Pre-extraction drying alters fecal testosterone measurements and variability in male Gyrfalcons (Falco rusticolus)

Non-invasive endocrine monitoring is an important tool in captive breeding programs for birds, however, hormone measurements from fecal samples remain sensitive to methodological decisions. Variation in fecal moisture content introduces systematic bias in hormone quantification and complicates the interpretation of reproductive patterns. Although fecal hormone analysis is widely used in avian studies, direct comparisons of how pre-extraction processing influences hormone measurements and the detection of seasonal endocrine patterns remain limited to cockerels and parrots. This study compared testosterone concentrations in fecal samples processed under two pre-extraction conditions (samples dried at 80°C for 48 h and non-dried groups), in adult male Gyrfalcons ( Falco rusticolus ) during the breeding season. 126 fecal samples were collected twice weekly over eight weeks from nine captive-bred males and analyzed using ethanol-based extraction followed by enzyme immunoassay (ELISA). Differences between groups and temporal patterns were assessed using generalized linear mixed-effects models. Testosterone concentrations were higher in dried than in non-dried samples (median: 181.20 vs. 71.09 ng/g; p < 0.001), with non-dried showing higher variability. Drying revealed substantial and variable water loss, identifying fecal moisture as a major source of measurement bias. Concentrations declined across the sampling period (p < 0.001), consistent with seasonal dynamics, and no significant interaction between processing condition and time was detected, indicating consistent temporal patterns across groups. These results demonstrate that sample processing influences fecal testosterone measurements and differences between dried and non-dried samples primarily reflect methodological rather than biological variation. Drying reduces moisture-driven variability and is therefore recommended as a standard approach for fecal hormone analysis in raptors.