Chinese Journal of Animal Nutrition >

2024 , Vol. 36 >Issue 9: 5883 - 5894

DOI: https://doi.org/10.12418/CJAN2024.500

RESEARCH PAPER

Effects of Dietary Supplementation with Clostridium butyrate or Sodium Butyrate on Intestinal Short-Chain Fatty Acids Contents, Digestive Enzymes Activities, Antioxidant Indexes and Intestinal Structure of Juvenile Largemouth Bass

  • HOU Dongqiang , 1, 2 ,
  • HU Junru 1, * ,
  • CHEN Bing 1 ,
  • PENG Kai 1 ,
  • LI Peijia 2 ,
  • HUANG Wen 1 ,
  • CAO Junming 3 ,
  • ZHAO Hongxia , 1, 4, **
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  • 1 Collaborative Innovation Center of Aquatic Sciences, Guangdong Key Laboratory of Animal Breeding and Nutrition, Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
  • 2 College of Fisheries, Guangdong Ocean University, Zhanjiang 524088, China
  • 3 Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
  • 4 Guangzhou Fishtech Fisheries Science and Technology Co., Ltd., Guangzhou 510640, China
** professor, E-mail:

* Contributed equally

Received date: 2024-01-16

  Online published: 2024-09-08

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Abstract

The purpose of this experiment is to study the effects of dietary supplementation with Clostridium butyrate (CB) or sodium butyrate (SB) on intestinal short-chain fatty acids contents, digestive enzymes activities, antioxidant indexes and intestinal structure of juvenile largemouth bass. Three iso-nitrogen (50%) and iso-lipid (9%) diets were prepared, which were the base diet (CON group), the base diet+10 mL/kg CB (CB group) and the base diet+1.0 g/kg SB (SB group), respectively. A total of 360 fish [(5.02±0.01) g] were randomly divided into 3 groups with 3 replicates per group and 40 fish per replicate. The experiment period was 56 days. The results showed as follows: 1) compared with the control group, the final weight and specific growth rate in CB group were significantly increased (P<0.05), but there was no significant difference in SB group (P>0.05). 2) Compared with the control group, the intestinal acetic acid content in CB group was significantly increased (P<0.05), and the contents of propionic acid, isobutyric acid and isovaleric acid in CB and SB groups were significantly increased (P<0.05). 3) Compared with the control group, dietary CB and SB supplementation significantly increased the activities of amylase, trypsin and lipase in the intestine (P<0.05), CB group significantly increased the activity of alkaline phosphatase in the intestine (P<0.05), and SB group significantly increased the activity of sodium-potassium-ATPase (P<0.05). 4) The intestinal catalase activity in CB and SB groups was significantly higher than that in control group (P<0.05). Compared with the control group, the malondialdehyde content in SB group was significantly decreased (P<0.05), and the total antioxidant capacity in CB group was significantly increased (P<0.05). 5) Compared with the control group, the villi width in the middle intestine in CB group was significantly increased (P<0.05), which alleviated intestinal inflammation. In conclusion, dietary CB significantly improve the growth performance of largemouth bass juvenile fish, and the addition of CB or SB significantly increase the intestinal short-chain fatty acid contents of largemouth bass, significantly improve the activities of digestive enzymes and antioxidant indexes, and improve the intestinal structure. The addition of 10 mL/kg CB has better effects on growth performance, antioxidant indexes and intestinal villus height.

Key words： largemouth bass; Clostridium butyricum; sodium butyrate; digestive enzymes; antioxidant enzymes; intestinal structure

Cite this article

HOU Dongqiang , HU Junru , CHEN Bing , PENG Kai , LI Peijia , HUANG Wen , CAO Junming , ZHAO Hongxia . Effects of Dietary Supplementation with Clostridium butyrate or Sodium Butyrate on Intestinal Short-Chain Fatty Acids Contents, Digestive Enzymes Activities, Antioxidant Indexes and Intestinal Structure of Juvenile Largemouth Bass[J]. Chinese Journal of Animal Nutrition, 2024 , 36(9) : 5883 -5894 . DOI: 10.12418/CJAN2024.500

水产养殖业的重要使命在于推动食品安全以及保护海洋资源,为人类提供优质蛋白质来源[1-2]。随着水产养殖业的迅猛发展,高密度集约化养殖导致的鱼类生存环境恶化引发了生理和免疫层面的应激反应以及病原体的侵袭,严重影响了肠道生态平衡[3-4]。此外,植物蛋白质中含有大量抗营养因子和不平衡的必需氨基酸,过量添加会导致鱼的肠道炎症、免疫力下降和鱼类生长缓慢[5⇓-7]。肠道由肠道屏障、免疫细胞和肠道菌群组成,功能包括营养物质的消化和吸收以及对抗外界环境的不利因素,肠道健康对于动物的整体健康具有重要意义[8⇓⇓-11]。
益生菌在改善水环境和动物的生长发育方面具有积极作用[12]。丁酸作为肠道上皮细胞主要的能量物质可改善肠道结构,同时进入细菌细胞内抑制有害菌增殖,促进益生菌繁殖[13]。糖类经过丁酸梭菌(Clostridium butyricum, CB)发酵可产生甲酸、乙酸和丁酸等短链脂肪酸,这些短链脂肪酸可治疗由肠道菌群紊乱引起的肠道炎症,促进肠道蠕动,也能被肠道所吸收[14-15]。研究表明,CB在改善凡纳滨对虾(Litopenaeus vannamei)肠道结构、增加丁酸等短链脂肪酸含量和修复肠道损伤等方面作用显著[16]。CB在维持肠道菌群平衡、提供营养元素和提高肠道消化酶活性方面起到重要作用[14]。丁酸钠(sodium butyrate, SB)作为丁酸与钠元素结合的产物,具有较好的稳定性和诱食效果[17],SB进入肠道后会水解成丁酸根离子(CH3-CH2-CH2-COO-)和钠离子(Na+),酸根离子被肠黏膜细胞吸收,为黏膜细胞的增殖提供能量[18]。研究表明,饲料中添加SB可促进草鱼(Ctenopharyngodon idella)[19]、大菱鲆(Scophthalmus maximus)[4]、黄颡鱼(Pelteobagrus fulvidraco)[20]和黄姑鱼(Nibea albiflora)[21]生长和改善肠道健康。饲料中添加2.5 g/kg SB显著提高了鲫鱼(Carassius auratus)增重率、特定生长率,并增加了前肠绒毛高度[22];在鲤鱼饲料中添加0.3 g/kg的SB提高了饲料转化率[23]。研究表明,CB或SB能够通过在肠道中释放或代谢产生丁酸,改善肠道环境促进生长,提高免疫和抗氧化功能增强机体抗环境应激能力[24-25],但两者对肠道健康的影响和作用机制是否相同仍不清楚。
大口黑鲈(Micropterus salmoides)具有适应性强、繁殖周期短、肉质鲜嫩等优点,深受渔民和消费者的喜爱[26-27]。我国大口黑鲈2022年总产量在60万t左右,其中广东省是大口黑鲈的主要养殖地,占中国国内总产量的60%以上[28]。水产养殖中病原菌侵害和植物蛋白质源中抗营养因子导致的肠道受损限制了大口黑鲈养殖的可持续发展,凸显了水产养殖需要可持续解决方案。深入研究能够保护鱼类肠道健康、改善肠道生态环境的绿色健康饲料添加剂,对于水产养殖业的可持续发展具有重要而深远的意义。本实验室前期研究发现,大口黑鲈饲料中CB的适宜添加量为10 mL/kg[29],SB的适宜添加量为1.0 g/kg[30]。因此,本试验通过在饲料中添加CB或SB,比较CB和SB对大口黑鲈肠道短链脂肪酸含量、肠道消化酶活性与抗氧化指标以及肠道结构的影响,为CB及SB在大口黑鲈饲料中的应用提供相关的理论依据。

1 材料与方法

1.1 试验饲料

本研究均按照广东省农业科学院实验动物使用指南进行,由广东省农业科学院动物实验伦理委员会正式批准 (SC-GDAAS-2021-032)。饲料以鱼粉和豆粕为主要蛋白质源,鱼油和豆油为脂肪源,配制成3种等氮(50%)和等脂(9%)的饲料,分别为基础饲料(对照组)、基础饲料+10 mL/kg CB(CB组)、基础饲料+1.0 g/kg SB(SB组)。CB是菌落数为8×108 CFU/mL的液态CB,SB含量为80%。饲料中CB菌落数实测值为1.5×109 CFU/kg,SB含量实测值为0.75 g/kg。原料经过研磨、过筛(60目),然后根据配方准确称重,并逐级混匀。将鱼油和豆油加入到混匀的原料中充分混合,然后分别将CB和SB加入到水中与原料混合,制成2 mm颗粒饲料(SLX-80双螺杆挤出机,华南理工大学机械厂),在烘干机中55 ℃干燥6 h,将制备好的饲料放置在-20 ℃备用。试验基础饲料组成及营养水平参考本实验室前期研究[30]。

1.2 试验设计及饲养管理

大口黑鲈鱼苗购自广州景龙渔业(中国广州)。将大口黑鲈幼鱼在室内循环系统中暂养7 d,每天投喂2次基础饲料。暂养结束后,将360尾均重为(5.02±0.01) g、健康的大口黑鲈幼鱼随机分为3组,分别饲喂3种饲料,每组3个重复,分配到9个玻璃纤维缸(300 L)中,每个缸中40尾鱼。试验期为56 d。每天喂食2次,并记录每个缸中鱼的饲料消耗量。在56 d的养殖试验中,水温在28~31 ℃,pH为7.3~7.7,氨氮含量≤0.02 mg/L,亚硝酸盐含量≤0.2 mg/L,溶解氧含量≥6.0 mg/L,光周期为自然光照。

1.3 采集样品

饲养试验结束后,待试验鱼被禁食24 h后,用40 mg/L的MS-222麻醉后称重和计数。每个缸取6尾鱼称体重、肠道重,并测量体长。分离鱼的腹部脂肪并称重,用于计算腹脂率(AFR)。每个缸取3尾鱼的肠道收集在5 mL冻存管中,先暂存在液氮中,然后保存在-80 ℃,用于测定肠道消化酶活性、抗氧化指标以及短链脂肪酸含量。将每缸中3尾鱼的肠道分为前肠、中肠和后肠,分别固定在10%福尔马林溶液中,然后用苏木精-伊红染色,使用标准组织学技术制作肠道切片。

1.4 指标测定

1.4.1 营养成分测定

饲料和鱼体中的水分、粗蛋白质、粗脂肪和粗灰分含量分别参考国标方法:干燥恒重法(GB 5009.3—2016)、凯氏定氮法(GB 5009.5—2016)、索氏抽提法(GB 5009.6—2016)和马弗炉550 ℃高温灼烧法(GB 5009.4—2016)测定。

1.4.2 生长性能和形体指标

相关计算公式如下:
特定生长率(SGR, %/d)=100×[ln终末鱼体重(g)-ln初始鱼体重(g)]/试验天数;
肝体比(HSI,%)=100×肝脏重(g)/鱼体重(g);
肥满度(CF, g/cm3)=100×鱼体重(g)/鱼体长(cm)3;
存活率(SR,%)=100×终末鱼总数量(尾)/初始鱼总数量(尾);
AFR(%)=100×腹脂重(g)/鱼体重(g);
饲料效率(FE,%)=100×[终末鱼总重(g)-初始鱼总重(g)]/总摄食量(g);
摄食率(FI,%)=100×总摄食量(g)/{[终末鱼总重(g)+初始鱼总重(g)]/2×试验天数(d)}。

1.4.3 肠道消化酶与刷状边缘酶活性以及抗氧化指标测定

根据制造商的说明,使用商业试剂盒(南京建成生物工程研究所)测定肠道淀粉酶(AMS)、胰蛋白酶、脂肪酶(LPS)、碱性磷酸酶(AKP)、γ-谷氨酰转移酶(γ-GT)、肌酸激酶(CK)、钠钾ATP酶(Na+/K+ ATPase)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、过氧化物酶(POD)、谷胱甘肽过氧化物酶(GSH-Px)活性及丙二醛(MDA)含量和总抗氧化能力(T-AOC)。

1.4.4 肠道短链脂肪酸含量测定

肠道短链脂肪酸含量由气相色谱-质谱仪(GC-MS)测定。取样品0.1 g左右于10 mL离心管中,加入2 mL水(10%磷酸水溶液),搅动让样品混匀,加入2 mL乙醚萃取5 min,然后在2 400×g离心15 min;离心后取乙醚相,再分别加入1 mL乙醚按相同的步骤提取2次,合并3次提取液,定容,放入GC-MS仪进行测定分析。

1.5 数据统计

使用SPSS 25.0软件对试验数据进行单因素方差分析(one-way ANOVA),差异显著时采用Duncan氏法进行组间多重比较,P<0.05表示差异显著。

2 结果与分析

2.1 生长性能和形体指标

由表1可知,与对照组相比,CB组终末体重、SGR和FE显著提高(P<0.05);与对照组和SB组相比,CB组FI显著降低(P<0.05)。各组间的初始体重、AFR、SR、CF和HSI无显著差异(P>0.05)。
表1 饲料中添加丁酸梭菌或丁酸钠对大口黑鲈生长性能和形体指标的影响

Table 1 Effects of dietary Clostridium butyrate or sodium butyrate on growth performance and body parameters of largemouth bass

项目
Items		 组别Groups
对照CON CB SB
初始体重IW/g 5.02±0.01 5.02±0.01 5.02±0.01
终末体重FW/g 50.54±1.47a 56.10±0.16b 52.39±0.94ab
特定生长率SGR/(%/d) 4.11±0.05a 4.30±0.01b 4.17±0.01ab
摄食率FI/% 3.56±0.07b 3.33±0.05a 3.63±0.04b
饲料效率FE/% 82.10±2.19a 89.50±1.26b 81.03±0.92a
肥满度CF/(g/cm3) 2.02±0.04 2.04±0.08 1.94±0.12
肝体比HSI/% 0.82±0.02 0.76±0.03 0.81±0.03
腹脂率AFR/% 1.31±0.11 1.53±0.14 1.39±0.08
存活率SR/% 99.33±0.67 100.00±0.00 100.00±0.00

同行数据肩标相同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 or no letter superscripts mean no significant difference (P>0.05), while with different letter superscripts mean significant difference (P<0.05). The same as below.

2.2 肠道短链脂肪酸含量

由表2可知,CB组肠道乙酸含量显著高于对照组(P<0.05);与对照组相比,CB组和SB组肠道丙酸、异丁酸和异戊酸含量显著提高(P<0.05)。各组间肠道正丁酸和正戊酸含量无显著差异(P>0.05)。
表2 饲料中添加丁酸梭菌或丁酸钠对大口黑鲈肠道短链脂肪酸含量的影响

Table 2 Effects of dietary Clostridium butyrate or sodium butyrate on intestinal short-chain fatty acid contents of largemouth bassmg/g

项目
Items		 组别Groups
对照CON CB SB
乙酸Acetic acid 1 212.37±132.72a 2 406.40±485.82b 1 605.73±114.08ab
丙酸Propionic acid 96.17±9.91a 158.65±28.95b 211.57±14.68b
正丁酸n-butyric acid 84.10±18.63 105.67±21.57 60.50±3.54
异丁酸iso-butyric acid 90.23±3.53a 123.07±1.43b 142.75±13.65b
正戊酸n-valeric acid 21.07±3.90 32.90±7.72 31.80±6.50
异戊酸iso-valeric acid 21.00±4.43a 54.50±6.57b 54.43±3.81b

2.3 肠道消化酶和刷状边缘酶活性

由表3可知,与对照组相比,饲料中添加CB和SB可显著提高肠道AMS、胰蛋白酶和LPS活性(P<0.05),CB组肠道AKP活性显著提高(P<0.05),SB组肠道Na+/K+ ATPase活性显著提高(P<0.05)。各组间肠道γ-GT和CK活性无显著差异(P>0.05)。
表3 饲料中添加丁酸梭菌或丁酸钠对大口黑鲈肠道消化酶和刷状边缘酶活性的影响

Table 3 Effects of dietary Clostridium butyrate or sodium butyrate on intestinal digestive enzyme and brush edge enzyme activities of largemouth bassU/mg prot

项目
Items		 组别Groups
对照CON CB SB
淀粉酶AMS 25.01±5.01a 60.94±3.95b 59.66±3.33b
胰蛋白酶Trypsin 634.81±62.08a 983.71±45.54b 1203.21±37.90c
脂肪酶LPS 21.52±0.99a 39.90±0.62b 36.53±2.48b
碱性磷酸酶AKP 54.65±2.14a 77.35±2.89b 65.01±5.59ab
钠钾ATP酶Na+/K+ ATPase 0.93±0.08a 1.51±0.14b 2.07±0.25c
肌酸激酶CK 0.64±0.09 0.86±0.02 0.85±0.14
γ-谷氨酰基转移酶γ-GT 21.84±1.86 29.65±0.48 33.56±5.69

2.4 肠道抗氧化指标

由表4可知,CB组和SB组肠道CAT活性显著高于对照组(P<0.05)。与对照组相比,SB组肠道MDA含量显著降低(P<0.05),CB组肠道T-AOC显著提高(P<0.05)。各组间肠道POD、SOD和GSH-Px活性无显著差异(P>0.05)。
表4 饲料中添加丁酸梭菌或丁酸钠对大口黑鲈肠道抗氧化指标的影响

Table 4 Effects of dietary Clostridium butyrate or sodium butyrate on intestinal antioxidant indexes of largemouth bass

项目
Items		 组别Groups
对照CON CB SB
过氧化氢酶CAT/(U/mg prot) 61.51±3.98a 86.99±1.33b 79.26±2.67b
过氧化物酶POD/(U/mg prot) 4.33±0.61 5.54±0.27 5.94±0.88
丙二醛MDA/(nmol/mg prot) 3.83±0.28b 2.85±0.14ab 1.91±0.42a
总抗氧化能力T-AOC/(U/mg prot) 1.39±0.16a 2.60±0.10b 1.56±0.01a
超氧化物歧化酶SOD/(U/mg prot) 218.62±8.83 282.61±31.66 271.44±42.89
谷胱甘肽过氧化物酶GSH-Px/(U/mg prot) 377.97±36.17 447.32±24.01 414.73±34.22

2.5 肠道结构

由表5可知,与对照组相比,CB组中肠绒毛宽度显著增加(P<0.05),CB组前肠、中肠和后肠肌层厚度有增加的趋势。
表5 饲料中添加丁酸梭菌或丁酸钠对大口黑鲈肠道结构的影响

Table 5 Effects of dietary Clostridium butyrate or sodium butyrate on intestinal structure of largemouth bassmm

项目
Items		 组别Groups
对照CON CB SB


前肠Foregut		 绒毛高度Villus height 0.36±0.01 0.37±0.03 0.37±0.07
绒毛宽度Villus width 0.10±0.01 0.11±0.01 0.12±0.01
肌层厚度Muscular thickness 0.28±0.02 0.31±0.07 0.30±0.04


中肠Midgut		 绒毛高度Villus height 0.50±0.04 0.50±0.06 0.46±0.04
绒毛宽度Villus width 0.10±0.01a 0.12±0.01b 0.11±0.01ab
肌层厚度Muscular thickness 0.20±0.02 0.22±0.06 0.19±0.02


后肠Hindgut		 绒毛高度Villus height 0.58±0.02 0.58±0.05 0.50±0.01
绒毛宽度Villus width 0.10±0.01 0.10±0.01 0.12±0.01
肌层厚度Muscular thickness 0.16±0.02 0.19±0.06 0.14±0.03
如图1所示,各组大口黑鲈黏膜层少量绒毛上皮细胞脱落,胞核固缩,胞浆嗜酸性增强(黑色箭头),固有层偶见淋巴组织小灶性浸润(红色箭头),绒毛数量丰富,固有层结构紧密,肌层肌纤维着色均匀,黏膜层绒毛丰富,形态正常,未见其他明显异常。
图1 大口黑鲈肠道组织结构

CON: CON组 CON group; CB:CB组 CB group; SB: SB组 SB group。

黑色箭头:胞质嗜酸性增强;红色箭头:淋巴细胞浸润。Black arrow: cytoplasmic eosinophilic enhancement; Red arrow: lymphocyte infiltration.

Fig.1 Intestinal structure of largemouth bass

3 讨论

3.1 饲料中添加丁酸梭菌或丁酸钠对大口黑鲈生长性能的影响

短链脂肪酸,俗称挥发性脂肪酸,包括乙酸、丙酸、丁酸、异丁酸、戊酸和异戊酸等,是肠道中极为重要的一类化合物,这些短链脂肪酸在维持肠道健康、调节免疫功能以及促进营养物质的吸收等方面起着至关重要的作用[31]。不同的短链脂肪酸在机体中的去向、分布以及生理作用有所不同,乙酸主要被外周器官和组织吸收,丙酸被肝脏代谢,丁酸被肠道吸收利用[32]。肠道细菌厌氧发酵产生短链脂肪酸,其含量增加与肠道菌群变化密切相关,肠道菌群种类间竞争使得优势细菌在肠道定植,而劣势细菌会被淘汰[33]。能够产丁酸的细菌以乳酸为底物,可以将乳酸转化为丁酸,丁酸可为乳酸菌等提供适宜环境[34]。在本研究中,饲料中添加CB可增加大口黑鲈肠道中的乙酸含量,此外,CB组和SB组肠道中丙酸、异丁酸和异戊酸含量显著提高。研究表明,CB可以显著增加肠道乙酸、丙酸和丁酸的含量[35]。短链脂肪酸及其盐被认为是安全的促生长剂,通过提高水生生物胃肠道消化吸收能力来促进动物的生长[36]。在饲料中添加CB可以提高大黄鱼(Larimichthys crocea)[37]和杂交罗非鱼(Oreochromis niloticus×O.aureus)[38]的生长性能,同样,SB在卵形鲳鲹幼鱼(Trachinotus ovatus)[39]、刺参(Apostichopus japonicus)[40]、黑鲷(Acanthopagrus schlegelii)[41]和彭泽鲫(Carassius auratus Pengze)[42]的研究中也得出相似结论。在本研究中,CB组大口黑鲈的终末体重、SGR和FE显著提高,CB组FI显著降低,表明CB可以提高大口黑鲈消化能力,促进营养物质的消化吸收,SB组与对照组相比生长性能指标无显著差异。CB在肠道中发酵产生的丁酸等短链脂肪酸可作为肠上皮细胞的能量物质,促进肠绒毛生长[43]。综上所述,CB通过增加肠道短链脂肪酸的含量促进肠道发育,从而提高大口黑鲈生长性能,与SB相比,CB在提高大口黑鲈生长性能方面效果更佳。

3.2 饲料中添加丁酸梭菌或丁酸钠对大口黑鲈肠道消化酶和刷状边缘酶活性的影响

肠道是鱼类最主要的营养物质消化和吸收的部位,消化酶活性是评估其消化功能的重要指标,消化酶活性增加可促进机体对营养物质的消化吸收[44⇓-46]。研究发现,在银鲳鱼(Pampus argenteus)饲料中添加益生菌可提高肠道消化酶活性[47]。Suzer等[48]在金头鲷(Sparus aurata, L.)饲料中添加乳酸杆菌,发现其显著提高了金头鲷幼鱼肠道消化酶的活性,从而提高饲料利用率。Tian等[49]研究发现,丁酸盐可显著提高草鱼的肠道消化酶活性。同样,饲喂军曹鱼(Lates calcarifer)添加丁酸盐的饲料,显著提高了鱼的肠道蛋白酶、脂肪酶和α-淀粉酶活性[50]。在本研究中,饲料中添加CB和SB可显著提高大口黑鲈肠道AMS、胰蛋白酶和LPS活性。CB在肠道中可产生消化性胞外酶(如蛋白酶、LPS、AMS等)和非淀粉多糖酶(如胶酶、纤维素酶、葡聚糖酶)[51]。CB组大口黑鲈肠道消化酶活性提高的原因与CB在肠道中产生多种消化酶有关,而SB组消化酶活性增强与丁酸刺激消化酶的分泌有关[23]。肠绒毛形成细刷结构,刷状缘上细胞含有丰富的酶(包括AKP、CK和Na+/K+ ATPase),提高了消化酶的效率。肠道AKP可水解单磷酸脂,在维持肠道黏膜屏障的稳定以及肠道功能方面起到重要作用[52]。Na+/K+ ATPase可调节细胞内外渗透压,为营养物质的运输提供势能[53]。本研究发现,大口黑鲈饲喂添加CB的饲料可显著提高肠道AKP活性,并且饲喂添加SB的饲料显著提高了Na+/K+ ATPase活性,表明CB和SB可提高刷状边缘酶活性,CB可提高肠黏膜稳定性,而SB在提高营养物质运输上更具优势,但其作用机制仍需进一步探究。与本试验得出结论一致,与对照组相比,饲喂黄颡鱼幼鱼添加SB的饲料可增加肠道消化酶和刷状边缘酶活性[20]。总之,CB和SB均可提高大口黑鲈肠道消化酶和刷状边缘酶活性,在改善肠道消化和吸收能力方面起到积极作用。

3.3 饲料中添加丁酸梭菌或丁酸钠对大口黑鲈肠道抗氧化指标的影响

鱼类的肠道是相对敏感的器官,容易受到有毒物质和氧化应激的影响,由于肠道与外界相连,使其更容易受到外界环境和饮食因素的影响[54-55]。鱼类抵御外界干扰主要依靠复杂的抗氧化系统,其中包括酶类,如GSH-Px、CAT、SOD以及POD,以及非酶类抗氧化物质,如还原型谷胱甘肽(GSH)来进行修复氧化损伤并清除体内的自由基,以维持健康状态[55-56]。MDA是脂质与活性氧氧化反应的有毒产物,其含量的升高会引起生物细胞受损、机体老化和疾病的发生[57-58]。研究表明,饲喂虹鳟包膜的SB可显著提高肠道的SOD、CAT和GSH-Px的活性[59]。另一项研究中,饲料中添加500和1 000 mg/kg SB组黄颡鱼肠道SOD、CAT和GSH-Px活性显著提高,显著降低了氨胁迫下黄颡鱼的死亡率,增强了抗氧化能力[60]。在CB的研究中发现,CB可显著降低罗非鱼肠道MDA含量[61],CB提高杂交石斑鱼(Epinephelus fuscoguttatus×E. lanceolatus)脾脏中CAT活性[62]。CB可在肠道中产生还原型辅酶Ⅰ或还原型辅酶Ⅱ过氧化物酶,可清除活性氧,增强肠道抗氧化能力[63]。本研究中,饲料中添加CB或SB显著提高了肠道CAT活性和T-AOC,SB显著降低MDA含量。结果表明,饲料中添加CB和SB有利于清除自由基,并且减轻大口黑鲈肠道氧化造成的损伤。

3.4 饲料添中加丁酸梭菌或丁酸钠对大口黑鲈肠道结构的影响

肠道机械屏障包括肠黏膜上皮细胞、细胞间紧密连接以及肠道的运动功能等,是最重要的物理屏障[64-65],丁酸等短链脂肪酸直接为肠上皮细胞提供能量,促进肠绒毛生长,增强肠道物理屏障[44]。保证肠道的完整性可有效防止有毒物质的侵袭[66]。丁酸是肠道上皮细胞利用效率最高的短链脂肪酸,占能量供求的70%[34]。CB可增加大菱鲆肠道上皮细胞的厚度、宽度和高度,也可显著上调紧密连接蛋白的表达量[67]。饲喂凡纳滨对虾不同剂量的CB发现,各添加组的肠道绒毛高度和肠壁厚度显著高于对照组[68]。类似研究发现,SB可改善尼罗罗非鱼(Oreochromis nilotica)幼鱼[69]和巨骨舌鱼(Arapaima gigas)[70]肠道绒毛的高度和宽度。本研究中,CB组中肠绒毛宽度显著增加,前肠、中肠和后肠肌层厚度有增加趋势。由肠道组织结构图可以看出,对照组肠绒毛胞浆嗜酸性增强,SB组固有层偶见淋巴组织小灶性浸润,CB组胞浆嗜酸性和淋巴组织浸润显著减少。结果表明,CB在减轻肠道炎症反应和促进细胞增殖方面作用显著。综上所述,饲料中添加CB显著提高了中肠绒毛宽度,并且减轻肠道炎症,肠道结构的改善与肠道中短链脂肪酸含量的增加密切相关。

4 结论

综上所述,饲料中添加CB显著提高了大口黑鲈终末体重、SGR和FE,显著提高了生长性能。饲喂CB和SB显著提高大口黑鲈肠道短链脂肪酸含量、肠道消化酶活性和抗氧化指标,增强了消化能力和抗氧化能力。CB增加了大口黑鲈肠道绒毛宽度,减轻了肠道炎症反应。本试验条件下,结合生长性能、肠道抗氧化指标和肠道结构综合评价,与SB相比,饲料中添加10 mL/kg的CB对提高大口黑鲈生长性能、肠道抗氧化酶活性以及增加肠道绒毛宽度作用更佳。

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