畜牧兽医学报  2021, Vol. 52 Issue (9): 2522-2533. DOI: 10.11843/j.issn.0366-6964.2021.09.015    PDF    
合生素对樱桃谷肉鸭肌肉品质、机体抗氧化性能和免疫功能的影响
王瑞秀, 陈中卫, 刘强, 庄苏     
南京农业大学动物科技学院, 南京 210095
摘要:旨在研究合生素替代日粮中抗生素对樱桃谷肉鸭肌肉品质、血清生化指标、机体抗氧化能力以及免疫功能的影响。本研究选择1日龄樱桃谷肉鸭540只,随机分为3组,每组6个重复,每个重复30只,分别饲喂基础日粮(对照组)、基础日粮+40 mg·kg-1杆菌肽锌(抗生素组)和基础日粮+1 000 mg·kg-1合生素(合生素组)。试验期为42 d,分为前期(1~14 d)和后期(15~42 d)。于试验期第14和42天,分别从每重复中选取1只接近平均体重的公鸭进行称重、屠宰,采集血样、胸肌、腿肌、免疫器官以及肠道黏膜样品用于血清生化指标、肌肉品质、免疫器官指数、抗氧化指标、肠道免疫相关基因等的测定。试验结果表明:1)日粮中添加合生素和抗生素显著降低了樱桃谷肉鸭宰后24 h胸肌和腿肌的蒸煮损失(P < 0.05)以及腿肌的亮度值(P < 0.05),显著提高了宰后24 h腿肌的红度值(P < 0.05)。2)与对照组相比,除抗生素能够显著提高樱桃谷肉鸭42 d脾脏指数(P < 0.05),日粮中添加抗生素或合生素对樱桃谷肉鸭的血清生化指标和免疫器官指数均无显著影响(P>0.05)。3)合生素组肉鸭14 d血清中谷胱甘肽过氧化物酶(GSH-Px)活力显著高于抗生素组与对照组(P < 0.05),42 d空肠黏膜中的超氧化物歧化酶(SOD)活力显著高于对照组(P < 0.05),14 d空肠黏膜中丙二醛含量显著低于对照组(P < 0.05);抗生素组和合生素组42 d空肠黏膜中GSH-Px活力均显著高于对照组(P < 0.05);与对照组和合生素组相比,日粮中添加抗生素显著提高了14 d回肠黏膜中GSH-Px活力(P < 0.05)。4)合生素组14 d空肠黏膜中Toll样受体3(TLR3) mRNA表达量显著高于对照组(P < 0.05),但与抗生素组间无显著性差异(P>0.05);14和42 d回肠黏膜中维甲酸诱导基因1(RIG-1) mRNA表达量均显著高于对照组和抗生素组(P < 0.05);与对照组相比,日粮中添加合生素和抗生素均能显著上调14 d回肠黏膜中TLR3和Toll样受体7(TLR7) mRNA相对表达量(P < 0.05)。综合以上结果指出,合生素能够作为一种抗生素替代品改善樱桃谷肉鸭的肌肉品质和抗氧化性能,提高肠道黏膜中免疫相关基因的表达,增强机体免疫调节能力。
关键词合生素    樱桃谷肉鸭    肌肉品质    血清生化指标    抗氧化能力    免疫    
Effects of Synbiotics on Meat Quality, Antioxidant Capacity and Immune Function of Cherry Valley Ducks
WANG Ruixiu, CHEN Zhongwei, LIU Qiang, ZHUANG Su     
College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China
Abstract: This study aimed to explore the effects of dietary supplementation of synbiotics, as a possible antibiotic substitute, on meat quality, serum biochemical indexes, antioxidant capacity and immune function of Cherry Valley ducks. A total of 540 one-day-old Cherry Valley ducks were randomly assigned to 3 groups with 6 replicates of 30 ducks each. The ducks were fed with basal diet free from antibiotics (control group), basal diets +bacitracin zinc (40 mg·kg-1, antibiotic group), and basal diet+synbiotic (1 000 mg·kg-1, synbiotic group). The experimental period lasted for 42 days, which consisted of starter period (1-14 d) and grower period (15-42 d). At day 14 and 42 of the experiment, one male duck whose weight was close to the average body weight from each replicate was randomly selected, weighed and slaughtered. Then blood, muscle, immune organs and intestinal mucosa samples were collected for the determination of serum biochemical indexes, muscle quality, immune organs indexes, antioxidant indexes, and the expression of intestinal immune-related genes. The results showed that: 1) Dietary supplementation of synbiotics or antibiotics significantly reduced the cooking loss of breast and thigh muscles (P < 0.05) and the brightness value of thigh muscle (P < 0.05), and significantly increased the redness value of thigh muscle at 24 h after slaughter (P < 0.05). 2) Compared with control group, except for the significant increase of spleen index at 42 d in antibiotic group (P < 0.05), dietary supplementation of synbiotic or antibiotic had no significant effect on the serum biochemical indexes and immune organ indexes of Cherry Valley ducks (P>0.05). 3) In synbiotic group, glutathione peroxidase (GSH-Px) activity in the serum at 14 d was higher than that in control and antibiotic groups (P < 0.05), the activity of superoxide dismutase (SOD) in jejunal mucosa at 42 d was higher and malonaldehyde contents in jejunal mucosa at 14 d was lower than those in the control group (P < 0.05). Compared with control group, dietary supplementation of synbiotic or antibiotic significantly increased jejunal mucosal GSH-Px activity at 42 d (P < 0.05), then ileal mucosal GSH-Px activity at 14 d in antibiotic group was higher than that in control and synbiotic groups (P < 0.05). 4) The mRNA expression of Toll-like receptor 3 (TLR3) in jejunum mucosa in synbiotic group at 14 d was higher than that in the control group (P < 0.05), but no significant difference compared with antibiotic group (P>0.05); the mRNA expressions of retinoic-acid-inducible gene I (RIG-1) in the ileal mucosa at 14 and 42 d in synbiotic group were significantly higher than that in control and antibiotic groups (P < 0.05); Compared with control group, dietary supplementation of synbiotic or antibiotic significantly upregulated mRNA expression of TLR3 and TLR7 in the ileal mucosa at 14 d (P < 0.05). Based on the above results, the synbiotic, as a substitute for antibiotics, could improve the meat quality, promote the antioxidant capacity, enhance the expression of immune-related genes in the intestinal mucosa and improve the immunomodulation of Cherry Valley ducks.
Key words: synbiotic    Cherry Valley ducks    meat quality    serum biochemical indexes    antioxidant capacity    immunity    

饲料中添加适量抗生素可以预防和治疗动物疾病、促进动物生长、提高饲料转化效率[1]。然而,抗生素的过度使用也会带来细菌耐药性、抗生素残留、环境污染等一系列问题,甚至威胁人类健康[2]。欧盟早在2006年就已禁止将抗生素作为生长促进剂应用到动物生产中[3]。我国于2020年7月1日起全面禁止动物饲料中添加除中草药外的其他促生长类药物生长促进剂[4]。然而,禁止抗生素使用也会带来动物生长过程中的一系列问题,如饲料转化率降低,疾病发生率提高等[5]。因此,寻找抗生素替代品已成为畜牧生产和饲料行业亟需解决的问题。近年来,人们已经发现了包括益生菌、益生元、合生素等众多绿色健康的饲料添加剂来减少甚至取代饲料中抗生素的添加使用。益生菌是一类有益的活性微生物,可改善肠道菌群的平衡[6]。日粮中添加益生菌可以增加动物肠道内有益菌数量,促使有益菌在肠道内大量繁殖,并通过产生抗菌物质、与有害菌竞争养分及黏附位点等方式来减少致病菌在肠道内定植[7-8]。益生元主要是功能性低聚糖,一般不能被动物肠道内的消化酶水解利用,也不会被动物肠道吸收,但能够选择性刺激肠道内有益菌生长、繁殖和激活代谢功能[9]。合生素又称合生元,是益生菌和益生元组合而成的生物制剂,具有益生菌和益生元的双重作用[10]。本试验中合生素含有的益生菌(丁酸梭菌和枯草芽孢杆菌)和益生元(低聚木糖和低聚壳聚糖)单独添加到动物日粮中都具有促进动物生长,提高抗氧化和免疫性能的作用[11-14]。更多的研究集中在这几种益生菌和益生元单独添加对畜禽生长、免疫以及肠道菌群的影响方面,但这几种物质结合而成的新型合生素产品的作用尚未见相关报道。众多研究表明,日粮中添加合生素能够提高肉鸡、猪和水产动物机体抗氧化和免疫功能,改善肠道健康,促进其生长,其饲喂效果类似甚至优于抗生素[15-17]。但是,关于合生素在水禽,尤其是肉鸭上的研究尚不多见。

本试验是在樱桃谷肉鸭日粮中添加由低聚木糖、低聚壳聚糖、丁酸梭菌和枯草芽孢杆菌组合而成的合生素,探讨合生素作为一种抗生素替代品对樱桃谷肉鸭的肌肉品质、血清生化指标、免疫器官指数、抗氧化能力和肠道黏膜中免疫相关基因表达的影响,为合生素在动物生产中的应用提供新的依据。

1 材料与方法 1.1 试验设计

选择1日龄樱桃谷肉鸭540只,随机分为3组:对照组、抗生素组和合生素组,每组6个重复,每个重复30只肉仔鸭,分别饲喂基础日粮(对照组)、基础日粮+40 mg·kg-1抗生素(杆菌肽锌,抗生素组)和基础日粮+1 000 mg·kg-1合生素(南京华牧动物科技研究所有限公司,合生素组)。每1 g合生素中含有100 mg低聚木糖、50 mg低聚壳聚糖、2×109 CFU丁酸梭菌和3×1010CFU枯草芽孢杆菌。基础日粮的组成和营养成分见表 1。试验共进行42 d,分为前期(1~14 d)和后期(15~42 d)。试验鸭采用网上平养,前3 d舍内温度保持在32~33 ℃,之后每周下降2~3 ℃直至舍内温度维持在25 ℃左右。试验期间所有鸭子按常规程序进行疫苗接种,饲喂颗粒饲料,自由采食和饮水,24 h连续光照。在试验第14和42天时,以重复为单位记录鸭子重量,并从每重复中选取1只接近平均体重的公鸭进行宰杀采样备用。

表 1 基础日粮组成及营养水平(风干基础) Table 1 Composition and nutrient levels of basic diet (air-dry basis)  
1.2 样品采集

血清:于试验期第14和42天从每重复挑选1只樱桃谷肉鸭颈静脉采血,血样静置一段时间后于3 500 r·min-1离心15 min分离血清,并保存在-20 ℃条件下待测相关指标。

免疫器官:于试验期第14和42天,肉鸭致死后迅速剖开腹腔,用镊子分离出胸腺、脾和法氏囊,剔除脂肪组织,吸干液体,剪取后称重。

肠道黏膜:于试验期第14和42天,肉鸭致死后,取出肠道组织,分离出空肠(十二指肠至卵黄囊憩室)和回肠(卵黄囊憩室至回盲结合处),然后用预冷的磷酸盐缓冲液冲洗肠道内容物,并沿肠道纵轴剪开,用载玻片刮取肠道黏膜置于冻存管中,液氮速冻后保存于-80 ℃冰箱中备用。

胸肌与腿肌:于试验第42天屠宰后,采集左侧胸肌、腿肌保存于4 ℃冰箱中用于肌肉品质指标测定。

1.3 指标测定

1.3.1 肌肉品质   采集的左侧胸肌、腿肌用于测定肌肉pH、肉色、滴水损失和蒸煮损失,具体测定方法参照《肉的食用品质客观评价方法》[18]

1.3.2 血清生化指标   血清中总蛋白(TP)、白蛋白(ALB)、尿素氮(BUN)、葡萄糖(GLU)、甘油三酯(TG)和总胆固醇(TC)的含量采用相应方法测定,并通过TP和ALB计算球蛋白(GLB)和白球比(A/G)。所用测定试剂盒均购自于南京建成生物工程研究所,具体测定步骤按照试剂盒说明书进行。

1.3.3 血清和肠道黏膜中抗氧化指标   血清和肠道黏膜中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活力以及丙二醛(MDA)含量采用南京建成生物工程研究所试剂盒测定,测定步骤按照对应试剂盒说明书进行。

1.3.4 免疫器官指数   根据公式:免疫器官指数(g·kg-1)=免疫器官质量(g)/活体体质量(kg),计算胸腺、脾脏和法氏囊器官指数。

1.3.5 肠道黏膜免疫基因   从-80 ℃冰箱中取出冷冻保存的肠道黏膜(空肠、回肠)样品,按照TRIzol试剂盒(TaKaRa)说明书提取总RNA,用NanoDrop ND-2000分光光度计(NanoDrop Technologies,Wilmington,DE,USA)检测RNA浓度。用PrimeScriptTM RT Master Mix (Perfect Real Time),20 μL反应体系进行反转录(TaKaRa)。然后按荧光定量测定:TB Green Premix EX Taq 10 μL (TaKaRa),上游引物0.4 μL,下游引物0.4 μL,50× ROX Reference Dye 0.4 μL,cDNA 2 μL,加ddH2O至20 μL。扩增条件:95 ℃预变性5 min;95 ℃ 10 s, 60 ℃ 34 s,40次循环;熔解曲线95 ℃ 15 s,60 ℃ 1 min,95 ℃ 15 s。以对照组鸭子肠道黏膜的基因表达量为基准,以β-actin作为内参基因,采用2-ΔΔCt法计算肠道黏膜中免疫基因维甲酸诱导基因I(RIG-1)、Toll样受体3(TLR3)、Toll样受体7(TLR7)的mRNA相对表达量。测定试剂盒均购自上海皓嘉科技发展有限公司。基因引物序列见表 2[19-20],并由生工生物工程(南京)股份有限公司合成。

表 2 PCR引物序列 Table 2 PCR primers sequences
1.4 数据统计分析

利用Microsoft Excel 2019对试验数据进行简单处理,采用SPSS 25.0统计分析软件对试验数据进行单因子方差分析(one-way ANOVA),组间差异分析方法采用Duncan’s法,P < 0.05为差异显著。试验结果表示为“平均值±标准误”。

2 结果 2.1 合生素对樱桃谷肉鸭肌肉品质的影响

表 3可知,与对照组相比,日粮中添加合生素和抗生素显著降低了樱桃谷肉鸭宰后24 h胸肌和腿肌的蒸煮损失(P < 0.05)以及腿肌的亮度值(P < 0.05),显著提高了腿肌的红度值(P < 0.05)。合生素和抗生素两组之间的各项指标之间并无显著差异(P>0.05)。

表 3 合生素对樱桃谷肉鸭宰后24 h肌肉品质的影响 Table 3 Effects of synbiotics on the meat quality of Cherry Valley ducks at 24 h after slaughter
2.2 合生素对樱桃谷肉鸭血清生化指标的影响

表 4可知,与对照组相比,合生素和抗生素对樱桃谷肉鸭14与42 d血清中TP、ALB、GLB、A/G、BUN、GLU、TC和TG均无显著影响(P>0.05)。

表 4 合生素对樱桃谷肉鸭血清生化指标的影响 Table 4 Effects of synbiotics on serum biochemical indexes of Cherry Valley ducks
2.3 合生素对樱桃谷肉鸭抗氧化指标的影响

表 5可知,14 d时,合生素组肉鸭血清GSH-Px活力显著高于对照组与抗生素组(P < 0.05),而空肠黏膜中MDA的含量显著低于对照组(P < 0.05),但与抗生素组无显著性差异(P>0.05);抗生素显著提高回肠黏膜中GSH-Px活力(P < 0.05)。42 d时,合生素组空肠黏膜中SOD活力显著高于对照组(P < 0.05),合生素与抗生素组空肠黏膜GPX-Px活力均显著高于对照组(P < 0.05)。

表 5 合生素对樱桃谷肉鸭抗氧化指标的影响 Table 5 Effects of synbiotics on the antioxidant capacity of Cherry Valley ducks
2.4 合生素对樱桃谷肉鸭免疫器官指数的影响

表 6可知,与对照组相比,抗生素组樱桃谷肉鸭42 d脾脏指数显著提高(P < 0.05),胸腺指数有一定的增加趋势(P=0.061)。合生素组樱桃谷肉鸭14和42 d的脾脏指数、胸腺指数和法氏囊指数数值上比对照组均略有提高,但差异不显著(P>0.05)。

表 6 合生素对樱桃谷肉鸭免疫器官指数的影响 Table 6 Effects of synbiotics on immune organ indexes of Cherry Valley ducks  
2.5 合生素对樱桃谷肉鸭肠道黏膜免疫相关基因表达量的影响

图 1可知,14 d时,合生素组肉鸭空肠黏膜中TLR3相对表达量显著高于对照组(P < 0.05),但与抗生素组无显著性差异(P>0.05),回肠黏膜中RIG-1的mRNA表达量显著高于对照组和抗生素组(P < 0.05),抗生素与合生素组回肠黏膜中TLR3和TLR7的mRNA相对表达量均显著高于对照组(P < 0.05),且两组之间差异不显著(P>0.05)。此外,日粮中添加合生素显著提高了42 d樱桃谷肉鸭回肠黏膜中RIG-1的相对表达量(P < 0.05)。

试验结果均表示为“均值±标准误”,每组重复数为6。图柱上附不同字母表示差异显著(P < 0.05) The results data are represented as "Mean±SEM", there are 6 replicates in each group. Different letters on the column indicate significant difference (P < 0.05) 图 1 合生素对樱桃谷肉鸭肠道黏膜免疫相关基因相对表达量的影响 Fig. 1 Effects of synbiotics on the relative mRNA expressions of immune-related genes in intestinal mucosa of Cherry Valley ducks
3 讨论 3.1 合生素对樱桃谷肉鸭肌肉品质的影响

肌肉pH、肉色、滴水损失和蒸煮损失是反映肌肉品质的重要指标。肉色包括亮度值、红度值和黄度值,是评价动物肌肉质量的重要感官指标。一般情况下,一定范围内,肌肉的红度值越大,亮度值和黄度值越小,肉品质越好[21]。本试验中,合生素和抗生素显著升高了樱桃谷肉鸭腿肌的红度值,降低了亮度值,因此,可以认为两者均具有改善腿肌肉色的作用。这种作用可能与肌肉中色素含量以及肌红蛋白的形态等有关[22]。肌肉pH反映了宰后动物机体糖原的酵解速度,一定范围内的pH与肌肉系水力呈现正相关关系[23]。而系水力直接影响肌肉的质地、风味、多汁性和营养状态,具有重要的经济价值[24]。蒸煮损失、滴水损失是用来衡量肌肉系水力的重要指标。已有研究证实,合生素能够显著提高肉鸡胸肌pH,降低腿肌的蒸煮损失[25]。本试验结果表明,日粮中添加合生素和抗生素均显著降低了樱桃谷肉鸭胸肌和腿肌的蒸煮损失。综合以上结果说明,合生素可作为一种抗生素替代品,改善樱桃谷肉鸭的肌肉品质。

3.2 合生素对樱桃谷肉鸭血清生化指标的影响

血清TP由ALB和GLB组成,血清TP的含量高表明蛋白质的合成代谢旺盛,有利于机体对蛋白质的吸收和利用。血清BUN含量可以反映动物体内蛋白质代谢和氨基酸之间的平衡状况,BUN含量越低,动物机体蛋白质合成和氨基酸平衡情况越好[26-27]。GLU是机体主要的能源物质,血清GLU水平可反映机体对碳水化合物的吸收程度和营养水平[28]。血清中TG和TC含量高低可以反映动物机体中脂质代谢情况[29]。本试验中,日粮中添加合生素或抗生素对樱桃谷肉鸭的血清生化参数均无显著影响,这表明本试验所用合生素对樱桃谷肉鸭的蛋白质、脂质等营养物质的代谢影响较小。前人研究结果显示,日粮中添加合生素对动物机体蛋白质和碳水化合物等营养物质的吸收和代谢影响不大,但会对脂质代谢产生一定影响[30-33]。但合生素对动物机体脂质代谢的影响也有不同结果。苗晓微[34]发现,合生素能明显降低肉鸡血清中TC含量。相反地,孙鸣等[31]研究发现, 饲喂合生素能够提高仔猪血清中TC含量,促进其脂肪沉积。此外,动物血清中生化参数的高低还受年龄、发育阶段、饲料营养、内分泌状况等众多因素的影响。

3.3 合生素对樱桃谷肉鸭抗氧化性能的影响

自由基是在动物机体代谢过程中产生的物质,体内自由基过多会使动物机体产生氧化应激损伤。SOD和GSH-Px是畜禽体内主要的酶类抗氧化剂,可以清除体内过量的自由基[35];MDA则是脂质过氧化物的最终产物,反映机体的氧化损伤程度[36]。前人研究表明,日粮中添加合生素能提高肉鸡[37-39]、水产动物[40-41]等动物机体的抗氧化能力。本试验也表明,合生素能够提高樱桃谷肉鸭血清和肠道黏膜中SOD和GSH-Px活力,降低MDA含量,这表明日粮中添加合生素能够改善肉鸭抗氧化反应、降低细胞损伤。研究表明,合生素对动物机体的抗氧化功能可能与其抗菌功效有关[42],其组成成分益生菌(丁酸梭菌、枯草芽孢杆菌)对动物机体抗氧化能力的促进作用发挥主要作用[43-45]。益生菌可直接作为免疫佐剂,或通过促进肠道微生物菌群间接发挥免疫佐剂的作用,改善动物免疫功能[46]

3.4 合生素对樱桃谷肉鸭免疫器官指数的影响

法氏囊、胸腺和脾是家禽的重要免疫器官。胸腺是T淋巴细胞成熟的场所,参与细胞免疫。法氏囊是禽类特有的中枢免疫器官,是最早产生并提供B淋巴细胞的器官。脾作为禽类最大的外周免疫器官,主要产生抗体,参与激发免疫反应[47]。胸腺指数、法氏囊指数和脾脏指数是评价禽类机体免疫状态的常用指标,指数的提高意味着免疫系统成熟较快[48]。苗晓微[34]研究指出,合生素能够显著促进肉鸡免疫器官的生长发育,提高胸腺、脾脏和法氏囊指数。Chen等[42]研究表明,由低聚木糖和枯草芽孢杆菌、丁酸梭菌组成的合生素能够改善肉鸡胸腺指数,从而提高肉鸡的免疫功能。本试验中,除抗生素组42 d脾脏指数外,日粮中添加合生素虽然在统计上没有显著提高樱桃谷肉鸭的胸腺、脾脏和法氏囊指数,但数值上均高于对照组,这说明合生素具有提高肉鸭免疫器官生长的作用。除改善免疫器官指数外,也有报道合生素降低了肉仔鸡的胸腺、脾脏和法氏囊指数[49]。分析其原因,这可能与合生素的组成、剂量、饲养条件以及动物品种等因素有关。

3.5 合生素对樱桃谷肉鸭免疫相关基因表达量的影响

先天性免疫又称非特异性免疫,是机体抵御外来病原微生物感染的第一道屏障[50]。先天性免疫主要通过模式识别受体识别病原相关分子启动机体的免疫应答反应[20]RIG-1、TLR3和TLR7是重要的模式识别受体。郝光恩[51]研究认为,枯草芽孢杆菌通过提高樱桃谷肉鸭胸腺和脾中的模式识别受体RIG-1、TLR3和TLR7的基因表达量,促进下游相关细胞因子和抗病毒蛋白的表达,清除入侵机体的病原微生物。研究发现,TLR3可以诱导IL-6、IL-8等的表达[52]TLR7能刺激IFN-α的快速上调[53],而RIG-1也具有使IFN-β等细胞因子表达提高的作用[54]。细胞因子是一种具有免疫调节作用的小分子物质,在先天性免疫和适应性免疫过程中发挥重要作用[55]。本试验研究发现,除14 d空肠RIG-1外,合生素组肉鸭空肠和回肠黏膜中RIG-1、TLR3和TLR7的mRNA表达量数值上均有不同程度的上调。因此,本研究表明,肉鸭日粮中添加合生素能够调节机体内免疫识别因子,提高机体的免疫调节能力。

4 结论

本研究结果表明,由低聚木糖、低聚壳聚糖、枯草芽孢杆菌和丁酸梭菌组成的合生素能够改善樱桃谷肉鸭肌肉品质,提高其抗氧化能力和肠道黏膜中免疫相关基因的表达,增强机体的免疫调节能力。

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(编辑   郭云雁)