Metritis and the uterine disease microbiome are associated with long-term changes in the endometrium of dairy cows†.

Cows with metritis (uterine disease) during the first 1 to 2 weeks postpartum have lower pregnancy rates when inseminated later postpartum (typically >10 weeks). We hypothesized that metritis and the disease-associated uterine microbiome have a long-term effect on endometrial gene expression. Changes in gene expression may inform a mechanism through which disease lowers pregnancy rates. A total of 20 cows were enrolled at 1 to 2 weeks postpartum to either metritis (clinical disease; n = 10) or healthy (control; n = 10) groups and randomly assigned to be slaughtered at approximately 80 and 165 dpp (mid-lactation). The microbiome of the reproductive tract was sampled to confirm the presence of pathogens that are typical of metritis. In addition to the original clinical diagnosis, study cows were retrospectively assigned to uterine-disease and control groups based on the composition of their microbiome. There was no effect of early postpartum uterine disease on the uterine microbiome at mid-lactation (time of slaughter). Nonetheless, early postpartum metritis and the disease microbiome were associated with a large number of differentially-expressed genes at mid-lactation primarily in the caruncular compared with the inter-caruncular endometrium. Gene enrichment analysis identified oxidative phosphorylation as the primary pathway increased in caruncular endometrium of diseased cows whereas growth factor signaling pathways were reduced. The current study demonstrated that metritis and a uterine disease microbiome leave a sustained imprint on gene expression in the caruncular endometrium that may explain lower fertility in cows with postpartum uterine disease.

Evaluation of cervical and uterine size, at 4 weeks postpartum, as a predictor of subsequent fertility in Jersey cattle.

Uterine and cervical size of Holstein dairy cows is reported among reasons for a decline in dairy cow fertility. Therefore, the objectives of this study were to (a) determine whether size of the cervix and uterus at 4 weeks postpartum impacted subsequent fertility at first service in Jersey cattle, (b) determine whether progesterone level at 4 weeks postpartum impacted cyclicity and (c) the association of the presence of corpus luteum and uterus and cervix size. Body condition scores at calving, presence of postpartum diseases, parity number and milk weights were taken from lactating Jersey dairy cows (N = 147) for 28 days postpartum. During the fourth week postpartum, a blood sample was obtained for progesterone concentration, and transrectal ultrasonography was performed by a high-resolution ultrasound machine to determine cervical and uterine horn diameter, as well as ovarian structures measurements. Correcting for parity number, BCS at calving, presence of diseases and milk yield, cows with a cervix >2.54 ± 0.63 cm and uterine horn >2.25 ± 0.59 cm were less likely to become pregnant at first service (p = .04 and p = .003, respectively). The cows with larger cervix had a trend to be less likely to have a corpus luteum present at the 4th week of lactation (p = .067). Cows with larger uterine horn size were less likely to have a corpus luteum present at the 4th week of lactation (p = .015). It is concluded that a larger cervix and/or uterus during the postpartum was associated negatively with fertility and cyclicity in Jersey cows.

Opportunities and limitations of milk mid-infrared spectra-based estimation of acetone and ß-hydroxybutyrate for the prediction of metabolic stress and ketosis in dairy cows.

Subclinical (SCK) and clinical (CK) ketosis are metabolic disorders responsible for big losses in dairy production. Although Fourier-transform mid-infrared spectrometry (FTIR) to predict ketosis in cows exposed to great metabolic stress was studied extensively, little is known about its suitability in predicting hyperketonemia using individual samples, e.g. in small dairy herds or when only few animals are at risk of ketosis. The objective of the present research was to determine the applicability of milk metabolites predicted by FTIR spectrometry in the individual screening for ketosis. In experiment 1, blood and milk samples were taken every two weeks after calving from Holstein (n = 80), Brown Swiss (n = 72) and Swiss Fleckvieh (n = 58) cows. In experiment 2, cows diagnosed with CK (n = 474) and 420 samples with blood ß-hydroxybutyrate [BHB] <1.0 mmol/l were used to investigate if CK could be detected by FTIR-predicted BHB and acetone from a preceding milk control. In experiment 3, correlations between data from an in farm automatic milk analyser and FTIR-predicted BHB and acetone from the monthly milk controls were evaluated. Hyperketonemia occurred in majority during the first eight weeks of lactation. Correlations between blood BHB and FTIR-predicted BHB and acetone were low (r = 0.37 and 0.12, respectively, P < 0.0001), as well as the percentage of true positive values (11.9 and 16.6%, respectively). No association of FTIR predicted ketone bodies with the interval of milk sampling relative to CK diagnosis was found. Data obtained from the automatic milk analyser were moderately correlated with the same day FTIR-predicted BHB analysis (r = 0.61). In conclusion, the low correlations with blood BHB and the small number of true positive samples discourage the use of milk mid-infrared spectrometry analyses as the only method to predict hyperketonemia at the individual cow level.

The microbiome of the pregnant uterus in Holstein dairy heifers and cows assessed by bacterial culture and 16S ribosomal RNA gene sequencing.

Introduction: The possibility that there is a resident and stable commensal microbiome within the pregnant uterus has been supported and refuted by a series of recent studies. One element of most of the initial studies was that they were based primarily on 16S rRNA gene sequencing from bacteria. To account for this limitation, the current study performed both bacterial culture and 16S rRNA gene sequencing in a side-by-side manner (e.g., same tissues isolated from the same animal). Methods: The uteruses of 10 mid-pregnant (156 ± 5 d of gestation) Holstein heifers and cows were collected following slaughter. The external surface of the reproductive tract (positive control for contamination during tissue collection) as well as tissues within the pregnant uterus (placentome, inter-cotyledonary placenta, inter-caruncular endometrium, amnionic fluid, allantoic fluid, fetal abomasum content, and fetal meconium) were sampled for bacterial culture and 16S rRNA gene sequencing. Results: There were 87 unique bacterial species cultured from the external surface of the pregnant reproductive tract (contamination control) and 12 bacterial species cultured from pregnancy tissues. Six out of 10 cattle (60%) exhibited bacterial growth in at least one location within the pregnant uterus. For the metataxonomic results (16S rRNA gene sequencing), a low targeted microbial biomass was identified. Analyses of the detected amplicon sequence variants (ASV) revealed that there were: (1) genera that were prevalent on both the external surface and within the pregnant uterus; (2) genera that were prevalent on the external surface but either not detected or had very low prevalence within the pregnant uterus; and (3) genera that were either not detected or had low prevalence on the external surface but found with relatively high prevalence within the pregnant uterus. Conclusion: There are a small number of viable bacteria in the pregnant uterus. The 16S rRNA gene sequencing detected a microbial community within the pregnant uterus but with a low biomass. These results are consistent with recent studies of the pregnant bovine uterus and leave open the question of whether there is adequate microbial mass to significantly affect the biology of the normal healthy bovine pregnancy.

Systemic antibiotic treatment of cows with metritis early postpartum does not change the progression of uterine disease or the uterine microbiome at 1 month postpartum.

Background: Postpartum uterine disease (metritis) is common in dairy cows. The disease develops within 1 week after calving and is associated with microbial dysbiosis, fever, and fetid uterine discharge. Cows with metritis have a greater likelihood of developing endometritis and infertility later postpartum. Antibiotic treatment is used to relieve symptoms of metritis but the capacity of antibiotic treatment to improve fertility later postpartum is inconsistent across published studies. We hypothesized that an antibiotic has only a short-term effect on the uterine microbiome and does not change the progression of disease from metritis to endometritis. To test this hypothesis, we studied the effects of systemic antibiotic given to cows diagnosed with metritis and healthy cows early postpartum on the development of endometritis and the uterine microbiome at 1 month postpartum. Results: Cows diagnosed with metritis were compared to healthy ones in a 2 × 2 factorial design, where they were either treated with an antibiotic (ceftiofur hydrochloride) at 7 to 10 days postpartum or left untreated. Cows were slaughtered at one month postpartum and the uterus was assessed for endometritis (presence of purulent material in the uterine lumen and inflammation in the endometrium) and uterine samples were collected for bacteriology and metagenomics (16S rRNA gene sequencing). As expected, the uterine microbiome at disease diagnosis had dysbiosis of typical metritis pathogens (e.g., Fusobacterium, Bacteroides, and Porphyromonas) in diseased compared with healthy cows. At one month postpartum, there was a tendency for more endometritis in metritis cows compared with healthy but antibiotic treatment had no effect on endometritis prevalence regardless of the original disease diagnosis. Likewise, when bacteria were cultured or sequenced, there were a greater number of species (culture) or amplicon sequence variants (ASV; sequencing) in the uterine lumen of cows with metritis. However, antibiotic treatment had no effect on the prevalence of cultured species or the composition of the detected ASV. The uterine microbiome at 1 month postpartum was associated with the clinical observation of the uterus (endometritis or healthy). Conclusions: Early postpartum antibiotic treatment only provides temporary resolution of uterine dysbiosis that is not sustained long-term. Failure to resolve the dysbiosis is associated with a greater prevalence of endometritis in cows with metritis, and the occurrence of endometritis significantly impacts fertility later postpartum.

Establishment of the uterine microbiome following artificial insemination in virgin heifers.

Introduction: The concept of a sterile uterus was challenged by recent studies that have described the microbiome of the virgin and pregnant uterus for species including humans and cattle. We designed two studies that tested whether the microbiome is introduced into the uterus when the virgin heifer is first inseminated and whether the origin of the microbiome is the vagina/cervix. Methods: The uterine microbiome was measured immediately before and after an artificial insemination (AI; Study 1; n = 7 AI and n = 6 control) and 14 d after insemination (Study 2; n = 12 AI and n = 12 control) in AI and non-AI (control) Holstein heifers. A third study (Study 3; n = 5 Holstein heifers) that included additional negative controls was subsequently conducted to support the presence of a unique microbiome within the uterus despite the low microbial biomass and regardless of insemination. Traditional bacteriological culture was performed in addition to 16S rRNA gene sequencing on the same samples to determine whether there were viable organisms in addition to those detected based on DNA sequencing (16S rRNA gene sequence). Results and discussion: Inseminating a heifer did not lead to a large change in the microbiome when assessed by traditional methods of bacterial culture or metataxonomic (16S rRNA gene) sequencing (results of Studies 1 and 2). Very few bacteria were cultured from the body or horn of the uterus regardless of whether an AI was or was not (negative control) performed. The cultured bacterial genera (e.g., Bacillus, Corynebacterium, Cutibacterium, Micrococcus, Staphylococcus, and Streptococcus) were typical of those found in the soil, environment, skin, mucous membranes, and urogenital tract of animals. Metataxonomic sequencing of 16S rRNA gene generated a large number of amplicon sequence variants (ASV), but these larger datasets that were based on DNA sequencing did not consistently demonstrate an effect of AI on the abundance of ASVs across all uterine locations compared with the external surface of the tract (e.g., perimetrium; positive control samples for environment contamination during slaughter and collection). Major genera identified by 16S rRNA gene sequencing overlapped with those identified with bacterial culture and included Cutibacterium, Staphylococcus, and Streptococcus.