The response of grey mouse lemurs to acute caloric restriction before reproduction supports the ‘thrifty female hypothesis’ Free

Reproduction and survival are common examples used to depict the concept of trade-offs: by investing energy in reproduction, the individual ultimately impacts its own survival chances (Blomquist, 2009; Cox et al., 2010; Doughty and Shine, 1997). In the majority of birds and mammals, the succession of seasons has shaped the necessity of producing offspring in a limited time and during the most energetically favourable period, i.e. during summer or the wet season (Ball and Ketterson, 2008; Conover, 1992; Fournier et al., 1999). As reproduction is one of the most energy-consuming physiological processes (Clutton-Brock et al., 1989; Speakman, 2008), some species have developed energy-saving mechanisms. Temporal heterothermy is primarily seen as a phenomenon preventing decreased survival chances during winter, when food availability is low and climate conditions are challenging (Geiser, 2004; Jastroch et al., 2016). By decreasing their metabolic rate and body temperature in a controlled manner – a process called torpor – some organisms manage to prevent excessive fat loss during winter (Geiser, 1998; Melvin and Andrews, 2009). The idea that heterothermy might be used to maximize reproductive success in the long-term by saving energy to allocate later to reproduction is much less described in the literature than the effects of energy reserve allocation on survival. However, torpor has direct costs that could impact short-term survival, similar to any other stress response (Bieber et al., 2014; Burton and Reichman, 1999; Luis and Hudson, 2006; Prendergast et al., 2002; Wei et al., 2018); in this context, the ‘torpor optimization hypothesis’ states that torpor should be used only if fat stores are necessary for survival or reproduction (Humphries et al., 2003).

In parallel, males and females do not share the same reproductive schedule: males produce high stocks of spermatozoa in the winter before mating, while females ovulate or engage in gestation and lactation during spring. Consequently, female mammalian reproduction is seen as a more pressing process for selecting energy-saving mechanisms because of the high energy expenses allocated to lactation after the winter season (Clutton-Brock et al., 1989; Jonasson and Willis, 2011). Moreover, as the survival chances of animals with short life spans are uncertain, reproductive opportunities are sparce and must be optimized (Sæther et al., 2004), especially in unpredictable environments (Dewar and Richard, 2007).

Because of the costs of torpor and the seeming sex imbalance in energy allocation to reproduction, the ‘thrifty female hypothesis’ (TFH) was proposed by Jonasson and Willis (2011). In the little brown bat (Myotis lucifugus), they showed that females retain more fat during the winter than males, which ultimately confers an advantage in resisting white nose syndrome (Jonasson and Willis, 2011). If a link exists between seasonal reproduction and the occurrence of thrifty phenotypes in females, sex differences in energy balance should be evident in other mammal species capable of hibernation or torpor. In fact, in polar bears (Ursus maritimus), hibernation and denning are facultative, except in gestating females (Lennox and Goodship, 2008). Moreover, adult male ground squirrels exhibit shorter torpor bouts than females (Gur and Gur, 2015), but the effect on body mass has not been reported. Another example of an effect of sex is the differential phenology of hibernation and reproductive investment between male and female Tasmanian echidnas (Nicol et al., 2019), in relation to male body condition and female constraint to hatch under favourable environmental conditions. In contrast, female common hamsters hibernate for shorter periods than males, an uncommon feature compared with most hibernators that seems to be linked to differential strategies to manage energy balance prior to the reproductive period (Siutz et al., 2016). Here, we focused on confirming the TFH in grey mouse lemurs, a small nocturnal primate from Madagascar, and a seasonal breeder that is well described to use torpor (Giroud et al., 2008; Vuarin et al., 2015).

Evidence for another case of a thriftier female has been shown in grey mouse lemurs, as males and females exhibit strong differences in their body mass fluctuations over winter. Torpor events lasting several days have been observed in wild females, which were rather ‘fat’, but not in males (Schmid and Ganzhorn, 2009). Indeed, while males begin to lose weight around the middle of the season concomitantly with testicular growth initiation (Perret and Aujard, 2001; Terrien et al., 2017), females maintain fat storage until summer, when mating occurs. Moreover, females were shown to use deeper and longer torpor bouts than males to face food rarefaction at the onset of winter in field conditions (Vuarin et al., 2015). However, in a previous study under laboratory conditions, even if wintering males and females expressed strong specificities in their thermic profiles, we did not observe a difference in energy balance between sexes under either a control ration or 40% caloric restriction (Noiret et al., 2024).

We hypothesized that the expression of the female thrifty phenotype during winter could occur under more energetically challenging conditions. Here, we subjected the animals to intense caloric restriction of 80% for 2 weeks during the second half of the winter, when males are known to reactivate spermatogenesis (Perret and Aujard, 2001), and thus, we subjected them to an abrupt change in caloric intake. Indeed, initiating and modulating torpor in response to caloric restriction is widely documented (Giroud et al., 2008; Vuarin et al., 2015). We equipped the mouse lemurs with temperature and activity loggers, monitored their body mass, and measured two markers of fitness [cortisol and oxidative DNA damage via 8-hydroxy-2'-deoxyguanosine (8-OHdG)] and their reproductive success. By putting the animals in a drastic yet safe caloric challenge, we expected to trigger sex-specific energy-saving mechanisms that are sufficient to observe differences in body mass loss between male and female grey mouse lemurs.

Twenty-four grey mouse lemurs, Microcebus murinus (Miller 1777), 12 males and 12 females, all aged 2–4 years and raised in good health in the breeding colony of Brunoy (MNHN, France, licence approval no. E91-114-1), were included in the experiment. These animals were tested at the end of the winter-like season, during weeks 17–21 of winter-like photoperiodic exposure (short day, SD; 10 h light per day), at the time when testis recrudescence occurs (Perret and Aujard, 2001). Animals were kept in individual cages in semi-isolation from the others (with visual, hearing and odorant interactions being possible between individuals) in climatic chambers for the duration of the experiment (1 month). The temperature and humidity were maintained constant (24–26°C and 55%, respectively). The lemurs were fed a fresh mixture (containing egg, concentrated milk, cereals, spicy bread, cream cheese and water) and banana, and were provided with water ad libitum. All described experimental procedures were approved by the Animal Welfare Board of the UMR 7179, the Cuvier Ethics Committee for the Care and Use of Experimental Animals of the Muséum National d'Histoire Naturelle and were authorized by the Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation (no. 14075-2018031509218574) and complied with the European ethics regulations for the use of animals in biomedical research.

We performed an integrative description of physiological parameters to decipher the energy balance of the animals, their metabolic activity and their oxidative status. For this, the animals were fed daily with a control ration (control treatment: control females N=6; control males N=6) of 22 g of mixture and 3 g of banana (24.48 kcal day⁻¹) for 1 week and measured for 4 consecutive days via indirect calorimetry (Oxymax, Columbus Instrument Inc., Columbus, OH, USA). At the end of this procedure, the animals' urine was sampled, which was always collected ∼3 h prior to lights off. The creatinine, cortisol and 8-OHdG levels in the urine samples were measured. After 1 day of recovery, the animals were either maintained on the control diet for 2 more weeks or fed daily with an 80% reduced ration compared with that of the control group (caloric restriction ‘CR’: CR females N=6; CR males N=6) of 4.90 kcal day⁻¹. Such conditions were designed to induce sufficient caloric stress to challenge the animals but not enough to put the animals at risk considering that the animals' body mass is high at this time of the year. They were weighed 3 times a week to monitor their body mass (BM in g) and measured once from the tip of the nose to the anus to establish their body length (in cm). Their reproductive state was monitored for morphological changes as previously described (Perret and Aujard, 2001). Males' testis size was rated at the beginning and the end of the experiment on a scale from 0 to 2 depending on testis size and consistency: 0=testes are in the abdominal cavity and scrotal sacs are loose; 0.5=testes descend and measure 1 cm together; 1=2 cm; 1.5=3 cm; 2=4 cm and their consistency becomes harder. At the end of the 2 week treatment, they underwent the same procedures as the control group (4 days of indirect calorimetry and urine sampling). The animals were returned to the control diet for several days to allow full recovery from the CR treatment and then returned to their original housing conditions in social groups. The reproductive status of the females was monitored until they reached oestrus via direct visual evaluation to establish the number of days to reach mating readiness after the transition to long days (LD: 14 h light per day).

We applied the same method as described previously (Noiret et al., 2020), using an automated Oxymax indirect calorimetry system (Columbus Instruments Inc.) set in push mode with subsampling. Briefly, the animals were placed in monitored cages for 4 consecutive days to measure their metabolic rate (MR, in ml O₂ kg⁻¹ h⁻¹), the data being generated by the Oxymax proprietary system based on Lighton's (2008) equation.

As the MR is known to be directly linked to the oxygen consumption rate V̇_O2 (Geiser et al., 2014), we used V̇_O2 to describe variation in the metabolic rate of the mouse lemurs. The water vapor was scrubbed using magnesium perchlorate prior to reaching the analysers. Analyses were based on V̇_O2 averaged over a day (under artificial light) and night (in the dark) to represent the mean MR during the resting and the active/feeding periods, respectively. Daily energy expenditure (DEE) was also calculated following the equation from Lusk's (1924) table: DEE=(3.815+1.232×RER)×V̇_O2×BM, were RER corresponds to the respiratory exchange ratio, calculated as the ratio between V̇_CO2 and V̇_O2 (V̇_CO2/V̇_O2).

Mouse lemurs had DSI telemetry captors (TA-F10, Data Sciences Instruments) implanted into the abdominal cavity to monitor both body temperature (T_b, in °C) and activity (counts min⁻¹). Surgical procedures were performed under general anaesthesia (0.5 mg 100 g⁻¹ valium; 2 mg 100 g⁻¹ ketamine; isoflurane maintenance) and operating analgesia (before surgery: 0.05 mg kg⁻¹ buprenorphine i.m. for 30 min, local cutaneous injection of lidocaine around the abdominal aperture; after surgery: renewal of buprenorphine 4 h later, followed by treatment with meloxicam p.o. for 2 days). The recovery time between surgical implantation and the beginning of the experiment was 2 weeks. At the end of the experiment, the implants were removed via the same procedure. This method allowed us to record individuals' T_b every 5 min. Body temperature was analysed as the minimal T_b registered each day (T_b,min, in °C) and was computed as the mean during the control and caloric restriction phases of the experiment. We also measured the duration of T_b under 33°C (torpor duration, TD, in min), a threshold often used in torpor analysis (Canale et al., 2011; Giroud et al., 2008). Activity was analysed as the mean activity during the day (when the light is on; i.e. when the animals are either resting or go into torpor) and the mean activity during the night (when the light is off; i.e. when the animals are supposedly active).

Cortisol (ng ml⁻¹; Cortisol Urine ELISA from LDN^®, ref. MS E-5100) and 8-OHdG (ng ml⁻¹; OxiSelect^TM Oxidative DNA Damage ELISA kit, Cell Biolabs Inc., ref. STA-320) were measured in duplicate from urine samples as indicators of the organisms' response to environmental stress (Miller et al., 1991) and oxidative stress-related DNA damage (Loft et al., 1993), respectively. The creatinine concentration (mg ml⁻¹) was used to normalize all urine measurements as an indicator of renal filtration activity (Microvue^TM Creatinine ELISA kit, Quidel^® Corporation, ref. 8009). The results are thus expressed in ng mg⁻¹ creatinine.

The results are presented as medians±interquartile range. No outliers were identified or removed from the datasets after testing with Dixon's Q test. Statistical analysis was conducted by the use of R software v.3.5.1 (http://www.R-project.org/), and tests were considered significant when P-values were less than 0.05. We applied linear mixed models (LMMs) with random individual effects (‘lme4’ v.1.1-35.1 package) to test the effect of sex, the phase of the experiment (before and after 2 weeks of caloric treatment, accounting for the effect of time at the end of winter), caloric treatment (control versus CR) and their interaction, as follows: Y=Phase×Treatment×Sex+(1|ID). The single model approach, including the triple interaction between Phase, Treatment and Sex was preferred to a model comparison one in order to answer our question of a sex-specific response to CR during the whole course of the experiment. Other variables, such as body size (for BM analysis), body mass or ‘Lot’ (origin from the general colony), were included in the models when they had a significant effect on the explained variable. Model fit was assessed by checking the normal distribution of residuals (‘performance’ v.0.10.3 package). The coefficients of determination R² (marginal and conditional; ‘MuMIn’ v.1.47.5 package) are reported in Table 1, along with ANOVA (type III) analysis of the models for each explained variable. Non-parametric pairwise Wilcoxon tests were used for post hoc analysis (pairwise non-parametric tests for caloric treatment effect on the same individuals, unpaired for sex effect or tests between groups). The results are presented (V statistics for paired analysis, W statistics for unpaired analysis, followed by P-values) along with medians and interquartile ranges.

At the beginning of the experiment, BM did not significantly differ between males and females (females 129±20.8 g, males 109±15.0 g; W=93, P=0.24), even when considering BM relative to body length (females 9.0±1.2 g cm⁻¹, males 8.3±1.3 g cm⁻¹; W=60, P=0.76), although males are shorter than females (females 14.3±0.3 cm, males 13.5±0.3 cm; W=102.5, P<0.001). The effect of CR treatment on BM significantly depended on sex (Phase:Treatment:Sex: χ²=7.52, P=0.0061; Fig. 1A, Table 1). Indeed, CR females lost approximately 13% of their initial BM, while males lost 21% of their initial BM (control females +1.8±1.8%, V=1.5, P=0.07; CR females −13.1±4.3%, V=21, P=0.018; control males +9.6±7.3%, V=1, P=0.13; CR males −21.6±3.9%, V=21, P=0.018). The proportion of BM loss between CR males and CR females was significantly different (W=36, P=0.0011).

Males and females tended to increase their TD differently in response to caloric treatment (Phase:Treatment:Sex: χ²=2.96, P=0.085; Fig. 1B, Table 1). Indeed, TD significantly increased in CR females (+650±675 min, V=21, P=0.016) but to a lesser extent in CR males (+178±323 min, V=10, P=0.0502), therefore being significantly greater in CR females than in CR males (W=29.5, P=0.039). In contrast, TD remained statistically unchanged for control individuals, which rarely experienced T_b under 33°C (control females 0±85 min, V=0, P=0.37; control males 0±68 min, V=0, P=1). Moreover, initial BM (and final BM) did not correlate with TD in CR individuals, either for males or for females (CR females: r=0.30, P=0.56; CR males: r=−0.22, P=0.76; Fig. 2). For control individuals, only 2 females with a lower BM entered torpor during the 2 weeks of the experiment (correlation test between initial BM and TD in control females: r=−0.96, P=0.0089), and one male with a higher BM (more than 30 g heavier) also entered torpor (control males: r=0.90, P=0.10).

T_b,min decreased in all CR animals but to a lesser extent in males as compared with females (CR females −22.2±15.3%, V=21, P=0.016; CR males −12.0±13.6%, V=1, P=0.031; Fig. 1C) and did not vary in control individuals (control females −1±7.9%, V=12, P=0.31; control males −2.5±2.1%, V=10, P=0.13). The variation in T_b,min after CR tended to differ between males and females, though not reaching the significance level (W=9, P=0.089). Considering the data distribution (non-Gaussian, negatively skewed), this effect was not confirmed with LMMs (Table 1: Phase:Treatment:Sex: χ²=0.75, P=0.39).

During the day, when the animals were resting, CR had a sex-specific effect on MR (Table 1, Phase:Treatment:Sex during the day: χ²=9.6, P=0.0019). Indeed, MR decreased in CR females but not in CR males (CR females −44.4±21.9%, V=21, P=0.016; CR males −8.4±28.9%, V=12, P=0.42; comparison of CR females and CR males: W=21, P=0.0011). In the control groups, females retained the same MR levels (control females −0.5±16.7%, V=12, P=0.82) while MR tended to decrease in males (control males −12.6±4.8%, V=15, P=0.063).

During the night, when the animals were active, MR significantly decreased only in the CR females (Table 1: Phase:Treatment:Sex: χ²=11.23, P<0.001; control females +4.9±12.5%, V=9, P=0.65; CR females −34.9±17.1%, V=21, P=0.016; control males −3.2±17.9%, V=8, P=0.50; CR males +9.0±10.2%, V=6, P=0.84; comparison of CR females and CR males: W=2, P=0.0043).

While the 24 h energy expenditure did not significantly change according to treatment, sex and phase (Table 1, Phase:Treatment:Sex: χ²=2.27, P=0.13), further analysis revealed that the DEE of females decreased more than that of males after 2 weeks of CR (CR females −44.8±14.5%, V=21, P=0.016; CR males −24.6±11.2%, V=21, P=0.016; comparison between CR females and CR males: W=7, P=0.047). The DEE levels for control individuals remained statistically unchanged during the 2 weeks of the experiment (control females 12±18.5%, V=12, P=0.91; control males 2.3±8.3%, V=15, P=1).

The mean level of activity during the night tended to differ between sexes depending on treatment (Table 1: Phase:Treatment:Sex: χ²=3.19, P=0.074; Fig. 1G), with a significant decrease in activity only in CR females according to non-parametric analysis (control females +31.5±16.5%, V=0, P=1; CR females −36.3±21.6, V=20, P=0.031; control males +10.3±38.8%, V=1, P=0.94; CR males +21.3±57.6%, V=8, P=0.72; comparison between CR females and CR males: W=8, P=0.066).

During the day, the mean activity tended to differ between sexes depending on treatment, though not reaching the significance level (Table 1: Phase:Treatment:Sex: χ²=2.77, P=0.096; Fig. 1F), with a tendency to a decrease in CR females only (control females +92.2±311.6%, V=2, P=0.94; CR females −42.8±49.8%, V=18, P=0.078; control males +29.5±164.0%, V=3, P=0.81; CR males −18.0±36.1%; V=15, P=0.22; comparison between CR females and CR males: W=12, P=0.20).

The cortisol levels did not change after CR in either sex (Fig. 3A, Table 1: Phase:Treatment: χ²=0.31, P=0.58; control females −7.7±54.5%, V=11, P=0.5; CR females −7.9±49.7%, V=9, P=0.41; control males 9.0±11.0%, V=10, P=0.3; CR males −22.2±59.2%, V=13, P=0.34; comparison between CR females and CR males: W=15, P=0.53). Oxidative damage tended to change with caloric treatment, though not reaching the significance level, with no effect of sex (Table 1: Phase:Treatment: χ²=2.87, P=0.091; Phase:Treatment:Sex: χ²=0.97, P=0.32; Fig. 3B; control females +14.6±22.6%, V=7, P=0.78; CR females −30.1±24.5%, V=15, P=0.031; control males +20.9±4.0%, V=3, P=0.91; CR males −23.9±21.3, V=21, P=0.016; comparison between CR females and CR males: W=12, P=0.33). Overall, the 8-OHdG concentrations tended to be greater before (females 32.6±19.2 ng mg⁻¹ creatinine, males 39.7±14.2 ng mg⁻¹ creatinine; Fig. 3B) than after the 2 weeks under CR (CR females 25.2±3.3 ng mg⁻¹ creatinine; CR males 33.1±7.9 ng mg⁻¹ creatinine), with only a tendency for an effect of sex after CR (before: W=46, P=0.12; Fig. 3B; after: W=37, P=0.067).

CR females did not delay their oestrus after entering the long day (LD) photoperiodic regimen, as each control and CR group showed oestrus approximately 17 days after the LD transition (control females 18±3.8 days, CR females 17±1.9 days, W=11, P=0.9). In males, the testes grew in size between the first and second phases of the experiment, but CR had no effect on it (+50±0.35% for both control and CR males, W=18.5, P=0.57).

The importance of the energy reserve in achieving reproductive success in spring should define the magnitude of torpor use upstream, with major effects of sex linked to differences in the agenda of energy allocation in reproduction between males and females (i.e. spermatogenesis versus gestation/lactation). Here, we explored thrifty phenotypes in grey mouse lemurs by examining differences in BM loss after CR, sex-specific T_b modulations and torpor use in relation to metabolism and activity patterns.

After 2 weeks of intense 80% caloric restriction, female grey mouse lemurs lost significantly less BM than males did, which confirms a sex-specific difference in energy balance, in accordance with our expectations of a thriftier phenotype in females, in accordance with what has been observed in little brown bats. Moreover, we showed that females used significantly longer torpor bouts than males under CR and animals under a control diet, regardless of their initial BM. In cheirogaleids, Blanco et al. (2022) showed that females spent more time torpid than males at an equivalent BM and better retained their fat storage (Blanco et al., 2022). In our study, initial BM did not correlate with daily TD in CR individuals after 2 weeks of treatment, either for males or for females. Moreover, males and females had equivalent BM at the beginning of the experiment, and females still ended up performing deeper and longer torpor bouts than did the males after 2 weeks of CR. In addition, only CR females showed a significant decrease in MR (V̇_O2) after the CR treatment, which was associated with T_b modulations and a decrease in day and night activity levels. Consequently, we showed that females decrease their metabolism in response to food shortage more than males and that this modulation had a relatively positive impact on their energy balance compared with that of males, which did not show a decrease in MR and lost more BM. Our results in grey mouse lemurs are consistent with those of the TFH, as it is defined in M. lucifugus: females have a greater capacity to preserve BM (energy reserves) than males by using longer torpor bouts, potentially as an adaptation to higher reproductive costs (i.e. gestation and lactation) (Jonasson and Willis, 2011).

The seasonal reproductive cycle of the little brown bat is very different from that of grey mouse lemurs. Indeed, the mating period of these chiroptera occurs at the end of summer, and females delay ovulation and fecundation by storing sperm in their sacs throughout the winter (Fenton and Barclay, 1980; Waiping and Fenton, 1988). This ‘non-selective mating’ process allows males to mate with ‘passive’ hibernating females during winter (Waiping and Fenton, 1988), a fact that underlines very clearly the difference between sexes in metabolic activity during the poor season. Hence, in theory, males no longer have to spare energy after they invested in reproduction, contrary to females, which would optimize energy savings until the end of winter by using more torpor, in accordance with the torpor optimization hypothesis (Humphries et al., 2003). Indeed, torpor should be used only if fat stores are required later for survival and/or reproduction considering the costs associated with a decreased metabolism during torpor episodes (from dehydration to memory loss, increased vulnerability to predators and temporary diminished immune function) (Bieber et al., 2014; Landes et al., 2020; Luis and Hudson, 2006; Wei et al., 2018). As a result of this sex-specific reproductive schedule, males lose more fat than females do during winter, which provides an advantage to females to surviving environmental energy-demanding challenges at the end of winter as they emerge from the hibernation period in better condition than males (Jonasson and Willis, 2011).

In contrast to little brown bats, grey mouse lemurs mate during the transition to the wet season in nature (Radespiel et al., 2006) and approximately 2 weeks after LD initiation under laboratory conditions (Perret and Aujard, 2001). Even if male grey mouse lemurs begin spermatogenesis earlier during the second half of winter to prepare for mating (Noiret et al., 2024; Perret and Aujard, 2001), we could not find evidence of a difference in MR between the sexes under the control situation, contrary to what observed in little brown bats (male bats spend ∼13% more energy over winter than females; Czenze et al., 2017; Jonasson and Willis, 2011). Therefore, as observed in male brown bats that can still have some reproductive activity during winter and can mate with ‘passive’ hibernating females (Pfeiffer and Mayer, 2013; Sato et al., 2023; Waiping and Fenton, 1988), male mouse lemurs invest in their reproduction under unfavourable environmental conditions. Consequently, we propose that the reason for the female thrifty phenotype should not be restricted to an adaptation to higher reproductive costs for females in spring/summer, as it could also be the result of greater allocation of male energy to reproduction during winter (and the double cost of a higher MR in the context of low food availability) with subsequent greater BM loss. In other words, males with less use of torpor would not necessarily be selected to avoid the physiological costs of torpor, as stated in the ‘torpor optimization hypothesis’ (Humphries et al., 2003; Jonasson and Willis, 2011), but would instead be a consequence of increased metabolism because of reproductive activity resuming earlier than that of females. Similar observations of sex-specific torpor use during winter or thriftier phenotypes in females were previously made in various hibernating animals, such as bears (Lennox and Goodship, 2008), marmots (Tafani et al., 2013), ground squirrels (Gur and Gur, 2015; Michener and Locklear, 1990), dwarf lemurs (Blanco et al., 2022, 2021) and grey mouse lemurs (Noiret et al., 2024; Vuarin et al., 2015). Hence, the TFH could be extended to other seasonally breeding mammals using torpor. Indeed, spermatogenesis involves shorter torpor bouts to maintain an optimum metabolism and temperature to promote gamete production (Barnes et al., 1986; Gagnon et al., 2020). In bats, even if mating during winter does not involve spermatogenesis (testes have regressed and testosterone levels are low) but rather spermatozoa storage (Sato et al., 2023), metabolic arousal is required, although the fitness pressure should be less important than for mouse lemurs, as they only ‘occasionally’ mate during this winter period. Both strategies, however, most likely reduce torpor use to increase reproductive success, even more so considering that males of both species allocate considerable energy to reproduction as they engage in sperm competition (Aslam et al., 2002; Harcourt et al., 1981; Pfeiffer and Mayer, 2013). Finally, even if torpor has costs, as stated in the ‘torpor optimization hypothesis’, it also has benefits, especially for surviving winter, in food-depleted environments or in other energy-demanding situations (such as infection by pathogens or unpredicted food shortages during spring) (Geiser, 1998, 2004; Keil et al., 2015).

Additionally, because we strongly linked the TFH with seasonal breeding, one may oppose the observation of thriftier females in non-seasonal breeders, generally domesticated species that lost photoperiodicity in a seasonally buffered environment as a result of human activity (Blottner and Jewgenow, 2007; Setchell, 1992). Indeed, compared with male rats, female laboratory rats exhibit greater thermogenic functionality of their brown adipose tissue and greater resistance to weight loss (Rodriguez-Cuenca et al., 2002; Valle et al., 2007), features that highly resemble thrifty phenotypes and are shared with female seasonal breeders (Gur and Gur, 2015; Lennox and Goodship, 2008; Terrien et al., 2010). As male and female rats endure rapid and continuous spermatogenic and oestrous cycles, respectively, throughout the year (approximately 50 days for a male and 4–5 days for a female with no seasonal discontinuity) (Lohmiller and Swing, 2006), we believe that the thrifty phenotype is a sex-specific adaptation to delayed reproductive allocation and large energy expenses in females, which would be an inherited residue – still beneficial – in non-seasonal breeders.

In a previous experiment, chronic 40% CR throughout winter induced deeper and longer torpor bouts in female grey mouse lemurs (Noiret et al., 2024), but both sexes still lost equivalent body mass. In comparison, the present 80% intense CR was considered an acute stress compared with that experienced by animals fed the control ration during their upstream winter before the start of the experiment. The acuteness of food shortages could thus be the factor triggering male and female differences in energy balance. Moreover, few control individuals decreased their body temperature to under 33°C: only two females that had a lower BM than the other control females, and one male, which was ∼30 g heavier. Hence, under a control diet (under which the control groups did not lose BM), initial BM correlated positively with TD in females only, which is consistent with the ‘torpor optimization hypothesis’ and the results of Blanco and colleagues (2022). However, caloric restriction triggered the use of torpor in males and females regardless of their initial BM, and TD did not correlate with BM, contrary to the findings of Blanco et al. (2022), who also decreased the ambient temperature to induce hibernation in dwarf lemurs. However, it remains difficult to compare experimental food shortages to the energetic challenges in nature, which can be drastic yet variable in intensity or quality (Radespiel et al., 2006). Moreover, grey mouse lemurs are a female-dominant species (Hohenbrink et al., 2016; Kraus et al., 2008; Ostner et al., 2003), and male- and female-specific ecology can introduce many environmental biases influencing energy balance, such as nest quality (Radespiel et al., 1998), group size (Radespiel et al., 2001) and access to food (Génin, 2003), which can increase the difficulty of measuring raw intersexual variability in energy management. Nonetheless, body mass fluctuations between males and females were previously observed in the wild, with males gaining less body mass than females at the onset of winter or losing more body mass during the dry season (Dammhahn and Kappeler, 2008; Fietz, 1998; Schmid, 1999; Vuarin et al., 2015).

Thus, an acute CR during short days would not necessarily be a stressor for animals coming from a highly seasonal and hypervariable environment such as Madagascar (Dewar and Richard, 2007). Indeed, the present 80% CR did not result in cortisol elevation compared with a 60% CR applied in post-reproductive LD-acclimated animals (Noiret et al., 2020). Nonetheless, even if CR led to a decrease in oxidative damage in urine in both sexes, males tended to present higher levels of 8-OHdG, as was observed in the 60% CR study (Noiret et al., 2020). In addition to their lower body mass in late winter and considering the high energy costs they face during spring in the wild (for territoriality, finding mates), the survival chances of male grey mouse lemurs may be reduced compared with those of females. In nature, male survival at the onset of summer was shown to be 16% inferior to that of females, but this effect is usually attributed to more ‘risky’ behaviours in males (Kraus et al., 2008), with territorial defence, male-to-male competition and exposure to predators. Under TFH, torpor in seasonally breeding females would thus benefit both reproduction and survival, while in males, torpor would ultimately impact survival as they have to reactivate to invest in reproduction. Using deep torpor in response to CR for males during late winter, when spermatogenesis is supposed to occur, would ultimately impair fitness, as this would negatively affect breeding success.

Even if recent observations of wild populations do not show any sex bias, we cannot exclude this phenomenon in the future, as increasingly unpredictable environmental events are expected to occur with global warming, which could challenge sexes differently (Canale and Henry, 2010; Easterling et al., 2000), depending on the nature of the stressor or environmental cues. While we expect that female grey mouse lemurs would have an advantage in resisting food rarefaction compared with males, by performing longer or deeper torpor bouts, other species with a different biology have shown that females were also more responsive to thermal cues. In the Northern hemisphere, earlier permafrost thawing for the past decades has been responsible for earlier termination of hibernation in females contrary to that in males in an Artic ground squirrel (Chmura et al., 2023), and a heat wave led to earlier emergence from hibernation in female Richardson's ground squirrels, while ∼60% of the males were not yet ready for mating (Kucheravy et al., 2021). These situations, coming from females' sensitivity to seasonal cues for the reactivation of their reproductive axis (Simonneaux, 2020; Tolla and Stevenson, 2020), led to a mismatch in reproductive readiness, which could increase further in the future and have consequences on populations. In regard to the consequences that a sex bias in mortality or a reproductive mismatch could have for population extinctions (Grayson et al., 2014; Le Galliard et al., 2005; Wedekind, 2002), it is important to assess more acutely any sex differences in energy-saving strategies and their impact on fitness to better predict the future of wild populations and act appropriately.

We provided evidence for thriftier phenotypes in female than in male grey mouse lemurs after intense CR in late winter, at the onset of the reproductive period. This phenomenon is probably linked to a sex-specific difference in energy allocation to reproduction due to seasonality, the fact that males invest in reproduction earlier than females, and a sex-specific adaptation in energy-saving mechanisms linked to greater selective pressures concerning reproductive success in females. From an evolutionary point of view, the TFH means that by using torpor, females can promote both reproduction and survival in a seasonal context, while males have to endure a more classic unbalanced trade-off: as they invest in reproduction, they ultimately disadvantage their own survival. Males showing poorer energy balance before mating, especially in the context of climate change, could face greater losses than females, leading to a sex-biased population with an increased extinction risk.

The expert animal care and management provided by A. Anzeraey, L. Dezaire, S. Gondor, I. Hiron and M. Perret is gratefully acknowledged.