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.