The succession of fecal bacterial community and its correlation with the changes of serum immune indicators in lambs from birth to 4 months

YlN Xue-jiao,Jl Shou-kun,DUAN Chun-hui,TlAN Pei-zhi,JU Si-si,YAN Hui,ZHANG Ying-jie,LlU Yue-qin

College of Animal Science and Technology,Hebei Agricultural University,Baoding 071000,P.R.China

Abstract Early bacterial colonization and succession within the gastrointestinal tract have been suggested to be crucial in the development of host immunity.In this study,we have investigated the changes in live weight and concentrations of selected serum parameters in relation to their fecal bacterial communities as determined by high throughput sequencing of the 16S rRNA gene over the same period in lambs.The results showed that lambs’ growth performance,the serum parameters,fecal bacterial community and fecal bacterial functions were all affected (P＜0.05) by age of the lambs.Similarity within age groups of fecal microbiota was lower in the preweaning period and increased sharply (P＜0.05) after weaning at 60 days.The similarity between the samples collected from birth to 90 days of age and those collected at 120 days of age,increased (P＜0.05) sharply after 30 days of age.Some age-associated changes in microbial genera were correlated with the changes in concentrations of immune indicators,including negative (P＜0.05) correlations between the relative abundance of Lachnospiraceae UCG-010,Eubacterium coprostanoligenes group,Ruminococcaceae UCG-005,Ruminococcaceae UCG-009,Ruminococcaceae UCG-013,Ruminiclostridium 6,Ruminococcaceae UCG-008,and Oscillibacter with serum concentrations of lipopolysaccharide(LPS),D-lactate dehydrogenase (DLA),immunoglobulin (IgA,IgM,and IgG),and cytokines (interleukin-1β (IL-1β),IL-6,IL-12,and IL-17),tumor necrosis factor-α (TNF-α),and the relative abundance of these genera increased from 45 days of age.In conclusion,these results suggested that the age-related abundances of particular genera were correlated with serum markers of immunity in lambs,and there might be a critical window in the period from birth to 45 days of age which provide an opportunity for potential manipulation of the fecal microbial ecosystems to enhance immune function.

Keywords: fecal bacteria,establishment,sheep,serum parameter,early life

1.lntroduction

Intestinal microbes play critical roles in absorption and metabolism of elements in food (Dill-McFarlandet al.2017),differentiation of the intestinal epithelium tissues(Sommer and B?ckhed 2013),host immunity (Ivanovet al.2009),and maintenance of the intestinal mucosal barrier function (Chenget al.2018).Meanwhile,the gastrointestinal environment may support the growth,reproduction,and longevity of the bacterial community(Browneet al.2016;Selber-Hnatiwet al.2017).Changes in the gut environment from diet and host physiology,as well as ingested microbes,may create competition for resources that affect the resident microbiota and reshape the microbial community,which in turn may affect host physiology (Selber-Hnatiwet al.2017).

Maintaining a healthy gastrointestinal microbiota is vital for the health and productivity of ruminants.In ruminants,microbiota modifications are associated with stimulation of the early innate immune system (Arrietaet al.2014;Schwaigeret al.2020) and have been linked to the prevention of mastitis development (Raultet al.2020).For humans,the first 6 months after birth are identified as a ‘window of opportunity’ (Penderset al.2007;Hillet al.2017) to manipulate neonatal immunity,where the immune system is not well-established and much more responsive to external and environmental factors than seen in the adult and may characterize the individual throughout life (Rodriguezet al.2015).Disrupting early gut community succession may lead to dysbiosis in which the community loses key taxa,diversity,and/or metabolic capacity (Laforest-Lapointe and Arrieta 2017).Unraveling the links between the succession patterns of the gut microbiome and the development of immune function will be a key to improve our understanding of the importance of the host’ microbiome for the development of the immune system.Although the ecological patterns of microbial succession in the early period of ruminants have been partly defined in previous studies (de Oliveiraet al.2013;Dill-McFarlandet al.2017;Guoet al.2020),the taxonomic and functional features and their role in the development of the host immunity remains unknown.

Although the concept of a gradual establishment of the gut bacterial community is well accepted,the details of this process remain to be clarified.In this study,we hypothesized that there might be a vital period,i.e.,a window of opportunity,during which changes in the gut microbiome might influence the development of the immune system.Therefore,the present study was conducted with lambs to examine colonization of the fecal bacterial community and concomitant changes in serum indicators of immune function from birth to 4 months of age.The information obtained could enable the development of interventions aimed at maintaining and/or improving the host immune system to facilitate the prevention of disease.

2.Materials and methods

2.1.Animals,diets,and experimental design

The study was conducted between January and August 2019 in the Weizun Farm (Hengshui,China).It used a total of 10 newly born female lambs (Hu sheep breed,singletons,(2.87±0.28) kg body weight,BW) that were raised under the conventional system used for commercial sheep herds in China.After birth,the lambs were housed together with their mothers in individual pens and provided with starter feedab libitumfrom day 15(Table 1).They were weaned by removing ewes from the pens at day 60,and offered a mixed ration (Table 1) at 0730 and 1500 h each day with approximately 5% feed refusal.Ewes and lambs had free access to fresh water throughout the study.The lambs were weighed before the morning feeding on days 0,30,60,90,and 120 to determine the average daily gain (ADG) (Fig.1).

Table 1 Nutrient composition of experimental diets (dry matter basis)

2.2.Sample collection

Blood samples (5 mL) were collected by jugular venepuncture from each lamb before the morning feeding on 30,60,90,and 120 days of age (Fig.1).The sampleswere centrifuged at 3 000×g for 15 min at 4°C to gather serum,which was stored at -20°C until analysis.

Fig.1 Experimental design and sampling schedule.There are 10 lambs used in our study.

Fecal digesta samples were obtained from each lamb on 9 occasions as follows: the first within 24 h after birth,then at 3,10,20,30,45,and 60 days of age before weaning,then at the ages of 90 and 120 days after weaning (Fig.1).These samples were collected directly from the rectum approximately 2 h after the morning feeding by the use of sterile gloves and sterile cotton swabs.The samples were placed in sterile containers,immediately snap-frozen in liquid nitrogen,and stored at -80°C until DNA extraction.Ten replicates were measured for each indicator.

2.3.Serum measurements

Activities of superoxide dismutase (SOD),total antioxidant capacity (T-AOC),and glutathione peroxidase (GSH-Px)in serum were measured using commercial colorimetric assay kits (Nanjing Jiancheng Biology Co.,Nanjing,China) according to the manufacturer’s instructions.Serum malondialdehyde (MDA) concentration was measured by a thiobarbituric acid test using a commercial kit (Nanjing Jiancheng Biology Co.).Serum levels of immunoglobulins (IgG,IgA,and IgM),cytokines(interleukin-1β (IL-1β),IL-6,IL-12,and IL-17),tumor necrosis factor-α (TNF-α),diamine oxidase (DAO),lipopolysaccharide (LPS),and D-lactic acid (DLA) were determined with ELISA test kits (Nanjing Jiancheng Biology Co.) according to the manufacturer’s instructions.

2.4.DNA extraction and sequencing

Genomic DNA in rectal digesta was obtained using the DNA Isolation Kit (MoBio Laboratories,Carlsbad,CA,USA) following the manufacturer’s instructions.The purity and quality of the genomic DNA were determined using 1% agarose gel electrophoresis and spectrophotometry.The extracted DNA was stored at-20°C for further analysis.The barcoded primers 338F(5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R(5′-GGACTACHVGGGTWTCTAAT-3′) were used to amplify the V3-V4 region of the 16 rRNA gene (Munyakaet al.2015).PCRs were performed in triplicate using a 25-μL mixture containing 12.5 μL of KAPA 2G Robust Hot Start Ready Mix,1 μL of forward primer (5 μmol L-1),1 μL reverse primer (5 μmol L-1),5.5 μL of ddH2O,and 5 μL of DNA (total template quantity was 30 ng).Cycling parameters were 95°C for 5 min;followed by 95°C for 45 s,55°C for 50 s,and 72°C for 45 s with a final extension of 72°C for 10 min.Amplicons were extracted from 2% agarose gels and purified using the QIAquick Gel Extraction Kit (QIAGEN,Germany).Deep sequencing of purified products was performed on an Illumina Miseq platform (Caporasoet al.2012) at Allwegene Company (Beijing,China).

2.5.Bioinformatics analysis

The raw data were screened initially;short (shorter than 230 bp) or low quality reads (quality score lower than 20)were removed by QIIME1 (version 1.8.0).Clean,pairedend sequences were merged using FLASH (version 2.7.1).Sequences were classified into operational taxonomic units (OTUs) using a threshold of 97% similarity (Edgar 2013).Species richness (Chao1 index) and diversity(Shannon diversity index) were chosen for alpha diversity and calculated by QIIME1 (version 1.8.0) (Caporasoet al.2010).Heat maps were generated using the package‘pheatmap’ of R (version 4.0.0) Software.The Tax4Fun2 method was applied for predicting the functional profiles of bacterial communities based on the 16S rRNA gene sequence using package ‘Tax4Fun2’ by R (v4.0.0)Software (Wemheueret al.2020).The 16S rRNA function prediction was categorized into the Kyoto encyclopedia of genes and genomes (KEGG).Identification of enterotypes which means the microbiota hold the reproducible patterns of variation and the variation can be separated into clusters was performed as described by previous studies (Arumugamet al.2011,2014).

2.6.Randomforest analysis

To acquire the best discriminant performance of microbial taxa across lamb’s ages,we regressed (Subramanianet al.2014) the relative abundances of bacterial taxa at the genus level against the ages of the lambs using default parameters of the R (Breiman 2001) implementation of the algorithm (R package ‘RandomForest’,ntree=1 000,using default mtry of p/3,where p is the number of taxa of genus) (Liaw and Wiener 2002).Lists of genera ranked by RandomForests in order of feature importance were determined by set seed.The number of important marker taxa was then identified by using 10-fold cross-validation implemented with the rfcv() function in the R package‘RandomForest’ with 10 repeats.After the minimum cross-validation error was obtained,we chose the number that stabilized against the cross-validation error curve as marker taxa correlating with ages of the lambs.

2.7.Data availability

The datasets generated from the current study are available in the Genome Sequence Archive repository(http://gsa.big.ac.cn),under accession numbers PRJCA002881.

2.8.Statistical analysis

The dissimilarity of microbial communities among different age groups was performed and visualized by R (v4.0.0)Software using Principal coordinates analysis (PCoA) based on Bray-Curtis distance.The microbiota Bray-Curtis similarity was calculated based on OTUs.An analysis of similarity(ANOSIM) was performed to compare the fecal bacterial communities in lambs at different ages.Relationships between microbiota and the serum parameters were analyzed using Spearman’s rank correlation test.

The results were analyzed as a completely randomized design.Individual lamb considered as an experimental unit for every analysis.All statistical analyses were carried out using R (v4.0.0) Software.Data on BW,ADG,and serum parameters were evaluated by ANOVA after checking independency,normality,and homogeneity:

whereYiis the dependent parameter,μis the overall mean,Xiis the age effect andeiis the residual error.We used the orthogonal comparison method to analyze the linear or quadratic development trend of the growth performance and serum biochemical parameters with the age.When significant effects were detected between ages,means were compared by Duncan’s multiple range test.Comparisons between pathways of groups were performed using the Kruskal-Wallis test.P-value＜0.05 was considered to indicate statistical significance.

3.Results

3.1.Growth performance and serum parameters

The initial BW of lambs was (2.87±0.28) kg and the linear increases in BW with age are shown in Table 2.The highest (P＜0.05) ADG was found between 30 to 60 days of age,and the lowest ADG (P＜0.05) was found between 60 to 90 days of age (Table 2).

Results for the serum parameters are presented in Table 3.The concentrations of IgG,IgM,IgA,IL-1β,IL-6,IL-12,TNF-α,LPS,and DLA showed a quadratic (P＜0.05)and linear (P＜0.05) decrease with age,with the highest value in 30 days of age (P＜0.05).Compared to other age groups,the concentration of serum GsH-Px was lower(P＜0.05) at 120 days of age,both linear and quadratic effects were significant (P＜0.05).The concentration of IL-17 and MDA decreased linearly (P＜0.05) with increasing age.Meanwhile,antioxidant indicators of serum SOD and T-AOC increased (P＜0.05) from 30 to 60 days and then decreased (P＜0.05) thereafter,and the quadratic effect was significant (P＜0.05).There was no significant (P＞0.05)difference among ages in mean serum DAO concentration.

Table 2 Effect of age on growth performance of lambs

Table 3 Effect of age on serum biochemical parameters of lambs

3.2.Changes in fecal microbiota and taxonomic composition

High throughput sequencing was used to study the composition of rectal microbiota at different ages of the lambs.After removal of chimeras,filtered high-quality sequences were grouped into 69 533 OTUs.The alpha(Chao1 and Shannon indices) and beta diversities were calculated.These alpha diversity indices showed a higher richness,diversity in older lambs (Fig.2-A and B).To further investigate the taxonomic composition of the bacteria,we compared the relative abundance of fecal microbiota at the phylum level (Fig.2-C).The dominant phyla present in the fecal samples were Firmicutes(～69%),Bacteroidetes (～18%) and Proteobacteria(～9%).The relative abundance of Firmicutes and Bacteroidetes increased with age of the lambs,and that of Proteobacteria decreased (Fig.2-C).The phylum Spirochaetae was more abundant after weaning.Furthermore,the bacterial composition similarity comparison of fecal samples determined by PCoA and ANOSIM indicated a lower degree of similarity before 30 days of age,whereas from 45 days of age,the sample dots clustered together indicating an increase in similarity(Fig.2-D).Analysis of similarity (ANOSIM) comparisons also showed the changes (P＜0.05,R=0.4192) in the fecal microbiota of lambs at different ages.

3.3.The stability of fecal microbiota over time

The within-group similarity comparison based on the Bray-Curtis distance showed significant age-dependent changes (Fig.3-A).While the alpha diversity continued to increase,the within-group similarity between individual lambs increased significantly (P＜0.05) after weaning(Fig.3-A),indicating high stability in the bacterial communities at this stage of growth.

Furthermore,as shown in Fig.2-D,the lambs’ fecal samples apparently clustered together with a limited dispersion in the PCoA graph after 60 days.In order to emphasize the process of gradual approximation to the mature fecal bacterial community structure of lambs,we calculated the similarity between the fecal bacteria of samples collected from birth to 90 days of age and those collected at 120 days of age (Fig.3-B).According to our results,the similarity sharply increased after 30 days of age,reaching a maximum (P＜0.05) at 90 daysvs.120 days (Fig.3-B),indicating that the composition of the lambs’ fecal microbial ecosystem moved closer to the mature situation.

To characterize the stability of an individual’s bacteria,we determined the average Bray-Curtis distance between adjacent age groups.The results disclosed that the bacterial composition of each individual’s fecal microbiota changed over time.We found that fecal microbiota similarity between 0 and 3 days of age and between 30 and 45 days was lower than at other stages,whereas the similarity of 90 and 120 days was higher (P＜0.05) than the other groups (Fig.3-C).Our results showed that large differences in community composition occur in the period after birth and at the time after feeding starter,whereas a stable bacterial community was established after weaning.

As shown in Fig.2-C,Firmicutes,Bacteroidetes and Proteobacteria were the predominant phyla.For Bray-Curtis similarity,within these phyla,the similarity increased(P＜0.05) with the age of the lambs,and the Firmicutes and Bacteroidetes were more stable components of the microbiota than Proteobacteria (Fig.3-D).

Fig.2 Effect of age on fecal bacterial communities at different timepoints.A and B,species richness estimates (Chao1) (A) and diversity indices (Shannon) (B) for the 9 sampling time points are presented.C,relative abundances of bacterial phyla across 9 lambs’ age groups.D,constrained ordination of Bray-Curtis dissimilarity based on relavie abundances of bacteria measured using principal coordinates analysis (PCoA).There are 10 lambs used in our study.

Fig.3 Changes in fecal bacterial diversity as the lambs aged.The Bray-Curtis similarity within groups (A),between samples (B) collected at the time point from birth to 90 days of age and those collected at the 120 days of age,the similarity between adjacent time points (C),and the Bray-Curtis similarity based on the 3 dominant phyla (D).Data are expressed as mean±SE.There are 10 lambs used in our study.

For maturation of the fecal microbiota,these findings indicated that the microbial community was unstable and changed rapidly before weaning in lambs and they had revealed 2 potentially important periods,one from 0 to 3 days of age and the other from 30 to 45 days of age.

3.4.Enterotype in different ages

Use of enterotypes classifier following Arumugam’s method (Arumugamet al.2014) enabled the fecal microbiota of lambs to be clustered into 4 enterotypes:enterotype 1 was dominated byBacteroides;enterotype 2 was dominated byEscherichia-Shigella;enterotype 3 was dominated byRuminococcaceae UCG-005and enterotype 4 was dominated byLactobacillus.The main enterotypes at birth of the lambs (day 0) were enterotypes 1 and 2,but at 3 days of age,the main emerotype was enterotype 4,then this declined and was eventually superseded by enterotype 3 after 30 days(Fig.4-A).PCoA analysis demonstrated the differences in enterotypes of the bacterial communities (Fig.4-B).

Fig.4 Enterotypes shift with age increase.A,the relative abundances of enterotypes among age groups of lambs.B,Scatterplot from principal coordinates analysis (PCoA) based on bacterial Bray-Curtis distance in each sample.There are 10 lambs used in our study.

3.5.Age-discriminatory bacteria in rectal digesta of lambs

To characterize fecal microbiota maturation across ages of lambs,the relative abundance of genus was regressed against each lamb’s age using the Random Forests machine learning algorithm.All bacterial taxa were ranked by the order of ‘a(chǎn)ge-discriminatory importance’.After calculating the importance of each bacterial taxon with the mean squared error (MSE) and 10-fold crossvalidation,26 bacterial taxa at the genus level were defined as biomarker taxa in the model in which the cross-validation error curve stabilized (Fig.5-A).Most age-discriminatory bacteria belonged to Firmicutes (20/26)and Bacteroidetes (4/26) at the phylum level (Fig.5-A).All age-discriminatory bacterial taxa were highly agedependent (Fig.5-B).Of these age-discriminatory bacterial taxa,2 taxa (out of 26) had higher relative abundance before 20 days of age (LachnoclostridiumandEnterococcus),13 taxa increased with the age of the lambs (Anaerosp-orobacter,Treponema 2,Ruminiclostridium 1,Rikenellaceae RC9 gut group,Prevotellaceae UCG-004,Ruminococcaceae UCG-010,Ruminococcaceae UCG-009,Ruminiclostridium 6,Ruminococcaceae UCG-013,LachnospiraceaeAC2044 group,Eubacterium coprostanoligenes group,Ruminococcaceae UCG-005,andOscillibacter),7 taxa had higher relative abundance between 45 and 60 days of age (Coprococcus 3,Coprococcus 1,Eubacterium hallii group,Parasutterella,Parabacteroides,Lachnospiraceae UCG-010,andRuminococcaceae UCG-008),4 taxa had higher relative abundance after 20 days of age(Ruminococcaceae UCG-004,Alistipes,Holdemania,andRuminococcus torques group).The relative abundance of the genus,Eubacterium coprostanoligenes group,Eubacterium hallii group,Coprococcus 1,Prevotellaceae UCG-004,Lachnospiraceae AC2044 group,Ruminococcaceae UCG-005,Ruminococcaceae UCG-009,Ruminococcaceae UCG-013,Ruminococcaceae UCG-010,Coprococcus 3,Ruminiclostridium 6,Ruminococcaceae UCG-008,andOscillibacterwere all increased from 45 days of age.

Fig.5 Bacterial taxonomic biomarkers of lambs’ age and the fecal microbiota gradually stabilize.A,the top 26 biomarkers of bacterial genus were identified by applying RandomForests regression of their relative abundances in lambs against chronological age.Ranking in descending order of importance to the accuracy of the model was to determine biomarker taxa.The insert illustrates the result of 10-fold cross-validation error.B,heatmap of changes over ages showing the relative abundances of the top 26 age-predictive biomarkers for bacterial genus.There are 10 lambs used in our study.

3.6.Function prediction of fecal microbiota

We performed Tax4Fun2 analysis and found 33 KEGG pathways (level 2),9 of which differed according to age of the lambs (Fig.6).Signaling molecules and interaction,development,metabolism of other amino acids were higher (P＜0.05) at birth.Signal transduction,transport and catabolism had high (P＜0.05) relative abundance between 20 to 45 days of age.Excretory system,glycan biosynthesis and metabolism had high (P＜0.05) relative abundance at the age of 3 days.The pathway of the Nervous system had low (P＜0.05) relative abundance at birth.

Fig.6 Shifts in fecal bacterial functional profiles as the lambs aged.Heatmap showing the relative abundances of the KEGG pathways (level 2) against chronological age of lambs.＊,P＜0.05.There are 10 lambs used in our study.

3.7.Correlation of age-related bacterial genera with serum immune markers

Spearman correlation was established to explore the relationships between age-related genera and the serum immune markers (Table 4).The relative abundance ofLachnospiraceae UCG-010,Eubacterium coprostanoligenes group,Ruminococcaceae UCG-005,Ruminococcaceae UCG-009,Ruminococcaceae UCG-013,Ruminiclostridium 6,andOscillibacterhad negative (P＜0.05) Spearman correlations with serum concentrations of LPS,DLA,immunoglobulin (IgA,IgM,and IgG) and cytokines (IL-1β,IL-6,IL-12,IL-17,and TNF-α).Meanwhile,relative abundances ofEnterococcusandLachnoclostridiumwere positively (P＜0.05) correlated with serum concentrations of LPS,DLA,immunoglobulin(IgA,IgM,and IgG) and cytokines (IL-1β,IL-6,IL-12,IL-17,and TNF-α).Abundances ofParabacteroidesandRuminococcaceae UCG-004were negatively (P＜0.05)correlated with levels of TNF-α and immunoglobulins (IgA,IgM,and IgG).

4.Discussion

The mammalian gastrointestinal microbiota plays an important role in diagnosing,treating,and ultimately preventing disease,as an indicator of and contributor to host health (Faithet al.2013).Previous studies have illustrated the importance of the early gut microbiome for neonatal immune system development and disease pathogenesis (Arrietaet al.2014).A window of opportunity occurs in early life,during which changes in gut microbial colonization can result in immune dysregulation that predisposes susceptible hosts to disease (Laforest-Lapointe and Arrieta 2017).Although the ecological patterns of microbial succession in the early life of ruminants have been partly defined in previous studies (de Oliveiraet al.2013;Dill-McFarlandet al.2017;Yinet al.2022),the taxonomic and functional features during this stage remain unknown.The current study was investigated to determine the links between the process of gut microbial colonization and the age-dependent immune functions that relied on them.Our hypothesis that there might be a critical period,during which changes in the gut microbiome might influence the development of the immune system has been supported by the results of this study.Our results indicated that the immunity of lambs changed in an age-dependent manner that was correlated with several age-related changes in fecal bacterial taxa,thus there may be a critical period from birth to 45 days of age in which to address the relatively low abundance of critically important bacterial genera.This highlighted the importance of exploring the interactions between the complex microbial ecology and the host immune system,and it may be possible to manage the key microbial taxa for maintaining and/or improving the development of the immune system and preventing disease.

The gut microbiota provides an important layer of defense against invasion by pathogenic microorganisms(Kosiewiczet al.2011).The serum immunoglobulin (IgA,IgM,and IgG),cytokines (IL-1β,IL-6,IL-12,IL-17,and TNF-α),and intestinal permeability indicators (DLA,LPS,and DAO) have been widely used as sensitive indicators of immune function (Yinet al.2021).The gut bacteria can produce several immunoglobulins (Lécuyeret al.2014;Kochet al.2016),and alter the levels of inflammatory cytokines in the intestines or circulation (Genget al.2020).In our study,the serum concentrations of several immune indicators were decreased with the increasing age of the lambs,indicating that the immunocompetence gradually established as the lambs aged.In this study,we found several bacterial taxa that have a strong connection with both growth and the immunocompetence of the host.We found the generaEnterococcusandLachnoclostridiumwere positively correlated with the serum levels of immune markers in the lambs.These genera are potentially pathogenic:Enterococcushave high relative abundance in disease states (Zhanget al.2020),andLachnoclostridiumis a biomarker for cancer (Lianget al.2020).In contrast,genera from the family Lachnospiraceae (Lachnospiraceae UCG-010andLachnospiraceae AC2044 group),one of the major taxonomic groups of gut microbiota,have been associated with the maintenance of gut health (Vojinovicet al.2019).The members of the Ruminococcaceae and Lachnospiraceae families have been reported to be taxonomic markers of healthy quarters in mastitis-free mammary glands (Falentinet al.2016).These findings may be the result of these two families being vital for producing short-chain fatty acids (Maet al.2020) which can regulate immune cell function (Correa-Oliveiraet al.2016;Laforest-Lapointe and Arrieta 2017).In our study,the age-related genera,Lachnospiraceae UCG-010,Lachnospiraceae AC2044 group,Ruminococcaceae U C G-0 0 5,Ruminococcaceae UCG-009,a n dRuminococcaceae UCG-013all correlated negatively with serum concentrations of the immune markers.Our present findings illustrated the importance of particular gut microbial taxa and their temporal succession patterns in the development of immunity in lambs.

With the increasing age of the lambs,their immunocompetence became gradually established as the result of increases in abundance of favorable gut microbes that stimulated the development of immunity and probably led to reductions in populations of potential pathogens.For instance,the relative abundances of potentially pathogenic genera,EnterococcusandLachnoclostridium,were higher before 10 days of age.Whereas members of the Ruminococcaceae and Lachnospiraceae families,potential health biomarkers,had an increased relative abundance from 45 days of age.The similarity analysis revealed that the gut microbiota of the lambs was relatively unstable before 45 days of age and changed rapidly about this time.These results support the proposal that this stage of development may be a critical period that provides a‘window of opportunity’ for potential intervention.During this ‘window of opportunity’,the lambs’ gut harbors a highly variable and increasingly diverse microbial community of low resilience,which renders it easily disrupted by disturbances (Yassouret al.2016),and the health biomarkers were relative low,thus this window provides a unique opportunity for potential manipulation of such a complex microbial ecosystem.In addition,our result indicated that the lowest ADG occurred during 60 to 90 days of age,a month after weaning.During the weaning period,the lambs were completely consumed solid feed and separated from their mothers,which caused physiological and psychological stress to metabolism (Nematiet al.2015),gut microbiota (Liet al.2022),and immune response (Zhanget al.2018),finally,affected the growth performance of lambs.Thus,we inferred that the lowest daily gain between 60 and 90 days of age was due to weaning stress,demonstrating the importance to mitigate the negative effects of weaning.

In this study,we found that several gut bacteria,such as Ruminococcaceae and Lachnospiraceae,may play an important part in the establishment process of host immunity for lambs.Ruminococcaceae and Lachnospiraceae have been described by others as important immune indicators (Correa-Oliveiraet al.2016;Laforest-Lapointe and Arrieta 2017),but we suggest they may also be key players in the development of immunity.Further study into the microbial ecology and metabolism of these bacteria should disclose mechanistic underpinnings of their host-health associations and enable their development as therapeutics.

5.Conclusion

The immune response of lambs is the highest at 30 days of age,due to the high concentrations of immunoglobulins,cytokines and intestinal permeability indicators in 30 days of age.The gut bacterial community in lambs was eventually superseded by enterotype 3 after 30 days indicating the generaRuminococcaceae UCG-005may play important role in the maturity of lambs.Besides,our study demonstrates that several age-related bacterial genera appear to be important in the establishment process of both the rectal bacterial community and host immune function,especially the members of the Ruminococcaceae and Lachnospiraceae families.For lambs,there may be a critical period from birth to 45 days of age in which to address the relatively low abundance of critically important bacterial genera.It could lead to the development of effective interventions aimed at maintaining and/or improving immune system development and disease prevention in growing lambs.

Acknowledgements

This study was supported by the China Agriculture Research System of MARA and MOF (CARS-38),the National Key Research &Development Program of China (2018YFD0502100) and the Scientific Research Foundation of Hebei Agricultural University,China(YJ201825).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Ethical approval

The animal study was reviewed and approved by the Animal Care and Use Committee of Hebei Agricultural University,China (Project ID: YJ201825).