The objective of this study was to evaluate the progression of the uterine microbiota from calving until establishment of metritis. Uterine swabs (n ‫؍‬ 72) collected at 0, 2, and 6 ؎ 2 days postpartum (dpp) from 12 metritic and 12 healthy cows were used for metagenomic sequencing of the 16S rRNA gene on the Illumina MiSeq platform. A heat map showed that uterine microbiota was established at calving. The microbiota changed rapidly from 0 to 6 ؎ 2 dpp, with a decrease in the abundance of Proteobacteria and an increase in the abundance of Bacteroidetes and Fusobacteria, which were dominant in metritic cows. Uterine microbiota composition was shared; however, metritic and healthy cows could be discriminated using relative abundance of bacterial genera at 0, 2, and 6 ؎ 2 dpp. Bacteroides was the main genus associated with metritis because it was the only genus that showed significantly greater abundance in cows with metritis. As the abundance of Bacteroides organisms increased, the uterine discharge score, a measure of uterine health, worsened. Fusobacterium was also an important genus associated with metritis because Fusobacterium abundance increased as Bacteroides abundance increased and the uterine discharge score worsened as the abundance increased. The correlation with uterine discharge score and the correlation with Bacteroides or Fusobacterium showed that other bacteria, such as Helcoccocus, Filifactor, and Porphyromonas, were also associated with metritis. There were also bacteria associated with uterine health, such as "Candidatus Blochmannia," Escherichia, Sneathia, and Pedobacter. Metritis is a huge concern for the dairy industry worldwide because it is highly prevalent (25 to 40%) and negatively affects the productivity, survival, and welfare of dairy cows (1). Diverse bacteria, including anaerobes and facultative anaerobes, were observed in the uteri of dairy cows within the first 2 weeks postpartum, but they were naturally cleared out within 60 days postpartum (dpp) (1). Culture-based studies observed that Escherichia coli, Trueperella pyogenes, Fusobacterium necrophorum, and Bacteroides spp. (e.g., Prevotella melaninogenica, formerly Bacteroides melaninogenicus) were commonly associated with endometritis or pyometra (1-3).Although culture-based studies have laid out the foundation of our understanding of the uterine microbiota, previous studies might have underestimated the microbial complexity of the intrauterine environment of cows postpartum, given that less than 1% of the microorganisms in many environments are readily cultured under standard laboratory conditions (4). In recent years, cultureindependent techniques such as clone library sequencing (5, 6) and pyrosequencing (7, 8) have been used to characterize the uterine microbiota of cows with metritis (5-7) and endometritis (7,8). Sequencing using the Illumina platform allows for deeper sequencing than has previously been feasible even with pyrosequencing (9). Indeed, evaluating the rarefaction curves from previous 16S rRNA sequencing studies ...

Two experiments were conducted to characterize blood concentrations of minerals and acid-base status after oral dosing of Ca salts and to determine the effects of oral Ca on mineral and metabolic status and incidence diseases. The hypotheses were that administration of oral Ca as CaCl2 and CaSO4 maintains blood total Ca (tCa) concentrations ≥2.125 mM and reduces the incidence of diseases in early lactation. In experiment 1, 18 Holstein cows on the day of calving were assigned to receive a single dose of 0, 43, or 86g of Ca as an oral bolus. Blood was sampled before and after treatments to characterize acid-base status and concentrations of minerals. In experiment 2, 450 Holstein cows considered of low (LRM; normal calving) or high risk (HRM; dystocia, twins, stillbirth, retained placenta, vulvo-vaginal laceration, or a combination of these) of metritis (primiparous-LRM=84; primiparous-HRM=84; multiparous-LRM=138; multiparous-HRM=138) on the day of calving were blocked by parity and then randomly assigned to control, no Ca supplementation; 86g of Ca on d 0 and 1 postpartum (CaS1); or 86g of Ca on d 0 and 1 postpartum followed by 43g/d on d 2 to 4 postpartum (CaS4). Blood was sampled before and 30 min after treatment on d 0, and 30 min after treatments on d 1 to 4, and d 7 and 10 for determination of concentrations of minerals and metabolites and blood acid-base responses. Disease incidence was evaluated for the first 30 DIM. Concentrations of ionized Ca (iCa) increased for 2h in cows supplemented with 43g of Ca and fewer than 8h in cows supplemented with 86g of Ca. The changes in iCa concentrations from pretreatment to 30 min after 86g of Ca supplemented on d 0 were 0.11±0.03 mM in multiparous cows and 0.25±0.03 mM in primiparous cows. Oral Ca reduced the incidence of subclinical hypocalcemia (SCH; tCa <2.125mM) in the first 4 d in the experiment (control=69.3%; CaS1=57.5%; CaS4=34.2%). Calcium supplementation decreased the prevalence of SCH on d 0 and 1 postpartum in all cows. Stopping oral Ca in CaS1 on d 1 postpartum, however, caused a rebound in SCH on d 2 to 4 postpartum in primiparous cows. Oral Ca increased the incidence of metritis (control=22.7%; CaS1=34.8%; CaS4=32.8%), primarily because of an increase in LRM primiparous cows (control=17.9%; CaS1=35.7%; CaS4=42.9%). Oral Ca increased morbidity in primiparous cows (control=38.1%; CaS1=61.8%; CaS4=60.3%) but had no effect on multiparous cows (control=38.2%; CaS1=35.1%; CaS4=30.1%). Large doses of oral Ca as salts of chloride and sulfate in the first days postpartum should be avoided in primiparous cows and used only in cows at risk of clinical hypocalcemia.

The objectives of this experiment were to determine the effects of oral Ca supplementation on milk yield, body condition, pregnancy per artificial insemination (P/AI), and days to pregnancy in Holstein cows considered to be of low (LRM; no calving assistance, live singleton without retained placenta) or high (HRM; at least one of the following: dystocia, twins, stillbirth, retained placenta, or vulvo-vaginal laceration) risk of developing metritis. The hypotheses were that oral Ca supplementation during the early postpartum period would reduce the loss of body condition and improve lactation performance and reproduction. Four hundred fifty Holstein cows were blocked by parity as primiparous cows (n=174) or multiparous cows (n=276). Within parity, a cow considered at HRM was matched with a cow of LRM and the pair was randomly assigned to control (no Ca supplementation), 86g of Ca on d 0 and 1 postpartum (CaS1), or 86g of Ca on d 0 and 1 postpartum followed by 43g/d on d 2 to 4 postpartum (CaS4). Body condition was scored at calving and 32 d postpartum and estrous cyclicity was evaluated at 38 and 52 d postpartum. Milk yield was recorded daily. Multiparous cows were classified as above or below the mean 305-d mature equivalent milk yield based on production in the previous lactation. Reproductive performance was evaluated for the first 210 d postpartum. Body condition did not differ among treatments, and cows lost on average 0.44 units of body condition in the first month of lactation. Calcium supplementation did not affect milk yield in the first 5 mo postpartum. For multiparous cows, Ca supplementation was beneficial to milk yield in the first 30 DIM in cows of greater production potential, but detrimental to multiparous cows with below average production potential. Calcium supplementation to primiparous cows reduced P/AI at first (control=55.8, CaS1=31.5, CaS4=37.0%) and all artificial inseminations (control=48.5, CaS1=34.6, CaS4=38.5%); however, Ca supplementation to multiparous cows improved P/AI at the first (control=32.1, CaS1=38.6, CaS4=41.3%) and all artificial inseminations (control=28.1, CaS1=35.3, CaS4=40.5%). These responses in P/AI to Ca supplementation resulted in extended median days to pregnancy (control=75, CaS1=100, CaS4=94 d) and smaller proportion of pregnant cows (control=89.3, CaS1=83.9, CaS4=83.9%) in primiparous cows, but shorter days to pregnancy (control=115, CaS1=94, CaS4=94 d) and increased proportion of pregnant cows in multiparous cows (control=67.0, CaS1=77.2, CaS4=74.3%). Risk of metritis depressed most measures of reproduction evaluated in the experiment. Results indicate that responses to oral Ca supplementation are conditional on parity and production potential of cows. Oral Ca supplementation was detrimental to reproduction in primiparous cows. On the other hand, Ca supplementation benefited reproduction in multiparous cows and milk yield in the cohort of multiparous cows of greater production potential.

ObjectiveThis study aimed to evaluate bacterial and host factors causing a fever in cows with metritis. For that, we investigated uterine microbiota using a metagenomic sequencing of the 16S rRNA gene (Study 1), and immune response parameters (Study 2) in metritic cows with and without a fever.Principal Findings (Study1)Bacterial communities were similar between the MNoFever and MFever groups based on distance metrics of relative abundance of bacteria. Metritic cows showed a greater prevalence of Bacteroidetes, and Bacteroides and Porphyromonas were the largest contributors to that difference. A comparison of relative abundance at the species level pointed to Bacteroides pyogenes as a fever-related species which was significantly abundant in the MFever than the MNoFever and Healthy groups; however, absolute abundance of Bacteroides pyogenes determined by droplet digital PCR (ddPCR) was similar between MFever and MNoFever groups, but higher than the Healthy group. The same trend was observed in the total number of bacteria.Principal Findings (Study2)The activity of polymorphonuclear leukocyte (PMN) and the production of TNFα, PGE2 metabolite, and PGE2 were evaluated in serum, before disease onset, at 0 and 3 DPP. Cows in the MNoFever had decreased proportion of PMN undergoing phagocytosis and oxidative burst compared with the MFever. The low PMN activity in the MNoFever was coupled with the low production of TNFα, but similar PGE2 metabolite and circulating PGE2.Conclusion/SignificanceOur study is the first to show a similar microbiome between metritic cows with and without a fever, which indicates that the host response may be more important for fever development than the microbiome. Bacteroides pyogenes was identified as an important pathogen for the development of metritis but not fever. The decreased inflammatory response may explain the lack of a febrile response in the MNoFever group.

The main objective of this study was to evaluate the efficacy of intrauterine administration of chitosan microparticles (CM) in curing metritis in dairy cows. A secondary objective was to evaluate the effects of metritis treatments on milk yield, survival, and reproductive performance. Cows with a fetid, watery, red-brownish vaginal discharge were diagnosed with metritis. Holstein cows (n = 826) with metritis from 3 dairies located in northern Florida were blocked by parity (primiparous or multiparous) and, within each block, randomly assigned to one of 3 treatments: CM (n = 276) = intrauterine infusion of 24 g of CM dissolved in 40 mL of sterile distilled water at the time of metritis diagnosis (d 0), 2 (d 2), and 4 (d 4) d later; ceftiofur (CEF; n = 275) = subcutaneous injection of 6.6 mg/ kg ceftiofur crystalline-free acid in the base of the ear at d 0 and d 3; Control (CON; n = 275) = no treatment applied at metritis diagnosis. All groups could receive escape therapy if condition worsened. Cure was considered when vaginal discharge became mucoid and not fetid. A group of nonmetritic (NMET; n = 2,436) cows was used for comparison. Data were analyzed by generalized linear mixed and Cox's proportional hazard models. Cows in CM and CON had lesser risk of metritis cure on d 12 than cows in CEF (58.6 ± 5.0 vs. 61.9 ± 4.9% vs. 77.9 ± 3.9, respectively). The proportion of cows culled within 60 days in milk (DIM) was greater for cows in CM than for cows in CEF and CON (21.5 ± 2.7 vs. 9.7 ± 1.9 vs. 11.3 ± 2.0%, respectively). Treatment did not affect rectal temperature or plasma nonesterified fatty acids, β-hydroxybutyrate, and haptoglobin concentrations. Milk yield in the first 60 DIM differed for all treatments, and it was lowest for CM (35.8 ± 0.3 kg/d), followed by CON (36.8 ± 0.3 kg/d) and CEF (37.9 ± 0.3 kg/d). The hazard of pregnancy up to 300 DIM was lesser for CM than CEF (hazard ratio = 0.62; 95% CI: 0.50-0.76), for CM than CON (hazard ratio = 0.77; 95% CI: 0.62-0.95) and for CON than CEF (hazard ratio = 0.80; 95% CI: 0.65-0.99). Culling was greater, and milk yield and fertility were lesser for CEF than NMET. In summary, CM did not improve the cure of metritis, and was detrimental to milk yield, survival, and fertility compared with CON. In contrast, CEF increased the cure of metritis, milk yield, and fertility compared with CM and CON. Finally, the negative effects of metritis on milk yield culling and fertility could not be completely reversed by CEF.