Vertical Effects Of Maternal Microbiota On The GIT Microbiome And Gut Function Of Offspring During Pregnancy And Lactation Period In Female Goats

Gut microbiota plays significant functional roles,such as gut development,digestion,and absorption,as well as immune responses in young animals.Maternal microbiota,diet,environmental factors,and early management greatly influence the gut microbiota composition and function of offspring.This further influence and causes changes in nutrient utilization and immune responses in young animals.Vertical transmission of maternal microbes can influence microbial colonization in early life.However,the extent of maternal transmission from different parts,the types of target microbes selected by offspring,and the effects of vertical transmission of breast milk microbes on the composition and function of gut microbes in young goats are still unclear.This greatly limits the early health intervention of young animals at the microbe’s level,as well as the development and application of early milk replacer and starter feeding for young animals.This study used goats as an animal model to systematically investigate the succession pattern of gut microbes and the metabolite profiles that mediate maternal physiological responses from non-pregnancy,gestation to lactation period,and mapping of metabolites that mediate maternal physiological responses.This study also revealed the rules of the effect of the vertical transmission of the maternal microbes on the gut microbiota composition of the offspring.Further,the early weaning and breast milk deficient goat models revealed the mechanism of early intervention that affects the hindgut microbiota composition,and mediates hindgut inflammation and lipid metabolism disorders.Our main results are as follows:I.Succession patterns of maternal gut microbiota and metabolites during pregnancy and lactationTo elucidate the relationship between the gut microbiome,metabolism,and reproductive hormones during non-pregnancy,pregnancy,and lactation stages,18 healthy female goats were selected and investigated.Serum and fecal samples from 18 pregnant goats during non-pregnancy(Baseline),0d(Pre＿D0),60d(Pre＿D60),and 120d(Pre＿D120)of pregnancy,as well as 0d(Lac＿D0),14d(Lac＿D14),and 56d(Lac＿D56)of lactation were collected for 16 S r RNA gene sequencing and untargeted metabolomic analysis.We found that the gut microbiota was drastically altered from pregnancy to lactation(ANOSIM;R=0.504,P=0.001),with Anaerovoracaceae being strongly correlated with decreased fecal estradiol and progesterone(R>0.6,P<0.01).In addition,we generated a metabolome atlas of gut and serum from non-pregnancy to lactation.The serum and fecal metabolome alterations during the entire reproductive cycle revealed specific metabolic fingerprints,characterizing various stages of pregnancy and lactation.Concentrations of gamma-aminobutyric acid,3-hydroxypropionic acid,indole-3-carboxylic acid,and N-acetylvaline were significantly lower in the gut of pregnant goats compared with non-pregnant goats(P<0.05),and the concentrations of Carnitine C5:0,2’-deoxyinosine and deoxyadenosine were significantly increased(P<0.05).Serum concentrations of thromboxane B2,serotonin,N-formylmethionine,and 20-carboxyarachidonic acid were significantly elevated in the third trimester(P<0.05).Next spearman correlation analysis was used and the results showed that Anaerovoracaceae was correlated with fecal and serum landmark metabolism at each stage of pregnancy and lactation(|R|>0.8,P<0.01).These microbes may be closely related to the regulation of physiological hormones and metabolic changes during pregnancy.II.Convergence patterns of vertical transmission of maternal microbes affect the gut microbial composition of offspring in goat and sheep modelsTo further elucidate the patterns that maternal microbes vertically affect the gut microbiota composition of offspring during parturition and lactation,goats and sheep were used as models.Five healthy pregnant Tan sheep and 6 healthy cashmere goats at 2-3 years old were selected.Additionally,in this study,five lambs and six goat kids were obtained.Five lambs and three goat kids were sampled after birth,and another 3 kids were sucked with their mothers to 3d and were sampled at 3d.All samples were collected for amplicon sequencing.The microbiota was detected in the placenta,amniotic fluid,and vagina of the mother,as well as in the rumen,abomasum,and meconium of newborn lambs/kids after parturition.It was revealed that the microbiota that belongs to the phylum Firmicutes,Actinobacteria,Proteobacteria,and Bacteroidetes were dominant.A low number of microbes in the rumen,abomasum,and meconium of newborn lambs/kids originated from the placenta,amniotic fluid,and vagina of the mother.Furthermore,the vertical transmission rules of the maternal microbes were similar in goats and sheep.The microbes of Romboutsia,Escherichia-Shigella,and Alistipes strains had strong maternal vertical transmission.After the lamb/kids are born,the strains of Fusobacterium necrophorum＿subsp,Streptococcus,Escherichia-Shigella,and Peptostreptococcaceae have colonized the gastrointestinal tracts by the way of vertical transmission in breast milk.III.Correlation analysis of nutrient composition changes and microbial composition in goat milk during lactationAfter birth,milk microbes are the most direct means of vertical transmission of maternal microbes.To reveal the relationship between changes in microbial composition in goat milk and changes in its nutritional components,we selected eighteen healthy pregnant goat,and milk samples during lactation were collected on the 0d,7d,14 d,28d,and 56 d of lamb birth to determine the nutritional and microbial compositions.The composition and abundance of microbial communities in goat milk were significantly affected by the age of lactation.This was mainly divided into three stages from 0 to 56 d of lactation,namely:D0-D7;D14-D28 and D28-56(ANOSIM,R=0.434,P=0.001).In addition,specific bacteria were present in goat colostrum,mainly including related strains of Shewanella and Faecalibacterium.Subsequently,according to the differences in goat milk microbial,the milk could be divided into three types from D0 to D56 days of lactation,which are defined as Type 1,Type 2,and Type 3.Type 1 was mainly the microbial typing of colostrum(D0and D7),Type 2 was mainly for D14 and D28-day-old milk microbial typing,and Type 3was mainly for D56-day-old milk microbial typing.Microbial phenotypes in Type 1 were mainly stress tolerance,gram-negative,and biofilm formation.The microbial phenotypes in Type 3 were mainly facultative anaerobes and gram-positive.Combined with Spearman correlation analysis,it was found that the content of milk protein was significantly positively correlated with the relative abundance of Bacillus,Salmonella,Faecalibacterium,and Achromobacter.Taken together,the abundance of microbes in goat milk with the age of lactation may be related to the changes in nutrient components.IV.Gut microbiota derived metabolites contribute negatively to colon lipid metabolism in a milk replacer-fed goat modelThe vertical transmission of active substances and microbiota in breast milk is crucial for maintaining intestinal homeostasis in offspring.In this study,milk replacer-fed goat kids were used as models to further investigate the effect of vertical milk transmission of Type 2on the colon microbial composition,metabolic characteristics,and epithelial physiological response.A total of 10 female Tibetan goat kids were selected to breast milk ad libitum for 3d postpartum.On the 4 d after birth,the kids were randomly divided into two groups.The breast milk(BM)group continued to feed on breast milk and the milk replacer(MR)group was fed commercial milk replacer.The experimental period was 28 days.We found that milk replacer feeding reduced the growth performance of goat kids(P<0.05)and decreased the concentrations of TC and NFFA in serum and liver(P<0.05).Additionally,it up-regulated the expression of genes,such as CCL25,PRAP1,FABP2,RBP2,APOC3,ABCG5,and ABCG8 in the colonic epithelium of goat kids.We also found that milk replacer feeding negatively regulates the colon epithelial lipid absorption and expression of cholesterol transport metabolic pathways.Moreover,milk replacer feeding resulted in dramatic changes in the composition and function of the core microbes in the colon of goat kids.The breast milk feeding was mainly enriched with Clostridium,Lachnoclostridium,Eubacterium,and Flavonifractor,as well as Faecalibacterium in the colon content of goats.Milk replacer feeding significantly reduces secondary bile acids in colon contents and serum,especially lithocholic acid,beta-murine,urocanic acid,and 2,3-deoxydeoxycholic acid,etc.It also significantly down-regulated the expression of fatty acid elongation and fatty acid metabolism pathways in serum.Subsequent transplantation of goat colonic microbes as donor microbes into C57/6J mice revealed that the recipient mice of milk replacer-fed goat microbes had significantly lower cholesterol concentrations in the liver.The concentration of free fatty acids in the liver and serum was significantly reduced in the mouse model.Nevertheless,the gene expression of ABCG5,ABCG8,and FABP2 in the colon was significantly up-regulated(P<0.05)and the ability of colonic lipid metabolism was significantly reduced.This study confirmed that milk replacer feeding induced colonic lipid metabolism disorders with colonic microbial disturbances in goat kids.V.Gut microbiota derived metabolites contribute negatively to hindgut barrier function development in the early weaning goat modelTo investigate the effects of the disruption of vertical milk transmission of Type 3 milk on hindgut microbial composition,metabolic characteristics,and physiological responses of the intestinal epithelium,early weaned goat kids were used as models.A total of 18 female Tibetan goat kids were selected for weaning on the 28 th and 60 th days postpartum,respectively.All kids were weaned at postnatal day 28 and started to be fed starter feed,and all lambs were fed to 75 d.We found that early weaning significantly increased the hindgut inflammatory responses in the colonic epithelium and up-regulated the expression of pro-apoptotic genes(Bax,Caspase-9,and Caspase-3;P<0.05).Additionally,it decreased the expression of colonic genes of the intestinal barrier proteins ZO-1 and Claudin-1(P<0.05).Early weaning significantly altered the microbial composition and metabolites of the hindgut and significantly increased the number of B.acidifaciens in the hindgut(P<0.001).It also decreased the number of B.uniformis in the hindgut(P<0.001).The Spearman correlation analysis found that the abundance of B.acidifaciens was significantly negatively correlated with lysophosphatidylcholine.Similarly,the chemokine signaling,IL-17 signaling,and PPAR signaling pathway were upregulated in the colonic mucosa of the early-weaned goats.By applying intestinal microbiota transplantation from goats to defaunated C57/6J mice,we confirmed that the caecal microbiota of D28 goat kids increased the relative abundance of B.acidifaciens and significantly up-regulated the expression of Bax,GPR109 A,GPR43,SLC27A2,FABP6,NR1H3(P<0.05).It also decreased the expression of ZO-1 and claudin-1protein in the mouse jejunum and colon(P<0.05).These results propose that the hindgut microbiota and metabolites mediate the decreased of tight junction protein expression during early weaning,and the relative abundance of B.acidifaciens was negatively correlated with the hindgut barrier gene expression.This study shows how weaning stress can affect key host-microbe interaction regulators in the hindgut in a lysophosphatidylcholine dependent and independent manner.In conclusion,this study systematically elucidates the succession patterns of gut microbes in goats from non-pregnancy,pregnancy,and lactation stages,and constructs a metabolite map of the gut and serum of goats throughout the reproductive cycle.This study utilized young ruminant models to reveal the rules of the parturition process and the vertical transmission of maternal microbes in lactating goats,affecting the composition of the gut microbes of the offspring,and further used model animals to reveal that the imbalance of gut microbes in goat kids mediates hindgut lipid metabolism disorders and inflammation.This study screened potential probiotics that can inhibit the occurrence of gut inflammation and positively regulate the lipid metabolism of the hindgut.This study can provide potential functional probiotics for the development of high-efficiency and healthy milk replacers/starter feeds for young ruminants.This study provides a theoretical basis for early manual intervention strategies for young ruminants.