Does pupil area reflect physiological heat stress in hair ewe lambs?

Heat stress compromises livestock productivity and animal welfare, driving the need for non-invasive monitoring technologies under a warming climate. We assessed pupillometry and ocular thermography as heat-stress indicators in six nulliparous Santa Inês × Dorper ewe lambs exposed to 26, 29 and 34 °C at 65% relative humidity in a climatic chamber under standardized lighting. Pupillary area (PA), and ocular surface temperature over the pupillary region (OSPT) were assessed by digital image analysis and infrared thermography, respectively. From PA we derived a pupillary area index (PAI = PA/ baseline PA at 26 °C). Thermoregulatory responses, including respiratory rate (RR), heart rate (HR), rectal temperature (RT), and serum cortisol were also measured. In this preliminary study, PA increased by 16.05 mm² (20.7%) from 26 to 34 °C, mirrored by a 21% increase in PAI, while OSPT increased by 3.34% ( P = 0.012). Likewise, RR and HR increased progressively across the thermal treatments ( P < 0.001), whereas RT ( P = 0.013) and cortisol ( P = 0.010) increased at 34 °C. Repeated-measures correlation (rrm) analysis revealed strong positive associations between PA and RR (rrm = 0.86), HR (rrm = 0.83), and cortisol concentration (rrm = 0.77) (all P < 0.001), and a moderate association with RT (rrm = 0.58, P < 0.01). OSPT was strongly correlated with RT (rrm = 0.82) and RR (rrm = 0.74) (both P < 0.001). Mixed-effects regression analyses confirmed that PA remained positively associated with OSPT, RR, HR, RT, hemoglobin concentration, and cortisol. In conclusion, pupillary dynamics and ocular surface temperature over the pupillary region are associated with the thermoregulatory responses of heat-stressed hair ewes. Further studies are needed to validate these findings under field conditions, evaluate potential threshold values, and develop automated monitoring systems for practical farm applications.