Associations of parity with health disorders and blood metabolite concentrations in Holstein cows in different production systems.

Data were obtained from studies in Australia, Canada, and the United States using individual cow data from 28,230 Holstein cows to evaluate associations between parity and disease. Our goal was to develop understanding of disease risks for cows of differing parity. We hypothesized that there would be increased risks of disease and changes in metabolite concentrations with increased parity. Parity ≥5 represented 2,533 cows or 9.0%, parity 4 was 9.8% (2,778), parity 3 as 19.0% (5,355), parity 2 as 28.1% (7,925), and parity 1 was 34.1% (9,639) of the sample. Of these cows, 15.5% were in Australia, 14.7% in Canada, and 69.8% in the United States. Lactational incidence (LI) risk of clinical hypocalcemia increased with parity from 0.1% for parity 1 to 13% for parity ≥5 cows. The marked increase suggests profound differences in metabolism with increased parity. The LI of clinical mastitis was 17.4%. The odds of mastitis increased with parity to 2.5 times greater in parity ≥5 than in parity 1. The LI of lameness increased with parity; specifically, the odds of lameness was 5.6 times greater for parity ≥5 than parity 1. Dystocia incidence was 8.7% and greatest for parity 1 cows. The LI of retained placenta was 7.4% and increased with parity, with the odds for parity ≥5 2.3 times greater than for parity 1. The LI of metritis was 10% and of endometritis 14%, with the greatest odds in parity 1. The LI of clinical ketosis was 3.3% with a marked increase in odds with parity. The prevalence of subclinical ketosis was 26.8% with only cows in parity 1 having lower odds than other parities. Parity ≥5 cows had greater odds (odds ratio = 1.7) of respiratory disease than parity 1 cows, which were lesser than other parities. Metabolite concentrations were evaluated in 5,154 Holstein cows in the precalving, calving, and immediate postcalving data sets. Metabolic measures near peak lactation provided 1,906 observations. Concentrations of ß-hydroxybutyrate (BHB) and nonesterified fatty acids increased with parity on d 1 to 3 of lactation and at peak lactation. On d 1 to 3 after calving differences in glucose, nonesterified fatty acids, and BHB indicated a greater reliance on mobilized lipid to export energy to peripheral tissues as BHB for greater parity cows. Differences in concentrations among parity groups were marked at times, for example >0.20 mM in Ca for parity 1 and 2 to parity ≥5 and >0.33 mM for all older parities compared with parity 1 for P on the day of calving. The marked increase suggests profound differences in metabolism with increased parity are probably influenced, in part, by increased production. We found marked differences in concentrations of metabolites with parity that are consistent with reduced reproduction, health, and body condition for higher parity cows. These unfavorable differences in metabolism in Ca, P, glucose, and cholesterol concentrations for higher parity cows also complement the often-substantial differences in disease risk with parity and suggest a need to carefully consider the parity structure in study design. Managers and advisors will need to consider methods to reduce risk of health disorders tailored to cows of different ages.

Metabolic alterations in dairy cattle with lameness revealed by untargeted metabolomics of dried milk spots using direct infusion-tandem mass spectrometry and the triangulation of multiple machine learning models.

Lameness is a major challenge in the dairy cattle industry in terms of animal welfare and economic implications. Better understanding of metabolic alteration associated with lameness could lead to early diagnosis and effective treatment, there-fore reducing its prevalence. To determine whether metabolic signatures associated with lameness could be discovered with untargeted metabolomics, we developed a novel workflow using direct infusion-tandem mass spectrometry to rapidly analyse (2 min per sample) dried milk spots (DMS) that were stored on commercially available Whatman® FTA® DMPK cards for a prolonged period (8 and 16 days). An orthogonal partial least squares-discriminant analysis (OPLS-DA) method validated by triangulation of multiple machine learning (ML) models and stability selection was employed to reliably identify important discriminative metabolites. With this approach, we were able to differentiate between lame and healthy cows based on a set of lipid molecules and several small metabolites. Among the discriminative molecules, we identified phosphatidylglycerol (PG 35:4) as the strongest and most sensitive lameness indicator based on stability selection. Overall, this untargeted metabolomics workflow is found to be a fast, robust, and discriminating method for determining lameness in DMS samples. The DMS cards can be potentially used as a convenient and cost-effective sample matrix for larger scale research and future routine screening for lameness.

Effects of oral treatment with chondroitin sulfate and glucosamine in an experimental model of metacarpophalangeal osteoarthritis in horses.

BACKGROUND: Combined chondroitin sulfate (CS) and glucosamine (GlcN) has been widely used in oral formulations to prevent and treat osteoarthritis. CS is effective for controlling pain in osteoarthritic patients, whereas GlcN can stimulate glycosaminoglycan synthesis, thus reducing extracellular matrix degradation. Although several studies have been published on this topic, the effectiveness of treatment with oral CS and GlcN remains uncertain. The objective of this study was to analyze the progression of experimentally induced osteoarthritis in horses and verify the effectiveness of an oral compound based on CS and GlcN to treat and/or modulate this disease. The study analyzed the metacarpophalangeal joint of the left thoracic limb of 16 horses divided into two groups, with eight horses treated with CS and GlcN in the treated group (GT) and eight untreated horses in the control group (GC). Chondral lesions were induced through arthroscopy, which was defined as time-point zero (T0). Physical, ultrasonographic, and radiographic examinations and synovial fluid biomarkers measurements were performed on days 0, 30, 60, 90, and 120. At the end of the experiment (T4), arthroscopy was performed again to macroscopically evaluate the joints and collect material for microscopic analysis. RESULTS: Significant differences were observed between groups in some evaluated parameters, such as visual lameness assessment, synovial concentrations of prostaglandin E2, and ultrasound examination. However, the GT still presented slightly improved results for joint flexion angle, analysis of lameness using sensors, and histopathological analysis of chondral repair tissue, however, without the statistical significance (p>0.05). CONCLUSIONS: The treatment was considered effective in the clinical modulation of experimental osteoarthritis, with improvement of some parameters in the GT. However, this type of treatment may not be entirely effective to change the catabolic process in articular cartilage and the progressive induced chondral damage.

Recombinant fibroblast growth factor-18 (sprifermin) enhances microfracture-induced cartilage healing.

Posttraumatic osteoarthritis is a disabling condition impacting the mostly young and active population. In the present study, we investigated the impact of intra-articular sprifermin, a recombinant truncated fibroblast growth factor 18, on the outcome of microfracture treatment, a widely used surgical technique to enhance cartilage healing at the site of injury. For this study, we created a cartilage defect and performed microfracture treatment in fetlock joints of 18 horses, treated joints with one of three doses of sprifermin (10, 30, or 100 µg) or with saline, hyaluronan, and evaluated animals functional and structural outcomes over 24 weeks. For primary outcome measures, we performed histological evaluations and gene expression analysis of aggrecan, collagen types I and II, and cartilage oligomeric matrix protein in three regions of interest. As secondary outcome measures, we examined animals' lameness, performed arthroscopic, radiographic, and computed tomography (CT) scan imaging and gross morphology assessment. We detected the highest treatment benefit following 100 µg sprifermin treatment. The overall histological assessment showed an improvement in the kissing region, and the expression of constitutive genes showed a concentration-dependent enhancement, especially in the peri-lesion area. We detected a significant improvement in lameness scores, arthroscopic evaluations, radiography, and CT scans following sprifermin treatment when results from three dose-treatment groups were combined. Our results demonstrated, for the first time, an enhancement on microfracture outcomes following sprifermin treatment suggesting a cartilage regenerative role and a potential benefit of sprifermin treatment in early cartilage injuries.

Long-term, multidomain analyses to identify the breed and allelic effects in MSTN-edited pigs to overcome lameness and sustainably improve nutritional meat production.

Beef and mutton production has been aided by breeding to integrate allelic diversity for myostatin (MSTN), but a lack of diversity in the MSTN germplasm has limited similar advances in pig farming. Moreover, insurmountable challenges with congenital lameness and a dearth of data about the impacts of feed conversion, reproduction, and meat quality in MSTN-edited pigs have also currently blocked progress. Here, in a largest-to-date evaluation of multiple MSTN-edited pig populations, we demonstrated a practical alternative edit-site-based solution that overcomes the major production obstacle of hindlimb weakness. We also provide long-term and multidomain datasets for multiple breeds that illustrate how MSTN-editing can sustainably increase the yields of breed-specific lean meat and the levels of desirable lipids without deleteriously affecting feed-conversion rates or litter size. Apart from establishing a new benchmark for the data scale and quality of genome-edited animal production, our study specifically illustrates how gene-editing site selection profoundly impacts the phenotypic outcomes in diverse genetic backgrounds.

Nerve growth factor in the equine joint.

Nerve growth factor (NGF) is a neurotrophin with many functions. In humans, it is involved in inflammation, nerve growth, apoptosis and pain signalling. Increased concentrations of NGF in synovial fluid has been shown in humans and dogs with osteoarthritis. Despite osteoarthritis being a common problem in horses, no studies have previously been published on NGF in the equine joint. The aim of this study was to quantify NGF in equine synovial fluid from healthy joints, acutely inflamed septic joints and joints with structural changes associated with osteoarthritis. A secondary aim was to identify the localisation of NGF and its two receptors, TrkA and p75NTR, in healthy and osteoarthritic articular cartilage. NGF concentrations in synovial fluid from osteoarthritic joints (n = 27), septic joints (n = 9) and healthy joints (n = 16) were determined by ELISA. In addition, articular cartilage from osteoarthritic and healthy joints was examined for NGF, TrkA and p75NTR using immunohistochemistry staining. NGF was present in equine synovial fluid and articular cartilage. Compared to synovial fluid from healthy joints, NGF concentration was higher in synovial fluid from joints with structural osteoarthritic changes (P = 0.032) or acute septic inflammation (P = 0.006). In articular cartilage with severe osteoarthritic changes, there was more abundant positive immunohistochemistry staining for NGF and its receptors than in normal articular cartilage. Further studies should focus on identifying precursor forms of NGF, and on receptor expression and downstream signalling of TrkA and P75NTR in health and disease.

Serum metabolomics identifies metabolite panels that differentiate lame dairy cows from healthy ones.

INTRODUCTION: Although much is known about lameness application of metabolomics technologies to better understanding its etiology and pathogenesis is of utmost interest. OBJECTIVES: The objective of this study was to investigate serum metabolite alterations in pre-lame, lame and post-lame dairy cows in order to identify potential screening serum metabolite biomarkers for lameness and better understand its pathobiology. METHODS: A combination of direct injection and tandem mass spectrometry (DI-MS/MS) with a reverse-phase liquid chromatography and tandem mass spectrometry (LC-MS/MS) analysis was performed in the serum of six cases of lameness and 20 healthy control cows (CON) at - 8 and - 4 weeks prepartum, at lameness diagnosis week, and at + 4 and + 8 weeks postpartum. RESULTS: Data indicated that pre-lame, lame, and post-lame cows experienced altered concentrations of multiple metabolites. It is interesting to note that throughout the 16-weeks of the study, 7 serum metabolites [e.g., diacyl-phosphatidylcholine (PC aa) C30:0, phosphatidylcholine acyl-alkyl (PC ae) C40:2, sphingomyelin (SM) (OH) C14:1, SM C18:0, isoleucine (Ile), leucine (Leu), and lysine (Lys)] differentiated CON cows from the lame ones. Furthermore, 4 metabolic pathways (i.e., Lys degradation, biotin metabolism, tryptophan (Trp) metabolism, and valine [(Val)-Leu-Ile degradation) were altered in cows with lameness during the onset and progression of the disease. CONCLUSION: Multiple metabolite and pathway alterations were identified in the serum of pre-lame, lame, and post-lame cows that through light into the pathobiology of the disease and that can be used as potential biomarker sets that can predict the risk of lameness in dairy cows.

Milk Metabotyping Identifies Metabolite Alterations in the Whole Raw Milk of Dairy Cows with Lameness.

The objective of this study was to evaluate whether whole raw milk originating from Holstein dairy cows affected by lameness alters its composition. A total of 20 healthy control cows and 6 cows diagnosed with lameness were selected out of 100 sampled cows in a nested case control study at 2 weeks postpartum, and whole raw milk samples were collected and analyzed with direct inject/liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance. In total, 168 metabolites were identified and quantified using an in-house mass spectrometry library. A total of 35 of the identified metabolites decreased versus control cows. Only two metabolites (i.e., sn-glycero-3-phosphocholine and phosphatidylethanolamine ae C42:1) were increased in the milk of lame cows. In conclusion, milk metabotyping of lame cows revealed significant changes in multiple milk components, including amino acids, lipids, and biogenic amines. Most of the milk compounds identified as altered were lowered, suggesting deflection of nutrients from the mammary gland to the host needs for healing lameness-associated pathological processes.

Proteomic profiling of proteins in the dorsal horn of the spinal cord in dairy cows with chronic lameness.

Chronic lameness affects bovine welfare and has a negative economic impact in dairy industry. Moreover, due to the translational gap between traditional pain models and new drugs development for treating chronic pain states, naturally occurring painful diseases could be a potential translational tool for chronic pain research. We therefore employed liquid chromatography tandem mass spectrometry (LC-MS/MS) to stablish the proteomic profile of the spinal cord samples from lumbar segments (L2-L4) of chronic lame dairy cows. Data were validated and quantified through software tool (Scaffold® v 4.0) using output data from two search engines (SEQUEST® and X-Tandem®). Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) analysis was performed to detect proteins interactions. LC-MS/MS identified a total amount of 177 proteins; of which 129 proteins were able to be quantified. Lame cows showed a strong upregulation of interacting proteins with chaperone and stress functions such as Hsp70 (p < 0.006), Hsc70 (p < 0.0079), Hsp90 (p < 0.015), STIP (p > 0.0018) and Grp78 (p <0.0068), and interacting proteins associated to glycolytic pathway such as; γ-enolase (p < 0.0095), α-enolase (p < 0.013) and hexokinase-1 (p < 0.028). It was not possible to establish a clear network of interaction in several upregulated proteins in lame cows. Non-interacting proteins were mainly associated to redox process and cytoskeletal organization. The most relevant down regulated protein in lame cows was myelin basic protein (MBP) (p < 0.02). Chronic inflammatory lameness in cows is associated to increased expression of stress proteins with chaperone, metabolism, redox and structural functions. A state of endoplasmic reticulum stress and unfolded protein response (UPR) might explain the changes in protein expression in lame cows; however, further studies need to be performed in order to confirm these findings.

Cardiac vagal tone, plasma cortisol, and dehydroepiandrosterone response to an ACTH challenge in lame and nonlame dairy cows.

We studied the adrenocortical and vagal tone responses to a single ACTH challenge in lame (n = 9) vs nonlame (n = 9) dairy cows. Cows were paired according to parity, days in milk, and milk yield. Plasma cortisol and dehydroepiandrosterone concentrations and cardiac vagal tone response (high-frequency component of heart rate variability) were compared after intravenous ACTH administration. Baseline, minimum or maximum, amplitude of the response and area under the response curve were compared. No difference was detected between groups in the cortisol response. Dehydroepiandrosterone was irresponsive to ACTH treatment, and concentrations did not differ between lame and nonlame cows. Vagal tone decreased in response to the ACTH treatment. High frequency component of heart rate variability was lower in the lame group at all sampling times. Lameness was associated with delayed return to baseline. We concluded that the adrenal response capacity is not influenced by lameness, which supports the concept of lameness being a chronic intermittent rather than a chronically persistent stressor. Dehydroepiandrosterone concentrations were not proven to be useful indicators of hypothalamus-pituitary axis dysfunction in cattle. A decreased vagal contribution to heart rate variability-possibly coupled with increased sympathetic modulation-was observed in lame cows, which suggests that lameness affects the mechanisms underlying the action of ACTH on cardiovascular activity.

Changes in saliva proteins in two conditions of compromised welfare in pigs: An experimental induced stress by nose snaring and lameness.

The aim of this study was to identify biological pathways and proteins differentially expressed in saliva of pigs in two conditions of compromised welfare: an acute stress consisting of restraint with a nose snare and in pigs with lameness which is a highly frequent problem in the swine industry. For this purpose, high-resolution quantitative proteomics based on Tandem Mass Tags labelling was used. Four proteins showed significant differences in the conditions of compromised welfare, namely cornulin, the heat shock protein 27 and lactate dehydrogenase (LDH), that showed significant increases, whereas immunoglobulin J chain showed a significant decrease. LDH, which was the protein that showed the highest differences, was selected for validation and clinical evaluation as a diagnostic biomarker. Significant changes in this protein were observed between pigs restrained with a nose snare and pigs with lameness compared with healthy pigs when measured with available commercial assays in a larger population of pigs. In conclusion, this study reports that in situations of compromised welfare on farm, such as acute stress and lameness in pigs, there are changes in proteins and metabolic pathways in saliva, and describes a series of proteins that could potentially be used as biomarkers for both short term acute stress and longer term chronic stress of lameness. These biomarkers would have the advantage of being measured in saliva by a noninvasive and not stressful collection sampling procedure.

Lameness and lying behavior in grazing dairy cows.

Lameness is a serious welfare issue for dairy cows. To date, the majority of studies have focused on its effect on health and behavior at the herd level. The objectives of this study were to identify (1) between-cow and (2) within-cow changes in lying behavior associated with consistent and changing lameness status in grazing dairy cows. Previous studies of lying behavior in grazing dairy cows have not considered the effect of precipitation, so a third aim was to determine the effect of precipitation on lying behavior. A total of 252 dairy cows from 6 pasture-based farms in southern Brazil were gait scored weekly to assess lameness using a 5-point scale [1-5, numerical rating score (NRS)] for 4 consecutive weeks. Cows were considered to have consistent lameness if they were scored as lame (NRS ≥3) on each of the 4 visits and considered to have a changing lameness status if scored as being nonlame (NRS <3) on at least 1 of the 4 visits. Cows classified as having a changing lameness status were further classified as developed, recovered, or inconsistent. Lying behavior (daily lying time, mean lying bout duration, and daily number of lying bouts) was recorded continuously for 3 wk using leg-mounted accelerometers. Cow-level variables included parity, days in milk, and body condition score. Regional precipitation and temperature were recorded hourly. Because only 1 primiparous cow was identified as lame at each of the 4 visits, the between-cow analysis of lameness was run on multiparous cows only. The overall prevalence of clinical lameness on the first visit was 39%, with development and recovery rates of 16 and 10% over the 4 visits, respectively. The between-cow effect of consistent lameness status on daily lying time and number of lying bouts was dependent on precipitation; consistently lame cows had reduced lying time and lying bouts on days with rain compared with days without rain. There was no within-cow effect of changing lameness status on any of the lying behaviors. Precipitation was associated with decreased daily lying time, increased mean lying bout duration, and decreased daily number of lying bouts. The results of this research provide the first evidence that the effect of consistent lameness status on lying behavior is associated with rainfall in grazing dairy cows. Future work measuring lying behavior of grazing dairy cows should include precipitation as a covariate.

Correlation between synovial fluid cytokine concentrations and limb function in normal dogs and in dogs with lameness from spontaneous osteoarthritis.

OBJECTIVE: To determine the relationship between synovial biomarker concentrations and severity of lameness and to assess the ability to differentiate normal from osteoarthritic joints with synovial biomarker concentrations. STUDY DESIGN: Prospective clinical study. SAMPLE POPULATION: Twelve hounds with no evidence of osteoarthritis (OA) and 27 client-owned dogs with unilateral lameness and joint pain in a single joint from naturally occurring OA. METHODS: Enrollment in the OA group required a history of lameness, radiographic evidence of OA on orthogonal joint radiographs, and ≥6% gait asymmetry between contralateral limbs. The concentrations of 14 synovial OA biomarkers in synovial samples obtained after gait analysis were measured with enzyme-linked immunosorbent assays and compared between normal and OA joints. RESULTS: Concentrations of monocyte chemoattractant protein (MCP)-1, substance P, interleukin (IL)-6, IL-8, KC-like, matrix metalloproteinase (MMP)-1, and MMP-3 were greater (P ≤ .05) in OA than in normal joints. The concentrations of bradykinin and tissue inhibitors of metalloproteinase-4 were decreased in OA compared with normal joints. Monocyte chemoattractant protein 1 was identified as the most accurate marker to distinguish OA from normal joints. No correlation was detected between any OA biomarker concentration, individually or in combination, and severity of gait asymmetry at the walk. CONCLUSION: Differences in proinflammatory and anti-inflammatory biomarkers were detected between OA and normal joints, but no relationship was identified between biomarker concentrations and gait asymmetry in dogs with OA. CLINICAL IMPACT: This information will help guide future studies to elucidate how factors such as disease chronicity, severity, and etiology affect these relationships.

Space-use patterns highlight behavioural differences linked to lameness, parity, and days in milk in barn-housed dairy cows.

Lameness is a key health and welfare issue affecting commercial herds of dairy cattle, with potentially significant economic impacts due to the expense of treatment and lost milk production. Existing lameness detection methods can be time-intensive, and under-detection remains a significant problem leading to delayed or missed treatment. Hence, there is a need for automated monitoring systems that can quickly and accurately detect lameness in individual cows within commercial dairy herds. Recent advances in sensor tracking technology have made it possible to observe the movement, behaviour and space-use of a range of animal species over extended time-scales. However, little is known about how observed movement behaviour and space-use patterns in individual dairy cattle relate to lameness, or to other possible confounding factors such as parity or number of days in milk. In this cross-sectional study, ten lame and ten non-lame barn-housed dairy cows were classified through mobility scoring and subsequently tracked using a wireless local positioning system. Nearly 900,000 spatial locations were recorded in total, allowing a range of movement and space-use measures to be determined for each individual cow. Using linear models, we highlight where lameness, parity, and the number of days in milk have a significant effect on the observed space-use patterns. Non-lame cows spent more time, and had higher site fidelity (on a day-to-day basis they were more likely to revisit areas they had visited previously), in the feeding area. Non-lame cows also had a larger full range size within the barn. In contrast, lame cows spent more time, and had a higher site-fidelity, in the cubicle (resting) areas of the barn than non-lame cows. Higher parity cows were found to spend more time in the right-hand-side area of the barn, closer to the passageway to the milking parlour. The number of days in milk was found to positively affect the core range size, but with a negative interaction effect with lameness. Using a simple predictive model, we demonstrate how it is possible to accurately determine the lameness status of all individual cows within the study using only two observed space-use measures, the proportion of time spent in the feeding area and the full range size. Our findings suggest that differences in individual movement and space-use behaviour could be used as indicators of health status for automated monitoring within a Precision Livestock Farming approach, potentially leading to faster diagnosis and treatment, and improved animal welfare for dairy cattle and other managed animal species.

Claw horn lesions in mid-lactation primiparous dairy cows under pasture-based systems: Association with behavioral and metabolic changes around calving.

The objectives of this study were to describe the lying behavior of primiparous dairy cows under pasture-based systems during the pre- and postcalving period and characterize the association of lying behavior and analytes related to energy metabolism during this period with claw horn disruption lesion development later in lactation. Our convenience sample included 39 primiparous Holstein cows from 3 commercial farms that were assessed for body condition score (BCS; 5-point scale, 0.25-point increments) and had blood collected at wk -3, -2, -1, 1, 2, and 3 relative to calving date. Blood samples were assayed for nonesterified fatty acids, ß-hydroxybutyrate (BHB), and cholesterol concentrations. Electronic data loggers (HOBO Pendant G Acceleration, Onset Computer Corporation, Bourne, MA) recorded lying behavior at 1-min intervals from 3 wk before calving to 3 wk after calving. Starting at 4 wk after calving and until 16 wk after calving, cows were examined for claw lesions at approximately 4-wk intervals. Sole lesions and white line lesions were scored on a 0 to 10 scale. Of the 39 primiparous cows, 19 cows scored 0 at all exams during the entire study period and 20 cows had at least 1 severe lesion (score ≥4) between 8 and 16 wk after calving. Time spent lying before calving averaged 10.3 ± 0.3 h/d, but declined to 7.3 ± 0.3 h/d after calving (least squares means ± standard error). At calving, we noted an increase in the number of lying bouts (12.9 ± 0.45 bouts/d) compared with the pre- and postcalving averages of 11.6 (±0.53) and 9.1 (±0.47) bouts, respectively. Cows that developed claw lesions later in mid lactation spent less time lying down than cows without lesions during wk 3 after calving compared with healthy cows (7.29 ± 0.22 vs. 8.51 ± 0.16 h/d). Lesion cows had fewer lying bouts per day, and these bouts were of longer duration than no-lesion cows after calving. Increased odds of lesion were found to be associated with shorter lying times and fewer number of lying bouts during wk 3 (odds ratio = 1.23). Nonesterified fatty acids (747 ± 58 vs. 990 ± 86.85 µmol/L) and BHB (0.77 ± 0.06 vs. 0.60 ± 0.04 mmol/L) concentrations during wk 1 were greater in cows that developed claw lesions relative to cows that did not develop lesions. The BHB concentrations also remained higher in wk 2 for cows that developed claw lesions (0.63 ± 0.04 vs. 0.46 ± 0.03 mmol/L) compared with cows that did not develop any lesions. Cows that developed lesions experienced greater losses in BCS from wk -3 to 3 than cows without lesions (0.74 ± 0.01 and 0.61 ± 0.01 BCS change, respectively). In summary, changes in lying behavior and energy metabolic status after calving were associated with claw horn disruption lesions in mid-lactation primiparous cows under pasture-based systems.

Effects of medical ozone upon healthy equine joints: Clinical and laboratorial aspects.

OBJECTIVE: The aim of this study was to verify whether transient inflammatory reactions induced by intra-articular medicinal ozone administration affect joint components, by in vivo evaluation of inflammatory (prostaglandin E2, Substance P, Interleukin-6, Interleukine-1, Tumor Necrosis Factor), anti-inflammatory (Interleukin-10) and oxidative (superoxide dismutase activity and oxidative burst) biomarkers and extracellular matrix degradation products (chondroitin sulphate and hyaluronic acid) in synovial fluid. METHODS: The effects of medicinal ozone were analyzed at two ozone concentrations (groups A and B, 20 and 40 µg/ml, respectively), using oxygen-injected joints as controls (group C); each group received ten treatments (15 ml gas per treatment). Physical evaluation, evaluation of lameness, ultrasonography, and synovial fluid analysis were performed. RESULTS: All joints presented mild and transient effusion throughout the study. Group B exhibited the highest lameness score on day 14 (P<0.05), detected by the lameness measurement system, probably because of the higher ozone concentration. All groups exhibited increased ultrasonography scores on day 14 (P < 0.05). Groups A and B exhibited increased proteins concentrations on day 21 (P<0.05). There was no change in hyaluronic acid concentration or the percentage of high-molecular weight hyaluronic acid throughout the experiment. Chondroitin sulfate concentrations decreased in group B, and did not change in group A and C, indicating that neither treatment provoked extracellular matrix catabolism. Cytokine and eicosanoid concentrations were not significantly changed. CONCLUSIONS: The ozonetherapy did not cause significant inflammation process or cartilage degradation, therefore, ozonetherapy is safe at both evaluated doses.

Short communication: Is hair cortisol a potential indicator for stress caused by chronic lameness in dairy cows?

The objective of this study was to evaluate hair cortisol concentration as an indicator for stress caused by chronic lameness in dairy cows. Sixty-eight cows were scored for lameness for 4 consecutive weeks. The hair of the tail switch was clipped at the beginning of the study and regrown hair was clipped after 4 wk. Hair samples were analyzed for cortisol concentration. Animals with 2 consecutive locomotion scores ≥3 or with an overall mean score >1.5 were classified as lame. After pair matching lame and nonlame cows, considering days in milk, lactation number, and milk yield, and excluding cows with less than 20 mg hair sample for analysis, 21 lame and 21 nonlame cows were included in the analysis. The mean hair cortisol concentration in this study was 2.32 ± 0.35 pg/mg (mean ± standard deviation). Cortisol concentration from hair regrown in the study period was 2.38 ± 0.95 and 2.26 ± 1.35 pg/mg for lame and nonlame cows (n = 21), respectively; we found no difference in mean cortisol level of primiparous and multiparous cows. Based on these data, hair cortisol concentration was not a useful indicator to differentiate cows with chronic lameness and healthy cows.

Changes in concentrations of haemostatic and inflammatory biomarkers in synovial fluid after intra-articular injection of lipopolysaccharide in horses.

BACKGROUND: Septic arthritis is a common and potentially devastating disease characterized by severe intra-articular (IA) inflammation and fibrin deposition. Research into equine joint pathologies has focused on inflammation, but recent research in humans suggests that both haemostatic and inflammatory pathways are activated in the joint compartment in arthritic conditions. The aim of this study was to characterize the IA haemostatic and inflammatory responses in horses with experimental lipopolysaccharide (LPS)-induced joint inflammation. Inflammation was induced by IA injection of LPS into one antebrachiocarpal joint of six horses. Horses were evaluated clinically with subjective grading of lameness, and blood and synovial fluid (SF) samples were collected at post injection hours (PIH) -120, -96, -24, 0, 2, 4, 8, 16, 24, 36, 48, 72 and 144. Total protein (TP), white blood cell counts (WBC), serum amyloid A (SAA), haptoglobin, iron, fibrinogen, thrombin-antithrombin (TAT) and d-dimer concentrations were assessed in blood and SF. RESULTS: Intra-articular injection of LPS caused local and systemic signs of inflammation including increased rectal temperature, lameness and increased joint circumference and skin temperature. Most of the biomarkers (TP, WBC, haptoglobin, fibrinogen and TAT) measured in SF increased quickly after LPS injection (at PIH 2-4), whereas SAA and d-dimer levels increased more slowly (at PIH 16 and 144, respectively). SF iron concentrations did not change statistically significantly. Blood WBC, SAA, haptoglobin and fibrinogen increased and iron decreased significantly in response to the IA LPS injection, while TAT and d-dimer concentrations did not change. Repeated pre-injection arthrocenteses caused significant changes in SF concentrations of TP, WBC and haptoglobin. CONCLUSION: Similar to inflammatory joint disease in humans, joint inflammation in horses was accompanied by an IA haemostatic response with changes in fibrinogen, TAT and d-dimer concentrations. Inflammatory and haemostatic responses were induced simultaneously and may likely interact. Further studies of interactions between the two responses are needed for a better understanding of pathogenesis of joint disease in horses. Knowledge of effects of repeated arthrocenteses on levels of SF biomarkers may be of value when markers are used for diagnostic purposes.

A proteomics-based identification of putative biomarkers for disease in bovine milk.

The objective of this study was to identify and characterize potential biomarkers for disease resistance in bovine milk that can be used to indicate dairy cows at risk to develop future health problems. We selected high- and low-resistant cows i.e. cows that were less or more prone to develop diseases according to farmers' experience and notifications in the disease registration data. The protein composition of milk serum samples of these high- and low-resistant cows were compared using NanoLC-MS/MS. In total 78 proteins were identified and quantified of which 13 were significantly more abundant in low-resistant cows than high-resistant cows. Quantification of one of these proteins, lactoferrin (LF), by ELISA in a new and much larger set of full fat milk samples confirmed higher LF levels in low- versus high-resistant cows. These high- and low-resistant cows were selected based on comprehensive disease registration and milk recording data, and absence of disease for at least 4 weeks. Relating the experienced diseases to LF levels in milk showed that lameness was associated with higher LF levels in milk. Analysis of the prognostic value of LF showed that low-resistant cows with higher LF levels in milk had a higher risk of being culled within one year after testing than high-resistant cows. In conclusion, LF in milk are higher in low-resistant cows, are associated with lameness and may be a prognostic marker for risk of premature culling.

Differences in leukocyte profile, gene expression, and metabolite status of dairy cows with or without sole ulcers.

Sole ulcers are one of the most severe pathologies causing lameness in dairy cows and are associated with abnormal behavior and impaired production performance. However, little is known about how or whether lameness caused by sole ulcers affects the cow systemically. This study compared hematology profile, leukocyte gene expression, and physiological responses [metabolite, cortisol, the endogenous steroid hormone dehydroepiandrosterone (DHEA), and haptoglobin concentrations] of cows with sole ulcers and healthy cows. Twelve clinically lame cows (lame) were identified as having at least one sole ulcer and no other disorder, and matched with a cow that had good locomotion and no disorders (sound), using days in milk, liveweight, body condition score, and diet. Blood samples were taken from all 24 cows within 24h of sole ulcer diagnosis. Leukocyte counts were obtained using an automated cell counter, cortisol and DHEA concentration by ELISA, and plasma haptoglobin, urea, total protein, creatine kinase, and glucose were analyzed on an Olympus analyzer. Expression of 16 genes associated with lameness or stress were estimated using reverse transcription-PCR. Data were analyzed using the MIXED procedure in SAS software (version 9.3; SAS Institute Inc., Cary, NC). Lame cows had a higher neutrophil percentage, a numerically lower lymphocyte percentage, and tended to have a higher neutrophil:lymphocyte ratio than sound cows. Serum cortisol and DHEA concentrations were higher in lame than in sound cows. Lame cows also tended to have higher haptoglobin and glucose levels than sound, as well as higher protein yet lower urea levels. Sound cows tended to have higher relative expression of the gene coding for colony-stimulating factor 2 than lame, but in all other cases where differences were detected in cytokine gene expression (IL-1α, IL-1ß, CXCL8, and IL-10), relative gene expression in sound cows tended to be, or was, lower than in lame. Relative expression of MMP-13, GR-α, Fas, haptoglobin, and CD62L were, or tended to be, higher in lame than sound cows. A high neutrophil:lymphocyte ratio in combination with higher cortisol levels in cows with ulcers is indicative of physiological stress. Moreover, increased DHEA and a higher cortisol:DHEA ratio, as well as a tendency for higher haptoglobin levels and increased haptoglobin mRNA expression, are indicative of systemic inflammation. Increased cytokine mRNA expression indicates activation of the immune system compared with healthy cows. Increased expression of MMP-13 mRNA has been found in cows with impaired locomotion and thus could be implicated in development of claw horn disorders.