Identification of Predictive Biomarkers of Lameness in Transition Dairy Cows.

The aim of this study was to identify with a high level of confidence metabolites previously identified as predictors of lameness and understand their biological relevance by carrying out pathway analyses. For the dairy cattle sector, lameness is a major challenge with a large impact on animal welfare and farm economics. Understanding metabolic alterations during the transition period associated with lameness before the appearance of clinical signs may allow its early detection and risk prevention. The annotation with high confidence of metabolite predictors of lameness and the understanding of interactions between metabolism and immunity are crucial for a better understanding of this condition. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with authentic standards to increase confidence in the putative annotations of metabolites previously determined as predictive for lameness in transition dairy cows, it was possible to identify cresol, valproic acid, and gluconolactone as L1, L2, and L1, respectively which are the highest levels of confidence in identification. The metabolite set enrichment analysis of biological pathways in which predictors of lameness are involved identified six significant pathways (p < 0.05). In comparison, over-representation analysis and topology analysis identified two significant pathways (p < 0.05). Overall, our LC-MS/MS analysis proved to be adequate to confidently identify metabolites in urine samples previously found to be predictive of lameness, and understand their potential biological relevance, despite the challenges of metabolite identification and pathway analysis when performing untargeted metabolomics. This approach shows potential as a reliable method to identify biomarkers that can be used in the future to predict the risk of lameness before calving. Validation with a larger cohort is required to assess the generalization of these findings.

Predictive modelling of deviation from expected milk yield in transition cows on automatic milking systems.

The transition period is a pivotal time in the production cycle of the dairy cow. It is estimated that between 30% and 50% of all cows experience metabolic or infectious disease during this time. One of the most common and economically consequential effects of disease during the transition period is a reduction in early lactation milk production. This has led to the utilisation of deviation from expected milk yield in early lactation as a proxy measure for transition health. However, to date, this analysis has been used exclusively for the retrospective assessment of transition cow health. Statistical models capable of predicting deviations from expected milk yield may allow producers to proactively manage animals predicted to suffer negative deviations in early lactation milk production. The objective of this retrospective cohort study was first, to explore the accuracy with which cow-level production and behaviour data collected on automatic milking systems (AMS) from 1-3 days in milk (DIM) can predict deviation from expected 30-day cumulative milk yield in multiparous cows. And second, to assess the accuracy with which predicted yield deviations can classify cows into groups which may facilitate improved transition management. Production, rumination, and physical activity data from 31 commercial AMS were accessed. A 3-step analytical procedure was then conducted. In Step 1, expected cumulative yield for 1-30 DIM for each individual cow-lactation was calculated using a mixed effect linear model. In Step 2, 30-Day Yield Deviation (YD) was calculated as the difference between observed and expected cumulative yield. Lactations were then assigned to one of three groups based on their YD, RED Group (0% YD). In Step 3, yield, rumination, and physical activity data from days 1-3 in lactation were used to predict YD using machine learning models. Following external validation, YD was predicted across the test data set with a mean absolute error of 9%. Categorisation of animals suffering large negative deviations (RED group) was achieved with a specificity of 99%, sensitivity of 35%, and balanced accuracy of 67%. Our results suggest that milk yield, rumination and physical activity patterns expressed by dairy cows from 1-3 DIM have utility in the prediction of deviation from expected 30-day cumulative yield. However, these predictions currently lack the sensitivity required to classify cows reliably and completely into groups which may facilitate improved transition cow management.

Predicting lameness in dairy cattle using untargeted liquid chromatography-mass spectrometry-based metabolomics and machine learning.

Lameness in dairy cattle is a highly prevalent condition that impacts on the health and welfare of dairy cows. Prompt detection and implementation of effective treatment is important for managing lameness. However, major limitations are associated with visual assessment of lameness, which is the most commonly used method to detect lameness. The aims of this study were to investigate the use of metabolomics and machine learning to develop novel methods to detect lameness. Untargeted metabolomics using liquid chromatography-mass spectrometry (LC-MS) alongside machine learning models and a stability selection method were utilized to evaluate the predictive accuracy of differences in the metabolomics profile of first-lactation dairy cows before (during the transition period) and at the time of lameness (based on visual assessment using the 0-3 scale of the Agriculture and Horticulture Development Board). Urine samples were collected from 2 cohorts of dairy heifers and stored at -86°C before analysis using LC-MS. Cohort 1 (n = 90) cows were recruited as current first-lactation cows with weekly mobility scores recorded over a 4-mo timeframe, from which newly lame and nonlame cows were identified. Cohort 2 (n = 30) cows were recruited within 3 wk before calving, and lameness events (based on mobility score) were recorded through lactation until a minimum of 70 d in milk (DIM). All cows were matched paired by DIM ± 14 d. The median DIM at lameness identification was 187.5 and 28.5 for cohort 1 and 2, respectively. The best performing machine learning models predicted lameness at the time of lameness with an accuracy of between 81 and 82%. Using stability selection, the prediction accuracy at the time of lameness was 80 to 81%. For samples collected before and after calving, the best performing machine learning model predicted lameness with an accuracy of 71 and 75%, respectively. The findings from this study demonstrate that untargeted LC-MS profiling combined with machine learning methods can be used to predict lameness as early as before calving and before observable changes in gait in first-lactation dairy cows. The methods also provide accuracies for detecting lameness at the time of observable changes in gait of up to 82%. The findings demonstrate that these methods could provide substantial advancements in the early prediction and prevention of lameness risk. Further external validation work is required to confirm these findings are generalizable; however, this study provides the basis from which future work can be conducted.

Use of qualitative behavioural assessment to investigate affective states of housed dairy cows under different environmental conditions.

In addition to the reduction of suboptimal welfare, there is now a need to provide farmed animals with positive opportunities to provide confidence that they have experienced a life worth living. Diversification of the environment through environmental enrichment strategies is one suggested avenue for providing animals with opportunities for positive experiences. The provision of more stimulating environmental conditions has been widely implemented in other animal production industries, based on evidenced welfare benefits. However, the implementation of enrichment on dairy farms is limited. In addition to this, the relationship between enrichment and dairy cows' affective states is an under-researched area. One specific welfare benefit of enrichment strategies which has been observed in a number of species, is increased affective wellbeing. This study investigated whether the provision of different forms of environmental enrichment resources would impact the affective states of housed dairy cows. This was measured by Qualitative Behavioural Assessment, currently a promising positive welfare indicator. Two groups of cows experienced three treatment periods; (i) access to an indoor novel object, (ii) access to an outdoor concrete yard and (iii) simultaneous access to both resources. Principal component analysis was used to analyse qualitative behavioural assessment scores, which yielded two principal components. The first principal component was most positively associated with the terms "content/relaxed/positively occupied" and had the most negative associations with the terms 'fearful/bored'. A second principal component was most positively associated with the terms "lively/inquisitive/playful" and was most negatively associated with the terms "apathetic/bored". Treatment period had a significant effect on both principal components, with cows being assessed as more content, relaxed and positively occupied and less fearful and bored, during periods of access to additional environmental resources. Similarly, cows were scored as livelier, more inquisitive and less bored and apathetic, during treatment periods compared to standard housing conditions. Concurrent with research in other species, these results suggest that the provision of additional environmental resources facilitates positive experiences and therefore enhanced affective states for housed dairy cows.

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.