Improved uterine immune mediators in Holstein cows supplemented with rumen-protected methionine and discovery of neutrophil extracellular traps (NET)

S. L. Stella, D. A. Velasco-Acosta, C. Skenandore, Z. Zhou, A. Steelman, D. Luchini, F. C. Cardoso

Research output: Contribution to journalArticlepeer-review


During the transition from prepartum to early lactation, dairy cows often experience negative energy balance (NEB) that may result in reproductive stress and decreased fertility. The objective of this study was to observe the effects of rumen-protected methionine (RPM) on plasma amino acid concentrations, uterine cytology, immunohistochemistry (IHC) of glutathione peroxidase 1 (GPX) and superoxide dismutase 1 (SOD), and to confirm neutrophil extracellular trap (NET) formation. Multiparous Holstein cows (n = 20) were randomly assigned to two treatments starting at 21 d before calving until 73 days in milk (DIM). Treatments were: CON (n = 9, no supplementation, TMR with a Lys:Met = 3.5:1) and MET (n = 11, TMR + Smartamine® M with a Lys:Met = 2.8:1). Uterine endometrial biopsies, uterine cytology, and blood samples from the coccygeal artery or vein were collected at 15, 30, and 73 DIM. Blood plasma samples were analyzed for amino acids and metabolites. Uterine biopsies were analyzed for NET formation, neutrophil numbers, as well as GPX and SOD by IHC. Additionally, uterine cytology was analyzed for polymorphonuclear neutrophil (PMN) to epithelial cell percentage. Cows in CON had lower methionine plasma concentrations (18.05 ± 2.0 μM) than cows in MET (30.39 ± 1.6 μM). Cows in CON had greater cystine plasma concentrations (3.62 ± 0.3 μM) than cows in MET (2.8 ± 0.3 μM). No treatment differences were observed for SOD or GPX in the endometrium. Cows in CON tended to have a high score for positively immunolabeled GPX cells at 15 DIM than cows in MET. No treatment differences were observed for the percentage of PMN in uterine cytology, number of neutrophils, or extent of NET formation in the endometrium. A treatment by time interaction was observed for PMN percentage and the number of neutrophils: cows in MET tended to have greater PMN percentages than cows in CON at 15 DIM which decreased for subsequent days and cows in MET had greater neutrophil numbers in the endometrium at 30 DIM than cows in CON. In conclusion, dietary supplementation of RPM altered plasma amino acid concentrations and increased neutrophil infiltration in the postpartum period, suggesting improved uterine immunity.

Original languageEnglish (US)
Pages (from-to)116-125
Number of pages10
StatePublished - Jul 1 2018


  • Immunohistochemistry
  • Methionine
  • Neutrophil extracellular trap
  • PMN

ASJC Scopus subject areas

  • Small Animals
  • Food Animals
  • Animal Science and Zoology
  • Equine


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