No abstract

We tested the hypothesis that the uterus of virgin heifers and pregnant cows possessed a resident microbiome by 16S rRNA gene sequencing of the virgin and pregnant bovine uterus. The endometrium of 10 virgin heifers in estrus and the amniotic fluid, placentome, intercotyledonary placenta, cervical lumen, and external cervix surface (control) of 5 pregnant cows were sampled using aseptic techniques. The DNA was extracted, the V4 hypervariable region of the 16S rRNA gene was amplified, and amplicons were sequenced using Illumina MiSeq technology (Illumina Inc., San Diego, CA). Operational taxonomic units (OTU) were generated from the sequences using Qiime v1.8 software, and taxonomy was assigned using the Greengenes database. The effect of tissue on the microbial composition within the pregnant uterus was tested using univariate (mixed model) and multivariate (permutational multivariate ANOVA) procedures. Amplicons of 16S rRNA gene were generated in all samples, supporting the contention that the uterus of virgin heifers and pregnant cows contained a microbiome. On average, 53, 199, 380, 382, 525, and 13,589 reads annotated as 16, 35, 43, 63, 48, and 176 OTU in the placentome, virgin endometrium, amniotic fluid, cervical lumen, intercotyledonary placenta, and external surface of the cervix, respectively, were generated. The 3 most abundant phyla in the uterus of the virgin heifers and pregnant cows were Firmicutes, Bacteroidetes, and Proteobacteria, and they accounted for approximately 40, 35, and 10% of the sequences, respectively. Phyla abundance was similar between the tissues of the pregnant uterus. Principal component analysis, one-way PERMANOVA analysis of the Bray-Curtis similarity index, and mixed model analysis of the Shannon diversity index and Chao1 index demonstrated that the microbiome of the control tissue (external surface of the cervix) was significantly different from that of the amniotic fluid, intercotyledonary placenta, and placentome tissues. Interestingly, many bacterial species associated with postpartum uterine disease (i.e., Trueperella spp., Acinetobacter spp., Fusobacteria spp., Proteus spp., Prevotella spp., and Peptostreptococcus spp.) were also present in the uterus of virgin heifers and of pregnant cows. The presence of 16S rRNA gene sequence reads in the samples from the current study suggests that the uterine microbiome is established by the time a female reaches reproductive maturity, and that pregnancies are established and maintained in the presence of a uterine microbiome.

Although cowside testing strategies for diagnosing hyperketonemia (HYK) are available, many are labor intensive and costly, and some lack sufficient accuracy. Predicting milk ketone bodies by Fourier transform infrared spectrometry during routine milk sampling may offer a more practical monitoring strategy. The objectives of this study were to (1) develop linear and logistic regression models using all available test-day milk and performance variables for predicting HYK and (2) compare prediction methods (Fourier transform infrared milk ketone bodies, linear regression models, and logistic regression models) to determine which is the most predictive of HYK. Given the data available, a secondary objective was to evaluate differences in test-day milk and performance variables (continuous measurements) between Holsteins and Jerseys and between cows with or without HYK within breed. Blood samples were collected on the same day as milk sampling from 658 Holstein and 468 Jersey cows between 5 and 20 d in milk (DIM). Diagnosis of HYK was at a serum β-hydroxybutyrate (BHB) concentration ≥1.2 mmol/L. Concentrations of milk BHB and acetone were predicted by Fourier transform infrared spectrometry (Foss Analytical, Hillerød, Denmark). Thresholds of milk BHB and acetone were tested for diagnostic accuracy, and logistic models were built from continuous variables to predict HYK in primiparous and multiparous cows within breed. Linear models were constructed from continuous variables for primiparous and multiparous cows within breed that were 5 to 11 DIM or 12 to 20 DIM. Milk ketone body thresholds diagnosed HYK with 64.0 to 92.9% accuracy in Holsteins and 59.1 to 86.6% accuracy in Jerseys. Logistic models predicted HYK with 82.6 to 97.3% accuracy. Internally cross-validated multiple linear regression models diagnosed HYK of Holstein cows with 97.8% accuracy for primiparous and 83.3% accuracy for multiparous cows. Accuracy of Jersey models was 81.3% in primiparous and 83.4% in multiparous cows. These results suggest that predicting serum BHB from continuous test-day milk and performance variables could serve as a valuable diagnostic tool for monitoring HYK in Holstein and Jersey herds.