Chinese Journal of Animal Nutrition >

2023 , Vol. 35 >Issue 6: 3800 - 3811

DOI: https://doi.org/10.12418/CJAN2023.353

RESEARCH PAPER

Effects of Clostridium butyricum on Growth Performance, Meat Quality and Skeletal Muscle Development of Lambs

  • CHEN Xiaoyu , 1, 2 ,
  • DOU Lu 1, 2 ,
  • HUA Xiaoqing 3 ,
  • LIU Chang 1, 2 ,
  • YANG Zhihao 1, 2 ,
  • SUN Li’na 1, 2 ,
  • SU Lin 1, 2 ,
  • ZHAO Lihua 1, 2 ,
  • JIN Ye , 1, 2, *
Expand
  • 1 College of Food Science and Engineering, Inner Mongolia Agricultural University, Hohhot 010018, China
  • 2 Integrative Research Base of Beef and Lamb Processing Technology, Ministry of Agriculture and Rural Affairs, Hohhot 010018, China
  • 3 Ordos Agricultural and Livestock Product Quality and Safety Center, Ordos 017000, China
*professor, E-mail:

Received date: 2022-12-26

  Online published: 2023-06-08

Fold

Abstract

This experiment aimed to investigate the effects of dietary Clostridium butyricum on growth performance, meat quality and skeletal muscle development of lambs. Eighteen crossbred ewes of Dorper×Small-Tailed Han sheep were selected and randomly divided into control group (CON group) and Clostridium butyricum group (CB group), and each group had nine sheep. Sheep in the CON group were fed a basal diet, while those in the CB group were fed the basal diet supplemented with Clostridium butyricum (viable count was 5.0×108 CFU/g), and the supplemental level was 5 g/(d·sheep). The pre-test period lasted for 7 days, and the formal test period lasted for 90 days. The growth performance of each group was measured, and the biceps femoris muscle was taken to measure the meat quality, muscle fiber morphology, the activities of related enzyme and the expression of related gene of skeletal muscle development after slaughter. The results showed as follows: 1) the final body weight and average daily gain of lambs in the CB group were significantly higher than those in the CON group (P<0.05), and the feed to gain ratio was significantly lower than that in the CON group (P<0.05). 2) The pH24, brightness (L*) value, yellowness (b*) value, cooking loss and shear force of lambs in the CB group were significantly lower than those in the CON group (P<0.05), and the redness (a*) value was significantly higher than that in the CON group (P<0.05). 3) Compared with the CON group, the biceps femoris muscle weight and muscle fiber cross-sectional area in the CB group were significantly higher than those in the CON group (P<0.05). 4) Compared with the CON group, the activities of branched-chain amino acid transaminase (BCAT) and branched-chain α-keto acid dehydrogenase (BCKDH) in biceps femoris muscle in the CB group were extremely significantly increased (P<0.01). 5) Compared with the CON group, the mRNA relative expression levels of insulin-like growth factor-1 (IGF-1) and serine/threonine-protein kinase 1 (Akt1) in skeletal muscle were significantly up-regulated in the CB group (P<0.05), while the mRNA relative expression levels of eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) and Forkhead box protein O3 (FoxO3) were extremely significantly down-regulated (P<0.01), and the mRNA relative expression levels of muscle atrophy gene F-box (MAFbx) and muscle-specific ring finger protein 1 (MuRF1) were significantly down-regulated (P<0.05); in addition, the mRNA relative expression level of myogenin (MyoG) in skeletal muscle was significantly up-regulated in the CB group (P<0.05), while the mRNA relative expression levels of myostatin (MSTN) and calpain (CAPN-1) were significantly down-regulated (P<0.05), and the mRNA relative expression level of calpastatin (CAST) was extremely significantly down-regulated (P<0.01). Overall, dietary Clostridium butyricum can enhance the growth performance and meat quality of lambs and promote the growth and development of skeletal muscle, and the mechanism may be related to the serine/threonine-protein kinase (Akt) signaling pathway, MyoG/MSTN system and CAPN-1/CAST system.

Key words： Clostridium butyricum; lamb; growth performance; meat quality; skeletal muscle development

Cite this article

CHEN Xiaoyu , DOU Lu , HUA Xiaoqing , LIU Chang , YANG Zhihao , SUN Li’na , SU Lin , ZHAO Lihua , JIN Ye . Effects of Clostridium butyricum on Growth Performance, Meat Quality and Skeletal Muscle Development of Lambs[J]. Chinese Journal of Animal Nutrition, 2023 , 35(6) : 3800 -3811 . DOI: 10.12418/CJAN2023.353

肉类是优质蛋白质、维生素及矿物质等营养物质的良好来源,肉品质包含色泽、嫩度、pH等。近年来,随着生活质量的改善,大众对肉类的品质要求日益提高。国家颁布的一系列禁牧休牧条例使得肉羊的饲养方式逐渐由传统放牧转变为舍饲或半舍饲,而饲养方式的转变会导致羊肉营养价值及肉品质发生不同程度的劣化现象,因此通过饲粮提升舍饲羊肉品质成为众多科技工作者的研究热点。丁酸梭菌是一种有益的革兰氏阳性芽孢杆菌,能够发挥产丁酸、形成孢子、调节肠道菌群、增加机体有益微生物数量等多种益生作用[1]。丁酸梭菌作为益生菌已被广泛应用于动物饲粮中,研究表明,丁酸梭菌可在畜禽肠道中定植,能够改变瘤胃微生物组成并加速体内新陈代谢反应,有助于改善畜禽对饲粮中营养物质的消化吸收[2],以提高动物的生长性能、饲料转化效率及肉品质。Cao等[3]在研究丁酸梭菌对仔猪生长性能的影响时发现,丁酸梭菌可通过提高仔猪的平均日增重改善其生长性能;Hossain等[4]研究发现,在饲粮中补充丁酸梭菌可降低鸡肉的亮度(L*)值,改善肉品色泽;李怀宇[5]研究发现,丁酸梭菌不仅可以显著提高肉鸡的平均日增重、改善肉色等肉品质指标,还可提高肉鸡的免疫能力及抗氧化能力。
近年来,畜禽骨骼肌生长发育备受关注,骨骼肌不仅是动物运动过程中关键的肌肉组织,还是体内葡萄糖和脂肪酸氧化的重要场所[6],其生长发育能够增强动物的产肉能力,因此深入探讨绵羊骨骼肌生长和发育机制对羊肉产业发展有着重要意义。机体蛋白质沉积主要表现为骨骼肌质量的提高,研究表明骨骼肌的生长发育受到多种信号因子的调节[7]:1)丝氨酸/苏氨酸蛋白激酶(serine/threonine-protein kinase,Akt)是多种信号级联反应的重要节点,能够通过激活哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)促进动物体内蛋白质合成,抑制叉头框蛋白O3(Forkhead box protein O3,FoxO3),缓解机体蛋白质降解[8];2)骨骼肌发育也受肌细胞生长素(myogenin,MyoG)及肌肉生长抑制素(myostatin,MSTN)等骨骼肌生长因子的调节[9];3)钙蛋白酶-1(calpain-1,CAPN-1)/钙蛋白酶抑制蛋白(recombinant calpastatin,CAST)系统主要调控肌原纤维蛋白的降解,对肌肉发育同样有着重要作用[10]。目前关于丁酸梭菌的研究多集中于其对单胃动物生长性能、抗氧化能力及肉品质的影响方面,而对于反刍动物,尤其是通过丁酸梭菌调控其骨骼肌发育的影响目前尚未见相关报道。因此,本试验拟在绵羊饲粮中补充丁酸梭菌,探究丁酸梭菌对绵羊生长性能、肉品质及骨骼肌发育的影响,并进一步探究其潜在机制,以期为后续通过饲粮改善绵羊生长性能和提升羊肉品质提供理论依据和参考。

1 材料与方法

1.1 试验设计

选取来自内蒙古呼和浩特市和林格尔县养殖场健康无病且体重相近的3月龄杜泊×小尾寒羊杂一代母羊共18只,采用完全随机试验设计将其分为对照组(CON组)和丁酸梭菌组(CB组),每组9只。CON组饲喂基础饲粮(以玉米、玉米青贮为主),基础饲粮组成及营养成分见表1;CB组在CON组饲粮的基础上补充丁酸梭菌(活菌数为5.0×108 CFU/g),补充量为5 g/(d·只),预先将丁酸梭菌与玉米青贮混合,再与其他饲料原料充分混合均匀后饲喂绵羊。预试期为7 d,正试期为90 d。试验期间各组绵羊均自由采食及饮水,饲养环境条件一致。
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)%

项目 Items 含量 Content
原料 Ingredients
玉米 Corn 44.10
玉米青贮 Corn silage 37.00
豆粕 Soybean meal 15.60
石粉 Limestone 0.60
磷酸氢钙 CaHPO4 0.20
碳酸钙 CaCO3 0.30
食盐 NaCl 0.70
赖氨酸 Lysine 0.60
预混料 Premix1) 0.90
合计 Total 100.00
营养水平 Nutrient levels2)
代谢能 ME/(MJ/kg) 12.20
粗蛋白质 CP 15.70
中性洗涤纤维 NDF 26.50
酸性洗涤纤维 ADF 17.40
粗纤维 Crude fiber 8.00
粗灰分 Ash 7.70
钙 Ca 2.40
总磷 TP 0.55
赖氨酸 Lys 0.60

1)预混料为每千克饲粮提供The premix provided the following per kg of the diet:VA 20 000 IU,VB2 4 mg,VB6 10 mg,VD3 6 000 IU,VE 75 IU,Se 0.30 mg,Fe 35 mg,CuSO4 25 mg,ZnO 36 mg,Mn 20 mg。

2)代谢能为计算值,其余为实测值。ME was a calculated value, while the others were measured values.

1.2 样品采集

试验结束后屠宰各组绵羊,屠宰前需断水2 h,禁食24 h,然后按照穆斯林阿訇屠宰法进行屠宰。屠宰后每组取9只羊的股二头肌测定肉品质相关指标,同时取5 g股二头肌并将其切分成小块置于无菌无酶冻存管内,标明序号后快速投入液氮罐内速冻,后转移至-80 ℃冰箱保存,用于测定骨骼肌发育相关酶活性和相关基因mRNA相对表达量。然后将股二头肌顺肌纤维方向修剪成小块,放置于4%多聚甲醛内固定24 h以上,用于肌纤维组织形态的测定。

1.3 指标测定

1.3.1 生长性能指标

从试验开始到结束期间每30 d称量并记录绵羊晨饲前的空腹体重,期间详细记录绵羊每日饲喂量和剩料量,计算生长性能指标。

1.3.2 肉品质指标

屠宰之后取股二头肌测定45 min和静置排酸24 h后的pH,记为pH0和pH24。同时在屠宰1 h内对股二头肌的色泽进行测定,测定结果以L*值、红度(a*)值、黄度(b*)值表示。将50 g左右股二头肌样品放入塑料密封袋内,于80 ℃水浴锅内升温至样品中心温度达70 ℃后拿出,等待温度降低到室温后顺着肌肉纤维的方向切割成3 cm×1 cm×1 cm的小肉条后测定剪切力。取一块股二头肌样品,记录样品煮前质量(m1),放入80 ℃水浴锅中升温至样品中心温度达70 ℃,取出待温度降低至室温后,用滤纸轻轻地拭干样品表层水分并记录其煮后质量(m2),蒸煮损失按以下公式进行计算:
蒸煮损失(%)=[(m1-m2)/m1]×100。

1.3.3 肌纤维横截面积和直径

4%多聚甲醛固定后的股二头肌经脱水、包埋后制成4 μm的石蜡切片。石蜡切片脱蜡复水后,按照苏木精-伊红(HE)染色试剂盒说明书进行染色。染色结束后于显微镜下对切片进行拍照,利用Image-Pro Plus软件测量肌纤维横截面积和直径。所用试剂盒购自北京索莱宝科技有限公司。

1.3.4 骨骼肌发育相关酶活性

采用双抗体夹心法测定骨骼肌中支链氨基酸转氨酶(branched chain aminotransferase,BCAT)、支链α-酮酸脱氢酶(branched-chain α-ketoacid dehydrogenase,BCKDH)活性,采用上海酶联生物科技有限公司生产的酶联免疫吸附测定(ELISA)试剂盒按照说明书进行检测。

1.3.5 骨骼肌发育相关基因相对表达量

使用TRIzol法提取股二头肌总RNA,并用微量分光光度计测定总RNA的质量及浓度,RNA完整性采用1%(质量分数)的琼脂糖凝胶电泳检测[11]。参照反转录试剂盒制备cDNA,保存于-20 ℃冰箱。内参基因及目的基因的引物序列参照NCBI中提供的序列进行设计(表2),均由生工生物工程(上海)股份有限公司合成。实时荧光定量PCR程序为:95 ℃、30 s;95 ℃、5 s,57 ℃、30 s,72 ℃、30 s,共45个循环;72 ℃、10 min。以胰岛素样生长因子-1(insulin-like growth factor-1,IGF-1)、Akt1、mTOR、p70核糖体蛋白S6激酶(p70 ribosomal proteinS6 kinase,p70S6K)、真核翻译起始因子4E结合蛋白1(eukaryotic translation initiation factor 4E binding protein 1,4EBP1)、FoxO3、肌肉特异性环指蛋白1(muscle ring-finger protein 1,MuRF1)、肌肉萎缩基因F-box(muscle atrophy gene F-box,MAFbx)、MyoG、MSTN、CAPN-1和CAST为目的基因,β-肌动蛋白(β-actin)为内参基因,采用2-ΔΔCt法计算各目的基因的mRNA相对表达量。
表2 实时荧光定量PCR所用引物序列

Table 2 Primer sequences used for real-time fluorescent quantitative PCR

基因
Genes		 引物序列
Primer sequence
(5'—3')		 产物长度
Product size/bp		 GenBank登录号
GenBank accession
number
β-肌动蛋白
β-actin		 F:CATTCACGAAACTACCTT
R:CTGCTTGCTGATCCACAT		 262 NM_001009784.3
胰岛素样生长因子-1
IGF-1		 F:TCCTCCTCGCATCTCTTCTATCTGG
R:CGCACACGAACTGGAGAGCATC		 118 XM_012159659.4
丝氨酸/苏氨酸蛋白激酶1 Akt1 F:GCAGCATCGTGTGGCAGGAC
R:GTCTTGGTCAGGTGGCGTAATGG		 140 NM_001161857.1
哺乳动物雷帕霉素靶蛋白
mTOR		 F:GCAGCAACAGCGAGAGTGAGG
R:GGAACGGAAGAAGCCTTGGACAG		 137 XM_027975551.2
p70核糖体蛋白S6激酶
p70S6K		 F:GGAAGAACTGCTGGCTCGGAAG
R:CCTGGTTGGCACTTTCACTGAGAG		 149 XM_042255326.1
真核翻译起始因子4E结合蛋白1
4EBP1		 F:CGGAAGTTCCTGATGGAGTGTCG
R:GCAGGTGATTCTGGCTGGCTTC		 130 XM_004021776.5
叉头框蛋白O3
FoxO3		 F:TTGGTCCCTCGTCTCTGAACTCC
R:CATCGCTGTGGCTGAGTGAGTC		 147 XM_042253149.1
肌肉特异性环指蛋白1
MuRF1		 F:AATGCCTTGTTCTCTGGTTCGCTAG
R:ACTCTGGCTGTCCTGGGAATGTAG		 84 XM_004005100.5
肌肉萎缩基因F-box
MAFbx		 F:GAGGACCGGCTCTTATGGAAG
R:ACAAGATTAATCGCTTGCGGATCT		 76 XM_004011657.5
肌细胞生长素
MyoG		 F:GGACCCTACAGATGCCCACA
R:GGTTTCATCTGGGAAGGCCG		 94 NM_001174109.1
肌肉生长抑制素
MSTN		 F:GAGAGATGACAGCAGCGACG
R:AGACTCCGTGGGCATGGTAA		 85 NM_001009428.3
钙蛋白酶-1
CAPN-1		 F:TCGCCTCCCTTACCCTCAATGAC
R:CCACATCCACCCACTCACCAAAC		 126 NM_001127267.1
钙蛋白酶抑制蛋白
CAST		 F:CTCAGACACGTCATCGCCTC
R:TCTTTGTTGTCACGCGCAGA		 132 NM_001009788.1

1.4 数据统计分析

试验数据经过Excel 2010整理后,使用SPSS 22.0软件进行单因素方差分析(one-way ANOVA),结果用平均值±标准差表示。P<0.05表示差异显著,P<0.01表示差异极显著。

2 结果与分析

2.1 饲粮添加丁酸梭菌对绵羊生长性能的影响

由表3可知,CB组绵羊的终末体重及平均日增重均显著高于CON组(P<0.05),料重比显著低于CON组(P<0.05)。
表3 饲粮添加丁酸梭菌对绵羊生长性能的影响

Table 3 Effects of dietary Clostridium butyricum on growth performance of lambs

项目
Items		 组别 Groups
CON CB
初始体重 Initial BW/kg 27.75±1.52 27.18±2.38
第30天体重 BW of day 30/kg 35.12±3.45 37.63±3.19
第60天体重 BW of day 60/kg 39.60±3.21 43.17±2.90
终末体重 Final BW/kg 43.16±4.66b 50.11±3.95a
平均日增重 ADG/(kg/d) 0.15±0.01b 0.20±0.03a
干物质采食量 DMI/(kg/d) 1.20±0.10 1.30±0.08
料重比 F/G 7.96±0.94a 6.20±0.66b

同行数据肩标小写字母不同表示差异显著(P<0.05)。下表同。

In the same row, values with different small letter superscripts mean significant difference (P<0.05). The same as below.

2.2 饲粮添加丁酸梭菌对绵羊肉品质的影响

由表4可知,CON组与CB组绵羊肉的pH0无统计学差异(P>0.05),CB组羊肉的pH24、L*值、b*值、蒸煮损失及剪切力均显著低于CON组(P<0.05),a*值则显著高于CON组(P<0.05)。
表4 饲粮添加丁酸梭菌对绵羊肉品质的影响

Table 4 Effects of dietary Clostridium butyricum on meat quality of lambs

项目
Items		 组别 Groups
CON CB
pH0 6.78±0.23 6.70±0.15
pH24 6.16±0.12a 5.99±0.13b
亮度值 L* value 36.57±1.99a 33.78±1.55b
红度值 a* value 16.15±0.53b 17.90±1.56a
黄度值 b* value 3.65±0.37a 2.95±0.45b
蒸煮损失 Cooking loss/% 33.85±0.85a 30.74±1.74b
剪切力 Shear force/N 74.44±6.50a 61.26±7.86b

2.3 饲粮添加丁酸梭菌对绵羊骨骼肌发育的影响

2.3.1 饲粮添加丁酸梭菌对绵羊骨骼肌肌纤维组织形态的影响

如图1所示,饲粮添加丁酸梭菌可使绵羊股二头肌肌纤维间隙和肌细胞横截面积明显增大。由图2可知,CB组股二头肌重量和肌纤维横截面积显著高于CON组(P<0.05),肌纤维直径也高于CON组,但差异不显著(P>0.05)。
图1 饲粮添加丁酸梭菌对绵羊股二头肌肌纤维组织形态的影响

Fig.1 Effects of dietary Clostridium butyricum on muscle fiber morphology in biceps femoris muscle of lambs

图2 饲粮添加丁酸梭菌对绵羊股二头肌质量、肌纤维横截面积和肌纤维直径的影响

*表示差异显著(P<0.05),**表示差异极显著(P<0.01)。下图同。

Fig.2 Effects of dietary Clostridium butyricum on biceps femoris muscle mass, muscle fiber cross-sectional area and muscle fiber diameter of lambs

* mean significant difference (P<0.05), and ** mean extremely significant difference (P<0.01). The same as below.

2.3.2 饲粮添加丁酸梭菌对绵羊骨骼肌发育相关酶活性的影响

如图3所示,CB组股二头肌中BCAT、BCKDH活性均极显著高于CON组(P<0.01)。
图3 饲粮添加丁酸梭菌对绵羊骨骼肌发育相关酶活性的影响

Fig.3 Effects of dietary Clostridium butyricum on skeletal muscle development related enzyme activities of lambs

2.3.3 饲粮添加丁酸梭菌对绵羊骨骼肌发育相关基因mRNA相对表达量的影响

如图4所示,与CON组相比,饲粮添加丁酸梭菌显著上调了绵羊骨骼肌中IGF-1和Akt1的mRNA相对表达量(P<0.05),显著下调了MuRF1和MAFbx的mRNA相对表达量(P<0.05),极显著下调了骨骼肌中4EBP1和FoxO3的mRNA相对表达量(P<0.01)。
图4 饲粮添加丁酸梭菌对绵羊骨骼肌中Akt信号通路基因mRNA相对表达量的影响

Fig.4 Effects of dietary Clostridium butyricum on mRNA relative expression levels of Akt signaling pathway genes in skeletal muscle of lambs

如图5所示,与CON组相比,CB组绵羊骨骼肌中MyoG的mRNA相对表达量显著上调(P<0.05),MSTN、CAPN-1的mRNA相对表达量显著下调(P<0.05),CAST的mRNA相对表达量极显著下调(P<0.01)。
图5 饲粮添加丁酸梭菌对绵羊骨骼肌中关键生长因子基因mRNA相对表达量的影响

Fig.5 Effects of dietary Clostridium butyricum on mRNA relative expression levels of critical growth factor genes in skeletal muscle of lambs

3 讨论

3.1 饲粮添加丁酸梭菌对绵羊生长性能的影响

动物生长性能是最直观的指标,直接影响其经济效益。李文茜[12]研究发现,在同一饲养条件下给犊牛饲粮补充丁酸梭菌,丁酸梭菌可分泌活性酶降解饲粮中的营养成分,提高犊牛对养分的消化吸收,进而通过提高饲粮利用率显著提高其平均日增重。同样,Cai等[13]也通过试验证实山羊在饲粮中补充丁酸梭菌可显著提高平均日增重,改善热应激山羊的生长性能。本试验中,饲粮中补充丁酸梭菌饲喂绵羊90 d后,绵羊的终末体重及平均日增重显著增加,与上述研究结果相一致,同时发现料重比显著降低,说明绵羊对营养成分的吸收利用率得到提高,分析原因可能是丁酸梭菌代谢生成丁酸等短链脂肪酸(short-chain fatty acids,SCFA),可调节肠道内的pH,降低有害微生物的繁殖,使消化酶充分发挥作用[14],提高营养物质的消化吸收率;此外,丁酸梭菌还可产生维生素、氨基酸、淀粉酶、纤维素酶及木聚糖酶等多种物质,改善瘤胃的内环境,提高反刍动物的生长性能[15]。本团队前期试验发现,丁酸梭菌可改变绵羊瘤胃微生物组成,提高厚壁菌门的丰度,降低变形菌门的丰度[16]。因此,丁酸梭菌可通过促进纤维物质降解或抑制有害微生物繁殖使机体更好地汲取养分,从而对绵羊的生长发育起到积极作用。

3.2 饲粮添加丁酸梭菌对绵羊肉品质的影响

色泽、pH、剪切力和蒸煮损失均会对肉类的适口性产生影响,同时也是影响肉品质的重要因素。pH可反映动物体内糖酵解速率,与嫩度等其他肉品质指标密切相关。之前已有学者证实益生菌有降低畜禽肌肉pH24的趋势,如杜瑞等[17]和窦露等[18]均通过试验表明益生菌的补充可能改变绵羊体内糖酵解速率,进而降低羊肉的pH。本试验中,CB组pH24较CON组显著降低,表明丁酸梭菌可加快绵羊体内糖酵解速率。肉品色泽是感官评定最直观的指标,有研究指出,益生菌可提高动物体的抗氧化能力,进而改善肌肉色泽[19]。本试验中,CB组的股二头肌L*值和b*值均显著低于CON组,a*值则显著高于CON组,表明丁酸梭菌可改善羊肉的色泽,这与Meng等[20]的研究结果一致。蒸煮损失可反映肌肉的保水性,并在很大程度上影响肉品嫩度、营养成分等指标。Česlovas等[21]研究发现,饲粮中添加益生菌可降低猪肉的蒸煮损失并提高肉质保水性。本试验中,CB组的蒸煮损失显著低于CON组,这说明丁酸梭菌的添加使得羊肉保水性更好。嫩度是影响消费者对肉品评价的重要指标,与剪切力呈负相关关系。本试验结果显示,CB组的股二头肌剪切力较CON组显著降低,说明丁酸梭菌的添加改善了羊肉的嫩度,究其原因可能是丁酸梭菌所分泌的脂肪酶可将大颗粒脂质分解为小分子,还可调控脂肪代谢相关基因的表达,提高机体对脂肪的消化吸收,增加肌内脂肪含量,从而改善羊肉嫩度[22]。

3.3 饲粮添加丁酸梭菌对绵羊骨骼肌发育的影响

3.3.1 饲粮添加丁酸梭菌对绵羊骨骼肌肌纤维组织形态的影响

畜禽出生后,肌纤维数量维持恒定不再增加,骨骼肌发育表现为肌纤维横截面积和直径的增大以及肌肉重量的增加[23]。本研究发现,饲粮添加丁酸梭菌显著增大了绵羊的骨骼肌重量和肌纤维横截面积。申文雪等[24]研究表明,丁酸梭菌可显著提高肉鸡的腿肌重,与本试验结果一致,推测其原因可能是丁酸梭菌代谢生成SCFA、次级胆汁酸和调节肌肉功能的氨基酸等物质,这些物质可通过影响蛋白质代谢过程进而调节骨骼肌质量,提高绵羊的生产性能[25]。因此,为进一步探究丁酸梭菌对绵羊骨骼肌发育的影响,本试验测定了骨骼肌发育相关酶活性及相关基因的mRNA相对表达量。

3.3.2 饲粮添加丁酸梭菌对绵羊骨骼肌发育相关酶活性的影响

支链氨基酸(BCAA)是骨骼肌中含量最丰富的游离氨基酸,也是组成肌肉蛋白质的基本底物和重要的调节因子。研究表明,BCAA中的亮氨酸及其代谢产物(α-酮异已酸和β-羟基-β-甲基丁酸)可增强动物体对氨基酸的吸收,还可以减缓骨骼肌内蛋白质的降解速率,加快体内蛋白质沉积,从而提高骨骼肌的生长发育[26]。BCAT和BCKDH是骨骼肌中2个典型的BCAA代谢酶,能够参与调节BCAA分解代谢,当动物体内存在大量的BCAA时,BCKDH通过去磷酸化增加酶的活性,优先分解代谢亮氨酸[27]。本试验中,CB组骨骼肌中BCKDH和BCAT活性均极显著高于CON组,因此推测丁酸梭菌的添加加快了BCAA的分解代谢,这一过程使得亮氨酸的代谢产物富集,进一步激活蛋白质翻译的起始,加快蛋白质沉积,从而促进骨骼肌生长发育,提高肌肉重量[28]。

3.3.3 饲粮添加丁酸梭菌对绵羊骨骼肌发育相关基因表达的影响

畜禽骨骼肌发育受众多信号因子的调节,IGF-1是调控骨骼肌发育的典型细胞因子之一,能够促进肌肉细胞的增殖和分化[29],同时也是Akt上游的一个调节器。本试验中,丁酸梭菌的添加显著提高了骨骼肌中IGF-1的mRNA相对表达量,丁酸梭菌进入肠道后产生SCFA,SCFA可增加IGF-1含量[30],IGF-1诱导激活Akt信号通路进而促进蛋白质合成,对骨骼肌发育起到积极作用。
Akt1是畜禽体内存在的一种丝氨酸/苏氨酸蛋白激酶,可双向调控蛋白质代谢,与骨骼肌发育密切相关[31]。mTOR在肌纤维形成过程中起着关键作用,位于Akt信号通路下游,通过调节p70S6K和4EBP1介导编码核糖体蛋白mRNA的翻译起始,其中,p70S6K是mTOR信号通路的正向调控因子,4EBP1是负向调控因子,两者共同作用增加骨骼肌中蛋白质的合成量[32],从而诱导骨骼肌生长发育。本研究中,与CON组相比,CB组骨骼肌中mTOR和p70S6KD的mRNA相对表达量有所上调,Akt1的mRNA相对表达量显著上调,4EBP1的mRNA相对表达量显著下调,表明饲粮中补充丁酸梭菌可诱导Akt/mTOR信号通路增强蛋白质合成,这可能是导致绵羊骨骼肌重量增加的原因。李玉鹏等[33]研究表明,丁酸梭菌通过mTOR信号通路上调紧密连接蛋白的表达。Liu等[34]也发现丁酸梭菌可提高小鼠体内Akt、mTOR和p70S6K的磷酸化水平。因此,丁酸梭菌促进骨骼肌发育可能与Akt/mTOR信号通路增强蛋白质合成有关。
FoxO3也是Akt信号通路下游重要调节因子之一,当饲粮营养水平发生改变时,Akt通过磷酸化抑制FoxO3蛋白的活性,进而调节骨骼肌蛋白质降解过程,影响机体骨骼肌发育。MAFbx和MuRF1是动物体内蛋白质降解的关键标志物,两者表达下降时蛋白质降解速率减缓,抑制骨骼肌萎缩[35]。已有文献报道,给小鼠口服益生菌后可降低肌肉中MuRF1和MAFbx的表达,防止小鼠骨骼肌萎缩[36]。本试验中,与CON组相比,CB组骨骼肌中FoxO3的mRNA相对表达量极显著下调,MuRF1和MAFbx的mRNA相对表达量显著下调,这与前人研究结果一致,表明丁酸梭菌能够有效调控蛋白质降解相关基因表达,进而抑制骨骼肌蛋白质的降解。因此,丁酸梭菌促进绵羊骨骼肌发育可能与Akt/FoxO3信号通路抑制蛋白质降解有关。
MyoG属于肌决定因子家族,在肌肉生长发育和细胞分化方向起着关键作用,有研究表明MyoG基因缺乏可能导致动物体内肌肉无法正常生成[23]。MSTN是调控肌肉萎缩的因子,对肌肉生长发育具有抑制作用[37]。MyoG表达量升高有助于骨骼肌发育,而MSTN表达量下调能够促进MyoG发挥作用,提高骨骼肌重量。本试验中,与CON组相比,CB组骨骼肌中MyoG的mRNA相对表达量显著上调,MSTN的mRNA相对表达量显著下调,表明丁酸梭菌可有效促进骨骼肌生长发育。因此,丁酸梭菌促进绵羊骨骼肌发育可能与MyoG/MSTN系统有关。
CAPN-1属于钙蛋白酶系统,在肌肉蛋白质降解过程中发挥着限速酶的作用,能够降解大量的肌原纤维蛋白及骨架蛋白,调节蛋白质沉积,从而影响骨骼肌发育[38]。CAST是钙蛋白酶抑制蛋白,调节骨骼肌中CAPN-1的活性,从而促进骨骼肌发育[39]。本试验中,与CON组相比,CB组骨骼肌中CAPN-1和CAST的mRNA相对表达量均显著下调,提示骨骼肌中蛋白质降解速率放缓,可能导致蛋白质沉积增强。因此,丁酸梭菌促进骨骼肌发育可能与CAPN-1/CAST系统增强绵羊骨骼肌内蛋白质沉积有关。

4 结论

① 饲粮添加丁酸梭菌显著增加了绵羊的终末体重和平均日增重,降低了料重比,改善了生长性能。
② 饲粮添加丁酸梭菌可改善绵羊股二头肌的色泽,提高其嫩度及保水性,对羊肉品质有显著的改善作用。
③ 丁酸梭菌可能通过介导Akt信号通路和调节MyoG/MSTN系统与CAPN-1/CAST系统改善绵羊骨骼肌发育。

References

Publishing order | Descend order by publishing year | Descend order by cited within
[1]
ARIYOSHI T, HAGIHARA M, TAKAHASHI M, et al. Effect of Clostridium butyricum on gastrointestinal infections[J]. Biomedicines, 2022, 10(2):483.

DOI

[2]
HAN Y S, TANG C H, LI Y, et al. Effects of dietary supplementation with Clostridium butyricum on growth performance,serum immunity,intestinal morphology,and microbiota as an antibiotic alternative in weaned piglets[J]. Animals, 2020, 10(12):2287.

DOI

[3]
CAO G T, TAO F, HU Y H, et al. Positive effects of a Clostridium butyricum-based compound probiotic on growth performance,immune responses,intestinal morphology,hypothalamic neurotransmitters,and colonic microbiota in weaned piglets[J]. Food & Function, 2019, 10(5):2926-2934.

[4]
HOSSAIN M M, BEGUM M, KIM I H. Effect of Bacillus subtilis,Clostridium butyricum and Lactobacillus acidophilus endospores on growth performance,nutrient digestibility,meat quality,relative organ weight,microbial shedding and excreta noxious gas emission in broilers[J]. Veterinární Medicína, 2016, 60(2):77-86.

DOI

[5]
李怀宇. 丁酸梭菌对肉鸡生长性能、屠体品质的影响及其机理研究[D]. 硕士学位论文. 杭州: 浙江大学, 2021.

LI H Y. Effects of dietary supplementation of Clostridium butyricum on growth performance and carcass traits in broilers and its mechanism[D]. Master’s Thesis. Hangzhou: Zhejiang University, 2021. (in Chinese)

[6]
孙燕勇, 付绍印, 祁云霞, 等. 绵羊骨骼肌生长发育调控基因研究进展[J]. 中国畜牧兽医, 2019, 46(5):1429-1438.

SUN Y Y, FU S Y, QI Y X, et al. Research advances on regulation genes of growth and development of skeletal muscle in sheep[J]. China Animal Husbandry & Veterinary Medicine, 2019, 46(5):1429-1438. (in Chinese)

[7]
KOBAYASHI J, UCHIDA H, KOFUJI A, et al. Molecular regulation of skeletal muscle mass and the contribution of nitric oxide:a review[J]. FASEB bioAdvances, 2019, 1(6):364-374.

DOI

[8]
KANG P, WANG X Y, WU H T, et al. Glutamate alleviates muscle protein loss by modulating TLR4,NODs,Akt/FOXO and mTOR signaling pathways in LPS-challenged piglets[J]. PLoS One, 2017, 12(8):e0182246.

DOI

[9]
TANNENBAUM G S, GURD W, LAPOINTE M. Leptin is a potent stimulator of spontaneous pulsatile growth hormone (GH) secretion and the GH response to GH-releasing hormone[J]. Endocrinology, 1998, 139(9):3871-3875.

PMID

[10]
王伟. CAPN1基因和CASP9基因在牛骨骼肌形成中的功能作用研究[D]. 硕士学位论文. 洛阳: 河南科技大学, 2017.

WANG W. Study on the function of CAPN1 gene and CASP9 gene in bovine skeletal muscle formation[D]. Master’s Thesis. Luoyang: Henan University of Science and Technology, 2017. (in Chinese)

[11]
白艳苹, 侯艳茹, 苏琳, 等. 乳酸菌诱导线粒体生物发生对绵羊肌纤维特性和肉品质的影响[J]. 农业工程学报, 2021, 37(10):269-276.

BAI Y P, HOU Y R, SU L, et al. Effects of Lactobacillus induced mitochondrial biogenesis on muscle fiber properties and meat quality of sheep[J]. Transactions of the Chinese Society of Agricultural Engineering, 2021, 37(10):269-276. (in Chinese)

[12]
李文茜. 丁酸梭菌对犊牛及育成牛生长性能、血液指标和瘤胃发酵的影响[D]. 硕士学位论文. 哈尔滨: 东北农业大学, 2019.

LI W Q. Effects of Clostridium butyricum on growth performance,blood index and rumen fermentation in calves and bred cattle[D]. Master’s Thesis. Harbin: Northeast Agricultural University, 2019. (in Chinese)

[13]
CAI L Y, HARTANTO R, XU Q B, et al. Saccharomyces cerevisiae and Clostridium butyricum could improve B-vitamin production in the rumen and growth performance of heat-stressed goats[J]. Metabolites, 2022, 12(8):766.

DOI

[14]
DUAN Y F, DONG H B, WANG Y, et al. Effects of the dietary probiotic Clostridium butyricum on intestine digestive and metabolic capacities,SCFA content and body composition in Marsupenaeus japonicus[J]. Journal of Ocean University of China, 2018, 17(3):690-696.

DOI

[15]
阳田恬, 刘梦瑶, 张静, 等. 丁酸梭菌的特性及其生物学功能[J]. 饲料工业, 2022, 43(6):59-64.

YANG T T, LIU M Y, ZHANG J, et al. The characteristics and biological functions of Clostridium butyricum[J]. Feed Industry, 2022, 43(6):59-64. (in Chinese)

[16]
王威皓, 窦露, 康乐天, 等. 日粮添加丁酸梭菌对小尾寒羊脂肪酸代谢和肉品质的影响[J/OL]. 食品科学:1-13[2023-01-20]. http://kns.cnki.net/kcms/detail/11.2206.TS.20221110.2207.064.html.

WANG W H, DOU L, KANG L T, et al. Effects of dietary Clostridium butyricum on fatty acid metabolism and meat quality of small-tailed Han sheep[J/OL]. Food Science:1-13[2023-01-20]. http://kns.cnki.net/kcms/detail/11.2206.TS.20221110.2207.064.html. in Chinese)

[17]
杜瑞, 靳烨, 王柏辉, 等. 益生菌对苏尼特羊胃肠道菌群、代谢物及肉品质的影响[J]. 食品科学, 2020, 41(11):14-21.

DOI

DU R, JIN Y, WANG B H, et al. Dietary probiotics affect gastrointestinal microflora and metabolites and consequently improves meat quality in Sunit lambs[J]. Food Science, 2020, 41(11):14-21. (in Chinese)

DOI

[18]
窦露, 刘畅, 巴吉木色, 等. 日粮添加乳酸菌对苏尼特羊生长、肉品质、风味物质和抗氧化能力的影响[J]. 食品科学, 2021, 42(1):25-32.

DOU L, LIU C, BA J M S, et al. Effects of dietary lactic acid bacteria on growth,meat quality,flavor compounds and antioxidant capacity of Sunit sheep[J]. Food Science, 2021, 42(1):25-32. (in Chinese)

DOI

[19]
吴红翔, 欧阳捡云, 刘三凤, 等. 木寡糖益生素和益生素对广东麻鸡屠宰性能及肌肉品质的影响[J]. 江西农业大学学报, 2006, 28(6):899-902.

WU H X, OUYANG J Y, LIU S F, et al. Effect of xylooligosaccharides probiotics and probiotics on slaughter performance and meat quality in Guangdong hemp chicken[J]. Acta Agriculturae Universitatis Jiangxiensis, 2006, 28(6):899-902. (in Chinese)

[20]
MENG Q W, YAN L, AO X, et al. Influence of probiotics in different energy and nutrient density diets on growth performance,nutrient digestibility,meat quality,and blood characteristics in growing-finishing pigs[J]. Journal of Animal Science, 2010, 88(10):3320-3326.

DOI

[21]
ČESLOVAS J, VIGILIJUS J, ALMANTAS Š, et al. The effect of probiotics and phytobiotics on meat properties and quality in pigs[J]. Veterinarija ir Zootechnika, 2005, 29(51):80-84.

[22]
LIU Y H, LI Y Y, FENG X C, et al. Dietary supplementation with Clostridium butyricum modulates serum lipid metabolism,meat quality,and the amino acid and fatty acid composition of Peking ducks[J]. Poultry Science, 2018, 97(9):3218-3229.

DOI

[23]
刘向东. 限饲对绵羊骨骼肌发育和胶原沉积影响及机制的研究[D]. 硕士学位论文. 晋中: 山西农业大学, 2017.

LIU X D. Study on mechanisms and effects of restricted-diet on skeletal development and collagen accumulation in lambs[D]. Master’s Thesis. Jinzhong: Shanxi Agricultural University, 2017. (in Chinese)

[24]
申文雪, 刘金松, 范小燕, 等. 丁酸梭菌替代金霉素对肉鸡腿肌肉品质和抗氧化功能的影响[J]. 微生物学报, 2022, 62(4):1464-1472.

SHEN W X, LIU J S, FAN X Y, et al. Effect of Clostridium butyricum as a replacement of chlortetracycline on quality and antioxidation of broiler leg muscle[J]. Acta Microbiologica Sinica, 2022, 62(4):1464-1472. (in Chinese)

[25]
GIRON M, THOMAS M, DARDEVET D, et al. Gut microbes and muscle function:can probiotics make our muscles stronger?[J]. Journal of Cachexia,Sarcopenia and Muscle, 2022, 13(3):1460-1476.

DOI

[26]
ZHENG L F, WEI H K, HE P L, et al. Effects of supplementation of branched-chain amino acids to reduced-protein diet on skeletal muscle protein synthesis and degradation in the fed and fasted states in a piglet model[J]. Nutrients, 2016, 9(1):17.

DOI

[27]
段叶辉. 低蛋白日粮平衡BCAAs比例调控10-30 kg猪肌肉组织蛋白质与脂质代谢的机制研究[D]. 博士学位论文. 北京: 中国科学院大学, 2017.

DUAN Y H. Study on the mechanism of balanced BCAAs ratio in low protein diet regulating protein and lipid metabolism in muscle tissue of 10-30 kg pigs[D]. Ph.D.Thesis. Beijing: University of Chinese Academy of Sciences, 2017. (in Chinese)

[28]
王彬, 李奇. 亮氨酸的代谢及营养生理作用研究进展[J]. 饲料研究, 2012(1):14-16.

WANG B, LI Q. Progress in leucine metabolism and nutritional physiology[J]. Feed Research, 2012(1):14-16. (in Chinese)

[29]
FLORINI J R, EWTON D Z, COOLICAN S A. Growth hormone and the insulin-like growth factor system in myogenesis[J]. Endocrine Reviews, 1996, 17(5):481-517.

DOI PMID

[30]
YAN J, HERZOG J W, TSANG K, et al. Gut microbiota induce IGF-1 and promote bone formation and growth[J]. Proceedings of the National Academy of Sciences of the United States of America, 2016, 113(47):E7554-E7563.

[31]
吕欣, 周达岸. PI3K/AKT信号通路对骨骼肌再生的影响研究进展[J]. 中国运动医学杂志, 2020, 39(11):908-912.

LV X, ZHOU D A. Research progress on the effect of PI3K/AKT signal pathway on skeletal muscle regeneration[J]. Chinese Journal of Sports Medicine, 2020, 39(11):908-912. (in Chinese)

[32]
WEI X Y, LUO L F, CHEN J Z. Roles of mTOR signaling in tissue regeneration[J]. Cells, 2019, 8(9):1075.

DOI

[33]
李玉鹏, 李海花, 郭晓飞, 等. 丁酸梭菌介导mTOR信号通路调控猪肠上皮细胞炎症反应的分子机制[J]. 中国饲料, 2021(23):24-29.

LI Y P, LI H H, GUO X F, et al. The molecular mechanism of Clostridium butyricum mediated mTOR signaling pathway regulating the inflammatory response of porcine intestinal epithelial cells[J]. China Feed, 2021(23):24-29. (in Chinese)

[34]
LIU M, XIE W J, WAN X Y, et al. Clostridium butyricum protects intestinal barrier function via upregulation of tight junction proteins and activation of the Akt/mTOR signaling pathway in a mouse model of dextran sodium sulfate-induced colitis[J]. Experimental and Therapeutic Medicine, 2020, 20(5):10.

[35]
LIU P J, HU Y S, WANG M J, et al. Nutrient weight against sarcopenia:regulation of the IGF-1/PI3K/Akt/FOXO pathway in quinoa metabolites[J]. Current Opinion in Pharmacology, 2021, 61:136-141.

DOI

[36]
BINDELS L B, BECK R, SCHAKMAN O, et al. Restoring specific lactobacilli levels decreases inflammation and muscle atrophy markers in an acute leukemia mouse model[J]. PLoS One, 2012, 7(6):e37971.

DOI

[37]
LEE S J, MCPHERRON A C. Myostatin and the control of skeletal muscle mass:commentary[J]. Current Opinion in Genetics & Development, 1999, 9(5):604-607.

[38]
GE L H, XU Y S, XIA W S, et al. Synergistic action of cathepsin B,L,D and calpain in disassembly and degradation of myofibrillar protein of grass carp[J]. Food Research International, 2018, 109:481-488.

DOI

[39]
NORTH M K, FRYLINCK L, HOFFMAN L C. The physical and biochemical changes in springbok (Antidorcas marsupialis) longissimus thoracis et lumborum and Biceps femoris muscle during ageing[J]. Meat Science, 2015, 110:145-152.

DOI

Options
Outlines

/

〈 〉

京ICP备17050989号-2
Copyright © Chinese Journal of Animal Nutrition, All Rights Reserved.
Tel: 010－62817823　E-mail: cjan@vip.163.com
Powered by Beijing Magtech Co., Ltd.