Integrating uterine microbiome and metabolome to advance the understanding of the uterine environment in dairy cows with metritis.

BACKGROUND: Metritis is a prevalent uterine disease that affects the welfare, fertility, and survival of dairy cows. The uterine microbiome from cows that develop metritis and those that remain healthy do not differ from calving until 2 days postpartum, after which there is a dysbiosis of the uterine microbiome characterized by a shift towards opportunistic pathogens such as Fusobacteriota and Bacteroidota. Whether these opportunistic pathogens proliferate and overtake the uterine commensals could be determined by the type of substrates present in the uterus. The objective of this study was to integrate uterine microbiome and metabolome data to advance the understanding of the uterine environment in dairy cows that develop metritis. Holstein cows (n = 104) had uterine fluid collected at calving and at the day of metritis diagnosis. Cows with metritis (n = 52) were paired with cows without metritis (n = 52) based on days after calving. First, the uterine microbiome and metabolome were evaluated individually, and then integrated using network analyses. RESULTS: The uterine microbiome did not differ at calving but differed on the day of metritis diagnosis between cows with and without metritis. The uterine metabolome differed both at calving and on the day of metritis diagnosis between cows that did and did not develop metritis. Omics integration was performed between 6 significant bacteria genera and 153 significant metabolites on the day of metritis diagnosis. Integration was not performed at calving because there were no significant differences in the uterine microbiome. A total of 3 bacteria genera (i.e. Fusobacterium, Porphyromonas, and Bacteroides) were strongly correlated with 49 metabolites on the day of metritis diagnosis. Seven of the significant metabolites at calving were among the 49 metabolites strongly correlated with opportunistic pathogenic bacteria on the day of metritis diagnosis. The main metabolites have been associated with attenuation of biofilm formation by commensal bacteria, opportunistic pathogenic bacteria overgrowth, tissue damage and inflammation, immune evasion, and immune dysregulation. CONCLUSIONS: The data integration presented herein helps advance the understanding of the uterine environment in dairy cows with metritis. The identified metabolites may provide a competitive advantage to the main uterine pathogens Fusobacterium, Porphyromonas and Bacteroides, and may be promising targets for future interventions aiming to reduce opportunistic pathogenic bacteria growth in the uterus.

Evaluating differences in milk production, reproductive performance, and survival associated with vaginal discharge characteristics and fever in postpartum dairy cows.

The objective was to assess differences in productive and reproductive performance, and survival associated with vaginal discharge characteristics and fever in postpartum dairy cows located in Western and Southern states of the U.S.A. This retrospective cohort study included data from 3 experiments conducted in 9 dairies. Vaginal discharge was evaluated twice within 12 DIM and scored on a 5-point scale. The highest score observed for each cow was used for group assignment (VD group) as follows: VD 1 and 2 (VD 1/2; n = 1,174) = clear mucus/lochia with or without flecks of pus; VD 3 (n = 1,802) = mucopurulent with < 50% pus; VD 4 (n = 1,643) = mucopurulent with ≥50% of pus or non-fetid reddish/brownish mucous, n = 1,643; VD 5 = fetid, watery, and reddish/brownish, n = 1,800. All VD 5 cows received treatment according to each herd's protocol. Rectal temperature was assessed in a subset of VD 5 cows, and subsequently divided into Fever (rectal temperature ≥39.5°C; n = 334) and NoFever (n = 558) groups. A smaller proportion of cows with VD 5 (67.6%) resumed ovarian cyclicity compared with VD 1/2 (76.2%) and VD 4 (72.9%) cows; however, a similar proportion of VD5 and VD 3 (72.6%) cows resumed ovarian cyclicity. A smaller proportion of VD 5 (85.8%) cows received at least one artificial insemination (AI) compared with VD 1/2 (91.5%), VD 3 (91.0%), or VD 4 (91.6%) cows. Although we did not detect differences in pregnancy at first AI according to VD, fewer cows with VD 5 (64.4%) were pregnant at 300 DIM than cows with VD 1/2 (76.5%), VD 3 (76.2%), or VD 4 (74.7%). Hazard of pregnancy by 300 DIM was smaller for VD 5 compared with VD 1/2, VD 3, or VD 4 cows. A greater proportion of VD 5 cows were removed from the herd within 300 DIM compared with other VD groups. There was 760 kg lesser milk production within 300 DIM for VD 5 compared with VD 2, VD 3, and VD 4, whereas VD 2, VD 3, and VD 4 had similar milk production. We did not detect an association between fever at diagnosis of VD 5 and reproductive performance or milk production. A greater proportion of VD 5 cows without fever were removed from the herd by 300 DIM compared with VD 5 cows with fever. Differences in productive and reproductive performance, and removal of the herd were restricted to fetid, watery, and reddish/brownish vaginal discharge, which was independent of fever.

Association between prepartum body condition score and prepartum and postpartum dry matter intake and energy balance in multiparous Holstein cows.

The objectives of this retrospective observational study were to investigate the association between body condition score (BCS) at 21 d before calving with prepartum and postpartum dry matter intake (DMI), energy balance (EB), and milk yield. Data from 427 multigravid cows from 11 different experiments conducted at the University of Florida were used. Cows were classified according to their BCS at 21 d before calving as FAT (BCS ≥4.00; n = 83), MOD (BCS 3.25 to 3.75; n = 287), and THIN (BCS ≤3.00; n = 57). Daily DMI from -21 to -1 and from +1 to +28 DIM was individually recorded. Energy balance was calculated as the difference between net energy for lactation consumed and required. Dry matter intake in FAT cows was lesser than in MOD and THIN cows both prepartum (FAT = 9.97 ± 0.21, MOD = 11.15 ± 0.14, THIN = 11.92 ± 0.22 kg/d) and postpartum (FAT = 14.35 ± 0.49, MOD = 15.47 ± 0.38, THIN = 16.09 ± 0.47 kg/d). Dry matter intake was also lesser for MOD cows compared with THIN cows prepartum, but not postpartum. Energy balance in FAT cows was lesser than in MOD and THIN cows both prepartum (FAT = -4.16 ± 0.61, MOD = -1.20 ± 0.56, THIN = 0.88 ± 0.62 Mcal/d) and postpartum (FAT = -12.77 ± 0.50, MOD = -10.13 ± 0.29, THIN = -6.14 ± 0.51 Mcal/d). Energy balance was also lesser for MOD cows compared with THIN cows both prepartum and postpartum. There was a quadratic association between BCS at 21 d before calving and milk yield. Increasing BCS from 2.5 to 3.5 was associated with an increase in daily milk yield of 6.0 kg and 28 d cumulative milk of 147 kg. Increasing BCS from 3.5 to 4.5 was associated with a decrease in daily milk yield of 4.4 kg and 28 d cumulative milk of 116 kg. In summary, a moderated BCS at 21 d before calving was associated with intermediate DMI and EB pre- and postpartum but greater milk yield compared with thinner and fatter cows. Our findings indicate that a moderated BCS is ideal for ensuring a successful lactation.

Blood metabolomics and impacted cellular mechanisms during transition into lactation in dairy cows that develop metritis.

The objective of this study was to identify metabolites associated with metritis and use them for identification of cellular mechanisms affected during transition into lactation. Holstein cows (n = 104) had blood collected in the prepartum period (d -14 ± 6 relative to calving), at calving (d 0), and at the day of metritis diagnosis (d 7 ± 2 after calving). Cows with reddish or brownish, watery, and fetid discharge were diagnosed with metritis (n = 52). Cows with metritis were paired with herdmates without metritis (n = 52) based on days in milk. The metabolome of plasma samples was evaluated using untargeted gas chromatography time-of-flight mass spectrometry. Univariate analyses included t-tests and fold change analyses. Metabolites with false discovery rate adjusted P ≤ 0.10 on t-tests were used for partial least squares discriminant analysis coupled with permutational analysis using 2,000 permutations. Metabolites with false discovery rate adjusted P ≤ 0.10 on t-tests were also used for enriched pathway analyses and identification of cellular processes. Cows that developed metritis had affected cellular processes associated with lower amino acid metabolism in the prepartum period, greater lipolysis, cell death, and oxidative stress at calving and at metritis diagnosis, and greater leukocyte activation at calving, but lower immune cell activation at metritis diagnosis. In summary, cows that developed metritis had plasma metabolomic changes associated with greater lipolysis, oxidative stress, and a dysregulated immune response which may predispose cows to metritis development.

Unraveling the immune and metabolic changes associated with metritis in dairy cows.

The objective was to unravel the peripartum immune and metabolic changes associated with metritis in Holstein cows. Holstein cows (n = 128) had blood collected at -14, 0, 3, and 7 d relative to parturition (DRP). Flow cytometry was used to evaluate blood leukocyte counts, proportions, and activation. Total cells, live cells, single cells, monocytes (CD172α+/CD14+), polymorphonuclears (CD172α+/CD14-/SSChigh), B-cells (CD21+/MHCII+), CD4+ T-cells (CD4+), CD8+ T-cells (CD8+), and γδ T-cells (γδTCR+) were evaluated. Both CD62L and CD11b were used as markers of cell activation. Major histocompatibility complex class II was used as a marker of antigen presentation in monocytes. A Milliplex Bovine Cytokine/Chemokine 08-plex kit was used to evaluate plasma concentrations of IFN-γ, IL-1α, IL-1ß, IL-4, IL-6, IL-8, IL-10, and tumor necrosis factor-α. The body weight (BW) change prepartum was calculated as the difference between calving BW and prepartum BW divided by the number of days between measurements. Plasma fatty acids (FA) were measured at -14 and 0 DRP using untargeted gas chromatography with time-of-flight mass spectrometry. Data were analyzed by ANOVA for repeated measures. Cows that developed metritis (n = 57) had greater prepartum BW, prepartum BW loss, and greater FA concentrations at calving. Plasma FA at calving was positively correlated with IL-1ß. Cows that developed metritis had persistent systemic inflammation, which was demonstrated by greater B-cell activation, greater pro-inflammatory cytokine concentrations, and greater cell damage pre- and postpartum. Postpartum, we observed greater polymorphonuclear cell activation and extravasation but lesser monocytes and CD4+ T-cells activation and extravasation, which suggests postpartum immune tolerance. Greater prepartum adiposity in cows that developed metritis may lead to systemic inflammation pre- and postpartum and immune tolerance postpartum, which may lead to failure to prevent bacterial infection, and development of puerperal metritis.

Flow cytometry panels for immunophenotyping dairy cattle peripheral blood leukocytes.

Many aspects of the bovine immune system remain poorly characterized, which poses an obstacle to improving dairy cow health. Herein, we describe two flow cytometry panels that included antibodies against CD8α, CD4, TCR-δ, CD172α, CD14, MHCII, CD21, CD62L, and CD11b. These panels were used to characterize the phenotype of leukocyte subpopulations from the peripheral blood of 30-day old Holstein calves and Holstein cows at 260 d of gestation and calving. No leukocyte subset differences were found between the pre- and post-partum cows. However, calf leukocytes presented a higher proportion of CD3+ lymphocytes, γδ T-cells, CD8+ γδ T-cells, and monocytes when compared with mature cows. Conversely, cow lymphocytes had a higher proportion of CD4+ and CD8+ T-cells, and B-cells than calf lymphocytes. The proportion of CD4+ T-cells and B-cells expressing CD62L was greater in calves than in cows, while cow B-cells expressed greater levels of CD11b than calf B-cells. In contrast, calf polymorphonuclear cells (PMN) and monocytes expressed greater levels of CD11b compared to cows. Moreover, calf monocytes expressed higher levels of MHCII compared with those of cows. Collectively, our data provides a resource to better understand the bovine immune system as well as immune-related diseases that affect dairy cattle.