Food supplements modulate changes in leucocyte numbers in breeding male ground squirrels
School of Biological Sciences, University of Nebraska, Lincoln, NE 68506-0118, USA e-mail: gbachman{at}unl.edu
Accepted 22 April 2003
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Summary |
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Key words: cost of breeding, immunosuppression, Spermophilus beldingi, ground squirrel, leucocyte, hibernation
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Introduction |
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At least three hypotheses might explain suppressed immune function in
breeding males. First, immune function might be depressed by elevated
testosterone titers during breeding
(Boonstra and Boag, 1992;
Boonstra et al., 2001b
;
Bradley, 1987
;
Folstad and Karter, 1992
;
Grossman, 1985
;
Tang-Martinez and Taylor,
1998
). Second, reproductive competition might impose stress on
males, resulting in immune suppression through elevated glucocorticoid titers
(Boonstra et al.,
2001a
,b
;
Bradley, 1987
;
Buchanan, 2000
). Third,
immunosuppression might result from the diversion of energy or nutrients
towards reproductive activities and away from immune function
(Sheldon and Verhulst, 1996
).
Although this third hypothesis often implies intervening hormonal control and
forms a basis of adaptive explanations for endocrine-based immunosuppression
(Boonstra and Boag, 1992
;
Deerenberg et al., 1997
;
Gustafsson et al., 1994
;
Nordling et al., 1998
;
Wedekind and Folstad, 1994
),
energy limitations can also directly cause immunosuppression. Immune function
is known to be depressed by specific nutrient deficits as well as by
starvation (Cunningham-Rundles,
1993
; Jain, 1993
;
Jose and Good, 1973
;
Lochmiller et al., 1993
).
Nutrient or energy limitation in breeding males is suggested by mass losses,
which may reflect both increased energy expenditure on reproductive activities
and reduced food intake (Andersson,
1994
). However, the role of lowered food intake on changes in
immune function during breeding is unknown.
If resource limitation is an important variable determining the extent of
immunosuppression, the impact is most likely to be found in animals that mate
when food availability is low. This situation is typical for male Belding's
ground squirrels living at high elevations in western USA. Males emerging from
hibernation are usually unable to forage due to extensive snow cover. As the
snow melts and foraging opportunities increase, females emerge and, within a
few days, come into estrus for a few hours each. This presents males with a
time and energy allocation conflict. Mate acquisition competes with foraging
for time and uses energy needed to restore the complete function of
physiological systems, including the immune system, which atrophied during
hibernation (Lyman and Chatfield,
1955; Shivatcheva and
Hadjioloff, 1987
; Sidky et
al., 1972
; Spurrier and Dawe,
1973
). Failure to maintain adequate immune function might be
particularly costly because males acquire numerous wounds during the breeding
period (Sherman and Morton,
1984
). I investigated the effect of food availability on immune
function by provisioning a set of males in the field just prior to breeding
and following their subsequent performance through the breeding period
relative to an unprovisioned control group. I assessed the status of the
immune system from leucocyte (white cell) counts. While this is an indirect
assessment of immune function, low leucocyte counts are predictive of a less
effective immune system and have been used previously as a proxy for immune
function (Boonstra et al..
2001a
,b
;
Gustafsson et al., 1994
;
Voigt, 2000
).
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Materials and methods |
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Provisioning
I began provisioning on 12 May, by which time four males had been located.
The first male was assigned a treatment (provisioned or control) based on a
coin toss, and subsequent treatment assignments were alternated. The last two
males were added to the experiment on 18 May. One pair of males who were
always found sharing a burrow or swapping burrows were assigned the same
treatment (control). By otherwise alternating the assignment of provisioned
and control treatments according to capture sequence, I hoped to evenly
distribute time since emergence between treatments and, by doing so, to evenly
distribute any variable, such as age, that co-varied with emergence. Only
adult males were used in this study; yearling males were distinguished and
eliminated by size and the lack of scrotal testes
(Morton and Gallup, 1975). The
first yearling male was captured on 26 May. I studied 12 control and 11
provisioned males, although all are not represented in every sample. To
provision a male, I placed approximately 20 g of the bait mixture into a
burrow after using radiotracking to both confirm his location and establish
that no other study male was in the same burrow. Provisioning ended on 25 May
after male activity and ranging increased prior to the first mating.
Data collection
I weighed each male to the nearest 0.1 g (Ohaus Scout, 400/.1, Pine Grove,
NJ, USA) and collected a blood sample soon after his first appearance above
ground (Sample 1, 9-18 May). Subsequent samples were collected as follows:
Sample 2, 26 May (four days prior to the first known mating); Sample 3, 10
June (two days prior to the last known mating); Sample 4, 26 June; Sample 5,
15 July (when summer fattening began); and Sample 6, 31 July (Figs
1,
2A). On sample days, males were
captured in the morning at or near their burrows. Within 15 min of capture,
the trap was covered and moved to a protected location off the snow and out of
the wind. Squirrels were transferred from the trap to a mesh bag and
anesthetized with an inhalant anesthetic (isoflurane; Abbot Laboratories,
North Chicago, IL, USA) to facilitate handling. Within 5 min of removal from
the trap, we weighed each squirrel, checked for wounds and collected a thin
film blood smear from toenails that were clipped short enough to obtain a
small droplet from the blood supply in the nail. Previously clipped nails were
easily identifiable and were not clipped again. We preferentially used the 1st
and 5th digit of the hind feet as these were the smallest. When needed,
bleeding was stopped with direct pressure to the cut together with Kwik-stop
styptic powder (Gimborn Pet Specialities, Atlanta, GA, USA). Up to 2 h elapsed
between trapping and sampling, but there was no consistent bias in trapping or
handling times across individuals or treatments. Behavioral data were recorded
from 09.00 h to as late as 18.30 h on 20 days from the start of provisioning
through the breeding period. Male location and activity were recorded at least
every 15 min, with a closer watch being kept on males near an estrous
female.
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Analysis of blood smears
Blood smears were stained with Geimsa solution (J. T. Baker/Mallinkrodt,
Baker Inc., Phillipsburg, NJ, USA) and examined under oil immersion at
100x magnification (Zeiss Axiostar, Thornwood, NY, USA). I was able to
identify the following leucocytes based on the morphology of these cells in
standard laboratory rodents (Voigt,
2000): basophil, eosinophil, lymphocyte, monocyte and neutrophil.
Leucocytes other than lymphocytes and neutrophils were extremely rare
collectively, averaging 1.4±0.2% across all samples, and were therefore
excluded from the analysis. Most lymphocytes and neutrophils are in storage,
with relatively fewer in circulation at any given time
(Goldsby et al., 2000
;
Jain, 1993
). Lymphocytes are
responsible for the specific immune response, while the phagocytic neutrophils
form a 'first line of defense', being associated with non-specific immune
responses. I distinguished between immature (banded) neutrophils, which have
an elongated, band-shaped nucleus, and mature (segmented) forms, in which the
nucleus is segmented into multiple lobes; banded neutrophils are not as
phagocytically active as the segmented forms
(Jain, 1993
). Leucocyte counts
were obtained by recording the number and kind of each cell type as well as
the number of erythrocytes encountered in a single pass across the slide, then
standardizing the leucocyte numbers to 100 000 erythrocytes. To reduce biases
in counts due to uneven distribution of cell types across the smears, I began
searching for cells off the ends of the smear, and the search path moved from
one long axis of the slide to another
(Voigt, 2000
). Counts were
made by eight observers who were trained and tested on standards. Each datum
is the average of counts from two observers.
Statistical analysis
The primary focus of the analysis was four samples taken from
near-emergence from hibernation to approximately two weeks after breeding in
late June. Cell counts for Samples 5 and 6 were not significantly different
from those of Sample 4. I evaluated the effects of treatment and sample time
on the leucocyte counts using mixed models repeated-measures ANOVAs (SAS) that
included an autoregressive term to remove any dependence in leucocyte scores
between successive samples. For analyses with significant interactions, I used
t-tests to evaluate the effect of treatment within sample periods
(unpaired) and differences between sample periods within treatments (paired),
and the resulting P values were Bonferroni-adjusted. All leucocyte
measures were square-root transformed to remove right skew prior to analysis,
but the figures plot untransformed values. Descriptive statistics are reported
as means ± S.E.M.
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Results |
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Control males
During breeding (Samples 2 and 3), control males had fewer circulating
white blood cells than at any other time, suggesting suppressed immune
function at this time. The total leucocyte count dropped by half from
emergence (Sample 1) to the beginning of the breeding period (Sample 2),
although not significantly, remained low through the breeding period (Samples
2-3) and increased significantly, more than trebling, after breeding was over
(Sample 4; Fig. 2A). Counts
remained elevated for the rest of the summer (see Materials and methods). This
pattern was matched by changes in the lymphocytes and neutrophils
(Fig. 2B). Changes in total
neutrophil numbers were mainly due to changes in the number of mature,
segmented neutrophils (Fig.
2C), which more than quadrupled after the end of breeding (Samples
3-4). The mean proportion of segmented (mature) neutrophils increased steadily
over time (Kendall's tau=0.79, d.f.=5, P=0.003).
Provisioned males
There were substantial differences between control and provisioned groups
in the numbers and kinds of circulating white blood cells over time.
Provisioned males were fed from emergence (Sample 1) until four days before
the first mating (Sample 2). During this time, their total leucocyte counts
increased significantly while controls decreased
(Fig. 2A; change from Sample 1
to 2, t9=3.532, P=0.006). As a result,
provisioned males entered breeding (Sample 2) with three times as many
leucocytes as controls (Fig.
2A). Leucocyte numbers in provisioned males decreased slightly
during breeding but were still nearly twice as high as controls at the end of
breeding (Sample 3). As with controls, counts tended to increase after
breeding (Samples 3-4; Fig. 2A)
and the two groups had similar leucocyte counts by Sample 4, two weeks after
the last mating.
The provisioned and control males also differed in how the sub-populations of leucocytes changed. In provisioned males, trends in lymphocyte numbers over time followed the pattern for total leucocytes although no differences were significant. However, due to the pre-breeding decline of lymphocyte numbers in control males (see above), provisioned males began breeding with over three times as many lymphocytes as controls (Sample 2; Fig. 2B), reflecting the increase in provisioned lymphocyte numbers relative to controls (change from Sample 1 to 2, t9=2.605, P=0.03). The neutrophil count in provisioned males increased significantly from emergence to the onset of breeding (change from Sample 1 to 2, t9=3.162, P=0.01), when it was significantly higher than that of controls (Fig. 2B). Subsequent changes in neutrophil counts in provisioned males were not significant. As with controls, provisioned males showed little change in the total number of immature, banded neutrophils over time (Fig. 2C). Therefore, the significant increase in the total number of neutrophils as breeding began (Sample 2) appears to be due to an increase in the number of segmented neutrophils. Provisioned males began breeding with significantly more segmented neutrophils than did controls and maintained these numbers throughout breeding (Fig. 2C). In short, provisioning abolished the trend for a pre-breeding decline in lymphocyte numbers seen in control males and advanced the increase in lymphocytes and neutrophils from after breeding to before.
Mass effects
Mean male mass decreased from emergence through breeding, increased slowly
for approximately a month and then increased rapidly in preparation for
hibernation (Fig.
1;Checker, 2001).
Temporal changes in leucocyte counts from emergence through breeding generally
followed changes in mass (Figs
1,
2); however, body mass was not
significantly correlated with leucocyte numbers in any sample period.
Provisioned males gained mass from Sample 1 to 2 relative to controls as both absolute mass (provisioned mean gain = 21.9±8.7 g; control mean loss = -23.0±5.8 g; t10=3.903, P=0.003, N=12) and as a proportion of initial mass (provisioned proportional gain = 0.1±0.04 g; control proportional loss = -0.09±0.02 g; t10=3.810, P=0.003, N=12). While leucocyte numbers were not related to mass at Sample 2 (P=0.26), there was a positive correlation between the change in mass since the first sample and the change in neutrophil numbers (Fig. 3), which reflects the impact of the provisioning treatment on both mass and cell numbers (see below). While the changes in total leucocyte numbers and lymphocytes both exhibited a positive trend with the change in mass, the relationships were not significant (change in mass vs change in leucocyte count, Sample 1 to 2, P=0.11, r=0.484, N=12; change in mass vs change in lymphocyte count, Sample 1 to 2, P=0.25, r=0.368, N=12). During the breeding period (Samples 2-3), provisioned males tended to lose more mass than did controls (40±9 g vs 8.9±11.3 g: t11 =2.183, P=0.052, N=13), and after breeding both groups gained mass at similar rates (Samples 3-4; provisioned = 8.9±8.1 g day-1; control = 8.9±5.4 g day-1). During breeding, cell numbers were not related to mass change.
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Male activity
Before matings began, males were active for a mean of 4.4±0.3 h,
increasing to 7.2±0.4 h early in the breeding period (paired
t17=8.783, P=0.0001, N=18), with no
difference between control and provisioned groups. Similarly, foraging time
did not differ between control and provisioned groups. Overall, foraging
increased significantly during the breeding period, from a mean of
0.54±0.1 h day-1 before breeding to 2.4±0.2 h
day-1 during breeding (t17=11.014,
P=0.0001, N=18). During the breeding period, provisioned
males were involved in significantly more agonistic encounters, spending a
mean of 51±7 min in fights or chases, in contrast to 33±2 min
for control males (t16=2.124, P=0.05,
N=18). Mating success, however, did not differ between the treatment
groups (provisioned, 3.9±0.4 copulations; control, 3.7±0.6
copulations; range 1-5). All males had received numerous wounds by the end of
the breeding period.
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Discussion |
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The allocation of limited energy and nutrients during breeding may be
particularly important for mammals, such as ground squirrels, that breed
shortly after emerging from hibernation. The size and function of many organs,
including organs associated with immune function, are reduced during
hibernation (Lyman and Chatfield,
1955; Morton and Parmer,
1975
; Shivatcheva and
Hadjioloff, 1987
; Sidky et
al., 1972
). The limited food available in early spring
(Morton, 1975
;
Morton and Sherman, 1975
) must
be directed to the recovery of organ function as well as breeding activity.
Like most ground squirrels, Belding's do not cache food. Of the >20
Spermophilus species, only four are thought to cache when suitable
food items are available (S. saturatus, S. richardsonii, S.
columbianus and S. parryii;
Wilson and Ruff, 1999
;
Michener and Locklear, 1990
)
and, with the exception of S. parryii
(Buck and Barnes, 1999
), only
the males do so, using the stores primarily to support testes development and
spermatogenesis and possibly to maintain condition in early spring. Whether
these caches are sufficient in energy or nutrient content to also support the
development of immune function raises another dimension to the costs and
benefits of caching in hibernating sciurids.
In squirrels, reactivation of lymphoid tissue begins before above-ground
activity commences (Shivatcheva and
Hadjioloff, 1987; Sidky,
1972
), but this study shows that a mature complement of leucocytes
has not been established at this time. The subsequent development of the
leucocyte population is dependent upon access to food. Although control males
foraged more during breeding, they appeared to allocate their resources to
fueling reproductive activities and did not increase mass or leucocyte numbers
until after breeding. The provisioning experiment demonstrates that males have
the capacity to improve one aspect of immune function, circulating white cell
numbers, nearly one month earlier than in controls. An important implication
from this study is that immunosuppression may not be an obligatory cost of
reproduction for males. In good habitats or with sufficient time to forage,
males can potentially maintain both immune function and breeding effort by
increasing net energy intake. A key allocation problem for males may therefore
be whether to spend time acquiring mates vs nutrients, and the cost
of insufficient intake may be reduced immune function. This provides an
alternative, or at least a contributing, mechanism for reduced immune function
in breeding male mammals. While all mammals do not face conditions as extreme
as those encountered by these squirrels, many lose mass during breeding
(Andersson, 1994
), suggesting
that energy allocation to immune function could be at risk.
Previous studies have proposed that immune suppression in male mammals
during breeding is a consequence of elevated titers of either stress hormones,
particularly glucocorticoids, or testosterone
(Boonstra and Boag, 1992;
Boonstra et al.,
2001a
,b
;
Buchanan, 2000
;
Grossman, 1985
;
Nelson and Demas, 1996
). While
there is little question that endocrine changes during breeding are
immunosuppressive, these effects alone are unlikely to account for the
different leucocyte profiles in the provisioned and control males in this
study.
Stress associated with breeding can lead to endocrine changes that reduce
the number of circulating leucocytes
(Buchanan, 2000;
Jain, 1993
;
Voigt, 2000
). Endocrine
changes associated with acute stress remove lymphocytes from circulation by
moving them to marginal pools and tissues, which can enhance immune responses
(Dhabhar and McEwen, 1997
,
1999
), while chronic stress
leads to increased lympholysis, as well as increasing the retention of
neutrophils in circulation, and is ultimately immunosuppressive
(Baxter and Forsham, 1972
;
Dhabhar and McEwen, 1997
,
1999
). In the present study,
differential exposure to acute stressors such as trapping and handling or
aggressive social interactions cannot account for the lower breeding leucocyte
counts in control males (see Materials and methods;
Boonstra and Singleton, 1993
;
Kenagy and Place, 1999
;
Moberg, 1985
). Similarly, all
males were active in breeding, which has been shown to be a source of chronic
stress leading to higher cortisol titers and lower white cell counts in male
Arctic ground squirrels (S. parryii; Boonstra et al.,
2001a
,b
).
While the drop in lymphocyte numbers seen in control males at the onset of
breeding is consistent with changes induced by chronic stress, an increase in
neutrophil numbers did not occur (Fig.
2B,C). Similarly, although provisioned males reduced total
leucocyte numbers during the breeding period, the trend was to decrease both
lymphocytes and neutrophils, and neither decline was significant on its own
(Fig. 2B,C). Therefore, stress
responses are insufficient to explain both the higher leucocyte counts in
provisioned males and the pattern of changes in leucocyte numbers during
breeding.
The immunosuppressive effect of testosterone has attracted attention as a
potential mediator of a trade-off between immune function and dominance in
male competition (Tang-Martinez and
Taylor, 1998; Wedekind and
Folstad, 1994
). Not surprisingly, testosterone levels are elevated
in male ground squirrels during the mating period
(Barnes, 1986
;
Boonstra et al., 2001a
). As
with the stress hormones, the greater nutritional status of provisioned males
at the start of breeding may ameliorate much of the immunosuppressive effects
of testosterone. Provisioned males, on average, were involved in more
agonistic encounters than were control males and they tended to be among the
first males to mate with a female, suggesting that they were either dominant
or that they were better able to locate an estrous female (G.C.B., unpublished
data). How their behavior relates to androgen levels is unknown, but if
androgen levels correlate with these indicators of dominance, provisioned
males would be predicted to have a lower white cell count than controls. This
is clearly not the case (Fig.
2A) and, although the provisioned males tended to reduce leucocyte
counts, the controls still maintained the lowest counts during breeding.
Although the immunosuppressive effects of glucocorticoids and androgens do
not readily explain the relatively low leucocyte numbers in the control males,
the immuno-enhancing effect of leptin
(Fantuzzi and Faggioni, 2000)
may have a role in the differences between control and provisioned groups. The
effects of leptin on the immune system include the proliferation of T cells
(Lord et al., 1998
) and
possibly neutrophils (Fantuzzi and
Faggioni, 2000
; Laharrague et
al., 2000
). Leptin is produced in a number of tissues including
gastric mucosa and adipose (Bado et al.,
1998
). As the increase in body mass observed in the provisioned
males was mostly in lean tissue (G.C.B., unpublished data), an increase in
leptin from adipose tissue is unlikely. However, feeding is a stimulus that
can lead to an increase in leptin secretion by the gastric mucosa
(Bado et al., 1998
) as well as
an increase in the growth of the digestive tract in early spring. Increased
leptin in peripheral circulation together with the growth of lymphoid tissue
in the digestive tract
(Cunningham-Rundles, 1993
;
Shivatcheva and Hadjioloff,
1987
) are possible important mediators of the increased leucocyte
numbers observed in the provisioned males.
A net fitness benefit could result from immune suppression if mounting an
immune response or maintaining an immune system during breeding uses resources
that could otherwise be allocated to reproduction
(Buchanan, 2000;
Raberg et al., 1998
;
Sheldon and Verhulst, 1996
;
Wedekind and Folstad, 1994
).
The potential for a trade-off between immune function and reproduction is
indicated by studies that document reduced immune function during breeding
seasons in contrast to non-breeding periods
(Boonstra et al., 2001b
;
Festa-Bianchet, 1989
;
Nelson and Demas, 1996
) or
those that demonstrate a negative correlation between reproductive effort and
immune status across individuals during the same breeding period
(Deerenberg et al., 1997
;
Gustafsson et al., 1994
;
Nordling et al., 1998
),
although all studies do not show this trade-off
(Lozano and Ydenberg, 2002
).
While much work emphasizes the role of energy, non-energy nutrients are also
important for the development and maintenance of immune function. Restriction
of protein and carotenoids can lead to a reduction in the size of lymphoid
organs and the number and function of leucocytes and can affect the immune
response (Cunningham-Rundles,
1993
; Gonzalez et al.,
1999
; Jose and Good,
1973
; Lochmiller et al.,
1993
; Nordling et al.,
1998
). Therefore, it is reasonable to anticipate co-variation
between energy and nutritional state and immune function, particularly under
conditions of limited resource availability.
If limited resources can be a proximate cause of an inability to maintain
immune function during breeding, then greater access to resources could allow
an individual to avoid the trade-off. The implication of glucocorticoid- and
testosterone-induced immunosuppression during breeding is that
immunosuppression will covary with the endocrine titers. In this study,
provisioned males prepared for and participated in breeding alongside control
males but, because of the provisioning, they were able to increase their
peripheral leucocyte numbers. Similarly, access to food may in part explain
the results of field studies that do not find notable reductions in immune
function (Boonstra and Singleton,
1993; Hasselquist et al.,
1999
; Nelson and Demas,
1996
) and in laboratory studies with ad libitum food
where the resulting ability of animals to maintain mass or condition has been
cited as a factor contributing to a lack of immune suppression
(Svensson et al., 1998
).
The lower leucocyte count in control males was not related to mating
success. Control males foraged more and engaged in fewer agonistic behaviors
but, in this study, the degree to which this affected the ability of males to
acquire mates was not significant. If the lower cell numbers reflect an
important reduction in immune capacity then males may be making a trade-off
during breeding, favoring reproductive effort over survival, as has been
proposed for a variety of animals (Boonstra
and Boag, 1992; Boonstra et
al., 2001b
; Bradley,
1987
; Deerenberg et al.,
1997
; Festa-Bianchet,
1989
; Gustafsson et al.,
1994
; Nordling et al.,
1998
; Sheldon and Verhulst,
1996
). The extent to which the low leucocyte counts in breeding
male Belding's ground squirrels reflect impaired immune function to the kind
of injuries that males face during breeding and how this affects survival or
future reproductive success await additional work.
Funding for data collection and analyses was provided by NSF IBN9982698 and the University of Nebraska-Layman Foundation. Procedures were covered by a UNL animal care protocol. D. Dawson and the Valentine Eastern Sierra Reserve provided logistical support. Thanks to R. Gibson, D. Wylie and two anonymous reviewers for advice and comments. Special thanks to the students who contributed to data collection and analysis: J. Caverzagie, N. Checker, A. Echternacht, M. Glathar, P. Grimm, J. Hale, E. Hauan, S. Jesseau, S. Krienert, J. Reddish, C. Smith, J. Swanson and B. Thacker.
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