Investigation of Uterine Fluid Extracellular Vesicles' Proteomic Profiles Provides Novel Diagnostic Biomarkers of Bovine Endometritis.

Cow uterine infections pose a challenge in dairy farming, resulting in reproductive disorders. Uterine fluid extracellular vesicles (UF-EVs) play a key role in cell-to-cell communication in the uterus, potentially holding the signs of aetiology for endometritis. We used mass spectrometry-based quantitative shotgun proteomics to compare UF-EV proteomic profiles in healthy cows (H), cows with subclinical (SE) or clinical endometritis (CLE) sampled at 28-35 days postpartum. Functional analysis was performed on embryo cultures with the exposure to different EV types. A total of 248 UF-EV proteins exhibited differential enrichment between the groups. Interestingly, in SE, EV protein signature suggests a slight suppression of inflammatory response compared to CLE-UF-EVs, clustering closer with healthy cows' profile. Furthermore, CLE-UF-EVs proteomic profile highlighted pathways associated with cell apoptosis and active inflammation aimed at pathogen elimination. In SE-UF-EVs, the regulation of normal physiological status was aberrant, showing cell damage and endometrial repair at the same time. Serine peptidase HtrA1 (HTRA1) emerged as a potential biomarker for SE. Supplementation of CLE- and SE-derived UF-EVs reduced the embryo developmental rates and quality. Therefore, further research is warranted to elucidate the precise aetiology of SE in cattle, and HTRA1 should be further explored as a potential diagnostic biomarker.

Indirect Sensing of Subclinical Intramammary Infections in Dairy Herds with a Milking Robot.

This study determined the impact of subclinical intramammary infections (IMIs), such as the major and minor udder pathogens (MaPs and MiPs), on the somatic cell count (SCC) in cow milk and investigated the possibilities of indirect sensing of the udder pathogens using the mastitis detection index (MDi) (DeLaval, Tumba, Sweden). The MDi incorporates quarter-level milk electrical conductivity, blood in milk, and milking interval. The case group (n = 21; MDi ≥ 1.4) was compared with the control group (n = 24; MDi < 1.4) for the presence of IMIs. The microbiological investigation of udder quarter foremilk samples was performed two times with an interval of 10 to 14 days. The case and control groups differed in terms of the occurrence of MaPs and MiPs in milk. During the continuous subclinical IMI and the episodic MaP infection, a higher SCC was detected compared with the episodic MiP infection or quarters without IMI. The novel finding of this study was that by using the milk quality sensor for the sensing of subclinical IMIs, there was an indication for the successful detection of episodic MaPs. However, the sensing of the continuous subclinical IMIs was not possible in the current study and still needs to be investigated.

Uterine Fluid Extracellular Vesicles Proteome Is Altered During the Estrous Cycle.

Uterine environment is tightly and finely regulated via various signaling pathways mediated through endocrine, exocrine, autocrine, juxtacrine, and paracrine mechanisms. In utero signaling processes are paramount for normal and abnormal physiology which involves cell to cell, cells to gametes, cells to embryo, and even interkingdom communications due to presence of uterine microbiota. Extracellular vesicles (EVs) in the uterine fluid (UF) and their cargo components are known to be mediators of in utero signaling and communications. Interestingly, the changes in UF-EV proteome during the bovine estrous cycle and the effects of these differentially enriched proteins on embryo development are yet to be fully discovered. In this study, shotgun quantitative proteomics-based mass spectrometry was employed to compare UF-EV proteomes at day 0, 7, and 16 of the estrous cycle to understand the estrous cycle-dependent dynamics. Furthermore, different phase UF-EVs were supplemented in embryo cultures to evaluate their impact on embryo development. One hundred fifty-nine UF-EV proteins were differentially enriched at different time points indicating the UF-EV proteome is cycle-dependent. Overall, many identified pathways are important for normal uterine functions, early embryo development, and its nutritional needs, such as antioxidant activity, cell morphology and cycle, cellular homeostasis, cell adhesion, and carbohydrate metabolic process. Furthermore, the luteal phase UF-EVs supplementation increased in vitro blastocyst rates from 25.0 ± 5.9% to 41.0 ± 4.0% (p ≤ 0.05). Our findings highlight the importance of bovine UF-EV in uterine communications throughout the estrous cycle. Interestingly, comparison of hormone-synchronized EV proteomes to natural cycle UF-EVs indicated shift of signaling. Finally, UF-EVs can be used to improve embryo production in vitro.

Characterization of bovine uterine fluid extracellular vesicles proteomic profiles at follicular and luteal phases of the oestrous cycle.

Extracellular vesicles (EV) have been identified in uterine fluid (UF), however the bovine UF-EV profile during different phases of the oestrous cycle has not yet been established. Therefore, we compared the UF-EV, and their protein profile at follicular and luteal phases of the oestrous cycle. UF samples were collected from healthy uteri of six live and six slaughtered cows at follicular or luteal phases. Isolation of EV was performed using tangential flow filtration followed by size exclusion chromatography. EV were characterized by nanoparticle tracking analysis (NTA), fluorescence NTA, zeta potential, and transmission electron microscopy. Mass-spectrometry was used to evaluate EV protein profile from live cows. Particle concentrations (mean ± SD) were higher (P < 0.05) at follicular than at luteal phase in both live (1.01 × 108 ± 1.66 × 107 vs 7.56 × 107 ± 1.80 × 107, respectively) and slaughtered cows (1.17 × 108 ± 2.34 × 107 vs 9.12 × 107 ± 9.77 × 106, respectively). The proportion of fluorescently labelled EV varied significantly between follicular and luteal phases across live (28.9 ± 1.9% vs 19.3 ± 2.8%, respectively) and slaughtered cows (26.5 ± 6.3% vs 27.3 ± 2 .7%, respectively). In total, 41 EV proteins were differentially expressed between the phases. Some of the proteins were involved in reproductive processes, cell adhesion and proliferation, and cellular metabolic processes. The results indicated differences in bovine UF-EV concentration and protein profile at follicular and luteal phases, which would suggest that EV modulate uterine microenvironment across the oestrous cycle. Further research is needed to understand the effect of EV changes throughout the oestrous cycle.

Individual and Combined Effects of Diseases and Cytological Endometritis on Reproductive Performance and Culling of Dairy Cows: Preliminary Results.

The objective of this study was to evaluate the effect of calving-related disorders/clinical diseases (DIS) and cytological endometritis (CYTO) on the reproductive performance and culling of dairy cows. In a total of 119 multiparous Holstein cows, DIS were recorded and CYTO was diagnosed at 40 ± 2 d in milk. Onset of luteal activity was defined as the first postpartum measurement of milk progesterone >5 ng/mL. A dummy variable containing all four possible permutations between DIS and CYTO statuses was created. The pregnancy rates after first artificial insemination were 57.7, 42.9, 23.0 and 15.8% for the DIS−/CYTO−, DIS+/CYTO−, DIS−/CYTO+ and DIS+/CYTO+ groups, respectively. The hazard of pregnancy was affected by DIS−/CYTO+ and DIS+/CYTO+ health statuses (hazard ratio (HR) = 0.43 and 0.29, respectively), whereas DIS+/CYTO− and DIS−/CYTO− cows had a similar hazard to pregnancy. The hazard of onset of luteal activity was affected by DIS+/CYTO+ health status (HR = 0.45), but not by DIS+/CYTO− and DIS−/CYTO+ health statuses. Compared with DIS−/CYTO− cows, DIS−/CYTO+ and DIS+/CYTO+ cows had 4.24 and 5.57 times the odds of being culled, respectively. Culling was not affected by DIS+/CYTO− health status. In conclusion, CYTO+ status, irrespective of DIS status, decreased the pregnancy rate. When DIS+ and CYTO+ were combined, they had an additive negative effect. Our preliminary findings suggest that the far-reaching consequences of clinical diseases on fertility and culling can be mediated through the development of CYTO. Further investigations including a larger number of cows are required to confirm these results.

Ovarian follicular response to oestrous synchronisation and induction of ovulation in Norwegian Red cattle.

BACKGROUND: Oestrous synchronisation of cattle has been widely applied to accomplish simultaneous ovulation in animals and facilitate timed artificial insemination. The main aim of this study was to investigate the ovarian follicular growth and ovulatory response to oestrus and ovulation synchronisation in Norwegian Red heifers and cows. Oestrous cycles in 34 heifers and 10 cows from 4 herds were synchronised with two PGF2α analogue treatments 11 days apart, followed by GnRH analogue treatment for induction of ovulation. Thereafter, the ovaries were examined by ultrasonography at 3 h intervals until ovulation. RESULTS: The luteolytic effect of the PGF2α analogue was verified in 9 of 10 cows by progesterone contents in milk. Maximum physical activity of the cows occurred on average 69 h after PGF2α analogue treatment. An ovulatory response was recorded in 95.5% (42/44) of the animals. A significant difference in follicle size at ovulation was found between 2 of the herds. Animals with medium sized and large follicles and heifers aged > 16 months ovulated earlier than other animals. CONCLUSIONS: The applied sequence of treatments in the study was shown to be effective in synchronizing and inducing ovulation within a relatively narrow time interval in the Norwegian Red heifers and cows, consistent with findings in other cattle breeds.

Relationships between plasma insulin-like growth factor-1 and insulin concentrations in multiparous dairy cows with cytological endometritis.

Cytological endometritis (CYTO) is a uterine inflammation characterised by the percentage of polymorphonuclear neutrophils in endometrial cytology. This observational study evaluated the association of blood plasma insulin-like growth factor-1 (IGF-1) and insulin concentrations at -2 weeks prepartum, +1 week and +3 weeks postpartum with the development of CYTO in 119 multiparous Holstein cows. Overall CYTO prevalence was 30.3 per cent. Multivariable logistic regression model revealed the odds of developing CYTO were 3.54 times (P=0.018) greater in cows with week -2 prepartum IGF-1 concentrations less than 74.6 ng/ml and 4.41 times (P=0.004) higher in cows with week +1 postpartum IGF-1 concentrations less than 13.2 ng/ml than the odds of this health outcome in cows with IGF-1 concentrations at least 74.6 and13.2 ng/ml, respectively. Additionally, cows with body condition score (BCS) up to 2.75 at week -2 prepartum and cows experiencing calving-related disorders and/or ill health had higher risk for CYTO compared with cows with BCS=3.50-3.75 (OR=6.8, P=0.049) and cows without health complications (OR=3.1, P=0.030). Insulin was not a significant predictor for CYTO in the model. Our findings provide further evidence that reduced dairy cow fertility associated with low plasma concentrations of IGF-1 is in part mediated through the inflammatory status of the uterus.

Treatment of dairy cows with PGF2α or NSAID, in combination with antibiotics, in cases of postpartum uterine inflammation.

BACKGROUND: The aim of the study was to test the effect of two treatments in cases of acute puerperal metritis (APM) and clinical metritis (CM). METHODS: Cows with APM and CM (n = 40)) were matched according to plasma fibrinogen levels (Fb) into three groups. Two negative control groups D (n = 11) and E (n = 17) were composed of healthy cows. The proportion of animals with APM and CM was similar within the groups. Treatment was started on the 3rd day postpartum (PP). In group A (n = 15), intramuscular (i.m.) administration of ceftiofur was used for five days in combination with flunixin for three days. Group B (n = 15) received i.m. administration of ceftiofur for five days followed by two injections of prostaglandin F2α, with an interval of 8 h, on the 8th day PP. Group C (n = 10) served as a control group with no treatment. The general health status, body temperature (BT) and vaginal discharge were evaluated daily. Endometrial biopsies for bacteriology were taken once a week for seven weeks PP. Blood samples for the analysis of acute phase proteins were collected once a week for six weeks PP. Samples for progesterone analysis were taken twice a week for seven weeks PP. Fertility performance data were recorded. RESULTS: The area under the curve of BT was higher in group B than in group D cows (P < 0.05). No differences were found for vaginal discharge. There were no differences in bacterial growth, start of ovarian activity or serum amyloid-A or fibrinogen levels among the groups. The haptoglobin concentration was higher in the first and second weeks PP in group B compared with the other groups (P < 0.05). The number of days open was higher in group A than in both groups B and D (P < 0.05). The pregnancy rate after the first two services was higher (P < 0.05) in groups B and D than in groups A and C. The number of services per pregnancy was lower in group B than in group C (P < 0.05). CONCLUSIONS: Regardless of more severe uterine inflammation found in animals from group B, these cows showed the same fertility parameters as healthy animals.

Assessment of progesterone profiles and postpartum onset of luteal activity in spring calving Hereford beef suckler cattle.

BACKGROUND: Reproduction is the single greatest factor limiting beef cattle production. Previous research on beef suckler luteal activity has largely focused on the mechanisms, and duration, of postpartum anoestrus. However, the temporal pattern of luteal activity after resumption of post-partum ovarian activity, and the impact of pattern type on days open (DO) in purebred beef suckler cows, are unknown. METHODS: Progesterone concentration was measured in milk samples taken thrice weekly from 120 lactations, in 87 animals, on 3 farms, over two years. Onset of luteal activity (OLA) was defined as the first day milk progesterone concentration exceeded 3 ng/ml for two successive measurements, or exceeded 5 ng/ml once. It was defined as delayed if it occurred more than 61 days postpartum. A short initial luteal phase consisted of progesterone concentrations which exceeded 3 ng/ml for fewer than 4 sequential measurements. Temporal progesterone patterns were classified as: 1) Normal cyclicity; 2) Cessation of luteal activity; 3) Prolonged luteal activity; 4) Erratic phase: failure to conform to 1, 2 or 3. Data concerning parity, previous calving interval, breeding values, calf birth and 200-d weight were obtained from the Norwegian Beef Cattle Recording System database. RESULTS: The mean (SD) OLA was 41 d (20). Parity and calf birth weight were inversely correlated with OLA. Delayed OLA occurred in 14.4% of lactations. A short first luteal phase occurred in 61.5% of lactations, but this was unrelated to irregular luteal phase occurrence, pregnancy or DO. Irregular luteal phases occurred in 22% of lactations. The irregularities were: prolonged luteal phase (11%); cessation of luteal activity (5%); erratic luteal activity (6%). Early OLA was associated with prolonged luteal phases. DO was positively correlated with irregular luteal phases and negatively correlated with calf 200-d weight. CONCLUSIONS: This study demonstrates that irregular luteal phases negatively affect reproductive performance in purebred beef suckler cattle. A moderate incidence of irregular luteal phases was seen in the study population. Whilst a positive relationship was seen between OLA and DO, unfavourable associations between early OLA and incidence of irregular luteal phases should be considered when developing breeding programmes.

Effect of yeast culture on milk production and metabolic and reproductive performance of early lactation dairy cows.

BACKGROUND: The main objective of this study was to estimate the effect of supplementation with Saccaromyces cerevisiae (SC) (Yea-Sacc 1026) on milk production, metabolic parameters and the resumption of ovarian activity in early lactation dairy cows. METHODS: The experiment was conducted during 2005/2006 in a commercial tied-house farm with an average of 200 milking Estonian Holstein Friesian cows. The late pregnant multiparous cows (n = 46) were randomly divided into two groups; one group received 10 g yeast culture from two weeks before to 14 weeks after calving. The groups were fed a total mixed ration with silages and concentrates. Milk recording data and blood samples for plasma metabolites were taken. Resumption of luteal activity was determined using milk progesterone (P4) measurements. Uterine bacteriology and ovarian ultrasonography (US) were performed and body condition scores (BCS) and clinical disease occurrences were recorded. For analysis, the statistical software Stata 9.2 and R were used to compute Cox proportional hazard and linear mixed models. RESULTS: The average milk production per cow did not differ between the groups (32.7 +/- 6.4 vs 30.7 +/- 5.3 kg/day in the SC and control groups respectively), but the production of milk fat (P < 0.001) and milk protein (P < 0.001) were higher in the SC group. There was no effect of treatment on BCS. The analysis of energy-related metabolites in early lactation showed no significant differences between the groups. In both groups higher levels of beta-hydroxybutyrate (BHB) appeared from days 14 to 28 after parturition and the concentration of non-esterfied fatty acid (NEFA) was higher from days 1-7 post partum (PP). According to US and P4 results, all cows in both groups ovulated during the experimental period. The resumption of ovarian activity (first ovulations) and time required for elimination of bacteria from the uterus did not differ between the groups. CONCLUSION: Supplementation with SC had an effect on milk protein and fat production, but did not influence the milk yield. No effects on PP metabolic status, bacterial elimination from the uterus nor the resumption of ovarian activity were found.

Relationships between luteal activity, fertility, blood metabolites and body condition score in multiparous Estonian Holstein dairy cows under different management.

The objective was to compare the relationships between luteal activity and fertility, and relate these parameters to metabolic indices and body condition changes in multiparous Estonian Holstein cows on two commercial dairy farms under different management and levels of production and nutrition (higher, H, n=54 (71 lactations) and lower, L, n=39 (39 lactations)). For statistical analysis cows were categorized according to their milk progesterone (P4) profiles as follows: normal ovarian function; delayed start of cyclicity (DC) (interval from calving to first luteal response (P45 ng/ml up to and more than 50 d respectively, followed by regular cyclicity); cessation of luteal activity (prolonged interluteal interval, P4<5 ng/ml, with a duration of 14 d between two adjacent luteal phases); prolonged luteal activity (P4 levels 5 ng/ml for 20 d without preceding insemination). The Mixed procedure of the SAS system was used to compare milk production traits, blood metabolites (ketone bodies, non-esterified fatty acids, total cholesterol) and aspartate aminotransferase, body condition scores (BCS) and fertility parameters between the two farms, and also fertility parameters between the farms within P4 categories. Differences in milk fat/protein ratio, ketone body levels and BCS indicated a deeper negative energy balance (NEB) during the first month after calving on farm L. On both farms nearly 50% of the recently calved dairy cows suffered from ovarian dysfunction during the post-partum period. Delayed start of cyclicity was the most prevalent abnormal P4 profile, 25% and 28% on farms H and L, respectively. Prolonged luteal activity accounted for one-third of atypical ovarian patterns on farm H, and cessation of luteal activity on farm L. On farm L, DC cows had lower BCS values from day 10 to day 90 after calving compared with normal cows (P<0.01) and cows lost more BCS (1.2 units) during the 40 d after calving than normal resumption cows (0.75 units; P<0.05). On farm H with moderate NEB the delayed start of ovulation post partum did not impair subsequent reproductive performance.

The effects of ovarian function on estrus synchronization with PGF in dairy cows.

Milk progesterone concentration (P4), milk yield, milk composition, ovarian structures and pregnancy status were studied in 108 cows treated with two doses of PGF 14 days apart and inseminated at fixed time (TAI) 80-82 h later. The synchronization protocol was started at 70+/-1.4 days after parturition. Milk P4 profiles revealed that anestrus, failure of luteolysis following treatment with PGF and failure to ovulate following luteolysis were the main reasons for low pregnancy rate with TAI. Anestrous cows had a higher percentage of milk fat (P<0.05) and higher fat to protein ratio (P<0.01), and cows that did not undergo luteolysis had higher milk yield (P<0.05) and lower percentage of milk protein (P<0.05) than cows that responded to PGF treatment. Cows that did not undergo luteolysis and cows that did not ovulate following luteolysis had lower milk P4 during the luteal phase preceding the second PGF injection (P<0.01 and P<0.05, respectively). Pregnancy rates 24 and 47 days after TAI in cows that responded as expected to the synchronization treatment were 62% and 54%, respectively. Pregnancy was precluded in non-responsive cows. The largest follicle at the time of TAI in cows experiencing late embryonic mortality was smaller (P=0.02) than in cows that successfully maintained pregnancy. Results suggest that a primary reason for low pregnancy rate in dairy cows after administration of PGF and TAI is inappropriate ovarian function prior to, or following treatment.