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Background:Recent studies underscored that divergence in residual feed intake (RFI) in mature beef and dairy cattle is associated with changes in ruminal microbiome and metabolome profiles which may contribute,at least in part,to better feed efficiency.Because the rumen in neonatal calves during the preweaning period is underdeveloped until close to weaning,they rely on hindgut microbial fermentation to breakdown undigested diet components.This leads to production of key metabolites such as volatile fatty acids (VFA),amino acids,and vitamins that could potentially be absorbed in the hind-gut and help drive growth and development.Whether RFI divergence in neonatal calves is associated with changes in hindgut microbial communities and metabolites is largely unknown.Therefore,the objective of the current study was to determine differences in hindgut microbiome and metabolome in neonatal Holstein heifer calves retrospectively-grouped based on feed efficiency as most-efficient (M-eff) or least-efficient (L-eff) calves using RFI divergence during the preweaning period.Methods:Twenty-six Holstein heifer calves received 3.8 L of first-milking colostrum from their respective dams within 6 h after birth.Calves were housed in individual outdoor hutches bedded with straw,fed twice daily with a milk replacer,and had ad libitum access to a starter grain mix from birth to weaning at 42 d of age.Calves were classified into M-eff [n =13;RFI coefficient =-5.72 ± 0.94 kg DMI (milk replacer + starter grain)/d] and L-eff [n =13;RFI coefficient =5.61 ± 0.94 kg DMI (milk replacer + starter grain)/d] based on a linear regression model including the combined starter grain mix and milk replacer DMI,average daily gain (ADG),and metabolic body weight (MBW).A deep sterile rectal swab exposed only to the rectum was collected immediately at birth before colostrum feeding (i.e.,d 0),and fecal samples at d 14,28,and 42 (prior to weaning) for microbiome and untargeted metabolome analyses using 16S rRNA gene sequencing and LC-MS.Microbiome data were analyzed with the QIIME 2 platform and metabolome data with the MetaboAnalyst 4.0 pipeline.Results:No differences (P ＞ 0.05)in body measurements including body weight (BW),body length (BL),hip height (HH),hip width (HW),and wither height (WH) were detected between M-eff and L-eff calves at birth and during preweaning.Although milk replacer intake did not differ between groups,compared with L-eff,M-eff heifers had lower starter intake (P ＜ 0.01) between d 18 to 42 of age,whereas no differences (P ＞ 0.05) for ADG,cumulative BWG,or body measurements were observed between RFI groups during the preweaning period.Microbiome and metabolome profiles through the first 42 d of age indicated greater hindgut capacity for the production of energy-generating substrates (butyrate and propionate) and essential nutrients (vitamins and amino acids) in heifers with greater estimated feed efficiency.Conclusion:Despite consuming approximately 54.6％ less solid feed (cumulative intake,10.90 vs.19.98 ± 1.66 kg) from birth to weaning,the microbiome-metabolome changes in the hindgut of most-efficient heifers might have helped them maintain the same level of growth as the least-efficient heifers.