Blood parameters

We collected a blood sample from each individual two days before the start and on the last day of the experimental treatment, where females were sampled in a random order. We immediately collected blood upon removing the female from her cage (mean blood sample duration 4.4 ± 2.0 min) in order to measure baseline corticosterone (CORT) levels (Romero and Wikelski, 2001). We recorded body temperature at the time of blood sampling using an infrared thermometer (Raytek Raynger MX2) at a standard distance and angle (300 mm and 45°, therefore covering a 6-mm area) with a constant emissivity coefficient (0.95). Body temperature was measured right before the blood samples when snakes were undisturbed within their thermogradient. This method gives close estimates of cloacal temperature measurements in vipers without the negative side effects of handling stress (Shine et al., 2002). We collected blood samples (150 μl) through cardiocentesis using a 1-ml syringe and a heparinized 29-gauge needle. Immediately after sampling, we allocated a small amount of blood into two 10-μl micro-capillary tubes and centrifuged them. We measured haematocrit (Hct; length of red blood cells/length of total sample) in duplicate for each blood sample. Next, for each sample, we transferred the remaining blood into Eppendorf tubes (0.5 ml) and centrifuged them for 3 minutes at 2000 × g. Plasma was stored at −28°C until laboratory analyses. We measured plasma osmolality from 10 μl aliquots using a vapour pressure osmometer (± 3 mOsm kg⁻¹; Vapro2, Elitech group). We determined plasma CORT concentrations (ng ml⁻¹) following a well-established radioimmunoassay protocol (see Dupoué et al., 2016 for details). Samples were run in three assays (intra-assay variation: 7.58%, inter-assay variation: 8.84%). We examined the oxidative status of females by measuring the concentration of reactive oxidative metabolites (ROMs) as an index of oxidative damage (Costantini, 2016) and antioxidant capacity (OXY) as an index of defence (Costantini, 2011). We used d-ROMs colorimetric kits (MC003, Diacron International, Italy) to evaluate the activity of organic hyperoxides and OXY-absorbent test kits (MC435, Diacron International Italy) to assess the non-enzymatic ability of diluted plasma samples (1:100) to neutralize an oxidant attack from hypochlorous acid. We determined intra-plate coefficients of variations in ROMs (1.2%) and OXY (2.7%) using a pooled plasma sample three times in each 96-well plate (1 plate for ROMs, 1 for OXY).