Evaluation of Skeletal Muscle Autolysis in Boa constrictor: A Histomorphological and Molecular Approach for Estimating the Post-Mortem Interval in Captive Reptiles

Estimating the early post-mortem interval (PMI) remains a persistent challenge in forensic and veterinary practice, particularly for poikilothermic species such as snakes, where the influence of ambient temperature is still poorly characterized. This study investigated early PMI estimation in Boa constrictor by tracking morphological, immunohistochemical, and molecular changes in skeletal muscle over a defined post-mortem interval, under controlled temperature and humidity conditions. Specimens of varying body size were sampled at successive time points, and skeletal muscle tissue was processed for histopathological evaluation (weighted average of autolysis scores and glycogen concentration), immunohistochemical assessment of desmin and fibrinogen localization, and molecular analysis of RNA integrity (RIN). Histological and molecular parameters both declined in a time- and temperature-dependent manner, and several regression models — linear, quadratic and logarithmic — were fitted and compared to describe this decline. The quadratic model consistently provided the best fit for both histological and RNA-based parameters, capturing an initial rapid decline followed by a progressive deceleration consistent with the known biphasic nature of post-mortem tissue degradation; it was further used to estimate the vertex point marking the boundary of the most informative early PMI window. Among the parameters tested, the weighted average of histological scores emerged as the strongest single predictor of PMI, while RIN provided a complementary, independent line of evidence that closely tracked the histological trend. Preliminary immunohistochemical findings suggested a time-dependent shift in fibrinogen distribution, though this observation was based on a limited number of samples and requires further validation, while desmin did not yield any results, most likely due to a lack of cross-reactivity. Body size showed a measurable but secondary influence on degradation kinetics relative to time and temperature. Overall, this work provides a preliminary, quantitative reference framework for estimating early PMI in Boa constrictor and represents a step toward extending validated thanatochronological tools to reptilian forensic and veterinary practice, an area where such methods remain scarce despite their legal and welfare relevance to a growing population of captive and CITES-protected species.