Milk microbiome in the first month of lactation and at weaning from ewes supplemented with zinc pre- and postpartum

Authors: KNUTH, RYAN - University Of Wyoming; PAGE, CHAD - University Of Wyoming; STEWART, WHITNEY - University Of Wyoming; HUMMEL, GWENDOLYNN - University Of Wyoming; WOODRUFF, KELLY - University Of Wyoming; WHALEY, JAELYN - University Of Wyoming; SPRINGER, ALEXIS - University Of Wyoming; AUSTIN, KATHLEEN - University Of Wyoming; Murphy, Thomas - Tom; BISHA, BLEDAR - University Of Wyoming; CUNNINGHAM-HOLLINGER, HANNAH - University Of Wyoming

Submitted to: Journal of Animal Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/16/2024
Publication Date: 6/12/2024
Citation: Knuth, R.M., Page, C.M., Stewart, W.C., Hummel, G.L., Woodruff, K.L., Whaley, J.R., Springer, A.L., Austin, K.J., Murphy, T.W., Bisha, B., Cunningham-Hollinger, H.C. 2024. Milk microbiome in the first month of lactation and at weaning from ewes supplemented with zinc pre- and postpartum. Journal of Animal Science. 102. Article skae163. https://doi.org/10.1093/jas/skae163.
DOI: https://doi.org/10.1093/jas/skae163

Interpretive Summary: Mastitis is an important disease with economic and welfare implications in both clinical and subclinical states. This research described the microbial diversity and taxonomy of milk collected from clinically healthy Rambouillet (WF; n = 9) and Hampshire (BF; n = 4) primiparous ewes in a longitudinal study involving differing dietary zinc concentrations [1× National Academics of Sciences, Engineering, and Medicine (NASEM) recommendations, CON; 3× NASEM recommendations, ZnTRT]. Milk was collected weekly during the first 3 wk of lactation and at weaning, and somatic cell counts (SCC) were classed (low, medium, high). Mastitis pathogens were among the most relatively abundant via amplicon sequencing, including Staphylococcus, Mannheimia, Corynebacterium, and Pseudomonas. Breed, Zn treatment, and SCC class effects on milk microbiome a-diversity and ß-diversity changes and taxonomy were assessed. These effects and their two-way interactions were limited but variable in early lactation samples and not evident in weaning samples. Notably, BF ewe milk samples had increased Faith’s phylogenetic diversity and increased Shannon’s entropy during early lactation, and CON milk samples had a reduced rate of compositional change than ZnTRT samples. These results support the existence of a milk microbiome that is variably affected by breed, increased dietary zinc concentrations, and SCC class.

Technical Abstract: Mastitis is an important disease with economic and welfare implications in both clinical and subclinical states. The aim of this research was to sequence the hypervariable V4 region of the 16S rRNA gene to describe the microbial diversity and taxonomy of milk from clinically healthy ewes (Rambouillet, WF = 9; Hampshire, BF = 4). Experimental ewes represented a subset of a larger study assessing the impacts of divergent dietary zinc (Zn) concentrations [1× National Academics of Sciences, Engineering, and Medicine (NASEM) recommendations = CON or 3× NASEM recommendations = ZnTRT] throughout late gestation and lactation. Milk was collected at four periods during early lactation (18 – 24 h, 7 d, 14 d, and 21 d postpartum) and at weaning (84 ± 14 d postpartum). Somatic cell counts (SCC) were quantified, averaged, and classed (low: < 500 × 103; medium: 500 × 103 – 100 × 104; high: > 100 × 104 cells/mL). Milk samples (n = 67) were sequenced to identify bacteria and archaea; the most abundant phyla were Actinobacteria, Bacteroidetes, Cyanobacteria, Euryarchaeota, Firmicutes, Fusobacteria, Lentisphaerae, Proteobacteria, Spirochaetes, Tenericutes, Saccharibacteria TM7, and Verrucomicrobia. Mastitis pathogens were among the most relatively abundant genera, including Staphylococcus, Mannheimia, Corynebacterium, and Pseudomonas. Effects of breed, dietary Zn concentration, SCC class, and their two-way interactions on milk microbiome diversity and taxonomy were assessed within early lactation (using a repeated measures model) and weaning samples. Alpha-diversity metrics included Pielou’s evenness, Faith’s phylogenetic diversity, and Shannon’s entropy indices. The main and interactive effects between Zn treatment, breed, SCC class, and period were variable in early lactation and not evident in weaning samples. Milk from BF ewes had increased Faith’s phylogenetic diversity and Shannon’s entropy, and differed in unweighted UniFrac composition (P = 0.10). Milk from CON ewes had a reduced rate of composition change through early lactation (P = 0.02) indicating greater microbiome stability than ZnTRT ewe milk. These results support that milk is not sterile, and breed, dietary Zn concentration, and SCC class variably affect the milk microbiome. Findings from the current study provide important foundational insights into the effects of increased dietary Zn supplementation on longitudinal changes in the milk microbiome and associations with mammary gland health and mastitis.