transferred into 2 samples were coll
2.7 PHYSIOLOGICAL MEASURES
2.7.1 Blood sampling
Adults and nestlings (aged 7 - 1 4 days) were blood sampled under Home Office licence (PPL 30/1961) for the purpose of analysing T levels (see 2.7.4) and molecular sexing nestlings (see 2.7.5). Blood samples (approximately 100 pi), were taken from each bird by lightly pricking the brachial vein in the wing with a sterile needle, drawing blood into 2 - 3 microcapillary tubes (50 pi capacity) and expelling into 1.5 ml plastic collecting tubes. All birds were weighed after blood sampling with a pesola balance (accuracy 0.25 g). During fieldwork in 2003 and 2004, blood samples were kept at around 0 °C for a maximum of 5 h by transferring the plastic tubes onto ice packs stored inside a cool bag. At the field laboratory, blood samples were centrifuged in a bench top centrifuge (Boeco) at 1300 rpm for 15 min. Plasma was transferred into a labelled 0.5 ml microcentrifuge tube to be later analysed for T. Red blood cells (RBCs) were retained in the original collecting tube for molecular sexing. Between April - July in 2003 and 2004, plasma and RBCs were stored at -4 °C in the field laboratory until transported back to Cardiff University on dry ice, to be stored at -20 °C until further analysis.
2.7.2 Faecal sampling
In addition to blood sampling, non-invasive faecal sampling was also used as a technique to assess androgen levels excreted by adults and nestlings. In the field, nestlings were stimulated to produce a faecal sample directly into a 1.5 ml plastic tube, by holding the nestling above the tube and gently squeezing the abdomen. Adult faecal samples were collected during the 2005 validation studies by placing plastic sheets in the bottom of each bird cage. Whenever possible, directly touching faecal samples by hand was avoided to stop the transfer of hormones and contaminants into the sample. Faecal samples were kept at around 0 °C for a maximum of 5 h in the field by transferring the plastic tubes onto ice packs stored inside a cool bag. Between April - July in 2003 and 2004, faecal samples were stored at -4 °C in the field laboratory until transported back to Cardiff University on dry ice, to be stored at -80 °C until freeze dried. All faecal samples were stored in a freeze dried state until further analysis.
2.7.3 Faecal T extraction
Faecal samples were extracted over 2 days.
Day 1: To extract faecal androgens, freeze dried faecal samples were homogenised with a pestle and mortar, before 0.01 g of the homogenate was extracted with 2.5 ml of 90 % ethanol in a 10 ml conical flask by shaking on an orbital shaker (IKA ® KS 130 basic) for 60 min. The contents of each flask were vortexed, decanted into a test tube and centrifuged at 4500 rpm for 20 min. Approximately 2 ml of supernatant was transferred using a pipette into a second test tube, which was put onto a hotblock under air to dry at 37 °C. The pellet in the first test tube was re-suspended in 1.25 ml 90 % ethanol, vortexed (V400 Multitube vortexer, Alpha Laboratories) for 1 min and centrifuged again at 4500 rpm for 20 min. All supernatant was then added to the second test tube on the hotblock and left to dry (4 - 5 h). Once evaporated, 4 ml dichloromethane was added to the second test tube to remove the dried hormones from the sides of the glass tube, and vortexed for 3 min. Test tubes were stored at 4 °C in a fridge overnight.
Day 2: The contents of each test tube were mixed by vortexing for 3 min and shaking on an orbital shaker for 30 min. The supernatant was transferred to a third test tube and left to dry under air on a hotblock (2 - 3 h). Once evaporated, the dried androgens sticking to the inside of the third test tube were dissolved in 0.3 ml of assay buffer solution, transferred to a 1.5 ml plastic tube and stored at -20 °C until assayed for androgen concentration by T radioimmunoassay (see 2.7.4).
2.7.4 Testosterone radioimmunoassay (T-RIA)
2.7.4.1 Theory underlying radioimmunoassay techniques
Androgen concentrations in plasma and faecal samples taken from birds were measured by direct radioimmunoassay (Parkinson & Follett 1995). Radioimmunoassay (RIA) is a technique which is used to measure small quantities of materials (e.g. hormones) of unknown concentration circulating in the blood (Parkinson & Follett 1995). During a hormone radioimmunoassay, an antigen-antibody complex is formed when a plasma or faecal sample containing the hormone (unlabelled antigen), is incubated in an assay tube with a fixed amount of antibody and some additional hormone labelled with an
isotope (labelled antigen). A separate reference hormone standard, containing: a known concentration of unlabelled hormone, radiolabeled hormone and a fixed amount of antibody, is incubated at the same time. The underlying principle of hormone radioimmunoassay is the competitive inhibition of binding of the labelled antigen to antibody by unlabelled antigen. Both labelled and unlabelled hormones in the sample and standard assay tubes bind and unbind with the antibody (the number of antigens out number antibody binding sites), until a state of equilibrium is reached. Because a fixed amount of antibody is present in each assay tube, the amount of labelled hormone bound to the antibody (the measured radioactivity) is a function of the concentration of unlabelled hormone in either the standard or the sample tube. The higher the concentration of unlabelled hormone, the lower the quantity of labelled hormone bound to the antibody and, therefore, a high activity reading for a given sample will mean a low hormone concentration. By comparing how much each labelled antigen binds to the antibody between sample and standard curve concentrations (which is linear between upper and lower cut off points), the concentration of hormone in the sample can be estimated.
2. 7.4.2 T-RIA protocol
Total androgen concentration was measured in faecal and plasma samples collected between 2003 - 2005 by T radioimmunoassay using anti-T anti-serum (code 8680- 6004, Biogenesis, UK) and [125I]-T label (code 07-189126, ICN, UK) (Parkinson & Follett 1995) at Cardiff University. In each radioimmunoassay, 50 - 100 extracted faecal samples or plasma samples were processed over 3 days. Also included in each assay were 3 controls (including: blanks, total counts and no hormones), as well as standards of a known T concentration to calculate a calibration curve and inter-assay variation.
Day 1: To set up faecal and plasma samples, 20 pi aliquots of extracted faeces or plasma samples were added to 2 microcentrifuge tubes using a Hamilton syringe. To set up 3 controls, 20 pi of assay buffer solution was added to 12 (3 x 4) microcentrifuge tubes using a Hamilton gun. To set up standard samples for a calibration curve, microcentrifuge tubes were labelled 1 - 12 in triplicate, before 20 pi of assay buffer solution was added to tubes 2 -1 1 and 20 pi of assay standard (20 ngmF1) was added to