Effects of Wash Protocol and Contamination Level on Concentrations of Cortisol and Dehydroepiandrosterone (DHEA) in Swine Hair

SIMPLE SUMMARY: Quantifying the hormones cortisol and dehydroepiandrosterone (DHEA) in swine hair is of increasing interest to evaluate long-term stress and resilience. Because swine hair is often contaminated with varying amounts of excrement, it needs to be decontaminated with a solvent prior to hair hormone extraction to rid the hair of potential external contaminants. However, it is unknown how contamination influences hair hormone concentrations, and if current wash protocols are effective in removing contamination. The goals of this study were thus, to determine if wash solvents (methanol versus isopropanol), contamination level (none, mild, or severe), and the number of washes (one, three, or five) influenced hair cortisol and DHEA concentrations. This study showed that hair cortisol, but not DHEA concentrations were reduced when external contamination was present, and that methanol was more effective at removing external contamination compared to isopropanol. There were also decreasing concentrations of cortisol and DHEA within the hair and wash solvent with an increasing number of washes. Thus, it is recommended not to use contaminated hair for hormone analysis, and to wash swine hair with a minimum of three 3 min methanol washes prior to analysis. ABSTRACT: The effect of washing procedure and contamination level on the concentrations of cortisol and dehydroepiandrosterone (DHEA) in swine hair was explored over two studies. Hair shaved from finisher pigs (n = 8) and sows (n = 8, cortisol study 1 only) was split into two treatments (two hair samples/pig) to receive either three isopropanol or methanol washes, and two paired subsamples of hair were contaminated with feces and urine, mildly or severely. Samples were further subdivided and received one, three, or five methanol washes. Hormone concentrations were quantified from the hair and wash solvent, and the ratio of hormones in the solvent to that in the hair calculated. When grouping sow and grower hair together for analysis, hair cortisol concentrations were 13% greater after three isopropanol washes compared to methanol (22.84 ± 3.12 vs. 19.77 ± 2.64 pg/mg, respectively). When analyzing sow and grower hair separately, sow hair cortisol concentrations were 20% higher following three isopropanol washes compared to methanol washes (22.06 ± 5.21 vs. 27.72 ± 5.65 pg/mg), with no differences in grower pig hair concentrations. The solvent cortisol concentrations did not differ with wash solvent. No differences were seen for DHEA. Contamination level did not influence hormone concentrations. Hair cortisol concentrations were 24% higher after one wash compared to five washes (11.98 ± 1.47 vs. 9.05 ± 0.92 pg/mg), whereas the solvent cortisol concentrations were 80% and 84% higher after one wash compared to three and five washes, respectively (21.09 ± 4.04 vs. 4.21 ± 1.62 vs. 3.36 ± 1.32 pg/mg). The solvent–hair cortisol ratio was 65% and 73% higher following one wash compared to three and five washes (1.36 ± 0.80 vs. 0.47 ± 0.12 vs. 0.37 ± 0.14). Hair DHEA concentrations were 39% higher after one wash compared to five washes (42.39 ± 6.87 vs. 26.02 ± 5.69 pg/mg). The solvent DHEA concentrations, and the solvent–hair ratio for DHEA were 94% and 98% and 92% and 98% higher going from one wash to three and five washes, respectively (solvent: 5.07 ± 0.26 vs. 0.28 ± 0.12 vs. 0.12 ± 0.09 pg/mg and solvent–hair ratio: 0.13 ± 0.006 vs. 0.010 ± 0.004 vs. 0.003 ± 0.002). Following three methanol washes, the non-contaminated hair had 46% and 48% higher hair (17.47 ± 1.12 vs. 9.35 ± 0.80 vs. 9.05 ± 1.06 pg/mg) and a 76% and 72% higher solvent (16.31 ± 8.07 vs. 3.92 ± 0.50 vs. 4.50 ± 2.31 pg/mg) cortisol concentration compared to mild and severely contaminated hair, respectively. Wash solvent influences cortisol concentrations in swine hair, but not DHEA. Contaminated swine hair should be avoided in analyses when possible.