# Milk replacer feeding once or twice a day did not change the ruminal metabolomic profile and the microbial diversity of dairy calves from birth to weaning

A. Zened, C. Julien, L. Cauquil, Géraldine Pascal, C. Canlet, M.

Tremblay-Franco, D. Ali-Haimoud-Lekhal, F. Enjalbert, Corine Bavourthe,
Sylvie Combes

► To cite this version:

A. Zened, C. Julien, L. Cauquil, Géraldine Pascal, C. Canlet, et al. Milk replacer feeding once or twice a day did not change the ruminal metabolomic profile and the microbial diversity of dairy calves from birth to weaning. Journal of Dairy Science, 2024, 107 (8), pp.5574-5586. 10.3168/jds.2023-24327. hal-04667561

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# Milk replacer feeding once or twice a day did not change the ruminal metabolomic profile and the microbial diversity of dairy calves from birth to weaning

A. Zened,<fnref n="1" />,<fnref n="2" />* C. Julien,<fnref n="2" /> L. Cauquil,<fnref n="2" /> G. Pascal,<fnref n="2" /> C. Canlet,<fnref n="3" /> M. Tremblay-Franco,<fnref n="3" />

D. Ali-Haimoud-Lekhal,<fnref n="1" />,<fnref n="2" /> F. Enjalbert,<fnref n="2" /> C. Bavourthe,<fnref n="2" /> and S. Combes<fnref n="2" />

## ABSTRACT

In commercial dairy production systems, feeding calves once daily could be an alternative to reduce labor expenses. Several studies comparing once-a-day (OAD) versus twice-a-day (TAD) milk feeding systems have not evidenced differences in calf growth, rumen development, blood parameters or health scores, but effect on ruminal microbiota remains to be investigated. The objective of this study was to determine the effects of OAD or TAD on the establishment of the ruminal microbiota and its metabolic activity. Sixteen male calves (45.9 ± 5.7 kg at birth) were involved in the trial from birth to weaning (63 d). After the colostrum phase, 2 feeding programs based on a milk replacer were tested and calves were allocated to these programs on d 5. To study the establishment of the bacterial community, ruminal fluid was obtained from each calf 1 h after the morning meal at 7, 35, and 63 d of age. The ruminal metabolome was evaluated at a 7-d interval from d 1 to d 63. Ruminal microbiota and metabolite profiles were characterized by 16 S rRNA gene sequencing- and by ¹H nuclear magnetic resonance spectroscopy, respectively. Our results showed that feeding milk replacer once or TAD did not change the ruminal microbiota and metabolites of dairy calves from birth to weaning. Microbial data showed that diversity and richness increased with age, suggesting a shift from a heterogeneous and less diverse community after birth (d 7) to a more diverse but homogeneous community at 35 and 63 d. These findings suggest that feeding milk OAD can be successfully applied to a calf feeding system without compromising microbial establishment and functions.

**Key words:** dairy calf, feeding frequency, rumen, microbiota, metabolic profile

## INTRODUCTION

In dairy farms, the period between birth and weaning is expensive and time-consuming for the farmers. It is an unproductive phase that lasts 2 mo, in which the milk feed is usually based on a milk replacer (MR). This period also requires time for care and rigorous inspections to prevent diseases. The most common milk feeding program (FP) used for dairy calves is twice daily. It has been reported that feeding only once a day (OAD) decreases labor by 39%, compared with feeding calves twice daily (Galton and Brakel, 1976).

None of the studies that compared OAD versus twice-a-day (TAD) milk FP have shown differences in growth performances, starter intake, rumen development of calves (Ackerman et al., 1969; Galton and Brakel, 1976; Julien et al., 2015; Saldana et al., 2019), blood constituents (Stanley et al., 2002), health scores (Kehoe et al., 2007; Hulbert et al., 2011; Saldana et al., 2019), or behavior (Mushtaq et al., 2024), indicating that feeding milk OAD can be performed without apparent detrimental effects. This change in feeding strategy takes place during a crucial developmental period of the young ruminant. From birth to 3 wk of age, calves are considered as preruminants. During that time, the rumen is anatomically and physiologically underdeveloped (Davis and Drackley, 1998) and the diet, composed exclusively of milk or MR, bypasses the rumen and arrives into the abomasum via reticular groove. The beginning of starter intake (at ~3 wk of age) allows the transition from mono-gastric to ruminant. All these events lead to a series of anatomical and physiological shifts in the gastrointestinal tract, including the establishment of a wide diverse microbial ecosystem. The dynamics of the establishment

<footnote n="1">¹INP-PURPAN, 31076, Toulouse, France</footnote>

<footnote n="2">²GenPhySE, Université de Toulouse, INRAE, ENVT, 31326, Castanet Tolosan, France</footnote>

<footnote n="3">³Toxalim, Université de Toulouse, INRAE, ENVT, INP-Purpan, UPS, 31027, Toulouse, France</footnote>
of the ruminal microbiota in calves is linked to age and diet change (Rey et al., 2014). The initial phase of colonization is composed essentially of facultative anaerobic bacteria (more than 50% of *Proteobacteria*). These will play an important role in the establishment of the ruminal environment, particularly for strictly anaerobic populations (Fonty et al., 1989). In the second phase, a significant change in the bacterial community takes place, which strictly anaerobic bacteria dominate (Fonty et al., 1989). The last stage of colonization depends on solid food intake, age at weaning and the presence or absence of surrounding adult animals (Rey et al., 2014).

The microbial colonization of the calf's digestive tract plays a major role in the development of host digestive health and function; thus, it is important to ensure its proper establishment. Most studies have focused on the effect of diet composition on the establishment of ruminal microbiota. However, consequences of changing feeding frequency to once daily have never been evaluated. This work was conducted to investigate the effects of 2 feeding programs based on a MR fed OAD or TAD on ruminal metabolites and microbial diversity of Holstein calves from birth to weaning. The results of this work complement the previously published data on growth and rumen fermentation and development of dairy calves (Julien et al., 2015).

# MATERIALS AND METHODS

## Animals and Diets

This work is a companion study to that of Julien et al. (2015) analyzing samples collected from their Holstein calves. This study was conducted at the Borret experimental farm (INP-ENSAT, Toulouse, France). At the time of the study, the experimental farm did not have an institutional animal care and use committee or equivalent, but all research animal activity was performed in compliance with federal, state, and local laws of the French Ministry of Agriculture for all animal housing and handling procedures (Anonymous, 1988).

Sixteen healthy male Holstein calves (45.9 ± 5.7 kg at birth) were involved in the trial from birth to weaning (63 d). Immediately after birth (d 1), they were separated from their mother and placed in individual pens (1.05 × 2.30 m, bedded with straw) without any contact with adult animals. At birth (d 1), they received colostrum in 2 equal meals. Then they received whole milk twice each day, 2 L at 0800 h and 2 L at 1600 h, until the morning of d 4. On the evening of d 4, they received an oral rehydratant (Vitactif sachet repas, Chasseneuil-du-Poitou, France) mixed in 2 L of water to manage a transition between whole milk and MR

distribution beginning on d 5. Two FP (OAD or TAD) were tested as of d 5 in the morning. Each feeding program was based on a MR formulated by the same supplier (Bonilait-Protéines, Chasseneuil-du-Poitou, France) with equivalent proportions of dairy proteins, but with different skim milk powder and whey protein contents. Both MR had similar chemical composition: on average 20.8% CP and 18.0% fat, on a DM basis. Calves were divided into 2 groups according to their birth weight (equivalent weights between the 2 groups): (1) OAD (n = 8): calves receiving 200 g/L of MR once a day, (2) TAD (n = 8): calves receiving 125 g/L of MR twice a day. The volumes distributed per calf and per meal in both groups varied with age: 2 L/meal from d 5 to d 7, 2.5 L/meal from d 8 to d 14, 3 L/meal from d 15 to d 21, 3.5 L/meal from d 22 to d 42, 3 L/meal from d 43 to d 49, 2.5 L/meal from d 50 to d 56 and 2 L/meal from d 57 to weaning (d 63). Considering the initial concentration of MR distributed in both groups, TAD calves received a higher amount of MR than OAD calves. From d 5 to weaning, a starter diet composed by 31% barley, 15% wheat, 13% husked sunflower cake, 10% corn, 10% maize germ meal, 7% sunflower cake, 5% wheat middlings, and traces of sugar cane molasses and rapeseed meal (17.5% of CP, 21.1% of NDF, 32.2% ADF, 4,283 kcal/kg of gross energy (GE), 4.3% of ether extract, on a DM basis) and wheat straw (4.5% of CP, 87.2% of NDF, 57.5% ADF, 4,381 kcal/kg of GE, on a DM basis) were provided ad libitum. Each calf had free access to water from d 3 to the end of the experiment (d 63). Water and starter diet were distributed individually, in separate buckets, in 2 meals at 0800 and 1600 h. The total quantity of MR distributed to calves of OAD and TAD groups were 34.1 and 40.9 kg/calf, respectively. Foods provided and refusals were recorded at each meal.

## Sample Collection, DNA Extraction, and Amplicon Preparation for Sequencing

Samples of ruminal fluid were obtained from each calf at 7 (d 7), 35 (d 35), and 63 (d 63) d of age. Ruminal fluid (~200 mL) was collected via a stomach tube (internal diameter 13 mm) 1 h after the morning milk meal and just before the distribution of straw and starter. The stomach tube was sterilized before use with 2% Steranios (Centravet 803910 disinfection of surgical and medical equipment). The collected ruminal fluid was filtered through a metal sieve (1.6-mm mesh) and immediately frozen at -20°C and then stored at -80°C for subsequent analysis.

The extraction and purification of total DNA from each sample was performed by a combination of mechanical (FastPrep, MP Biomedicals, Illkirch, France), chemical, and thermal lysis by using the QIAamp DNA Stool
Mini kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. The extracted DNA purity and quantity were determined with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). The DNA samples were amplified with 2 specific primers targeting the hypervariable regions V3–V4 on the 16S ribosomal RNA gene (460 bp on *Escherichia coli*). The specific bacterial primers used were forward 343F (TACGGRAGGCAGCAG, Liu et al., 2007) and reverse 784R (TACCAGGGTATCTAATCCT, Andersson et al., 2008). Each primer had a barcode sequence of 11 nucleotides on 5' end, which was unique for each sample. The PCR were performed with 1 µL of DNA (1–5 ng/µL), 0.5 µM of forward and reverse primers, 1× PCR buffer, 200 µM dNTP, 0.5 U of Isis DNA polymerase (MP Biomedicals, Illkirch, France) in a total volume of 100 µL. The amplification program was as follow: 2 min at 94°C followed by 32 denaturation cycles (succession of 30 s at 94°C, 30 s at 60°C and 30 s at 72°C) and a final extension step for 7 min at 72°C. Amplicons were purified with the MinElute PCR Purification Kit (QIAGEN GmbH, Hilden, Germany). The individual purified amplicon samples were checked on agarose gels 2% (wt/vol) and the quality and quantity of purified PCR products were assessed using a NanoDrop ND-1000 spectrophotometer.

The purified PCR products were quantified using the Quant-iT PicoGreen ds DNA Assay kit (Invitrogen, Saint Aubin, France) on an ABI Prism 7900HT sequence detection system (Applied Biosystems, Saint Aubin, France) and then pooled in equimolar amounts at 1,000 ng/µL. The pooled amplicons were pyrosequenced using 454 GS FLX Titanium sequencer (Lubbock, TX).

## Sequence Analysis

A total of 449,765 16S ribosomal DNA amplicon sequences were obtained and sorted based on their respective barcodes, representing the 46 collected ruminal samples. Using FROGS guidelines (Escudié et al., 2018), sequences were filtered by removing sequences that did not match both proximal PCR primer sequences (no mismatch allowed), with a short sequencing length (<380 nucleotides) or with at least one ambiguous base. Reads were clustered into operational taxonomic units (OTU) using SWARM (Mahé et al., 2014). Chimeric DNA sequences were detected using VSEARCH and removed. The average read length was 493 ± 38 nucleotides. Sequencing reads were deposited in the National Center for Biotechnology Information Sequence Read Archive (NCBI SRA PRJNA482520). The OTU taxonomic assignment was performed using the BLAST algorithm against the SILVA SSU Ref NR 132 database, where species

with a pintail less than 80 were removed (Pruesse et al., 2007).

## NMR Spectroscopy and Metabolite Identification and Quantification

One hour after the morning meal, samples of ruminal fluid were collected on each calf at a 7-d interval from d 1 to d 63 via a stomach tube (as described above). Ruminal fluid was filtered through a metal sieve and subsamples were immediately stored at -80°C for subsequent metabolomic analysis. Rumen fluid samples were thawed at room temperature and centrifuged at 4,000 × g for 20 min. An aliquot of 500 µL of supernatant was mixed with 200 µL of a standard buffer solution (pH = 7.40) prepared with D₂O and centrifuged at 5,000 × g for 10 min. Then, 600 µL of supernatant was transferred to a standard ¹H nuclear magnetic resonance (NMR) tube for NMR spectral analysis.

All NMR spectra were obtained on a Bruker DRX-600 Avance NMR spectrometer operating at 600.13 MHz for ¹H resonance frequency using an inverse detection 5 mm ¹H-¹³C-¹⁵N cryoprobe attached to a Cryoplatform (the preamplifier cooling unit). The NMR spectra were acquired at 300K using the Carr-Purcell-Meiboom-Gill spin-echo pulse sequence with presaturation, with a total spin-echo delay (2nτ) of 100 ms to attenuate broad signals from macromolecules. A total of 128 transients were collected into 32k data points using a spectral width of 20 ppm, and a relaxation delay of 5 s. The free induction decays were zero filled to 64k in the frequency domain and multiplied by an exponential weighting function corresponding to the line broadening (0.3 Hz) before Fourier transformation. Spectra were referenced to sodium trimethylsilyl propionate (TSP; 0.0 ppm) and manually corrected for phase and baseline distortions.

Spectral assignment was based on matching 1-dimensional and 2-dimensional (2D) data to reference spectra in a home-made reference database, as well as with other databases (http://www.brmb.wisc.edu and http://www.hmdb.ca), and reports in the literature (Ametaj et al., 2010, Saleem et al., 2013). Both a 2D ¹H-¹³C-HSQC (heteronuclear single quantum correlation) and a 2D ¹H-¹H-COSY (correlation spectroscopy) were used for structural confirmation. Metabolite concentrations were obtained on the 1-dimensional ¹H spectra. The integrals of different metabolites were taken relative to TSP, which serves as an internal reference for chemical shifts (0.0 ppm), as well as for concentration measurements.
## Statistical Analyses

All statistical analyses were carried out using R software, version 3.5.0 in RStudio software. Richness (observed OTU), Shannon, and InvSimpson α-diversity indexes were calculated using the phyloseq package of R (McMurdie and Holmes, 2013) and analyzed using a linear mixed model with FP and age as fixed effects and calf as random effect. The interaction between FP and age was not significant, and thus withdrawn from the model. Beta-diversity was investigated after calculation of the Bray-Curtis distance matrix that was visualized with a nonmetric multidimensional scaling (nMDS). Pairwise Bray-Curtis distance was used to assess interindividual variability within age groups. To check group differences an ADONIS pairwise test with the Bray-Curtis distance was carried out. Due to residuals non-normal distribution, taxonomic composition at phylum, family and genus levels was analyzed using Kruskal-Wallis nonparametric tests. The *P*-values were adjusted using the Benjamini-Hochberg method. Multiple pairwise comparisons between age groups were performed using Wilcoxon tests. The Metacoder package was used to visualize phylogenetic shifts in bacterial community data (Foster et al., 2017).

Eighteen outliers over the 160 samples for absolute contents of assigned metabolites were not considered for subsequent data analysis. Then, the absolute and relative concentrations of metabolites were analyzed using a nonparametric ANOVA. Multiple comparisons were compensated for using false discovery rate calculations with the Benjamini-Hochberg method.

A partial least square (PLS) analysis (R package mixOmics; Rohart et al., 2017) was used to explore the relationship between ruminal metabolome profiles and the most abundant microbial OTU (with a threshold of 0.0005%). The PLS correlations between OTU profiles and the metabolome were evaluated separately at 7 d and at 35 and 63 d of age with a threshold of 0.6.

## RESULTS

### Bacterial Diversity in the Rumen of Preweaning Dairy Calves

The number of observed OTU and α-diversity indices (Table 1) increased from d 7 to 35 (*P* < 0.001) and remained stable at 63 d. Feeding program had no effect on bacterial diversity. The nMDS (Figure 1A) of the Bray-Curtis distance matrix showed a great shift in community structure between 7 and 35 d (*P* < 0.001), afterward bacterial community was similar between 35 and 63 d. Interindividual Bray-Curtis distance calculations per age group revealed a great variability in the ruminal bacterial community between calves at 7 d of age compared with that at 35 and 63 d (Figure 1B, *P* < 0.001).

### Taxonomical Composition of Ruminal Bacterial Communities of Preweaning Dairy Calves

The frequency of feeding had no effect on ruminal bacterial composition (Supplemental Tables S1, S2, and S3, see Notes). Overall, 6 phyla were detected in the samples. Among them, *Bacteroidetes* was the dominant one (62%), followed by *Firmicutes* (19%), *Proteobacteria* (15%), and *Actinobacteria* (4%). *Fibrobacteres* and *Verrucomicrobia* were in minor proportions (less than 0.05%). An age effect was observed on *Proteobacteria* phylum relative abundance with a 2-fold increase from d 7 to 35 (*P* = 0.0024).

*Prevotellaceae* (55%) and *Bacteroidaceae* (6%) were the most abundant families, which belong to the *Bacteroidetes* phylum. In the *Firmicutes* phylum, *Lachnospiraceae* (7%), *Erysipelotrichaceae* (5%), *Streptococcaceae* (1.4%), and *Ruminococcaceae* (2.3%) were the most abundant families. The *Proteobacteria* phylum was represented mostly by *Succinivibrionaceae* (13%) and *Pasteurellaceae* (1%). *Atopobiaceae* (2.6%) was the most abundant family in the *Actinobacteria* phylum followed by *Actinomycetaceae* (1%) and *Bifidobacteriaceae* (0.5%) families.

Almost 81% of the sequences were assigned at genus level and spread across 76 genera. *Prevotella* (48%),

<table><caption>**Table 1.** Effects of feeding program (FP) based on 1 (OAD) or 2 (TAD) milk replacer daily meals and age-related differences in calf rumen bacterial α-diversity (mean ± SD)</caption><thead><tr><th rowspan="2">Diversity index</th><th colspan="2">FP</th><th colspan="3">Day</th><th colspan="2">P-value</th></tr><tr><th>OAD</th><th>TAD</th><th>7</th><th>35</th><th>63</th><th>FP</th><th>Day</th></tr></thead><tbody><tr><td>Observed</td><td>210 ± 57</td><td>222 ± 65</td><td>150 ± 51<sup>a</sup></td><td>240 ± 41<sup>b</sup></td><td>253 ± 21<sup>b</sup></td><td>0.418</td><td>&lt;0.001</td></tr><tr><td>Shannon index</td><td>4.03 ± 0.51</td><td>4.12 ± 0.72</td><td>3.56 ± 0.81<sup>a</sup></td><td>4.34 ± 0.26<sup>b</sup></td><td>4.32 ± 0.17<sup>b</sup></td><td>0.713</td><td>&lt;0.001</td></tr><tr><td>InvSimpson index</td><td>25 ± 10</td><td>29 ± 12</td><td>19.6 ± 13.1<sup>a</sup></td><td>33 ± 9.4<sup>b</sup></td><td>29 ± 6.8<sup>b</sup></td><td>0.235</td><td>&lt;0.001</td></tr></tbody></table>

<footnote><sup>a,b</sup>Within a row, mean values with a different superscript between days differ (*P* < 0.05).</footnote>
<figure><img src="image_3.png" /><figcaption>**Figure 1.** (A) Nonmetric multidimensional scaling (nMDS) 2-dimensional representation using Bray-Curtis distance of ruminal bacterial community of dairy calves receiving 1 (OAD) or 2 (TAD) milk replacer daily meals at 7, 35, and 63 d of age; Each point represents a sample plotted according to its bacterial composition and abundance. Samples with similar bacterial composition and abundance are close whereas a greater distance between 2 points infers a lower similarity between them. (B) Effect of age on ruminal bacterial community within-group dispersion as assessed by distance to centroid in Bray-Curtis distance matrix.</figcaption></figure>

Bacteroides (13%), and *Succinivibrionaceae* UCG-001 (11%) were the most abundant genera in the rumen of dairy calves.

Taxonomic profiles of the ruminal bacterial community revealed a strong shift between 7 and 35 d of age (Figure 2, Supplemental Table S2). Mainly *Actinomy cetaceae* (*P* < 0.001), *Bacteroidaceae* (*P* < 0.001), *Bifidobacteriaceae* (*P* = 0.0024), *Streptococcaceae* (*P* < 0.001), *Enterobacteriaceae* (*P* = 0.018), *Tannerellaceae* (*P* < 0.001), and *Pasteurellaceae* (*P* < 0.001) decreased while *Atopobiaceae* (*P* = 0.003), *Lachnospiraceae* (*P* = 0.016), and *Succinivibrionaceae* (*P* < 0.001) increased.

Among the most abundant genera, *Bacteroides*, *Strep- tococcus*, *Gallibacterium*, *Actinomyces*, and *Bifidobacte- rium* decreased and even disappeared between 7 and 35 d of age, especially *Bacteroides* whose relative abundance went from 19.4% at d 7 to 0% (Supplemental Table S3). *Prevotella*, *Lachnospiraceae* NK3A20 group, *Olsenella*, *Succinivibrionaceae* UCG-001, *Oribacterium*, *Rumino- coccaceae* UCG-014, and *Dialister* increased from d 7 to 35 (Supplemental Table S3).

## Ruminal Metabolomic Profile

A total of 26 metabolites (Figure 3A) were identified and quantified in the ruminal fluid of dairy calves from 1 to 9 wk of age. Feeding program had no effect on ruminal metabolites (adjusted *P* > 0.05). Hippurate, citrate, taurine and TMAO-betaine could only be quantified at birth with a concentration up to a maximum of 0.2 *mM* (Supplemental Table S4, see Notes). At birth, lactate represented ~52% of total ruminal metabolites assigned. Except for choline, formate and succinate, metabolite concentrations decreased or increased with age (Figure 3A). The changes in metabolite concentrations were observed mainly during the first 2 wk of life (Figure 3B). From wk 1 to weaning (wk 9), acetate and propionate represented the major part of ruminal metabolites: combined, they represented between 65% on wk 3 and 72% on wk 8 of ruminal metabolites.

## Relationship Between Ruminal Metabolites and Microbiota

To get insight into ruminal functioning, a PLS analysis (Figure 4) was performed between microbial composition
<figure><img src="image_4.png" /><figcaption>**Figure 2.** Phylogenetic tree indicating relative abundance relationship between taxa in ruminal microbiota of calves at different ages (7, 35, and 63 d). Differential colors of pairwise comparisons between ages highlighting significant taxonomic enrichments (Wilcoxon test, *P* < 0.05). The gray color of branches represents taxa that do not differ in abundance. Green branches are the taxa that increase at the ages indicated in the right side of the figure compared with those in the top (35 vs. 63 d of age, 7 vs. 63, and 7 vs. 35 d), and orange branches represent the opposite (35 vs. 7 d and 63 vs. 7 d).</figcaption></figure>

at the OTU level and the 22 major metabolites detected at 7, 35, and 63 d of age. As seen previously, the PLS plot of the individuals, using the ruminal metabolomics and microbiota data, revealed a clear separation between the 7-d-old calves on one side, and the 35- and 63-d-old ones on the opposite side (Figure 4A). According to PLS variable loading values, 7-d-old calf ruminal samples exhibited the highest formate concentration and, to a lesser extent, choline, succinate, lactate, and galactose concentrations (Figure 4B). The 7-d-old calf ruminal samples also showed the highest relative abundance of 4 OTU affiliated to *Actinomyces*, 1 OTU affiliated to

*Gallibacterium*, 1 OTU to *Parabacteroides*, 9 OTU to *Bacteroides*, and 2 OTU to *Streptococcus* (Figure 4C). At 35 and 63 d, metabolites concentrations (Figure 4B), including primarily VFA (propionate, acetate, valerate, and to a lesser extent, butyrate), AA (isoleucine, alanine, valine), and also amines (methylamine, dimethylamine), energy substrates, uracil, 3-hydroxyphenylpropionate, methanol, ethanol, and creatine-creatinine, were related only to the relative abundances of 10 OTU affiliated to *Prevotella* and 3 OTU belonging to the *Succinivibrionaceae* UCG-001 genus group (Figure 4C).
A

<figure><img src="image_5.png" /></figure>

B

<figure><img src="image_6.png" /><figcaption>**Figure 3.** (A) Ruminal metabolome of dairy calves from 1 to 9 wk of age. Heatmap representing the mean concentration (rows) for each week in each group (columns). The colors represent the Z-scores (row-scaled expression) from low (blue) to high values (red). Metabolites (rows) were clustered by the ward.D method. (B) Concentration differences of metabolites over 2 consecutive weeks (from W_0 to W_9, 2 by 2). Colors indicate the Kruskal-Wallis results (in orange: *P* < 0.05, in blue: *P* > 0.05, and in gray: undetected).</figcaption></figure>

Because the PLS analysis resulted in 2 age groups (7 vs. 35 and 63 d; Figure 4A), we further investigated the relationship between ruminal metabolites and microbiota using PLS separately in the 2 groups. At 7 d of age, correlations above 0.6 were observed between 90 OTU and 12 metabolites' concentrations (Figure 5 and Supplemental Table S5, see Notes). The concentration of succinate was positively associated with more than half of these out, which were affiliated to *Prevotella* (18 OTU), *Succinivibrionaceae* UCG-001 (16), *Oribacterium* (3 OTU), *Lachnospiraceae* NK3A20 group (4 OTU), and *Ruminococcaceae* UCG-014 (4 OTU). Trimethylamine concentrations were also associated with this latter OTU to a lesser extent.

A second cluster of metabolites comprising galactose, choline, glucose, formate and the 3 branched-chain AA (lysine, isoleucine, and valine) was correlated positively to 2 OTU affiliated to *Prevotella* and *Alloprevotella*.

Finally, a third cluster of metabolites concerned VFA that were mainly negatively correlated with *Prevotella* (11 OTU), *Erysipelotrichaceae* UCG-002 group (5 OTU), *Syntrophococcus* (4 OTU), and *Olsenella* (3 OTU).

At 35 and 63 d, no strong correlation could be evidenced between ruminal microbiota and ruminal metabolome. Only 4 OTU (*Syntrophococcus* and *Prevotella*) exhibited

correlations above the threshold of 0.6 (Supplemental Table S6, see Notes).

DISCUSSION

In dairy systems, as the first period of rearing is long, expensive, and time-consuming, switching from TAD to OAD feeding of calves after birth would be interesting for farmers to decrease labor and subsequent calf-rearing costs. Previous work stemming from the same experimental trial (Julien et al., 2015) showed no differences in animal performances between feeding once or TAD, despite lower MR intake in the OAD group. Indeed, OAD calves had numerically higher starter concentrate intake than TAD calves from wk 6, suggesting an ability of the calves to compensate for the low initial dry feed intake. This was in agreement with other studies testing the same arrangement (Ackerman et al., 1969; Kehoe et al., 2007; Hulbert et al., 2011; Saldana et al., 2019). The aim of the present study was to complement these previous observations by exploring for the first time the potential effect of a once- versus twice-daily FP of calves on ruminal microbiota and ruminal metabolome. Milk replacer feeding OAD or TAD did not change the ruminal microbiota diversity of dairy calves from birth
<figure><img src="image_7.png" /><figcaption>**Figure 4.** Partial least square (PLS) analysis results of the microbiota composition matrix and the metabolomics matrix measured from the same set of calf ruminal samples at 7, 35, and 63 d of age. (A) Projection plot of the individuals into the space spanned by the first 2 PLS components. (B) Loading values of the 30 first OTU and (C) of the 22 metabolites involved in the PLS calculation. Positive loading values are in red and negative in blue.</figcaption></figure>

to weaning. In our work, in both FP groups, 7 d after birth, the calves' ruminal microbiota was less diverse and more heterogeneous than at 35 and 63 d of age, suggesting a primary and random colonization shortly after birth followed by a strong shift to a mature community (Malthuge and Guan, 2017). At birth, the digestive tract is rapidly colonized by aerobic and facultative anaerobic bacteria, whose abundances decrease and are replaced by anaerobic taxa (Fonty et al., 1987; Minato et al., 1992). The initial inoculum is transmitted from the mother and surrounding environment (Mackie et al., 1999; Funkhouser and Bordenstein, 2013) leading to a high interindividual variability of the primary bacterial community

(Li et al., 2012; Jami et al., 2013; Dill-McFarland et al., 2017). This could have a hidden FP effect on early colonization of the ruminal microbiota. Additionally, despite the full functionality of the reticular groove during the first 2 to 3 wk of life (Castro et al., 2016), a little amount of milk (not exceeding 10 g/d) could enter the rumen and provide a fermentable substrate (Toullec and Guiloteau, 1989). But this amount may not be sufficient to induce microbial changes.

In both FP groups, several genera were observed at 7 d of age and then disappeared at 35 and 63 d. A sharp decrease of *Bacteroides* with age has already been reported in many studies (Wu et al., 2012; Jami et al., 2013; Rey et
<figure><img src="image_8.png" /><figcaption>**Figure 5.** Clustered image maps from the partial least square analysis applied to the 7-d calf ruminal microbiota and metabolome. Heatmap showing the correlation between operational taxonomic units (OTU; rows) and metabolites (columns) in the 7-d calf rumen. Colors represent the correlation scores from negative (blue) to positive values (red). The OTU (rows) were clustered by the complete method.</figcaption></figure>
al., 2014). Indeed, *Bacteroides* are facultative anaerobic bacteria, first colonizers of the gut (Le Huërou-Luron et al., 2010), that can settle quickly and are the most efficient bacteria to metabolize milk oligosaccharides (Vallès et al., 2014). Other less dominant genera, such as *Streptococcus*, *Gallibacterium Actinomyces*, and *Bifidobacterium* were observed only at 7 d after birth. Many authors have already reported the similar observations with several functional bacteria in the rumen, including cellulolytic and sulfate-reducing bacteria, during the first week of life (Wu et al., 2012; Rey et al., 2014; Malmuthuge et al., 2014; Castro et al., 2016; Meale et al., 2017). These bacteria are known to have many fermentative characteristics, suggesting a selection of ruminal bacteria driven by functional needs (Li et al., 2012), and confirm that pioneers colonize an open space and create the necessary conditions for more specialized late-coming organisms.

Our observations confirmed those of Rey et al. (2014) showing a high prevalence of *Prevotella* within the first week of life, but were different from the results of Li et al. (2012) and Wu et al. (2012) who observed this genus at low abundance (less than 5%) in the rumen of calves eating only milk. *Prevotella* has diverse metabolic capabilities including protein degradation, fermentation of starch and xylan (Purushe et al., 2010). In our study, the presence of *Prevotella*, 7 d after birth suggests an early development of the ruminal microbiome and its capacity to ferment a plant polysaccharide-based diet.

Taken together, our results suggest that this 7-d bacterial community is pioneering, specialized, and highly competitive, which would explain the lack of effect of our MR FP.

In both MR FP, between 7 and 35 d of age, there was an increase in diversity, commonly found in the literature, with a shift toward a more homogeneous and mature community. A dramatic change in α-diversity occurred at 35 d of age, compared with 7 d of age. Similar results were reported for calves by Li et al. (2012) between 14 and 42 d of age, and by Jami et al. (2013) in 1- and 3-d-old calves compared with 2-mo-old calves. These authors showed an effect of age on ruminal bacterial communities, with an increase in diversity and within-group similarity. In our study, a spectacular increase was observed for the genera *Prevotella* and *Succinivibrionaceae* UCG-001, which normally dominate rumen of lactating cows (Dill-McFarland et al., 2017). The high abundance of these taxa coincided with the period when calves have increased the solid feed intake (Julien et al., 2015), suggesting that the consumption of solid diet that stimulates the growth of rumen papillae, drives the diversity and the structure of ruminal microbiota toward that of adult animals. Moreover, *Lachnospiraceae* and *Ruminococcaceae* families had greater abundances in 35-d-old calves

compared with 7-d-old calves. These anaerobic bacteria are generally abundant in the gut of adult ruminants eating forage-based diets (Malmuthuge, 2016).

Finally, between 35-d-old and 63-d-old calves, there was no difference in α- and β-diversity, despite the increase in the solid feeds/MR ratio. These findings suggest that the shift in carbohydrate composition of the diet during the transition from MR to starter diet is the key in changing the rumen microbiota toward a mature ruminant state. Because many taxa indicative of the mature microbiota are already present in 35-d-old calves and did not change within a month, it could be hypothesized that a mature gastrointestinal microbiota could be reached at ~35 d of age. Altogether, these results suggest that nutritional manipulation to control ruminal microbiome shaping should be operated before 35 d of age. This hypothesis is supported by studies trying to modulate digestive microbiome establishment, such as strategy of weaning (Meale et al., 2015, 2016) or yeast supplementation before weaning (Xiao et al., 2016; Fomenky et al., 2018). In our study, the FP did not include nutritional modifications of the MR, and no effect was observed on ruminal microbiome establishment.

The MR FP had no effect on the ruminal metabolome profile. Regardless of the MR FP, our weekly serial sampling allowed us to evidence that the most important age-related changes in metabolome were observed within the first week of the calf's life. This result is in accordance with the study of Li et al. (2018) showing a change of small intestine metabolites across 3 time points (d 1, 42, and 70 after birth). However, our study did not show a clear difference between 35-d-old and 63-d-old calves. Accordingly, it was shown that ruminal fermentative and enzymatic activities were stabilized at 1 mo of age in calves (Rey et al., 2012; Jiao et al., 2015). As in all mammals, during the first weeks of life, before acquiring the ability to digest solid food, the diet of calves is exclusively composed of liquid feed, and notably the colostrum that provides most of the nutrients, such as AA playing a major role in the development of the neonatal gut, and especially in protein synthesis (Hammon and Blum, 1999).

Four metabolites including taurine and hippurate were only detected in the first week of the calves' life. These metabolites have already been identified in colostrum. Among free AA in cow colostrum, taurine has the highest level (Jacometo et al., 2017) and its concentration decreases markedly in mature milk (Zanker et al., 2000). Hippurate has already been found at high levels in bovine colostrum on d 1 and decreases with time (Settachaimongkon et al., 2021; O'Callaghan et al., 2021). Known as the most abundant microbial-host co-metabolites, hippurate is produced by conjugation between glycine and microbial benzoate in the liver and
kidney (Pallister et al., 2017) and might be uptake from blood to the mammary gland. Trimethylamine, dimethylamine and methylamine concentrations in ruminal fluid increased with age. Trimethylamine is a harmful metabolite exclusively derived from the microbiota, and notably through choline degradation, which is an essential nutrient found in high amounts in mammalian milk that plays a key role in neonatal development (Pinotti et al., 2002). Concentrations of several metabolites including glucose, galactose, uracil, ethanol, methanol, several short-chain fatty acids, and AA increased. Although many of these metabolites are substrates for beneficial microorganisms, and can be used by the calves, some of them are toxic and inflammatory. The most important change observed in ruminal metabolites, derived from carbohydrates' catabolism, mainly VFA (acetate, propionate, butyrate, valerate) which are markers of ruminal microbial fermentation. During the first week of life, ruminal fluid concentrations of VFA did not exceed 20 mmol/L (data previously published, Julien et al., 2015). They reached 100 mmol/L before weaning, in agreement with the results of Rey et al. (2012). The VFA are the promoters of rumen development and their absorption provides 70% of energy to the host (Bergman, 1990). It is well known that papillae development is stimulated by calf starter fermentation (Sander et al., 1959), which increases propionate and butyrate concentrations in the rumen (Warner et al., 1956; Stobo et al., 1966). The presence of high concentrations of various VFA before the availability of plant substrates (fiber, starch, and so on) suggests that at an early stage of development the rumen already exhibited an aptitude for carbohydrate and protein metabolism (Li et al., 2012). During the first week after birth, the VFA profile is associated with lactose and milk oligosaccharides' degradation into acetate and lactate (Shimomura and Sato, 2006; Jiao et al., 2015). The increase in 3-hydroxyphenylpropionate concentration with age has been linked to the bacterial degradation of plant polyphenols that can be further metabolized by the colonic microbiota to short-chain fatty acids, lactate, succinate, oxaloacetate, ethanol, CO2, and H2 (Moco et al., 2012).

To have insight into associations between the abundance of detected bacterial OTU and the observed ruminal metabolome, and to better characterize the ruminal ecosystem, a PLS analysis in calves regardless of their MR FP or age was carried out. This analysis confirmed the separation of the 7-d-old calves from the 2 other age groups and highlighted the strong correlations between metabolites and OTU only observed at 7 d of age. It appears that succinate is a central metabolite in calves' ruminal fluid, presenting strong correlations with genera from several taxonomic groups including the *Succinivibionaceae* family and the *Prevotella* genus. Indeed, it

has been shown that members of the *Succinivibronaceae* family have the ability to ferment carbohydrates into succinate and acetate (Santos et Thompson, 2014). Additionally, according to the literature-curated interspecies network NJS16 of the human gut microbiota (Sung et al., 2017), 18 species of the *Prevotella* genus were shown to export succinate as a result of extracellular macromolecule degradation or metabolization of imported small-molecule metabolites. In our study, 11 additional OTU belonging to the *Prevotella* genus were also negatively associated with the VFA metabolites.

Succinate is an intermediary metabolite in the pro-pionate production cascade, *Prevotella* OTU might also favor succinate accumulation in the 7 d-old calf rumen.

## CONCLUSIONS

Feeding dairy calves once daily from the fourth day of age to weaning (63 d) did not cause any difference in the ruminal microbiota and its metabolomic profile compared with TAD-fed calves, indicating that feeding milk OAD did not affect the colonization of ruminal bacteria. Taken together, our results show that the pioneer community and the process of ecological succession of species leading to a mature ruminal microbiota have robust properties that enable them to withstand changes in the MR frequency. Additionally, our findings showed a progressive development of the ruminal microbiota and metabolome with significant differences according to the age primarily between 7- and 35-d-old calves, whereas homogeneous ruminal bacterial communities were observed at 35 and 63 d.

## NOTES

This work was funded by the INRA PHASE Department (Toulouse, France). We acknowledge the farm crew at the Borret Dairy Farm (Poucharramet, France), specifically Brigitte Gestes. We thank Béatrice Gabinaud for technical assistance. We also thank Joseph Nakhle for his precious work as master student. Supplemental material for this article is available at https://doi.org/10.17632/5z9hs5k88h.1. This study was conducted at the Borret experimental farm (Toulouse, France). At the time of the study, the experimental farm did not have an institutional animal care and use committee or equivalent, but all research animal activity was performed in compliance with federal, state, and local laws of the French Ministry of Agriculture for all animal housing and handling procedures. The authors have not stated any conflicts of interest.

**Nonstandard abbreviations used:** 2D = 2-dimensional; FP = feeding program; GE = gross energy; MR
= milk replacer; nMDS = nonmetric multidimensional scaling; NMR = ¹H nuclear magnetic resonance; OAD = once a day; OTU = operational taxonomic units; PLS = partial least square; TAD = twice a day; TSP = sodium trimethylsilyl propionate.

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ORCIDS

A. Zened ● https://orcid.org/0000-0002-0331-940X

L. Cauquil ● https://orcid.org/0000-0002-0384-8811

G. Pascal ● https://orcid.org/0000-0002-5250-6594

F. Enjalbert ● https://orcid.org/0000-0001-6801-7261

S. Combes ● https://orcid.org/0000-0002-2945-4423