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First published online September 5, 2008
Journal of Experimental Biology 211, 2960-2968 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.017897
Hematological changes associated with egg production: direct evidence for changes in erythropoiesis but a lack of resource dependence?
Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia, Canada V5A 1S6
* Author for correspondence at present address: Women's Health Research Institute, Provincial Health Services Authority, Room E208–4500 Oak Street (Box 42), Vancouver, British Columbia, Canada V6H 3N1 (e-mail: ewagner3{at}cw.bc.ca)
Accepted 14 July 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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6%), red blood cell counts
(8%), and plasma hemoglobin concentration (9%) during egg
production, even on a high-quality ad libitum diet, consistent with
an effect of hemodilution associated with yolk precursor production. However,
our results provide strong support for the hypothesis that erythropoiesis is
transiently suppressed during egg-laying and that the recovery from anemia is
relatively long-lasting, extending through incubation and hatching periods.
Decreased hematocrit, red blood cell counts, and hemoglobin concentration did
not recover at clutch completion, but showed evidence of recovery to baseline
pre-breeding levels at hatching. More importantly, there was significant
time-dependent variation in the proportion of reticulocytes, which increased
at clutch completion but peaked at hatching 10–12 days after clutch
completion, and in mean red blood cell volume, which showed a significant
increase at clutch completion; consistent with enhanced production and release
of larger immature cells into the circulation following suppression of
erythropoiesis. Finally, we found no evidence for resource dependence of
anemia associated with egg production in relation to diet quality, i.e.
exogenous lipid and protein resources available to the laying female. This
study demonstrates that transient suppression of erythropoiesis and,
subsequently, increased reticulocytosis, are key components of reproductive
anemia in egg-laying females.
Key words: anemia, cost of reproduction, egg production, erythropoiesis, estrogen, zebra finch
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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; Harshman and Zera,
2007), this trade-off between current reproduction and future
fecundity and/or survival has traditionally been assumed to involve
reallocation of resources among different, competing physiological systems
(Stearns, 1992). More recently
it has been suggested that the reproductive process itself or the regulatory
(physiological) networks controlling reproduction (e.g.
Partridge et al., 2005;
Harshman and Zera, 2007) might
induce costs of reproduction. Hormones are particularly strong candidates for
generating as well as regulating such trade-offs because hormones can have
pleiotropic effects, both positive and negative, on multiple physiological
systems (e.g. Finch and Rose,
1995; Ketterson and Nolan,
1999; Williams et al.,
2005; Zera et al.,
2007). Here we focus on one potential physiological mechanism that
might mediate costs of reproduction: the development of anemia during egg
production in birds (Williams et al.,
2004a; Williams,
2005). Anemia – a reduction in hematocrit, hemoglobin and
red blood cell number (Campbell and Ellis,
2007) – during egg production has been documented in
numerous studies [(e.g. deGraw et al.,
1979; Jones, 1983;
Morton, 1994;
Gayathri and Hegde, 2006;
Wagner et al., 2008) and
references therein], and may persist into later stages of incubation and chick
rearing in some cases (Williams et al.,
2004a). In addition, Kalmbach et al.
(Kalmbach et al., 2004) showed
that experimentally increasing egg production in female great skuas
(Stercorarius skua) increased anemia, in terms of a greater reduction
in hematocrit and red blood cell number compared with control females. Since
anemia should reduce total oxygen carrying capacity of the blood and
compromise aerobic performance, this might explain negative effects of
increased egg production effort on subsequent reproductive stages (i.e.
incubation and chick rearing) that have been widely reported in birds (e.g.
Monaghan et al., 1998;
Nager et al., 2001).
Several potential mechanisms have been proposed to explain the development
of anemia during egg production. Decreased hematocrit might be an indirect
effect of estrogen-dependent hepatic production of yolk precursors and
mobilization of calcium ions (Morton,
1994; Salvante and Williams,
2002), osmotically active compounds that are transported in the
blood at high concentrations during egg production. This in turn may trigger
an increase in plasma volume due to osmotic movement of water from
extra-cellular spaces into the blood (e.g. hemodilution) to maintain plasma
osmolarity or viscosity at a constant level
(Reynolds and Waldron, 1999),
which would decrease hematocrit (red blood cells per unit plasma volume) but
not total cell number. Some authors have proposed that the reduction in
hematocrit reflects a transient suppression of erythropoiesis (e.g. red blood
cell production) during egg production in order to redirect energy to meet the
increased metabolic demands of the reproductive organs
(Ronald et al., 1968), or
because essential factors required for erythropoiesis are preferentially
allocated to the production of egg components
(Jones, 1983;
Gayathri and Hegde, 2006;
Kasprzak et al., 2006).
Alternatively, development of anemia might represent a direct, negative
pleiotropic effect of estrogen, which is present at high levels during egg
production and has essential reproductive functions
(Kalmbach et al., 2004;
Williams et al., 2004b;
Williams et al., 2005).
Estrogens inhibit the differentiation, proliferation and survival of white and
red blood cell precursors in the bone marrow
(Blobel and Orkin, 1996;
Medina et al., 2000;
Perry et al., 2000). Blocking
estrogen receptors using the anti-estrogen tamoxifen inhibits the development
of anemia in egg-laying birds supporting a role for estrogen-dependent
suppression of erythropoiesis in anemia
(Wagner et al., 2008). The
candidate mechanisms that have been proposed involve very different
predictions about (1) the time-course of development and recovery from anemia,
(2) whether specific changes would occur among a subset or all hematological
variables (hematocrit, hemoglobin concentration, red blood cell size and
number, proportion of immature red blood cells or reticulocytes; see
Table 1), and (3) the extent to
which development of anemia should be influenced by resource availability or
diet quality.
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In the present study, we tested predictions generated from these different,
non-mutually exclusive hypotheses for the causal mechanisms underlying anemia
associated with egg production using female zebra finches (Taeniopygia
guttata; see Table 1).
Specifically, if hemodilution is solely responsible for the observed decrease
in hematocrit, we predicted that, (1) red blood cell number and hemoglobin per
unit plasma volume would also decrease during egg production, but that these
changes would be reversed at clutch completion when plasma yolk precursor
concentration returned to non-breeding levels
(Salvante and Williams, 2002),
and (2) there would be no changes in mean red blood cell size in relation to
cell age [e.g. newly produced red blood cells are larger than mature cells
(Campbell and Ellis, 2007)] or
in the proportion of reticulocytes, since dilution per se would not
change rates of red blood cell turnover (production and degradation of cells).
By contrast, if anemia involved transient, estrogen-dependent suppression of
erythropoiesis, we predicted that (1) decreased hematocrit, cell number and
hemoglobin concentration would not recover at clutch completion since it takes
7–14 days for regenerative erythropoiesis to restore red blood cell
numbers following experimentally induced anemia
(Domm and Taber, 1946;
Clark et al., 1988), and (2)
there would be a marked increase in immature red blood cells (reticulocytes)
post-laying and a corresponding increase in mean cell volume [i.e.
reticulocytes are larger than mature cells
(Campbell and Ellis, 2007)].
Finally, if anemia during egg production is resource dependent, we predicted
that females on a low-quality diet (e.g. protein and lipid deficient) would
show larger decreases in hematocrit, red blood cell counts and hemoglobin
concentration compared with females on a high-quality diet.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Experimental protocol
Twenty-nine breeding pairs were randomly assigned to either a low-quality
(N=14) or high-quality diet treatment (N=15). Between
pairing and clutch completion, females on the low-quality diet continued to
receive the standard seed diet ad libitum (see above), while those on
the high-quality diet received the standard seed diet plus a daily egg food
supplement (6 g day–1, 20.3% protein:6.6% lipid). Initial
observations showed that hatching success was extremely low on the low-quality
diet, confirming that this diet was poor quality, so all pairs
receiving this diet were separated and returned to same-sex holding cages at
clutch completion. As such, we present data from the hatching and
chick-rearing periods for females receiving the high-quality diet only:
breeding pairs receiving the high-quality diet were permitted to incubate eggs
and rear chicks to fledging, with egg-food provided again during the
chick-rearing period. Pairs were left undisturbed from clutch completion until
the hatching period, at which point nest boxes were checked daily to determine
hatching success per clutch. Immediately after hatching, chicks were weighed
and marked with non-toxic dye to indicate hatch order, and then individually
banded at 8 days of age. The mass of each chick was recorded at 7, 10, 14 and
21 days post-hatch to monitor growth rates. At 30 days of age, final brood
size for each nest was recorded, and weight, tarsus length, and bill length of
each chick was measured. After a 3 month rest period, the same matched pairs
were bred again under the opposite diet regime than previously assigned, such
that repeated measures data was obtained for 23 pairs that initiated
egg-laying on both diet regimes (of the original 29 females two died in the
intervening rest period (both from the high-quality diet trial) and four
females either did not lay eggs or laid only one egg and were eliminated from
the second low-quality diet trial).
Blood sampling and hematological analysis
To monitor hematological parameters across the breeding cycle, female birds
were blood sampled at five intervals. (1) pre-breeding – at pairing
(N=29); (2) egg-laying – day of laying of first egg
(N=29); (3) clutch completion – after two consecutive days
without laying an egg (N=25); (4) hatching – the day the first
chick hatched (N=18); and (5) fledging – on average 21 days
post-hatching (N=12). For the diet-quality analysis we used blood
samples obtained for 23 females at prebreeding, the 1-egg stage, and clutch
completion only. All blood samples (50 µl) were collected within 3 min
of capture from the brachial vein (to avoid potential capture-related stress
effects) between the hours of 09.30 and 11.30.
Hematological variables were measured with standard techniques developed
for human blood and commonly used on birds
(Campbell and Ellis, 2007).
Hematocrit (Hct; %) was measured with digital calipers (±0.01 mm)
following centrifugation of whole blood for 3 min at 13,000 g.
Hemoglobin (Hb; g dL–1 whole blood) was measured using the
cyanomethemoglobin method (Drabkin and
Austin, 1932) modified for use with a microplate spectrophotometer
(BioTek Powerwave 340, BioTek Instruments, Winooski, VT, USA), using 5 µl
whole blood diluted in 1.25 ml Drabkin's reagent (D5941 Sigma-Aldrich Canada,
Oakville, Ontario, Canada) with absorbance measured at 540 nm. Intra- and
inter-assay coefficients were 1.7% and 3.9%, respectively. Erythrocyte counts
(RBC; number of cellsx106 µl–1) were
determined from duplicate samples (1 µl blood diluted 1/200 with modified
Natt and Herrick's solution (Natt and
Herrick, 1952; Robertson and
Maxwell, 1990) with an improved Neubauer hemocytometer (Fisher
Scientific, Ottawa, Ontario, Canada). The average variation among duplicate
RBC samples from the same bird was 6.9%, and measurement error (determined
from repeated sampling) was 8.9%, which is to be expected with this technique
(Campbell and Ellis, 2007).
From these measurements we calculated mean red cell volume (MCV; femtolitres
or fl) using the formula Hct/RBCx10=MCV
(Campbell and Ellis, 2007). The
proportion of reticulocytes (% Ret=number of immature red blood cells/total
red blood cellsx100) was estimated from whole blood smears after
supravital staining with new Methylene Blue (R4132, Sigma Aldrich Canada,
Oakville, Ontario, Canada). A total of 1000 red blood cells were counted per
slide, and reticulocytes were distinguished from mature erythrocytes by their
relatively larger size and less condensed chromatin
(Campbell and Ellis, 2007). Red
blood cells were classified as reticulocytes if at least five reticulum (RNA)
aggregations were visible in the cytoplasm or if a distinct ring of reticulum
was surrounding the nucleus (Fernandez and
Grindem, 2006). The same individual counted all blood smears
(E.C.W.), and slides were randomly coded prior to analysis so that the
examiner was blind to the identity of the female and reproductive stage being
scored. If blood samples collected at various stages were of insufficient
volume to permit all analyses listed above, priority was given to measuring
hematocrit (final sample sizes are listed in
Table 2).
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Statistical analysis
All statistical analyses were carried out using SAS software version 9.1
(SAS 2003). Separate
repeated-measures analyses were performed to (1) examine variation in body
mass and hematological parameters across the entire breeding cycle in females
on the high quality diet (pre-breeding, 1-egg, clutch completion, hatching and
fledging stages), and (2) examine the effect of diet quality on changes in
body mass and hematological parameters during egg-production (pre-breeding,
1-egg and clutch completion). To examine temporal variation in body mass and
hematological parameters, we used repeated-measures mixed linear models (MIXED
procedure) with reproductive stage included in the model as a fixed effect and
individual as a random effect. For the second analysis (effect of diet
quality), diet type and diet x stage were also included as fixed effects
in the model. For all repeated measures analyses, the denominator degrees of
freedom for tests of fixed effects and post-hoc tests were computed
using the Kenward-Roger method as recommended by Littell et al.
(Littell et al., 2006). The
between- and individual-variance components (e.g. random effects) were
calculated and evaluated via comparison with a Gaussian distribution
expected if the variance/covariance was equal to zero. Post-hoc tests
for differences between means were corrected for multiple comparisons using
the Tukey–Kramer adjustment formula. Clutch size was the only variable
that was not approximately normal in distribution (Kruskal–Wallis test;
UNIVARIATE procedure) and was therefore log-transformed prior to analyses. All
values presented are least square means ± s.e.m. unless otherwise
stated.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Body mass variation and mass independence of hematological variables
Body mass varied across the complete reproductive cycle for all females
(N=29) on the high-quality diet (F4,23=35.91,
P<0.0001; Fig. 1A)
as we have previously described (e.g.
Salvante and Williams, 2002;
Wagner et al., 2008). The
between-individual variance component estimate associated with the model was
0.61±0.22 (Z=2.77, P<0.003) and the
within-individual variance component was 0.49±0.08 (Z=6.28,
P<0.0001). In relation to diet quality, there was a highly
significant diet x reproductive stage interaction for change in body
mass between pre-breeding and clutch completion, i.e. the pattern of change in
body mass varied by diet (F2,53=7.96, P<0.001;
Fig. 1C). The
between-individual variance component estimate associated with the model was
0.58±0.23 (Z=2.51, P<0.015) and the
within-individual variance component was 0.75±0.06 (Z=12.85,
P<0.0001). On the high quality diet, mean body mass increased from
pre-breeding to the 1-egg stage, then decreased to levels significantly lower
than preceding stages at clutch completion (pre-breeding vs clutch
completion, t45=4.36, P<0.008; 1-egg
vs clutch completion t45=8.92,
P<0.0001 (Fig. 1C).
By contrast, there was no significant change in mean body mass across
reproductive stages on the low quality diet (P>0.05 for all
comparisons; Fig. 1C).
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As previous studies have found a relationship between body condition and
hematocrit (reviewed by Fair et al.,
2007), body mass was initially included as a covariate in all
repeated-measures analyses of hematological variables. However, body mass did
not significantly influence the variables of interest (P>0.1 for
all) except in the analysis of hematocrit across the complete reproductive
cycle (P=0.002). Therefore, body mass was retained as a covariate for
analysis of hematocrit and omitted from all other analyses of hematological
traits (hemoglobin concentration, red blood cell number, mean cell volume, and
reticulocyte count).
Variation in hematocrit
Hematocrit varied significantly with breeding stage
(F4,84=13.28, P<0.0001;
Fig. 1B), including mass as a
covariate in the model (F1,104=5.44, P=0.02). The
between-individual variance component estimate was 13.92±5.5
(Z=2.53, P=0.0026) and the within-individual variance
component was 10.51±1.28 (Z=8.24, P<0.0001).
Hematocrit decreased significantly from pre-breeding to the 1-egg stage
(t78=6.99, P<0.0001), and remained
significantly lower than pre-breeding levels at clutch completion
(t80=4.4, P<0.0003;
Fig. 1B). At hatching,
hematocrit was not significantly different from clutch completion or
pre-breeding values (P>0.15 for both) i.e. hematocrit appeared to
recover to `baseline' levels (Fig.
1B). However, at fledging, hematocrit decreased to levels
significantly lower than the preceding stage (hatching vs fledging:
t80=2.86, P<0.05) as well as pre-breeding
baseline levels (t81=4.42, P<0.0003;
Fig. 1B).
For the diet-quality experiment (N=23), there was a significant
diet x reproductive stage interaction for hematocrit
(F2,60=4.18, P<0.02;
Fig. 1D). However,
post-hoc pairwise analyses did not detect any significant differences
in hematocrit at any stage between the different diets (P
0.2 for
all comparisons), and change in hematocrit between breeding stages
was independent of diet quality for all pair-wise comparisons
(P0.10). After removing the interaction term from the model, the
main effects of diet quality (F1,22=1.24, P=0.28)
and body mass (F1,100=2.30, P=0.13) were not
significant, but reproductive stage had a highly significant effect on
hematocrit (F2,38=35.14, P<0.0001). The
between-individual variance component estimate associated with the model was
8.55±3.56 (Z=2.40, P<0.02) and the
within-individual variance component was 0.52±0.09 (Z=5.55,
P<0.0001). Among females on the high-quality diet, mean hematocrit
decreased significantly from pre-breeding birds to the 1-egg stage
(t43=5.12, P<0.0001), and remained
significantly lower than pre-breeding values at clutch completion
(t52=3.76, P<0.005;
Fig. 1D,
Table 3). On the low-quality
diet, hematocrit decreased significantly from pre-breeding to the 1-egg stage
(t42=7.05, P<0.0001), and again remained
significantly lower than pre-breeding values at clutch completion
(t46=7.13, P<0.001;
Fig. 1D,
Table 3).
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Variation in plasma hemoglobin concentration
Plasma hemoglobin concentration varied significantly with breeding stage
(F4,51=4.85, P<0.02;
Fig. 2A); the
between-individual and within-individual variance component estimates
associated with the model were 1.86±0.78 (Z=2.38,
P<0.01) and 2.27±0.27 (Z=8.25,
P<0.01), respectively. There were no significant differences in
hemoglobin concentration between the pre-breeding, 1-egg, clutch completion or
hatching stages (P>0.1 for all comparisons), but hemoglobin
concentration at the fledging stage was significantly lower than all other
stages (P<0.05) except clutch completion (P=0.5;
Fig. 2A). By contrast, for the
diet-quality analysis, the diet x reproductive stage interaction was not
significant (F2,51=0.45, P=0.64;
Fig. 2C). After removing the
interaction term from the model, the main effect of diet quality was not
significant (F1,32=0.50, P=0.48), but there was a
significant main effect of reproductive stage on variation in hemoglobin
concentration (F2,50=13.44, P<0.0001). The
between-individual and within-individual variance component estimates
associated with the model were –0.33±0.74
(Z=–0.44, P=0.66) and 0.44±0.10
(Z=4.44, P<0.0001) respectively. For the pooled diet
groups, hemoglobin concentration decreased from 16.19±0.35 mg
dl–1 in pre-breeding birds to 14.51±0.34 mg
dl–1 at the 1-egg stage (t47=4.49,
P<0.0001), and hemoglobin concentration remained significantly
lower than pre-breeding levels at clutch completion (14.33±0.36 mg
dl–1; t51=4.69,
P<0.0001).
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Mean red blood cell volume varied significantly with breeding stage (F4,78=3.13, P<0.02; Fig. 3A). The between-individual and within-individual variance component estimates were 133.76±58.97 (Z=2.27, P<0.025) and 255.89±42.06 (Z=7.93, P<0.0001), respectively. There were no significant differences found in mean cell volume among pre-breeding, 1-egg and hatching stages (P>0.05 for all comparisons; Fig. 3A). However, mean cell volume at clutch completion was significantly greater than pre-breeding mean cell volume (t77=–2.74, P<0.05), whereas mean cell volume at the fledging stage was significantly lower than mean cell volume at clutch completion (t82=3.13, P<0.025; Fig. 3A). For the comparison between diets, the interaction diet x reproductive stage was not significant (F2,48=0.14, P=0.871; Fig. 3C). After the interaction term was removed from the model, neither diet quality (F1,36=0.38, P=0.54) nor reproductive stage (F2,56=1.20, P=0.31) had a significant effect on variation in red blood cell volume. The between-individual and within-individual variance component estimates were 25.57±52.99 (Z=0.48, P=0.63) and 0.18±0.11 (Z=1.69, P=0.09), respectively.
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Effects of diet quality on reproductive output
On the high-quality diet (all females, N=29), mean egg size was
1.106±0.021 g, mean clutch size was 5.9±0.5 eggs and mean clutch
mass was 6.444±0.497 g. For those females that hatched chicks
(N=18; 62% hatching success), mean brood size at hatch was
3.1±0.3 chicks, and number of chicks fledged was 2.0±0.4.
Correlational analyses showed no relationship between hematological parameters
measured at pre-breeding, 1-egg or clutch completion stages and mean egg size,
clutch size or brood size at hatch and fledging stages (P>0.05 in
all cases), and no relationship between the change in hematological parameters
from pre-breeding to the 1-egg stage or from 1-egg to clutch completion stage
and reproductive traits (P>0.05 in all cases). Diet quality
significantly influenced reproductive output: females on the high-quality diet
produced larger clutches with larger mean egg mass
(Table 3). There was no
correlation between reproductive output and hematological parameters at any
stage or the change in these parameters across stages on either the high- or
low-quality diet (P>0.05 in all cases).
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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6%), red blood cell counts (8%), and plasma hemoglobin
concentration (9%) during egg production, even with a high-quality ad
libitum diet. It is likely that the initial decrease in these traits
involves hemodilution given the marked changes in plasma yolk precursor,
calcium and water balance at onset of laying
(Morton, 1994;
Reynolds and Waldron, 1999;
Salvante and Williams, 2002).
However, our results provide strong support for the hypothesis that
erythropoiesis is transiently suppressed during egg-laying and that the
recovery from anemia is relatively long-lasting, extending through incubation
and hatching periods. Decreased hematocrit, red blood cell counts and
hemoglobin concentration did not recover at clutch completion, but showed
evidence of recovery to baseline pre-breeding levels at hatching, consistent
with the estimated timescale for regenerative erythropoiesis following
experimentally induced anemia [7–14 days
(Domm and Taber, 1946;
Clark et al., 1988)]. More
importantly, there was significant time-dependent variation in (1) the
proportion of reticulocytes, which increased at clutch completion but peaked
at hatching 10–12 days after clutch completion, and (2) in mean red
blood cell volume, which showed a significant increase at clutch completion.
This is consistent with enhanced production and release of larger immature
cells into the circulation following suppression of erythropoiesis. Finally,
we found no evidence that development of anemia associated with egg production
was affected by diet quality, i.e. exogenous lipid and protein resources
available to the laying female.
Of note, there were two key differences between experiments regarding
temporal variation in hematological traits. In the analysis of temporal
variation across the entire reproductive cycle, we found that there was no
significant difference in mean hemoglobin concentration and reticulocyte
counts between pre-breeding, 1-egg and clutch completion stages, whereas in
the diet quality experiment, we found that these traits varied significantly
across these stages. Inconsistencies between the two analyses can be
attributed to differences in sample size (i.e. not all individuals were
included in the diet quality experiment) as well as the significant degree of
intra-individual variation in hematological traits typical of this population
(see Wagner et al., 2008).
A reduction in hematocrit during egg production has been reported in
numerous avian species (reviewed by
Williams et al., 2004a;
Wagner et al., 2008), ranging
from –1.5% in the great tit (Horak
et al., 1998a) to –10% in the red-billed quelea
(Jones, 1983) relative to
pre-breeding levels. Furthermore, Kalmbach et al.
(Kalmbach et al., 2004)
concluded that the extent of anemia was proportional to egg laying effort. The
magnitude of this change in hematocrit is comparable to putative `adaptive'
adjustments of hematocrit proposed to facilitate oxygen uptake and transfer
during periods of intense metabolic activity (reviewed by
Saino et al., 1997a;
Saino et al., 1997b). For
example, increased hematocrit (range 1–20%) is associated with
acclimatization to cold temperatures
(Kubena et al., 1972;
Carey and Morton, 1976), low
oxygen partial pressures (Jaeger and
McGrath, 1974; Keys et al.,
1986; Prats et al.,
1996), endurance exercise or migration
(Palomeque and Planas, 1978;
Soler et al., 1999;
Landys-Ciannelli et al., 2002)
and experimentally elevated flight costs
(Saino et al., 1997a;
Cuervo and De Ayala, 2005).
This supports that changes in hematocrit associated with egg production
(6% decrease) are biologically relevant and could potentially have a
negative impact on future reproduction via effects on oxygen carrying
capacity and aerobic capacity (Williams et
al., 2004a; Williams,
2005).
Reticulocytosis, the enhanced release of reticulocytes from the bone marrow
due to stimulatory effects of the hormone erythropoietin, is considered the
best single indicator of intensified erythropoiesis in response to the tissue
hypoxia associated with anemia (Fernandez
and Grindem, 2006). Although the proportions of reticulocytes
present at the prebreeding and 1-egg stages in our study were within the
reference range reported for small birds (10%)
(Campbell and Ellis, 2007), we
noted an approximate 5–7% increase in the proportion of reticulocytes
during later breeding stages (clutch completion and hatching) that is
consistent with the time-course of the reticulocyte response to anemia.
Reticulocytosis is initially observed 2–4 days after induction of anemia
(i.e. suppression of red blood cell production or blood loss), typically peaks
at 4–7 days, and gradually declines within 2–3 weeks of the
original insult (Fernandez and Grindem,
2006). To our knowledge, our study is the first to examine changes
in proportion of reticulocytes across the reproductive cycle, although
previous studies have investigated changes in the hematopoietic organs in this
context (reviewed by Kendall,
1995; Kalmbach et al.,
2004). For example, Jones
(Jones, 1983) documented that
the thymus became enlarged and actively produced red blood cells in the
red-billed quelea (Quelea quelea) during incubation, and suggested
that this functioned to augment erythropoiesis in the bone marrow and
compensate for anemia during egg-laying. The changes observed in mean red
blood cell volume in the present study were also consistent with the
hypothesis that erythropoiesis is suppressed during egg production. During
initial stages of egg-laying, estrogen would inhibit the production of new red
blood cells, but existing red blood cells would mature and splenic degradation
of senescent erythrocytes would presumably continue
(John, 1994). At clutch
completion the decrease in plasma estrogen to non-breeding levels
(Williams et al., 2004b;
Williams et al., 2005) would
release erythropoiesis from inhibition. This would result in a higher
proportion of reticulocytes in the plasma, which are larger due to less
condensed chromatin (Campbell and Ellis,
2007). In other words, fewer red blood cells would be present at
clutch completion, but a greater proportion of these cells would be of larger
and mean cellular volume would increase; again, consistent with a regenerative
response to anemia (Fernandez and Grindem,
2006). It is possible that recovery from anemia, with enhanced and
sustained levels of erythropoiesis during incubation, is regulated by high
plasma prolactin levels that are present during incubation
(Sockman et al., 2006), as
prolactin promotes erythropoiesis in mice
(Jepson and Lowenstein, 1964),
and has been characterized as a hematopoietic growth factor
(Constantinescu et al., 1999;
Welniak et al., 2001).
In the present study, we found evidence for a further reduction in
hematocrit and hemoglobin concentration at the end of the chick-rearing period
(fledging), though at a lower level than that seen during laying. In general,
reductions in hematocrit during chick rearing have been attributed to a
decline in body condition of provisioning parents and/or reallocation of
resources to meet increased energetic demands of chick rearing
(Morton, 1994;
Pap, 2002), although these
correlative results contrast with the fact that hematocrit is positively
correlated with experimentally enlarged brood sizes in the great tit
(Horak et al., 1998b).
Although the energetic demands of chick rearing are presumably moderated in
captive species (e.g. absence of predators, reduced foraging costs, ad
libitum food), metabolic rate is still significantly higher in
captive-breeding zebra finches during chick rearing compared with pre-breeding
or incubating birds (Vezina et al.,
2006). However, an alternate explanation in our study is that the
decrease in hematocrit at fledging may have been due to a second phase of
estrogen-dependent anemia and suppression of eythropoiesis in females
preparing to lay a second clutch (i.e. re-laying). Data from a follow-up study
(E.C.W. and T.D.W., unpublished data) support this: hematocrit did not
decrease linearly throughout the chick-rearing period as would be expected if
the energetic demands of chick provisioning were negatively impacting maternal
body condition. Rather, hematocrit remains at a constant level during chick
rearing, and does not show a decrease until the end of the fledging period,
which is when females would be initiating egg production for a second clutch
(E.C.W. and T.D.W., unpublished data).
We predicted that if reproductive anemia functioned to allow reallocation
of nutrients or energy to egg production, then hematological changes should be
more marked in females bred on a protein- and lipid-deficient diet, where the
demand for endogenous reserves would presumably be greater. Although there was
a significant interaction between diet quality and reproductive stage in the
overall model for hematocrit, we could not detect any statistically
significant differences in mean hematocrit in relation to diet quality within
any breeding stage, or in the change in hematocrit between breeding
across stages. In addition, diet quality had no effect on hemoglobin
concentration or red blood cell number although these traits also decreased
significantly during egg-laying. Therefore, we found little evidence to
support that diet quality affected the development or extent of anemia during
egg production. To our knowledge, no other studies have examined whether diet
quality (e.g. protein and lipid content) has an effect on the degree of change
in hematological parameters during egg production, although effects of diet
restriction on hematology have been investigated in commercial species
(Kubena et al., 1972;
Garcia et al., 1986;
Maxwell et al., 1990). Food
restriction is a common practice for improving biological and economical
performance in the domestic fowl, and only severe food restriction programs
cause hematological parameters to deviate from the `normal' range
(Maxwell et al., 1990).
However, there is another possible explanation for the apparent lack of effect
of resource availability on hematological traits in our study: diet quality
did have a significant effect on primary reproductive effort. Females on the
high-quality diet produced bigger clutches of larger eggs, and these eggs were
likely of better quality (as indicated by the low hatching success rate for
eggs laid on the low-quality diet). This suggests that when diet quality was
poor, egg-laying females might have traded-off reproduction for hematological
status, maintaining hematocrit, hemoglobin and red blood cell number at some
minimum functional level at a potential cost of reduced reproductive
investment. Our body mass data support this idea: although on the high-quality
diet females were significantly heavier at the 1-egg stage, there was no
difference in mean body mass at clutch completion between trials, suggesting
that females were not in poorer condition at cessation of laying on the
low-quality diet. Maintenance of erythropoiesis at the cost of reduced
reproductive output and egg/offspring viability has been demonstrated
experimentally in egg-laying Japanese quail fed an iron-deficient diet
(Garcia et al., 1986). In
comparison to control females, when iron-deficient quail were provided with a
radio-labeled iron supplement, a significantly higher amount of labeled iron
was detected in the hematopoietic organs (i.e. bone marrow, liver, spleen) and
erythrocytes, but no difference was found in amount of labeled iron found in
the oviduct or eggs (Garcia et al.,
1986). In other words, although iron deposited in the yolk is
essential for embryo development and postnatal survival, once iron was
provided to iron-deficient females it was preferentially diverted to restoring
red blood cell production and hematocrit levels in the laying female herself
and not to reproductive output (Garcia et
al., 1986).
In conclusion, our study demonstrates that transient suppression of
erythropoiesis and, subsequently, increased reticulocytosis are key components
of reproductive anemia in egg-laying females. Moreover, reticulocytosis, which
is a classic physiological response associated with `remission' or recovery
from anemia is prolonged in egg-laying females (see also
Kalmbach et al., 2004)
extending into incubation and chick rearing. Thus, reproductive anemia could
provide a physiological basis for cost of egg production underlying the
negative `carry-over' effects of increased egg production effort on subsequent
reproductive stages (i.e. incubation and chick rearing) that have been widely
reported in birds (e.g. Monaghan et al.,
1998; Nager et al.,
2001). Future work should focus on linking these physiological
changes in hematology to fitness, and confirming that this connectivity
operates via effects on oxygen-carrying capacity and aerobic
capacity, preferably using experimental manipulation of hematological
parameters in egg-laying females.
LIST OF ABBREVIATIONS
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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