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Volume 45 Issue 10
Oct.  2021
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Effects of dietary sodium deoxycholate on the growth, glucose metabolism and intestinal microbiota of largemouth bass (Micropterus salmoides)

  • Corresponding author: ZHANG Meiling, mlzhang@bio.ecnu.edu.cn
  • Received Date: 2021-03-03
    Accepted Date: 2021-04-10
    Available Online: 2021-08-30
  • Bile acids play an important role in glucose metabolism, lipid metabolism, and they can also help to maintain the liver health and intestinal homeostasis in animals. In recent years, bile acids have been widely used as new additives in aquatic feeds. However, so far, most of the bile acid products used in aquaculture are mixed bile acids. There are many different kinds of bile acids, and different bile acids have different effects on the growth and metabolism of fish. Therefore, it is necessary to evaluate the effects of different bile acids on fish growth and metabolism to select the appropriate type of bile acids for precise nutritional regulation. This experiment was conducted to evaluate the effect of sodium deoxycholate as a feed additive on Micropterus salmoides. Two experimental diets were formulated to contain different sodium deoxycholate levels of 0, and 300 mg/kg referred to as CON and SD, respectively. The effects of sodium deoxycholate on the growth condition, metabolic characteristics and intestinal microbiota were analyzed. M. salmoides (10.80 ± 0.12) g were cultured for 8 weeks. The results showed that sodium deoxycholate significantly increased the final body weight [(37.24 ± 0.64) g in the control group and (46.87 ± 1.44) g in the sodium deoxycholate group]. The body length was (12.42 ± 0.12) cm in the control group and (13.07 ± 0.14) cm in sodium deoxycholate group. The condition factor (CF) was (1.95 ± 0.06) g/cm3 in the control group and (2.09 ± 0.03) g/cm3 in the sodium deoxycholate group, but there was no significant difference in viscera index (VSI) or hepatosomatic index (HSI). Sodium deoxycholate had no effect on the total lipid and crude protein of whole fish. Sodium deoxycholate significant increased the expression level of gluconeogenesis-related and glycolysis-related genes in liver and muscle, and the sodium deoxycholate promoted the glycogen accumulation in muscle [(0.31 ± 0.03) mg/g in control group and (0.46 ± 0.03) mg/g in sodium deoxycholate group] by increasing the activity of glycogen synthase, but there was no significant difference in the liver glycogen content between these two groups. In addition, sodium deoxycholate significantly increased bile acids content in gallbladder [(199.4 ± 12.72) μmol/L in control group, (341.1 ± 8.52) μmol/L in sodium deoxycholate group]. Our results indicated that sodium deoxycholate can promote the bile acid synthesis mainly by down-regulating the expression level of fxr gene and up-regulating the expression level of cyp7a1 gene in the liver. The abundance of Firmicutes and Bacteroidetes decreased while Actinobacteria increased in M. salmodides fed with sodium deoxycholate at the phylum level. All these results suggested that sodium deoxycholate can be used as a feed additive for M. salmodides to promote the growth condition, and increase muscle glycogen accumulation and bile acid synthesis.
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Effects of dietary sodium deoxycholate on the growth, glucose metabolism and intestinal microbiota of largemouth bass (Micropterus salmoides)

    Corresponding author: ZHANG Meiling, mlzhang@bio.ecnu.edu.cn
  • Lab of Aquaculture Nutrition and Environment Health, College of Life Sciences, East China Normal University, Shanghai 200241, China

Abstract: Bile acids play an important role in glucose metabolism, lipid metabolism, and they can also help to maintain the liver health and intestinal homeostasis in animals. In recent years, bile acids have been widely used as new additives in aquatic feeds. However, so far, most of the bile acid products used in aquaculture are mixed bile acids. There are many different kinds of bile acids, and different bile acids have different effects on the growth and metabolism of fish. Therefore, it is necessary to evaluate the effects of different bile acids on fish growth and metabolism to select the appropriate type of bile acids for precise nutritional regulation. This experiment was conducted to evaluate the effect of sodium deoxycholate as a feed additive on Micropterus salmoides. Two experimental diets were formulated to contain different sodium deoxycholate levels of 0, and 300 mg/kg referred to as CON and SD, respectively. The effects of sodium deoxycholate on the growth condition, metabolic characteristics and intestinal microbiota were analyzed. M. salmoides (10.80 ± 0.12) g were cultured for 8 weeks. The results showed that sodium deoxycholate significantly increased the final body weight [(37.24 ± 0.64) g in the control group and (46.87 ± 1.44) g in the sodium deoxycholate group]. The body length was (12.42 ± 0.12) cm in the control group and (13.07 ± 0.14) cm in sodium deoxycholate group. The condition factor (CF) was (1.95 ± 0.06) g/cm3 in the control group and (2.09 ± 0.03) g/cm3 in the sodium deoxycholate group, but there was no significant difference in viscera index (VSI) or hepatosomatic index (HSI). Sodium deoxycholate had no effect on the total lipid and crude protein of whole fish. Sodium deoxycholate significant increased the expression level of gluconeogenesis-related and glycolysis-related genes in liver and muscle, and the sodium deoxycholate promoted the glycogen accumulation in muscle [(0.31 ± 0.03) mg/g in control group and (0.46 ± 0.03) mg/g in sodium deoxycholate group] by increasing the activity of glycogen synthase, but there was no significant difference in the liver glycogen content between these two groups. In addition, sodium deoxycholate significantly increased bile acids content in gallbladder [(199.4 ± 12.72) μmol/L in control group, (341.1 ± 8.52) μmol/L in sodium deoxycholate group]. Our results indicated that sodium deoxycholate can promote the bile acid synthesis mainly by down-regulating the expression level of fxr gene and up-regulating the expression level of cyp7a1 gene in the liver. The abundance of Firmicutes and Bacteroidetes decreased while Actinobacteria increased in M. salmodides fed with sodium deoxycholate at the phylum level. All these results suggested that sodium deoxycholate can be used as a feed additive for M. salmodides to promote the growth condition, and increase muscle glycogen accumulation and bile acid synthesis.

  • 胆汁酸作为胆汁的主要成分,在动物的葡萄糖代谢[1]、脂肪代谢[2]、肝肠健康[1]和肠道内环境稳态的维持[3]等方面发挥着重要作用。近年来,胆汁酸作为一种新型添加剂广泛应用到水产饲料中。有研究表明,在饲料中添加600 mg/kg的胆汁酸复合物[主要含有58.3%猪脱氧胆酸(hyodexyocholic acid, HDCA)、17.4%的鹅脱氧胆酸(chenodeoxycholic acid,CDCA)、10.0%的甘氨熊去氧胆酸(glycoursodeoxycholic acid, GUDCA)],可以提高异育银鲫(Carassius auratus gibelio)的生长性能和抗氧化能力[4];在饲料中添加450 mg/kg的胆汁酸可以显著提高军曹鱼(Rachycentron canadum)的生长性能和饲料利用率[5];在饲料中添加300 mg/kg的胆汁酸复合物[含有8%的猪胆酸(hyocholic acid, HCA)、70.9%的HDCA和20.2%的熊去氧胆酸(ursodeoxycholic acid, UDCA)]不仅可以增加大口黑鲈(Micropterus salmoides)的增重率和特定生长率,还可以缓解高糖饲料所诱导的肝脏纤维化[6];在饲料中添加900 mg/kg的胆汁酸可以显著提高大菱鲆(Scophthalmus maximus)的增重率、饲料系数以及肠道消化酶活性[7]。但是到目前为止,这些研究主要关注混合型胆汁酸对水产动物生长的影响,而且在不同研究中所使用的胆汁酸复合物组成差异较大,不同种类胆汁酸对鱼类生长及代谢的影响尚不清楚。

    动物体内胆汁酸的种类很多,按照其来源可以分为初级胆汁酸和次级胆汁酸。其中初级胆汁酸在肝脏中合成,之后随着胆汁流入肠道,经肠道菌群的脱羟基作用转化为次级胆汁酸[8]。在不同物种之间,胆汁酸的组成大不相同。如人的初级胆汁酸以胆酸(cholic acid,CA)和CDCA为主[9],CA相对应的次级胆汁酸为脱氧胆酸(deoxycholic acid, DCA),与CDCA对应的次级胆汁酸为石胆酸(lithocholic acid, LCA)和UDCA,其中UDCA在小鼠(Mus musculus)中是初级胆汁酸[10-11]。在小鼠中,初级胆汁酸以CA、β-鼠李酸(β-muricholic acid, βMCA)为主,其中βMCA在其他动物体内的含量很低,在肠道菌群的作用下转化为ωMCA[12]。在猪(Sus scrofa)体内,CDCA和HCA是主要的初级胆汁酸,HCA可在肠道菌群的作用下转化为HDCA[13]。在草鱼(Ctenopharyngodon idella)体内,胆汁中主要的初级胆汁酸为CA和CDCA,次级胆汁酸为DCA;肠道中的初级胆汁酸主要为CA,次级胆汁酸则为DCA和UDCA[14]。在大菱鲆体内,胆汁中主要的初级胆汁酸为牛磺胆酸(taurocholic acid, TCA)和牛磺鹅去氧胆酸(taurochenodeoxycholic acid, TCDCA),次级胆汁酸为牛磺β-鼠胆酸(tauro-beta-muricholic acid, TβMCA)和牛磺ω-鼠胆酸(tauro-omega-muricholic acid, TωMCA)[15]。在高等动物中的研究表明,不同种类胆汁酸的功能存在较大差异[16-18],但是在水产动物上仍缺乏相关的研究。

    目前混合型胆汁酸在水产饲料中广泛使用,由于组成复杂多样,混合型胆汁酸中发挥功效的具体成分难以确定,此外,有关单一胆汁酸对鱼类影响方面的研究也鲜有报道。因此,评估不同种胆汁酸对鱼体生长及代谢的影响,对于选用合适的胆汁酸类型以实现精准营养调控具有重要帮助。有研究表明,在饲料中添加300 mg/kg的胆汁酸复合物(含有8%的HCA,70.9%的HDCA和20.2%的UDCA)可以使大口黑鲈获得最佳生长性能[19]。因为胆汁酸通常以胆汁酸盐的形式存在,所以本研究主要评估饲料中添加300 mg/kg的脱氧胆酸钠(sodium deoxycholate, SD)对大口黑鲈生长、代谢以及肠道菌群的影响,研究成果将为进一步扩展胆汁酸盐在水产养殖中的应用提供重要的理论基础。

1.   材料与方法
  • 以进口鱼粉、鸡肉粉、豆粕为蛋白源,以豆油为脂肪源,面粉和木薯淀粉为主要糖源配制基础饲料(表1)。分别在基础饲料中添加0和300 mg/kg的脱氧胆酸钠,配制成对照组(CON)和脱氧胆酸钠添加组(SD)饲料。饲料原料经研磨粉粹后过60目筛网,按照逐级放大法将各种原料充分混合,之后加入豆油和水混匀。使用水产饲料专用膨化机将混匀的饲料挤压成条,在50 °C下将饲料水分烘干至10%以下,用饲料粉碎机将饲料制备成直径约3 mm的颗粒饲料,用自封袋包装后置于−20 °C冰箱保存备用。

    成分
    ingredients
    对照组饲料
    control
    diet
    脱氧胆酸钠组饲料
    sodium
    deoxycholate diet
     进口鱼粉 imported fish meal 45 45
     鸡肉粉 chicken powder 9 9
     豆粕 soybean meal 12 12
     面粉 flour 11 11
     谷朊粉 gluten 2 2
     木薯淀粉 cassava starch 8 8
     豆油 soybean oil 6 6
     乌贼膏 squid ointment 4 4
     磷酸二氢钙 Ca(H2PO4)2 1.5 1.5
     预混料 premix 1.5 1.5
     脱氧胆酸钠 sodium deoxycholate 0 0.03
    营养成分 proximate
     水分/% moisture 7.66 7.49
     灰分/% crude ash 10.82 11.04
     粗蛋白质/% crude protein 50.45 50.04
     粗脂肪/% crude lipid 13.24 13.38
    注:维生素预混料(mg/kg)含维生素A 500 000 IU,维生素D3 50 000 IU,维生素E 2 500 mg,维生素K3 1 000 mg,维生素B1 5 000 mg,维生素B2 5 000 mg,维生素B6 5 000 mg,维生素B12 5 000 μg,肌醇25 000 mg,泛酸10 000 mg,胆碱100 000 mg,烟酸25 000 mg,叶酸1 000 mg,生物素250 mg,维生素C 10 000 mg;矿物质预混料(g/kg)含碳酸钙314.0 g,磷酸二氢钾469.3 g,硫酸镁147.4 g,氯化钠49.8 g,葡萄糖酸铁10.9 g,硫酸锰3.12 g,硫酸锌4.67 g,硫酸铜0.62 g,碘化钾0.16 g,氯化钴0.08 g,钼酸铵0.06 g,亚硒酸钠0.02 g
    Notes:vitamin premix (mg/kg) [vitamin A 500 000 IU, vitamin D3 50 000 IU, vitamin E 2 500 mg, vitamin K3 1 000 mg, vitamin B1 5 000 mg, vitamin B2 5 000 mg, vitamin B6 5 000 mg, vitamin B12 5 000 μg, inositol 25 000 mg, pantothenic acid 10 000 mg, choline 100 000 mg, niacin 25 000 mg, folic acid 1 000 mg, biotin 250 mg, vitamin C 10 000 mg; mixed minerals (g/kg) contain CaCO3 314.0 g, KH2PO4 469.3 g, MgSO4·7H2O 147.4 g, NaCl 49.8 g, Fe(II)gluconate 10.9 g, MnSO4·H2O 3.12 g, ZnSO4·7H2O 4.67 g, CuSO4·5H2O 0.62 g, KI 0.16 g, CoCl2·6H2O 0.08 g, NH4 molybdate 0.06 g, NaSeO3 0.02 g

    Table 1.  Formulation of experimental diets %

  • 实验所用的大口黑鲈购自广州市添发鱼苗发展有限公司,鱼苗运至华东师范大学生命科学学院实验基地暂养1个月,暂养期间投喂商品饲料。挑选状态良好,初始体质量为(10.80±0.12) g的鱼随机分到6个250 L的养殖桶中,每个桶30尾鱼,分成2个处理(对照组和脱氧胆酸钠组),每个处理3个重复。

    养殖实验历时8周,在自然光周期和环境温度下进行,采用经过充分曝气的自来水,在室内进行养殖。饲料投喂量为体质量的3.5%,每2周称1次体质量以调整投喂量。养殖期间,水温为26 °C~29 °C,溶解氧为5.0~7.5 mg/L。每天用虹吸管清除粪便和污水,日换水量为100%,养殖期间定期清洗养殖桶。

  • 8周实验结束后,将实验鱼饥饿8 h,从每个桶中取4尾实验鱼,共计12尾。用丁香酚麻醉后,逐一称重并测量体长。利用1 mL无菌注射器从尾椎静脉取血,4 °C静置12 h,之后进行低温离心 (3 000 r/min,4 °C,10 min),取上层血清,于−80 °C保存。随后解剖取内脏团、肝脏称重,用于形体指标测定,在大致相同的位置切取小块肝脏组织固定在4%多聚甲醛中,用于制备PAS(Periodic Acid-Schiff stain)染色切片,剩下的肝脏冻于−80 °C。取胆囊、肠道、肠道内容物以及背部一侧的肌肉立即置于−80 °C中保存。取另一侧肌肉样品固定在环保型GD固定液(主要成分为甲醛溶液、冰醋酸和无水乙醇)中,用于制备PAS染色切片。

  • 根据实验测定的数据计算大口黑鲈的肥满度、脏体比、肝体比。

    肥满度(condition factor,CF,g/cm3) =($ {W_{\rm{B}}}/ $$ L_{\rm{B}}^3 $)×100;

    脏体比(viscerosomatic index,VSI,%)=(WV/WB)×100%;

    肝体比(hepatosomatic index,HIS,%)=(WI/WB)×100%;

    式中,WB为体质量(g);LB为体长(cm),WV为内脏团质量(g),WI为肝脏质量(g)。

  • 每个处理组取6尾实验鱼,使用丁香酚麻醉后置于−20 °C冰箱以供全鱼体成分分析。水分采用105 °C烘干法,灰分采用550 °C灼烧法至恒重,采用凯氏定氮法(FOSS kjeltec 8200)测定粗蛋白,采用氯仿-甲醇法测定总脂肪。

  • 肌肉和肝脏中的糖原采用南京建成生物工程研究所有限公司生产的相关试剂盒测定,糖原合成酶 (GCS) 以及糖原磷酸化酶a (GPa) 均采用北京百奥莱博科技有限公司生产的相关试剂盒测定。肝脏、胆囊、肠道和血清中的总胆汁酸也采用相应的试剂盒测定。

  • 将固定在4%的多聚甲醛中的肝脏组织和环保型GD固定液中的肌肉组织送往武汉塞维尔生物技术有限公司制作切片,PAS染色,采用尼康显微镜进行拍照观察糖原染色情况。

  • 使用北京艾德莱生物科技有限公司的TRIpure试剂提取组织总RNA,使用FastQuant RT Kit with gDNA试剂盒将RNA反转录成cDNA。根据Zhang等[20]、Yu等[6]的研究设计了相关基因的特异性定量PCR引物(表2)。内参基因选取β-actinef1α (二者Ct值取算术平方根作为内参)。采用10倍梯度稀释法确定引物的扩增效率为95%~105%。PCR程序:95 °C 10 min;95 °C 5 s,60 °C 15 s,40个循环;绘制熔解曲线以确定引物的特异性。mRNA的相对表达量采用2−ΔΔCt法计算。

    引物
    primers
    引物序列5′-3′
    primers sequence 5′-3′
    产物长度/bp
    product length
    pepck-2-F ATTCCCTTCAGTATGGGTCCT 142
    pepck-2-R CTTTGACCGACTTCCTCACC
    fbpase-1b-F CTTCACCTCCTGTGTGCTTG 182
    fbpase-1b-R CAGCTGGCTCACCATCTGTA
    g6pase-like-F GGGAGTCCAGGTGTGTGTCT 182
    g6pase-like-R CAGCGAAGGAGGTCAAGAAG
    hk-4-like-F CATCACCTTCCTCGTCTCGG 107
    hk-4-like-R ACCTCCTGTCGCTATCACTCC
    Pfkpa-F ACAACACACCAACTGACACCT 175
    Pfkpa-R GCAGCATCGAGCAGAACATGA
    cyp7a1-F TTCAGTGTGGGGTCGTTGGG 153
    cyp7a1-R CTGGGCTTCACAGGCTAACACC
    fxr-F AGCCGAAAGATGCCCAACG 118
    fxr-R GGAGTTCACCGATGCTTTTG
    β-actin-F TGCTGAGCGTGAGATTGTG 193
    β-actin-R GAAGGTCGGAAGGAAGGGA
    ef1α-F TGCTGCTGGTGTTGGTGAGTT 147
    ef1α-R TTCTGGCTGTAAGGGGGCTC

    Table 2.  Sequences of the primers used in this study

  • 将每个处理取6个肠道内容物样品送至上海派森诺生物科技有限公司进行DNA提取和测序分析。提取DNA之后用细菌16S rRNA的V3~V4区特异性引物进行扩增,引物序列为F: 5′-ACTCCTACGGGAGGCAGCA-3′,R: 5′-GGACTACHVGGGTWTCTAAT-3′。利用Illumina平台对菌群DNA片段进行双端(Paired-End)测序。采用DADA2法对测序数据进行过滤、去噪、拼接和去嵌合体等。对原始数据的ASV进行聚类分析,基于ASVs和注释结果进行样品物种复杂度分析、组间物种差异分析等。所有样本的原始数据已经提交GenBank数据库中(PRJNA705114)。

  • 数据经Excel 2019软件处理后,用GraphPad Prism 7.0软件进行分析,采用t检验进行对照组和脱氧胆酸钠组组间差异显著性分析,P<0.05表示差异显著。实验数据均以平均值±标准误(mean±SE)来表示。

2.   结果
  • 饲料中添加脱氧胆酸钠显著增加了大口黑鲈的终末体质量、体长以及肥满度(CF)(P<0.05),但是对脏体比和肝体比没有显著影响。大口黑鲈的粗蛋白含量和总脂肪含量在2组之间没有显著差异。与对照组相比,脱氧胆酸钠组肝糖原含量没有显著变化,但肌糖原含量显著增加(P<0.05)(图1)。

    Figure 1.  Effects of sodium deoxycholate on growth performance, body composition and glycogen of M. salmoides

  • 为了进一步验证脱氧胆酸钠对肌糖原的影响,将大口黑鲈的肌肉进行石蜡切片、PAS染色观察。相比于对照组,脱氧胆酸钠组的大口黑鲈肌糖原含量明显增多(图2-a)。脱氧胆酸钠组肌肉中与糖原合成相关的GCS活性显著高于对照组(P<0.05)(图2-b),而与糖原分解相关的GPa的活性在2组之间没有显著差异(图2-c)。此外,本研究还检测了肌肉中与糖酵解相关的己糖激酶基因(hk-4-like)、磷酸果糖激酶基因(pfkpa)的表达以及与糖异生相关的磷酸烯醇式丙酮酸羧激酶基因(pepck-2)、葡萄糖-6-磷酸酶基因(g6pase-like)、果糖-1,6-二磷酸酶基因(fbpase-1b)等基因的表达。结果发现,脱氧胆酸钠组g6pase-like的表达显著高于对照组(P<0.05),hk-4-likepfkpapepck-2和fbpase-1b的表达则无显著差异(图2-d),说明饲料中添加脱氧胆酸钠可以促进肌肉糖异生并增加糖原合成酶的活性,促进肌肉中糖原的积累。

    Figure 2.  Effects of sodium deoxycholate on glucose metabolism of M. salmoides muscle

  • 为进一步确认脱氧胆酸钠对肝脏糖代谢的影响,将大口黑鲈肝脏进行石蜡切片、PAS染色观察,并测定了GCS和GPa的活力以及糖酵解、糖异生关键酶基因的表达情况。结果发现,脱氧胆酸钠组和对照组肝脏中糖原的含量没有明显差异(图3-a)。肝脏中GCS和GPa的活性在2组之间也无显著的变化(图3-bc)。饲料中添加脱氧胆酸钠显著上调了fbpase-1b的表达(P<0.05)(图3-d)。上述结果表明,在饲料中添加脱氧胆酸钠虽然可以调节肝脏中的糖异生,但由于糖原合成与分解相关的酶活并未受到显著的影响,因此肝脏中糖原含量在2组间并没有显著差异。

    Figure 3.  Influence of sodium deoxycholate on glucose metabolism of M. salmoides liver

  • 为了探究脱氧胆酸钠是否会影响大口黑鲈的胆汁酸代谢,本实验测定了不同组织中胆汁酸的含量。结果发现,脱氧胆酸钠组的胆囊中的总胆汁酸含量显著高于对照组(P<0.05)。在肝脏、肠道内容物、血清中2组之间的总胆汁酸含量没有显著的差异,但相比于对照组,脱氧胆酸钠组中肠道和血清中总胆汁酸含量均有上升的趋势 (图4)。

    Figure 4.  Effects of sodium deoxycholate on the bile acid metabolism of M. salmoides

    胆汁酸经典合成途径限速酶基因(cyp7a1)的表达结果显示,脱氧胆酸钠组cyp7a1的表达显著高于对照组(P<0.05)(图4-e)。同时,脱氧胆酸钠组胆汁酸受体基因 (fxr)的表达显著低于对照组(P<0.05)(图4-f)。说明大口黑鲈摄食添加脱氧胆酸钠的饲料会造成肝脏中fxr的表达下调以及cyp7a1的表达上调,进而促进胆汁酸的合成并造成胆囊中胆汁酸的积累。

  • 考虑到肠道菌群与胆汁酸代谢密切相关[18],我们利用16S rRNA测序技术分析了2组大口黑鲈肠道菌群的组成。通过分析各类群在门水平上的相对丰度发现,对照组的优势菌为软壁菌门(Tenericutes, 35.78%)、变形菌门(Proteobacteria, 17.68%)、放线菌门(Actinobacteria, 7.10%)和拟杆菌门(Bacteroidetes, 13.07%),脱氧胆酸钠组的优势菌为放线菌门(51.65%)、软壁菌门(17.07%)和变形菌门(15.52%)。与对照组相比,脱氧胆酸钠组的厚壁菌门(Firmicutes)和拟杆菌门的丰度显著降低,放线菌门的丰度显著增加(P<0.05)(图5-a)。通过主坐标分析(图5-b)发现,对照组和脱氧胆酸钠组的菌群组成存在在明显的差异。46个ASV的热图分析显示,在对照组和脱氧胆酸钠组间有17个ASV发生了显著的变化,其中与脱氧胆酸钠组相比,拟杆菌门的7个ASV和厚壁菌门的6个ASV显著降低,放线菌门的1个ASV和变形菌门的3个ASV显著增加(P<0.05)(图5-c)。

    Figure 5.  Influence of sodium deoxycholate on the microbiota composition of M. salmoides

3.   讨论
  • 胆汁酸是胆固醇的代谢产物,在肝脏中合成并存储于胆囊中,对机体的脂肪代谢起着重要的调控作用[18]。本实验在饲料中添加脱氧胆酸钠可以显著提高大口黑鲈终末体质量,这与在饲料中添加一定量的混合型胆汁酸对军曹鱼[5]、大口黑鲈[6]、大菱鲆[7]、吉富罗非鱼(Oreochromis niloticus GIFT)幼鱼[21]生长的影响是一致的。肥满度是反应鱼体生长情况的指标,在本研究中,脱氧胆酸钠的添加显著增加了大口黑鲈的肥满度,说明脱氧胆酸钠的添加可以改善大口黑鲈的生长状况,这与在齐口裂腹鱼(Schizothorax prenanti)幼鱼[22]上的研究结果基本一致。有研究发现,在饲料中添加一定浓度的胆汁酸复合物可以显著提高草鱼肝胰脏以及肠道各段的蛋白酶、脂肪酶和淀粉酶的活性[23],进而促进草鱼对饲料中营养物质的吸收利用,促进草鱼的生长。向枭等[24]的研究表明,在高脂饲料中添加胆汁酸复合物可以显著提高齐口裂腹鱼幼鱼肝脏以及肠道的蛋白酶和脂肪酶的活性。但在本研究中,饲料中脱氧胆酸钠的添加对大口黑鲈消化酶活性并没有显著影响,这可能与脱氧胆酸钠的添加剂量有关。因此在今后的实验中,还需检测多个剂量梯度的脱氧胆酸钠添加对鱼体代谢的系统性影响。

    本研究发现,在饲料中添加脱氧胆酸钠可以显著增加肌肉中糖原的积累,有文献报道,胆汁酸可以通过FXR介导的信号通路来调控宿主的糖代谢[1],FXR与糖异生密切相关,如胆酸可以激活小鼠的FXR并通过诱导小异二聚体伴侣(small heterodimer partner, SHP)。抑制糖异生基因的表达同时降低血糖水平[25]。在本实验同样发现,脱氧胆酸钠显著降低了大口黑鲈肝脏中fxr的表达量,促进肝脏中fbpase-1b和肌肉中g6pase-like的表达显著上调。脱氧胆酸钠引起的糖异生基因表达的上调并没有造成血糖的变化,但增加了肌糖原的含量,通过检测发现脱氧胆酸钠显著增加了肌肉中GCS的活性。由此可见,脱氧胆酸钠可能促进糖异生并通过增强GCS的活性,提高肌肉对血糖的储存利用。

    FXR是调节胆汁酸稳态的重要受体[26-28],可以通过抑制cyp7a1活性来负反馈调控胆汁酸合成[29]。本研究中,在饲料中添加脱氧胆酸钠显著下调了fxr的表达,使其对CYP7A1酶的抑制作用减弱,增加了胆汁酸的合成,促进了胆囊中胆汁酸的积累。但也有研究表明,在饲料中添加胆汁酸复合物对大口黑鲈fxr的表达没有影响[30]。在高等动物中的研究发现FXR受体对不同胆汁酸的响应存在一定差异[1,18],在鱼类中是否有类似的规律,还需要深入的研究。

    在进食时,胆汁酸会从胆囊分泌到肠道中,并且在肠道菌群的作用下被进一步代谢[18],而肠道菌群的组成与胆汁酸稳态密切相关[31]。有研究发现,在饲料中添加LCA增加了草鱼肠道中的变形菌门和厚壁菌门的相对丰度,减少了梭杆菌门的相对丰度[32];也有研究发现,饲料中添加胆汁酸复合物减少了草鱼肠道中厚壁菌门和放线菌门的丰度,提高了拟杆菌门丰度[33]。赵盼月[34]研究发现,低剂量的胆汁酸复合物会增加欧洲鳗鲡(Anguilla anguilla)肠道中厚壁菌门和拟杆菌门的相对丰度,高剂量的胆汁酸复合物会降低厚壁菌门和拟杆菌门的相对丰度。在本研究中,饲料中添加脱氧胆酸钠显著改变了肠道菌群的组成并显著降低了大口黑鲈肠道中厚壁菌门和拟杆菌门的相对丰度,增加了放线菌门的相对丰度。这说明不同种类、不同添加剂量的胆汁酸对鱼类肠道菌群的影响差异较大。肠道菌群在宿主应对外界环境变化过程中发挥着重要调节作用[35],同时会影响宿主的生长[36]与代谢[37]。但由于鱼类的固有菌群与高等生物不同,并且在鱼类中肠道微生物功能的研究尚处于起步阶段,因此,脱氧胆酸钠添加以后大口黑鲈肠道中微生物组成的变化是否与脱氧胆酸钠促进大口黑鲈生长相关目前尚不清楚,后续可以通过菌群转接等实验来对肠道微生物的功能进行进一步确认。

4.   结论
  • 本研究探讨了饲料中添加单一的脱氧胆酸钠对大口黑鲈生长、糖代谢、胆汁酸代谢和肠道菌群的影响。结果表明,在饲料中添加300 mg/kg的脱氧胆酸钠可以促进大口黑鲈的生长、促进肌糖原的积累、增加胆汁酸的合成及影响肠道菌群组成结构。在水产养殖中,饲料中添加一定量的胆汁酸有一定的促生长作用,但胆汁酸的组成复杂,不同胆汁酸种类对鱼类生长的影响并不是一致的。胆汁酸的种类、添加剂量、鱼的种类、发育阶段以及饲料组成都可能影响胆汁酸对鱼类的作用效果,因此,不同种类的胆汁酸对不同鱼种生长的影响需要深入系统的研究。

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