Assessing the Relationship Between Beef Calf Grimace Scores and Circulating Haptoglobin During Castration

Alternative methods of assessing pain, such as facial grimace analysis, are essential for those that do not communicate verbally. Pain can be self-reported, but quantification through observation is essential for non-verbal populations (e.g., human infants). These observer-reported grimace scales have been extended, adapted, and validated for various non-human animals (e.g., mice, cats, pigs), but few studies have focused on applying and validating these scales in cattle. Accordingly, the objective of this research was to explore the relationship between facial grimace scores and circulating haptoglobin to support validation of an emerging pain scale in beef calves. Haptoglobin is an inflammation-induced plasma protein that plays an important regulatory role within acute-phase reactions, which occur in response to acute tissue damage. Beef calves (n = 10) were separated from their dam and entered a squeeze chute where their testes were manually stimulated to simulate a sham castration. After 40 mins, they re-entered the chute and blood was collected, then they were continuously video recorded as they exited. After 72 h during which calves grazed with their dam (to return to baseline), calves underwent the same procedures as described above excepting a surgical castration. Still images were captured from video recordings at both timepoints, and trained observers (n = 3) scored six facial action units (FAU) to calculate overall facial grimace score. Intraclass correlation was 1.0 for each observer, and interclass correlation was 0.78. Plasma was separated from other blood components via centrifugation, and circulating haptoglobin was analyzed using a Novus Colorimetric ELISA Kit. Absorbance was measured spectrophotometrically and sample concentrations were calculated from a standard curve determined through best-fit regression analysis (r2=0.97). Data were analyzed with SAS using a repeated-measures mixed model to test the effect of procedure (sham versus real) on overall facial grimace score and haptoglobin, with calf as the subject. Pearson correlation analysis was conducted to explore the relationship between overall facial grimace score and haptoglobin concentrations. Facial grimace score did not differ (P = 0.15) between sham (0.29±0.03) versus actual castration (0.23±0.03), but circulating haptoglobin was affected (P ≤ 0.01) such that concentrations were 21.4 µg/mL for sham and 48.5 µg/mL for real castration. There was no correlation between overall facial grimace score and circulating haptoglobin (r=-0.09, P = 0.43). These findings suggest that the current facial grimace scale may not accurately reflect pain experienced by beef calves during castration but is perhaps affected by stress resulting from handling or separation from the dam. Further, there is no correlation between circulating haptoglobin and the established facial grimace scale for calves. More research is needed to refine and validate facial grimace scales in calves, ideally with circulating biomarkers known to be associated with pain.