The energy budget, that is, a sum of processes associated with energy intake and expenditure, has enormous relevance to individual fitness since it integrates all aspects of the life of organisms. The changes in the energy budget during the lifespan are related to requirements and the use of time and space (Withers et al., 2016). Thus, partitioning the available energy according to short-term priorities at a specific moment allows animals to survive and optimizes long-term reproductive success (Wade and Schneider, 1992). In endotherms, the development of thermoregulatory mechanisms after birth is the determinant of the adult’s fitness. The development of functional mechanisms that ensure thermal homeostasis is one of the main requirements that must be achieved before parental care independence (Thompson, 1992). Defined by their physiological maturity, different strategies for the development of endotherm pups have been proposed to exist between altriciality and precociality extremes (Hill, 1992). The distinction between altricial and precocial development is related to the time at which pups become furred, possess capable eyesight, coordinate their locomotor abilities, or, particularly, reach endothermy (McClure and Randolph, 1980). Precocial pups can use physiological thermoregulation at an early stage, whereas altricial pups show a low capacity to control their body temperature (T_b) due to the lack of functional thermogenic mechanisms, losing heat rapidly when they face an ambient temperature (T_a) below thermoneutral zone (TNZ; Hill, 1992). In the case of altricial pups, the requirement for warmth might be provided by the exogenous source of heat. Direct physical contact of pups with their parents might help to overcome the thermal imbalance. Thus, when physiological thermogenic capacity is limited, behavioral thermoregulation could restrict heat loss, for example, either by huddling with other members of the litter (Geiser and Kenagy, 1990), by behavioral thermotaxis (Farrell and Alberts, 2007), or by remaining in the nest (Antinuchi and Busch, 2001). Total thermogenic capacity is defined as the maximum amount of heat that can be produced to maintain a constant T_b and can be approximated by cold-induced maximum metabolic rate (MMR). The components of total thermogenic capacity are shivering. Shivering thermogenesis involves rapid, involuntary contractions of skeletal muscles to produce heat, related to biochemical-mechanical processes at the cellular level, such as inefficiency of biochemical pathways or friction among myofibrils (refer to Hohtola, 2004). Unlike ST, NST has specialized tissues to produce heat without any muscle contraction. Brown adipose tissue (BAT) is the main source of NST (Janský, 1973; Foster and Frydman, 1979; but see Nowack et al., 2017 for a discussion about muscle-mediated NST). BAT is a highly vascularized tissue with a large number of mitochondria and respiratory enzymes and is found in discrete areas in the body, mostly surrounded by the main organs (Cannon and Nedergaard, 2004). The activation of BAT depends on the release of norepinephrine (NE) by the sympathetic nervous system, and its functioning is related to uncoupling the oxidative phosphorylation in BAT’s mitochondria. The presence of uncoupling proteins (UCP), particularly UCP1, short-circuits the electron transport chain circuit, producing heat (Oelkrug et al., 2015; Chouchani et al., 2019).

Depending on the pup’s development strategy, the onset of functional total thermogenic capacity can vary (Brück and Hinckel, 1996). While thermogenic mechanisms in precocial pups are well developed right after birth, complete development takes days in altricial pups (Berthon et al., 1994; Farrell and Alberts, 2007; Canals et al., 2009; Lezama-García et al., 2022). For example, in rats, behavioral control of T_b is important during the first days of life; by the second week, with the increase of body mass and thermal insulation, the appearance of ST enhances thermogenic capacity (Sant Anna and Mortola, 2003). In the white-footed mouse Peromyscus leucopus, NST appears by day 10, whereas ST was observed around 2 weeks after birth (Hill, 1976; Robertson et al., 2019). Deer mice Peromyscus maniculatus from a low-altitude population can shiver at day 14, but only NST is observed in individuals from a high-altitude population at this age (Robertson and McClelland, 2019). Given the variation between altricial species, it is difficult to establish a temporal pattern of postnatal development of different thermogenic mechanisms, but it is generally recognized that, in mammal pups, shivering is not functional at early stages, but NST tends to be active. Deposits of BAT are well developed and matured only a few days after birth, contrary to muscles, which show a slow development during ontogeny (Lagerspetz, 1966; Robertson and McClelland, 2019).

In this general framework, inhabiting a relatively thermally stable environment can diminish the cost of thermoregulation. Some mammalian species live in underground burrows that can buffer cold external T_as and its fluctuation. In particular, subterranean rodents occupy almost all available habitats, from cold to tropical, from mountain slopes to deserts, inhabiting tunnel systems parallel to the surface. Although subterranean burrows buffer T_a and protect against predators, they are stressful in most cases because they are humid, dark, stagnant, and lack most clues available for orientation (Burda et al., 2007). Among rodents, different degrees of adaptation to the use of burrows are present (Begall et al., 2007), with some species adapted to digging and living almost exclusively underground (i.e., subterranean) and some other species also adapted to digging but with extensive use of surface (i.e., fossorial). Either subterranean or fossorial rodents, underground ecotope had determined the observed convergence in morpho-physiological features observed between both groups of rodents (e.g., low mass-specific minimal metabolic rate, a wide range of thermoneutrality, high thermal conductance, and low Tb; McNab, 1966; Vleck, 1979). Different studies have been carried out for evaluating the total thermogenic capacity, ST, and NST in adults of subterranean and fossorial species, finding significant variability in the type and proportion of the mechanisms on total thermogenic capacity. While in some South American Ctenomyid species, a combination of NST and ST is observed (e.g., C. porteusi), in other species, only ST is present (C. tuconax; Luna et al., 2019). Similarly, in African bathyergids, the cost of NST varies depending on the body mass and sociality (Luna et al., 2021). However, no studies have been performed to evaluate the ontogeny of total thermogenic capacity and the nature of thermogenic mechanisms involved in pups of subterranean or fossorial species.

The genus Ctenomys is distributed in several environments in the southern cone of South America, being one of the most speciose genera among subterranean and fossorial rodents, including species from ∼100 g (C. pundti) to more than 1,000 g (C. conoveri; Reig et al., 1990). Particularly, Ctenomys talarum is a solitary fossorial rodent that lives in the coastal grasslands of Argentina (Antenucci and Busch, 1992). Similar to other Ctenomys species, tunnel systems show a linear pattern with no differences in total burrow length between sexes, feeding mainly on aerial plant parts near the burrow openings (Antenucci and Busch, 1992; Comparatore et al., 1995). Males of C. talarum established a dominance hierarchy among them, monopolizing mating activity, and consequently, dominant male burrows occupied central distribution surrounded by several female burrows (Busch et al., 1989; Antenucci and Busch, 1992). The reproductive season lasted ~9 months, beginning in June–July (austral winter) and continuing until February (austral summer). Birth takes place from August through early December after a long gestation period (~100 days; Malizia and Busch, 1991; Fanjul et al., 2006). Females produced as many as two litters per reproductive season, with a mean litter size of 4 (Busch et al., 1989). For C. talarum, the cost of acceding to new food patches made through the construction of new tunnel systems is relatively high (Luna et al., 2002).

During the ontogeny, C. talarum pups show an initial altricial phase, represented by a period of high thermal vulnerability. During lactation (~10 days), pups are not capable of maintaining T_b on their own, depending, therefore, on their mother to thermoregulate. After this period, pups enhance their thermoregulatory capacity and start eating solid food. At approximately 30–40 days after birth, pups reach the T_b of adults and become behaviorally and physiologically independent. At this age, the weaning period starts, and pups show active exploratory behavior. Around day 60, solitary pups disperse aboveground from their maternal burrow and construct their burrow (Malizia et al., 1995; Zenuto et al., 2002; Antenucci et al., 2003; Cutrera et al., 2003; Baldo et al., 2014). In this context, the aim of this study was to evaluate the thermogenic capacity development of pups of the fossorial rodent Ctenomys talarum. We also evaluated the presence of BAT during ontogeny and the timing of mitochondrial uncoupling proteins related to BAT-mediated NST. We hypothesized that no thermogenic capacity is attained during the altricial phase of their development (~day 10), increasing their thermogenic capacity after weaning, before the dispersal period. Furthermore, we expect that NST is completely functional after day 10, reaching the value of the adult during this period.

When RMR and NST_max were compared, MR was affected by age (F_5,66 = 108.28, p < 0.001) and treatment (F_1,66 = 33.95, p < 0.001). However, no significant effects of the interaction between age and treatment (F_5,66 = 1.92, p = 0.1) were found (Figure 1; Table 1). There is an increment in MR from day 2 until day 10 [P_{(day 2–day 10)} = 0.008, P_{(day 6–day 10)} < 0.001]. When pups were older than day 10, age affected MR [P_{(day 10–day 30)} < 0.005, P_{(day 15–day 30)} = 0.01]. Moreover, differences in MR were found between day 6 and day 30 [P_{(day 6–day 30)} < 0.005]. When MMR and NST_max were compared, we found age differences (F_5,31 = 93.21, p < 0.001) and treatment (F_1,30 = 36.01, p < 0.001). The interactions between age and treatment were also different (F_5,30 = 11.55, p < 0.001, Figure 1; Table 1). There is an increment in MMR from day 2 to day 10 [P_{(day 2–day 10)} = 0.01, P_{(day 6–day 10)} = 0.006], with values of MMR between day 2 and day 6 being similar (p = 0.99). After day 10, MMR was different from the following days [P_{(day 10–day 15)} = 0.99, P_{(day 10–day 30)} = 0.99, P_{(day 10–day 60)} = 0.81]. After day 15, due to the relatively lower value at day 30, MMR was different only from day 60 [P_{(day 15–day 30)} = 0.99, P_{(day 15–day 60)} = 0.004, P_{(day 30–day 60)} = 0.002]. When comparing MMR with NST_max, differences between days were observed. In this sense, MMR was higher than NST_max at day 15 (p = 0.002), day 30 (p = 0.049), and day 60 (p < 0.001). Similarly, as no differences were observed between RMR and NST_max, MMR was different from RMR between days (F_5,66 = 168.05, p < 0.001), treatment (F_1,66 = 124.13, p < 0.001), and the interaction between days and treatment (F_5,66 = 45.48, p < 0.001, Figure 1; Table 1). MMR was higher than RMR at day 15 (p = 0.002), day 30 (p = 0.049), and day 60 (p < 0.001). For comparative purposes, in Figure 2A, physiological variables were expressed in mass-specific MR.

When comparing T_b before the exposure to cold conditions to estimate MMR or NE injection, neither the exposure alone nor their interaction with age was different (F_1,31 = 1.49, p = 0.23 and F_5,31 = 1.52, p = 0.21, respectively), whereas T_b between days was different (F_1,31 = 17.64, p < 0.001; Table 2). Body temperature at day 60 was higher than those observed at days 2, 6, 10, and 15 (all, p < 0.001) and also T_b at day 30 compared to day 2 (p = 0.01) and day 6 (p = 0.03). After cold exposure, ΔT_b was similar between days (F_5,31 = 0.88, p = 0.51). After NE injection, ΔT_b on day 2 was lower than ΔT_b on all other days (F_5,31 = 8.58, p < 0.001; Table 2). Interscapular BAT mass increased linearly with body mass. The relationship of iBAT mass with body mass was iBAT mass = 0.009 M – 0.011 (R² = 0.89, F_1,39 = 293.25, p < 0.001, Figure 3). UCP1 and COXII content in iBAT were similar between days (T = 5.88, df = 5, p = 0.32 and T = 8.96, df = 5, p = 0.11, respectively; Table 3). The activity of the COX complex in iBAT was similar at all ages (F_5,32 = 1.12, p = 0.37, Table 3).

To attain thermal independence before the dispersal period, pups must fully acquire thermoregulatory capacity (Hill, 1992). The main objective of this study was to determine the thermogenic development of pups of the fossorial C. talarum, stablishing the onset of thermogenic capacity and its components. To the best of our knowledge, this is the first study to establish the postnatal development of non-shivering thermogenesis (NST) and cold-induced maximum metabolic rate (MMR) in a fossorial species. Pups of C. talarum showed a biphasic pattern of mass-specific RMR during their development. Mass-specific RMR increases until day 10, with a subsequent decline to day 60, confirming the pattern found by Zenuto et al. (2002). In this species, acquisition of thermoregulatory capacity takes days, since the onset of homeothermy is observed around day 30, a period in which pups showed T_b of adults (Zenuto et al., 2002; Cutrera et al., 2003; Baldo et al., 2014). Notably, no thermogenic mechanism is observed until day 10, and NST_max does not differ from RMR up to day 60.

In altricial species, it has been described that the first thermogenic mechanism to appear is NST (Robertson and McClelland, 2019; Lezama-García et al., 2022), involving the maturation of BAT a few days after birth (Robertson et al., 2019). In pups of the golden hamster Mesocricetus auratus, the onset of NST occurs around 14 days after birth, whereas in the Djungarian hamster Phodopus campbelli, the onset of NST occurs after day 9 (Newkirk et al., 1995). Despite the high metabolic activity of BAT, the common rat showed a weak NST on day 5 after birth (Spiers and Adair, 1986). In the golden Syrian hamster Mesocricetus auratus, thermogenic capacity develops within a week after birth and is determined by qualitative changes in mitochondrial function related to the initiation of UCP synthesis (Houštěk et al., 1990). In newborn rats, NST capacity appears after weaning, but UCP1 is detectable in iBAT just after birth, reaching its maximum value on day 10 (Porras et al., 1990). Interestingly, C. talarum pups showed a complete absence of NST until day 60, but this does not imply the complete absence of BAT. Interscapular BAT patch is directly recognizable, increasing in size with age but never exceeding 1% of the body mass. Neonatal exposure to cold can establish modifications in NST capacity, induced by changes in neuronal stimulation and content of UCP in BAT (Morrison et al., 2000; Sant Anna and Mortola, 2003). However, even though pups of C. talarum can be exposed to cold when isolated from the mother and siblings, UCP1 content does not vary between days. In the same way, different molecular markers of the oxidative capacity of BAT ensure NST (Klingenspor, 2003) does not vary between days. For example, the activity of cytochrome c oxidase (COX), a marker enzyme for the mitochondrial membrane, and the content of COXII, commonly used to estimate respiratory capacity in BAT mitochondria (Klaus et al., 1988; Klingenspor et al., 1996), remain similar between days. Although unlikely, given the relative thermal stability of burrows, it would be worth establishing whether acclimatization to low T_as by mothers might affect the magnitude of pup’s thermogenesis, as has been observed in the surface-dwelling altricial leaf-eared mouse Phyllotis darwini during the first days of life (Canals et al., 2009).

Day 60 corresponds to the beginning of the period of dispersion of this species, which occurs on the surface (Malizia et al., 1995). Interestingly, the size of iBAT and the amount of UCP1, as well as markers of the oxidative capacity of BAT in adults, are similar to that in juveniles (Luna et al., 2012). Due to the complete maturation of endocrine and neural pathways (Symonds et al., 2012; Symonds, 2013; Lezama-García et al., 2022), changes in the efficiency of BAT-related NST might establish the functional NST after day 60. Complete maturation of neuro-endocrine pathways would allow an increased capacity for fatty acid oxidation and tricarboxylic acid cycle activity to uncouple oxidative phosphorylation to produce heat (Chouchani et al., 2019). For example, in P. maniculatus, a delay in the onset of NST in pups reared at high altitude was associated with a delay in the maturation of nervous and O₂ supply systems (Robertson et al., 2019; Robertson and McClelland, 2019; Velotta et al., 2020).

As proposed, ST matures later than NST because of the slow development of muscles (Robertson and McClelland, 2019; Lezama-García et al., 2022). In C. talarum pups, shivering was characteristically observed over the first days (Baldo et al., 2014) but appeared to be fully functional after day 15. Aerobic-resistant muscles that generate repeated contractions could be mainly recruited to produce heat; thus, the proportion of this type of muscle can establish ST potential (Hohtola, 2004). Muscles of altricial rodents are relatively immature at birth, and they mature due to changes in muscle fiber structure and hypertrophy (White et al., 2010). From day 10, offspring of C. talarum start to show exploratory behaviors, wandering the tunnel systems and beginning to scratch and push the soil (Vassallo, 2006; Echeverría et al., 2016). The progression of digging behavior has particular energetic requirements to loosen, remove, and break down the soil to construct a new burrow during dispersal. Similar to ST, aerobic-resistant muscles are used during digging activities (Alvarez et al., 2004). Thus, muscular work performed during digging could also be exploited as a source of heat via ST (Luna and Antinuchi, 2007). The onset of functional ST overlaps with the beginning of the exploration of tunnels near the thermally stable nest, tunnels that usually show T_as below TNZ (Cutrera and Antinuchi, 2004). In this context, even though it is not clear how muscle maturation occurs during the first days of life, the capacity for ST can be associated with an increase in digging capacity. Furthermore, cold acclimation in adult rodents was proposed to lead to muscle training and an increase in ST capacity (Shefer and Talan, 1998). As digging becomes more efficient, ST might also do so. Interestingly, from day 15 until dispersal, total thermogenic capacity relies entirely on ST, increasing its specific energetic cost due to the progressive decrease in mass-specific RMR and the constancy of total thermogenic capacity.

The total thermogenic capacity of pups from day 10 is similar to those observed in adults (Luna et al., 2012). The consistency of mass-specific MMR over the lifespan of this species addresses important questions about the mechanistic meaning of physiological adjustments within upper and lower metabolic limits. A fixed metabolic ceiling determines that any energetic modification must be within the aerobic scope. The net aerobic scope represents the capacity of aerobic processes above minimal metabolism (see Nespolo et al., 2017 for a discussion). For example, unlike juveniles, adults of C. talarum show a functional NST that represents ~40% of the total thermogenic capacity (Luna et al., 2012). Thus, from day 10, when the metabolic ceiling is established, there is an increase in the net aerobic scope given the progressive decrease in mass-specific RMR. Any energy input intended for other processes, in addition to growth, must be within metabolic limits. In fact, the part of the energy cost of NST in adults could be related to the difference between mass-specific RMR at day 60 (1.75 mL O₂/gh; this study) and in adults (0.89 mL O₂/gh; Luna et al., 2012). Such metabolic trade-offs between different energy-consuming mechanisms might ensure a balanced daily energy expenditure, hence survival. Maximal metabolism is an ecologically relevant trait (Bozinovic and Rosenmann, 1989), and although it appears to be infrequently reached during life, maximal metabolism can be affected by natural selection (Rezende et al., 2004). It is not the focus of this study to evaluate which factor determines mass-specific MMR constancy, but it has been proposed that there would have been an evolutionary limitation on reaching high MMR values (Luna et al., 2012, 2019) because of limitations in respiratory and cardiovascular systems imposed by burrow’s atmosphere (i.e., low O₂ and high CO₂). Therefore, the value of maximal metabolism might be independent of the physiological mechanism that elicits it. In adults of C. talarum, cold-induced MMR also does not vary after acclimation to cold, being similar to exercise-induced MMR (see Luna et al., 2012). As proposed, further studies are needed to assess the consequences of fixed ceilings and net aerobic scope in subterranean and fossorial species when compared to surface-dwelling species (Luna et al., 2015, 2019).

Heat balance in endotherms results from the integration of a coordinated system of heat production and loss (Naya et al., 2013). One of the main characteristics of underground rodents is their high thermal conductance (see McNab, 1966). Thus, not only changes in thermogenic capacity but also thermal insulation are important to maintaining thermal homeostasis (Withers et al., 2016). Early development in altricial pups represents a stage of dependence on external thermal sources (MacArthur and Humphries, 1999; Antinuchi and Busch, 2001; Zepeda et al., 2018), which mostly depends on thermal resources provided by the mother and behavioral thermoregulation, such as huddling with their siblings (Zenuto et al., 2002). The effectiveness of huddling depends not only on the reduction of the individual’s exposed surface but also on the augmented heat transfer between littermates and the mother (Webb et al., 1990; Gilbert et al., 2007). In the altricial P. darwini, a strong maternal effect on the thermoregulatory development of pups was observed until day 7 (Canals et al., 2009). McClure and Randolph (1980) found in the eastern wood rat Neotoma floridana pups that poor insulation after birth is related to the maximization of heat transfer. In C. talarum, as no thermogenic mechanisms are observed until day 10, behavior can alleviate the need for these energetically costly mechanisms. Until day 10, hairless pups spend most of the time in contact with their mother and siblings (Cutrera et al., 2003; Baldo et al., 2014). Zenuto et al. (2002) established that the increase in mass-specific RMR until day 10 was related to the increase in fur coverage and the increase in the intake of solid food after day 7. After day 10, pups remained huddled for shorter periods of time due to the increment of agonistic aggressive behavior between them (Cutrera et al., 2003; Baldo et al., 2014). Thus, the onset of ST also coincides with the time that pups start to spend less time huddled and become furrier, increasing their insulation. Therefore, thermoregulatory benefits associated with the underground environment might allow a short period where behavioral strategies are important to avoid thermogenic costs. During lactation, pups might invest energy to accelerate the production of new tissue and its maintenance (Hill, 1992), without compromising their thermal balance. The thermally stable environment of the nest and behavior might lead to a delay in the onset of ST, hence independent thermoregulation, until the period in which pups find new thermal environments by exploring and digging nearby tunnels.

In conclusion, although pups of C. talarum were altricial at birth (Zenuto et al., 2002; Cutrera et al., 2003; Baldo et al., 2014), they maintained behaviorally their thermal balance during lactation. Until day 10, pups are dependent on their mother to obtain heat, remaining in the nest where they commonly huddle with their littermates (Cutrera et al., 2003; Baldo et al., 2014). During this period, resting metabolism increases, but no thermogenic mechanisms were observed. As most of the energy is destined for growth, pups at this age showed impossibility to maintain T_b when confronted with T_as below adults TNZ. From day 10, mass-specific RMR showed a progressive decrease and MMR became fixed, establishing a specific increment of ST until day 60. Postnatal development of musculature related to digging could be employed as a heat source when pups start to explore and dig in the surrounding of the nest, but it appears to be inefficient until day 30 when pups achieved adult T_b. No functional NST was observed in pups until day 60 besides the presence of UCP1 and other molecular markers (e.g., COX activity) of NST in BAT. Attaining complete thermal independence is critical after day 60 since juveniles disperse at this age (Malizia et al., 1995). During their aboveground dispersal, juveniles must dig a new burrow or eventually find an empty burrow, so they are exposed to aboveground climatic fluctuations. Interestingly, in adults, NST complies with around 40% of the total thermogenic capacity (Luna et al., 2012), so, at some point during or after dispersal, NST becomes functional.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by the Institutional Committee for the Care and Use of Laboratory Animals (number 2555-06-14 RD141/15; CICUAL), Facultad de Ciencias Exactas y Naturales (FCEYN), and Universidad Nacional de Mar del Plata (UNMDP).

FL designed the study. CDA supported the respirometric experimental procedures. JS-S and JO supported the molecular experimental procedures. FL and CDA analyzed the data, prepared the manuscript, and created figures and tables. All authors contributed to the article and approved the submitted version.

This project was supported by the Consejo Nacional de Investigaciones Científicas y Técnicas of Argentina (CONICET, PIP3164) and the Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación (Agencia I+D+i, PICT 3785).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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