Regular monitoring of the estrous cycle in rats for a sizeable experimental study

Abstract

Background: Hormonal fluctuations in female rats pose a challenge to their inclusion in experimental studies, resulting in their underrepresentation in biomedical research. However, monitoring the estrous cycle is essential for assessing reproductive cyclicity and detecting pathological changes. This study presents a standardized and optimized protocol for monitoring the regular estrous cycle in rats, addressing the labor-intensive nature and subjectivity of traditional vaginal cytology. Materials and Methods: The method involves systematic animal handling and sample collection, followed by crystal violet staining and microscopic examination to identify distinct cellular features of the four estrous phases: proestrus, estrus, metestrus, and diestrus. Results: Key improvements include pre-study acclimatization, standardized lavage collection volume, mixing samples for uniform smears, and preparing duplicate slides. These adjustments significantly reduced errors in estrus staging from 33.3 to 0.04% and cut procedure time by approximately 30%. Conclusion: The refined protocol improved data quality, reliability, and researcher confidence, proving effective for large experimental groups and minimizing animal stress. This comprehensive guide serves as a valuable resource for developing practical Standard Operating Procedures (SOPs) in laboratories conducting estrous cycle monitoring.