Short- and long-term effects of litter size manipulation in a small wild-derived rodent

Abstract

Iteroparous organisms maximize their overall fitness by optimizing their reproductive effort over multiple reproductive events. Hence, changes in reproductive effort are expected to have both short- and long-term consequences on parents and their offspring. In laboratory rodents, manipulation of reproductive efforts during lactation has however revealed few short-term reproductive adjustments, suggesting that female laboratory rodents express maximal rather than optimal levels of reproductive investment as observed in semelparous organisms. Using a litter size manipulation (LSM) experiment in a small wild-derived rodent (the common vole; Microtus arvalis), we show that females altered their reproductive efforts in response to LSM, with females having higher metabolic rates and showing alternative body mass dynamics when rearing an enlarged rather than reduced litter. Those differences in female reproductive effort were nonetheless insufficient to fully match their pups’ energy demand, pups being lighter at weaning in enlarged litters. Interestingly, female reproductive effort changes had long-term consequences, with females that had previously reared an enlarged litter being lighter at the birth of their subsequent litter and producing lower quality pups. We discuss the significance of using wild-derived animals in studies of reproductive effort optimization.

1. Introduction

A central tenet of life-history theory is that current investments in reproduction are traded-off against future reproductive performances and survival [1]. Consequently, iteroparous organisms are expected to maximize their overall fitness by optimizing their reproductive effort over multiple breeding events. An important tool to investigate parental reproductive effort optimization is the use of litter size manipulation (LSM) experiments [2]. LSM experiments are classical in wild bird studies [3], and although results are often in agreement with life-history theory (i.e. parents subjected to increased reproductive effort in a given season are paying a cost in terms of survival or reproduction in subsequent seasons), these results can nevertheless be complex and sex-specific [4]. In small mammals, LSM studies are typically restricted over a single reproductive event [5] preventing a detailed examination of parental reproductive effort optimization. In those studies, litter size enlargement has usually no or little effect on the reproductive effort of lactating females while this effort can be decreased through litter size reduction [5–7]. Those results suggest that, similar to semelparous species, female rodents might express maximal levels of investment in their current reproduction rather than optimize reproductive investment over several breeding attempts. Because most small rodents are short-lived, natural selection could favour maximal levels of investment in current reproduction in populations where females are likely to reproduce only once in their lifetime.

Most of our knowledge on reproductive optimization in small mammals comes from laboratory animals. However, laboratory breeding-schedules are prone to selecting individuals that display maximal investment in their first reproduction event. Hence, it cannot be excluded that the observed lack of change in reproductive effort in response to litter size enlargement is foremost a feature of laboratory rodents. In wild rodents, there are only a handful of studies that have investigated reproductive costs over multiple breeding attempts and their results are mixed. In red squirrels (Tamiasciurus hudsonicus) and Columbian ground squirrels (Urocitellus columbianus), experimentally enlarging litters resulted in smaller weanlings but did not compromise female future survival [8–10]. In bank voles (Myodes glareolus), LSM had no effect on weanling production, but females rearing enlarged litters had lower survival and future fecundity [11]. However, because those studies were performed under natural conditions, it remains unclear whether long-term reproductive costs were mediated by extrinsic factors (e.g. food shortage preventing females from rearing an enlarged litter because of their energy requirements) or intrinsic variations in female reproductive effort optimization. So far, the best evidence of long-term effects of reproduction in rodents comes from laboratory-strain female mice (Mus musculus) that combined the energy demands of pregnancy and lactation [12]. In this study, females that were both pregnant and lactating during their first reproductive attempt delayed implantation at the start of their second pregnancy, and gave birth to more numerous but lighter pups compared with females with non-overlapping litters [12]. However, a replicate study using wild-derived mice gave contrasting results, with females being concurrently pregnant and lactating during their first attempt producing heavier pups at their second attempt [13].

In this study, to overcome the pitfalls of using laboratory animals in LSM experiments and to avoid confounding factors of LSM in the wild, we performed an LSM experiment in a small wild-derived rodent, the common vole (Microtus arvalis), kept under laboratory conditions for two generations (F₂). We investigated females’ reproductive optimization by manipulating the size of their first litter and testing subsequent consequences on their second, un-manipulated, reproductive attempt. We predicted that changes in females’ reproductive effort in response to LSM (measured as changes in females’ resting metabolic rate (RMR) and body mass) should translate into changes in reproductive performance during their second reproductive attempt.

2. Material and methods

We created 14 pairs of females that gave birth on the same day (defined as day 0). Within each pair, we created reduced (−2 pups) and enlarged (+2 pups) litters at day 2 by exchanging three pups from the reduced litter against one pup from the enlarged litter. The body masses of females and their pups were measured at days 2, 10 and 21. Pups were weaned at day 21. Common vole pups feed only on maternal milk between days 0 and 10 and gradually start eating dry food thereafter ([14] and M Lehto Hürlimann 2010 personal observation). Thus, we separately investigated the effects of LSM on early (2–10 days) and late (10–21 days) changes in female body mass and pup growth rates. Pup weaning mass is positively linked to survival in wild rodents [10] and to life expectancy in our captive population of voles (for details, see the electronic supplementary material). Hence, we used pup weaning mass as a proxy of offspring quality. At day 15–17, we measured females’ RMR using a respirometry system (for details, see the electronic supplementary material). The same females were paired with a new male 19–85 days after they had weaned their manipulated litter (median = 48 days). This variation in time intervals between the first and the second pairing is explained by the fact that, when setting-up the LSM experiment, females were distributed among three different pairing sessions separated by a monthly interval between sessions. However, for their second reproduction, when investigating the long-term consequences of the LSM experiment, all females were paired on the same date. Litter sizes were not manipulated during the second reproductive attempt. Intervals between pairings did not differ between treatments (table 1c), and the inclusion of this covariate in the statistical analyses does not affect our conclusions.

Table 1.

Short- and long-term effects of litter size manipulation experiment on female voles and their offspring. Differences between groups were tested using Markov Chain Monte Carlo (MCMC) simulations. Sample sizes (N), MCMC means, 95% highest posterior density (HPD) intervals and p-values are reported. Significant (p < 0.050) differences are written in italics.

	reduced litters			enlarged litters
variable	N	mean [95% HPD]		N	mean [95% HPD]		p-value
(a) pre-manipulation breeding parameters in females
age at pairing (days)	14	139.90	[104.25; 172.17]	14	127.22	[102.42; 153.15]	0.434
body mass at pairing (g)	14	22.28	[17.26; 27.50]	14	19.34	[15.74; 23.08]	0.247
duration of gestation (days)	14	20.72	[20.08; 21.39]	14	20.71	[20.23; 21.23]	0.995
body mass gain during gestation (%)	14	29.43	[19.11; 40.57]	14	33.31	[25.47; 41.45]	0.457
body mass at day 2 (g)	14	26.67	[23.27; 30.16]	14	23.91	[21.41; 26.44]	0.114
litter size, day 0	14	3.79	[3.07; 4.43]	14	3.22	[2.74; 3.71]	0.100
proportion of sons, day 0	14	0.40	[0.18; 0.63]	14	0.52	[0.36; 0.68]	0.269
(b) short-term effects of litter size manipulation on female and pup phenotypes
body mass change, day 2–10 (%)	13	−3.97	[−11.38; 3.09]	14	7.26	[2.17; 12.30]	0.003
body mass change, day 10–21 (%)	12	−9.21	[−15.92; −2.65]	14	−19.66	[−24.31; −14.97]	0.006
body mass, day 21 (g)	12	24.05	[20.74; 27.33]	14	20.55	[18.25; 22.85]	0.040
resting metabolic rate, day 16 (ml O2/h/g)	12	2.25	[1.72; 2.78]	14	2.80	[2.43; 3.17]	0.039
litter size, day 21	12	1.84	[1.10; 2.57]	14	5.07	[4.57; 5.59]	<0.001
pup mean early growth rate, day 0–10 (g/day)	13	0.53	[0.41; 0.65]	14	0.32	[0.24; 0.40]	0.003
pup mean late growth rate, day 10–21 (g/day)	12	0.66	[0.51; 0.82]	14	0.56	[0.45; 0.67]	0.211
pup mean body mass, day 21 (g)	12	14.87	[12.65; 16.94]	14	11.59	[10.13; 13.06]	0.004
(c) interval between reproductive events
time interval from weaning to the next pairing (days)	8	50.73	[29.34; 72.75]	12	50.44	[36.02; 64.28]	0.962
body mass change from weaning to the next pairing (%)	8	23.41	[0.49; 45.36]	12	15.09	[1.75; 28.82]	0.441
(d) effects of litter size manipulation on female and pup phenotypes in the subsequent reproductive event
body mass at pairing (g)	8	31.26	[23.66; 39.04]	12	23.92	[19.13; 28.47]	0.065
duration of gestation (days)	8	20.30	[19.61; 20.99]	12	20.66	[20.24; 21.09]	0.276
body mass gain during gestation (%)	8	16.20	[1.66; 30.59]	12	32.20	[23.12; 41.20]	0.030
body mass at day 2 (g)	8	34.29	[28.47; 39.88]	12	28.48	[25.10; 32.01]	0.049
litter size, day 0	8	4.14	[2.74; 5.60]	12	4.58	[3.73; 5.50]	0.519
proportion of sons, day 0	8	0.69	[0.41; 0.97]	12	0.63	[0.46; 0.81]	0.647
body mass change, day 2–10 (%)	8	−10.07	[−16.11; −4.71]	12	−4.88	[−8.25; −1.32]	0.069
body mass change, day 10–21 (%)	8	−10.90	[−21.19; −0.27]	9	−16.30	[−23.04; −9.38]	0.288
body mass, day 21 (g)	8	27.32	[22.03; 32.69]	9	22.67	[19.24; 26.48]	0.086
litter size, day 21	8	4.15	[2.63; 5.58]	12	4.25	[3.36; 5.18]	0.896
pup mean early growth rate, day 2–10 (g/day)	8	0.59	[0.52; 0.67]	12	0.47	[0.43; 0.52]	0.010
pup mean late growth rate, day 10–21 (g/day)	8	0.63	[0.46; 0.80]	12	0.54	[0.44; 0.65]	0.299
pup mean body mass, day 21 (g)	8	14.86	[12.91; 16.82]	12	12.70	[11.43; 13.90]	0.034

Statistical analyses were performed using linear mixed models in R (v. 2.15.1) using the function lmer from the package lme4 [15]. We entered LSM (enlarged versus reduced litter) as fixed factor and mating pair as random factor. This latter factor accounts for spatial and temporal heterogeneity in the mating pairs (for details, see the electronic supplementary material). We used Markov Chain Monte Carlo (MCMC) simulations to compute estimates and to test their significance with the function pvals.fnc from the package languageR [16].

3. Results

Before LSM, there were no differences between treatments in female phenotypes and reproductive biology (table 1a).

LSM was successful: females with an enlarged litter weaned on average 3.2 pups more than females with a reduced litter (table 1b). Two out of the 14 females with a reduced litter failed to wean pups, whereas all the females with an enlarged litter weaned at least one pup (Fisher's exact test: p = 0.49). Female body mass dynamics during lactation differed between treatments, with females raising an enlarged litter that ended up lighter at weaning (table 1b). Females with an enlarged litter had also higher RMR. In addition, their pups grew more slowly during the early growth phase and were consequently lighter at weaning (table 1b).

Twelve out of the 14 females with an enlarged litter and eight out of the 12 successful females with a reduced litter successfully re-mated (Fisher's exact test: p = 0.37). There was no difference between treatments in the time interval and female body mass change between the first and the second pairing (table 1c).

When delivering their second litter, females having previously reared an enlarged litter were lighter than females having previously reared a reduced litter, despite having gained more weight during gestation (table 1d). There was no difference in litter sizes between treatments (table 1d). Pups reared by females that had previously reared an enlarged litter grew more slowly during the early growth phase and were lighter at weaning (table 1d). Three out of 12 females from the enlarged treatment died within 48 h preceding day 21, whereas all of the eight females from the reduced treatment survived up to day 21 (Fisher's exact test: p = 0.24). Exclusion from the analyses of the three litters with dead females led to lower significance levels, but it did not alter the fact that pups were growing at slower rates when raised by females that had reared enlarged litters (electronic supplementary material).

4. Discussion

Our results show that wild-derived voles kept under laboratory conditions were capable to some extent of adjusting their current reproductive effort in response to LSM. This is clear from the higher RMR of females rearing enlarged rather than reduced litters. There were also clear between-treatment differences in lactating females’ body mass trajectories suggesting that lactating effort may vary over the time course of pups’ development and not peak at the same time point for females rearing reduced versus enlarged litters. Consistently with previous LSM experiments on laboratory or wild rodents [5–11], pups from enlarged litters grew at slower rates especially early in life when feeding only on maternal milk. Those pups were consequently lighter indicating that females were presumably less able to fully accommodate their offspring needs when weaning an enlarged rather than a reduced litter. LSM experiments in laboratory rodents have shown that beyond a critical litter size, females can no further increase their food intake, consequently leading to decreased milk production and pup growth [5,6,14]. In the common vole, lactating females are known to reach an asymptote in food intake beyond a litter size of six pups [14]. In our study, no female weaned more than six pups, although we occasionally observe litter sizes of seven or eight pups in our laboratory population, indicating that effort in our LSM remained within natural ranges of reproductive investment. Thus, the slower developmental rate of pups in enlarged litters was probably not merely mediated by females reaching a metabolic ceiling but also by changes in females’ reproductive allocation strategy. Because laboratory breeding schedules are prone to select for large litters (especially during the first reproductive attempt), laboratory rodents might encompass a biased sample of individuals selected for expressing maximal levels of reproductive investment and little optimization of their resource allocation over multiple reproductive events. Indeed, rodents recently derived from the wild on average produce smaller litters and show lower increase in metabolic rate during lactation than rodents maintained in the laboratory for many generations [17]. In our study, voles were kept in the laboratory for only two generations. Hence, it is possible that females recently derived from the wild might still exhibit moderate investment in current reproduction, and in turn greater scope for optimization of their effort over multiple reproductive events.

At their second reproduction, female voles that had previously reared a reduced litter were heavier at birth, and produced pups that grew faster during lactation and ended up bigger at weaning. Because body mass at weaning has a strong influence on life expectancy [10], even under laboratory conditions (see the electronic supplementary material), our results support the existence of long-term fitness consequences of LSM in a small mammal species. In conclusion, this study is the first to demonstrate in a small wild-derived rodent that lactating females may adjust their reproductive effort in response to altered reproductive demand with effects on future reproduction. As in birds [3], it validates that also in mammals the optimization of reproductive investments over multiple events can be shaped by intrinsic constraints (see also [2]).

Data accessibility

Data available from the Dryad Digital Repository: http://dx.doi.org/10.5061/dryad.ms700.

Funding statement

We are grateful to the Swiss NSF for financial support (grant no. 31003A_124988 to P.B.).