W. Heuwieser

Evaluation of an electronic cowside test to detect subclinical ketosis in dairy cows.

The objective of this study was to determine the diagnostic performance of an electronic beta-hydroxybutyrate (BHBA) hand-held meter (Precision Xtra) for use in dairy cattle. Specific objectives were to compare the electronic BHBA meter with serum BHBA concentrations determined photometrically and 2 commonly used chemical cowside tests (Ketostix, Ketolac) and to evaluate accuracy in a field study employing 35 investigators. Of the 196 blood samples collected in experiment 1, 17 (8.7%) contained > or =1,200 micromol of BHBA/L of blood and 10 (5.1%) contained > or =1,400 micromol of BHBA/L of blood. Pearson correlation coefficients were highly significant for all tests. The highest correlation coefficient (0.95) was found between measurements of whole blood BHBA determined with the Precision Xtra test and the serum BHBA concentrations determined photometrically. Correlation coefficients between serum BHBA and BHBA in urine using Precision Xtra and Ketostix, and milk using Ketolac were lower. The Precision Xtra test was both 100% sensitive and specific at > or =1,400 micromol of BHBA/L of whole blood. Using milk and urine, positive and negative predictive values were considerably lower for both chemical tests as well as for the electronic meter. In the second study undertaken with 35 bovine veterinary practices, 926 blood samples were collected. In this study, the Precision Xtra test had sensitivities of 88 and 96% at 1,200 and 1,400 micromol of BHBA/L of whole blood, respectively. Specificities were 96 and 97%, respectively. Level of agreement was lower in the second study employing multiple investigators. Considerable differences in variance occurred among investigators. We conclude that the electronic hand-held BHBA measuring system using whole blood is a useful and practical tool to diagnose subclinical ketosis. Sensitivity and specificity are excellent for a cowside test and higher than 2 commonly used chemical dipsticks (Ketostix and Ketolac).

Prevalence of subclinical ketosis and relationships with postpartum diseases in European dairy cows

Subclinical ketosis (SCK) is defined as concentrations of β-hydroxybutyrate (BHBA) ≥ 1.2 to 1.4 mmol/L and it is considered a gateway condition for other metabolic and infectious disorders such as metritis, mastitis, clinical ketosis, and displaced abomasum. Reported prevalence rates range from 6.9 to 43% in the first 2 mo of lactation. However, there is a dearth of information on prevalence rates considering the diversity of European dairy farms. The objectives of this study were to (1) determine prevalence of SCK, (2) identify thresholds of BHBA, and (3) study their relationships with postpartum metritis, clinical ketosis, displaced abomasum, lameness, and mastitis in European dairy farms. From May to October 2011, a convenience sample of 528 dairy herds from Croatia, Germany, Hungary, Italy, Poland, Portugal, Serbia, Slovenia, Spain, and Turkey was studied. β-Hydroxybutyrate levels were measured in 5,884 cows with a handheld meter within 2 to 15 d in milk (DIM). On average, 11 cows were enrolled per farm and relevant information (e.g., DIM, postpartum diseases, herd size) was recorded. Using receiver operator characteristic curve analyses, blood BHBA thresholds were determined for the occurrence of metritis, mastitis, clinical ketosis, displaced abomasum, and lameness. Multivariate binary logistic regression models were built for each disease, considering cow as the experimental unit and herd as a random effect. Overall prevalence of SCK (i.e., blood BHBA ≥ 1.2 mmol/L) within 10 countries was 21.8%, ranging from 11.2 to 36.6%. Cows with SCK had 1.5, 9.5, and 5.0 times greater odds of developing metritis, clinical ketosis, and displaced abomasum, respectively. Multivariate binary logistic regression models demonstrated that cows with blood BHBA levels of ≥ 1.4, ≥ 1.1 and ≥ 1.7 mmol/L during 2 to 15 DIM had 1.7, 10.5, and 6.9 times greater odds of developing metritis, clinical ketosis, and displaced abomasum, respectively, compared with cows with lower BHBA blood levels. Interestingly, a postpartum blood BHBA threshold ≥ 1.1 mmol/L increased the odds for lameness in dairy cows 1.8 (95% confidence interval: 1.3 to 2.5) times. Overall, prevalence of SCK was high between 2 to 15 DIM and SCK increased the odds of metritis, clinical ketosis, lameness, and displaced abomasum in European dairy herds.

Endometrial expression of selected transcripts involved in prostaglandin synthesis in cows with endometritis

Several cytokines and prostaglandins play an important role in preparing the endometrium for implantation and mediating pro-inflammatory events. The aim of the present study was to examine mRNA expression of interleukin 1alpha (IL-1alpha), interleukin receptor antagonist (IL-1-RN), cytosolic prostaglandin E synthase (cPGES), microsomal PGES (mPGES-1 and mPGES-2) and lipocalin-type PGDS (L-PGDS) in the bovine endometrium. Endometrial epithelium samples were collected ex vivo from cows with different status of health at day 21-27 postpartum on a dairy farm. Three groups (n=9 animals each) were defined: (1) healthy cows with no signs of endometritis (control group), (2) cows with subclinical endometritis, and (3) cows with signs of clinical endometritis. Oestrous cycle-dependent mRNA expression pattern was investigated using bovine endometrial epithelial cells from healthy uteri collected at the abattoir. These uteri were classified into post-ovulatory, early-to-mid luteal, late luteal or pre-ovulatory phase (n=8 animals for each cycle phase). After collecting endometrial epithelium using the cytobrush-method, mRNA analysis was performed by real-time RT-PCR. L-PGDS, IL-1alpha and IL-1-RN mRNA were expressed significantly higher (P<0.05) in the endometrium of cows with subclinical or clinical endometritis compared with healthy cows. A twofold lower cPGES mRNA expression (P<0.05) was detected in cows with subclinical endometritis compared to healthy cows. L-PGDS and IL-1-RN mRNA expression was increased (P<0.05) after ovulation compared with the pre-ovulatory or luteal phase, respectively. These results support the hypothesis that a dys-regulated cytokine and/or prostaglandin profile in the uterus could be induced by subclinical endometritis or clinical endometritis.

Prevalence of bovine subclinical endometritis 4 h after insemination and its effects on first service conception rate

The objective of this study was to investigate the prevalence of subclinical endometritis 4h after AI and its effect on first service conception rate (FSCR) in dairy cows. A total of 201 Holstein-Friesian cows with no signs of clinical endometritis were examined 4h after first AI for signs of subclinical endometritis. Endometrial samples were collected from the uterus using the cytobrush technique. The proportion of polymorphonuclear cells (PMN) in the cytological sample was used to characterize an inflammation of the endometrium. Cows were categorized into three groups according to the proportion of PMN in the sample. Cows with 0% PMN (n=115) were assigned to group Zero, cows with >0-15% PMN (n=59) to group Medium, and cows with >15% PMN (n=27) to group High. Pregnancy diagnosis was performed between days 38-44 after AI by palpation of the uterus and its contents per rectum. The FSCR was significantly higher in group Medium than in groups Zero and High (57.6% vs. 39.1% and 29.6%). Statistical analysis revealed an interaction between parity and PMN group. Primiparous cows were at higher risk of being classified into group Medium than multiparous cows (OR=2.27, P=0.01). Primiparous cows in group Zero had lower odds of pregnancy after first AI than primiparous cows in group Medium (OR=0.3, P=0.02). A comparison with cows that were not examined for subclinical endometritis showed that the collection of endometrial samples itself had no effect on FSCR.

Rumination and its relationship to feeding and lying behavior in Holstein dairy cows

The objective of this study was to describe the relationship between rumination and feeding and lying behavior in dairy cows. Rumination time was monitored electronically using HR-Tags (SCR Engineers Ltd., Netanya, Israel). Feeding time and dry matter intake (DMI) were monitored using Insentec feed bins (Insentec BV, Marknesse, the Netherlands). All measures were collected in 2-h periods for 42 mature Holstein cows for a minimum of 9 d in the early dry period. Pearson correlation was used to describe associations, among 2-h periods within cow, first examining the relationship within a single period, and then modeling how this relationship changes when a lag of 2, 4, or 6h was imposed. Periods when cows spent more time ruminating were associated with lower feeding times and lower DMI (r = -0.71 and r = -0.72, respectively), likely because cows were unable to feed and ruminate simultaneously. The correlations with rumination time changed from negative to positive when lags of 2, 4, and 6h were modeled (r = -0.09, 0.24, and 0.15, and r = -0.16, 0.23, and 0.17 for feeding time and DMI at lags of 2, 4, and 6h, respectively). These results indicate that following periods of high feeding times and intakes, cows spent more time ruminating. This relationship peaks at approximately 4h after feeding. Periods of rumination were also associated with time spent lying down. Cows that spent more time ruminating per day, spent less time feeding (r = -0.34) and rumination times did not relate to DMI (r = 0.11). These data indicate that rumination time can be used to estimate within-cow variation in feeding behavior and intake, but daily summaries of rumination behavior are a poor indicator of DMI.

A clinical approach to determine false positive findings of clinical endometritis by vaginoscopy by the use of uterine bacteriology and cytology in dairy cows

Clinical endometritis in dairy cows is defined as mucopurulent or purulent vulvar discharge 21 days or more after parturition. The diagnosis of clinical endometritis is commonly based on vaginal examination. Techniques to reduce the proportions of false negative findings have been described. This paper discusses a clinical approach to determine the proportion of false positive findings that might occur by vaginal inspection. The consequences of false positive findings in dairy practice are unnecessary or inadequate treatments. In research, incorrect diagnoses have an impact on the interpretation of studies on the diagnosis and treatment of clinical endometritis. The objective of the present study was to compare intrauterine bacteriology and endometrial cytology in cows diagnosed with clinical endometritis with findings obtained by vaginoscopy. Clinical endometritis was defined as mucopurulent or purulent vulvar discharge. On two commercial dairy farms, cows were examined 21 to 28 d postpartum. Uterine samples (n = 230) were collected from cows with clinical endometritis with the cytobrush technique to determine the proportion of polymorphonuclear neutrophils (PMN) and to culture smears for aerobic bacteria. Two threshold values for the proportion of PMN (5 and 18%) were chosen as possible indicators for an inflamed endometrium. Common uterine pathogens A. pyogenes and E. coli were found in 33.5 and 10.4% of the samples, respectively. With increasing vaginal discharge score, proportion of samples positive for A. pyogenes increased significantly. The proportion of cows exceeding the thresholds for PMN increased with vaginal discharge score and the presence of A. pyogenes. Considering only the presence of aerobic uterine pathogens and a proportion of PMN above the threshold values of 5 and 18% as indicative for endometritis, a proportion of 17.3 and 28.5%, respectively, of diagnoses by vaginoscopy were false positive.

Influence of stage of lactation and milk production on conception rates after timed artificial insemination following Ovsynch.

Conception rates after timed artificial insemination (TAI) are of paramount importance for the success of protocols based on synchronization of ovulation. Stage of lactation and milk production level are known factors that influence dairy cow fertility. It was the objective of this study to analyse the effect of stage of lactation and milk production level on conception rates and pregnancy rates by 200 days in milk (DIM) in dairy cows synchronized with the Ovsynch protocol (Day -10, Day -1: 0.1 mg of D-Phe6-gonadorelin, Day -3: 0.5 mg of cloprostenol, Day 0: AI). A total of 1,288 dairy cows were assigned to two groups and classified in three production levels (high, average, low). Cows of all milk production levels in Group 1 (Simultaneous Ovsynch, SO) were synchronized with the Ovsynch protocol simultaneously for TAI between 73 and 81 DIM. In Group 2 cows with average milk production were synchronized at the same time as Group 1, while low producing cows were synchronized 3 weeks earlier and high producing cows were synchronized 3 weeks later than Group 1, respectively. First service conception rates (FSCRs) were lower (P<0.05) in cows synchronized earlier than in cows of the same production level synchronized later (low production: 14.4% (22/153) versus 34.5% (51/148); high production: 28.2% (40/142) versus 41.4% (53/128)). Milk production level had no significant impact on conception rates after TAI in cows synchronized at the same stage of lactation. At 200 DIM fewer cows with high production level were pregnant than cows with average or low production (P<0.05). This effect was independent of the stage of lactation at the initiation of Ovsynch. Endometritis at a postpartum examination did not influence conception rates after TAI. In conclusion, stage of lactation, but not milk production level, has a major influence on conception rates after TAI. Early AI after Ovsynch is less efficient and therefore its return on investment should be evaluated carefully.

Time-dependent mRNA expression of selected pro-inflammatory factors in the endometrium of primiparous cows postpartum.

BackgroundInflammatory processes and infections of the uterine wall must be accepted as a physiological event in dairy cows after calving. This might result in clinical or subclinical endometritis which is assumed to impair reproductive performance in the current lactation. Several cytokines and acute phase proteins have been discussed as local and systemic mediators of these inflammatory processes. The aim of the present study was to investigate the endometrial mRNA expression of the chemokine CXC ligand 5 (CXCL5), interleukin 1β (IL1B), IL6, IL8, tumour necrosis factor alpha (TNF), prostaglandin-endoperoxide synthase 2 (PTGS2) and haptoglobin (HP) in the postpartum period.MethodsEndometrial samples were obtained from primiparous cows (n = 5) on days 10, 17, 24, 31, 38 and 45 postpartum (pp) using the cytobrush technique. Cytological smears were prepared from cytobrush samples to determine the proportion of polymorphonuclear neutrophils (PMN). Total RNA was extracted from endometrial samples, and real-time RT-PCR was performed.ResultsA time-dependent mRNA expression of the investigated factors was found for the course of the postpartum period. In detail, a significantly higher expression of these factors was observed on day 17 pp compared to day 31 pp. Furthermore, the proportion of PMN peaked between days 10-24 pp and decreased thereafter to low percentages (< 5%) on day 31 pp and thereafter. In addition, CXCL5, IL1B, IL8 and HP mRNA expression correlated significantly with the proportion of PMN (P < 0.05). A significantly higher CXCL5, IL1B, IL6, IL8, PTGS2 and TNF mRNA content was observed in samples from cows with an inflamed endometrium compared with samples from cows with a healthy endometrium (P < 0.05).ConclusionsThese results show that inflammatory cytokines and acute phase proteins are expressed in the bovine endometrium in a time-related manner during the postpartum period, with a significant expression peak on day 17 pp as a possible mucosal immune response in the uterus. The evaluation of the expression patterns of such candidate genes may reveal more information than only determining the percentage of PMN to judge the severity of an inflammation.

Evaluation of the Metricheck device and the gloved hand for the diagnosis of clinical endometritis in dairy cows

The objective of this study was to compare 2 surrogate vaginal examination methods (i.e., gloved hand and a vaginal device) with vaginoscopy as a reference method for diagnosing clinical endometritis in dairy cows. Holstein-Friesian cows (n = 1,002) in 2 commercial dairy herds in Germany were examined for endometritis at 21 to 27 d in milk (DIM) by using 1 of 3 vaginal diagnostic methods. Vaginal examinations were performed either with a speculum (reference method), a vaginal device (Metricheck, Simcro, New Zealand), or a gloved hand. Vaginal discharge adhering to the diagnostic tool was classified according to a vaginal discharge score ranging from 0 to 3 (where 0 = translucent mucus, 1 = mucus containing flecks of white or off-white pus, 2 = less than 50% white or off-white mucopurulent material, and 3 = greater than 50% white or yellow pus that may be sanguineous). Cows with vaginal discharge scores of 1 to 3 received 500 microg of cloprostenol after examination and again 14 d later (35 to 41 DIM). The prevalence of endometritis in both herds was 40.6 and 40.3%, respectively. With the Metricheck device, significantly more cows were diagnosed as affected with endometritis than by examination with a speculum or a gloved hand (47.5 vs. 36.9 and 36.8%). Binary logistic regression for the risk of conception after first AI as an outcome variable, with vaginal discharge score, diagnostic method, and farm as covariates, revealed a significant effect of degree of endometritis, but not of the diagnostic methods. Survival analyses for the hazard of insemination and pregnancy within 200 DIM, respectively, revealed a significant effect of degree of endometritis, herd, and parity, but not of the diagnostic tool. It can be concluded that any one of the 3 vaginal examination methods can be used interchangeably, without a negative effect on reproductive performance.

Innate immunity and inflammation of the bovine female reproductive tract in health and disease

Mammalian reproductive physiology and the development of viviparity co-evolved with inflammation and immunity over millennia. Many inflammatory mediators contribute to paracrine and endocrine signalling, and the maintenance of tissue homeostasis in the female reproductive tract. However, inflammation is also a feature of microbial infections of the reproductive tract. Bacteria and viruses commonly cause endometritis, perturb ovarian follicle development and suppress the endocrine activity of the hypothalamus and pituitary in cattle. Innate immunity is an evolutionary ancient system that orchestrates host cell inflammatory responses aimed at eliminating pathogens and repairing damaged tissue. Pattern recognition receptors on host cells bind pathogen-associated molecular patterns and damage-associated molecular patterns, leading to the activation of intracellular MAPK and NFκB signalling pathways and the release of inflammatory mediators. Inflammatory mediators typically include the interleukin cytokines IL1β and IL6, chemokines such as IL8, interferons and prostaglandins. This review outlines the mechanisms of inflammation and innate immunity in the bovine female reproductive tract during health and disease condition.