This study was conducted with free-living Adélie penguins during the guard stage of chick rearing when adults alternate between taking foraging trips or guarding the young (21st December 2018-14th January 2019) at Dumont d'Urville station, Terre Adélie, East Antarctica (66°40′S; 140°01′E). We captured 58 adults (24 females and 34 males) on the nest, when both adults were attending the nest prior to a changeover. Only one member of a pair was ever sampled, to reduce disturbance time to the nest.
This study took advantage of a blood sample protocol required for a doubly labelled water (DLW) experiment that necessitated two initial blood samples, one taken after capture and a second one taken after a calibration period in which the animals must be held in captivity (Hicks et al., 2020 (link)). Specifically, upon capture, individuals were blood sampled within 3 min of first handling from the tarsus vein. This sample served as a background sample providing the unstressed situation. After blood sampling, birds were weighed to the nearest gram, flipper measured to the nearest millimetre, and injected with 0.3 ml of DLW per kg of body weight into the pectoral muscle [see Hicks et al. (2020) (link) for details]. For future identification, birds were marked with a unique identifying code printed on a piece of marine tape rolled around their back feathers. This entire handling period took about 15 min (study mean) and several previous studies have shown that 15 min of handling evokes a stress response as measured by increases in corticosterone in this species (Cockrem, 2013 (link); Cockrem et al., 2006 (link), 2008 (link)) and also in this population (Marciau et al. under review ). After this handling period, birds were placed in a contained area outside the lab for the DLW to equilibrate (for between 1.6 and 2.7 h). The contained area measured 2×2.80×5.20 m, birds were always placed in the contained area with another penguin to calm them and the space was filled with a layer of snow for comfort and cooling. A second blood sample was taken after the equilibrium period. This sample served as our handling time sample. Hold duration was calculated as the time between the first capture and the second blood sample. Whole blood was kept on ice in Eppendorf tubes for up to 10 min before being centrifuged (10,000 rpm, 10 min) and plasma stored at −80°C until analysed. The molecular sexing of all individuals was carried out at the service Analyses Biologiques of the Centre d'Etudes Biologiques de Chizé (CEBC). DNA extraction was conducted with 2 µl of pellet (red blood cells) and using a chelex resin (Chelex 100 Molecular Biology Resin, Bio-Rad; 10%) associated with Proteinase K (PK) as written in the manufacturer's instructions. We then performed a polymerase chain reaction (PCR) with amplification of the CHD gene following a standard procedure validated on penguins (Lee et al., 2010 (link)).
This study took advantage of a blood sample protocol required for a doubly labelled water (DLW) experiment that necessitated two initial blood samples, one taken after capture and a second one taken after a calibration period in which the animals must be held in captivity (Hicks et al., 2020 (link)). Specifically, upon capture, individuals were blood sampled within 3 min of first handling from the tarsus vein. This sample served as a background sample providing the unstressed situation. After blood sampling, birds were weighed to the nearest gram, flipper measured to the nearest millimetre, and injected with 0.3 ml of DLW per kg of body weight into the pectoral muscle [see Hicks et al. (2020) (link) for details]. For future identification, birds were marked with a unique identifying code printed on a piece of marine tape rolled around their back feathers. This entire handling period took about 15 min (study mean) and several previous studies have shown that 15 min of handling evokes a stress response as measured by increases in corticosterone in this species (Cockrem, 2013 (link); Cockrem et al., 2006 (link), 2008 (link)) and also in this population (Marciau et al. under review ). After this handling period, birds were placed in a contained area outside the lab for the DLW to equilibrate (for between 1.6 and 2.7 h). The contained area measured 2×2.80×5.20 m, birds were always placed in the contained area with another penguin to calm them and the space was filled with a layer of snow for comfort and cooling. A second blood sample was taken after the equilibrium period. This sample served as our handling time sample. Hold duration was calculated as the time between the first capture and the second blood sample. Whole blood was kept on ice in Eppendorf tubes for up to 10 min before being centrifuged (10,000 rpm, 10 min) and plasma stored at −80°C until analysed. The molecular sexing of all individuals was carried out at the service Analyses Biologiques of the Centre d'Etudes Biologiques de Chizé (CEBC). DNA extraction was conducted with 2 µl of pellet (red blood cells) and using a chelex resin (Chelex 100 Molecular Biology Resin, Bio-Rad; 10%) associated with Proteinase K (PK) as written in the manufacturer's instructions. We then performed a polymerase chain reaction (PCR) with amplification of the CHD gene following a standard procedure validated on penguins (Lee et al., 2010 (link)).
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