**Background:** The auditory brainstem response (ABR) is a non-invasive electrophysiological test used to assess auditory function in both humans and animals. It is a critical tool in translational audiology for evaluating hearing loss therapies, diagnosing auditory nerve and brainstem dysfunction, and monitoring cochlear implant performance. However, data heterogeneity across studies often hinders translation from animal research to clinical practice. This heterogeneity arises from three domains: animal-, equipment-, and experiment-related factors. This paper synthesizes knowledge on performing ABR in experimental animals, focusing on mice and rats, to provide universal recommendations for planning, preparing, and conducting ABR recordings.
**Methods:** The authors reviewed the literature and their own experience to develop structured recommendations. The paper is organized into three main sections: planning the experiment, preparing ABR recordings, and performing ABR recordings. Each section addresses the three domains (animal, equipment, experiment) with specific guidance. The planning section covers species/strain selection (e.g., C57Bl/6J mice develop age-related hearing loss at 6 months, CBA/J at 20 months), sex as a biological variable (both sexes should be included), age-dependent susceptibility (e.g., noise susceptibility window in CBA/J mice begins at 15 days and remains high until 3 months), sample size justification (power analysis recommended; typically 5–10 animals per group), and inclusion/exclusion criteria. The equipment section emphasizes calibration, oscilloscope testing, and electrode impedance (<3 kΩ). The experiment section details stimulus parameters (click vs. tone burst, polarity, repetition rate of 21/s, 2-1-2 tone burst, 2.5 ms in mice, 256–1024 averages, analysis time 10 ms, filters: high-pass 300 Hz, low-pass 3 kHz), anesthetic choice (isoflurane vs. ketamine/xylazine), acclimation (at least one week), and stress reduction.
**Key Results:** The paper does not present original experimental data but synthesizes existing knowledge. Key findings include: (1) Strain differences in ABR thresholds and waveforms exist (e.g., Sprague-Dawley rats have lower hearing thresholds than Long-Evans rats at 2–8 kHz; Sprague-Dawley rats have higher wave IV amplitude than Wistars). (2) Age-related hearing loss onset varies by mouse strain (C57Bl/6J: 6 months; CBA/J: 20 months; DBA/2J: 3 weeks; Balb/C: 10 months). (3) Pigmentation affects inner ear morphology and hearing (melanin protects against noise-induced hearing loss). (4) Sex differences affect hearing and metabolism (male CBA/Ca mice are more susceptible to high-fat diet effects on hearing). (5) Anesthetic choice influences ABR results (isoflurane elevates hearing thresholds in Long-Evans rats compared to ketamine/xylazine). (6) Electrode impedance >3 kΩ reduces recording quality. (7) Body temperature decrease of 0.5°C or more significantly alters ABR latencies and amplitudes.
**Clinical Implications:** Standardizing ABR protocols in animal research is essential for improving data reproducibility and facilitating translation to clinical audiology. By controlling for animal strain, sex, age, anesthesia, and equipment parameters, researchers can reduce variability and increase the reliability of preclinical findings. These recommendations align with the ARRIVE guidelines and aim to reduce the number of animals used while enhancing the validity of results. Ultimately, this will accelerate the development of therapies for hearing loss and auditory disorders, benefiting both human and veterinary medicine.