Study system

We bred adult zebra finches (2–3 years old) from the colonies at the Max Planck Institute for Ornithology in Seewiesen, Germany. Each of three experimental rooms consisted of three aviaries (1 × 2 × 2 m), each housing 7–8 pairs of birds. Each aviary was provided with 12 wooden nest boxes and ad libitum nesting materials, seeds, commercial finch egg food and water. In addition, birds were provided with fresh vegetables and hard-boiled eggs twice weekly throughout the experimental period. Our experimental birds were the offspring of these breeding adults. Each aviary produced an average of 29 offspring (16–42), with a total of 263 offspring from all treatment groups. Offspring that died before they reached 120 days post-hatch were not included in the experiment. Animal housing and care was all in accordance with European and local laws governing the care and use of laboratory animals (Council of Europe Treaty ETS-123). All experimental procedures were approved by and done under license from the Government of Upper Bavaria (Regierung von Oberbayern), licence number 55.2–1–54-2532-51-2013.

Experimental treatment

To determine if typical city traffic noise affects telomere dynamics in juvenile birds, we designed three noise exposure treatments: 1) the parents were exposed to noise during breeding, egg-laying and nestling care periods (PNoise), 2) juvenile birds were exposed to noise exposure from fledging throughout the sensory motor learning period, 18–120 days post-hatch (JNoise), and 3) a control group that was not exposed to noise at any time point (NoNoise) (Additional file 1: Table S1). Thus, the offspring in the PNoise treatment group were not exposed to traffic noise after fledging, and the offspring in the JNoise treatment were not exposed before fledging, nor were their parents. The PNoise group had a total of 95 offspring from 32 broods, the JNoise group 59 from 17 broods, and the control group (NoNoise) had 109 offspring from 35 broods. The difference in sample size between treatments is because the parents of the treatments PNoise and NoNoise bred twice, once in PNoise and once in NoNoise treatments. To control for potential effects of breeding experience we considered the number of breeding rounds in the statistical analysis (see below).

Noise playback consisted of 80, 5-min long recordings of street traffic noise, which was recorded at several busy intersections in Munich, Germany during April 2013. During the daylight hours (06:30–20:30), the 80 recordings were played continuously, in randomized order, with playback levels (measured at the position of the nest boxes) averaging between 65 and 85 dB(A) re 20 μPa. Nighttime playback (20:30–06:30) consisted of randomized playback of 40 noise recordings, which were less dense in the rate of passing than the daytime recordings and were reduced in overall amplitude, with playback level averages ranging between 45 and 75 dB(A). Therefore, noise playback mimicked typical urban noise patterns, according to published noise maps [33]. We played noise from a laptop computer to an array of 12 pairs of amplified portable speakers (Hama Sonic Mobil 400 Alu PS1032), with 4 pairs arranged above each of the three aviaries in the room. Noise playback was run using a script written in MatLab (version 7.5.0; Natick, MA, USA; www.mathworks.com) to randomize playback during day and night. For the PNoise group, playback of noise began 4 weeks before the introduction of nesting materials and nest boxes and continued until the median juvenile in the room had fledged (the date when half of the offspring had fledged). For the JNoise group, the noise playback began when the median juvenile was 18 days post-hatch, and continued until all juveniles had reached 120 days.

Telomere measurement

Blood samples were collected by brachial venipuncture for each bird at 21 and 120 days post-hatch to measure telomere length and loss rate. Telomere length at 25 days has previously been shown to be positively related to lifespan in zebra finches [17]. Blood was collected into heparinized capillary tubes (1.4 × 75 mm), transferred into Eppendorf tubes, and centrifuged to separate the cells from the plasma. The cells were then stored at − 80 °C until DNA extraction. We analyzed samples for 263 birds in total, 137 females, and 126 males, at both ages. We used the DNeasy Blood and Tissue kit (Qiagen) to extract genomic DNA from the red blood cells following the manufacturer’s protocol. We used a NanoDrop 8000 spectrophotometer (Thermo Scientific) to measure the quantity of the DNA. To measure relative telomere length we used quantitative PCR (Stratagene MX3000P), as described in [34], and adapted to zebra finches [35].

The relative telomere length of each sample was measured by calculating the ratio (T/S) of telomere repeat copy number (T) to single control gene copy number (S), relative to a reference sample. As the control gene, we used the Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The following forward and reverse primers were used to amplify the telomere: Tel1b (5′-CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT-3′), Tel2b (5′-GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT-3′) and zebra finch-specific GAPDH sequences: GAPDH-F (5′-AACCAGCCAAGTACGATGACAT-3′), GAPDH-R (5′-CCATCAGCAGCAGCCTTCA-3′). The telomere and GAPDH reactions were carried out on different plates, the number of PCR cycles required for the products to accumulate enough fluorescent signals to cross a threshold was determined. The detailed description of the conditions of the PCR can be found in [35]. A standard curve was included to measure the efficiencies of the reactions on every plate. The reference sample was from a zebra finch that was 21 days old at the time of collection. The efficiencies were within an acceptable range (plate mean ± SD GADPH 100.82 ± 3.31; telomere 91.68 ± 6.47) in all cases. All samples, including the standard curve, were run in triplicate, and average values were used to calculate the relative T/S ratios for each sample relative to the reference sample (for details see [17]). All of the samples of an individual were run on the same plate, i.e. the samples from each individual, taken on day 21 and 120 were run in the same plate in triplicate. In total, 23 plates were run for telomeres and GAPDH. The mean ± SD intraplate coefficient of variation of the Ct values was calculated per plate by dividing the standard deviation by the mean of the 20 ng concentration wells in the standard curve, multiplied by 100 (3 replicates). As a result, we got 1.99 ± 1.00 intraplate variation for the telomere assays and 0.15 ± 0.09 for the GAPDH assays, respectively. The average interplate variation for the ΔCt values was 3.96% and was calculated using the standard deviation value of ΔCt of the 20 ng wells of the standard curve of all plates divided by the mean, multiplied by 100.

Paternity analysis

To account for possible genetic effects on telomere loss, we considered the identity of parents in the analysis (see below). Since there is typically a considerable amount of extra pair young in captive zebra finch colonies [36], genetic paternity analysis is necessary to reliably assign parentage. To this end, all offspring were genotyped at 11 highly polymorphic microsatellite markers [37] and parentage was assigned by exclusion.

Statistics

All statistical analyses were performed with R 3.1.1 (R Core Team 2013). We fitted linear mixed-effects models to analyze our data, using the “lmer” function (package lme4). Additionally, we used the “sim” function (package arm) to simulate the posterior distribution of the model parameters and values were extracted based on 2000 simulations [38]. The statistical significance of fixed effects and interactions were assessed based on the 95% credible intervals (CI) around the mean (estimate). We considered an effect to be “significant” in the frequentist’s sense (p < 0.05) when the 95% CI did not overlap zero [39]. Telomere length (log-transformed) was set as the dependent variable, treatment (NoNoise, PNoise, JNoise), age when the sample was taken (21 or 120 days old), sex, mass of every individual at 21 and 120 days (mass) and breeding round as independent factors. Breeding round is the number of times the adults have reproduced. The individual ID, the ID of the genetic parents, and the aviary (to account for effects of the common aviary) were included as random effects. Genetic parentage was determined by exclusion using the R package SOLOMON [40]. The model used in the paper included interaction between treatment and age and was compared to other models using the Akaike Information Criterion (AIC), REML was set to FALSE [41]. The repeatability was calculated based on random effects of the model, for the individual repeatability parents ID were not taken into account.