DOI: 10.5061/dryad.1rn8pk10g
title: Senescence and the stress axis in male ground squirrels

description: A critical time in the life of a male occurs at reproduction when his
 behavior, physiology, and resources must be brought to bear for the
 central purpose of his life. We ask whether reproduction results in
 dysfunction of the stress axis, is linked to life history, and causes
 senescence. We assessed if deterioration in the axis underlies variation
 in reproductive lifespan in males of 5 species of North American ground
 squirrels whose life history varies from near semelparity to iteroparity.
 The most stressful and energy-demanding time occurs in spring during the
 intense 2–3 week breeding competition just after arousal from hibernation.
 We compared their stress axis functioning before and after the mating
 period using a hormonal challenge protocol. We found no evidence of stress
 axis dysfunction nor was there a relationship between reproductive
 lifespan and stress axis functional deterioration.  Moreover,
 there was no consistent relationship between free cortisol levels and
 downstream measures. Thus, stress axis function was not traded off to
 promote reproduction and conclude that it is a pre-requisite for life.
 Hence, it functions as a constraint and does not undergo senescence.
description: Study areas and field procedures  We trapped
 prebreeding (within a few days of first emerging from hibernation) and
 postbreeding males (2–3 weeks later, same site). For the 3 western species
 (AGS, CGS, and RGS) logistics (i.e. ~2500 km between the AGS and RGS
 sites), asynchronous timing of emergence and reproduction, and a narrow
 window of reproduction within a species permitted us to trap only them in
 2007 and the 2 eastern species (FGS, TLGS) in 2008.  In 2007, we trapped
 RGS at Kinsella, Alberta (53°N 111°33’W) on March 25–27 and April 15–16,
 AGS at the Pelly River Ranch, Yukon (62°50’N 137°18’W) on April 9–12 and
 April 27–30, and CGS at Kananaskis, Alberta (51°2’N 115°2’W) on April
 22–24 and May 7–9, respectively. In 2008, we trapped TLGS on April 15–19
 and May 5–11 and FGS on May 6–11 and May 28–30 near Portage La Prairie,
 Manitoba (TLGS at 50°7’N 98°23’W, FGS at 50°12’N 98°14’W). 
 Animals were trapped by placing burrow traps or cage traps
 (Tomahawk #102, Tomahawk Live Trap Company, Tomahawk, WI, USA) in or next
 to burrows that animals were seen to have entered. Traps were monitored at
 least every 30 minutes and captured males were then kept in cage traps
 covered with a pillowcase in a shaded, quiet location until trapping was
 completed. We started trapping soon after animals emerged (07h00–09h00)
 and continued trapping for at most 4 hours or until we had as many animals
 as we could process at one time (6–9 animals).  Field
 Hormonal and Sampling Procedures Trapped animals were
 brought to a field laboratory, placed in a cool, quiet location, covered
 with pillowcases, and left for at least 1 hour to habituate before
 initiating the hormonal challenge procedure. One animal at a time was
 removed from its trap, weighed, and anesthetized with isoflurane (IsoFlo,
 Abbott Laboratories, Saint Laurent, QC, Canada).  We took an initial 0.6
 mL blood sample (BASE) and injected 0.1 or 0.4 mg/kg dexamethasone (DEX)
 (Sabex, Montreal, Canada), then returned the animal to the covered trap
 and processed the next animal.  From the BASE sample, we obtained 4
 subsamples: 0.3 ml for a complete blood count (Vita-tech Veterinary
 Laboratory Services, Markham, ON, Canada); 75 µl microhematocrit tubes for
 hematocrit (packed red blood cell volume, %); a drop for glucose levels
 (mg/dL, FreeStyle glucose meter, Abbott Laboratories, Alameda, CA, USA);
 and the rest for hormone and free fatty acid measurements. The latter
 sample was centrifuged at 8800g for 5 min, and the plasma from the
 hematocrit tubes was pooled with it and frozen at -20°C.
 The DEX tests the negative feedback to the pituitary and inhibits
 ACTH release. As we found no difference in DEX response to the different
 doses in 2007 (unpublished data), we administered 0.1 mg/kg to all animals
 in 2008. Two hours after the DEX injection, we anesthetized the animal
 again and took a 0.2-0.3 mL blood sample (“DEX” bleed) and then injected 4
 IU/kg of adrenocorticotropic hormone (ACTH; Synacthen Depot, Novartis
 Pharmaceuticals Canada Inc, Dorval, Quebec) into the thigh muscle to
 directly stimulate adrenal GC production.  Post-ACTH blood samples of 0.2
 mL each were taken at 30, 60 and 120 minutes (“P30”, “P60” and “P120”
 bleeds). Glucose was measured at each sampling point. Blood samples were
 centrifuged and plasma collected and frozen at -20°C and, on return to the
 university, stored at -80°C. After the P120 bleed, the animal was
 euthanized by anesthetic overdose and decapitated.  On
 necropsy, we dissected out and weighed both adrenals (nearest mg),
 abdominal fat (fat that was easily removed from the abdominal cavity and
 did not include fat in the mesentery: nearest 0.1g), both testes (nearest
 0.01g), and eye lens. Adrenal mass was a rough indicator of adrenal
 capacity based on the expectation that adrenal hypertrophy would occur
 from the prebreeding to the postbreeding period reflecting greater
 capacity to produce GCs.  Abdominal fat was a measure of a key energy
 reserve and expected to decline markedly. Testes mass was a measure of the
 capacity to produce sperm and testosterone and expected to decline
 markedly from the prebreeding to the postbreeding period. Age may be a key
 determinant in how individuals respond to stress and we attempted to age
 our squirrels by measuring their eye lens weights and by sectioning their
 femurs and jaws. To quantify the intensity of
 inter-male aggression during the breeding season, we examined the skin
 externally and internally for evidence of wounding. The degree of wounding
 was scored according to the number of punctures (bite marks that
 penetrated the skin, usually only visible from the inside of the hide) and
 externally visible wounds (tears in the skin that exposed the muscle below
 or had scabbed over). Our scoring categories were:  “None” for no wounds,
 “Minimal” for &lt;10 punctures and &lt;2 cm2 of externally visible
 wounding, “Moderate” for ≥ 10 punctures and/or &gt;2
 cm<sup>2</sup> but &lt;7 cm<sup>2</sup> of
 externally visible wounding, and “Severe” for ≥
 7 cm<sup>2</sup> of externally visible wounding and/or
 significant damage to muscle, testes, or eyes. Because FGS were not
 abundant, we did not euthanize the prebreeding animals. As a result, their
 wound scores were based solely on an external examination. Thus, we also
 do not have prebreeding adrenal, abdominal fat, or testes mass for
 FGS. Laboratory procedures We
 measured total cortisol levels by radioimmunoassay (RIA) in all species.
 For FGS and TLGS we switched to a commercial cortisol kit (Diasorin
 GammaCoat Cortisol Kit, Stillwater, MN, USA) using kit instructions. To
 cross-validate levels between the two RIA methods, we used 18 plasma
 samples (levels ranging from 22 ng/mL to 452 ng/mL) and obtained the
 cortisol levels measured by our initial protocol and by the kit protocol.
 These were highly correlated (r<sup>2</sup> = 0.97), though
 the Diasorin kit tended to give values about 7 ng/mL higher than our
 initial protocol. This difference might be attributable to loss from the
 dichloromethane extraction procedure in our initial protocol, which was
 not used with the Diasorin kit. Initial tests revealed
 that cortisol is the dominant GC in RGS and CGS, with corticosterone being
 undetectable. A similar pattern was found in AGS. However, we found that
 TLGS had corticosterone levels that were typically 10–20% those of their
 cortisol levels, and that FGS had corticosterone levels around 30–40% of
 their cortisol levels. Thus, in both of these species, we measured
 corticosterone levels. Because plasma samples were limited for some
 individuals, we only measured corticosterone levels in 5 prebreeding and 5
 postbreeding individuals from both species, for all 5 hormone challenge
 plasma samples (BASE, DEX, P30, P60, and P120). We calculated
 corticosterone as a percentage of total GC at each bleed in these animals.
 We used these means to estimate amount of corticosterone in the remaining
 animals. To estimate free GC concentrations (i.e.
 hormone not bound to CBG) we measured maximum corticosteroid binding
 activity (MCBC). The equilibrium dissociation constants (±SE) are needed
 for calculating MCBC and for AGS, RGS, CGS, TLGS and FGS these were 4.04 ±
 0.42 nM, 5.4 ± 0.56 nM, 4.73 ± 0.29 nM, 17.8 ± 2.3 nM, and 26.9 ± 10.0 nM,
 respectively (unpublished data). For TLGS and FGS, we assumed that the
 dissociation constants of CBG for cortisol and corticosterone were equal
 and that the two GCs could be treated as a single GC pool.
 To assess the overall adrenal capacity to respond to the ACTH
 injection, we calculated the area under the free GC response curve (the
 AUC). AUC was measured from the DEX bleed through to the last bleed, using
 the DEX bleed GC concentration as a baseline. It provided an integrated
 measure of the animals’ sensitivity and capacity to produce GC. To measure
 the “intrinsic restraint” of the HPA axis, we used the ratio of the
 maximum free GC level in response to ACTH to the BASE bleed free GC.
 Because ACTH directly stimulates the adrenals, the maximum free GC levels
 are not constrained by lack of drive from the hypothalamus or pituitary or
 by negative feedback. In contrast, the free GC level at BASE was the
 result of ongoing stimulation of the HPA axis as a result of capture
 stress but modulated by negative feedback of the GCs at the level of the
 pituitary. The ratio of these two levels (i.e. maximum/BASE) represents a
 measure of the degree to which the HPA axis produces a less-than-maximal
 GC response at the BASE bleed (i.e. “intrinsic restraint”). The higher the
 ratio, the stronger the intrinsic restraint.
 Testosterone levels (ng/mL) were measured. The assay had a mean
 recovery of [1,2,6,7-3H] testosterone added to plasma of 96.5 ± 0.7%
 (range 92–102%), with a detection limit of 10 pg/25 mL. It had an intra-
 and inter-assay coefficient of variation of 5% and 7.1%, respectively.
 Free fatty acids (FFA) were measured; they had an intra- and inter-assay
 coefficient of variation of 6.6 and 10.0%, respectively.
description: Statistical analysis All data were analyzed
 using SAS 9.2 (SAS Institute Inc., Cary, NC, USA). For simple comparisons
 among species, we used one-way ANOVAs and the conservative Tukey-Kramer
 multiple comparison post-hoc test. For our regressions of postbreeding GC
 levels against mean reproductive lifespan and probability of surviving
 more than one breeding season (PROC GLM), we did not test for or control
 for phylogenetic non-independence.  In part, our sample size precluded
 using phylogenetic contrasts:  tests for phylogenetic independence perform
 poorly on small datasets and if our species were not independent, our
 sample size would be reduced by one in order to do the contrast.  Our
 decision is further justified by the fact that we selected very closely
 related species, and because previous comparative studies of GC levels
 have found that much more diverse species groupings are nonetheless
 phylogenetically independent. For the prebreeding and
 postbreeding comparisons, data were first examined for normality. Where
 significantly non-normal data could not be normalized with a
 transformation, we used the Wilcoxon-Mann-Whitney non-parametric
 two-sample test using the EXACT option to generate a Monte Carlo-based
 exact P-value. Because we released prebreeding Franklin’s ground squirrels
 and some of our postbreeding samples came from recaptured individuals, we
 analyzed differences between pre- and postbreeding periods using a mixed
 model in SAS PROC MIXED, with subjects as a repeated measure. All other
 species comparisons used PROC TTEST; where pre- and postbreeding data had
 non-homogeneous variances based on the folded-F test, we use
 Satterthwaite-adjusted degrees of freedom for the t-tests (which can
 result in degrees of freedom that are not whole numbers). Unless otherwise
 specified, we present means with 95% confidence intervals.