Fig 1.
Principal Coordinate Analysis (PCoA) plots for calves, grouped according to body weight, before and after exposure to stress.
(A) and (B) represent bacterial diversities in calves before and after dehorning, respectively; (C) and (D) represent bacterial diversities in calves before and after castration, respectively. Each sphere represents one sample. Red spheres represent heavy-weight and blue spheres represent light-weight calves. The input for PCoA was the OTU table containing the rarefied number of sequences observed in each OTU for each sample. The p-value obtained in Bray-Curtis distance comparisons was Bonferroni-corrected and a significance threshold of p-value < 0.05 was used in all statistical analyses.
Fig 2.
Shannon index before (Day0) and after (Day3) calves were exposed to the sham procedures or stressors (dehorning/castration).
(A) and (B), scatter plot of Shannon index (mean ± SEM) for SHM calves (grouped according to body weight) at Day 0 and Day 3 of sham-dehorning (n = 8) and sham-castration (n = 7), respectively. (C) and (D), scatter plot of Shannon index for stressed calves (grouped according to body weight) at Day 0 and Day 3 of dehorning and castration, respectively. Shannon diversity measures were compared between light and heavy calves using the Student’s T-test with a significance threshold of p-value < 0.05.
Fig 3.
Differential abundance analysis of OTUs (family-level) between heavy- and light-weight calves after dehorning (A) and castration (B).
OTU tables were rarefied to the sample containing the lowest number of sequences in each analysis. OTUs were assigned at the family level, and families with relative abundance of more than 0.1% of the total were used in DAA analysis. For multiple comparisons an estimate of the FDR was calculated to determine differentially abundant families with a significance threshold of p-value < 0.05. The families represented by the red bars are relatively more abundant in the light-weight calves; the families represented by the blue bars are relatively more abundant in the heavy-weight calves at Day 3 after dehorning (A) and castration (B).
Fig 4.
The Firmicutes: Bacteroidetes (F: B) ratio and concentration of Proteobacteria between calf groups (based on body weight) before and after exposure to stress.
The F: B ratio was determined by the ratio of total number of sequences representing phyla Firmicutes and Bacteroidetes, compared among weight groups by ANOVA with a significance threshold of p-value < 0.05. The number of sequences representing the phylum Proteobacteria in each sample was Log transformed and then compared among weight groups by ANOVA with a significance threshold of p-value < 0.05. Statistical analyses and graph preparation were done using the GraphPad Prism (Version 7.0c) software. (A), F: B ratio between heavy- and light-weight calves before (Day 0) and after (Day 3) dehorning. (B), F: B ratio between heavy- and light-weight calves before (Day 0) and after (Day 3) castration. (C), abundance of phylum Proteobacteria (Log10 number of sequences representing Proteobacteria) between heavy- and light-weight calves before (Day 0) and after (Day 3) dehorning. (D), abundance of phylum Proteobacteria (Log10 number of sequences representing Proteobacteria) between heavy- and light-weight calves before (Day 0) and after (Day 3) castration.