The objective of this study was to evaluate the use of corpus luteum (CL) color doppler ultrasonography (CD) for early pregnancy diagnosis in Bos taurus beef replacement heifers. Beef heifers (n = 183) from two locations were exposed to a 7-d CO-Synch + CIDR protocol followed by fixed-time artificial insemination (day 0). On days 20 and 22, B-mode and CD ultrasonography were performed to evaluate CL morphometries and blood perfusion, respectively. Heifers were considered non-pregnant when CL area was < 20 mm2 or estimated luteal blood perfusion was ≤ 25%. Conventional ultrasonography on day 29 was utilized to determine pregnancy status and considered the gold standard method for pregnancy diagnosis. Pregnant heifers had greater CL diameter, CL area, and CL volume when compared to non-pregnant heifers on days 20 and 22 (P < 0.001). Additionally, percentage of central, peripheral, and total luteal blood perfusion, as well as the respective blood perfusion scores were greater (P < 0.001) in pregnant compared with non-pregnant heifers on both day 20 and 22. Sensitivity, specificity, positive predicted value (PPV), negative predicted value (NPV), and accuracy for CD on day 20 were 100, 70, 86, 100, and 90, respectively. Sensitivity, specificity, PPV, NPV, and accuracy for CD on day 22 were 100, 76, 90, 100, and 92, respectively. Pairwise comparison of receiver operating characteristics curve analysis indicated no differences (P = 0.47) between CD on days 20 and 22 (area under the curve = 0.82 and 0.84, respectively). In conclusion, CD successfully detected most non-pregnant replacement heifers on day 20 and 22, while false negative results were absent (NPV = 100%).

This experiment investigated the effects of diet composition on rumen, vaginal, and uterine microbiota of beef heifers. Fifteen rumen-cannulated, pubertal Angus-influenced heifers were used in a replicated 3 x 3 Latin square design (28-d periods and 21-d washout intervals). Dietary treatments included diets based on (as-fed) 100% grass hay (HF), 61% grass hay + 39% corn-based concentrate (INT), or 25% grass hay + 75% corn-based concentrate (HG). Treatments were offered individually to heifers once daily at 2% BW. Heifers also received 280 g/d of a mineral mix containing melengestrol acetate. Rumen, vaginal, and uterine samples were collected on d 0 and 28 of each period for bacterial profiling of the 16S rRNA gene and pH measurement. Data were analyzed using the MIXED procedure of SAS using results from d 0 as independent covariates, and heifer as the experimental unit. Ruminal pH on d 28 was greater (P < 0.01) in HF compared with INT and HG, and greater (P < 0.01) in INT compared with HG. Uterine and vaginal pH on d 28 did not differ among treatments (P > 0.10). In the rumen, Bacteriodetes was the most abundant phylum with the relative abundance being significantly greater (P < 0.01) in HF and INT (64.9 and 62.6%, respectively) when compared with HG (53.9%). Prevotella was the most abundant genus of bacteria within the rumen but did not differ (P > 0.10) by dietary treatment. While there were no significant differences (P > 0.10) for bacterial phyla in vaginal samples, Prevotella was the most abundant genus with the relative abundance being significantly greater (P < 0.01) for the HG (18.62%) when compared with HF and INT (7.78 and 10.35%, respectively). There were no significant differences (P > 0.10) for bacterial phyla in uterine samples, and while Prevotella was the most abundant, it was unaffected (P > 0.10) by diet. Therefore, diet impacted rumen microbiota and appears to have an influence on vaginal microbiota of beef heifers.

Female sex steroid hormones have been shown to interact with cytokines within the immune system to help influence fertility. Previous research has indicated that shifts in commensal bacteria are associated with compromised reproductive performance during the postpartum period, yet evaluation of how the female sex hormone progesterone (P4) influences these microbial shifts is not fully understood. The objectives of this study were to determine 1) the relationship between P4 concentrations and cytokine concentrations, and 2) the relationship between P4 concentrations and the reproductive microbiome. Twenty Bos indicus-influenced beef cows (~60 days postpartum and free of health issues) were weighed, body condition scored and then subjected to the Bos indicus prostaglandin 5 day (d) + CIDR protocol beginning on d -8 and timed artificially inseminated on d 0. Blood samples were collected on d-3, -1, 0, and 28. Cytokines and reproductive microbiome were evaluated using vaginal and uterine flushes collected on d -3 and d-1. Experimental groups were assigned by d 28 pregnancy status, determined by transrectal ultrasonography, resulting in Open (n = 13) and Pregnant (n = 7) females. Bacterial community analyses were conducted targeting the V4 hypervariable region of the 16S rRNA gene. Plasma P4 concentrations on d -3 and d -1 were quantified per manufacturers’ instructions using a double-antibody RIA kit. Cytokine analyses of plasma and flushes were conducted using the RayBiotech Quantibody Bovine Cytokine Array Q1 kit per the manufacturer’s instructions. Statistical analyses were conducted using the GLM procedure and CORR procedure in SAS. Plasma P4 concentrations did not differ by pregnancy status (P > 0.05) but did decrease from d -3 to d -1 (0.89 vs. 0.08 ± 0.23 ng/mL, respectively; P = 0.02). There were no significant correlations between plasma P4 and plasma cytokines (P > 0.05). Interferon-alpha (IFNα) and P4 concentrations were positively correlated in Open and Pregnant uterine flushes (r = 0.41; P = 0.01). Concentrations of P4 interleukin (IL)-1F5 were negatively correlated in Open and Pregnant vaginal flushes (r = -0.32; P = 0.04). The relative abundance of genus Methanobrevibacter in uterine flushes was positively correlated with P4 concentrations in both experimental groups (r = 0.35; P = 0.02). Within vaginal flushes, there was a tendency for a negative correlation between the relative abundance of genus Bacteroides and P4 concentrations (r = -0.31; P = 0.05). Alternatively, there is a positive correlation between P4 concentration and relative abundance of genus Streptococcus (r = 0.47; P = 0.01) and genus Methanobrevibacter (r = 0.37; P = 0.02) in vaginal flushes. These results suggest there may be relationships between several cytokines and circulating P4 concentrations; therefore, potentially suggesting immunomodulatory activities of P4. Overall, this research alludes to P4 having a vital role in reproductive microbial shifts and associated immune responses. Further investigation on the local effects of P4 within the reproductive tract on immune responses and bacterial shifts could provide greater insight into mechanisms to improve fertility in beef cattle.