Blood collecting procedures and sex determination
The first blood samples were drawn from nestlings 41 - 66 d (mean = 52.4, SD = 5.7) after their estimated hatch, corresponding to the beginning of hacking. The second samples were drawn when nestlings were between 86 - 108 d old (mean = 97.6, SD = 5.5), just prior to their release (Table 1). A total of 55 nestlings, 26 males and 29 females, were double sampled. Intervals between repeat samples of the same individual were between 42 - 47 d (mean = 44.0, SD = 2.0).
Blood samples were collected between 11:00 AM and 15:00 PM to avoid variations in blood parameters due to circadian rhythms (Garcia-Rodriguez et al. 1987b; Ferrer 1990; Ferrer et al., 1994). Blood samples were collected from the cutaneous ulnar (brachial) vein with the birds cast in dorsal recumbency. Blood was collected in a 10 ml plastic syringe (Becton Dickinson S.A., Spain) attached to a 23 gauge 1” needle (Monoject, USA). Three blood smears were made using a slide-on-slide technique. One ml of blood was transferred to a di-potassium ethylene diamine tetra acetate (EDTA) anticoagulant tube (Teklab UK) for ‘DNA’ sexing. Birds were subsequently sexed at a (‘DNA’) molecular level according to methods described by Ogden et al. (2015). An additional 1 ml EDTA tube was filled and submitted for hematological analysis. A 1 ml tube containing lithium heparin (Teklab, UK) as an anticoagulant was filled for blood lead analysis. The remaining sample was transferred to a 6 ml lithium heparin tube (Becton Dickinson S.A., Spain) and submitted for biochemical analyses. All samples were kept in a cooler box at approximately 4° C for a maximum of 8 h prior to submission for laboratory analysis. Samples arrived within 24 h at Greendale Veterinary Diagnostics, Surrey, UK, where biochemical and hematologic analyses were performed. Blood lead analysis was performed at Veterinary Laboratory Agencies (VLA) UK. Packed cell volumes and total plasma protein levels were also examined within three hours of collection at the Veterinary Centre, University of Edinburgh, UK.