We used juvenile mice that were the offspring of wild-caught mice in Austria. We provided bedding, wood wool, tissues and cardboard tubes as nesting materials. Each experimental cage consisted of three standard (type II) mouse cages connected with plastic tubes to increase space (0.115
). Water and food (Altromin rodent diet 1324) were available ad libitum. The animals were kept under 12
h light/dark cycle with an ambient temperature of 23±3°C and relative humidity of 65±10%. The experiment was conducted from March to September 2005, and the procedures were approved by the Austrian Ministry of Education, Science and Cultures' Animal Care and Use Committee.
At four weeks of age, we assigned the animals to one of the three experimental groups. We housed females in large cages together with (i) one male and removed all offspring at 21 days of age to reduce interbirth intervals (reproduction treatment, REP), (ii) one male and we left female offspring to increase density (crowding treatment, CRD), or (iii) no males but one sister to avoid isolation stress (control, CTRL). We used the mean of two control females for our statistical analyses. To control for potential familial effects, we systematically assigned females from four-sister litters placing two in the control group and one each in treatment groups (each group was replicated 10 times). We did not include a similar control for males because males fight violently.
We collected blood to obtain DNA before the onset (before any births) and after termination of the experiment. To obtain DNA, we bled mice at 6, 14 and 16 weeks of age (75
μl each) and the blood was pooled (sample 1), stored in equal amounts of 50 mM EDTA and kept at −70°C for later DNA extraction. The experiment was terminated when the animals were six months old (mean age: 183.4±0.6 days). The animals were anaesthetized using ether, euthanized by rapid decapitation and blood was collected in 50
mM EDTA and frozen at −70°C (sample 2). We extracted DNA using a commercial kit (DNeasy Tissue Kit, Qiagen) and measured the mean telomere length of white blood cells using a real-time PCR method (Cawthon 2002
) adapted for mice (Callicott & Womack 2006
). A specially designed oligonucleotide primer set hybridizes to the TTAGGG and CCCTAA repeats and selectively amplifies telomeric DNA: longer telomeres lead to quantifiable acceleration of amplification. The amplification of telomeric DNA is compared to the amplification of a single-copy gene DNA of the same sample. We used MapK1 as the single-copy gene with the following primer sequences: Mapk1-F: 5′-GCT TAT GAT AAT CTC AAC AAA GTT CG-3′ and Mapk1-R: 5′-GAT GTT CTC ATG TCT GAA GCG-3′. All the samples were compared to one reference standard (relative telomere measurements), and the value of sample 1 (before treatment) was then subtracted from the value of sample 2 (after treatment). Negative values indicate telomeric attrition and positive values a gain. The procedure was carried out on an ABI (Applied Biosystems International) 7300 real-time PCR machine.
Before conducting statistical tests (SPSS 12.01), we confirmed that all assumptions were met. Since we had a priori
predictions for the direction of telomere changes over time (attrition) and among groups (CTRL<(REP, CRD)), we used isotonic regression to test for among-group differences (Gaines & Rice 1990
), directed Dunnett's pairwise multiple t
-tests for females and a directed t
-test for male differences (Rice & Gaines 1994
). To test whether telomeres changed length over time, we compared the mean values with the 0 reference value by using one-sample t
-tests. We predicted telomere attrition over time (mean telomere length change<0) and used directed tests. We obtained the critical values for each directed test from the p
values of the corresponding one-tailed test, by using γ
=0.8 as a pragmatic conventional value (Rice & Gaines 1994
). Using two-sided tests does not change the interpretation of the results, except that the observed increase for telomeres in males of reproduction group becomes significant. Averages are shown as mean±s.e.m. Sample sizes between the experimental groups differ because two females and two males of the reproduction treatment and one female of the crowding treatment died during the experiment.