We compared the hair cortisol levels of lactating dairy cows in a cold- and a warm-temperate region out of four climatic zones in Japan. We simultaneously investigated the effects of calving number, lactation period and month of hair sampling. Hair of nine Holstein lactating cows chosen from each region (i.e. 18 cows per sampling) was sampled in March, June, September and December. Number of calvings (1, 2, ≥3) and lactation duration (early: <100, middle: 101-200, and late: >201 days) were balanced between regions. Cortisol was extracted from hair by methanol, and its level was determined with a cortisol immunoassay kit. A multi-way analysis of variance revealed that the effects of month of hair sampling (P < 0.001) and its combination with region (P < 0.05) were significant. In a multiple comparison test, significant differences (P < 0.01) in hair cortisol level (pg/mg of hair) were found between June (13.0 ± 1.0) and the other 3 months, and between September (1.6 ± 0.2) and December (4.5 ± 0.3). The rise in cortisol level from March to June was more intense in the cold-temperate region. These results demonstrate the necessity of considering seasonal variations in each climatic region when we use hair cortisol level as an indicator of stress.
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