Arousal from hibernation increases blood oxygen saturation in 13-lined ground squirrels Open Access

Hibernators evade winter's energetic demands through metabolic suppression. In ground squirrels, hibernation involves cycling between torpor and interbout euthermia (IBE). 13-Lined ground squirrels (Ictidomys tridecemlineatus) can experience up to 20 torpor bouts per season, lasting 6–15 days with 90% metabolic suppression and body temperatures (T_b) of 4–6°C (Muleme et al., 2006; Fig. 1A). Torpor is interrupted by arousals to IBE, where metabolism and T_b return to high values (similar to summer values) for 6–10 h before animals re-enter torpor (Fig. 1A). Low T_b alone typically induces a leftward shift in the haemoglobin–oxygen (Hb–O₂) equilibrium curve, as P₅₀, the partial pressure of O₂ at which haemoglobin is 50% saturated, decreases with temperature. During torpor, P₅₀ decreases in hibernating ground squirrels, corresponding with suppressed metabolism (Revsbech et al., 2013; Maginniss and Milsom, 1994; Revsbech and Fago, 2017). However, early in arousal, metabolic demand rises before T_b, increasing the risk of hypoxia, especially before shivering thermogenesis commences.

Hb concentration and haematocrit (Hct) are also important for O₂ delivery; data on these values vary. For instance, one study reported declines in red blood cell count, Hb and Hct during torpor compared with summer (Spurrier and Dawe, 1973), but later research found no significant changes (Cooper et al., 2016a). Blood ion and metabolite concentration can help contextualize haematological changes; high blood Na⁺ may indicate dehydration, leading to overestimates of Hb and Hct as a result of decreasing plasma volume. Moreover, decreases in blood glucose may indicate anaerobic metabolism from reduced O₂ availability (Abu et al., 2018).

During arousal, heart rate (f_H), cardiac output and ventilation increase to accommodate increased metabolic rate (Fig. 1B,C). Blood pressure also increases during arousal, rising from 60/30 mmHg (systolic/diastolic) in torpor to 140/100 mmHg during IBE (Lyman, 1960), and blood flow to the heart of 13-lined ground squirrels increases 8-fold (Bullard and Funkhouser, 1962). During torpor in 13-lined ground squirrels, coagulation factor activity and platelet count decline (Cooper et al., 2016b), and fibrinolysis is 3-fold faster (Bonis et al., 2019), indicating minimal resistance to blood flow early in arousal.

During arousal, a 2-fold increase in both ventilation (Sprenger and Milsom, 2022) and cardiac output (Popovic, 1964) likely correspond with rapid increases in O₂ delivery, which can increase oxidative damage in other animal models (Granger and Kvietys, 2015). Even 13-lined ground squirrels have higher oxidative damage to lipids and proteins in some tissues during IBE compared with torpor (Duffy and Staples, 2022). 13-Lined ground squirrel arousals are often considered similar to ischaemia–reperfusion (I–R) (Bonis et al., 2019; Otis et al., 2017; Kurtz and Carey, 2006). I–R is characterized by a restriction of tissue blood supply, followed by rapid restoration of blood flow and re-oxygenation, and is often associated with pathological conditions such as ischaemic stroke or myocardial infarction. In humans, an arterial oxygen saturation below 55% indicates ischaemia and severe O₂ deprivation to tissue (Yu et al., 2020). While ischaemic hypoxia can cause significant injury itself, the damage is exacerbated during the reperfusion event, when sudden increases in O₂ result in excess reactive oxygen species (ROS) production (Granger and Kvietys, 2015). Elevated ROS production rates can exceed antioxidant capacity, causing oxidative damage to cellular macromolecules, including proteins, lipids and DNA.

Resistance to I–R injury is a characteristic of hibernating mammals. For example, hepatic cells from the facultative hibernator Syrian hamster (Mesocricetus auratus) did not show DNA damage following I–R, whereas lab mice (C57BL/6) cells did (Pissas et al., 2025). At the whole-animal level, in the summer phenotype, arctic ground squirrels (Spermophilus parryii) survive haemorrhagic shock better than rats (Sprague–Dawley; non-hibernators) (Bogren et al., 2014). Within a species, the hibernation phenotype further enhances ischaemia–reperfusion tolerance. Livers from hibernating 13-lined ground squirrels tolerate cold ischaemia followed by warm reperfusion for longer and with less damage than livers from summer 13-lined ground squirrels or rats (Lindell et al., 2004). This resistance is hypothesized to persist because of acquired adaptations that allow small hibernators to endure dozens of arousals every hibernation season. However, the mechanisms of I–R resistance in hibernators remain unknown.

Despite the documented changes in cardiovascular function during arousal (Fig. 1C), whether tissues become hypoxic in 13-lined ground squirrels remains unclear. In torpid arctic ground squirrels, arterial O₂ tension (Pa_O2) and arterial Hb O₂ saturation (Sa_O2) are comparable to those in non-hibernating rats (Sprague–Dawley; Ma et al., 2005), indicating that reductions in O₂ delivery are matched by reductions in tissue O₂ demand in torpor. Conversely, lactate accumulates in red blood cells of arctic ground squirrels during torpor, suggesting anaerobic metabolism (Gehrke et al., 2019). Furthermore, in 13-lined ground squirrels, levels of hypoxia-inducible transcription factor-1 (HIF-1α) increase in skeletal muscle, brown adipose tissue and liver during torpor, indicating widespread hypoxia (Maistrovski et al., 2012).

While it has long been predicted that 13-lined ground squirrels experience a significant influx of O₂ during arousal when f_H (Fig. 1B), ventilation and blood pressure (Fig. 1C) increase rapidly, other variables, including T_b and metabolic rate, may confound this effect. We hypothesized that an increased O₂ demand during arousal is matched by increased O₂ supply and predicted that O₂ availability would be low in early arousal but increase as arousal progresses. To test this hypothesis, we measured Sa_O2 during the transition from torpor to IBE using pulse oximetry of blood flow through the carotid arteries. Although changes in Hct and/or Hb could also affect O₂ delivery during arousal, taking multiple blood samples from arousing animals was not feasible. We did, however, compare these values, as well as some blood ion and glucose concentrations, between 13-lined ground squirrels in deep torpor and in summer.

All procedures were performed at Western University (London, ON, Canada, 251 m above sea level) following an approved animal use protocol (2020-034), conforming to the Canadian Council on Animal Care guidelines. The 13-lined ground squirrels, Ictidomys tridecemlineatus (Mitchill 1821), were live-trapped in Carman, MB, Canada (49°30′N, 96 98°01′W) or bred in captivity, following established husbandry protocols (Vaughan et al., 2006). Husbandry protocols followed the methodology previously outlined in Duffy and Staples (2022). Summer 13-lined ground squirrels were kept at 22±3°C, with a photoperiod adjusted weekly to match the conditions in Carman. Winter 13-lined ground squirrels were transferred to walk-in environmental chambers where the ambient temperature (T_a) was decreased by 1°C per day until it reached 4±2°C, and the photoperiod was set to 24 h of darkness to replicate fossorial (burrow) conditions.

In hibernating animals, torpor bouts were tracked by monitoring the presence of un-shredded paper towels daily (Hutchinson et al., 2024). All squirrels used in this study were adults that had previously undergone at least one hibernation season. We used pulse oximetry to assay arousing 13-lined ground squirrels (three females and six males) in December 2022 and summer 13-lined ground squirrels (two females and two males) in June 2023. We collected blood samples from 17 torpid squirrels (nine females, eight males) in January 2024 and from a different group of 11 summer squirrels (six females, five males) in June 2024, which were also used for heart mass comparisons and separate studies on mitochondrial metabolism.

During arousal from torpor to IBE and in the stable summer state, we measured f_H and Sa_O2 using a MouseOx^® Plus pulse oximeter collar (Starr Life Sciences, Oakmont, PA, USA; Harvard Apparatus Canada, Saint-Laurent, QC, Canada). Pulse oximetry measures the Sa_O2 of Hb molecules in red blood cells transcutaneously based on the principle that oxygenated and deoxygenated Hb absorb differing amounts of red and near-infrared (IR) light (Chan et al., 2013). The MouseOx^® collar used in this study was secured around the ground squirrel's neck, obtaining a strong signal from the carotid arteries, which supply blood to the head. A limitation of this methodology is the reliance on stable f_H for accurate measurements. The MouseOx^® is optimized to work at f_H between 90 and 900 beats min⁻¹, but torpid 13-lined ground squirrels have f_H as low as 2–4 beats min⁻¹ (Fig. 2A; MacCannell et al., 2018). As a result, the collar could not identify f_H or Sa_O2 during torpor but began taking measurements when f_H reached approximately 100 beats min⁻¹ early in arousal.

We removed torpid 13-lined ground squirrels from a walk-in environmental chamber (5°C) to an adjacent procedure room. Measurements of Sa_O2 were made at an ambient temperature of 22°C, similar to the housing temperature of summer animals. Hair was removed from the neck using electric clippers and Nair^© hair remover for all animals. During arousal, T_b was measured continuously (once per second) throughout the experiment using a rectal probe, with data acquired using Expedata (Sable Systems, NV, USA). This procedure induced arousal in torpid animals. In summer 13-lined ground squirrels, T_b was measured at the beginning and end of the 40 min trials.

Torpid and a separate group of anaesthetized (5% isoflurane, 95% O₂) summer squirrels were killed with cervical dislocation, and blood was drawn using a cardiac puncture to the right ventricle. We measured Hb, Hct, Na⁺, K⁺, Ca²⁺ and glucose in venous whole blood using a portable blood analyser (i-STAT-1; Abbott Laboratories, Abbott Park, IL, USA) equipped with CG8+ cartridges. Although the i-STAT system can measure blood gases, the very low blood pressure and flow during torpor made it impractical to collect blood in a gas-tight fashion, and the samples visibly changed colour within the syringe during sampling, indicating a change in oxygenation. Therefore, we excluded the blood gas data.

T_b values were collected every second, and mean values for each 2.5 min interval were calculated. f_H and Sa_O2 were collected every 0.2 s, with mean and s.e.m. values also calculated in 2.5 min intervals. The residuals of these data were normally distributed (Shapiro–Wilks test; Fig. S1A,B). To visualize the relationships among T_b, f_H and Sa_O2, T_b was grouped in 1°C bins and analysed using non-linear regressions (second-order polynomial models) with least square fitting. Linear regression was also used to assess the effect of f_H on Sa_O2.

Haematological data were normally distributed (Shapiro–Wilks test; Fig. S1C–H). Unpaired, two-tailed t-tests compared torpor and summer heart masses and blood metrics (Hct, Hb, sodium, potassium, calcium and glucose). All data analysis and plots were generated using Graph Pad Prism (version 9.5.1).

Beyond the low Sa_O2 values early in arousal, Sa_O2 declined further to 34% as T_b warmed to 10°C (Fig. 2B; Fig. S2B). This decline suggested that the early stages of arousal represented a phase where demand for O₂, likely due to increased thermogenesis by brown adipose, outpaced O₂ delivery. This decline in Sa_O2 also coincided with a fairly constant f_H at the beginning of arousal (Fig. 2A; Fig. S2A). This delay in f_H increase likely results from depressed myocardial contraction and relaxation kinetics at low temperatures, even though 13-lined ground squirrel hearts exhibit robust function at low temperatures that render non-hibernator hearts highly arrhythmic (Caprette and Senturia, 1984; Fedorov et al., 2005). Interestingly, the early decline in Sa_O2 during arousal found here aligns closely with another recent study in 13-lined ground squirrels, which found hyperventilation at the same T_b during arousal, suggesting decreased blood CO₂ (Sprenger and Milsom, 2022). Additionally, the low T_b values experienced early during arousal would have challenged O₂ offloading at the tissues, amplifying tissue hypoxia.

Throughout 80 min of arousal, Sa_O2 increased by 260%, which suggested 13-lined ground squirrels experienced hypoxia–reoxygenation. In IBE, when breathing room air, 13-lined ground squirrel Sa_O2 (87%) was similar to that of arctic ground squirrels (83%; Ma et al., 2005). In the wild, summer 13-lined ground squirrel burrow O₂ levels are estimated to be 18.9% (Studier and Procter, 1971), compared with 20.9% in well-mixed air. To our knowledge, O₂ levels in winter burrows have not been measured. However, snow cover could inhibit gaseous mixing in winter, so ambient O₂ levels may be even lower during the hibernation season, further exacerbating hypoxia challenges. In other hypoxia-tolerant species, such low Sa_O2 levels have only been reported in animals exposed to severely hypoxic environments. For example, in high-altitude deer mice, ∼65% Sa_O2 was reached only when breathing 8% O₂ (Ivy and Scott, 2021), and in naked mole rats, ∼40% Sa_O2 was reached only when breathing 3% O₂ (Pamenter et al., 2019).

Heart mass was similar between torpor and summer 13-lined ground squirrels (T₁₅=0.09, P=0.93; Fig. 3A). Body mass-corrected heart mass was 29.8% greater in torpor than in summer (T₁₅=5.70, P<0.0001; Fig. 3B) due to the corresponding 29.8% lower body mass in torpor than in summer (T₁₅=7.72, P<0.0001; Fig. 3C). Whole-blood Hct in torpor (49.76±1.01%) was 8.8% higher (T₂₆=2.60, P=0.015; Fig. 3D) than summer values (45.82±1.07%), and Hb concentration showed similar trends (Fig. 3E).

Many experiments have previously characterized Hb concentration in hibernators with mixed results (reviewed in Milsom and Jackson, 2011). Our Hct results align closely with a recent study in 13-lined ground squirrels (Cooper et al., 2016a). The modest increase in torpor compared with summer is unlikely to affect blood flow significantly on its own, but Spurrier and Dawe (1973) noted that Hct tended to rise during arousal. This Hct increase, accompanied by low blood temperature early during arousal, could increase blood viscosity, further slowing rates of O₂ delivery to tissues.

Whole blood collected during torpor had 6.7% lower Na⁺ concentrations than in summer (T₂₇=5.94, P<0.0001; Fig. 3F). K⁺ concentration did not differ between groups (T₂₄=0.39, P=0.27; Fig. 3G), but Ca²⁺ was 17.8% higher in torpor than in summer (T₂₇=2.45, P=0.021; Fig. 3H). Finally, glucose concentrations from torpid blood were 54.3% lower than in summer blood (T₂₇=11.26, P<0.0001; Fig. 3I).

It seems that the modest increase in Hct and Hb during torpor cannot be attributed to dehydration, as plasma Na⁺ levels were actually lower in this state, consistent with recent reports which indicate that 13-lined ground squirrel blood osmolarity declines during torpor despite the absence of water intake (Feng et al., 2019). We found that Ca²⁺ levels were higher in torpor than in summer, even without a dietary source. This finding supports the hypothesis that hibernators have developed mechanisms to maintain calcium homeostasis (Arfat et al., 2020). Blood glucose levels were significantly lower during torpor compared with those in summer (Fig. 3I), and these values align closely with previous measurements in 13-lined ground squirrel blood (Burlington and Klain, 1967; Nizielski et al., 1986). Low glucose levels may suggest a reliance on anaerobic metabolism. However, the evidence of lactate accumulation during torpor varies by species and blood sample type; for example, lactate accumulates in the red blood cells of arctic ground squirrels during torpor (Gehrke et al., 2019), while concentrations remained low in whole blood of golden-mantled ground squirrels (Callospermophilus lateralis; Maginniss and Milsom, 1994) and in serum of 13-lined ground squirrels (Feng et al., 2019). Instead, the blood glucose levels likely reflect that, during hibernation, 13-lined ground squirrels do not typically feed; the concentrations we report for torpid 13-lined ground squirrels resemble those found in fasted rats (Arnall et al., 1988). However, the values we report for summer 13-lined ground squirrels are similar to those of diabetic rats (Yang et al., 2020), which is expected because of the high body fat content in summer 13-lined ground squirrels (MacCannell et al., 2017).

The resistance of hibernators to I–R has been attributed to many physiological adaptations that likely evolved as protection from O₂ fluctuations during their torpor–IBE cycles, illustrated in Fig. 2B. Ground squirrel cardiac I–R tolerance is well defined in the literature (Bonis et al., 2019; Han et al., 2022; Xie et al., 2021; Salzman et al., 2017; reviewed in Drew et al., 2013).

Low glucose levels during torpor may indicate glycogenesis to supply energy during early arousal. One study found that by the third day of torpor, glycogen levels in 13-lined ground squirrel hearts were significantly higher than in summer, correlating with improved maintenance of cardiac function following ex vivo I–R in torpor (Heinis et al., 2015). Higher cardiac glycogen may help heart function during the early, hypoxic stages of arousal. Cardiac mitochondrial respiration is suppressed by 30% during torpor (Brown and Staples, 2014), which may help to conserve O₂. ROS production during O₂ fluctuations is also minimized in hibernator heart mitochondria by increased uncoupling proteins (Ballinger et al., 2017) to dissipate proton motive force and avoid reoxygenation-associated oxidative damage. There is also a hypothesis that repeated arousals themselves resemble ischaemic preconditioning, a condition in which previous bouts of tissue hypoxia prime tissues to resist later I–R insult (reviewed in Bhowmick and Drew, 2019). It is possible that repeated arousal throughout the hibernation season mimics preconditioning and mitigates oxidative stress by upregulating heat shock proteins and the HIF-1α pathway (reviewed in Jankovic et al., 2024).

The authors thank Dr Graham Scott for sharing his MouseOx^® equipment and software. We also thank Sharla Thompson, Lauren Rego and Colleen Barghout for their help with animal care, and Marina Zhang for her help with haematological processing. Thanks to two anonymous referees for their constructive comments that helped to improve the manuscript.