Influence of stable lighting on the circadian clock in hair follicle cells and impact on sleep behaviours in horses

All organisms utilise an endogenous timing system to predict and adapt to environmental changes that ultimately aids survival. Light signals are captured by the mammalian eye and transmitted to the master clock located in the hypothalamic suprachiasmatic nucleus (SCN). Time-of-day information is then transmitted throughout the body from the SCN, such that tissue-specific functionality occurs with 24-h rhythmicity and in synchrony with the environment. Hair follicles are an easily accessible tissue for studying the circadian clock and have previously been used to assess circadian synchrony in horses. Modern husbandry of horses requires them to spend a significant amount of time indoors, often in suboptimal lighting conditions and with frequent nighttime interactions. The aim of this thesis was to test the hypothesis that equine sleep behaviours will improve, with greater synchronicity of circadian rhythms, in riding school horses housed under lighting designed to support optimal circadian function. The influence of two lighting systems on equine sleep behaviours and circadian rhythmicity was evaluated by examining clock gene expression in a peripheral tissue using mRNA analysis and sleep and activity behaviours in stabled horses. To ensure hair follicle RNA stability during transportation from the United Kingdom to Ireland, study 1 was conducted to assess the effects of sample storage in RNAlater® at different ambient temperatures for one week before cryopreservation. Mane hair samples were collected from four horses at a single time point and one sample from each horse assigned to storage conditions A) -20º C, B) 4º C and C) 19º C. One-way ANOVA revealed no difference in RNA concentration (A:516 +/-125 ng/ml, B:273+/-93 ng/ml, C:476+/-176 ng/ml;P = 0.4293) or RNA Integrity Number (RIN) (A:9.5 +/-0.19, B:9.8+/-0.09, C:9.2+/-0.35 RIN; P = 0.3193) between the test groups. The results confirmed that cool or room temperature conditions would not compromise RNA integrity and permitted the sample transport for study 2. Study 2 assessed the impact of two different lighting systems on sleep patterns and circadian clock gene expression in hair follicles in stabled horses using a crossover design. Ten riding school horses were exposed to each of two lighting systems: a customized LED lighting system (comprising blue-enriched daytime light and dim red nighttime light) and a standard lighting system (fluorescent daytime lighting and darkness at night) for four weeks. On week 4 of each experimental period, video footage of horses in their stables was recorded for 72 hrs (days 1-3) and hair sample collected at 4-h intervals for 52 hrs (days 4-6). Behavioural parameters were based on an ethogram and time allotments during each 24-h period for each horse were calculated for total sleep duration, total recumbency, sternal recumbency, lateral recumbency, wakefulness, standing, out-of-stable duration and unknowns. There was no effect of lighting condition on total sleep, total recumbency, lateral recumbency, sternal recumbency, wakefulness, standing, out of stable and unknown behaviour (P>0.05). The lighting period (Day versus Night) had a significant effect on total sleep (P<0.01), total recumbency (P<0.01), wakefulness (P<0.01), and standing (P<0.05). No differences were observed for either lighting condition for the same lighting period for total sleep and total recumbency (P>0.05). Significantly higher wakefulness duration by Day was observed for the LED lighting condition with higher wakefulness (P<0.05), but no differences were found for the fluorescent lighting condition for Day vs Night (P>0.05). Quantitative polymerase chain reaction detected a significant overall effect of time for clock genes Per2 and Dbp (P<0.004; P<0.0023), but no effect of treatment or time by treatment interaction. Cosinor analysis detected significant 24-h rhythmicity for both Per2 and Dbp (P<0.01) in both control and treatment groups.