (CAT) in serum and liver of laying hens in FLHS model group were significantly decreased (P<0.05), and the malondialdehyde (MDA) content in serum and liver was significantly increased (P<0.05); compared with FLHS model group, the activities of T-SOD and CAT in serum and liver in experimental groups Ⅰ and Ⅱ were significantly increased (P<0.05), and the MDA content in serum and liver was significantly decreased (P<0.05). In conclusion, dietary 0.1% to 0.2% oleanolic acid can alleviate the liver injury in Hy-Line brown laying hens with FLHS induced by high-energy and low-protein diet during the peak laying period to a certain extent, enhance immune function, and strengthen the body’s antioxidant capacity, thereby improving their performance.

Key words： oleanolic acid; laying hens; FLHS; performance; serum biochemical indices; antioxidant function

Cite this article

WU Liyang , ZENG Fajiao , ZHOU Xiaoling , WANG Bin , GAO Shufeng , ZHOU Yinghua , ZENG Ao , SHU Yan . Effects of Dietary Oleanolic Acid on Performance, Serum Biochemical Indices and Antioxidant Function of Laying Hens with Fatty Liver Hemorrhagic Syndrome Induced by High-Energy and Low-Protein Diet[J]. Chinese Journal of Animal Nutrition, 2025 , 37(7) : 4494 -4506 . DOI: 10.12418/CJAN2025.368

蛋鸡脂肪肝出血综合征(fatty liver hemorrhagic syndrome,FLHS)是一种由体内脂质代谢紊乱引发的营养代谢疾病[1],常见于产蛋高峰、体况良好的笼养型蛋鸡群,特别是饲喂高能饲粮的笼养蛋鸡群[2]。该病症在临床上无明显症状,通常表现为肥胖,发病时蛋鸡会出现食欲不振、产蛋率突然下降,在遇到外界刺激时,病鸡可能因肝脏破裂而突然死亡。据报道,FLHS是规模笼养蛋鸡场中导致产蛋高峰期缩短的关键因素之一[2-3],因FLHS死淘的鸡群占养殖场蛋鸡死亡数的40%~70%[4]。目前,FLHS尚无法完全通过遗传育种和饲养管理等手段进行有效控制,随着集约化养殖规模的不断扩大,FLHS发病率呈逐年上升趋势,这给家禽业造成了严重的经济损失,成为蛋鸡产业的难点问题。
齐墩果酸是一种存在于多种植物的天然五环三萜类化合物,如女贞子、西洋参、连翘、三七和人参等[5],具有抗氧化、抗炎抑菌、保肝、抗高血脂和抗癌等多种生物活性[6-8]。然而,尽管齐墩果酸在多个领域展现出广泛的应用研究,但有关其在蛋鸡生产中的应用研究尚未见到。鉴于齐墩果酸的保肝护肝和降脂效果,推测其可能对缓解蛋鸡肝脏的脂质代谢紊乱具有积极作用,进而可能对蛋鸡的生产性能产生积极影响。本试验通过饲喂高能低蛋白质饲粮人工诱导蛋鸡发生FLHS,探究饲粮添加齐墩果酸对FLHS蛋鸡生产性能、血清生化指标和抗氧化功能的影响,并探讨齐墩果酸对蛋鸡FLHS的预防作用,旨在为预防蛋鸡FLHS提供新的理论依据和实践指导,以期降低蛋鸡养殖业损失,推动家禽业健康发展。

1 材料与方法

1.1 试验试剂和仪器

主要试剂:齐墩果酸为市售产品,纯度为95%;超纯RNA提取试剂盒等购自江苏康为世纪生物科技股份有限公司和南京诺唯赞生物科技股份有限公司;总超氧化物歧化酶(T-SOD)活性等生化指标检测试剂盒购自南京建成生物工程研究所。
主要仪器:实时荧光定量PCR仪(ABI 7500,Thermo Fisher Scientific,美国),酶标仪(Multiskan Sky,Thermo Scientific,美国),电感耦合等离子体发射光谱仪(iCap 7200 Thermo Scientific,美国),分光光度计(NanoDrop® ND-2000,Thermo Fisher Scientific,美国)等。

1.2 试验设计及饲养管理

本试验按照《实验动物 福利伦理审查指南》(GB/T 35892—2018)执行,获得湖南省微生物研究院学术委员会批准(批准编号为202201),试验在湖南湘乡市某蛋鸡场进行。
选取26周龄、体重和产蛋率相近的健康海兰褐蛋鸡200羽,随机分为4组,每组5个重复,每个重复10只鸡。对照组饲喂玉米-豆粕型基础饲粮,FLHS模型组饲喂高能低蛋白质饲粮,试验Ⅰ组和试验Ⅱ组分别饲喂添加0.1%和0.2%齐墩果酸的高能低蛋白质饲粮。基础饲粮参考《鸡饲养标准》(NY/T 33—2004)配制,高能低蛋白质饲粮的能量和蛋白质水平参照文献[9]配制,饲粮组成及营养水平见表1。饲粮中粗蛋白质含量参照GB/T 6432—2018中方法进行测定,粗脂肪含量参照GB 5009.6—2016中方法进行测定,钙和总磷含量参照GB 5009.268—2016中方法进行测定,代谢能以及蛋氨酸和赖氨酸含量参照《鸡饲养标准》(NY/T 33—2004)中国禽用饲料成分及营养价值表进行计算。
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		 基础饲粮
Basal diet		 高能低蛋白质饲粮
High-energy and low-protein diet
原料 Ingredients
玉米 Corn 61.50 68.00
豆粕 Soybean meal 25.58 15.00
大豆油 Soybean oil 0.50 4.00
石粉 Limestone 10.24 10.21
磷酸氢钙 CaHPO4 0.56 0.80
氯化钠 NaCl 0.32 0.32
氯化胆碱 Choline chloride 0.10 0.10
赖氨酸 Lys 0.06 0.37
蛋氨酸 Met 0.14 0.20
预混料 Premix1) 1.00 1.00
合计 Total 100.00 100.00
营养水平 Nutrient levels2)
代谢能 ME/(MJ/kg) 11.01 12.14
粗蛋白质 CP 15.10 11.40
粗脂肪 EE 2.70 5.70
钙 Ca 3.80 3.80
总磷 TP 0.41 0.40
蛋氨酸 Met 0.39 0.39
赖氨酸 Lys 0.87 0.84

1)预混料为每千克饲粮提供 The premix provided the following per kg of diets:VA 10 000 IU,VD 3 500 IU,VK 3 mg,VE 30 mg,VB1 2.4 mg,VB2 8 mg,VB5 12 mg,VB6 5 mg,VB12 25 μg,叶酸 folic acid 1.5 mg,烟酸 niacin 45 mg,生物素 biotin 0.2 mg,Cu 10 mg,Fe 75 mg,Mn 90 mg,Zn 80 mg,I 0.4 mg。
2)粗蛋白质、粗脂肪、钙和总磷为实测值,其余均为计算值。CP, EE, Ca and TP were measured values, while the others were calculated values.

试验预试期10 d,正试期90 d。预试期饲喂基础饲粮,根据产蛋率、蛋重及采食量等数据进行适当调整,使各重复差异不显著(P>0.05)。鸡群采用3层全阶梯式单笼饲养,全程饲喂粉料,自由采食和饮水,早晚人工照明补光,光照时间共计16 h/d。每天10:00和16:00固定时间捡蛋并记录,其他饲养管理按常规操作进行。

1.3 检测指标及方法

1.3.1 生产性能

正试期间,以重复为单位记录每日产蛋个数、蛋重及采食量等,并以重复为单位计算产蛋率、日产蛋量、平均蛋重、平均日采食量、料蛋比以及死淘率。计算公式如下:
产蛋率(%)=总产蛋数/蛋鸡存栏数;
日产蛋量(g)=日产蛋量/蛋鸡存栏数;
平均蛋重(g)=总产蛋重/总产蛋数;
平均日采食量(g)=(饲喂料总量-剩余料总量)/每个重复蛋鸡数;
料蛋比=总饲粮消耗量/总产蛋重;
死淘率(%)=100×试验期蛋鸡死淘数/试验开始时蛋鸡总数。

1.3.2 肝脏病理变化

正试期结束时,禁食12 h后,每重复随机选择1只鸡(每组5只鸡),采集肝脏,用生理盐水反复清洗后,固定于4%甲醛溶液中,依照标准流程制作石蜡切片(石蜡切片机,HY-3016)并进行苏木精-伊红(HE)染色,制作冰冻切片(冰冻切片机,Leica CM3050 S)并进行油红O染色。通过显微镜检查,以观察肝脏病理变化。

1.3.3 血清生化指标

正试期结束时,禁食12 h后,每重复随机选取体重相近的2只鸡,通过前颈静脉采血,室温下静置2 h后在台式低速离心机(TD5Z)上离心(1 000×g,10 min),吸取血清样品分装并保存于-80 ℃超低温冰箱中,用于后续指标的测定。
采用全自动生化分析仪(Catalyst One®,IDEXX)测定血清葡萄糖(GLU)、总蛋白(TP)、白蛋白(ALB)、球蛋白(GLB)、尿素(Urea)、肌酐(CR)、尿酸(UA)含量和白球比(ALB/GLB)以及谷丙转氨酶(ALT)、谷草转氨酶(AST)活性,采用磷酸甘油氧化酶-过氧化物酶(GPO-PAP)比色法测定血清甘油三酯(TG)含量,采用胆固醇氧化酶-过氧化物酶(COD-PAP)比色法测定血清总胆固醇(TC)含量,采用双试剂直接比色法测定血清高密度脂蛋白胆固醇(HDL-C)和低密度脂蛋白胆固醇(LDL-C)含量。

1.3.4 炎症指标

1.3.4.1 血清炎症因子含量

采用酶联免疫吸附试验(ELISA)法,通过酶标仪测定血清白细胞介素-1β(IL-1β)、白细胞介素-4(IL-4)、白细胞介素-6(IL-6)、白细胞介素-8(IL-8)、白细胞介素-10(IL-10)、白细胞介素-18(IL-18)和肿瘤坏死因子-α(TNF-α)含量。

1.3.4.2 肝脏炎症基因表达

在无菌条件下快速取出肝脏放入液氮中,置于-80 ℃超低温冰箱中保存。检测时取出冻存肝脏组织样本按质量与体积比1:9(g:mL)加入预冷灭菌生理盐水混合,60 Hz匀浆5 min,之后收集上清液待检测。肝脏总RNA提取采用超纯RNA提取试剂盒按照说明书进行操作,将RNA溶液储存在-80 ℃冰箱中,防止降解。采用紫外吸收法检测RNA浓度和纯度,然后采用HiScript Ⅲ 1st Strand cDNA Synthesis Kit (+gDNA wiper)进行cDNA反转录,操作按产品说明书进行。
选择甘油醛-3-磷酸脱氢酶(GAPDH)为内参基因,核因子-κB(NF-κB)、诱导型一氧化氮合酶(iNOS)、Toll样受体4(TLR4)、髓样分化因子88(MyD88)和GAPDH引物参考文献[9-10]报道,引物由生工生物工程(上海)股份有限公司合成,引物序列见表2。
表2 引物序列

Table 2 Primer sequences

基因
Genes		 引物序列
Primer sequences (5'—3')
核因子-κB NF-κB F:AGGTGGTCCCTAAGTTCCGTG
R:TTTGCCTCTTGGTGCGTTTC
诱导型一氧化氮合酶 iNOS F:ATCTACAGGTATTTGATGCTCGTG
R:TTCTGGATCTTGGCCGTTTG
Toll样受体4 TLR4 F:GGAGGTTGTAGATTTGAGTGAG
R:AGATGGGACATAACATGAGTTT
髓样分化因子88 MyD88 F:GAGTTGGAGCAAACGGAGTT
R:TTGGTGCAAGGATTGGTGTA
甘油醛-3-磷酸脱氢酶 GAPDH F:CCTCTCTGGCAAAGTCCAAGTG
R:GGTCACGCTCCTGGAAGATAGT
利用合成的cDNA作为模板,依照AceQ Universal SYBR qPCR Master Mix试剂盒操作指南,在实时荧光定量PCR仪上进行扩增,反应流程包括:初始95 ℃,10 min;随后进行40个PCR循环(95 ℃,15 s;60 ℃,60 s)。为了鉴定PCR产物的特异性,扩增后进行PCR产物的熔解曲线分析,温度设置为60~99 ℃。以GAPDH作为内参基因,采用2-△△Ct法计算目标基因mRNA相对表达量。

1.3.5 血清和肝脏抗氧化指标

取肝脏组织样本,按质量与体积比1:9(g:mL)加入预冷生理盐水,并在冰水浴条件下机械匀浆处理,离心(350×g,10 min)后,采用移液器转移上层清液至新离心管中以供后续检测。
分别取上述肝脏组织上清液和1.3.3中分离的血清,依据相应试剂盒说明,采用WST-1法测定T-SOD活性,采用紫外分光光度法测定过氧化氢酶(CAT)活性,采用硫代巴比妥酸(TBA)法测定丙二醛(MDA)含量。

1.4 数据处理与统计分析

采用Excel 2019对试验数据进行初步计算和整理,然后采用SPSS 24.0软件进行单因素方差分析(one-way ANOVA),并采用Duncan氏法进行多重比较检验,结果数据采用平均值和均值标准误(SEM)表示,P<0.05表示显著差异。

2 结果与分析

2.1 饲粮添加齐墩果酸对FLHS蛋鸡生产性能的影响

由表3可知,各组间蛋鸡平均日采食量、平均蛋重和死淘率无显著差异(P>0.05)。与对照组相比,高能低蛋白质饲粮组(FLHS模型组、试验Ⅰ组和试验Ⅱ组)蛋鸡产蛋率和日产蛋量显著降低(P<0.05),FLHS模型组和试验Ⅰ组料蛋比显著提高(P<0.05),试验Ⅱ组料蛋比差异不显著(P>0.05)。与FLHS模型组相比,试验Ⅰ组和试验Ⅱ组蛋鸡产蛋率和日产蛋量显著提高(P<0.05),料蛋比显著降低(P<0.05)。
表3 饲粮添加齐墩果酸对FLHS蛋鸡生产性能的影响

Table 3 Effects of dietary oleanolic acid on performance of FLHS laying hens

项目
Items		 对照组
Control
group		 FLHS模型组
FLHS model
group		 试验Ⅰ组
Experimental
group Ⅰ		 试验Ⅱ组
Experimental
group Ⅱ		 均值
标准误
SEM		 P值
P-value
产蛋率 Laying rate/% 90.39c 83.25a 85.91b 87.26b 0.272 <0.001
日产蛋量 Daily egg production/g 55.71c 50.61a 52.79b 53.21b 0.176 <0.001
平均日采食量 ADFI/g 120.29 119.80 119.56 118.20 0.323 0.119
平均蛋重 Average egg weight/g 61.63 60.82 61.45 60.97 0.160 0.229
料蛋比 F/E 2.16a 2.36c 2.26b 2.22ab 0.016 <0.001
死淘率 Mortality/% 2.00 6.00 2.00 2.00 0.020 0.493

同行数据肩标相同字母或无字母表示差异不显著(P>0.05),不同字母表示差异显著(P<0.05)。下表同。

In the same row, values with the same letter or no letter superscripts indicated no significant difference (P>0.05), while with different letter superscripts indicated significant difference (P<0.05). The same as below.

2.2 饲粮添加齐墩果酸对FLHS蛋鸡齐墩果酸对肝脏病理变化的影响

如图1所示,对照组蛋鸡肝细胞索清晰可见,无脂肪滴;与对照组相比,FLHS模型组出现了典型的肝脏脂肪变性的表现,可以看见脂肪空泡体积大、数量较多,由此判断造模成功;试验Ⅰ组和试验Ⅱ组肝细胞脂肪空泡体积减小、数量减少,相对FLHS模型组有明显的恢复迹象。
图1 饲粮添加齐墩果酸对FLHS蛋鸡齐墩果酸对肝脏病理变化的影响

Fig.1 Effects of dietary oleanolic acid on liver pathological changes of FLHS laying hens (20×)

2.3 饲粮添加齐墩果酸对FLHS蛋鸡血清生化指标的影响

由表4可知,各组间蛋鸡血清AST活性以及Urea、UA、LDL-C和GLU含量均无显著差异(P>0.05)。与对照组相比,除试验Ⅰ组血清HDL-C含量显著提高(P<0.05)外,试验Ⅰ组和试验Ⅱ组血清ALT活性、白球比以及TP、ALB、GLB、CR、TG和TC含量均无显著差异(P>0.05)。与FLHS模型组相比,试验Ⅰ组和试验Ⅱ组蛋鸡血清ALT活性、白球比以及CR、TG和TC含量显著降低(P<0.05),血清TP、ALB和GLB含量显著提高(P<0.05);试验Ⅰ组血清HDL-C含量显著提高(P<0.05)。
表4 饲粮添加齐墩果酸对FLHS蛋鸡血清生化指标的影响

Table 4 Effects of dietary oleanolic acid on serum biochemical indices of FLHS laying hens

项目
Items		 对照组
Control group		 FLHS模型组
FLHS model
group		 试验Ⅰ组
Experimental
group Ⅰ		 试验Ⅱ组
Experimental
group Ⅱ		 均值
标准误
SEM		 P值
P-value
谷丙转氨酶 ALT/(U/L) 3.88a 5.13b 4.07a 3.67a 0.196 0.018
谷草转氨酶 AST/(U/L) 221.70 309.70 233.83 229.10 14.510 0.084
总蛋白 TP/(g/L) 43.93b 34.17a 45.43b 45.93b 1.572 0.007
白蛋白 ALB/(g/L) 15.45b 13.40a 15.33b 15.57b 0.333 0.049
球蛋白 GLB/(g/L) 28.48b 20.77a 30.10b 30.37b 1.298 0.010
白球比 ALB/GLB 0.54a 0.65b 0.52a 0.51a 0.020 0.042
尿素 Urea/(mmol/L) 0.85 1.40 1.14 0.88 0.080 0.210
肌酐 CR/(μmol/L) 15.43a 29.33b 13.97a 13.43a 2.037 0.001
尿酸 UA/(μmol/L) 178.33 172.67 166.33 201.67 12.710 0.827
甘油三酯 TG/(mmol/L) 4.51a 8.07b 4.78a 3.90a 0.643 0.031
总胆固醇 TC/(mmol/L) 1.97a 3.02b 2.17a 1.99a 0.153 0.012
高密度脂蛋白胆固醇
HDL-C/(mmol/L)		 0.81a 0.91a 1.31b 0.91a 0.062 0.001
低密度脂蛋白胆固醇
LDL-C/(mmol/L)		 0.66 0.95 0.66 0.76 0.057 0.257
葡萄糖 GLU/(mmol/L) 10.60 10.89 12.75 11.55 0.437 0.340

2.4 饲粮添加齐墩果酸对FLHS蛋鸡炎症指标的影响

2.4.1 饲粮添加齐墩果酸对FLHS蛋鸡血清炎症因子含量影响

由表5可知,各组间蛋鸡血清IL-1β和IL-4含量无显著差异(P>0.05)。与对照组相比,FLHS模型组血清IL-6、IL-8、IL-18和TNF-α含量显著提高(P<0.05),血清IL-10含量显著降低(P<0.05);试验Ⅱ组血清IL-6含量显著提高(P<0.05),血清IL-8含量显著降低(P<0.05)。与FLHS模型组相比,试验Ⅰ组和试验Ⅱ组蛋鸡血清IL-6、IL-8、IL-18和TNF-α含量显著降低(P<0.05),血清IL-10含量显著提高(P<0.05)。
表5 饲粮添加齐墩果酸对FLHS蛋鸡血清炎症因子含量影响

Table 5 Effects of dietary oleanolic acid on serum inflammatory factor contents of FLHS laying hensng/L

项目
Items		 对照组
Control
group		 FLHS模型组
FLHS model
group		 试验Ⅰ组
Experimental
group Ⅰ		 试验Ⅱ组
Experimental
group Ⅱ		 均值
标准误
SEM		 P值
P-value
白细胞介素-1β IL-1β 133.68 140.89 128.76 133.05 2.044 0.198
白细胞介素-4 IL-4 172.71 158.39 159.53 171.79 2.785 0.113
白细胞介素-6 IL-6 38.81a 47.56c 41.35ab 42.40b 0.998 0.002
白细胞介素-8 IL-8 166.84b 183.21c 164.83b 150.05a 3.728 0.003
白细胞介素-10 IL-10 52.59b 44.65a 55.35b 54.31b 1.297 <0.001
白细胞介素-18 IL-18 179.34ab 207.01c 174.25a 186.76b 3.890 <0.001
肿瘤坏死因子-α TNF-α 60.31a 74.38b 62.98a 60.70a 1.757 0.001

2.4.2 饲粮添加齐墩果酸对FLHS蛋鸡肝脏炎症基因表达的影响

由表6可知,与对照组相比,FLHS模型组、试验Ⅰ组和试验Ⅱ组蛋鸡肝脏NF-κB、iNOS、TLR4和MyD88 mRNA相对表达量均显著提高(P<0.05)。与FLHS模型组相比,试验Ⅰ组和试验Ⅱ组蛋鸡肝脏NF-κB、iNOS、TLR4和MyD88 mRNA相对表达量显著降低(P<0.05)。
表6 饲粮添加齐墩果酸对FLHS蛋鸡肝脏炎症基因表达的影响

Table 6 Effects of dietary oleanolic acid on expression of liver inflammation genes of FLHS laying hens

项目
Items		 对照组
Control
group		 FLHS模型组
FLHS model
group		 试验Ⅰ组
Experimental
group Ⅰ		 试验Ⅱ组
Experimental
group Ⅱ		 均值
标准误
SEM		 P值
P-value
核因子-κB NF-κB 1.02a 3.11c 2.36b 2.07b 0.231 <0.001
诱导型一氧化氮合酶 iNOS 1.05a 4.26d 2.52c 2.06b 0.354 <0.001
Toll样受体4 TLR4 1.00a 2.83d 2.51c 2.06b 0.211 <0.001
髓样分化因子88 MyD88 1.02a 7.30d 2.55c 2.04b 0.730 <0.001

2.5 饲粮添加齐墩果酸对FLHS蛋鸡血清和肝脏抗氧化指标的影响

由表7可知,与对照组相比,FLHS模型组蛋鸡血清T-SOD和CAT活性显著降低(P<0.05),血清MDA含量显著提高(P<0.05);试验Ⅰ组血清T-SOD活性显著提高(P<0.05)。与FLHS模型组相比,试验Ⅰ组和试验Ⅱ组蛋鸡血清T-SOD和CAT活性显著提高(P<0.05),血清MDA含量显著降低(P<0.05)。
表7 饲粮添加齐墩果酸对FLHS蛋鸡血清和肝脏抗氧化指标的影响

Table 7 Effects of dietary oleanolic acid on serum and liver antioxidant indices of FLHS laying hens

项目
Items		 对照组
Control
group		 FLHS模型组
FLHS model
group		 试验Ⅰ组
Experimental
group Ⅰ		 试验Ⅱ组
Experimental
group Ⅱ		 均值
标准误
SEM		 P值
P-value
血清 Serum
总超氧化物歧化酶 T-SOD/(U/mL) 145.61b 126.79a 161.66c 145.95b 3.998 0.001
过氧化氢酶 CAT/(U/mL) 2.66b 1.55a 2.76b 2.86b 0.201 0.040
丙二醛 MDA/(nmol/mL) 1.55a 3.53b 2.08a 2.06a 0.259 0.010
肝脏 Liver
总超氧化物歧化酶 T-SOD/(U/mg prot) 96.38b 85.85a 103.65c 106.27c 2.539 0.004
过氧化氢酶 CAT/(U/mg prot) 15.36b 13.13a 18.81c 16.60b 0.695 <0.001
丙二醛 MDA/(nmol/mg prot) 0.64a 1.07b 0.46a 0.66a 0.079 0.009
与对照组相比,FLHS模型组肝脏T-SOD和CAT活性显著降低(P<0.05),肝脏MDA含量显著提高(P<0.05);试验Ⅰ组肝脏T-SOD和CAT活性显著提高(P<0.05),试验Ⅱ组肝脏T-SOD活性显著提高(P<0.05)。与FLHS模型组相比,试验Ⅰ组和试验Ⅱ组蛋鸡肝脏T-SOD和CAT活性显著提高(P<0.05),肝脏MDA含量显著降低(P<0.05)。

3 讨论

3.1 FLHS模型的构建

FLHS是蛋鸡在产蛋高峰期易发且呈渐进性发展的一种营养代谢性疾病,主要是由于蛋鸡体内脂质代谢异常而引发,已成为笼养蛋鸡因非感染性疾病死亡的主要原因,给家禽养殖业造成了巨大的经济损失。因此,调节蛋鸡肝脏脂质代谢,以维持肝脏正常功能在生产中具有重要意义。建立蛋鸡FLHS模型是研究FLHS发病机制及其防治措施的关键前提,目前制备FLHS蛋鸡模型主要采用2种方法:一种方法是通过向蛋鸡肌肉注射β-雌二醇,该方法能够显著提升蛋鸡肝脏脂肪含量[11],然而此法对蛋鸡刺激较大,容易造成死亡,且操作过程相对复杂,不利于大规模应用于模型制备;另一种更为常用的方法是饲喂高能低蛋白质饲粮,邢玉娟等[12]通过制备由74%基础饲粮、6%胆固醇、14%猪油和5%蔗糖等组成的高能饲粮,成功诱导蛋鸡发生了FLHS,这种方法简单易行,对试验蛋鸡刺激小,因此成为当前制备FLHS蛋鸡模型的主要方法。
本试验采用代谢能为12.14 MJ/kg、粗蛋白质含量为11.40%的玉米-豆粕型高能低蛋白质饲粮,对26周龄海兰褐商品蛋鸡进行了为期90 d的饲养。饲养结束后,FLHS模型组蛋鸡肝脏呈现肿大、质脆易碎的特点,表面颜色灰黄。在20倍显微镜下观察石蜡切片后的HE染色结果发现,FLHS模型组蛋鸡肝脏中肝索痕迹变得模糊,细胞核出现溶解迹象,且存在明显的脂肪变性,表现为透明空泡的形成,这验证了FLHS蛋鸡模型的成功构建。此外,肝脏功能指标血清ALT和AST活性的检测结果显示,FLHS模型组蛋鸡这2项指标与对照组相比均有所提高,这进一步证实了FLHS蛋鸡模型的成功制备。在构建FLHS蛋鸡模型的同时,齐墩果酸干预处理后,蛋鸡肝脏中的脂肪空泡数量明显减少,且空泡体积显著缩小,肝脏功能指标血清ALT和AST活性也有所下降,表明齐墩果酸对高能低蛋白质饲粮诱导的蛋鸡FLHS具有较好的预防作用,为后续开发FLHS的有效防治措施提供了参考。

3.2 饲粮添加齐墩果酸对FLHS蛋鸡生产性能的影响

长期饲喂高能低蛋白质饲粮会增加蛋鸡肝脏的代谢压力,造成肝脏脂肪积累,诱发蛋鸡FLHS,从而导致蛋鸡生产性能下降。裴碧璇[13]通过饲喂12周高脂饲粮使蛋鸡患上FLHS后发现,蛋鸡产蛋率和平均日采食量显著降低,料蛋比显著提高。本研究发现,高能低蛋白质饲粮可显著影响蛋鸡产蛋率、日产蛋重和料蛋比。前人研究表明,饲粮添加吡咯喹啉醌[13]、银杏叶提取物[14]及菊粉[15]等添加剂可逆转高脂饲粮导致的产蛋率降低情况,这表明在饲粮中适当使用添加剂来预防或缓解FLHS是可行的。本研究发现,在高能低蛋白质饲粮中添加0.1%~0.2%齐墩果酸,相较于FLHS模型组,蛋鸡产蛋率和日产蛋量显著提高,其中0.2%齐墩果酸添加组料蛋比恢复至饲喂基础饲粮的蛋鸡生产水平,这说明齐墩果酸能逆转FLHS造成的负面影响,具有改善蛋鸡生产性能的作用,这与戴兴兴等[16]的研究结果基本一致。

3.3 饲粮添加齐墩果酸对FLHS蛋鸡血清生化指标的影响

血清生化指标是评估机体代谢状态和健康状况的重要依据。ALT和AST作为肝脏健康的关键指标,在肝脏细胞受损时会释放到血液中,导致血清中这2种酶的活性升高[17-18]。本试验观察到,FLHS模型组蛋鸡血清ALT活性显著高于对照组,而在高能低蛋白质饲粮中添加0.1%~0.2%齐墩果酸则能显著降低血清ALT活性,这表明齐墩果酸有助于减轻肝脏损害并保护肝脏。尽管FLHS模型组蛋鸡血清AST含量与其他组无显著差异,但齐墩果酸的添加仍表现出降低血清AST活性的趋势,这进一步支持了齐墩果酸对肝脏的保护作用。除了肝脏功能指标外,血清TP、ALB、GLB、Urea、CR及UA等指标反映了动物体内蛋白质合成和分解代谢的动态平衡[19]。本试验发现,FLHS模型组蛋鸡血清TP、ALB和GLB含量与对照组相比显著降低,而0.1%和0.2%齐墩果酸添加组则与对照组无显著差异,这表明齐墩果酸能够改善蛋鸡的蛋白质代谢,提高其蛋白质合成能力。Urea和CR是评估肾小球滤过功能的重要指标,当肾脏受损时,会引起血清Urea和CR含量升高[20]。本试验发现,饲粮添加齐墩果酸还降低了蛋鸡血清Urea和CR含量,表明齐墩果酸有助于减轻肾脏的损害。在脂质代谢方面,血清TC、TG、HDL-C和LDL-C含量与脂质代谢过程紧密相关[21],作为血液中脂质的重要组分,血清TC和TG含量降低,意味着脂肪的利用效率更高[21-23]。本试验发现,FLHS模型组蛋鸡血清TC和TG含量显著高于对照组,而饲粮添加0.1%~0.2%齐墩果酸则能有效降低上述指标,且饲粮添加0.1%齐墩果酸还能显著提高血清HDL-C含量,并降低血清LDL-C含量,这表明齐墩果酸能够通过调节血脂水平来改善蛋鸡的脂质代谢。本试验的结果与前人研究结果一致,李雪飞等[24]研究证实齐墩果酸可以显著降低高脂饮食大鼠血清TC、TG和LDL-C含量;研究还发现,灌胃给予50 mg/(kg·d)齐墩果酸或富含该化合物的乙醇提取物能有效预防由21%猪油组成的高脂饮食引起的大鼠血清TG和磷脂含量的升高[25];同时,Pan等[26]报道,对饲喂含1%胆固醇和5%猪油的致动脉粥硬化饮食的新西兰白兔给予50 mg/(kg·d)齐墩果酸28 d,逆转了血清TC、TG和LDL-C的上调;Gamede等[27]研究发现,齐墩果酸显著降低了糖尿病前期大鼠的体重以及血浆TG和LDL-C含量,并显著提高了血浆HDL-C含量。这些研究结果表明,无论是在蛋鸡还是其他动物模型中,齐墩果酸都具有显著的降脂和保护肝脏的作用。

3.4 饲粮添加齐墩果酸对FLHS蛋鸡炎症指标的影响

肝脏中的枯否氏细胞(Kupffer’s cells,KCs),作为巨噬细胞的一种,展现出了高度的可塑性,能够根据外界刺激和内部环境的变化,通过不同的激活途径转化为M1或M2型巨噬细胞,这2种类型的巨噬细胞在机体的免疫反应和组织修复中扮演着截然不同的角色。M1型巨噬细胞通常被称为“经典激活”的巨噬细胞,它们的主要功能是促炎。在受到外界刺激时,M1型巨噬细胞会释放大量的促炎细胞因子,如IL-1β、IL-6、TNF-α以及iNOS等,这些因子在触发和加剧炎症反应中起着关键作用。相比之下,M2型巨噬细胞则更多地参与组织修复和细胞碎片的清除,它们通过释放抗炎因子和生长因子来促进组织的恢复和再生[28]。在高脂饮食诱导的大鼠非酒精性脂肪肝模型中,肝脏的KCs向M1型极化,这种极化状态的改变导致了肝脏内促炎因子的过度释放,诱发“促炎性细胞因子风暴”,进而引发了严重的肝脏损伤[29]。齐墩果酸的抗炎症作用已在其他模型中有所报道。细胞试验表明,齐墩果酸可以抑制由IL-1β引起的人滑膜细胞WS982细胞IL-6、IL-8和基质金属蛋白酶-1(MMP-1)表达上升[30]。Long等[31]在大鼠模型的试验结果表明,齐墩果酸可降低大鼠肾脏发生缺血再灌注损伤后促炎因子干扰素-γ(IFN-γ)、IL-6及抗髓过氧化物酶(MPO)抗体的释放,促进抗炎因子IL-10的分泌。刘昆梅等[32]研究发现,高剂量齐墩果酸能有效减少IL-1β和TNF-α的产生,并显著延长癫痫大鼠的癫痫发作时间间隔,同时减少其对癫痫的易感性。An等[33]研究了齐墩果酸对链脲佐菌素(streptozotocin,STZ)诱导的糖尿病雄性SD大鼠颈动脉损伤的影响,结果表明给予100 mg/(kg·d)齐墩果酸能够改善其GLU代谢,降低血清TNF-α、IL-1β、IL-6和IL-8含量。本试验结果显示,与对照组相比,FLHS模型组蛋鸡肝脏中M1型KCs标志物iNOS mRNA相对表达量显著提高,这进一步证实了M1型极化在肝脏损伤中的关键作用。同时,齐墩果酸干预能够显著降低蛋鸡肝脏iNOS mRNA相对表达量,这表明齐墩果酸能够抑制KCs向M1型的极化,从而减轻肝脏的炎症反应。此外,齐墩果酸还能够抑制多种促炎因子的释放,如IL-6、IL-8、IL-18和TNF-α等,这些因子的减少进一步缓解了肝脏的炎症反应。NF-κB是细胞炎症的关键通路,它调控着多种炎症因子的表达[34]。在炎症环境下,NF-κB信号通路相关蛋白的表达会显著上调。研究表明,齐墩果酸能够降低矽肺模型大鼠血清TNF-α含量,并通过抑制NF-κB蛋白表达来减缓矽肺炎症[35]。活化的NF-κB蛋白不仅可以促进巨噬细胞活化和浸润,诱导细胞凋亡[36];还可以调控靶基因p53,激活凋亡蛋白促进细胞凋亡。郗艳丽等[37]研究表明,给予齐墩果酸脂质体后,能够降低顺铂模型小鼠肾脏组织NF-κB和p53的mRNA水平,显著改善顺铂引起的小鼠肾脏损伤,并降低小鼠细胞凋亡的可能性。邓莉等[38]研究表明,齐墩果酸能抑制TNF-α激活的人成纤维细胞样滑膜细胞产生IL-6和IL-1β,同时降低与炎症反应相关的NF-κB信号传导途径核转录。本试验结果显示,FLHS模型组蛋鸡肝脏NF-κB、TLR4和MyD88 mRNA相对表达量较对照组显著提高,而齐墩果酸处理后,肝脏NF-κB、TLR4和MyD88 mRNA相对表达量显著降低,说明齐墩果酸能通过抑制NF-κB信号转导,有效减缓炎症发生,这也表明齐墩果酸的抗炎作用与其对NF-κB信号通路的抑制密切相关。

3.5 饲粮添加齐墩果酸对FLHS蛋鸡抗氧化功能的影响

超氧化物岐化酶(SOD)是细胞内的一种关键抗氧化酶,能够帮助清除自由基;CAT能够将对生物体有害的过氧化氢转换为水和氧气;而MDA是由活性氧(ROS)导致的脂质过氧化所产生的氧化物。MDA含量的升高可能会改变肝细胞的细胞膜通透性,引发肝脏损伤[39]。T-SOD和CAT被认为是构成机体天然抗氧化防御系统的核心[40]。研究表明,对大鼠肾脏发生缺血再灌注引发的氧化损伤模型使用齐墩果酸干预,能显著提高大鼠肾脏SOD和CAT活性,并降低肾脏MDA含量[31]。
此外,齐墩果酸还具有降低Toll样受体9(TLR9)、IL-18和NF-κB等炎症因子含量的作用,并能够通过减少MDA的生成来改善STZ诱导的糖尿病大鼠的炎症和氧化损伤[41]。李晓红等[42]研究也指出,齐墩果酸能够显著提高氯化汞诱导L02肝细胞T-SOD活性,降低MDA含量,从而有效改善氯化汞对肝细胞造成的氧化损伤。本试验中,与对照组相比,FLHS模型组蛋鸡血清和肝脏T-SOD和CAT活性均显著降低,且血清和肝脏MDA含量均显著提高;而当饲粮添加齐墩果酸后,血清和肝脏T-SOD和CAT活性得到了显著提升,同时MDA含量也显著降低。这一结果充分说明,齐墩果酸能够通过增强抗氧化酶活性,有效减轻氧化应激对机体的不良影响。

4 结论

在本试验条件下,饲粮添加0.1%~0.2%齐墩果酸可一定程度上缓解高能低蛋白质饲粮诱导的产蛋高峰期海兰褐蛋鸡FLHS肝脏损伤,提升免疫功能,增强机体抗氧化能力,从而改善其生产性能。

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