Experimental design and treatments
The calf trial, and all associated procedures for sampling and measurement taking, was approved by the AgResearch Grasslands Animal Ethics Committee, Palmerston North, New Zealand, application number 14249. Transportation of animals was conducted according to the Dairy Cattle Code of Welfare .
Full details of the calf trial have been described by Groenendijk et al. (2018) . In brief, Kiwi cross (Holstein-Friesian x Jersey) calves born on Farmway Farm, Rongotea or Ohau Dairies, Horowhenua, New Zealand during the 2017 spring calving season were collected twice daily (e.g. 1–14 h after birth) from the calving paddocks. Calves received 2 L of first-milking colostrum at the time of collection (within 15 h of birth) and then 2 L twice daily until 2 days old. The calves were kept on their source farms for a minimum of 4 days old and were then transported to a dedicated calf-rearing facility at Farmway Farm.
All calves were manually fed whole milk using an artificial teat attached to a bottle (2 L in the morning and 2 L in the afternoon) from 3 to 7 days of age, and were trained to use automated milk feeders in stalls (CalfSMART, Palmerston North, New Zealand) twice daily from 8 to 10 days of age, fed 4 L/d milk replacer during this time.
Calves (n = 199) with no apparent sign of illness were allocated to the 3 treatments (LA (n = 67), HA (n = 65) and ADLIB (n = 66)), balanced for source farm, date of birth and body weight. A commercially available milk replacer (Ancalf, NZAgbiz Ltd., Hamilton, New Zealand) was diluted in lukewarm water (150 g/L) and fed to all calves using automated milk feeders (CalfSMART, Palmerston North, New Zealand). All calves had ad libitum access to a pelleted calf-starter (20% CP pellets, SealesWinslow Limited, Tauranga, New Zealand) by automated feeder, and clean drinking water. From the third week of the trial, calves were given ad libitum access to ryegrass hay. The nutritional composition for all feeds is provided in Table S3. Daily feed DM intake (milk replacer, calf starter and total) and nutrient intakes (ME and CP) were calculated for the pre-weaning period (d 0 to 83 on the study) for each calf from data collected on the automated feeders. Water and hay intakes were not measured.
All calves were vaccinated for prevention of leptospirosis and major clostridial diseases (Ultravac 7in1, Zoetis, Auckland, New Zealand) between 4 and 8 weeks of age, and all calves received antibiotics (Alamycin LA300, Norbrook, Auckland, New Zealand) on the same date as prevention for pneumonia. Calves were monitored and scored for the incidence of scours (faecal score of 2 or greater as described in the Calf Health Scorer criteria of the School of Veterinary Medicine, University of Wisconsin, Madison, USA) during the first 3 weeks on trial, after which, scouring was negligible. Calf body weight and dimension measurements were performed where body dimensions were taken of all calves at the start of the study and at weaning. Hip and wither height was determined using a measurement stick and heart girth measurement (the smallest circumference behind the forelegs) was taken using a measuring tape while the animal was standing on a flat surface and with head in an upright position. Blood serum protein levels were determined using the method described in Groenendijk et al. .
Faecal sample collection and short chain fatty acid analysis
From days 67 ± 3 on trial, just prior to weaning, faecal samples were manually collected from calves and frozen on dry ice, then transported to the laboratory for storage at − 85 °C. Faecal samples were thawed on ice and used for SCFA analysis and DNA extraction. Approximately 1 g of material was weighed and diluted with 50% (v/w) phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4; pH 7.4), then centrifuged at 16,000 g for 10 min, 4 °C. A 270 μL aliquot of the supernatant was mixed with 30 μL of internal standard solution (20 mM 2-ethylbutyrate in 20% (v/v) phosphoric acid). Samples were frozen at − 20 °C, then thawed and centrifuged as above, prior to analysis by gas chromatography. Each supernatant sample (200 μL) was vigorously mixed with 100 μL concentrated HCl, and extracted twice with 800 μL diethyl ether for 1 min each. The supernatant extracts were pooled into a 2 ml vial and 800 μL of extract was derivatized with 100 μL of N-methyl-N-t-butyldimethylsilyltrifluoroacetamide (Sigma-Aldrich, St. Louis, MO, USA). The mixture was heated in a crimp top GC vial for 20 min at 80 °C and left for 48 h at room temperature to ensure complete derivitisation. Samples were analysed using a Shimadzu GC-2010 gas chromatograph (Shimadzu Corp., Kyoto, Japan) with a barium ionization detector 2010 (Shimadzu Corp.) and AOC 6000 autosampler (Shimadzu Corp.) and a Zebron ZB-5MS 30 m × 0.25 mm I.D. × 0.25 μm film capillary column (Phenomenex, Torrance CA, USA). Helium was used as carrier gas in conjunction with a He purifier (Valco Instruments Co. Inc., Houston TX, USA). Split injections (1 μL) were made with a ratio of 20:1 split, with column helium flow rate of 21.36 mL/min. Injector and detector temperatures were both 240 °C and column temperatures were programmed initially at 50 °C for 2 min, than increased to 130 °C with 5 °C per minute, followed by 15 °C per minute to 240 °C. The SCFA analyses were performed for every second animal when ranked by pre-weaning ADG within each treatment group, to ensure that the calves across the entire range of weight gain within each treatment group were represented in the subset of samples tested. For the LA, HA and ADLIB treatments, 33, 26 and 28 samples were analysed, respectively.
Total DNA was extracted from ca. 250 mg faecal sample using a commercial kit (Nucleospin Soil; Macherey-Nagel; Düren, Germany) according to the manufacturer’s instructions. To maximise DNA yield, a combination of buffer SL1 and enhancer solution SX was used, and physical disruption of the faecal sample was performed for 4 min at full speed using a Mini-Beadbeater-96 (Biospec Products, Bartlesville, USA). An addition DNA washing step was also included when the DNA was bound to the silica membrane. DNA was eluted with 50 μL elution buffer SE and stored at − 20 °C. All samples were further dialyzed for up to 4 h using 0.025 μm pore size membrane filters (MilliporeSigma, Burlington MA, USA) over double-distilled water. DNA concentration and purity was measured using an ND-1000 spectrophotometer (NanoDrop Technologies Inc., Wilmington DE, United States). Negative DNA extractions were not performed as the faecal samples are dense in microbial biomass, the risk of significant DNA contamination from the extraction kit was deemed neglible. Each batch of samples processed for DNA extractions and SCFA analyses was taken from across multiple sample collection dates, which minimised any potential confounding effect of collection date and processing date.
16S rRNA gene sequencing and analysis
The faecal bacterial communities were profiled by amplifying and sequencing the V4 region of the 16S rRNA gene for all DNA samples and negative controls (i.e. PCR reactions without DNA template), as previously described using the Illumina MiSeq platform (250-bp, paired end) . In brief, the forward F515 primer which has an eight-nucleotide barcode unique to each sample and a two-nucleotide linker sequence (5′-NNNNNNNNGTGTGCCAGCMGCCGCGGTAA-3′) and the reverse R806 primer (5′-GGACTACHVGGGTWTCTAAT-3′) were used in the 16S rRNA gene amplification. PCR reactions were conducted in triplicate in 15 μL reactions containing 1× GoTaq Green Mastermix (Promega, Madison, WI, USA), 1 mM MgCl2 and 2 pmol of each primer. The PCR amplification conditions included an initial denaturation step of 2 min at 94 °C, followed by 25 cycles of 94 °C for 45 s, 50 °C for 60 s, and 72 °C for 90 s, followed by a final extension step at 72 °C for 10 min. Triplicate reactions were subsequently combined and purified using a PCR purification column (QIAGEN, Hilden, Germany) and submitted to the DNA Technologies & Expression Analysis Core at UC Davis for sequencing on an Illumina MiSeq platform. Sequence data were quality filtered as previously described  and loaded into QIIME2 (version 2019.1)  using the default workflow. Briefly, reads were demultiplexed using barcode sequence associated with individual sample using Sabre software (sabre pe) (https://github.com/najoshi/sabre). Demultiplexed sequences were then processed through the DADA2 pipeline for quality control and the feature table was constructed at amplicon sequence variant (ASV) level. Eight samples (2 LA, 3 HA, 3 ADLIB) had < 3000 reads and were excluded from the feature table and further analysis. The sequence negative control samples averaged 133 reads per sample and were also omitted from further analysis. A naïve Bayesian pre-trained classifier for the V3-V4 region of 16S rRNA gene using the Silva 132 99% OTUs from 515F/806R region of sequences’ database was used for assigning taxonomic classifications to ASVs. The taxonomic composition of the samples was then summarised using the associated metadata. Bacterial data were retained after filtering non-bacterial taxa (e.g. eukaryotic and archaeal reads). Genus-level read abundances were analysed for alpha diversity using Shannon diversity and Chao1 index metrics with subsampling at 3000 reads per sample, and with the mean of 10 iterations used for diversity values. Data were converted into bacterial relative abundances for downstream statistical analyses.
The bacterial community composition data were analysed in R version 3.6.1  implemented in RStudio V1.2.1335 . PCoA on Bray-Curtis dissimilarity matrices  and permutation tests for homogeneity of multivariate dispersions (using 999 permutations) were conducted using the VEGAN R package . Analysis of similarities (ANOSIM)  was also performed using VEGAN R package . Permutational multivariate analysis of variance (PERMANOVA) was performed using base functions in R and then PERMANOVA. Least significant difference (LSD) post hoc analysis were performed using R package, agricolae . The R package rstatix (https://rpkgs.datanovia.com/rstatix/) was used to perform Kruskal-Wallis rank sum and Wilcoxon rank sum tests. Canonical correlation analysis was performed using the MixOmics R package  to correlate microbiome community structures with parameters associated with calf growth and performance , and faecal SCFA data. Heatmaps were generated in R. Correlation network data was visualized using the igraph package for R  and Cytoscape V3.5.1 .