Barbara Anne Murphy

Fifty-two horses aged >15 years, diagnosed with pituitary pars intermedia dysfunction (PPID), and displaying hypertrichosis were recruited via an online survey of PPID horse owners. From mid-December, group T (n = 29) wore Equilume^® light masks extending photoperiod to 15 h daily, while group C1 (n = 23) remained under natural photoperiod. As 85% (44/52) of recruited study horses received pergolide medication, a second unmedicated PPID research herd (C2; n = 17) was recruited and remained under natural photoperiod. Hair coat samples, shedding and body condition scores were collected monthly by owners for 13 months and analysed by the research team. Data related to management, coat condition and PPID clinical signs were collected using bimonthly questionnaires (BMQ). Time (p < 0.001), group (p = 0.025) and time-by-group interaction (p = 0.005) affected hair length. Group differences were attributable to shorter hair lengths in C2, and no differences in hair length occurred between T and C1 (p > 0.05). Time affected shedding scores (p < 0.001) which was advanced by one month in T (p < 0.05). In group T, BMQ responses showed improved coat condition in April (p = 0.035), decreased fat coverage in April and June (p < 0.05), and increased energy/alertness in February (p = 0.022). Improvements in coat condition (p = 0.043), clinical signs of PPID (p = 0.018), and general quality of life (p = 0.035) were reported in T only in a final study questionnaire. Blue light treatment merits further investigation as a complementary treatment for PPID.

MicroRNAs (miRNAs) are essential regulators of gene expression, yet few comprehensive databases exist for miRNA expression in non-model species, limiting our ability to characterize their roles in gene regulation, development, and disease. Similarly, isomiRs - length and sequence isoforms of canonical miRNAs with potentially altered regulatory targets and functions - have received even less attention in non-model species, including the horse, leaving a critical gap in our understanding of their biological significance. To address these challenges, we developed an open-source, containerized pipeline for identifying and quantifying miRNAs and isomiRs (FARmiR: Framework for Analysis and Refinement of miRNAs), and an associated interactive browser (AIMEE: Animal IsomiR and MiRNA Expression Explorer). AIMEE was developed to make miRNA expression data more accessible and user-friendly, a feature often lacking from other expression atlases. These tools were developed using equine data but can be readily extended to other species. Using these tools, we aggregated 461 small RNA-seq datasets, spanning 61 distinct tissues, integrating data from public repositories, an American Quarter Horse cohort, and the Functional Annotation of ANimal Genome (FAANG) consortium Thoroughbred samples, predicting 5,781 miRNAs and isomiRs. This work represents the largest systematically curated atlas of equine miRNA expression to date, providing a valuable resource that will enhance our understanding of miRNA and isomiR functions in tissue-specific regulation and ultimately improve biomarker discovery, functional genomics, and precision veterinary medicine.

Fifty-two horses aged >15 years, diagnosed with pituitary pars intermedia dysfunction (PPID), and displaying hypertrichosis were recruited via an online survey of PPID horse owners. From mid-December, group T wore Equilume® light masks extending photoperiod to 15 hours daily (n=29), while group C1 (n=23) and a second unmedicated PPID research herd (C2; n=17) remained under natural photoperiod. Hair lengths, shedding and body condition scores were assessed monthly for 13 months. Data related to management, coat condition and PPID clinical signs were collected using bimonthly questionnaires (BMQ). Time (p< 0.001), group (p=0.025) and time-by-group interaction (p=0.005) affected hair length. Group differences were attributable to shorter hair lengths in C2, and no differences in hair length occurred between T and C1 (p>0.05). Time affected shedding scores (p< 0.001) which was advanced by one month in T (p< 0.05). In group T, BMQ responses showed improved coat condition in April (p=0.035), decreased fat coverage in April and June (p< 0.05), and increased energy/alertness in February (p=0.022). Improvements in coat condition (p=0.043), clinical signs of PPID (p=0.018), and general quality of life (p=0.035) were reported in T only in a final study questionnaire. Blue light treatment merits further investigation as a complementary treatment for PPID.

Modern horse husbandry involves significant time spent indoors, often in suboptimal lighting conditions and with frequent night-time disturbances by humans for management purposes. The aim of this study was to investigate the influence of a customised light-emitting diode (LED) lighting system and a standard fluorescent lighting fixture on equine sleep behaviours, circadian rhythmicity and spontaneous blink rates in horses. Ten riding school horses experienced two stable lighting conditions for four weeks each in a cross-over study running from January to March, 2023. The treatment lighting consisted of an LED system that provided timed, blue-enriched white polychromatic light by day and dim red light at night, and control lighting was a fluorescent tube that was turned on and off manually morning and evening. During week 4 of each experimental period, spontaneous blink rate was recorded twice for 30 min, behaviour of horses in their stables was recorded continuously for 72 h, and hair samples for circadian clock gene analysis were collected at 4-h intervals for 52 h. No differences were detected for total sleep, lateral or sternal recumbency, wakefulness, standing, standing sleep, or spontaneous blink rate (P > 0.05), between lighting conditions. The lighting period (Day versus Night) influenced total sleep (P < 0.01), total recumbency (P < 0.01), wakefulness (P < 0.01), and standing sleep (P < 0.05) in both conditions. For the treatment condition only, higher wakefulness was recorded during Day (P < 0.05). An overall effect of time for clock genes PER2 and DBP was detected (P < 0.01), but there was no effect of treatment, or time by treatment interaction. Cosinor analysis detected significant 24-h rhythmicity for PER2 and DBP (P < 0.01) in both lighting conditions. Results imply that dim red light at night does not negatively impact normal sleep patterns or circadian rhythmicity, and provide evidence supporting further research to better understand the role of blue-enriched LED light at promoting increased wakefulness during daytime in stabled horses.

Hair follicles provide an easily accessible tissue for interrogating gene expression for multiple purposes in mammals. RNAlater® is a liquid storage solution that stabilises and preserves cellular RNA, eliminating the need to immediately process or freeze tissue specimens. The manufacturer advises storage of samples at 2-8°C overnight before transfer to –20°C. This study aimed to evaluate RNA integrity in hair follicle samples collected from horses, stabilized in RNAlater®, and stored under three short-term storage conditions. Mane hair samples complete with follicles were collected from four horses at a single time point. Approximately 15 hairs were placed in each of three 2 mL tubes containing 0.75ml RNAlater® solution. Test group A was stored at 4°C for 24-h, then decanted and stored at -20°C. Test groups B and C were stored at 4°C and 19°C (room temperature) respectively for 7 days, then decanted and stored at -20°C. RNA was isolated from all samples and RNA quantity and quality were measured. 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 = 0.2) or quality (A:9.5 +/-0.19, B:9.8+/-0.09, C:9.2+/-0.35 RIN; P = 0.46) between the test groups. There were no group differences in mean Cycle Threshold values from qPCR validation assays confirming high-quality template cDNA. The results suggest that storage of hair follicles for one week in RNAlater® at cool or room temperature conditions will not compromise RNA integrity and will permit extended transport times from remote sampling locations without the need for freezing.

Equine headshaking syndrome is a poorly understood neuropathic pain condition presenting as uncontrollable shaking, flicking, or striking of the head. Therapeutic options are limited, and treatments are only partially successful. Currently, epidemiological information on headshaking in the Southern Hemisphere is lacking. An online survey was circulated to Australian owners of headshaking horses to collect information on triggers, symptoms, seasonality, treatments, and perceived treatment efficacy. The responses (n = 216) showed the mean age at symptom onset as 9.6 (±4.7) years. More geldings were affected than mares (76% vs. 24%), and symptom onset occurred later in geldings compared to mares (10.1 ± 4.7 vs. 7.9 ± 4.0 years; p < 0.01). Bright sunlight, wind, and high pollen were the most commonly reported triggers (61%, 46% and 40%, respectively), and seasonal onset of symptoms was reported by 54% of respondents. In total, 71% of respondents reported using two or more treatments. The most common treatments were supplements (68%), nose nets (63%), light-blocking masks (48%), bodywork (48%) and pharmaceutical compounds (38%). Overall, treatments were considered ineffective by 33% of respondents. The findings were in agreement with surveys from the Northern Hemisphere. Of note was the perception of bright light as a primary trigger, alongside the reported low treatment efficacy of light-blocking masks. Seasonal intensification of symptoms and its relationship to day length merits further exploration.

Hair follicles provide an easily accessible tissue for interrogating gene expression in mammals. RNAlater® is a liquid storage solution that stabilises and preserves cellular RNA, eliminating the need to immediately process or freeze tissue specimens. The manufacturer advises storage of samples at 2-8°C overnight before transfer to –20°C. This study aimed to evaluate RNA integrity in hair follicle samples collected from horses, stabilized in RNAlater®, and stored under three short-term storage conditions. Mane hair samples complete with follicles were collected from three horses at a single time point. Approximately 15 hairs were placed in each of three 2 mL tubes containing 0.75ml RNAlater® solution. Test group A was stored at 4°C for 24-h, then decanted and stored at -20°C. Test groups B and C were stored at 4°C and 19°C (room temperature) respectively for 7 days, then decanted and stored at -20°C. RNA was isolated from all samples and RNA quantity and quality were measured. 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 quality (A:9.5 +/-0.19, B:9.8+/-0.09, C:9.2+/-0.35 RIN; P = 0.3193) between the test groups. There were no group differences in mean Cycle Threshold values from qPCR validation assays confirming high-quality template cDNA. The results suggest that storage of hair follicles for one week in RNAlater® at cool or room temperature conditions will not compromise RNA integrity and will permit extended transport times from remote sampling locations without the need for freezing.

Hair follicles (HF) represent a useful tissue for monitoring the circadian clock in mammals. Irregular light exposure causes circadian disruption and represents a welfare concern for stabled horses. We aimed to evaluate the impact of two stable lighting regimes on circadian clock gene rhythmicity in HF from racehorses. Two groups of five Thoroughbred racehorses in training at a commercial racehorse yard were exposed to standard incandescent light or a customized LED lighting system. The control group received light from incandescent bulbs used according to standard yard practice. The treatment group received timed, blue-enriched white LED light by day and dim red LED light at night. On weeks 0 and 20, mane hairs were collected at 4 h intervals for 24 h. Samples were stored in RNAlater at −20 °C. RNA was isolated and samples interrogated by quantitative PCR for the core clock genes: ARNTL, CRY1, PER1, PER2, NR1D2, and the clock-controlled gene DBP. Cosinor analyses revealed 24 h rhythmicity for NR1D2 and PER2 and approached significance for CRY1 (p = 0.013, p = 0.013, and p = 0.051, respectively) in week 20 in the treatment group only. No rhythmicity was detected in week 0 or in week 20 in the HF of control horses. Results suggest that lighting practices in racehorse stables may be improved to better stimulate optimum functioning of the circadian system.

Journal of Animal Science and Technology (JAST) is a peer-reviewed, open access journal publishing original research, review articles and notes in all fields of animal science. Topics covered by the journal include: genetics and breeding, physiology, nutrition of monogastric animals, nutrition of ruminants, animal products (milk, meat, eggs and their by-products) and their processing, grasslands and roughages, livestock environment, animal biotechnology, animal behavior and welfare.