Reduce, Replace, Refine: Determining A Posteriori Reference Intervals for Biochemistry in Hermann's Tortoise (Testudo hermanni).

Biochemical and hematological analyses are important for the assessment of animal health. However, for most wild species their use is hindered by the scarcity of reliable reference intervals. Indeed, collecting body fluids (e.g., blood, urine) in free-ranging animals is often technically challenging. Further, sampling many individuals would be essential to consider major sources of variations, such as species, populations, sex, age, and seasons. One alternative, according to the reduction, refinement, and replacement framework, is to establish reference intervals a posteriori using literature survey and unpublished data. We produced reference intervals for free-ranging Hermann's tortoises (Testudo hermanni), using analyses performed on blood samples collected in previous studies and conservation programs conducted in the field between 2010 and 2016 in southern France (n=195 individuals). Thirteen parameters were analyzed: packed-cell volume, blood concentrations of corticosterone, testosterone, glycemia, cholesterol, triglyceride, urea, uric acid, calcium, sodium, potassium, asparagine aminotransferase (AST), and alanine aminotransferases (ALT). Reference intervals for subgroups defined by sex and season were relevant for corticosterone, triglyceride, and calcium (sex) and cholesterol (season). Comparing our results with those obtained in captive individuals in Germany, except for urea and AST levels the intervals from both free-ranging versus captive tortoises were similar, suggesting that reference intervals established from captive individuals may be suitable for free-ranging populations in this species.