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Alterations in rumen microbiota via oral fiber administration during early life in dairy cows

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Animals and diets

The animal experiments were conducted in accordance with the Guidelines for Animal Experiments and Act on Welfare and Management of Animals, Hokkaido University, and all experimental procedures were approved by the Animal Care and Use Committee of Hokkaido University. All animal experiments were carried out in accordance with ARRIVE guidelines. Twenty newborn female Holstein calves with an average birth weight of 37.1 ± 1.0 kg (mean ± standard error) were randomly allocated to either the control or treatment group at birth. All calves were housed individually in separate calf hutches containing sawdust bedding. Feeding and managing of animals until weaning at 50 d of age was performed as described previously17. After supplementing colostrum at birth, calves in both groups were fed 4 L of pasteurized whole milk (44.2% crude protein [CP] and 29.3% fat on a dry matter [DM] basis) as a transition milk during the first week since birth. From 8 days until weaning at 50 days of age, milk replacer (28.0% CP and 18.0% fat on a DM basis) was fed twice daily at 0830 and 1600 h. Water, calf starter (22.9% CP, 11.0% neutral detergent fiber [NDF], 5.6% acid detergent fiber [ADF], 6.2% crude ash, and 3.0% ether extract on a DM basis), and chopped Timothy hay (3.4% CP, 53.1% NDF, 34.2% ADF, 4.3% crude ash, and 1.7% ether extract on a DM basis) were provided for ad libitum intake from 3 days of age. In addition to voluntary intake of solid diets, the calves in the treatment group were orally administered with a mixture of ground Timothy hay and psyllium (4.4% CP, 78.6% NDF, 5.8% ADF, 3.9% crude ash, and 0.3% ether extract on a DM basis) from 3 days until weaning at 50 days of age. Timothy hay was ground for oral administration using a Wiley grinder (WM-3, Irie Shokai) with a 2-mm screen. To improve the handling of the treatment diet for oral administration, we incorporated psyllium, which is a dietary fiber that primarily improves gastrointestinal conditions in humans and can be incorporated in oral electrolyte solution supplemented to neonatal calves38. As a treatment diet, ground Timothy hay (50 g) and psyllium (6 g) were mixed with 200 mL of water. Owing to the adhesiveness of psyllium, the treatment diet formed a “hay ball” and showed slight stickiness, which facilitates swallowing by calves. At 3–7 days of age, one hay ball (50 g of fibrous diet) was orally administered after morning milk feeding. From 8 days of age to weaning, an additional hay ball was fed immediately after evening milk feeding (100 g fibrous diet per day).

After weaning, animals in both dietary groups were merged into the same herd and managed on the same farm under identical conditions. From 9 months of age until calving, heifers were fed a ration containing Timothy hay, alfalfa hay, fescue hay, and concentrate. After calving, the cows were fed a diet for lactating cows, as described in Supplementary Table S8. Diets comprised a total mixed ration and were fed twice daily at 0900 and 1600 h. All animals had ad libitum access to water and mineral blocks throughout the experiment. Daily milk production for each cow was measured for the first 30 days of the lactation period and the average values for each dietary group on a weekly and monthly basis were calculated. Milk yield for four animals in each dietary group were not recorded due to health problems including mastitis and displaced abomasum symptoms after calving.

In this study, all animals (n = 20) were maintained until 9 months of age, without severe problems. Owing to health problems, several animals were excluded from the experiment before parturition as follows: three animals (one in the control group and two in the treatment group) at 60 days before the expected calving date and one animal in the control group at 21 days before the expected calving date. One animal in the control group (15 days after calving) and two animals in the treatment group (calving day) were diagnosed with displaced abomasum symptoms and were excluded from further sampling. Owing to technical problems, samples were not collected from three animals aged 7 days in the treatment group and one animal aged 21 days in the control group. All other samples (n = 176) were obtained at the target sampling points.

Sampling of rumen contents

Rumen contents were collected orally using a stomach tube. The stomach tube and the sample collection flask were thoroughly cleaned using water between sample collections from individual animals; the first fraction of the sample was discarded to avoid contamination from the previous sample and saliva. All samples were collected at 4 h after morning feeding. Rumen contents were collected at 7, 21, 35, 49, and 56 days, and at 9 months of age, 60 and 21 days before the expected calving date, at calving day, and 21 days after calving. The pH was measured using a pH meter (pH meter F-51; Horiba, Kyoto, Japan) immediately after sampling. Samples were collected in a sterile 50 mL tube and immediately placed on ice, followed by storage at − 30 °C until use.

Chemical analysis

Rumen contents (1.0 g) were centrifuged at 16,000×g at 4 °C for 5 min, and the supernatant was collected. The SCFA content was analyzed using a gas chromatograph (GC-14B; Shimadzu, Kyoto, Japan) as described previously39. In brief, the supernatant of the rumen contents was mixed with 25% meta-phosphoric acid at a 5:1 ratio, incubated overnight at 4 °C, and centrifuged at 10,000×g at 4 °C. The supernatant was then mixed with crotonic acid as an internal standard and injected into a gas chromatograph equipped with an ULBON HR-20 M fused silica capillary column (0.53 mm i.d. × 30 m length, 3.0 µm film; Shinwa, Kyoto, Japan) and a flame-ionization detector. d/l-lactic acid levels were measured using a commercial assay kit (Megazyme International Ireland, Wicklow, Ireland) according to the manufacturer’s instructions. NH3-N levels were measured via the phenol-hypochloride reaction method40 using a microplate reader at 660 nm (ARVO MX; Perkin Elmer, Yokohama, Japan).

DNA extraction and rumen microbiota profiling via amplicon sequencing

Total DNA was extracted and purified using the repeated bead-beating plus column method41. Rumen contents (0.25 g) were homogenized using sterile glass beads (0.4 g; 0.3 g of 0.1 mm and 0.1 g of 0.5 mm) and cell lysis buffer (1 mL; 500 mM NaCl, 50 mM Tris–HCl [pH 8.0], 50 mM ethylenediaminetetraacetic acid (EDTA), and 4% sodium dodecyl sulfate). The lysates were then incubated at 70 °C for 15 min, and the supernatant was collected for further processing. Bead-beating and incubation steps were repeated once, and all supernatants were combined. Total DNA was precipitated using 10 M ammonium acetate and isopropanol, followed by purification using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany). The DNA concentration was quantified using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and adjusted with Tris–EDTA buffer to the appropriate concentration.

For a comprehensive analysis of rumen bacterial communities, the MiSeq sequencing platform (Illumina, San Diego, CA, USA) was used. Total DNA obtained from the rumen contents was diluted to a final concentration of 5 ng/μL and subjected to PCR amplification of the V3-V4 regions of the 16S rRNA gene using the primer sets S-D-Bact-0341-b-S-17 (5′-CCTACGGGNGGCWGCAG-3′) and S-D-Bact-0785-a-A-21 (5′-GACTACHVGGGTATCTAATCC-3′)42. The PCR mixture consisted of 12.5 μL of 2× KAPA HiFi HotStart Ready Mix (Roche Sequencing, Basel, Switzerland), 0.1 μM of each primer, and 2.5 μL of DNA (5 ng/μL). PCR amplification was performed according to the following program described previously9: initial denaturation at 95 °C for 3 min; 25 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s; and a final extension step at 72 °C for 5 min. Amplicons were purified using AMPure XP beads (Beckman-Coulter, Brea, CA, USA) and subjected to sequencing on the Illumina MiSeq platform (Illumina) using the MiSeq Reagent Kit v3 (2 × 300 paired-end).

Data obtained from amplicon sequencing using the MiSeq platform were analyzed using QIIME2 version 2019.443. Paired reads were filtered, dereplicated, merged, and chimera-filtered using the q2-dada2 plugin44 to generate ASVs. Taxonomic classification of the ASVs was performed at the phylum, class, order, family, and genus levels using the SILVA 132 99% operational taxonomic units, full length, seven level taxonomy classifier (silva-132-99-nb-classifier.qza). Sequenced data were processed further and analyzed using R software version 3.6.245. ASV and taxonomy tables generated using QIIME2 were imported into R and merged with the sample metadata using the Phyloseq Bioconductor packages46. ASVs identified as Archaea, chloroplasts, and mitochondria were excluded. All samples were rarefied to a sampling depth of 16,805 reads, which was the smallest number of reads observed per sample in the filtered ASV table. Alpha diversity indices including Chao1, ACE, Shannon, and Simpson indices were calculated using the phyloseq function “estimate_richness”. PCoA was performed to determine differences in the microbial community structure based on the Bray–Curtis dissimilarity matrices at the genus level using the Phyloseq package. Venn diagrams were generated using ASVs showing mean relative sequence abundances of > 0.1% in either the control or the treatment groups at each sampling point. The relative abundance of each bacterial taxon was calculated by dividing the number of reads assigned to each taxon by the total number of reads. Taxa with an average relative abundance > 0.1% in > 50% of samples in either the control or treatment group during at least one sampling point were used for the analysis. Hierarchical cluster analysis of bacterial genera determined via amplicon sequencing at 21 days after calving and the weekly and monthly average milk yield for the first 30 days of lactation period was performed using the distances calculated from Spearman’s correlation and average linkage clustering.

Quantification of target bacterial species/groups using real-time PCR

The relative abundance of known ruminal bacterial species and groups, including the total bacteria, F. succinogenes, R. flavefaciens, Ruminococcus albus, Butyrivibrio spp., Prevotella spp., Selenomonas ruminantium, Megasphaera elsdenii, Treponema spp., Streptococcus bovis, Anaerovibrio lipolytica, and Ruminobacter amylophilus, was quantified using real-time PCR. Amplification was performed using a Light Cycler 480 system (Roche Applied Science, Mannheim, Germany) with a KAPA SYBR Fast qPCR Kit (Roche Sequencing, Basel, Switzerland) and the respective primer sets (Supplementary Table S9). The standards used for the real-time PCR were prepared as described previously47. Briefly, plasmid DNA containing the respective target bacterial 16S rRNA gene sequence was obtained by PCR cloning using the species/genus-specific or bacterial universal primer sets. The concentration of the plasmid was determined with a spectrometer. Copy number of each standard plasmid was calculated using the molecular weight of nucleic acid and the length (base pair) of the cloned standard plasmid. Ten-fold dilution series ranging from 1 to 108 copies were prepared for each target and run along with the samples. The respective genes were quantified using standard curves obtained from the amplification profile of the dilution series of the plasmid DNA standard (Supplementary Table S9). The PCR cycling conditions and reaction mixture were the same as those reported previously48. The relative abundance of each bacterial target was expressed as the proportion (%) of the abundance of the 16S rRNA genes of each bacterial target relative to that of the total bacteria.

Statistical analysis

All data were sorted based on animal age into two sets, from 7 to 56 days of age and from 9 months of age to 21 days after calving, and analyzed separately. Data on fermentation parameters and bacterial abundance quantified via real-time PCR were analyzed using a repeated measures model using GraphPad Prism software version 9.1 (GraphPad Software, San Diego, CA, USA) with the fixed effects of dietary group, age, and diet × age interaction, and the random effect of animals within the groups. The Greenhouse–Geisser correction was used where sphericity was violated. If the P-value for the treatment effect was < 0.1, then Tukey’s method was used for multiple comparisons of the means. The effects of dietary group, age, and diet × age interaction on the bacterial taxon abundance determined via amplicon sequencing were assessed using the Poisson regression model in the lme4 package49 in R software. Dunn’s test was used for multiple comparisons, and P-values were adjusted using the Benjamini and Hochberg method50. Although the treatment effect was found to be significant for several bacterial genera, the post hoc test did not reveal any differences between the control and treatment groups at each sampling point (Supplementary Tables S3, S4). Therefore, the bacterial taxa determined via amplicon sequencing were further compared between the dietary groups at each sampling point using the negative binomial model implemented as the Wald test in the DESeq2 Bioconductor package in the R software51. A false discovery rate threshold of 0.15 was used to determine significance. Bray–Curtis dissimilarity matrices were calculated and visualized as PCoA using Phyloseq Bioconductor packages in R. permutational multivariate analysis of variance (PERMANOVA) tests were performed based on the Bray–Curtis dissimilarity matrices to assess the effect of dietary group, age, and diet × age interaction on microbial composition using the Vegan package version 2.5-752 in R software.


Source: Ecology - nature.com

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