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Electronic supplementary material
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a) General methods
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We used the generation F2 of common voles (Microtus arvalis) whose founder parents (i.e.
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generation F0) were live trapped in twelve different sites in the canton Vaud, Switzerland, and transferred
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within 24 hours in an animal room at the University of Lausanne, Switzerland. Voles were housed
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individually in polycarbonate cages (42.5 x 26.6 x 18.5 cm) with constant 14 h light: 10 h dark cycle and a
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constant temperature of 22°C ± 1°C. Cages contained sawdust, hay and a flower pot as cover. Water and
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food pellets were available ad libitum, and apples and endives were offered two times a week. For the
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pairing, one male was transferred for one week into the cage of the female. Pups were born 19 to 23 days
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after the beginning of pairing (median 21 d). We measured female body mass at the beginning of pairing,
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two weeks after pairing, at the day of birth (day 0), when the pups were ten days old and when they were
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weaned at the age of 21 days. We estimated the duration of pregnancy as the number of days elapsed
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between the start of pairing and the day of birth of the pups.
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For purposes independent from the present study, founder parents were captured in sites divided in
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two altitude categories (high altitude >1300 m a.s.l. versus low altitude <600 m a.s.l.). We took in
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consideration this point by always pairing male and female from the same altitude category and by
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manipulating litter sizes among pairs of females from the same altitude category. In total, we created 10
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and 4 pairs of females from low and high altitude categories, respectively. When putting in place the litter
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size manipulation (LSM), females were distributed in three different mating sessions separated by an
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interval of ca. one month among sessions. This was done because some females were too young when the
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first mating session took place and because some females did not get pregnant during the first session.
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However, when addressing the long-term costs of LSM, that is when females had their second reproductive
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event, all the females were paired with a new male on the same date. In both enlarged and reduced
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treatments, 25% of females that successfully reproduced twice had their first pairing in the first session,
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50% in the second session and 25% in the third session. Therefore, long-term effects of our LSM treatment
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were not confounded with pairing dates. In the statistical analyses, we controlled for the fact that different
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pairs of females were paired on different days and they were coming from two different altitudinal
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categories by including the mating pair as a random factor in the statistical models.
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b) Measurements of resting metabolic rate
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Females were placed individually in a sealed polycarbonate cage (36.5 x 20.7 x 14.0 cm) without food one
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hour prior to the start of the measurements. We measured metabolic rate with SM-MARS-4 open flow
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system allowing the measurements of oxygen consumption (VO2) and carbon dioxide production (VCO2) of
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three animals in parallel (Sable Systems International, Las Vegas, USA). Measurement took place between
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7:30 and 17:00. The common vole has regular activity cycles spaced by ca. 150 min [1], and thus our
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measurements spanned both active and inactive periods. We recorded the activity of the voles with activity
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detectors (Sable Systems MAD-1) that were under the cages to ensure that the resting metabolic rate
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(RMR) values were recorded during an inactive period. Cages were in a dark climate chamber (EKOCTL 900
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P, Angelantoni Industrie, Massa Martana, Italy) set at 30oC, which is within the thermoneutral zone of the
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common vole [2]. The air was pulled from the cages with MFS-5 pumps with 1L/min rate to the Multiplexer
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(MUX) where the air sample (500ml/min) from one cage at a time was pumped with a subsampler (SS4) to
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the water vapour (RH-300)-, CO2 (CA-10)- and O2-analyzers (FC-10) in this order. The raw data was
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analysed using ExpeData software (Sable Systems International, Las Vegas, USA). One measuring cycle
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consisted of two 150s baseline readings (air from the climatic chamber) at the beginning and at the end of
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each cycle to control for baseline drift. In between two baselines readings, cages were sequentially
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measured 4 times for 150s. A sample was taken every second, and the mean value of the 45 last samples of
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every 150s period was used as single reading. In total three measuring cycles (in total 12 readings per cage)
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were recorded. The oxygen consumption (mL O2/h) was calculated according to the equation
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𝑉𝑂2 = 𝐹𝑅 ∗
(𝐹𝑖 𝑂2 − 𝐹𝑒 𝑂2 ) − 𝐹𝑖 𝑂2 ∗ (𝐹𝑒 𝐶𝑂2 − 𝐹𝑖 𝐶𝑂2 )
1 − 𝐹𝑖 𝑂2
2
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where FR = flow rate (mL/h), 𝐹𝑖 𝑂2 = Fractional concentration of O2 in incurrent air (baseline), 𝐹𝑒 𝑂2 =
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fractional concentration of O2 in excurrent air, 𝐹𝑖 𝐶𝑂2 = fractional concentration of CO2 in incurrent air,
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𝐹𝑒 𝐶𝑂2 = fractional concentration of CO2 in excurrent air. All values are corrected to standard temperature
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and pressure (STP) and corrected for water vapour pressure. As a measurement for RMR we computed the
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mean of the two lowest consecutive readings when a vole was inactive. Our measurement of RMR is
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defined as a lowest metabolic rate of inactive, lactating female measured at thermoneutrality in a not
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completely post-absorptive stage. Longer fasting time could lead to increased activity and reduced energy
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intake of the pups.
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c) Influence of weaning mass on survival in the laboratory
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Body mass at weaning is often viewed as an adequate measurement of fitness since heavier weanlings are
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often reported to enjoy higher post-weaning survival in the wild (e.g. [3]). Whether the same is true in the
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laboratory has however not been reported. We investigated whether body mass at weaning has an
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influence on life expectancy using a survival analysis on 1196 voles born in our animal room. Of these voles
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190 had died naturally in our animal room (median [25% and 75% quartiles] age at death: 95.0 days [59.8;
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131.3]), whereas 1006 were still alive or had been terminated before their natural death (median [25% and
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75% quartiles] age at termination or last observation: 169.0 days [118.0; 239.5]). We controlled for the fact
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that a large number of the individuals did not died naturally by using a right censoring procedure. Note
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here that 75% of the right censored individuals (i.e. still alive or terminated) where above 118 days. Thus,
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their inclusion in our survival analyses allowed us to accurately predict life expectancy up to 100 days at
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least, which is probably above the median life expectancy of voles in the field (mean survival time of 1457
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wild prairie vole M. ochrogaster that lived at least up to first re-capture was 65.6 days [4]). In the statistical
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analysis using the statistical package JMP 10.0 (SAS Institute Inc., Cary, NC), we entered the age at last
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observation (i.e. natural death, terminated, or still alive) as response variable and sex, body mass at
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weaning and their interaction as explanatory variables. We modelled their survival with a Frechet
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distribution since it fitted best the data following preliminary analyses with different distributions (i.e.
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exponential, Frechet, log normal, and Weibull distributions). In agreement with results in the wild, our
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results show that in the common vole body mass at weaning is a strongly significant positive predictor of
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life expectancy, at least up to 100 days, in the laboratory (mean ± SE = 0.06 ± 0.02, χ2 = 7.13, P = 0.008) (Fig.
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S1). Sex, either alone or in interaction with body mass at weaning, was non-significant (P = 0.53 and P =
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0.22, respectively). We have no information on the causes of death of voles that died naturally in the
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laboratory.
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d) Long-term effects of litter size manipulation without females that died at the end of lactation
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Three females that raised an enlarged litter in the first reproductive attempt died at the end of their second
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reproductive attempt. In the original analyses those females were included, except for female body mass
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change during the second half of lactation (days 10-21) and for body mass at weaning. Table S1 provides
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results when those three females are excluded from all the analyses on the long-term costs of
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reproduction. Analyses were performed as described in the main article. Although mean values reported in
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Tables 1 and S1 remain similar for the two treatments, the effect of pup mean body mass at day 21
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changed from being significant (P<0.05) in Table 1 to being marginally significant (P<0.08) in Table S1. This
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change significance is however likely explained by smaller sample sizes, and in turn weaker statistical
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power, rather than biased sampling leading to misleading results.
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Accordingly, in the common vole weaning age (i.e. when primary nutritional dependency on the
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mother ends) is thought to occur between ca. 17 and 20 days of age (e.g. 17.2 days in the PanTHERIA
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database [5] versus 20d in the AnAge database [6]). In our study, pups were separated from their mother at
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21 days of age, so pups were presumably no more lactated by their mother since 1 to 3 days when
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“weaned”. Females were found dead within 24h to 48h preceding “weaning” (we don’t know the exact
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time of death). Thus, time spent without maternal care was limited and occurred primarily when pups were
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already nutritionally independent. Note also that, among the three litters where mothers died at the end of
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lactation, two had not especially low values for pup mean body mass at day 21 (13.40g and 12.05g). Pups
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from three litters where mothers were still alive at weaning had a mean body mass lower than 12.05g.
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However, pups from the third litter with a dead mother at the end of lactation had a mean body mass at
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day 21 of 8.63g, while the second lowest pup mean body mass from this dataset was 10.45g. Excluding only
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this “outlier litter” from our analyses leads to a significant difference in pup mean body mass at day 21
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between reduced and enlarged litters (P = 0.048), further supporting the idea that our results are mainly
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dependent on statistical power. Mothers’ body mass at weaning was not included in the analyses for
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females that died at the end of lactation since corpses are known to lose fluids after death, and thus body
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mass of dead females is not comparable with live females. Finally, it is worth noting that, when the three
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litters where the mother died at the end of lactation are removed from the statistical analyses, the
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difference in pup mean early growth rate (day 2-10) between reduced and enlarged treatments remains
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highly significant. This is the period that coincides with the peak of lactation, and thus where female
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reproductive effort is expected to shape offspring development.
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References
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1. Gerkema MP, Daan S, Wilbrink M, Hop MW, van der Leest F. 1993 Phase control of ultradian feeding
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rhythms in the common vole (Microtus arvalis): the roles of light and the circadian system. J. Biol. Rhythms
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8, 151-171.
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2. Devevey G, Niculita-Hirzel H, Biollaz F, Yvon C, Chapuisat M, Christe P. 2008 Developmental, metabolic
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and immunological costs of flea infestation in the common vole. Funct. Ecol. 22, 1091-1098.
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3. Wauters L, Bijnens L, Dhont AA. 1993. Body mass at weaning and juvenile recruitment in the red squirrel.
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J. Anim. Ecol. 62, 280-286
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4. Getz LL, Simms LE, McGuire B, Snarski ME. 1997. Factors affecting life expectancy of the prairie vole,
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Microtus ochrogaster. Oikos 80, 362-370.
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5. Jones KE, Bielby J, et al. 2009. PanTHERIA: a species-level database of life history, ecology, and geography
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of extant and recently extinct mammals. Ecology 90, 2648-2648.
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6. Tacutu R, Craig T, Budovsky A, Wuttke D, Lehmann G, Taranukha D, Costa J, Fraifeld VE, de Magalhaes JP.
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2013. Human Ageing Genomic Resources: Integrated databases and tools for the biology and genetics of
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ageing. Nucl. Acids Res. 41: D1027-D1033.
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Figure S1. Predicted survival fit (solid line) and 95% confidence intervals (dashed lines) in relation to body
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mass at weaning in common voles kept under constant laboratory conditions (22°C, 14 h light: 10 h dark
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cycle).
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Age (days)
300
200
100
0
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10
14
18
22
Body mass at weaning (g)
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Table S1. Long-term effects of litter size manipulation experiment on female voles and their offspring,
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excluding from the analyses females that died at the end of lactation (N = 3 enlarged litters). Differences
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between groups were tested using Markov Chain Monte Carlo (MCMC) simulations. Sample sizes (N),
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MCMC means, 95% highest posterior density (HPD) intervals and P-values are reported. Significant
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(P<0.050) differences are written in boldface.
Reduced litters
N
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Mean [95% HPD]
Enlarged litters
N
Mean [95% HPD]
P-value
(a) Interval between reproductive events
Time interval from weaning to the next pairing (days)
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55.64
[31.64; 80.00]
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49.65
[33.41; 66.36]
0.583
Body mass from weaning to the next pairing (%)
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23.05
[-0.45; 46.25]
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12.88
[-2.96; 28.24]
0.3676
(b) Effects of litter size manipulation on female and pup phenotypes in the subsequent reproductive event
Body mass at pairing (g)
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31.18
[22.81; 40.12]
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Duration of gestation (days)
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20.30
[19.63; 20.98]
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23.62
[17.66; 29.23]
0.083
20.55
[20.10; 21.00]
0.432
Body mass gain during gestation (%)
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16.38
[2.66; 30.70]
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34.27
[24.73; 43.52]
0.016
Body mass at day 2 (g)
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34.34
[27.84; 40.74]
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28.31
[23.84; 32.61]
0.071
Litter size, day 0
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4.14
[2.55; 5.86]
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4.67
[3.51; 5.75]
0.501
Proportion of sons, day 0
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0.69
[0.38; 1.00]
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0.62
[0.42; 0.84]
0.663
Body mass change, day 2-10 (%)
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-10.09
[-15.99; -4.35]
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-3.89
[-7.77; 0.08]
0.039
Body mass change, day 10-21 (%)
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-10.86
[-21.44; -0.88]
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-16.27
[-22.88; -8.96]
0.273
Body mass, day 21 (g)
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27.31
[21.84; 32.90]
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22.68
[18.85; 26.23]
0.099
Litter size, day 21
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4.14
[2.51; 5.78]
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4.56
[3.44; 5.63]
0.588
Pup mean early growth rate, day 2-10 (g/day)
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0.59
[0.51; 0.67]
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0.46
[0.41; 0.51]
0.004
Pup mean early growth rate, day 10-21 (g/day)
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0.63
[0.45; 0.81]
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0.60
[0.48; 0.72]
0.698
Pup mean body mass, day 21 (g)
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14.87
[12.92; 16.90]
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13.14
[11.82; 14.48]
0.082
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