Telomere length analyses
Telomere length was measured in 322 individuals in blood samples using the TRF analysis following . Samples were distributed over 34 gels, and nestlings from the same brood were partly spread over gels with repeated samples from the same individual (ages 5 and 30 days) placed always in neighbouring lanes on a gel. Briefly, DNA was extracted using the CHEF Genomic DNA Plug kit (Bio-Rad, Hercules, CA) and digested overnight with proteinase K at 50ºC. About half of the digested DNA was simultaneously digested with Hind III (60U), Hinf I (30U) and Msp I (60U) for 18 h at 37ºC in NEB2 buffer. The digested DNA from the nucleated erythrocytes in each sample was then separated by pulsed-field gel electrophoresis at 14ºC for 24 h (3 V / cm, initial switch time 0.5 s, final switch time 7.0 s). The gels were dried (Bio-Rad model 538) and hybridized overnight using a 32P-end-labelled oligo (5’-CCCTAA-3’)4 that binds to the 3’ end-cap telomere overhang. Subsequently, a gel picture was obtained by detecting the radioactive signal (Cyclone Storage Phosphor System, PerkinElmer) with the use of a phosphor screen. Individual telomere length distributions for each sample were quantified through densitometry in the resulting gel picture using IMAGEJ v. 1.38x and following . For the present analyses, we used the averaged value of the individual telomere length size distributions. The intergel repeatability was measured as the coefficient of variation for a 29-day-old jackdaw control sample that was included in all 34 gels (6.01%), while the intragel repeatability is demonstrated by the high repeatability within-individuals (see results section).