In vivo dynamics of pro-inflammatory factors, mucins, and polymorph nuclear neutrophils in the bovine oviduct during the follicular and luteal phase.

Dynamic functional changes in the oviductal microenvironment are the prerequisite for the establishment of pregnancy. The objective of this study was to gain the first insights into oestrous cycle-dependent dynamics of polymorph nuclear neutrophils (PMN) and the mRNA abundance of selected genes and their correlations in the oviduct of living cows. Mini-cytobrush samples were taken from the oviducts of healthy heifers (n = 6) and cows (n = 7) during the follicular (FOL) and luteal phase (LUT) by transvaginal endoscopy. Total RNA was isolated from the samples and subjected to reverse transcription-quantitative PCR for selected pro-inflammatory factors, glycoproteins, and a metabolic marker. The percentage of PMN was determined by cytological examination. The mean PMN percentage was 2.8-fold greater during LUT than FOL. During LUT, significantly greater mRNA abundance of the pro-inflammatory factors IL1B, CXCL1, CXCL3, and CXCL8 was observed. The OVGP1 mRNA abundance was twice as high during FOL than in LUT. Pearson correlation, principal component analysis and heatmap analyses indicated characteristic functional patterns with strong correlations among investigated factors. Using this novel approach, we illustrate complex physiological dynamics and interactions of the mRNA expression of pro-inflammatory factors, mucins, OVGP1, and PMN in the oviduct during the oestrous cycle.

Short communication: Repeat breeder cows with fluid in the uterine lumen had poorer fertility.

The objective of the present study was to test the association between the presence of fluid in uterine lumen and the fertility of repeat breeder cows (RBC). Grazing Holstein cows that were clinically normal and were not pregnant after at least 3 consecutive inseminations were defined as RBC and included in the study (n = 358). A cut-point ≥2 mm of uterine lumen visible by ultrasonography was used as indicative of the presence of fluid in the uterus. The effect of this fluid on the fertility of RBC was assessed by mixed logistic regression. Twenty percent of the RBC had ≥2 mm of fluid in their uterus, and this finding was associated with a marked reduction in the odds of conception (odds ratio = 0.46). Additional intrauterine sampling revealed that 24.0% of RBC showed signs of subclinical endometritis [≥5% neutrophils in endometrial cytology (SCE)]. However, we found no agreement between the finding of uterine fluid and the diagnosis of SCE (kappa coefficient = 0.03, 95% confidence interval = -0.07 to 0.14). In conclusion, the presence of fluid (≥2 mm) in uterine lumen, detected by ultrasonography, was associated with lower fertility in RBC, but was not related to the diagnosis of SCE. Thus, ultrasonographic evaluation is an important tool to assist in assessment of RBC.

Endometrial mRNA expression of selected pro-inflammatory factors and mucins in repeat breeder cows with and without subclinical endometritis.

Repeat breeder cows (RBC) are defined as cyclic cows without clinical abnormalities that fail to conceive after at least three subsequent inseminations. Previous studies have elucidated cellular defence mechanisms in the bovine uterus but detailed information on inflammatory events of endometrial cells in RBC is still lacking. Thus, the objective of this study was to analyse endometrial mRNA expression of selected transcripts associated with uterine inflammatory processes. Cytobrush samples from 91 RBC and 11 synchronised heifers with no history of gynaecological abnormalities (controls, CON) were collected. The proportion of polymorphonuclear neutrophils in these samples was used for the diagnosis of subclinical endometritis (SE). Ultrasonography and progesterone blood concentrations were used to determine ovarian activity and the stage of the oestrous cycle. Total RNA was isolated from the cytobrush samples and subjected to reverse transcription-quantitative PCR for interleukins (IL) 1A, IL1B, IL6, IL8, chemokine CXL ligand (CXCL) 3, CXCL5, prostaglandin-endoperoxide synthase 2 (PTGS2), tracheal antimicrobial peptide (TAP) and mucin (MUC) 4, MUC5, MUC6, MUC12 and MUC16. CXCL3 mRNA was higher (2-fold) and PTGS2 mRNA lower (6-fold) expressed in RBC compared with CON (P < 0.05). After subdivision of RBC in animals with (RBC-SE) and without SE (RBC-noSE), these differences remained significant between RBC-noSE and CON. Higher mRNA abundances of IL1A and IL1B were found in RBC-SE compared with RBC-noSE (3- and 4-fold; P < 0.05). No differences in the mRNA expression of IL6, IL8, CXCL5 and TAP were observed between RBC-SE, RBC-noSE and CON. MUC4 and MUC12 mRNA was more highly expressed in RBC than in CON (P < 0.05). In RBC-noSE, a 5- and 14-fold higher MUC4 and MUC12 mRNA expression was noticed compared with CON (P < 0.05). A significantly lower mRNA expression of MUC5 and MUC16 (7- and 4-fold) was detected in RBC in the luteal phase compared with RBC in the follicular phase, whereas such a down-regulation was not observed for MUC4 and MUC12. In conclusion, we demonstrated different PTGS2 and CXCL3 mRNA expression between RBC and control heifers, which might be related to subfertility in RBC. Further studies are required to confirm that an unregulated MUC4 and MUC12 mRNA expression may contribute to subfertility of RBC. These findings provide a valid basis for further research on regulatory mechanisms of mRNA expression in subfertile cows.

The prevalence of subclinical endometritis and intrauterine infections in repeat breeder cows.

The objectives of this study were to assess the prevalence of subclinical endometritis and the presence of common uterine pathogens in repeat breeder cows. A total of 121 cows with three or more consecutive artificial inseminations without conception and no clinical signs of disease were defined as repeat breeder cows and were enrolled in this trial. Intrauterine samples were collected with the cytobrush technique to determine the prevalence of subclinical endometritis and bacteriologic infections. Blood samples were analyzed for concentrations of progesterone and estradiol in plasma to assess ovarian activity. Furthermore, breed, parity, history of calving and postpartum uterine infection, clinical findings of transrectal palpation, and backfat thickness were analyzed as potential factors for the prevalence of subclinical endometritis in repeat breeder cows. The prevalence of subclinical endometritis in repeat breeder cows was 12.7%; but common uterine pathogens, Escherichia coli and Trueperella pyogenes, were found in only one and three cows, respectively. Ovarian activity was determined in 95.0% of all cows. Recorded variables had no effect on the prevalence of subclinical endometritis in repeat breeder cows. In conclusion, subclinical endometritis and uterine infections linked to common pathogens were playing a minor role as a cause for repeat breeder cows in this study. Alternative reasons for failure to conceive in these cows are discussed.

Diversity and health status specific fluctuations of intrauterine microbial communities in postpartum dairy cows.

For the interpretation of clinical findings of endometritis and the development of disease prevention and intervention strategies a better understanding of the dynamics and interactions within intrauterine bacterial communities in healthy and diseased cows is required. To gain deeper insights into fluctuations within the uterine microbiota, intrauterine samples were collected from 122 cows at the day of calving, days 3, 9, 15, 21 and 28 postpartum. A total of 2052 bacterial isolates were identified by Fourier-transform-infrared spectroscopy. This culturomics-based approach showed that the aerobic uterine microflora comprised a huge diversity of bacteria belonging to 202 different species, representing 76 genera, with members of the genus Staphylococcus (24.2%) being predominant. On species level the uterine microflora was dominated by Trueperella pyogenes (13.2%), Escherichia coli (11.2%), Staphylococcus xylosus (5.4%), Bacillus pumilus (5.2%) and Streptococcus uberis (4.9%). Comparative analysis of uterine bacteria from cows with different vaginal discharge scores (VDS) revealed health status specific temporal microbial diversifications. Although the intrauterine flora of all VDS groups was dominated by T. pyogenes, E. coli and Staphylococcus spp., the relative number of bacteria differed between VDS groups. The presence of T. pyogenes on days 15 and 21 significantly increased the risk of VDS 2 and 3 at day 21, whereas Staphylococci at day 9 reduced the likelihood of VDS 3 (P<0.05). This study demonstrates that intrauterine bacterial infections are highly dynamic processes and that bacterial species follow specific patterns of progression, which require further research to decipher their potential role in uterine disease development.

Consultancy to dairy farmers relating to animal health and herd health management on small- and medium-sized farms.

The objectives of this study were to obtain information about animal health challenges for dairy farmers of small- and medium-sized herds and about the fields in which consultancy services should be improved. The hyperlink to an internet-based survey was sent to 9,021 farmers in Austria. The survey included questions about the participants and their farms, about who is consulting with the farmers with regard to animal health, feeding, sire selection, construction of barns and animal husbandry, about animal health issues farmers find most challenging, and about their demands for improved consultancy services. The questionnaire was completed anonymously. Analyses were stratified by milk yield (categorized) and whether farmers worked full-time or part-time. The overall response rate was 11.3% (n=1,018). The majority of farms kept less than 20 cows (54.0%) or 20 to 50 cows (40.1%). With regard to animal health, the veterinarian was the most important consultant for the majority of farmers (84.6%). On issues related to feeding, sire selection, and stable construction, the veterinarian was seen as a less important consultant than specialists in these fields (20.4, 11.6, and 7.9% suggested the veterinarian as an important consultant in these areas). The survey indicated that reproductive disorders, udder disease, poor conception rate, lameness, and calf diarrhea represent the most important challenges to farmers. Of these, concerns about calf diarrhea were affected by milk yield of the herds and management. More high- than low-yielding farms (11.7 vs. 6.4%) and more full-time than part-time managed herds (9.6 vs.4.3%) regarded calf diarrhea as an important problem. Farmers would welcome improved consultancy with regard to fertility, feeding, and sire selection. The results indicated which animal health issues farmers find particularly challenging and displayed which areas farmers require improved consultancy services. Veterinarians and organizations offering consultancy should take the results into consideration in improving or adapting their advisory services.