Chinese Journal of Animal Nutrition >

2024 , Vol. 36 >Issue 4: 2572 - 2585

DOI: https://doi.org/10.12418/CJAN2024.221

RESEARCH PAPER

Effects of Dietary Supplementation of Clostridium butyricum or Sodium Butyrate on Growth Performance, Serum Biochemical Indexes, Liver Immune and Antioxidant Capacity of Largemouth Bass

  • HOU Dongqiang , 1, 2 ,
  • LI Min 1, 2 ,
  • LI Peijia 2 ,
  • PENG Kai 2 ,
  • CHEN Bing 2 ,
  • HUANG Wen 2 ,
  • CAO Junming 2 ,
  • GUO Hui 1 ,
  • ZHAO Hongxia , 2
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  • 1 College of Fisheries, Guangdong Ocean University, Zhanjiang 524088, China
  • 2 Key Laboratory of Animal Nutrition and Feed in South China, Ministry of Agriculture and Rural Affairs, Guangdong Provincial Key Laboratory of Animal Breeding and Nutrition Research, Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
**professor, E-mail:

*Contributed equally

Received date: 2023-08-22

  Online published: 2024-04-15

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Abstract

The aim of this experiment was to investigate the effects of adding Clostridium butyricum (CB) or sodium butyrate (SB) to the diet on growth performance, serum biochemical indexes, liver immunity, antioxidant capacity and hypoxia stress of juvenile largemouth bass (Micropterus salmoides). Three kinds of iso-nitrogen (50%) and iso-lipid (9%) diets were prepared, which were the basal diet (Con), the basal diet supplemented with 10 mL/kg CB, and the basal diet supplemented with 1.0 g/kg SB. A total of 360 fish with body weight of (5.02±0.01) g were randomly divided into 3 groups and fed 3 kinds of diets with 3 replicates per group and 40 fish per replicate. The culture experiment was carried out for 8 weeks, and then hypoxia stress test was carried out. The results showed as follows: 1) compared with Con group, weight gain rate (WGR) and protein deposition rate (PDR) in CB group were significantly increased (P<0.05), and feed conversion ratio (FCR) in CB group was significantly decreased (P<0.05). Compared with Con group, the intestinal body index (ISI) in CB group was significantly increased (P<0.05). 2) Compared with Con group, the serum alkaline phosphatase (AKP) activity and triglyceride (TG) content in SB group were significantly decreased (P<0.05), the serum urea nitrogen content in CB and SB groups was significantly decreased (P<0.05), and the serum glucose (GLU) content in SB group was significantly increased (P<0.05). 3) Compared with Con group, dietary CB and SB supplementation significantly increased liver total protein (TP) content (P<0.05), and liver catalase (CAT), superoxide dismutase (SOD) and lysozyme (LZM) activities in CB and SB groups were significantly increased (P<0.05). The liver malondialdehyde (MDA) content in SB group was significantly lower than that in Con group (P<0.05). 4) Compared with Con group, the relative expression level of Toll-like receptor 22 (TLR22) gene in liver of SB group was significantly increased (P<0.05). The relative expression levels of interleukin-1β (IL-1β), interleukin-8 (IL-8) and myeloid differentiation factor 88 (MyD88) genes in CB and SB groups were significantly decreased (P<0.05), and the relative expression levels of insulin-like growth factor-1 (IGF-1) and growth hormone (GH) genes in CB group were significantly increased (P<0.05). 5) After 3 h of hypoxia stress, the cumulative mortality of Con, CB and SB groups was 68.75%, 31.25% and 35.42%, respectively. Compared with Con group, the cumulative mortality in CB group and SB group was significantly decreased (P<0.05). In conclusion, dietary supplementation of CB and SB can improve the growth performance, liver antioxidant enzyme activity, inhibit the expression of liver anti-inflammatory factors of largemouth bass, and improve the survival rate under hypoxia stress. Compared with SB, CB supplementation has better effects on growth performance, immunity and antioxidant of largemouth bass.

Key words： largemouth bass; Clostridium butyricum; sodium butyrate; growth performance; immunity; antioxidant

Cite this article

HOU Dongqiang , LI Min , LI Peijia , PENG Kai , CHEN Bing , HUANG Wen , CAO Junming , GUO Hui , ZHAO Hongxia . Effects of Dietary Supplementation of Clostridium butyricum or Sodium Butyrate on Growth Performance, Serum Biochemical Indexes, Liver Immune and Antioxidant Capacity of Largemouth Bass[J]. Chinese Journal of Animal Nutrition, 2024 , 36(4) : 2572 -2585 . DOI: 10.12418/CJAN2024.221

大口黑鲈(Micropterus salmoides)因其美味可口的肉质、迅猛的生长速度以及显著的经济价值,在中国市场受到热烈欢迎[1-2]。随着集约化大规模的养殖模式的推广,高密度的养殖导致水环境的恶化,并且应激反应增加,氧化应激和其他环境应激的因素包括环境改变、饮食、外源性致病菌和病毒[3⇓-5]。水中溶解氧降低产生的低氧应激对水产动物生长以及生理状态产生负面影响,低氧应激也是大口黑鲈养殖过程中常见的问题[6]。在低氧应激条件下,生物体会产生过量的活性氧,活性氧的积累可能导致蛋白质结构变性、脂质过氧化以及细胞损伤,最终导致细胞凋亡,发生氧化损伤,刺激自身免疫系统[7⇓-9]。在急性缺氧条件下,尼罗罗非鱼(Oreochromis nilotica)的肌糖原和肝糖含量显著降低,乳酸脱氢酶(LDH)活性增加,并对生长和免疫产生负面影响[10-11]。慢性缺氧可导致欧洲舌齿鲈(Dicentrarchus labrax)肝脏中的糖原含量降低,并增加乳酸含量,表明缺氧刺激了厌氧糖酵解途径[12]。低氧应激已被证明了可影响水产动物的健康,这一问题制约了大口黑鲈养殖的可持续发展。
短链脂肪酸(SCFAs,如甲酸、乙酸和丁酸等)进入肠道之后可改变肠道酸度并且可以影响矿物质的运输[13-14]。SCFAs还具有与元素螯合形成复合物的能力,另外,可促进胃肠道黏膜上皮细胞增殖,增大营养吸收面积,促进生长[15⇓-17]。丁酸梭菌(Clostridium butyricum,CB)是一种严格厌氧细菌,产生的丁酸被广泛用于改善生长性能、抗氧化和免疫[18⇓-20]。CB通过消化碳水化合物产生丁酸,丁酸可促进肠道结构发育、提高消化酶活性[21⇓⇓-24],通过降低肠道pH抑制致病菌的增殖[25],并且可刺激抗氧化酶和免疫球蛋白M的产生[26]。在银鲳(Pampus argenteus)[27]、大黄鱼(Larimichthys crocea)[28]、尼罗罗非鱼[29]和罗氏沼虾(Macrobrachium rosenbergii)[22]中的研究发现,添加107~1011 CFU/kg CB可显著改善生长性能和提高抗氧化能力。丁酸与钠元素螯合可产生丁酸钠(sodium butyrate,SB),具有较好的稳定性和适口性[30]。最近一些研究发现,SB在提高免疫力和抗氧化能力方面效果显著,可提高尼罗罗非鱼抗氧化酶活性[如超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-Px)][31],上调虹鳟鱼(Oncorhynchus mykiss)肠道SOD的表达水平,提高肠道消化酶活性以及降低肠道肿瘤坏死因子-α(TNF-α)的表达[18],此外,适当剂量的SB也可通过增强抗氧化能力和免疫反应来改善黄颡鱼(Pelteobagrus fulvidraco)幼鱼对氨应激的抵抗力[32]。
CB在肠道中的主要代谢产物为丁酸,而SB在肠道内主要也是释放丁酸,两者的作用机制相同,都是通过丁酸改善肠道环境促进生长、进入细菌内扰乱细菌细胞代谢最终导致细菌死亡[33]以及提高免疫相关基因的表达增强机体免疫[34]。根据本实验室先前开展的试验结果表明,大口黑鲈饲料中CB的适宜添加量为10 mL/kg[35]、SB的适宜添加量为1.0 g/kg。因此,本试验通过在饲料中添加CB或SB,研究CB和SB对大口黑鲈幼鱼生长、血清生化指标和抗低氧应激能力的影响,比较CB和SB在大口黑鲈养殖中的作用效果,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 试验材料

液态CB的菌落计数为8×108 CFU/mL;SB的纯度为80%。

1.2 试验饲料

基础饲料组成及营养水平见表1。试验所用蛋白质和脂质主要来自鱼粉、豆粕、鱼油和大豆油。配制成3种等氮(50%)和等脂(9%)的饲料,分别为基础饲料(Con)、基础饲料中添加10 mL/kg的CB的试验饲料、基础饲料中添加1.0 g/kg的SB的试验饲料。饲料中CB实测值为1.5×109 CFU/kg, SB实测值为0.75 g/kg。将所有成分研磨,并通过60目筛,根据配方准确称重,之后逐级混匀。将鱼油和豆油加入到混匀的原料中充分混合,然后将CB和SB加入到水中并与其他原料混合,混合后制成2 mm颗粒饲料(SLX-80双螺杆挤出机,华南理工大学机械厂),在55 ℃下干燥6 h,将制备好的饲料放置在-20 ℃备用。
表1 基础饲料组成及营养水平(干物质基础)

Table 1 Composition and nutrient levels of the basal diet (DM basis)%

项目 Items 含量 Contents
原料 Ingredients
鱼粉 Fish meal 35.00
大豆浓缩蛋白 Soy protein concentrate 9.00
血球蛋白粉 Haemoglobin powder 3.00
虾壳粉 Shrimp shell meal 5.00
豆粕 Soybean meal 20.00
棉籽蛋白粉 Cottonseed protein meal 9.00
木薯粉 Tapioca meal 9.00
鱼油 Fish oil 3.00
大豆油 Soybean oil 3.00
维生素预混料 Vitamin premix1) 1.00
矿物质预混料 Mineral premix2) 1.00
磷酸二氢钙 Ca(H2PO4)2 1.00
氯化胆碱 Choline chloride 0.20
海藻酸钠 Sodium alginate 0.80
合计 Total 100.00
营养水平 Nutrient levels3)
粗蛋白质 Crude protein 50.55
粗脂肪 Crude lipid 9.63
粗灰分 Ash 12.44

1)每千克维生素预混料含有 One kilogram of vitamin premix contained the following:VA 4 000 000 IU,VD3 2 000 000 IU,VE 30 g,VK3 10 g,VB1 5 g,VB2 15 g,VB6 8 g,VB12 0.02 g,烟酸 nicotinic acid 40 g,泛酸钙 calcium pantothenate 25 g,叶酸 folic acid 2.5 g,肌醇 nicotinic acid 150 g,生物素 biotin 0.08 g。

2)每千克矿物质预混料含有 One kilogram of mineral premix contained the following:MgSO4·H2O 12 g,NaCl 90 g, Cu 3 g, FeSO4·H2O 1 g,Ca(IO3)2 0.06 g, Met-Co 0.16 g, ZnSO4·H2O 10 g,NaSeO3 0.003 6 g。

3)营养水平为实测值。Nutrient levels were measured values.

1.3 试验设计与饲养管理

本试验是在广东省农业科学院动物科学研究所的室内循环水产养殖系统中进行。大口黑鲈鱼苗来自广州景龙渔业。购买后,适应试验环境7 d,在此期间每天2次喂食基础饲料。将360尾体重[(5.02±0.01) g]相近且健康的大口黑鲈鱼随机分配到9个玻璃纤维缸(300 L)中。每个缸40尾鱼,进行为期8周的养殖试验。将9个玻璃纤维缸分为3组,分别饲喂3种饲料,每组3个重复。每天在08:30和16:30饲喂2次,出现明显饱腹时停止喂食。每日称量剩余饲料重量,记录每个缸中鱼的每日食物消耗量以计算食物摄入量。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,光周期状态为12 h光照和12 h黑暗。

1.4 采集样品

养殖试验结束后禁食24 h,用40 mg/L MS-222麻醉并进行称重和计数。记录每个缸中鱼的总数量和总重量,用来计算生长性能相关指标。通过每个缸中6条鱼的称重和测量体长来计算肥满度。从每个缸中随机挑选3条鱼,并将其保存在-20 ℃冰箱以分析其体组成。使用1 mL注射器,从鱼的尾静脉抽血,并在室温下静置3 h凝结。将血液样品以3 500 r/min离心10 min,然后提取血清并保持在-80 ℃以备用。每个缸取3尾鱼的肝脏收集到在5 mL冻存管中,然后保存在-80 ℃,用于测定抗氧化酶活性和炎症相关基因表达。

1.5 指标测定

1.5.1 生长性能计算

生长性能计算公式如下:
增重率(WGR, %)=100×[终末体重(g)-初始体重(g)]/初始体重(g);
饲料系数(FCR)=总摄食量(g)/[(终末体重(g)-初始体重(g)];
蛋白质沉积率(PDR, %)=100×[终末体重(g)×终末鱼体蛋白质含量(%)-初始体重(g)×初始鱼体蛋白质含量(%)]/[饲料摄入量(g)×饲料蛋白质含量(%)];
脏体比(VSI,%)=100×内脏团重(g)/鱼体重(g);
肠体比(ISI,%)=100×鱼肠重(g)/鱼体重(g)。

1.5.2 血清生化指标测定

使用自动化学分析仪(Hitachi 7180,日本东京)测定血清葡萄糖(GLU)、甘油三酯(TG)、尿素氮(UN)、胆固醇(CHOL)、高密度脂蛋白胆固醇(HDL-C)和低密度脂蛋白胆固醇(LDL-C)含量及碱性磷酸酶(AKP)、谷丙转氨酶(ALT)、谷草转氨酶(AST)活性。

1.5.3 肝脏抗氧化及免疫指标测定

将肝脏样品匀浆并在4 ℃和2 500 r/min下离心15 min,收集上清液。肝脏总蛋白(TP)、丙二醛(MDA)含量,AKP、过氧化物酶(POD)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)和溶菌酶(LZM)活性以及总抗氧化能力(T-AOC)使用商业试剂盒(南京建成生物工程研究所)进行测定。

1.5.4 营养成分测定

饲料中的粗蛋白质、粗脂肪和粗灰分含量是根据AOAC(2019)[11]测定的。粗蛋白质含量采用半自动凯氏定氮测定系统测定,粗脂肪含量采用索氏抽提法测定。在550 ℃的马弗炉中燃烧6 h后测定粗灰分含量。

1.5.5 RNA提取和实时荧光定量PCR(RT-qPCR)分析

使用Trizol试剂盒(Invitrogen,美国)提取肝脏的总RNA,并使用1%琼脂糖凝胶电泳和分光光度仪分析评估其质量和数量。随后使用HiScript RT SuperMix试剂盒(Vazyme,中国))将RNA转录成cDNA。GenBank中大口黑鲈肝脏免疫和抗氧化相关基因的引物序列见表2。所选引物由上海生物工程技术有限公司合成。RT-qPCR在ABI 7500实时荧光定量PCR仪上进行,第1步预变性反应,95 ℃持续30 s,第2步循环反应,95 ℃持续10 s,60 ℃ 持续30 s,进行40个循环,第3步熔解曲线,95 ℃持续15 s,60 ℃持续60 s,95 ℃持续15 s。β-肌动蛋白是大口黑鲈研究中的非调节性参考基因,β-肌动蛋白基因表达很稳定,不受SB的显著影响。对于所有反应,基因表达样品一式三份测定,最后采用2-ΔΔCt方法分析目的基因相对表达量。
表2 引物序列

Table 2 Primer sequences

基因
Genes		 正向引物
Forward primer (5'—3')		 反向引物
Reverse primer (5'—3')		 GenBank登录号
GenBank accession
number
β-肌动蛋白
β-actin		 GGACACGGAAAGGATTGACAG CGGAGTCTCGTTCGTTATCGG XM_038695351.1
白细胞介素-1β
IL-1β		 CGTGACTGACAGCAAAAAGAGG GATGCCCAGAGCCACAGTTC XM_038696252.1
白细胞介素-8
IL-8		 CGTTGAACAGACTGGGAGAGATG AGTGGGATGGCTTCATTATCTTGT XM_038704088.1
白细胞介素-10
IL-10		 CGGCACAGAAATCCCAGAGC CAGCAGGCTCACAAAATAAACATCT XM_038696252.1
肿瘤坏死因子-α
TNF-α		 CTTCGTCTACAGCCAGGCATCG TTTGGCACACCGACCTCACC XM_038710731.1
Toll样受体22
TLR22		 TCGCTGTTCACCAATCTG TAGTTCTCCTCTCCATCTGT MN807054.1
髓样分化因子88
MyD88		 CTCAACCCCAAGAACACA CGAAGATCCTCCACAATG XM_038728827.1
胰岛素样生长因子-1
IGF-1		 CTTCAAGAGTGCGATGTGC GCCATAGCCTGTTGGTTTACTG DQ666526
生长激素 GH CCCCCAAACTGTCAGAACT ACATTTCGCTACCGTCAGG DQ666528

1.6 低氧应激

每个缸中剩余的16尾鱼进行低氧应激试验。每个缸中剩余的水量约为40 L,水面覆盖有薄膜,并关闭养殖系统的水循环,当鱼没有呼吸并对机械刺激没有反应时,鱼被认定已经死亡[37]。半数死亡的停止标准为比较对照组和试验组之间的生存差异提供了明确的统计基础。本研究在对照组鱼的累计死亡率(CMR)达到50%为低氧应激试验终止条件。试验进行2 h后,水中溶解氧含量保持在0 mg/L,当对照组中1/2的鱼死亡时,停止试验,并记录每个测试池中的死鱼数量。累计死亡率计算公式如下:
累计死亡率(%)=100×低氧应激后的死亡鱼总数/低氧应激前的鱼总数。

1.7 数据统计

试验数据使用平均值±标准误(n=3)表示。所有数据在SPSS 25.0统计软件中进行单因素方差分析,Duncan氏法进行组间多重比较,P<0.05被认为是差异显著。

2 结果与分析

2.1 生长性能

由表3可知,与Con组相比,CB组WGR和PDR显著提高(P<0.05),FCR显著降低(P<0.05),ISI显著增加(P<0.05)。各组间VSI无统计学意义(P>0.05)。
表3 饲料中添加CB或SB对大口黑鲈生长性能的影响

Table 3 Effects of dietary supplementation of CB or SB on growth performance of largemouth bass

项目
Items		 组别 Groups
Con CB SB
增重率 WGR/% 906.72±29.30a 1 017.30±3.65b 944.21±13.27a
饲料系数 FCR 1.22±0.03b 1.12±0.02a 1.23±0.01b
蛋白质沉积率 PDR/% 26.17±0.59a 30.16±0.17b 26.83±0.52a
脏体比VSI/% 5.94±0.36 6.21±0.41 6.17±0.54
肠体比 ISI/% 0.66±0.04a 0.84±0.01b 0.74±0.07ab

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 血清生化指标

由表4可知,与Con组相比,SB组血清AKP活性和TG含量显著降低(P<0.05),CB组和SB组血清UN含量显著降低(P<0.05,SB组血清GLU含量显著升高(P<0.05)。各组间血清CHOL、HDL-C、LDL-C含量与ALT、AST活性无显著差异(P>0.05)。
表4 饲料中添加CB或SB对大口黑鲈血清生化指标的影响

Table 4 Effects of dietary supplementation of CB or SB on serum biochemical indexes of largemouth bass

项目
Items		 组别 Groups
Con CB SB
碱性磷酸酶 AKP/(U/L) 83.94±9.81b 60.56±5.32ab 54.43±5.99a
甘油三酯 TG/(mmol/L) 2.49±0.19b 2.57±0.05b 1.84±0.14a
尿素氮 UN/(mmol/L) 7.48±0.28c 6.52±0.19b 4.98±0.23a
葡萄糖 GLU/(mmol/L) 4.23±0.09a 5.43±0.52ab 6.87±0.49b
胆固醇 CHOL/(mmol/L) 6.08±0.25 6.23±0.20 5.52±0.24
高密度脂蛋白胆固醇 HDL-C/(mmol/L) 2.99±0.20 2.99±0.09 2.56±0.08
低密度脂蛋白胆固醇 LDL-C/(mmol/L) 1.19±0.09 1.18±0.06 0.92±0.08
谷丙转氨酶 ALT/(U/L) 19.88±0.58 12.45±1.34 22.41±7.16
谷草转氨酶 AST/(U/L) 173.19±13.09 149.22±12.70 137.24±12.31

2.3 肝脏抗氧化及免疫指标

由表5可知,与Con组相比,饲料中添加CB和SB可显著提高肝脏TP含量(P<0.05)。CB组和SB组肝脏CAT、SOD和LZM活性显著提高(P<0.05)。SB组肝脏MDA含量显著低于Con组(P<0.05)。各组间肝脏AKP、GSH-Px活性和T-AOC无显著差异(P>0.05)。
表5 饲料中添加CB或SB对大口黑鲈肝脏抗氧化及免疫指标的影响

Table 5 Effects of dietary supplementation of CB or SB on liver antioxidant and immune indexes of largemouth bass

项目
Items		 组别 Groups
Con CB SB
总蛋白 TP/(g/L) 4.40±0.35a 6.48±0.26b 5.56±0.22b
碱性磷酸酶 AKP/(U/mg prot) 34.95±4.37 34.57±3.00 35.31±1.48
过氧化氢酶 CAT/(U/mg prot) 137.48±1.58a 192.28±8.83b 191.62±17.09b
过氧化物酶 POD/(U/mg prot) 1.86±0.23a 2.64±0.19b 1.54±0.05a
丙二醛 MDA/(nmol/mg prot) 4.50±0.70b 2.96±0.18ab 2.28±0.36a
总抗氧化能力 T-AOC/(U/mg prot) 0.57±0.16 1.62±0.07 0.97±0.56
超氧化物歧化酶 SOD/(U/mg prot) 179.30±4.48a 220.14±5.78b 185.17±9.06a
谷胱甘肽过氧化物酶 GSH-Px/(U/mg prot) 412.75±6.39 517.02±56.15 558.50±141.34
溶菌酶 LZM/(U/mg prot) 24.98±1.29a 36.65±1.11b 55.12±4.98c

2.4 肝脏炎症相关基因表达

由表6可知,与Con组相比,SB组肝脏Toll样受体22 (TLR22)基因相对表达量显著增加(P<0.05),CB组和SB组肝脏白细胞介素-1β(IL-1β)、白细胞介素-8(IL-8)和髓样分化因子88(MyD88)基因相对表达量显著降低(P<0.05),CB组肝脏生长激素(GH)基因相对表达量显著提高(P<0.05)。各组间肝脏肿瘤坏死因子-α(TNF-α)和白细胞介素-10(IL-10)基因相对表达量无显著差异(P>0.05)。
表6 饲料中添加CB或SB对大口黑鲈肝脏炎症相关基因表达的影响

Table 6 Effects of dietary supplementation of CB or SB on liver inflammation related gene expression of largemouth bass

项目
Items		 组别 Groups
Con CB SB
Toll样受体22 TLR22 0.19±0.06a 0.55±0.11ab 0.64±0.01b
肿瘤坏死因子-α TNF-α 0.81±0.02 0.62±0.05 0.70±0.14
白细胞介素-1β IL-1β 1.01±0.09b 0.54±0.11a 0.52±0.03a
白细胞介素-8 IL-8 2.24±0.11b 0.89±0.02a 1.22±0.22a
白细胞介素-10 IL-10 1.39±0.21 2.39±0.29 1.99±0.96
髓样分化因子88 MyD88 1.25±0.14b 0.42±0.02a 0.44±0.00a
胰岛素样生长因子-1 IGF-1 1.02±0.15 2.31±0.70 1.67±0.32
生长激素 GH 1.01±0.11a 1.85±0.12b 1.57±0.34ab

2.5 低氧应激累计死亡率

由表7可知,低氧应激3 h,Con、CB和SB组累计死亡率分别为68.75%、31.25%和35.42%,与Con组相比,CB组和SB组累计死亡率显著降低(P<0.05)。
表7 饲料中添加CB或SB对大口黑鲈低氧应激累计死亡率的影响

Table 7 Effects of dietary supplementation of CB or SB on cumulative mortality of largemouth bass under hypoxic stress%

时间
Time/h		 组别 Groups
Con CB SB
1 25.00±3.61 10.42±4.17 14.58±8.33
2 50.00±3.61b 20.83±4.17a 22.92±10.47a
3 68.75±3.61b 31.25±7.22a 35.42±11.02a

3 讨论

3.1 饲料中添加CB或SB对大口黑鲈生长性能的影响

本研究中,与Con组相比,饲料中添加CB显著提高了WGR和PDR,同时FCR显著降低。与本研究结果一致,在以往研究中,已经证明CB对鱼类的生长有益,包括鲈鱼[36]、南美白对虾(Penaeus vanname)[37]、黄颡鱼[21]、大黄鱼[28]、罗非鱼[38]和银鲳[39]。本研究中,与Con组相比,饲料中添加CB显著提高了ISI,表明CB显著增加大口黑鲈肠道长度,在促进肠道发育方面起到积极作用。胰岛素样生长因子-1(IGF-1)是一种蛋白质分子,主要作用于促进生长和细胞增殖,IGF-1在生物体内的功能和调控具有广泛影响,尤其在生长、代谢和细胞增殖方面发挥重要作用[40-41]。生长激素(GH)由IGF介导,在调节鱼类生长中起重要作用,鱼类的生长激素水平与生长速度密切相关[42-43]。本研究发现,在饲料中添加CB显着上调了肝脏IGF-1基因相对表达量,与Con组相比,CB组和SB组肝脏GH基因相对表达量无显著变化。因此,试验结果表明,饲料中添加CB可通过上调肝脏IGF-1基因相对表达量,从而提高的大口黑鲈生长性能。饲料中添加CB可显著提高大口黑鲈的生长性能,其原因可能与其提高饲料利用率、促进肠道发育和上调生长相关基因表达有关。

3.2 饲料中添加CB或SB对大口黑鲈血清生化指标的影响

通过检测血清中的不同生化分子含量或活性来评估动物的生理状态、营养代谢和疾病风险[44-45]。血清UN含量降低与氮利用效率提高有关[46],本研究发现,CB组和SB组可显着降低血清UN含量,表明饲料中添加CB和SB可提高氮的吸收以提高生长性能,同时减少氮的排放可减轻对环境的影响。试验结束后将鱼禁食24 h,饥饿是一种生物体的生存应激条件,鱼体可能通过调节代谢途径来适应这种短期的应激状态,以保证生存,引起鱼体内的一系列生理变化,其中包括血糖升高[47]。血清AKP活性升高表明细胞膜通透性和完整性受损,导致细胞损伤并反映出肝胆炎症的发生[48-49]。在本研究中,与Con组和CB组相比,SB组血清AKP活性和TG含量显著降低,GLU含量显著升高。而饲喂SB组的血清中GLU含量比CB组高,这也印证了本试验结论CB比SB抗氧化效果更佳。饲料中添加丁酸盐可显著提高红杂交罗非鱼血浆中GLU的含量[50]。同样,三倍体鲤鱼饲料中添加5.0 g/kg丁酸钠血清中GLU含量显著高于对照组[51]。与本研究结果一样,Chen等[52]发现饲料中添加SB降低了大口黑鲈肝脏中的TG含量,而在高脂肪饲料中添加SB降低了草鱼(Ctenopharyngodon idellus)的血清中TG含量[53]。而在黄颡鱼中,发现补充SB会增加血清TG含量[33]。综上所述,不同剂型SB、作用水产动物不同或养殖环境等因素差异可能会产生不同结果,而本试验中饲料中添加SB可促进大口黑鲈脂质代谢。ALT和AST是机体中重要的转氨酶,其活性可以反映肝脏的健康状况。通常,当血清ALT和AST活性升高表明肝脏损伤[54]。本研究中,饲料中添加CB和SB对大口黑鲈血清ALT和AST活性无显著影响,添加SB可降低肝脏中AKP活性,表明CB和SB对大口黑鲈肝脏无损害作用,且SB可改善肝脏健康。

3.3 饲料中添加CB或SB对大口黑鲈肝脏炎症因子表达的影响

在炎症反应中,细胞通过上调抗炎因子(如IL-10)的产生或下调促炎因子(如TNF-α、IL-8、IL-1β)的产生,可调节炎症反应的平衡,来限制炎症的程度和损害,避免过度和持续的炎症反应,从而减少对组织和器官的损害[55]。Toll样受体(TLR)识别并结合病原体相关分子模式(PAMP),结合之后,TLR与MyD88相互作用,激活的MyD88可激活NF-κB并诱导胞质促炎因子的产生[56]。本研究发现,SB组肝脏TLR22基因相对表达量显著增加,CB组和SB组肝脏IL-1β、IL-8和MyD88基因相对表达量显著降低,另外,与Con组相比,CB和SB组肝脏TNF-α基因相对表达量有降低趋势,IL-10基因相对表达量有升高趋势。与SB组相比,CB组肝脏促炎因子IL-1β、IL-8和TNF-α的基因相对表达量均低于SB组,同时,IL-10基因相对表达量高于SB组。TLR22和MyD88已被证明在草鱼[57]和断奶仔猪[58]的先天免疫反应中起重要作用。本试验结果表明,饲料中添加CB和SB可激活肝脏TLR22的表达,并抑制下游基因MyD88 mRNA转录。已经发现丁酸可抑制NF-κB的释放并阻止其从细胞质转移到细胞核,最终抑制促炎细胞因子的表达[59]。SB抑制NF-κB-p65信号通路,改善了2,4,6-三硝基苯磺酸诱导的小鼠肠道炎症[60]。SB可作用NF-κB-p65信号通路参与细胞促炎因子调节,提高了草鱼对肠炎的抵抗力[61]。在本研究中,CB和SB最终可能是通过TLR22-MyD88途径抑制NF-κB的释放,最终抑制促炎因子IL-1β、IL-8和TNF-α的表达,减轻了机体炎症反应。LZM是细胞内的一种细胞器,其中含有一系列水解酶,LZM在维持细胞的内环境稳定和清除垃圾物质方面起着关键作用,有助于身体的非特异性免疫防御[62-63]。本研究中,与Con组相比,CB组和SB组肝脏中LZM活性显著提高,与SB组相比,CB组肝脏LZM活性显著高于SB组。因此,饲料中添加CB和SB可通过TLR22-MyD88-NF-κB信号途径降低大口黑鲈肝脏炎症反应。与SB相比,CB在提高大口黑鲈抗炎症反应和免疫效果方面效果更佳。

3.4 饲料中添加CB或SB对大口黑鲈肝脏抗氧化指标的影响

氧化应激是细胞内活性氧生成超过抗氧化防御系统能力的情况,这些活性氧(包括自由基和其他氧化物)在细胞内引发氧化反应,可能对细胞内分子(如脂质、DNA和蛋白质)造成损伤,这些分子的氧化损伤可能导致细胞功能受损,甚至影响细胞的生存和健康,抗氧化酶在清除自由基和防止活性氧的产生方面发挥重要作用[31,64]。细胞内脂质与活性氧反应可导致脂质过氧化,MDA是脂质过氧化产物之一,可能对细胞和组织产生损害[65]。生物抗氧化防御系统中的关键酶包括:SOD、POD、GSH-Px和CAT等,当机体遭受氧化应激时,抗氧化酶会被激活,从而对抗活性氧的产生和损害,减少氧化应激对细胞的不利影响[66⇓-68]。本研究在大口黑鲈饲料中分别添加CB和SB,可显著提高肝脏中TP含量以及CAT、SOD和POD活性,SB组肝脏MDA含量显著低于Con组,但与CB组相比无显著差异,同时观察到肝脏T-AOC和GSH-Px活性有上升趋势,表明CB和SB可有效提高大口黑鲈抗氧化能力,并可降低脂质过氧化造成的损伤,与SB相比,CB在大口黑鲈肝脏抗氧化中的作用效果更佳。与本研究结果一致,在氨应激条件下,SB提高了黄颡鱼SOD、CAT和GSH-Px mRNA的表达水平,增强了黄颡鱼抗氧化能力[33]。研究表明,饲料中添加SB提高了热应激条件下尼罗罗非鱼中抗氧化酶(SOD、CAT和GSH-Px)的活性,同时降低了MDA含量[31]。本研究中,饲料中添加CB显著提高了大口黑鲈肝脏SOD、CAT和POD活性,这与杂交石斑鱼(Epinephelus fuscoguttatus ♀×E. lanceolatu ♂)中的研究结果[69]一致。综上所述,CB和SB对提高大口黑鲈抗氧化酶活性、增强其抗氧化能力具有积极作用,而CB在提高大口黑鲈抗氧化能力方面作用效果更佳。

3.5 饲料中添加CB或SB对大口黑鲈低氧应激的影响

水质、温度、氧含量、气候和光照等环境等因素的变化会破坏鱼类活性氧的动态平衡,可能导致氧化应激的加剧,从而影响鱼类的生理功能[70⇓⇓-73]。鱼类代谢需要有效水平的溶解氧,在集约化、高密度养殖环境中,水中的溶解氧是鱼类养殖的主要限制因素之一[74]。当鱼将能量用于应对某种特定的生存挑战,比如应对压力源,它们免疫力会降低[75],另外,缺氧可能对鱼的免疫系统造成损害并降低对致病性感染的抵抗力[76]。本研究中,饲料中补充CB和SB可显著降低大口黑鲈低氧应激条件下的累计死亡率,其中补充CB的大口黑鲈的累计死亡率为31.25%,抗低氧应激效果最佳。这可能是由于CB和SB提高了大口黑鲈体内抗氧化酶活性,并减少了炎症因子产生,从而降低了低氧应激条件下大口黑鲈死亡率,但其作用机制仍需进一步探索。综上所述,饲料中添加CB和SB可有效降低缺氧应激下大口黑鲈的累计死亡率,增强抗氧化能力,同等条件下CB对增强抗氧化能力效果最佳。

4 结论

本试验条件下,结合生长性能、血清生化指标、抗氧化酶活性和低氧应激累计死亡率指标综合评价,饲料中添加10 mL/kg CB的作用效果优于直接补充1.0 g/kg SB。

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