Sex-specific effects of calving season on joint health and biomarkers in Montana ranchers

Abstract

Background: Agricultural workers have a higher incidence of osteoarthritis (OA), but the etiology behind this phenomenon is unclear. Calving season, which occurs in mid- to late-winter for ranchers, includes physical conditions that may elevate OA risk. Our primary aim was to determine whether OA biomarkers are elevated at the peak of calving season compared to pre-season, and to compare these data with joint health survey information from the subjects. Our secondary aim was to detect biomarker differences between male and female ranchers.

Methods: During collection periods before and during calving season, male (n = 28) and female (n = 10) ranchers completed joint health surveys and provided samples of blood, urine, and saliva for biomarker analysis. Statistical analyses examined associations between mean biomarker levels and survey predictors. Ensemble cluster analysis identified groups having unique biomarker profiles.

Results: The number of calvings performed by each rancher positively correlated with plasma IL-6, serum hyaluronic acid (HA) and urinary CTX-I. Thiobarbituric acid reactive substances (TBARS), a marker of oxidative stress, was significantly higher during calving season than pre-season and was also correlated with ranchers having more months per year of joint pain. We found evidence of sexual dimorphism in the biomarkers among the ranchers, with leptin being elevated and matrix metalloproteinase-3 diminished in female ranchers. The opposite was detected in males. WOMAC score was positively associated with multiple biomarkers: IL-6, IL-2, HA, leptin, C2C, asymmetric dimethylarginine, and CTX-I. These biomarkers represent enzymatic degradation, inflammation, products of joint destruction, and OA severity.

Conclusions: The positive association between number of calvings performed by each rancher (workload) and both inflammatory and joint tissue catabolism biomarkers establishes that calving season is a risk factor for OA in Montana ranchers. Consistent with the literature, we found important sex differences in OA biomarkers, with female ranchers showing elevated leptin, whereas males showed elevated MMP-3.

Keywords: Biomarkers; Calving season; Inflammation; Joint pain; Osteoarthritis; Ranching; Sex differences.

Fig. 1

Study design to examine biomarkers and OA symptoms in ranchers before and during calving season

Fig. 2

Ensemble clustergrams by subject (top) and principal component analysis (PCA) loading plots (bottom). A Pre-season, B In-season, C Full study, and D Seasonal difference (in—pre). Ensemble clustering was performed in Python for pre-season, in-season, both seasons (“full study”), and seasonal difference (in-season – pre-season) of biomarker levels and rancher subject IDs using the single, complete, average, and ward linkage functions with the Euclidean distance, and the single, complete, and average linkage functions with the squared-Euclidean, cosine and Chebyshev distance. PCA biplots were generated in MATLAB to identify biomarkers with relatively large leverage within the dataset (“distinct”) for profiling and post-hoc statistics. In-season biomarker responses were more distinct than pre-season, with rancher subjects clustering together in more unique subclusters during calving season. MMP-3 and leptin were distinct in all PCAs, while C2C was only distinct during pre-season

Fig. 3

Distinct biomarker responses predicted by survey variables. A Pre-season, B In-season, C Full study, and D Seasonal difference (in – pre). One-way ANOVAs with Tukey’s multiple comparison were performed post-hoc in Minitab on distinct biomarkers identified by ensemble clustering and PCA. Minitab used adjusted p-values and f-values on a 0.05 significance level

Fig. 4

Sex-specific biomarker responses predicted by joint health outcomes and demographic information. A Pre-season, B In-season, and C Full study. Interval plots were produced in Minitab with 95% confidence intervals for the mean of each predictor level, pooled standard deviation for the intervals, and post-hoc ANOVA with Tukey’s group comparisons on a 0.05 significance level of adjusted p-values. Letters denote Tukey’s multiple comparison groups, e.g., statistically significant differences between groups, such as A vs. B but not A vs. AB. A subset of females reporting higher WOMAC scores demonstrated statistically significant elevations to in-season leptin and pre-season C2C levels. In C), pre-season MMP-3 responses were rank ordered by intensity with the upper and lower third profiled due to split responses of BMI. Female ranchers in the lowest third for pre-season MMP-3 levels were also in the highest third for pre-season leptin levels, and the opposite was true for males. Leptin trends may inversely correlate with MMP-3

Fig. 5

Significant predictors for seasonal biomarker and survey responses in a sample of ranchers. A Pre-season plasma biomarkers from one-way ANOVA, B Pre-season serum and urine biomarkers from one-way ANOVA, C Pre-season survey associations from ordinal logistic regression, D In-season plasma biomarkers from one-way ANOVA, E In-season serum and urine biomarkers form one-way ANOVA, and F In-season survey associations from ordinal logistic regression. One-way ANOVA and ordinal logistic regression were performed in Minitab on a 0.05 significance level