Whole-body microbiota of newborn calves and their response to prenatal vitamin and mineral supplementation

Abstract

Early life microbial colonization and factors affecting colonization patterns are gaining interest due to recent developments suggesting that early life microbiome may play a role in Developmental Origins of Health and Disease. In cattle, limited information exists on the early microbial colonization of anatomical sites involved in bovine health beyond the gastrointestinal tract. Here, we investigated 1) the initial microbial colonization of seven different anatomical locations in newborn calves and 2) whether these early life microbial communities and 3) serum cytokine profiles are influenced by prenatal vitamin and mineral (VTM) supplementation. Samples were collected from the hoof, liver, lung, nasal cavity, eye, rumen (tissue and fluid), and vagina of beef calves that were born from dams that either received or did not receive VTM supplementation throughout gestation (n = 7/group). Calves were separated from dams immediately after birth and fed commercial colostrum and milk replacer until euthanasia at 30 h post-initial colostrum feeding. The microbiota of all samples was assessed using 16S rRNA gene sequencing and qPCR. Calf serum was subjected to multiplex quantification of 15 bovine cytokines and chemokines. Our results indicated that the hoof, eye, liver, lung, nasal cavity, and vagina of newborn calves were colonized by site-specific microbiota, whose community structure differed from the ruminal-associated communities (0.64 ≥ R² ≥ 0.12, p ≤ 0.003). The ruminal fluid microbial community was the only one that differed by treatment (p < 0.01). However, differences (p < 0.05) by treatment were detected in microbial richness (vagina); diversity (ruminal tissue, fluid, and eye); composition at the phylum and genus level (ruminal tissue, fluid, and vagina); and in total bacterial abundance (eye and vagina). From serum cytokines evaluated, concentration of chemokine IP-10 was greater (p = 0.02) in VTM calves compared to control calves. Overall, our results suggest that upon birth, the whole-body of newborn calves are colonized by relatively rich, diverse, and site-specific bacterial communities. Noticeable differences were observed in ruminal, vaginal, and ocular microbiota of newborn calves in response to prenatal VTM supplementation. These findings can derive future hypotheses regarding the initial microbial colonization of different body sites, and on maternal micronutrient consumption as a factor that may influence early life microbial colonization.

Keywords: bovine; maternal; newborn; prenatal; vitamin and mineral supplementation; whole-body microbiota.

Figure 1

(A) Schematic overview of sampling regimen. Calves were born out of two groups of dams; one group was fed a basal diet plus commercial vitamin and mineral supplement (VTM) and the other was fed only the basal diet (CON) (n = 7/group). Newborn calves from each treatment group were sampled at 30-h after initial colostrum feeding. Samples were collected from each newborn for microbial characterization and consisted of hoof swabs, liver tissue, lung tissue, nasal cavity swabs, ocular swabs, ruminal fluid, ruminal tissue, and vaginal swabs. Created with BioRender.com. (B–G) Hoof microbiota of newborn calves born from dams received either vitamin and mineral (VTM) supplementation or no VTM supplementation received (CON) sampled at 30-h post initial colostrum feeding. (B) Non-metric multidimensional scaling (NMDS) plots of the Bray-Curtis dissimilarities of hoof swab samples. (C) Observed amplicon sequence variants (ASVs) of hoof swab samples. (D) Shannon diversity index of hoof swab samples. (E) Inverse Simpson diversity index of hoof swab samples. (F) Relative abundance (%) of top 7 most relatively abundant bacterial phyla of hoof swab samples; (G) Total bacterial abundance estimated by qPCR of hoof swab samples.

Figure 2

Liver tissue microbiota of newborn calves born from dams received either vitamin and mineral (VTM) supplementation or no VTM supplementation received (CON) sampled at 30-h after initial colostrum feeding (n = 7/group). (A) Non-metric multidimensional scaling (NMDS) plots of the Bray-Curtis dissimilarities. (B) Observed amplicon sequence variants (ASVs). (C) Shannon diversity index. (D) Inverse Simpson diversity index. (E) Relative abundance (%) of top 7 most relatively abundant bacterial phyla. (F) Total bacterial abundance estimated by qPCR.

Figure 3

Respiratory microbiota [Nasal (A–F) and lung (G–L) microbiota] of newborn calves born from dams received either vitamin and mineral (VTM) supplementation or no VTM supplementation received (CON) sampled at 30-h after initial colostrum feeding (n = 7/group). (A) Non-metric multidimensional scaling (NMDS) plots of the Bray-Curtis dissimilarities of nasal swab samples. (B) Observed amplicon sequence variants (ASVs) of nasal swab samples. (C) Shannon diversity index of nasal swab samples. (D) Inverse Simpson diversity index of nasal swab samples. (E) Relative abundance (%) of top 7 most relatively abundant bacterial phyla of nasal swab samples. (F) Total bacterial abundance estimated by qPCR of nasal swab samples. (G) Non-metric multidimensional scaling (NMDS) plots of the Bray-Curtis dissimilarities of lung tissue samples. (H) Observed amplicon sequence variants (ASVs) of lung tissue samples. (I) Shannon diversity index of lung tissue samples. (J) Inverse Simpson diversity index of lung tissue samples. (K) Relative abundance (%) of top 7 most relatively abundant bacterial phyla of lung tissue samples. (L) Total bacterial abundance estimated by qPCR of lung tissue samples.

Figure 4

Ruminal microbiota [Ruminal fluid (A–F) and ruminal tissue (G–L) microbiota] of newborn calves born from dams received either vitamin and mineral (VTM) supplementation or no VTM supplementation received (CON) sampled at 30-h after initial colostrum feeding (n = 7/group). (A) Non-metric multidimensional scaling (NMDS) plots of the Bray-Curtis dissimilarities of ruminal fluid samples. (B) Observed amplicon sequence variants (ASVs) of ruminal fluid samples. (C) Shannon diversity index of ruminal fluid samples. (D) Inverse Simpson diversity index of ruminal fluid samples. (E) Relative abundance (%) of top 7 most relatively abundant bacterial phyla of ruminal fluid samples. (F) Total bacterial abundance estimated by qPCR of ruminal fluid samples. (G) Non-metric multidimensional scaling (NMDS) plots of the Bray-Curtis dissimilarities of ruminal tissue samples. (H) Observed amplicon sequence variants (ASVs) of ruminal tissue samples. (I) Shannon diversity index of ruminal tissue samples. (J) Inverse Simpson diversity index of ruminal tissue samples. (K) Relative abundance (%) of top 7 most relatively abundant bacterial phyla of ruminal tissue samples. (L) Total bacterial abundance estimated by qPCR of ruminal tissue samples.

Figure 5

Bacterial genera that were differentially abundant in ruminal and vaginal microbiota between VTM and control calves (n = 7/group).

Figure 6

Ocular microbiota of newborn calves born from dams received either vitamin and mineral (VTM) supplementation or no VTM supplementation received (CON) sampled at 30-h after initial colostrum feeding (n = 7/group). (A) Non-metric multidimensional scaling (NMDS) plots of the Bray-Curtis dissimilarities. (B) Observed amplicon sequence variants (ASVs). (C) Shannon diversity index. (D) Inverse Simpson diversity index. (E) Relative abundance (%) of top 7 most relatively abundant bacterial phyla. (F) Total bacterial abundance estimated by qPCR.

Figure 7

Vaginal microbiota of newborn calves born from dams received either vitamin and mineral (VTM) supplementation or no VTM supplementation received (CON) sampled at 30-h after initial colostrum feeding (n = 7/group). (A) Non-metric multidimensional scaling (NMDS) plots of the Bray-Curtis dissimilarities. (B) Observed amplicon sequence variants (ASVs). (C) Shannon diversity index. (D) Inverse Simpson diversity index. (E) Relative abundance (%) of top 7 most relatively abundant bacterial phyla. (F) Total bacterial abundance estimated by qPCR.

Figure 8

Comparison of the overall microbial community structure, richness, diversity, composition, and total microbial abundance of microbiota associated with 8 different newborn calf body sites (N = 14). (A) Non-metric multidimensional scaling (NMDS) plots of the Bray-Curtis dissimilarities. (B) Overall observed amplicon sequence variants (ASVs). (C) Overall Shannon diversity index. (D) Total bacterial abundance estimated by qPCR. (E) Relative abundance (%) of most relatively abundant bacterial phyla.

Figure 9

Bacterial genera that were differentially abundant between different sampling sites (N = 14 per sample type). ND denotes not detected; Different lowercase letters indicate significantly different means (p < 0.05).

Figure 10

Observed cytokine concentration of newborn calves born from dams received either vitamin and mineral (VTM) supplementation or no VTM supplementation received (CON) sampled at 32 h after birth (n = 7/group).