Long term effects of artificial rearing before weaning on the growth performance,ruminal microbiota and fermentation of fattening lambs

HUANG Wen-qin,CUl Kai,HAN Yong,CHAl Jian-min,3,WANG Shi-qin,Lü Xiao-kang,DlAO Qi-yu,ZHANG Nai-feng

1 Institute of Feed Research,Chinese Academy of Agricultural Sciences/Key Laboratory of Feed Biotechnology of the Ministry of Agriculture and Rural Affairs,Beijing 100081,P.R.China

2 Guizhou Institute of Animal Husbandry and Veterinary Science,Guizhou 550005,P.R.China

3 Department of Animal Science,Division of Agriculture,University of Arkansas,Fayetteville,AR,72701,USA

Abstract Early life intervention is important to shape the gut microbiome profiles of adult animals due to the tremendous alteration of diet components.Nevertheless,there is still no unified understanding about its long-term effects in lambs.In this study,sixty 20-day-old lambs were assigned into ewe-rearing (ER) and artificial-rearing (AR) treatments to evaluate the effects of AR strategy on ruminal microbiota,fermentation,and morphology of pre-weaning lambs (from 20 to 60 days of age) and its long-term effects in the fattening stage (from 61 to 180 days of age).During the pre-weaning stage,ER lambs were breastfed and supplemented starter,while AR lambs were artificially fed with milk replacer and starter.During the fattening stage,all lambs in both treatments were fed with the same fattening diets.At 60,120 and 180 days of age,6 lambs from each group were slaughtered to collect rumen content and tissue samples.Compared with ER lambs,the dry matter feed intakes of AR lambs increased (P＜0.05) from 20 to 180 days of age,companying an increased average daily gain (ADG) from 61 to 120 days of age (P＜0.05) and from 121 to 180 days of age (0.05＜P＜0.1).Although there was no difference in short-chain fatty acid (SCFA,including acetate,propionate,and butyrate) between treatments before weaning (P＞0.05),it was higher (P＜0.05) in AR lambs compared with ER lambs at the fattening stage.The rumen keratin layer of AR lambs was thinner (P＜0.05) than that of ER lambs.Along with lamb growth from 60 to 180 days of age,the differences in rumen bacterial diversity between AR and ER treatments grew more distinct (P＜0.05).Compared with ER lambs,AR lambs increased (P＜0.05) rumen bacteria abundance,such as phylum Spirochaetes and genus Treponema at 60 days of age,phylum Actinobacteria and genus Succiniclasticum at 120 days of age,and phylum Proteobacteria at 180 days of age,but decreased genus Selenomonas from 60 to 180 days of age,and Anaerovibrio at 180 days of age.In summary,the early interventions before weaning could improve dry matter feed intake of lambs,which triggered robust rumen development and produced positive long-term effects on rumen fermentation and noticeable weight gain of fattening lambs.It suggests that the artificial rearing strategy is effective in improving rumen fermentation and microbial maturity of intensive fattening lambs.

Keywords:artificial rearing,lamb,rumen microbiome,rumen fermentation,growth

1.lntroduction

Under the industrialized or intensive sheep production system,it is a common practice for lambs to be breastfed up to 60 to 90 days with their dams (Sunet al.2018).However,the quantity and quality of breast milk are not likely to fully meet the nutritional requirements of lambs.The breast milk lactation of ewes peaks before 20 days postpartum,and then decreases gradually,resulting in a rapid decrease in the weight gain rate of lambs before weaning (Liang 2012).Many studies demonstrated that early weaning with artificial feeding milk replacer and starter feed could significantly improve weight gain and digestive physiology function in lambs when compared with those ewe reared (ER) during the pre-weaning period (Liet al.2018).The efficacy of the artificial-rearing(AR) strategy,that is,weaning the lamb from ewe milk to milk replacer and starter between 10-20 days old,and then stopping milk replacer when their starter intake reached 300 g d-1,was qualified in previous studies(Chaiet al.2017,2018).It has been reported that the AR strategy in newborn lambs could manipulate rumen microbial colonization and proliferation (Belancheet al.2019b;Lvet al.2019),which plays important roles in the digestion of enhanced starter intake to meet their nutrient requirements,and enhances the growth performance of lambs (Chaiet al.2017).In other words,the developing rumen in pre-weaning ruminants may provide a promising opportunity for the manipulation of such a complex microbial ecosystem (Yá?ez-Ruizet al.2015).Therefore,an early and high intake of solid feed should be important drivers not only for the growth performance but also for the structure of rumen microbiota and the fermentation patterns that initiate rumen epithelial development(Belancheet al.2019b;Lvet al.2019).

Except short-term physiological changes (Abeciaet al.2014a;Liuet al.2016),there is growing evidence that early nutritional interventions could have long-lasting consequences (Abeciaet al.2013;Chaiet al.2019a).In rat and human studies,the high insulin and IGF1 caused by high prenatal nutrition supply,including protein and other nutrients,increased both the later body weight and lipogenesis (Haschkeet al.2016).Similarly,improvement in plane of nutrition before weaning increased the postweaning feed efficiency of lambs (Bhattet al.2009).Furthermore,one study concluded that the early life modification of rumen archaeal populations in goat kids persisted over three months despite the treatment being removed after weaning (Abeciaet al.2013).It can be established that the early life diet of lambs is an important driver in shaping the rumen microbiome,which has a long-lasting implication (Lvet al.2019).However,there is still no unified understanding about the long-term effects of early nutritional intervention in lambs.A study based on grazing lambs showed that the pre-weaning nutritional intervention positively contributed to lamb growth rate during the fattening period (Belancheet al.2019a),but the persistence of the rumen microbiome was weak(Belancheet al.2019b).The inconsistent effect on the microbiome may be related to feeding systems,weaning time,rumen microbial indicators and post-weaning factors.There is still a general lack of understanding of the mechanism governing these processes such as host phenotypes and microbiome interactions,rumen fermentation and morphology relationship.Therefore,this study hypothesized that the AR system might cause a long-lasting effect on the rumen function and productivity of fattening lambs under intensive production systems through stimulating their feed intake and then shaping the rumen microbiota and fermentation of pre-weaning lambs.The objective of the present study was to evaluate the effect of early AR system on the growth performance,ruminal microbiota,fermentation,and morphology of lambs at the pre-weaning and fattening stages.

2.Materials and methods

2.1.Animals,experimental design and treatments

Sixty healthy Hu sheep male lambs ((8.26±2.14) kg of body weight (BW);(20±1) days of age) were randomly assigned into two rear systems:ewe-rearing (ER) and artificial-rearing (AR) treatments.Each treatment had 6 replicates with 5 lambs in each pen (3.6 m×2.1 m) as a replicate.The trial was consisted of 3 feeding stages including pre-weaning stage from 20 to 60 days of age(S1),early fattening stage from 61 to 120 days of age (S2),and later fattening stage from 121 to 180 days of age (S3).During the S1 stage,ER lambs were breastfed from 20 to 60 days of age,while AR lambs were weaned from ewe milk to milk replacer and starter feed from 20 days of age.According to Chaiet al.(2018),the milk replacer was stopped when lambs’ solid starter feed intake reached 300 g d-1for 3 consecutive days (around 50 days of age).In this study,all lambs were allowedad libitumaccess to pelleted starter feed from 20 to 60 days of age.During the S2 and S3 stages,all lambs were fed with fattening pellet diets.Water wasfreely accessed throughout the study.All lambs were weighed before morning meal on 20,60,120 and 180 days of age.The amount of daily feed offered and refused was recorded.Dry matter intake(DMI),average daily gain (ADG) and feed conversion rate(FCR,gain/feed) were calculated accordingly.

The milk replacer was formulated according to the chemical composition of ewe milk with ingredients of milk powder,whey powder,and minerals.The contents of crude protein (CP) and metabolizable energy (ME) were 25.59% and 12.60 MJ kg-1dry matter (DM),respectively.Feeding levels of milk replacer (MR) solids were adjusted to 2% of lambs’ BW.Lambs were fed thrice daily at 0800,1300 and 1800 h from 21 to 30 days of age and thereafter twice daily at 0800,and 1800 h until weaning.The mixture of 1 kg MR with 6 L of liquid milk of 38 to 40°C were fedviababy bottles.The pelleted starter (diameter,5 mm;length,4-6 mm) and fattening diets (diameter,7 mm;length,4-6 mm) were formulated according to the Chinese Feeding Standard of Sheep (NY/T 816-2004 2004).The ratio of forage to concentrate of the S2 and S3 fattening diets was 4:6 and 3:7,respectively.There was a transition period of 3-5 days between each feed change.Starter and fattening pellets were offered twice daily at 0800 and 1800 h.The formula and nutrient compositions of the starter and fattening pellets are presented in Table 1,and their chemical composition was analyzed according to AOAC (2010) except the neutral detergent fiber (NDF) and acid detergent fiber (ADF)whose measurement followed the method of Van Soestet al.(1991).

Table 1 Composition and nutrient levels of experimental diets (%,dry matter basis)

2.2.Sample collection and analysis

Six lambs from each treatment were selected and slaughtered at 60,120 and 180 days of age,respectively.All selected lambs had a BW that was close to the average weight of all lambs in the same pen and were slaughtered after fasting for 16 h.After slaughter,the rumen was dissected,and the content’s pH was measured by a portable pH meter (Orion 230 Aplus,1)The premix for starter contained the following per kg of diet:VA120 00 IU,VD2 000 IU,VE40 IU,Fe 30 mg,Mn 25 mg,Cu 20 mg,Zn 25 mg,Se 0.30 mg,I 0.50 mg,and Co 0.05 mg.The premix for fattening diet provided the following per kg of diet:VA4 000 IU,VD1 200 IU,VE20 IU,Cu 8 mg,Fe 48 mg,Mn 36 mg,Zn 32 mg,I 0.2 mg,Se 0.12 mg,and Co 0.4 mg.

2)ME,metabolizable energy;DM,dry matter;CP,crude protein;EE,ether extract;NDF,neutral detergent fiber;ADF,acid detergent fiber.NFC,non-fibrous carbohydrate.Nutrients were all measured values except ME and NFC.The ME was calculated according to Xionget al.(2012) and NY/T 816-2004 (2004);NFC=100-(NDF+CP+EE+Ash).Thermo Scientific,Beverly,MA,USA).A total of 2 mL of rumen contents was collected into plastic cryogenic vials and stored at -80°C for microbial analysis.About 50 mL of rumen contents was squeezed through 4 layers of gauze to collect 10 mL of rumen fluid into sterilized centrifuge tubes and stored at -20°C for rumen fermentation parameter analysis such as short chain fatty acid (SCFA) and ammonia nitrogen (NH3-N).To measure the SCFA concentration,the samples acidified by 25% HPO3were centrifuged at 5 600×g for 10 min.The component SCFA were separated when the liquid was filtered through a packed glass column,and then determined by gas chromatography (GC-17A;Shimadzu,Japan).The colorimetric technique based on Chaney and Marbach (1962) was used to analyze NH3-N concentration of the centrifuged samples.

After removing the rumen digesta,a portion (2 cm2) of rumen tissue was cut from the center of the cranial ventral sac and rinsed in saline (Lesmeisteret al.2004).Then,the rumen tissue was fixed in an embedding cassette,and put into 4% phosphate buffered paraformaldehyde solution.The fixed tissue was dehydrated,embedded,and sliced by a fully automatic dehydrator.Cuts of each block were isolated and stained with hematoxylin and eosin separately.Tissue cuts were observed at 40×magnification with a digital trinocular camera microscope(BA410 Digital,Motic Electric,Xiamen,China),the sections were selected according to the cuts content and observation requirements,and then the images were acquired using a microscopic camera system.Papillae length,papillae width,keratin layer thickness,muscle layer thickness and epithelium thickness were measured with image analysis software Image-Pro Plus 6.0 (Media Cybernetics,Silver Spring,MD,USA).

Bacterial community analysis of rumen content samples was carried out by Realbio Genomics Institute(Shanghai,China).Microbial DNA was extracted from rumen content by using the E.Z.N.A.Bacterial DNA Kit (Omega Bio-Tek,Norcross,GA,USA) according to the manufacturer’s instructions.Nanodrop 2000(Thermo Scientific,Wilmington,DE,USA) and 1%agarose gel electrophoresis were used to check the DNA concentration and quality.The forward primer (341F:5′-CCTACGGGRSGCAGCAG-3′) and the reverse primer(806R:5′-GGACTACVVGGGTATCTAATC-3′) were used to amplify the V3-V4 region of bacterial 16S rRNA genes by PCR.PCR amplification program consisted of the diluted genomic DNA which used the KAPA HiFi Hotstart ReadyMix PCR Kit (Kapa Biosystems,Wilmington,MA,USA) high fidelity enzyme to ensure amplification accuracy and efficiency.The library was constructed and quantified using Qubit?2.0 (Invitrogen,Carlsbad,CA,USA).After the quality was checked,the library was sequenced using Miseq PE250 Flatform (Illumina,Inc.,CA,USA) for paired end reads of 425 bp.Raw tags were checked on their rest lengths and average base quality by trimming barcodes and primers.The tags with a frequency more than 1 tended to be clustered into operational taxonomic units (OTUs),and each of the OTUs had a representative tag.OTUs were clustered at a level of 97% sequence similarity using UPARSE(http://drive5.com/uparse/) and Usearch (version 7.0) was used to identify and remove chimeric sequences.OTU profiling table and alpha/beta diversity analyses were also obtained by python scripts of QIIME v1.9.1 (the Knight and Caporaso labs,Flagstaff,AZ,USA).

2.3.Statistical analysis

Statistical analyses of the ADG,DMI,and FCR (each replicate pen served as an experimental unit),the rumen fermentation,morphology parameters,and logtransformed taxon abundance (each lamb served as an experimental unit) were performed using IBM SPSS Statistics 22.0 Software (IBM Corp.Armonk,NY,USA).A mixed model was specified as follows:

whereYijkis the dependent variable,μis the overall mean,Riis the fixed effect of the rearing system (i=AR or ER),Sjis the fixed effect of stages (i=20,60,120,or 190 days of age for BW,ori=S1,S2,or S3 for others),RSijis the interaction effect,ckis the random effect of replicates or lambs and εijkis the random residual error.Significant differences were detected,and the means were compared by the Holm-Sidak multiple comparisons correction test.P＜0.05 was declared as significant effects and trends occurred at 0.05＜P＜0.1.

Alpha-diversity of the rumen microbial data between treatments was tested using the Kruskal-Wallis test.Posthoc Dunn Kruskal-Wallis multiple comparisons with a Bonferroni adjustment were used to evaluate differences between treatments with boxplots made in R (‘ggpubr’packages).Beta diversity was visualized with a PCoA plot.The non-strict version of Linear discriminant analysis Effect Size (LEfSe),which determines significant taxa differing in at least one (and possibly multiple) class value(s)(Segataet al.2011),was used to identify the bacterial biomarkers of different treatments.Spearman correlations(r) were calculated to assess the relationships between the microbial taxa abundance and rumen fermentation and animal performance data.Correlations were defined as those with coefficients |r|≥0.3 andP＜0.05.

3.Results

3.1.Growth performance

Growth performance from the pre-weaning to fattening period is shown in Table 2.The rearing system and the interaction between rearing system and feeding stage had no effect (P＞0.05) on lamb BW,but the feeding stage affected (P＜0.05) lamb BW.The feeding stage and the interaction between rearing system and feeding stage had effect (P＜0.05) on lamb ADG,and the rearing system tended to affect (0.05＜P＜0.01) lamb ADG.The rearing system and feeding stage had effect (P＜0.05) on lamb DMI,but the interaction between rearing system and feeding stage did not affect (P＞0.05) lamb DMI.The rearing system,feeding stage,and their interaction affected (P＜0.05) lamb FCR.Specifically,the DMI increased (P＜0.05) but the BW had no change (P＞0.1) in AR lambs compared with ER lambs.The BW and DMI increased (P＜0.05) across treatments with lamb growth from 20 to 180 days of age.The ADG of AR lambs was similar (P＞0.1) before weaning,higher (P＜0.05)at the early fattening stage,and tended to be higher(0.05＜P＜0.1) at the later fattening stage compared with that of ER lambs.However,the FCR of AR lambs was lower (P＜0.05) before weaning,but similar (P＞0.1) at the early and later fattening stages compared with that of ER lambs.

3.2.Ruminal fermentation

In terms of rumen fermentation (Table 3),the ruminal pH,percentage of propionate,and the ratio of acetate to propionate of lambs were neither affected (P＞0.05) by the rearing system nor the interaction between rearing system and feeding stage,but were affected (P＜0.05) by the feeding stage.The ruminal percentage of acetate was affected (P＜0.05) by the feeding stage and the interaction between rearing system and feeding stage,but was not affected (P＞0.05) by the rearing system.The ruminal ammonia nitrogen,the concentration of total SCFA,acetate,propionate,and butyrate,and the percentage of butyrate were affected (P＜0.05) by the rearing system,feeding stage,and the interaction between rearing system and feeding stage.From 60 to 180 days of age,animals in both treatments increased (P＜0.05) the percentage of propionate but decreased (P＜0.05) the pH and the ratio of acetate to propionate.Compared with ER lambs,the ARlambs increased (P＜0.05) their rumen ammonia nitrogen concentrations at 60 days but not at 120 and 180 days of age.The rumen total SCFA,acetate and propionate concentrations of AR lambs were not affected (P＞0.05)at 60 days of age but increased (P＜0.05) at 120 and 180 days of age compared with that of ER lambs.The concentration and molar proportions of butyrate were not affected (P＞0.05) at 60 and 120 days of age but increased (P＜0.05) at 180 days of age.The rumen molar proportions of acetate of AR lambs were not affected(P＞0.05) at 60 and 120 days of age but decreased(P＜0.05) at 180 days of age compared with that of ER lambs.

Table 2 Effects of artificial rearing on the growth performance of lambs1)

Table 3 Effects of artificial rearing on rumen fermentation parameters of lambs1)

3.3.Rumen morphometric characteristics

The effects of rearing systems on morphometric characteristics are presented in Table 4.The rearing system and the interaction between rearing system and feeding stage had no effect (P＞0.05) on the rumen weight and volume while the feeding stage had effect (P＜0.05).The rearing system and the interaction between rearing system and feeding stage had no effect (P＞0.05) on the rumen papillae length and width while the feeding stage had effect (P＜0.05) on the papillae length and tended to affect (0.05＜P＜0.1) the papillae width.The rearing system had effect (P＜0.05) on the rumen keratin layer thickness while the feeding stage and the interaction between rearing system and feeding stage had no effect(P＞0.05).The rearing system and the interaction between rearing system and feeding stage had no effect (P＞0.05)on the rumen muscle layers thickness and epithelium thickness while the feeding stage had effect (P＜0.05) on the epithelium thickness and tended to affect (0.05＜P＜0.1)the muscle layer thickness.The rearing system lowered(P＜0.05) the rumen keratin layer thickness of AR lambs compared with ER lambs.Besides,the rumen volume in AR lambs tended to increase (0.05＜P＜0.1) compared with that in ER lambs.From 60 to 180 days of age,all lambs across treatments increased (P＜0.05) their rumen weight,volume,and rumen papillae length,while decreased(P＜0.05) their rumen epithelium thickness and tended to decrease (0.05＜P＜0.1) their papillae width and muscle layers thickness.

Table 4 Effect of artificial rearing on morphology of the rumen of lambs

3.4.16S rRNA analysis of bacterial communities

Using next-generation sequencing technology,1 258 192 reads across all samples with an average of 35 012±2 099 per sample were acquired.In terms of alpha-diversities of ruminal bacteria (Fig.1),the rearing system had no effect (P＞0.1) on the Observed Species,Chao1,Shannon and Simpson except that AR lambs decreased (P＜0.05)their Observed Species at 120 days of age compared with ER lambs.The feeding stage had effect (P＜0.05)on the Observed Species,Chao1,and Shannon,but did not affect the Simpson.The Observed Species,Chao1 and Shannon increased (P＜0.05) from 60 to 120 days ofage but decreased (P＜0.05) from 120 to 180 days of age in ER lambs.However,Observed Species,Chao1 and Shannon were observed to decrease (P＜0.05) from 120 to 180 days of age in AR lambs only.

Fig.1 Alpha-diversity indexes of ruminal bacteria in different ages of lambs across rearing systems (data are mean±SD,n=6 lambs per treatment).ER60,ER120,and ER180,60-,120-,and 180-day old ewe rearing lambs,respectively;AR60,AR120,and AR180,60-,120-,and 180-day old artificial rearing lambs,respectively.Significance levels:＊,P＜0.05;＊＊,P＜0.01.

Regarding beta-diversity measurements,the unweighted UniFrac distance between points showed the degree of sample similarity of bacterial communities(Fig.2).Distinct clusters in community structure between treatments were detected across age (P＜0.05).Samples between treatments were separated effectively(UnWeighted Unifrac ANOSIM,R=0.64,P=0.001)according to the PCoA plot (Fig.2-D).The AR60 and ER60 samples trended to form distinct clusters (ANOSIM,R=0.174,P=0.067;Fig.2-A) based on UnWeighted Unifrac distance.Along with lamb growth from 60 to 180 days of age,the distinction between AR and ER lambs became increasingly obvious (P＜0.05;Fig.2-A-C).

Fig.2 The unweighted UniFrac principal coordinate analysis (PCoA) indicating the effect of the rearing system and stage of ages on the beta-diversity of ruminal bacteria in lambs (data are mean±SD,n=6 lambs per treatment).AR60,AR120,and AR180,60-,120-,and 180-day old artificial rearing lambs,respectively;ER60,ER120,and ER180,60-,120-,and 180-day old ewe rearing lambs,respectively.

Next,the rumen core microbiome among treatments were examined (Fig.3).A total of 13 phyla were observed,seven of which were abundant over 0.1% of the total sequences in at least one group.On average,the phyla in both treatments including Bacteroidetes,Firmicutes and Proteobacteria accounted for over 93% of total abundance.At the genus level,a total of 104 genera were observed,32 of which were abundant over 0.1%as core microbiota.On average,the dominant genera,includingPrevotella,Succinivibrio,Succiniclasticum,andRuminococcus,accounted for over 50% of total abundance.

Fig.3 Effects of artificial rearing on phylum-level (A) and genera-level (B) composition of the rumen microbiota,represented as average relative abundance (n=6 lambs per treatment).AR60,AR120,and AR180,artificial rearing lambs at 60-,120-,and 180-day old,respectively;ER60,ER120,and ER180,ewe rearing lambs at 60-,120-,and 180-day old,respectively.

A supervised classification of the microbiota between treatments was carried out by utilizing the LEfSe analysis which is often used to identify the presence and effect size of biomarker bacteria in different treatments.A logarithmic LDA score cutoff of 2.0 was used to identify important taxonomic differences between treatments.The results suggested a remarkable difference in rumen microbiota between rearing systems and across stages of age (Fig.4).In particular,for genus level bacteria,the relative abundances of generaRuminobacter,Acidaminococcus,andMegasphaerain ER60 lambs,Fibrobacter,Treponema,Dialister,andClostridiumXIVbin AR60 lambs,ClostridiumXIVbandStreptophytain ER120 lambs,Succiniclasticum,Ruminococcus,Mogibacterium,Pseudoramibacter,Coprococcus,Olsenella,Intestinimonas,EubacteriumandMoryellain AR120 lambs,Butyrivibrio,SelenomonasandAnaerovibrioin ER180 lambs,andRoseburiain AR180 lambs were higher compared with other bacteria (LDA score (log10)＞2,Fig.4-A and B).

Fig.4 The abundance (log10(relative abundance)) of ruminal bacteria at all taxa levels for differentiating rearing systems and ages of lambs (n=6 lambs per treatment).A,clustering tree for LEfSe analysis,with different colors representing different groups,and nodes with different colors representing microbial groups that play an important role in the grouping represented by the color.Yellow nodes indicate microbial taxa that did not play an important role in the different groupings.The species names represented by the English letters in the figure are shown in the legend on the right.B,the LDA score obtained by LDA (linear regression analysis) of the microbial groups that have a significant effect in different groups (LDA threshold 2).ER60,ER120,and ER180,ewe rearing lambs at 60-,120-,and 180-day old,respectively;AR60,AR120,and AR180,artificial rearing lambs at 60-,120-,and 180-day old,respectively.

Mixed models were performed to find the interactions between rearing and stage (R×S) factors for the genera that were identified by the LEfSe analysis (Table 5).The rearing system,feeding stage and their interaction had effect (P＜0.05) on the relative abundance of phyla Proteobacteria and Spirochaetes.The rearing system and the interaction between rearing system and feeding stage did not affect (P＞0.05) the relative abundance of Fibrobacteres while the feeding stage affected (P＜0.05)it.The rearing system and the interaction between rearing system and feeding stage had effect (P＜0.05)on the relative abundance of Actinobacteria and the feeding stage tended to affect (0.05＜P＜0.1) it.Compared with ER lambs,the AR lambs enhanced (P＜0.05) their rumen abundances of Spirochaetes at 60 days of age,Actinobacteria at 120 days of age,and Proteobacteria at 180 days of age.The Fibrobacteres decreased (P＜0.05)from 60 to 180 days of age in both treatments.

At the genus level,the rearing system and the interaction between rearing system and feeding stage did not affect (P＞0.05) the relative abundance of generaRuminococcus,Acidaminococcus,ButyrivibrioandFibrobacter,while the feeding stage did (P＜0.05).The rearing system,feeding stage and their interaction had effect (P＜0.05) on the relative abundance of generaAnaerovibrioandTreponema.The rearing system did not affect (P＞0.05) the relative abundance ofSucciniclasticumbut the feeding stage and the interaction between rearing system and feeding stage did (P＜0.05).The rearing system affected (P＜0.05) the relative abundance ofSelenomonasbut the feeding stage and the interaction between rearing system and feeding stage did not (P＞0.05).The relative abundances of generaRuminococcus,Acidaminococcus,andFibrobacterdecreased (P＜0.05),whileButyrivibrioincreased (P＜0.05)with lamb growth from 60 to 180 days of age in both treatments.Compared with ER lambs,the AR lambs had lower (P＜0.05) abundances ofSelenomonas.The AR lambs enhanced (P＜0.05) their rumen abundances ofTreponemaat 60 days of age,Succiniclasticumat 120 days of age,but decreasedAnaerovibrioat 180 days of age compared with ER lambs.

3.5.Correlation between rumen microbiota and the phenotypes of fermentation and growth

To explore the relationship between the rumen microbiota that were abundant over 1% in at least one group and the phenotype of lambs such as growth performance and fermentation parameters with significant changes between treatments,the spearman correlation was calculated and shown in the heatmap (Fig.5).Correlation coefficients were labeled with asterisks if they were above 0.3 and theP-value was below 0.05.The total SCFA,butyrate,DMI,and rumen volumewere positively correlated (P＜0.05) withgeneraButyrivibrio,andAnaerovibrio,while they were negatively correlated (P＜0.05) with generaTreponema,Acidaminococcus,Fibrobacter,andRuminococcus.Although ADG and FCR had a positive correlation (P＜0.05) with generaTreponema,Acidaminococcus,andFibrobacter,they were negatively correlated(P＜0.05) with generaButyrivibrio,andAnaerovibrio.A positive correlation (P＜0.05)was observed between keratin layer thickness and generaRuminococcusandSelenomonas.A negative correlation (P＜0.05) was observed between ammonia nitrogen and generaSucciniclasticumandSelenomonas.

Fig.5 Relationship between rumen core microbiota and phenotypes of rumen fermentation and growth of lambs.Strong correlations are indicated by red and blue colors with 1 indicating a perfect positive correlation (dark red) and -1 indicating a negative correlation(dark blue),whereas weak correlations are indicated by yellow colors.ADG,average daily gain;DMI,dry matter intake;FCR,feed conversion rate.Spearman test:＊,P＜0.05;＊＊,P＜0.01.

4.Discussion

The hypothesis that the AR system might cause a long-lasting effect on the rumen function and productivity of fattening lambs under intensive production systems through stimulating their feed intake and then shaping the rumen microbiota and fermentation of pre-weaning lambs was partly supported by the results.Artificial rearing before weaning enhanced the DMI and rumen relative abundance of phylum Spirochaetes and genusTreponema,thinned rumen keratin layer and tended to enlarge the rumen volume of lambs.Furthermore,artificial rearing before weaning generated a positive long-term effect on the rumen fermentation function,including thinned rumen keratin layer,tended to enlarge rumen volume,and enhanced rumen SCFAs content at the whole fattening stage.But a complex effect on the rumen microbiota,including a district betadiversity,unchanged phyla Spirochaetes and genusTreponemaat the whole fattening stage,enhanced phylum Actinobacteria and genusSucciniclasticumat the early fattening stage,and enhanced phylum Proteobacteria at the later fattening stage were observed.Moreover,the growth performance of lambs was not strongly affected by the artificial-rearing system in the early weaning and fattening periods except ADG increased at the early fattening stage and tended to increase at the later fattening stage.It is concluded that an artificial-rearing strategy in the early life of young ruminants has long-term consequences on rumen microbiota and fermentation products.Therefore,these findings proved the mechanisms of early nutritional interventions in lambs and offered a foundation for studies aiming at improving ruminant health.

The diet is one of the key factors that drive the change of rumen microbiota structure (Carmodyet al.2015;Pazet al.2016).Consistent with one previous study(Chaiet al.2017),the AR lambs increased their DMI compared with ER lambs from 20 to 60 days of age in this study,suggesting that artificial rearing of lambs with milk replacer and starter feed can improve their adaptability to solid feed (Napolitanoet al.2002).Accordingly,the relative abundance of rumen genusTreponema(belong to phylum Spirochaetes) was higher in AR lambs than that in ER lambs.Lvet al.(2019) also found that lambs fed milk replacer+concentrate+alfalfa pellets increased their rumen abundance ofTreponemacompared with naturally reared lambs.Treponemadoes not utilize fiber directly.However,it helps other bacteria to digest cellulosic materials,such as whenTreponemaandFibrobactersynergistically break down the fiber components of plant feed (Zhanget al.2017;Xieet al.2018).Briefly,the high abundance of genusTreponemain AR lambs may indicate better fiber fermentation efficiency (Belancheet al.2017).

It has been reported that solid feeding significantly increased rumen development and enhanced SCFAs production (Connoret al.2013;Wanget al.2016).Although the concentration of SCFAs did not change significantly between treatments at weaning,the increased rumen volume (+28.2%,P=0.060) and thinned rumen keratin layer (-18.6%,P=0.001) were observed in AR lambs compared with ER lambs in this study.The increase of rumen volume and the thinner rumen keratin layer should be feedback to higher feed intake of solid feed in AR lambs (Connoret al.2013).Considering the increased rumen volume andTreponemaabundance,as well as the thinned rumen keratin layer,it can be inferred that AR lambs had a stronger capacity of SCFAs production and absorption than ER lambs,because both production and absorption are associated with SCFAs metabolism (Wanget al.2016;Xieet al.2018).

Noticeably,AR lambs at 60 days old had similar rumen microbial communities in comparison to ER lambs according to rumen alpha-and beta-diversity.This is possibly attributed to the fact that the artificial rearing system led to a higher solid feed intake and provided an important basis for proliferation and spread of rumen bacteria (Malmuthugeet al.2014).Indeed,the sooner solid feed consumption of ruminants occurs,the closer their rumen bacterial communities become to adults’because of the similar fermentation substrates (Mealeet al.2016).Nevertheless,AR lambs did not show expected higher weight gain and weaning weights in this study.It may be related to the physical and psychological stresses caused by weaning (Napolitanoet al.2008),which led to the resistance to nutrient digestion,metabolic efficiency and feed conversion rate in lambs (Belancheet al.2019a).

It has been reported that the microbial colonization of young ruminants was likely to be manipulated with longlasting effects on adult health (Yá?ez-Ruizet al.2010;Abeciaet al.2014b).As expected,the increasing trend of ADG and increased DMI were observed in the fattening period for AR lambs that consumed more solid feed before weaning in this study,indicating the disappearance of early weaning stress and the recovery of gastrointestinal function.Similarly,the improvement of pre-weaning plane of nutrition of lambs increased their post-weaning feed efficiency (Bhattet al.2009).The higher solid feed intake before weaning promoted the shift in microbiota towards mature rumen,which may be responsible for the long-term effects on growth performance (Mealeet al.2016,2017).When comparing the rate of increase in species richness between treatments,a milder process can be noted in AR lambs from 60 to 120 days,which may be related to a pre-maturing rumen (Mealeet al.2017).Subsequently,increasing differences in betadiversity between treatments at the fattening stage indicated the lasting effects of artificial rearing.Moreover,this study discovered that genera in phylum Firmicutes and Fibrobacteres had significantly changed between rearing systems,such asSucciniclasticum,Anaerovibrio,andSelenomonas.Most of them were related to fiber fermentation and SCFAs production.It was possibly due to the fact that the dry matter intake,the rumenTreponemaandFibrobacterabundance of AR lambs showed a sharp decline,while those of ER lambs showed a gradient drop from weaning to the end of fattening.The relative abundance ofSucciniclasticum,a fibrolytic bacteria common in the rumen,increased at the early fattening stage.Myeret al.(2017) also found higher abundances ofSucciniclasticumin lambs with greater ADF intake.Thus,it may indicate a metabolism advantage of the rumen of AR lambs.

The ingestion of solid feed is essential in developing the functionality of the rumen (Liet al.2012).The higher solid feed intake of AR lambs before weaning should be a potential driver for rumen fermentation during the fattening period.Interestingly,the rumen total SCFA,acetate,propionate and butyrate concentrations all increased during the whole fattening stage in AR lambs compared with those in ER lambs in this study.Therefore,it is reasonable to speculate that the SCFA yield is another important factor in boosting growth performance to produce a sustained effect.Further evidence should be the rumen keratin layer thickness,which was thinner in AR lambs compared with that in ER lambs during pre-weaning and early fattening stages in this study.One previous study (Xieet al.2020) reported that starter NDF might be beneficial to decrease the keratinization of the inner surface of the rumen because dietary fiber increased its ability in physically removing dying epithelial cells.The high solid feed intake with a high NDF intake before weaning should have a certain persistent effect on the low keratinization of rumen epithelium.Overall,these findings suggested that higher solid feed intake before weaning,elevated rumen SCFA yield and relative mature microbiota in AR lambs were the underlying causes that triggered robust rumen development in adulthood (Fig.6).

Fig.6 Putative schematic illustration of artificial rearing for rumen development of lambs and post effects in their fattening stage.NDF,neutral detergent fiber;SCFA,short-chain fatty acid;DMI,dry matter intake.↑,increase;↓,decrease.

Rumen species richness decreased across rearing systems from early to later fattening stage in this study.Previous studies have reported that high dietary concentrate levels can decrease the richness and diversity of ruminal bacteria (Qianet al.2018),and prevail the impact of host age on microbial composition(Malmuthuge and Guan 2017).However,there was an increase in abundance of phylum Proteobacteria and a decrease in abundance of Fibrobacteres and Spirochaetes.This might be attributed to highly soluble carbohydrates and low neutral and acid detergent fiber contents resulting from high concentrate feed consumed by lambs in the later fattening stage.Phylum Proteobacteria plays an important role in the fermentation and digestion of soluble carbohydrates (Cuiet al.2019),while Fibrobacteres and Spirochaetes play an important role in the fermentation of fibers.Dietary fermentable carbohydrates as the main energy source for bacteria led to an increase of starch-degrading bacteria in direct competition with fiber-degrading bacteria (Fernandoet al.2010;Petriet al.2013).Therefore,the rumen microbiota was mainly affected by the fattening diet rather than artificial rearing system in the later fattening stage,which was why the effect of artificial rearing on rumen bacteria and growth performance was only slightly noticeable at the later fattening stage.

5.Conclusion

Artificial rearing of lambs resulted in a slight weight loss before weaning,while led to a visible weight gain in the fattening period,especially in the early fattening period.Increased solid feed intake due to artificial rearing is an important driver not only for rumen microbiota development but also for stronger rumen fermentation and anatomical enlargement during the fattening period.Significant changes of rumen fibrolytic bacteria and short chain fatty acid yields of artificial rearing lambs could persist from pre-weaning to the fattening period,which contributes to rumen function and lambs’ productivity.Therefore,it has been suggested that the artificial rearing strategy is beneficial for lamb fattening.Nevertheless,further studies are needed,based on a better description of intrinsic associations among rumen microbiota,fermentation,and morphology development to clarify and fix the effect of artificial rearing on the productivity of lambs in the fattening period.

Acknowledgements

This research was funded by the National Natural Science Foundation of China (31872385),the Fundamental Research Funds for Central Non-profit Scientific Institution,China (Y2019CG08) and the National Key R&D Program of China (2017YFD0502001).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Ethical approval

The present experiment was conducted at Runlin Sheep Farm in Shandong Province,China,from August 2018 to January 2019.All experimental protocols were approved by the Animal Ethics Committee of the Chinese Academy of Agricultural Science (AEC-CAAS-20180601),and humane animal care and handling procedures were followed throughout the experiment.