Dynamic change of fungal community in the gastrointestinal tract of growing lambs

YIN Xue-jiao,JI Shou-kun,DUAN Chun-hui,TIAN Pei-zhi,JU Si-si,YAN Hui,ZHANG Ying-jie,LIU Yue-qin

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

Abstract Although fungal communities in the gastrointestinal tract have a significant role in animal health and performance,their dynamics within the tract are not well known. Thus,this study investigated fungal community dynamics in the rumen and rectum of lambs from birth to 4 mon of age by using IT1S rDNA sequencing technology together with the RandomForest approach to determine age-related changes in the fungal ecology. The results indicated that gastrointestinal fungal community composition,diversity,and abundance altered (P＜0.05) with the increasing age of the lambs. Two phyla,Ascomycota and Basidiomycota,dominated the samples. Similarity within age groups of the rumen fungi increased sharply after 45 days of age,while the similarity increased (P＜0.05) significantly after 60 days of age in the rectum.The age-related genera,Acremonium,Microascus,Valsonectria,Myrmecridium,Scopulariopsis,Myrothecium,Saccharomyces,and Stephanonectria,were presented in both ruminal and rectal communities,and their changes in relative abundance were consistent at both sites. The principal coordinates analysis showed significant differences(P＜0.05) between the fungal communities in the rumen and rectum. Our findings demonstrate that both the age of lambs and the gastrointestinal tract region can affect the composition of these fungal communities,and this provides new insight and directions for future studies in this research area.

Keywords: fungi,sheep,rumen,rectum,maturity

1.Introduction

The gastrointestinal tract of mammals harbors a complex community of microbiomes that is critical to host health,development and productivity,especially in early postnatal life (Yeoman and White 2014;Yanez-Ruizet al.2015). The gastrointestinal tract microbiome is a complicated microecosystem,which primarily contains bacteria,archaea,anaerobic fungi,and ciliated protozoa (Holmeset al.2012;Falonyet al.2016). Compared with adult microbiomes,the early establishment phases of the gastrointestinal microbiome are dynamic and are more susceptible to be influenced by environmental factors (Laforest-Lapointe and Arrieta 2017). However,the relevant published studies have mainly focused on the establishment of bacterial communities,with little attention to the fungi.

Fungi are present in most microbial ecosystems (Huseyinet al.2017;Liet al.2018),however,their functional role in the gastrointestinal tract of ruminants remains unclear. There are over 400 fungal species related to mammals,mainly affiliated to 3 major phyla: Ascomycota,Basidiomycota,and Chytridiomycota (Wheeleret al.2017). It has been indicated that fungi play a critical role in rumen fermentation (Langdaet al.2020),possess a wide range of biomass-degrading enzymes (Solomonet al.2016) that are essential to the lignocellulolytic capacity of herbivorous animals (Gruningeret al.2014). The previous study indicated that fungi play a vital role in methane production,microbial groups dominated by fungi outperformed bacterially dominated groups in terms of both methane release and the extent of cellulose degradation (Penget al.2021). Although fungi have smaller proportions than bacteria,they are important and immunomodulatory members of the gut microbiome(Laforest-Lapointe and Arrieta 2017),and they may be critical in both animals’ nutrient metabolism and intestinal health(Liet al.2018). Fungi in the intestine may stimulate innate immune responses of intestinal epithelial cells,respiratory and urinary systems,as well as skin tissues,they also contribute to the maturation of the mesenteric or intestinal lymphatic immune system (Liet al.2018). In spite of this,there has been very little investigation of the gastrointestinal fungal community dynamics in ruminants,especially in relation to the establishment of these communities within the gastrointestinal tract and their composition in different regions of the tract.

The composition,growth rate and diversity of microbial communities vary between different regions of the gastrointestinal tract (Liet al.2019;Guoet al.2020),probably due in part to the differences in the feed substrate presented at each site (McCannet al.2014). Consequently,the study of microbial community dynamics in the gastrointestinal tract that involve more than one region of the tract and cover a range of ages of the host animals may improve the understanding of fungal communities.

For bacterial communities,there have been studies of the colonization of the gastrointestinal tract of ruminants (Jamiet al.2013;Dill-McFarlandet al.2017),however,there is little similar information for the fungal communities. In this study,we hypothesized that the gastrointestinal fungal communities would change significantly over the growing period of lambs. Therefore,the present study was conducted to characterize the dynamics of fungal communities in rumen fluid and rectal digesta of lambs from birth to 4 mon of age. This study increases the understanding of the dynamic changes in the fungal ecosystem of lambs and provides guidelines to manipulate the fungal composition toward improving health and performance.

2.Materials and methods

2.1.Animals,diets,and experimental design

Experiments were performed between January and August 2019. The study used 10 female lambs (Hu sheep breed,singletons,(2.87±0.28) kg body weight (BW))that were born and reared at the experimental sheep farm facility located in Hengshui,China. The lambs were born in individual pens (3.0 m×0.8 m) within 1 wk;all with vaginal delivery. They were housed with their mothers and suckled in the same pens. From day 15,they had free access to a starter commercial compound. On day 60,they were weaned and thereafter offered a mixed ration at 0730 and 1500 h each day with approximately 5% feed refusal. Fresh water was freely available throughout the study period. The ingredients and nutrient composition of the diets are provided in Appendix A,which met the nutritional requirements of lambs (NY/T 816-2004,Ministry of Agriculture,China).

2.2.Sample collection

Rumen fluid samples were collected approximately 2 h after the morning feeding by using a flexible esophageal tube which was thoroughly cleaned with fresh warm water between individual lambs (Abeciaet al.2018). Rectal digesta samples were taken directly from the rectum by manual extraction using sterile gloves and sterile cotton swabs.

These samples were collected from each lamb at 9 time-points as follows: the first within 24 h after birth,then at days 3,10,20,30,45,60 (all before weaning),and at days 90 and 120 (post weaning) (Fig.1-A). All samples were immediately snap-frozen in liquid nitrogen and then stored at -80°C until processing for DNA extraction.

2.3.DNA extraction and sequencing

Genomic DNA was extracted from 0.5 g of the ruminal fluid and rectal digesta samples and was obtained using the Omega Stool DNA Kit (MoBio Laboratories,Carlsbad,CA,USA) by following the manufacturer’s instructions. The purity and quality of the extracted DNA were determined using 1% agarose gel electrophoresis and spectrophotometry. The extracted DNA was stored at -20°C until further analysis. The ITS rRNA genes were amplified with the primers ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2R(5′-GGACTACNNGGGTATCTAAT-3′). These primers also contained a set of 8-nucleotide barcode sequences that were unique to each sample. 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 of reverse primer (5 μmol L-1),5.5 μL of ddH2O,and 5 μL of DNA (total template quantity was 30 ng). The PCR protocol used was as follows: 95°C for 5 min;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 1% agarose gels and purified using the QIAquick Gel Extraction Kit (QIAGEN,Germany)according to the manufacturer’s instructions. Deep sequencing of purified products was performed on a Miseq platform (Caporasoet al.2012) at Allwegene Company (Beijing,China). After each run,image analysis,base calling and error estimation were performed using the Illumina Miseq PE300 Sequencing Platform (Illumina,Inc.,San Diego,CA,USA).

2.4.Analysis of sequencing data

Low-quality sequences (an average quality score＜20),shorter reads (lower than 230 bp),ambiguous bases and chimeras were filtered by QIIME1 (version 1.8.0). Clean,paired-end sequences with an overlap longer than 10 bp were merged using Vsearch (version 2.7.1). The representative sequence was classified into operational taxonomic units (OTUs) using a threshold of 97% similarity by Vsearch to determine the taxonomy of each ITS rRNA gene sequence. 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). Beta diversity indices were measured based on Bray-Curtis distance and visualized by Principal Coordinates Analysis (PCoA) in R (version 4.0.0). Nonmetric multidimensional scaling (NMDS) plots of the Bray-Curtis metric were calculated with square root transformed data in R (vegan package). Heat maps were generated using the package ‘pheatmap’ of R (version 4.0.0) Software. The co-occurrence of genera across fungi was determined by the package ‘igraph’ in R.

2.5.RandomForest analysis

We regressed (using the methodology of (Subramanianet al.2014) the relative abundances of fungal taxa at the genus level against the ages of lambs with the default parameters in the ‘RandomForest’ package in R (Breiman,2001) (ntree=10 000,mtry=p/3,where p is the number of taxa of genus or the number of pathways) (Liaw and Wiener 2002) in order to obtain the best discriminant performance of taxa across lamb ages. Lists of taxa were ranked by RandomForest in order of feature importance.The 10-fold cross-validation (the rfcv function in the R package ‘RandomForest’,100 repeats) was used to identify the number of marker taxa and pathway. The minimum cross-validation error was obtained,then we chose the number that stabilized against the crossvalidation error curve as marker taxa and pathway correlating with the ages of lambs (Zhanget al.2018).

2.6.Data availability statement

The datasets generated from the current study are available in the Genome Sequence Archive Repository(http://gsa.big.ac.cn),under accession numbers CRA004093.

2.7.Statistical analysis

All statistical analyses were carried out using R(version 4.0.0) Software,individual sheep served as an experimental unit. The alpha diversities were compared using the non-parametric two-samplet-test by the default number of Monte Carlo permutations (999). Analysis of similarity (ANOSIM) was performed to compare the fungal communities in the ruminal and rectal digesta at different ages of the lambs by using the Vegan package.The differences in the relative abundance of specific fungal taxa among multiple age groups were evaluated by ANOVA after checking normality and homogeneity.Multiple comparisons among age groups means were performed using Fisher’s protected least significant difference (LSD) test. Linear and quadratic polynomial contrasts were used to determine the age effect of fungal taxa. While thet-test was used to determine differences in alpha diversity (Chao1 and Shannon indexes) between rumen and rectum. Spearman correlation between parameters was performed with CORR procedures.P-value＜0.05 was considered to indicate statistical significance.

3.Results

3.1.Summary of fungal communities in rumen and rectum

To investigate the development of the gastrointestinal fungi in Hu sheep,we amplicon-sequenced rumen fluid and rectal digesta samples of the lambs from birth to 4 mon of age (Fig.1-A). The average number of highquality ruminal and rectal fungi operational taxonomic units (OTUs) obtained by ITS rRNA gene sequencing was 7 863 reads. The Chao1 index of the rumen fungal community increased dramatically (P＜0.05) from 0 to 20 days of age and then become relatively stable(Fig.1-B). The Shannon index of the ruminal fungal community was lower in comparison with other age groups of lambs at 3 days of age,it then increased continuously (P＜0.05) to 60 days,but decreased at 90 days (Fig.1-C). For the rectal fungal community,the Chao1 and Shannon indices were all increased (P＜0.05)with increasing age of the lambs (Fig.1-D and E).

3.2.Taxonomic composition of rumen fungal community change with age of lambs

Fifteen fungal phyla were identified in ruminal fluid of the lambs. However,Ascomycota predominated at all lamb ages (74.94% of total);with the next most dominant phyla being Basidiomycota (6.90%) and Neocallimasigomycota(3.35%) (Fig.2-A). By focusing on dominant fungi with a relative abundance＞1%,there were 5 dominant phyla and 11 dominant genera in the rumen (Tables 1 and 2). Among the dominant phyla in the rumen (Table 1),the relative abundance of Ascomycota reached the lowest (P＜0.05) at 0 day of age. Compared to other age groups,the relative abundance of Basidiomycota was higher (P＜0.05) at 120 days of age. The phyla Neocallimasigomycota obtained the highest (P＜0.05)abundance at 45 days of age. At the genus level,the increasing age increased (P＜0.05) the relative abundance ofWalleniaandSaccharomyces(Table 2). The relative abundance ofPiromyces,Fusarium,Colletotrichumreached the highest (P＜0.05) at 45 days of age. To examine differences in fungal taxonomic community composition and structure in the rumen,we provide the NMDS based on the Bray-Curtis distance (Fig.2-B).These showed that the fungal composition of the rumen fluid from 0 to 10 days was spatially separated from that at other lamb ages,and the samples clustered according to their particular feeding pattern,suggesting that each diet stage and lamb age group hosts its own distinct fungal community (Fig.2-B).

Table 1 Effect of age on rumen fungal bacterial community of lambs at phylum level (%)

Table 2 Effect of age on rumen fungal bacterial community of lambs at genus level (%)

As rumen microbes work synergistically to perform various metabolic activities in the rumen,we sought to determine the associative interactions between fungi using co-occurrence analysis based on the top 100 genera with 32 nodes. Associations are presented for different ages in Fig.2-C. Most of the genera were found to potentiate each other,except forScopulariopsisandMicoascuswhich were negatively (P＜0.05) correlated withFusarium. According to degree centrality,closeness centrality,and betweenness centrality,Sarocladiumwas the core genus. Seven clusters were positively correlated withSarocladium(Fusarium,Lectera,Bionectria,Stephanonectria,Valsonectina,Meyerozyma,andTrichoderma) (Fig.2-C).

The within-group similarity comparison,which was based on Bray-Curtis distance of the fungal communities among age groups,indicated age-dependent changes(Fig.2-D). The rumen fungal ecosystem of the lambs had a very low rate of inter-individual diversity at day 0,but this had increased dramatically at 3 days (Fig.2-D). There was little further change until 45 days of age after which it rose again steadily,being significantly highest (P＜0.05)at 120 days of age (Fig.2-D). The similarity of the fungal ecosystem at any stage to that of the mature pattern seen at 120 days of age,followed a steady increase from day 0,when there was little closeness,and had a steep incline after 45 days of age (Fig.2-E). The Bray-Curtis similarity between adjacent age groups describes the taxonomic stability of an individual’s fungi (Fig.2-F). The similarity was increased in an age-dependent manner;the values at 90 and 120 days being higher (P＜0.05) than at the earlier ages,and became stable gradually (Fig.2-F). These findings suggested that the rumen fungal colonization process has a clear temporal change that is divided into colonization (0 to 3 days of age),a transitional period (3 to 45 days of age),and a mature period (45 to 120 days of age).

3.3.Taxonomic composition of the rectal fungal community change with age of lambs

Fourteen fungal phyla were identified from the rectum. Fungi belonging to the phyla Ascomycota and Basidiomycota predominated accounting,respectively,for 76.84 and 3.01% of all taxa across ages (Fig.3-A).There were 5 dominant phyla and 12 dominant genera in the rectum (Appendices B and C). In the rectum,the phyla Ascomycota,Basidiomycota,and Mortierellomycota changed (P＜0.05) with increasing age (Appendix B). The NMDS analysis revealed that the fungal communities in the rectum at 9 time points are varied (Fig.3-B). The Bray-Curtis metric revealed obvious segregation within the fungal communities of the rectum in lambs up to 30 days of age but mingling of the communities thereafter (Fig.3-B).

The significant (P＜0.05) co-occurrence network of fungi had 29 nodes in the rectum of lambs.Moreover,Stephanonectriawas the core genus of lambs and positively correlated with other fungi(Valsonectria,Acremonium,Penicillium,Talaromyces,Pyrenocheatopsis,andMyrothecium) (Fig.3-C).

The within-group similarity was shown in Fig.3-D,the similarity was relatively low (P＜0.05) in 0 days of age and then increased from 0 to 3 days,then decreased from 3 to 30 days and increased thereafter. The similarity between the samples collected at each time and the samples collected last was the lowest (P＜0.05)in 3 days compared to the 120 days of age,and then increased with age (Fig.3-E). The increasing similarity between samples collected at each time point and those collected last indicated that the development process of rectal fungi is complicated (Fig.3-E) and this is supported by the complexity of the age-related changes in the similarity between adjacent age groups (Fig.3-F).The adjacent groups’ similarity was increased from the comparison of 0 and 3 days to 3 and 10 days,and then decreased,finally increased (P＜0.05) from the comparison of 30 and 45 days. The obtained findings demonstrated that the fungal colonization process of the rectum appeared to be generally more complicated than that in the rumen.

3.4.Age-discriminatory fungi in rumen fluid and rectal digesta of lambs

Application of the RandomForest algorithm to correlate rumen fungal composition with age of the lambs showed that the cross-validation error curve stabilized when 21 genera were used. These 21 genera(shown in Fig.4-A) are defined as biomarker taxa in the model. The age-discriminatory fungi mostly belonged to the phyla of Ascomycota(17/21),Basidiomycota (2/21),and Cyllamyces(1/21). Of these 21 agediscriminatory fungal taxa,6 taxa had the highest relative abundance at 60 days of age (Myrothecium,Valsonectria,Myrmecridium,Acremonium,Cryptococcus,andStephanonectria),5 taxa achieved this between 30 and 60 days of age(Bionectria,Thermomyces,Passalora,Cercospora,andFusarium),3 taxa increased abundance with the age of the lambs (Saccharomyces,Meyerozyma,andTomentella),the abundance of 3 taxa increased within the first 10 days after birth and decreased thereafter (unidentified,Microascus,andScopulariopsis),and 4 taxa increased in abundance between 20 and 30 days of age and then decreased (Cyllamyces,Phyllactinia,Dekkera,andWardomyces) (Fig.4-B).

For the rectal fungi,application of the RandomForest model also defined 21 genera as biomarker taxa,as shown in Fig.5-A where they are listed in descending order of importance. These mainly belonged to Ascomycota (20/21)and Basidiomycota (1/21) phyla.For these 21 fungal taxa,13 taxa increased abundance with age of the lambs (Acremonium,Valsonectria,Myrothecium,Talaromyces,Stephanonectria,Trichoderma,Myrmecridium,Pyrenochaetopsis,Xerochrysium,Colletotrichum,Penicillium,Aspergillus,andMyceliophthora),3 taxa had the highest relative abundance at 60 days of age (Bisifusarium,Holtermanniella,andSaccharomyces),and 5 taxa increased in abundance during the first 10 days after birth and then decreased thereafter (Microascus,Chaetomium,Scopulariopsis,Debaryomyces,andChrysosporium)(Fig.5-B).

3.5.The similarity of fungal communities in rumen fluid and rectal digesta of lambs

In the 20-and 45-days-old lambs,the Chao1 index for ruminal fungi was higher (P＜0.05) than that for rectal fungi(Fig.6-A). The Shannon index for rectal fungi was higher(P＜0.05) than that for ruminal fungi in lambs at 90 days of age (Fig.6-B). Twenty predominant genera (those with an average proportion ≥1%,based on all common genera)have been selected to illustrate alteration of the fungal community with increasing age of the lambs (Fig.6-C). In the rumen,the genusDebaryomyceswas predominant(P＜0.05) in samples from newborn and 3-days-old lambs(reaching 14.22%) compared with the older age groups.Several genera,includingMicroascusandAspergillushad higher (P＜0.05) abundances in samples from the 3-to 30-day-old lambs. With increasing lamb age,the abundance ofSaccharomycesincreased (P＜0.05). In rectal samples,the genusAspergillusincreased (P＜0.05)in abundance with age of the lambs. The generaDebaryomyces,ChaetomiumandChrysosporiumwere mainly (P＜0.05) present in the first 10 days of age andMicroascuswere more abundant (P＜0.05) in lambs from 3 to 20 days of age than that at other ages.

In the 0-to 30-days-old lambs,the fungal composition similarities in the rumen and rectum,assessed by PCoA and ANOSIM,were relatively diverse but clustered together after 45 days of age (Fig.6-D). Similarities,based on Bray-Curtis distance between ruminal and rectal fungal communities at each lamb age,increased (P＜0.05)from 0 to 10 days of age,decreased,then increased from 30 days of age (Fig.6-E). Overall,fungal community similarities in the rumen and rectum increased with increasing age of the lambs.

4.Discussion

Gastrointestinal microbial ecosystems are critical for the survival of the host (Chunget al.2012;Clementeet al.2012;Tremaroli and Backhed 2012) especially in ruminants (Maet al.2020;Newbold and Ramos-Morales 2020). Ruminal fluid and fecal digesta samples are generally used to provide information about the gut microbiota of ruminants (Klein-J?bstlet al.2014;De Mulderet al.2018;Chenet al.2020) and these have shown that microbial colonization of the gastrointestinal tract is beginning in utero (Biet al.2021),even before gut functions are well established (Jamiet al.2013;Yeomanet al.2018). However,the studies on microbial colonization of the gut have mainly focused on bacteria,the process of fungi establishment was still not fully investigated. Thus,it would be very useful to determine the development of fungi community in the rumen and rectum,investigate the key microbiota in the colonization process.We found that the succession process of the fungi can be divided into 3 phases: colonization (0-10 days of age),transition (10-45 days of age),and a relatively stable period of maturation (45-120 days of age). Meanwhile,the gastrointestinal fungal communities become more homogeneous with the lambs’ progress to maturity,some fungal genera were presented in both rumen and rectum(Acremonium,Microascus,Valsonectria,Myrmecridium,Scopulariopsis,Myrothecium,Saccharomyces,andStephanonectria),indicating that these genera may be important in the maturity process of the fungal community.Our hypothesis that gastrointestinal fungal communities would change significantly over the growing period of lambs has been supported by the results of this study. Our results provide scientific experimental bases for the role of fungi in the gastrointestinal tract of growing ruminants.

We used the Chao1 and Shannon indices,as recorded by others (Kumaret al.2015;Abeciaet al.2018),to determine the establishment of these fungal communities and expanded on this by measuring the Bray-Curtis similarity,which also assesses the establishment of a microbial community by examining differences in taxonomic composition and structure (Jiet al.2018;Querciaet al.2019). We found successive gradual changes of both alpha diversity and similarity for ruminal and rectal samples with age of the lambs. A gastrointestinal fungal community was rapidly established after birth of the lambs. The previous study suggested that the genusPiromycesin the rumen may be indicator of the rumen fungal development (Belancheet al.2019),the genusPenicilliumpresented in high levels within the animal and plant-based diet (Huseyinet al.2017),and high abundance of the generaPenicilliumandAspergilluswere found in growing children (Scheiet al.2017). Consistent with the previous studies,the 3 genera had high relative abundance in the growing period in our study,suggesting that these genera may play important role in metabolite nutrient and digest diet in ruminants.The genusSaccharomyces,which help the animals to digest carbohydrate-rich diets (Selber-Hnatiwet al.2017),had higher relative abundance from 60 days of age,it may be because the lambs consumed more solid diet and ingested yeast-containing foods with the increasing age.Our results showed that the process of fungal colonization in lambs can be sub-divided into 3 stages: from birth to 10 days of age is an initial colonization process,days 10 to 45 involve substantial taxonomic readjustment,and days 45 to 120 encompass a gradual stabilization of the fungal communities. This pattern agrees with that reported by others based on studies of bacteria in the growing lambs.For instance,at birth,the microbiota was highly influenced by microbes in the mothers’ vagina and skin,the external environment and colostrum (Guzmanet al.2015;Yeomanet al.2018). A second major period is when suckling infants transit from a milk only diet to solid feeds,this causes the infantile gut microbiota to undergo a change in composition to that of adults (Favieret al.2002) -a substantial transition phase. The final,third stage is a period of stabilization in which the gastrointestinal microbiota of young animals achieves the adult-type composition (Backhedet al.2015;Guoet al.2020).However,our results for gastrointestinal fungi,especially for the Bray-Curtis similarity and the relative abundances of the age-related genera,show some differences in lamb age-related changes of fungi between the rumen fluid and rectal digesta that indicate some regional differences in microbial community maturation across the gastrointestinal tract,as shown by Guoet al.(2020) for bacteria in calves.Based on the present and previous studies,there is a considerable window of opportunity (i.e.,from birth to about 45 days of age) in which it may be more effective for human to manipulate the gastrointestinal microbial communities of lambs that could benefit the health and growth performance of the animals.

Different gut segments contain specific microbial communities (Liet al.2019;Guoet al.2020;Huanget al.2020). Although the majority of fungi we recorded in the rumen and rectum belonged to just a few taxa (such as Ascomycota and Basidiomycota phyla),we observed significant fungal community differences in terms of alpha diversity,composition and age-related genera,between ruminal fluid and rectal digesta,and between lambs at different ages. Our study demonstrated that the fungal community may be affected by both the age of the lambs and the specific gastrointestinal tract sections,consistent with previous studies in ruminants (Guoet al.2020;Huanget al.2020). The differences in microbial communities between sections of the gastrointestinal tract may due to the regional variation in gut function,feed substrate and specific ecological environment of different gastrointestinal tract sections with the age increased(Sommer and Backhed 2013;Parkeret al.2018).

It is clear,especially from our Bray-Curtis similarity analysis,that the similarity between the rumen and rectum fungal communities increased with increasing age,though the ruminal fungal community of the lambs exhibited a greater diversity than that of the rectum.This indicates that the rumen offers a potentially rich source of microbes for more distal regions of the gastrointestinal tract,by transfer of digesta and,thus,the composition of the foregut communities can influence those of the hindgut (Jiet al.2018). Similar results are reported in humans,where oral microbial intake can affect the intestinal microbiota (Leeet al.2013). Besides,lambs in the present study,both ruminal and rectal samples contained age-related genera ofAcremonium,Microascus,Valsonectria,Myrmecridium,Scopulariopsis,Myrothecium,Saccharomyces,andStephanonectria,and these tended to have the same change way of relative abundance at both sites,indicating that these species of fungi are important contributors to gastrointestinal function in sheep. These results indicated that the microbiota import into the gut may affect the microbiota in the lower gut,which suggested that the modification of the foregut may also influence that in the hindgut. And the age-related genera shared by rumen and rectum may play a critical role in the maturation of ruminant fungal community,highlighting the need for further research on the role of fungi in the digestive system of ruminants.

5.Conclusion

The fungal communities studied in growing lambs demonstrated variation in taxonomic composition between the rumen and rectum,but the similarity increased with age. The succession process of the fungi can be divided into 3 phases: colonization,transition,and a relatively stable period of maturation. Initially,the gastrointestinal fungal communities are heterogeneous,but they become more homogeneous with the lambs’ progress to maturity.Some fungal genera were presented in both rumen and rectum,indicating that these genera may be important in the maturity process of the fungal community,and need further investigation to determine their importance for gastrointestinal function in growing sheep.

Acknowledgements

This work was supported by the China Agriculture Research System of MARA and MOF (CARS-38),the National Key R&D Program of China (2018YFD0502100),the Precision Animal Husbandry Discipline Group Construction Project of Hebei Agricultural University,China (1090064),and the Scientific Research Foundation of Hebei Agricultural University,China (YJ201825).

Declaration of 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 (YJ201825).

Appendicesassociated with this paper are available on http://www.ChinaAgriSci.com/V2/En/appendix.htm