Animal model and husbandry
This work was performed on bank voles (Myodes = Clethrionomys glareolus Schreber 1780) from generation 25 of an ongoing artificial selection experiment maintained at the Jagiellonian University (Poland). The rationale, history and protocols of the experiment have been presented in our earlier papers 28,34,35,39,40. Briefly, the colony was established based on about 320 wild voles captured in 2000 and 2001. After 5–6 generations of random breeding, the selection experiment has been started. In the selected, “aerobic” (A) lines the selection criterion is the maximum 1min rate of oxygen consumption (VO2swim), achieved during a swimming. The VO2swim values used as selection criteria are mass-adjusted (residuals from ANCOVA including also other covariates and cofactors). Four replicate lines for both selected and unselected control lines (C) are maintained (to allow valid tests of the effects of selection; Henderson, 1997), with 15–20 reproducing families in each of the 8 lines (which avoids excessive inbreeding). Since generation 18, VO2swim of the A lines exceeds that of the C lines by more than 60% (Supplementary Fig. S1b).
The animals were maintained in standard, polypropylene mouse cages with sawdust bedding, at constant temperature (20 ± 1°C) and photoperiod (16:8 light:dark; light phase starting at 2am). At the age of 17 days the animals were weaned, marked temporarily by fur clipping and kept in family groups. At the age of about 34 days, all individuals were marked permanently with mouse ear tags (model 10005-1; National Band and Tag, Newport, KY; mass 0.18 g) and later maintained in same-sex groups of three individuals in model 1264C cages or up to four individuals in a larger model 1290D cages (Tecniplast, Bugugiatte, Italy). The thick layer of sawdust allowed the animals to burrow under its surface, and it contained fragments large enough for the animals to gnaw on. Cages were changed every 5–14 days, depending on the number of animals in the cage, size of the cage and their cleanliness. Water and food (a standard rodent chow: 24% protein, 4% fat, 4% fiber; Labofeed H, Kcynia, Poland) were provided ad libitum. Every day all cages were visually inspected for presence of food and water or dead animals. The colony was under supervision of a qualified veterinary surgeon. During any kind of measurements, if symptoms of poor condition were observed in an animal (problems with breathing or moving, injury, etc.), it was removed from the experiment. Depending on judgment of the observer or animal care personnel, it was either allowed to recover or euthanized.
All the breeding, selection and experimental procedures were approved by the Local Ethical Committees in Krakow, Poland (decision no. 170/2014–1st Local Ethical Committee for Animal Experiments, Faculty of Pharmacy, Jagiellonian University Medical College in Kraków; 257/2017–2nd Local Institutional Animal Care and Use Committee, Institute of Pharmacology Polish Academy of Sciences in Kraków). In generation 25, the standard selection test was completed in 1026 individuals (849 from the A lines and 177 from the C lines), including all available A-line animals and typically one male and one female from each C-line family.
Selection test: swimming trial
The selection VO2swim test was performed at the age of 75–85 days, between 8:00 and 18:30 h, as described in our earlier reports 28,34,37. At the day of measurement, home cages were transferred from the housing room to the laboratory. An animal was removed from its cage shortly before the start of its measurement, weighed, and placed in a temporary container where it stayed for several minutes until the test started. The trial was performed in 15 cm diameter 3-liter glass jars (respirometric chambers) partly filled with water. To ensure that the metabolic response was related to physical activity rather than thermoregulatory burden, and to avoid a possibly confounding effect of reaction to cold stress, the water temperature was set at 38°C. The animal was gently placed on the surface of water and allowed to swim freely. The test lasted for up to 18 minutes, unless an individual began to drown or oxygen consumption rapidly decreased. After the swimming trial the animal was wiped with paper towel, returned to its home cage fitted with fresh bedding, and placed under a heating lamp which supported fur drying. The cages were then returned to the colony.
The VO2swim was measured simultaneously on two individuals in two parallel respirometric chambers, connected to two custom-built positive pressure open-flow respirometric systems. Some technical details of the respirometric systems (type of gas analyzers, mass-flow controllers, and air driers) changed across the subsequent generations of the selection experiment; here we present the details appropriate for generation 25, from which animals for the current experiment were sampled. Fresh air taken from outside of the building was dried with silica gel and pumped through the respirometric chambers at about 2,000 ml/min (STPD, measured before the chamber). The air flow was controlled with mass-flow controllers (GFC17 or GFC171S, Aalborg, Orangeburg, NY, United States) and the exact flow values were determined with high precision glass rotameters (ROTA model L2.5/100). A sample of excurrent air (about 200 ml/min) was dried with ND2 gas sample drier (Sable Systems, Las Vegas, NV, United States) and with chemical absorber (magnesium perchlorate), and directed to FC10 or FC10A oxygen analyzers, and CA-2A or CA-10A CO2 analyzers (Sable system, Las Vegas, NV, USA). We performed calibration of the gas analyzers according to the instruments manuals. The oxygen analyzers were calibrated at one-point against clean, dry air, assuming it has a concentration of 20.95% oxygen. For CO2 analyzers, the adjustment of zero was performed with respect to CO2-free dry air (CO2 was removed with Ascarite), and the adjustment of span with a reference calibration gas containing 0.95% CO2.
The analyzers recorded the gas concentrations at 1s intervals for approximately 21 minutes per animal, including the duration of the swimming trial, washout time after removing the animal from the chamber, and two 30-s baselines (initial and final). The rate of oxygen consumption (VO2) was calculated with the equation:
(1)
where V̇i is the incurrent air flow rate, and FiO, FeO, FiC, FeC are fractional concentrations (F) of oxygen (O) or CO2 (C) in incurrent (i) or excurrent (e) air. The equation is basically the same as Eq. 10.6 in 56, but it correctly accounts for the fact that CO2 concentration in incurrent air differs from zero. The VO2 values are then corrected for effective volume to achieve instantaneous rates 56, 57.
Experimental design overview
The experiment comprised two phases: three-week habituation, in which all animals were treated in the same way (days D21 to D0), and two-week experiment proper, in which the CMS procedure was applied to half of the animals (days D + 1 to D + 14; Table 1). All the work was performed in three nearly-balanced blocks, starting in 15-day intervals.
During the habituation period the animals could get accustomed to the daily weighing and fecal collection sessions that continued throughout the proper experiment. The habituation was performed on 49 A-line 69 C-line animals, sampled approximately equally from each of the replicate lines, one individual from a full-sib family. The animals were 54–64 days old at the beginning of this period. Every day the animals were handled, weighed and moved for 20 minutes to an empty cage, which enabled collection of freshly deposited feces. The procedures were suspended only on days D0, D + 7 and D + 14, when VO2swim was measured. The swimming trials followed the same procedure as in the selection test. After the D0 trial, 46 A-line and 48 C-line voles were sampled from those that completed the trial without incidents of drowning or diving attempts. These animals were assigned to two experimental groups, 47 animals each. One of the experimental groups was moved to a separate chamber where chronic mild stress (CMS) protocol was applied (Table 1). The second group remained undisturbed aside from the daily weighing and fecal collection that continued for both groups throughout the experiment. The swimming trials at D + 7 and D + 14 were performed on animals from both groups, in randomized order. On D7, D + 1, and D + 8 animals received weighed portions of approximately 105 g of standard food, and food remaining in feeder was weighed one day before each swimming trial.
Data were collected in three sets, representing three weeks, each concluded with a swimming trial: the last week of the habituation procedure (initial measures), and the first and the second week of the proper experiment. Five types of data were collected: the maximum and average rate of oxygen consumption during swimming (VO2swim and VO2avg), apparent food intake rate (FI) calculated from mass of food removed from feeder over 5 or 6 days preceding a swimming trial, and body mass (BMavg) and fecal corticosterone metabolites level (FCMs) 42, both representing four days preceding a swimming trial.
Habituation procedure
At day D22 the animals were weighed and separated into model 1264C cages (Tecniplast, Bugugiatte, Italy). Unlike rats or mice, bank voles are solitary in nature 58, and thus social isolation is unlikely to elicit stress-related disorders. Aside from being housed individually, the housing conditions were identical to that applied in the colony. Daily habituation sessions started at D21. In mice, 14 days of daily handling was sufficient for habituating the animals to human presence 43. The additional seven days we applied allowed collecting the initial data unbiased by the novelty of the daily routine and the experimental procedures applied afterwards.
Between 8:00 and 11:00am, each animal was removed from its home cage by neck scruff or in cupped hand, weighed in an opaque plastic cup (approximately 18 cm high and 8 cm in diameter) and placed in an empty, “sampling” cage of the same model as the housing cage, with no sawdust or access to food or water. After 20 minutes the animal was returned to its home cage. Feces deposited in the empty cage were collected starting from D2 for the first block and D7 for the second and third blocks.
Experimental groups establishment
During the habituation period two A-line animals died and three C-line ones were recognized as diabetic and excluded from the experiment (diabetes appears in a few percent of bank voles both in laboratory conditions and wild populations 59). The swimming trial performed at the end of the habituation (D0) was interrupted for four C-line animals which started to drown; another two C-line and one A-line animal were repeatedly diving which biased the measurement of their oxygen consumption. These seven animals were excluded from the experiment. The experimental blocks were balanced by excluding six excessive C-line animals, randomly chosen from over-abundant combinations of sex and replicate line. Thus, the proper experiment was performed on 48 C-line and 46 A-line voles (94 animals in total).
The animals were assigned to two treatment groups (comfort and CMS), 47 animals each. The assignment ensured that the number of animals of either sex from each replicate line was balanced between the groups. Within the replicate line × sex subgroup the assignment to the treatment groups was based on randomization blind with respect to all known or measured characters, particularly the age, mass or swimming performance.
Chronic mild stress (CMS) treatment
The daily manipulations introduced in the habituation procedure were continued on animals from both experimental groups throughout the experiment, and the order in which it was applied to the two groups within a given day was randomized. Animals from the CMS group were subjected to a series of mild stressors listed in Table 1. The procedure was adapted from protocols inducing the state of chronic stress in mice or rats 60–62. Two types of stressors were applied during each day. The series was arranged in a way that allowed the stressors applied during nighttime (18:00 to 2:00) and dawn (2:00 to 8:00) to be automated (disrupted light regime, intermittent noise or vibration), and those requiring manual operations were applied during daytime (8:00 to 18:00). Immediately after the daily handling, weighing and fecal collection the daytime stressor was initiated by either placing the animal in a new cage, containing no bedding or with a bedding thoroughly soaked with lukewarm water, or returned to the home cage but without access to water or food. After 8 hours the animals were returned to their home cages or access to food and water was restored, respectively to the type of stressor applied.
One CMS-group animal died in the first week of the experiment, and data collected from this individual were not used in the statistical analyses.
Feces collection and corticosterone metabolite analysis
Immediately after the animals were returned to their home cages, feces deposited in the “sampling” cages were collected using tweezers into empty Eppendorf tubes (0.5 ml, one tube per individual per session). Feces contaminated with urine were discarded, as bank vole urine contains varying amounts of corticosterone metabolites 63. The samples were stored on ice for up to 2h until frozen in -20°C.
After completion of all experimental procedures, the samples were dried overnight at 80°C. Because we observed a tendency for body mass to drop after the swimming trial, a decrease usually recovered over the following day or two, we assumed the fecal samples collected during the post-swimming recovery period could be biased by changes in the animal’s metabolism and food intake. Therefore, we excluded the samples collected during the two days following each swimming trial. The remaining samples were used to create three pools representing the four days preceding each of the three swimming trials (days D4 or D2 to D1, D + 3 to D + 6 and D + 9 to D + 13). The pooled samples were weighed, ground in a mortar, and a 50 mg subsample was placed in 1.5 ml Eppendorf tubes with 1 ml 80% methanol (POCh, Poland). The tubes were shaken for 30 minutes, then centrifuged for 10 min at 2500 g. A 0.5 ml aliquot of the supernatant was transferred to a separate Eppendorf tube and stored in -20°C. The trios of samples taken from an individual vole were extracted within the same batch, and the order in which these trios were extracted was randomized within blocks.
Fecal corticosterone metabolites in the extracts were measured using a 5α-pregnane-3β,11β,21-triol-20-one enzyme immunoassay, which measures metabolites with a 5α-3β,11β-diol structure. For a more detailed description of the used methods and the assay (e.g. antibodies and inter- and intra-assay variance), see 64. The method was validated and proven suitable for bank voles 63. In that particular validation experiment, it was shown that the intestinal gut passage time of corticosterone in bank voles is ca. 6–8h, from which it can be assumed that the samples collected in our experiment represented corticosterone released in the “early morning”, shortly after the onset of the light phase. All three samples of an individual were analyzed in the same batch, but the order of individuals and samples within individual was randomized.
Statistical analyses
Three sets of analyses were performed to test 1) effects of the habituation procedure, 2) differences between the comfort and CMS groups at the onset of the treatment, and 3) effects of the CMS treatment. The first set of analyses was performed on data from all individuals from generation 25 which completed the VO2swim test: 1026 individuals tested as a part of regular selection program (not included in the habituation procedure) and 100 included in this specific experiment (which passed the habituation procedure). This set contained data on animals’ body mass and rates of oxygen consumption (VO2swim, VO2avg) during their first swimming trial. The second and the third set of analyses were performed on data from the 93 individuals that completed the whole experiment. This data set contained data from measurements repeated three times per individual, taken during the last few days of the habituation, i.e., just before the experiment proper (“initial”), and the two weeks of experiment.
The analyses were performed with cross-nested mixed ANCOVA models, using SAS v. 9.4 (SAS Institute, Inc., Cary, NC, USA) Mixed procedure (with REML method of estimation and variance components restricted to positive values). All the models included selection direction (A vs. C lines) and sex as the main fixed factors, random effect of replicate line (nested within selection direction), and body mass as a covariate (unless body mass was the subject of analysis). The hierarchical structure of the statistical model (replicate lines nested in selection direction) is required to allow a proper distinction of the effects of selection from random genetic effects, such as the genetic drift 55. Models for the measures of swimming performance included respirometric system as additional fixed cofactor, and time at the start of measurement as an additional covariate. This basic model structure was further expanded to accommodate additional factors adequate for specific analyses:
1) In the first set of analyses the models included fixed factor determining if the animals was subject to the habituation procedure or not. The models included also a random factor of family (nested in replicate line) to properly handle non-independence of observations obtained on individuals from the same full-sib families, litter number of a particular family as a cofactor (the 1st, 2nd, and 3rd or further litter), and litter size as a covariate. As animals involved in the selection protocol are not weighed daily, the single body mass measured immediately before the swimming trial was used as a covariate.
2) The second analysis was performed for data obtained in the last few days of habituation period (just before applying CMS treatment): 4-day average body mass (BMavg), 6-day average daily food intake rate (FI) and 2- or 4-day average fecal corticosterone metabolites level (FCMs), and for the average and maximum swim-induced metabolic rates (VO2avg and VO2swim) measured at day D0. The models included fixed factor of the CMS treatment (CMS or comfort groups), an interaction between treatment and selection direction, a random factor of experimental block, and BMavg as covariate. Diagnostic graphs (histograms of residuals, residual vs quantile plots, and residuals vs predicted values plots) showed that values of FCMs were right-skewed, and were therefore log10transformed prior to the analyses.
3) The third, most important set of analyses was applied to similar traits as in the second set (BMavg, FI, FCMs), but measured in the final few days of the first and second week of the CMS treatment, and VO2swim and VO2avg measured on days D + 7 and D + 14 of the treatment. Unfortunately, despite best efforts put into group randomization, the second set of analyses revealed that the experimental groups (CMS vs comfort) varied in terms of swimming performance already in the “initial” week, before the CMS treatment was applied (see Table 3, Fig. 2, 3). Therefore, to correct for the discrepancy, the effect of CMS treatment on BMavg, FI, VO2swim and VO2avg was tested for values recalculated to represent percent change from the initial values (measured just before applying the treatment). Only for FCMs, which was right-skewed, the deviation was represented by log10transformed ratio of the inexperiment and initial values (which is equal to difference between log10transformed inexperiment and initial values of FCMs). The results obtained in the first and second week of the treatment were analyzed within one, repeated measures model. Therefore, in addition to the main effect of CMS treatment, the model included also the week of experiment (first or second) as a repeated measures (within-individual) fixed factor and a random effect of individual. The preliminary models assumed either equal or unequal residual variance (compound symmetry or unstructured covariance structure), and models with lower AIC (Akaike Information Criterion) were chosen for the final models.
In all the above analyses the initial models included interactions among the main fixed categorical factors (selection, sex, handling or experimental treatment and week, where applicable), between the main fixed factors and BMavg, and all the respective random interactions with the replicate line. The models were then step-wise reduced by removing non-significant interactions (p > 0.05). However, the interactions between selection direction and sex, and between selection direction, experimental treatment and week of experiment were a priori considered meaningful from the biological and experimental point of view, and were retained in the models irrespective of their significance. In the analyses for metabolic rates in the first set of analyses the interaction between selection direction and body mass was significant, and the final model had to retain heterogeneous slopes. Therefore, the effect of selection was tested for the average (23 g), minimal (16 g) and maximal (32 g) body mass relevant for both selection groups (using ”at” option in SAS “lsmeans” statement), but since it was highly significant in the entire range of body masses, only results adjusted for the average body mass are reported.
In all analyses the Satterthwaite’s approximation was used to calculate the effective degrees of freedom (df) for t tests or the denominator df for F tests (i.e., the df was computed from a combination of the dfs of respective random grouping effects and residual term, weighted by variance contribution of the terms; 65. Thus, the dfs could take any real value between df of the random factor and df of the residual term. Significance of the random effects was tested with the likelihood ratio (LR) test, based on results from models with the same structure as described above, but with variance components not restricted to positive values (“nobound” option in SAS Mixed procedure).
In set 3, several outliers were recognized and excluded from the analyses. Because values for these analyses were calculated from an initial and one of two measurements taken during the experiment, either one or both data points representing a particular individual were considered as outliers. The exclusion was applied to two data points from one individual in FI analysis, one data point in FCMs analysis, four data points from two individuals in VO2avg analysis and five data points from three individuals in VO2swim analysis (see Supplementary Fig. S2 and raw data set). The absolute values of studentized residuals of all excluded points were ≥ 2.7, and within excluded individuals the studentized residual of at least one of the points was ≥ 3.0.