灌胃和腹腔注射鼠李糖乳杆菌对妊娠期小鼠粪便菌群多样性的影响

doi:10.16431/j.cnki.1671-7236.2023.01.016

摘要/Abstract

摘要： 【目的】研究灌胃和腹腔注射鼠李糖乳杆菌（Lactobacillus rhamnosus，LGG）对妊娠期小鼠粪便菌群多样性的影响，为动物妊娠期生理及肠道健康提供理论依据。【方法】选择体重相近（23.33 g±1.55 g）、配种日期相同的SPF级妊娠昆明小鼠45只，随机分为3组，分别为对照组、试验Ⅰ组和试验Ⅱ组，每组15只。在相同饲养管理和饲粮营养水平下，从妊娠的第2天开始，对照组饲喂基础饲粮；试验Ⅰ组腹腔注射0.5 mL含有0.125 g LGG的灭菌生理盐水（连续14 d）；试验Ⅱ组灌胃0.5 mL含有0.125 g LGG的灭菌生理盐水（连续18 d），试验期21 d。所有小鼠于临产前1 d解剖，采集大肠粪便样品，提取各组粪便样本的基因组DNA，PCR扩增后进行文库构建，对合格文库应用NovaSeq 6000测序平台进行测序，采用Uparse软件（v 7.0.1001）以97%的一致性将序列聚类成为操作分类单元（OTUs）并筛选代表序列。采用Mothur方法与SILVA 132的SSUrRNA数据库进行物种注释分析，并统计门、科和属水平上的群落组成。使用QIQME 1.7.0软件进行Alpha多样性分析，计算Chao1、ACE、Shannon、Simpson指数，并进行LEfSe分析，利用FAPROTAX预测肠道菌群的功能。【结果】与对照组相比，试验Ⅱ组Chao1和ACE指数显著升高（P<0.05），试验Ⅰ和Ⅱ组小鼠Simpson指数降低（P>0.05）。与对照组相比，在门水平上，试验Ⅰ和Ⅱ组厚壁菌门升高；试验Ⅰ组变形菌门升高，试验Ⅱ组变形菌门降低；在科水平上，试验Ⅰ和Ⅱ组的毛螺菌科、乳酸菌科、韦荣球菌科和脱硫弧菌科升高；在属水平上，试验Ⅰ和Ⅱ组的乳酸杆菌属和未确定毛螺菌科属均升高。LEfSe分析结果显示，对照组有2种显著性的菌；FAPROTAX预测出35种肠道菌群的功能。【结论】在本试验条件下，灌胃LGG能够显著提高Chao1和ACE指数，且灌胃LGG组OTUs数量高于腹腔注射组；灌胃组小鼠厚壁菌门/拟杆菌门的比值、变形菌门、脱硫弧菌科和乳酸杆菌属较腹腔注射组降低，未确定毛螺菌科属升高。综合比较两种补喂方式，给妊娠小鼠灌胃LGG对妊娠期健康有促进作用。

关键词: 灌胃; 腹腔注射; 鼠李糖乳杆菌(LGG); 妊娠小鼠; 粪便菌群

Abstract: 【Objective】 The objective of this experiment was to study the effects of gastric and intraperitoneal injection of Lactobacillus rhamnosus (LGG) on the diversity of fecal flora in mice during pregnancy, and provide a theoretical basis for the physiological and intestinal health of animals during pregnancy.【Method】 45 SPF-grade pregnant Kunming mice with similar body weight (23.33 g±1.55 g) and the same breeding date were randomly divided into 3 groups, namely control group, test groups Ⅰ and Ⅱ, 15 in each group.Under the same feeding management and dietary nutrition level, starting from the second day of pregnancy, the control group was fed with basic diet, the test group Ⅰ was intraperitoneally injected 0.5 mL sterilized normal saline containing 0.125 g of LGG (continuous 14 days), and the test group Ⅱ was gavaged with 0.5 mL sterilized normal saline containing 0.125 g of LGG (continuous 18 days).The experiment lasted for 21 days. All mice were dissected 1 day before delivery, and large intestinal stool samples were collected.Genomic DNA was extracted from fecal samples of each group and amplified by PCR for library construction, the qualified libraries were sequenced using NovaSeq 6000 sequencing platform, Uparse software (v 7.0.1001) was used to cluster the sequences into operational taxa (OTUs) with 97% consistency and screen representative sequences.Species annotation analysis was performed using the Mothur method with the SSUrRNA database of SILVA 132, and community composition was counted at phylum, family and genus levels.QIQME 1.7.0 software was used for alpha diversity analysis, Chao1, ACE, Shannon, Simpson indexes were calculated, LEfSe analysis was performed, and FAPROTAX was used to predict the function of intestinal flora.【Result】 Compared with control group, Chao1 and ACE indexes in test group Ⅱ were significantly increased (P<0.05), the Simpson index of mice in test groups Ⅰ and Ⅱ was decreased (P>0.05).Compared with control group, at the phylum level, Firmicutes in test groups Ⅰ and Ⅱ were increased, Proteobacteria in test group Ⅰ was increased, and Proteobacteria in test group Ⅱ was decreased;At the family level, the levels of Lachnospiraceae, Lactate bacteria, Erysipelotrichaceae and Desulfovibrionaceae in test groups Ⅰ and Ⅱ were increased;At the genus level, Lactobacillus and undetermined Trichospiraceae were increased in test groups Ⅰ and Ⅱ. LEfSe analysis showed that there were two significant bacteria in control group.FAPROTAX predicted 35 functions for gut microbiota.【Conclusion】 Under the conditions of this study, LGG gavage could significantly improve Chao1 and ACE indexes, and the number of OTUs in LGG gavage group was higher than that in intraperitoneal injection group.The ratio of Firmicutes/Bacteroidetes, Proteobacteria, Desulvibriaceae and Lactobacillus in the gavage group were lower than those in the intraperitoneal injection group, while the undetermined Trichospiraceae were higher.A comprehensive comparison of the two supplementary feeding methods showed that gavage of LGG to pregnant mice could promote their health during pregnancy.

Key words: gavage; intraperitoneal injection; Lactobacillus rhamnosus (LGG); pregnancy mice; fecal bacteria

中图分类号:

S857.21

李佳豪, 李超, 臧长江, 孟军, 李宏坤, 贾怡琪, 麦丽克扎提·艾尔肯, 唐纳尔·托留别克. 灌胃和腹腔注射鼠李糖乳杆菌对妊娠期小鼠粪便菌群多样性的影响[J]. 中国畜牧兽医, 2023, 50(1): 151-160.

LI Jiahao, LI Chao, ZANG Changjiang, MENG Jun, LI Hongkun, JIA Yiqi, MAILIKEZHATI Aierken, TANGNAER Tuoliubieke. Effects of Gastric and Intraperitoneal Injection of Lactobacillus rhamnosus on Fecal Flora Diversity in Mice During Pregnancy[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 151-160.

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