Non-invasive wireless electroencephalographic recording of the sleep-wake cycle in freely moving reptiles, birds, and mammals: a novel methodology compatible with animal welfare.

Abstract

Animal welfare is increasingly assessed through the "Five Domains" framework, where monitoring brain activity via electroencephalography (EEG) is essential for objectively evaluating sleep-wake cycles and neurological health. However, traditional EEG studies in animals often require invasive procedures, anesthesia, and movement restriction, which compromise both animal welfare and natural behavior. To overcome these limitations, we developed a miniaturized wireless EEG device (24.8 × 24.8 × 8.2 mm; 5.2 g) with Bluetooth transmission, surface electrodes, and biocompatible adhesives. This system allows 10-12 h of recording without restricting movement while remaining compatible with animal welfare standards. We validated the methodology in three amniote species representing major vertebrate classes: Aldabra giant tortoise (), gentoo penguin (), and aardvark (). Recordings were conducted on conscious, freely moving animals in zoological facilities, and signals were analyzed using spectral frequency analysis. Three distinct EEG patterns were consistently identified across all species: active wakefulness, characterized by desynchronized high-frequency waves (0-30 Hz) and locomotor activity; NREM sleep or a homologous state, marked by synchronized high-amplitude, low-frequency waves (0.5-4 Hz); and REM sleep or a homologous state, defined by desynchronized high-frequency waves without locomotor activity. Fundamental brainwave frequencies (delta, theta, alpha, beta and gamma) were consistent and conserved across species, while amplitude varied according to anatomical differences. Interestingly, we observed specific patterns of EEG frequencies distribution in the three species, reflecting unique evolutionary spectral profiles, such as alpha dominance during aardvark wakefulness, theta profusion in penguin wakefulness and REM sleep, and delta massiveness in tortoise NREM sleep. This non-invasive methodology successfully distinguishes and records sleep-wake patterns in reptiles, birds, and mammals without surgical procedures, demonstrating that high-quality neurophysiological data can be obtained while adhering to animal welfare principles. The system maintained signal integrity within a 15-meter range, allowing for naturalistic behaviors in home enclosures. The technique opens new possibilities for longitudinal behavioral studies, detection of neurological disorders, and comparative sleep research in captive animals, representing a significant advance toward more ethical practices in animal neuroscience.