Clock Genes Expression in Peripheral Leukocytes and Plasma Melatonin Daily Rhythm in Horses

In mammals, behavioral and physiological processes display 24-hour rhythms that are regulated by the circadian system. In the present study, we investigated clock gene expression in peripheral leukocytes in horses. For this purpose, 10 Italian Saddle gelding horses (9-11 years old; 475 ± 28 Kg) were housed in individual boxes under natural photoperiod and natural environmental temperature. Blood samples were collected at 4-hour intervals over a 48-hour period. The day before the start of sampling, left jugular furrow of each horse was cannulated for the blood sample collection performed in heparinized tubes, for the assessment of melatonin concentration by means of radioimmunoassay and into PAX gene Blood RNA Tube for the assessment of clock genes by real-time RT-quantitative polymerase chain reaction (RTqPCR). Well-established melatonin showed a daily rhythm with nocturnal acrophase (day 1-21:30; day 2-21:40). All genes tested (Bmal1, Cry 1, Per 1, Per 2, and Per 3) except Clock showed daily rhythmicity of their expression in peripheral blood. Oscillations of Bmal1 and Per 2 were correlated with the oscillation of melatonin, which anticipated the acrophase of Bmal1 (day 1-01:29; day 2-01:00) and Per 2 (day 1-01:00; day 2-00:32) of about 3 hours. Our results support the presence of a cyclic transcription of clock genes in peripheral leukocytes in horses.