General and Comparative Endocrinology 179 (2012) 400–405
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General and Comparative Endocrinology
journal homepage: www.elsevier.com/locate/ygcen
Holding water steroid hormones in the African cichlid fish Pseudocrenilabrus
multicolor victoriae
Caitlin N. Friesen a,⇑, Lauren J. Chapman a, Nadia Aubin-Horth b,⇑
a
b
Department of Biology, McGill University, 1205 Docteur Penfield, Montreal, QC, Canada H3A 1B1
Département de Biologie, Institut de Biologie Intégrative et des Systèmes, Université Laval, 1030 Avenue de la Médecine, Québec, PQ, Canada G1V 0A6
a r t i c l e
i n f o
Article history:
Received 21 September 2011
Revised 24 July 2012
Accepted 27 July 2012
Available online 26 September 2012
Keywords:
Cortisol
Estradiol
Testosterone
Non-invasive
Hormone validation
Freshwater fish
a b s t r a c t
Measuring hormone levels multiple times on the same individual across different life stages or treatments
can facilitate our understanding of hormonal regulation of physiological and behavioral events. The conventional method of hormone measurement requires blood sampling, which is potentially lethal to small
individuals. In fishes, there is an alternative non-invasive method of hormone measurement using the
release of hormones across gill membranes from blood into holding water. Validation of this method is
required to evaluate its application value to different species. In the present study we used the maternal
mouth-brooding African cichlid fish, Pseudocrenilabrus multicolor victoriae to (i) investigate whether handling involved in using the holding water technique is a stressor by measuring excreted cortisol in male
and female P. multicolor handled one or multiple times, (ii) validate use of this technique by quantifying
the relationship between plasma and holding water measures of sex hormones in male P. multicolor, and
(iii) demonstrate the biological relevance of this technique using excreted levels of sex hormones in female
P. multicolor across different reproductive stages. Excreted cortisol and estradiol levels did not differ
between fish handled one or more times, suggesting that the repeated sampling approach over the breeding
cycle that we propose to use does not affect the excreted level of the hormone of interest. Measurements
from plasma and holding water samples were positively related for both testosterone and estradiol, indicating that the holding water technique is a reliable index of plasma hormone levels. Excreted sex hormone
levels varied with reproductive state, suggesting that the technique is a useful, non-invasive measure of sex
hormone levels in P. multicolor.
Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction
Repeated measurement of hormones allows us to investigate
patterns and consequences of hormonal variation, and to link these
changes to key physiological and behavioral processes. Individual
hormone levels are highly variable, so performing repeated measurements on the same individual can reveal patterns of variation
that would otherwise be masked. This allows researchers to explore marked hormonal fluctuations within an individual in response to time of day, physiological state, life stage, and/or
experimental treatment. Repeated measurements of hormones
have been conducted in mammals [10,23,28], birds [2], reptiles
[17,38], and fishes [1,3,4,7,22,27,35]; however, detailed time
⇑ Corresponding authors. Fax: +1 514 398 5069 (C. Friesen), fax: +1 418 656 7176
(N. Aubin-Horth).
E-mail addresses: [email protected] (C.N. Friesen), [email protected] (N. Aubin-Horth).
URLs:
http://biology.mcgill.ca/grad/caitlin/index.html
(C.N.
Friesen),
http://wikiaubinhorth.ibis.ulaval.ca/Main_Page (N. Aubin-Horth).
0016-6480/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ygcen.2012.07.031
course studies of hormone levels are limited due to challenges involved in obtaining repeated measures from a single individual. In
particular, the conventional method of measuring hormone concentrations from blood samples can be highly invasive, or even
lethal, for small individuals.
In fishes, an alternative, non-invasive method of hormone measurement has been developed and is becoming more widespread.
The holding water technique uses the passive release of free steroids across the fish’s gills from their blood into the surrounding
holding water [9,30,36]. The concentration of free steroids in holding water using this technique has been positively related to levels
of plasma steroids in a number of fish species including Oncorhynchus mykiss [8,30], Gasterosteus aculeatus [32], Astatotilapia burtoni
[15], and Amatitlania nigrofasciata [37]. The amount of free steroids
released into water samples, however, can vary considerably based
on physiological state (e.g., breeding versus non-breeding, stressed
versus non-stressed), hormone type, or the species of fish studied.
Experimental manipulations (e.g., confinement and handling)
occurring during the holding water technique can significantly increase concentrations of the stress hormone cortisol in some fish
C.N. Friesen et al. / General and Comparative Endocrinology 179 (2012) 400–405
species [24,25], and elevated levels of cortisol over time may impair the production and thus release of androgens [5,21,32]. As a
result, the holding water technique requires validation for use with
a new species to ensure reliable estimates of plasma hormone concentrations from water samples.
The goal of this study was to investigate the utility of the holding
water technique to measure the concentration of the sex hormones
testosterone and estradiol in the African cichlid, Pseudocrenilabrus
multicolor victoriae, a small, mouth-brooding haplochromine cichlid
found throughout the Lake Victoria basin of East Africa [11]. To
achieve this goal we addressed three issues: (i) stress effects of
experimental manipulations (handling and holding), (ii) technical
validation of the procedure, and (iii) biological validation. Experimental manipulations were investigated as a potential stressor by
measuring excreted levels of the stress hormone cortisol in males
and females that had experienced a holding water trial one or more
times prior to a final hormone sampling event. The technical validation quantified the relationship between waterborne levels and
plasma levels of sex hormones by measuring testosterone and estradiol in holding water and plasma samples from individual males that
were large enough to secure adequate plasma. Finally, the biological
validation examined the link between holding water hormones and
reproductive status by measuring excreted levels of the sex hormones testosterone and estradiol in females at different reproductive stages (pre-brooding, mid-brooding, and post-brooding).
2. Methods
2.1. Study organism
Male and female specimens of P. multicolor were live-captured
using minnow traps in the Lake Nabugabo region of Uganda, under
a Uganda National Council for Science and Technology Research
Permit. Specimens were live transferred to McGill University, Canada, and housed in 30-liter aquaria under conditions similar to
their natural environment (25 °C ± 0.2, 12L:12D). All fish were
fed flake food once daily in the morning, and the brooding status
of all females was assessed via visual inspection. All fish were handled in accordance with the guidelines of the Canadian Council on
Animal Care as administered by McGill University (McGill Animal
Care Protocol #5029).
2.2. Assay controls
To verify that no background hormones were present in our
holding water samples, negative controls were made by creating a
blank sample consisting only of clean unused aquarium water. This
water had the same composition as the fish tank water but was
taken from a reservoir that had never contained fish. To assess the
recovery rate of our steroid hormone extraction method combined
with the holding water technique, positive controls were made by
spiking water samples with known amounts of steroid hormone
and analyzing the recovery rate of the procedure. To assess consistency of our hormone measurements, inter- and intra-assay positive
controls were made by collecting holding water samples from 18 females at various reproductive states and combining these samples
to create pooled controls. The pooled samples were processed as described below then divided into aliquots and assayed at the start
and end of each EIA plate to determine the intra- and inter-assay
variability for each hormone.
2.3. Validation experiments
In this study, we conducted three separate experiments to validate the holding water technique of steroid measurement when
401
working with a new species in the laboratory, as recommended
by Scott et al. [31]. This included: an experiment to detect manipulation effects, a technical validation, and a biological validation.
2.3.1. Experimental manipulation effects
To determine whether repeated manipulations was a confounding factor in our experiment, we resampled individual fish and measured excreted stress and sex hormone levels (cortisol and estradiol,
respectively) at the start (day 1) and end (day 7) of a 1-week experiment with 15 adult male and female P. multicolor (2.39 ± 0.13 g
body mass) exposed to 1, 4, or 7 experimental manipulation events
(holding water trials) prior to final hormone sampling. Each manipulation event consisted of catching an individual fish from its tank
using a handheld net, placing the fish in a 400-mL beaker filled with
200 mL of water for 30 min, then returning the fish to its tank. If repeated handling is more stressful than testing P. multicolor only
once, we would expect to see an increase in average cortisol excretion rates in treatment groups exposed to 4–7 experimental manipulations relative to treatment groups exposed to only 1
manipulation. If the fish acclimate to the procedure, we predict a decline in cortisol levels with the increasing number of manipulations.
Since elevated stress levels can suppress sex hormone levels
[5,21,32], we would also expect a decrease in the average estradiol
excretion rate in treatment groups exposed to 4 or 7 experimental
manipulations if the handling is a significant stressor relative to
treatment groups exposed to only 1.
2.3.2. Technical validation
Measurements of excreted hormone levels in holding water
were validated by testing for a significant correlation between
waterborne and plasma hormone samples for the studied species,
as recommended by Scott et al. [31]. Holding water and plasma
samples were collected from 22 male P. multicolor (6.82 ± 0.42 g
body mass) to investigate the relationship between plasma and
waterborne testosterone and estradiol levels. Plasma samples were
collected immediately after holding water samples on the same day.
Male P. multicolor often reach a larger size than females, so males
were used for these comparisons in order to obtain sufficient
amounts of plasma.
2.3.3. Biological validation
To assess the biological relevance of the holding water technique, we measured excreted testosterone and estradiol levels
across different brooding stages in 9 female P. multicolor
(3.20 ± 0.24 g body mass). At the end of the 22-week biological validation study, the holding water and plasma measures of testosterone and estradiol were collected. We measured excreted sex
hormone levels at three distinct stages: (i) pre-brooding, (ii) brooding (female holds eggs and/or fry in her mouth), and (iii) postbrooding (all eggs and/or fry have been released).
2.4. Hormone measurement
2.4.1. Sample collection
All samples were collected in the morning (09:00–12:00) to
avoid diurnal effects. Holding water samples were collected in
400-mL beakers filled will 200 mL of clean unused aquarium water.
All glassware was cleaned with detergent, rinsed with ethanol, and
autoclaved before use. Fish were removed from their aquarium and
placed in individual beakers for 30 min. After the 30-min sampling
period, water samples were filtered, transferred to polypropylene
bottles, and immediately frozen at 20 °C until extraction. Immediately following the holding water sample collection, specimens
were euthanized in a buffered 1 g/L MS-222 solution for 1 min,
blood samples were collected, and the body length and mass were
recorded. Blood samples were collected from the caudal vein of
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C.N. Friesen et al. / General and Comparative Endocrinology 179 (2012) 400–405
euthanized fish using heparinized capillary tubes, and then immediately centrifuged at 13 000 RPM for 10 min before the separated
plasma layer was frozen at 80 °C until extraction.
2.4.2. Sample preparation
Solid phase extraction (SPE) was used to extract the hormones
from holding water and plasma samples. The protocol was comparable to similar experiments in other species [15,37]. Water samples
were thawed overnight to room temperature, and plasma samples
were thawed 30 min and then added to 15 ml of Millipore filtered
water. Extraction of steroid hormones from water was performed
using a Sep-Pack Plus C18 cartridge (Waters#WAT020515) attached
to a 12 sample vacuum manifold (VWR#CABJ9400). Each SPE
cartridge was primed by flushing 6 ml of 100% methanol followed
by 4 ml of autoclaved distilled water through the cartridge. SaintGobain Tygon tubing was used to draw the sample into the SPE
cartridge. The cartridge was then removed from the vacuum manifold, the ends were covered with Parafilm, and it was stored at
20 °C until eluted. On the day of elution, cartridges were removed
from the freezer and maintained at room temperature for at least
30 min. Steroid hormones were then eluted from cartridges
mounted on the vacuum manifold into 13 100 mm glass vials
by inserting a glass pipette into the cartridge and flushing through
two 2-ml aliquots of ethyl acetate using vacuum pressure. The use
of ethyl acetate as an elution solvent facilitated the measurement
of only the free fractions of steroids from the gills, which has been
shown to be a proxy of ‘‘physiologically active’’ steroids present in
the plasma [30,31]. The eluted solvent was immediately dried under
a constant stream of nitrogen gas using an Evap-o-rac drying rack
(Cole-Parmer#01610–15) immersed in a 35 °C water bath. The dried
steroid pellet was stored at 20 °C until resuspension for enzyme
immune-assay (EIA).
2.4.3. Enzyme immuno-assay (EIA)
Waterborne and plasma concentrations of testosterone,
estradiol, and cortisol were determined in separate assays using
commercial EIA kits from Cayman Chemicals, Ann Arbor, Michigan
(Testosterone #582701.1–96, Estradiol #582251.1–96, Cortisol
#582121.1–96). The dried samples were resuspended in 100% ethanol (representing 5% of the total volume) and 0.5 mL of the commercial EIA buffer provided, then stored at 20 °C for one week or
less on average. Dried samples were resuspended with 100% ethanol
in the resuspension buffer to increase recovery of steroids from
dried eluates [19]. EIA kits were run according to the manufacturer
instructions, and samples were assayed in triplicate.
2.5. Statistical analysis
Hormone levels were log10-transformed to normalize the data.
Data are presented as untransformed means ± standard error of
the mean (SEM). To test for experimental manipulation effects, a repeated measures analysis of variance was used to detect changes in
hormone levels by day (within-subjects factor) and treatment
(between-subjects factor). A significant day effect indicated that
hormone levels differed between the start and end of the experiment, and comparisons among treatments indicated whether hormone levels varied based on the frequency of experimental
manipulations (treatments included 1, 4, or 7 manipulations over
the course of one week). The Huynh–Feldt correction was used
when the assumption of sphericity was not met for the repeated
measures analysis. To analyze the results of our technical validation,
a linear regression was used to determine the relationship between
waterborne and plasma sex hormone concentrations. Finally, to
analyze the results of the biological validation, a repeated measures
analysis of variance was conducted to compare hormone levels
among reproductive stages (within-subjects factor). All analyses
were performed using SPSS Statistics 17.0 software with a level of
significance (a) of 0.05.
3. Results
3.1. Assay controls
Blank samples were included as negative controls to detect nonspecific binding of the antibody. Detected levels of cortisol, testosterone, and estradiol in blank samples were lower than all holding
water samples with the exception of two samples where the hormone levels were indistinguishable from baseline. Spiked samples
were included as positive controls to examine recovery rates of
added hormones. The average recovery rate for spiked samples
was generally quite low (testosterone: 22%, estradiol: 39%) but
consistent, and the coefficients of variation (CV) among spike replicates were 30% (testosterone, n = 4) and 19% (estradiol, n = 20).
The maximum acceptable CV among sample triplicates was considered to be 20%. In the few cases where the sample triplicate
CV exceeded 20%, the outlier of the triplicate was removed, and
the CV returned to less than 20%. The average CV among sample
triplicates was 7.5% (testosterone), 7.0% (estradiol), and 9.7% (cortisol) per plate when outlier samples were removed. The average
CV among sample triplicates was 8.6% (testosterone), 8.6% (estradiol), and 18.1% (cortisol) per plate when all samples (including
outliers) were considered. Pooled samples were included at the
start and end of each plate to determine inter- and intra-assay variability. Inter-assay variability was acceptable for all hormones
tested (testosterone: 14% CV, estradiol: 20% CV, cortisol: 10% CV),
as was intra-assay variability (within-plate sample coefficient of
variation averaged 17% for testosterone, 14% for estradiol, and
12% for cortisol). All holding water measures of hormones are presented as pg of hormone per ml of hormone collection water per
hour, and hormone values were not corrected for recovery rate.
3.2. Experimental manipulation effects
Repeated measures analysis of variance was used to detect
changes in hormone levels by day (within-subjects factor) and
treatment (between-subjects factor). All hormone measurements
were made using holding water samples because the mean body
size of specimens used in this particular study was too small to permit the measurement of hormones from blood. Repeated measures
analysis of variance detected no significant effect of day (F = 0.01,
df = 1,7, P = 0.94) or treatment (F = 0.84, df = 2,7, P = 0.47) on cortisol
levels, and no significant effect of day (F = 0.13, df = 1,7, P = 0.73) or
treatment (F = 0.41, df = 2,7, P = 0.68) on estradiol levels in P. multicolor. Body mass was also included in the analysis as a covariate and
was found to have no significant effect on either hormone. Overall
excretion rates averaged 1.79 ± 0.26 pg/ml/hr for cortisol and
1.63 ± 0.38 pg/ml/h for estradiol (Fig. 1).
3.3. Technique validation
Linear regression indicated a strong relationship between waterborne and plasma measures of both testosterone (R2 = 0.86, P < 0.01,
Fig. 2a) and estradiol (R2 = 0.65, P < 0.01, Fig. 2b) levels in male P.
multicolor. Plasma concentrations of testosterone averaged 61.47 ±
18.21 ng/ml (61474 ± 18209 pg/ml), whereas excretion rates of
waterborne testosterone averaged 104.48 ± 55.78 pg/ml/h. Plasma
concentrations of estradiol averaged 5.77 ± 1.17 ng/ml (5767 ±
1171 pg/ml), whereas excretion rates of waterborne estradiol averaged 1.57 ± 0.26 pg/ml/h. Plasma concentrations of testosterone
ranged from 560.5–381057.0 pg/mL, and plasma concentrations of
estradiol ranged from 334.0–20620.0 pg/mL. The detection limits
C.N. Friesen et al. / General and Comparative Endocrinology 179 (2012) 400–405
Fig. 1. Repeated measures of hormone levels measured at the start (day 1) and end
(day 7) of a 1-week experiment from individual P. multicolor in treatment groups
exposed to different frequencies of experimental manipulations. Handled (n = 5),
intermediate (n = 4), and naïve (n = 3) treatment groups were exposed to 7, 4, or 1
(respectively) experimental manipulation events prior to final hormone sampling.
Hormone levels were log10-transformed before analyses; means ± SEM are shown.
of the EIA kits used were 6 pg/ml and 20 pg/ml for testosterone and
estradiol, respectively.
3.4. Biological validation
Repeated measures analysis of variance indicated a significant effect of reproductive stage (within-subjects factor) on estradiol
(F = 6.75, df = 1.03, 8.22, P = 0.03) but not testosterone (F = 2.66,
df = 2, 16, P = 0.10). The overall trend of hormone levels across the
reproductive cycle was similar for testosterone and estradiol. Excretion rates of testosterone in females averaged 1.24 ± 0.37 pg/ml/h
during the pre-brooding stage, and then fell to 0.66 ± 0.06 pg/ml/h
during the brooding stage and 0.54 ± 0.08 pg/ml/h during the postbrooding stage (Fig. 3a). Excretion rates of estradiol in females averaged 3.52 ± 1.19 pg/ml/h during the pre-brooding stage, and then
fell to 0.41 ± 0.06 pg/ml/hr during the brooding stage and 0.51 ±
0.06 pg/ml/hr during the post brooding stage (Fig. 3b).
4. Discussion
The results of our study support the use of the water holding
technique with commercial EIAs as a noninvasive approach for steroid hormone measurement in P. multicolor. Early studies to detect
the release of fish hormones into holding water date back to the
1980s [33]. Since then, subsequent studies have shown that male
and female fish of various species release a wide range of steroid hor-
403
Fig. 2. (a) Testosterone and (b) estradiol levels in male P. multicolor (n = 22)
measured from holding water and plasma samples collected 30 min apart on the
same day. Hormone levels were log10-transformed before analyses. There is a
positive relationship between hormone levels recorded in water and plasma
samples for both testosterone (R2 = 0.86, P < 0.01) and estradiol (R2 = 0.65, P < 0.01).
Dotted lines represent average values of holding water hormone measures and the
solid line represents the corresponding plasma hormone level in pre- and postbrooding female P. multicolor (data presented in Fig. 3).
mones into their holding water [8,13]. Only a few studies have
obtained plasma and water measures from the same individuals at
the same time, but in such cases, a positive relationship has been observed for stress hormones such as cortisol [8,30,32,37] and sex hormones [12,15,32]. In our study, we found that waterborne sex
hormone levels of testosterone and estradiol correlated significantly
with plasma samples collected from the same individuals. Further,
we found no statistical effect on either stress or sex hormones (cortisol and estradiol) related to the experimental manipulations involved in the repeated collection of holding water samples. A
special concern when measuring excreted hormone levels from
fish-holding water is the measurement of cortisol levels, as the holding water sampling procedure may itself be a stressful event to the
test animals [37]. Elevated cortisol levels can inhibit digestion, energy storage, growth, and reproduction [6]. The cortisol excretion
levels observed in our experiment did not suggest any detectable effects of our experimental manipulations (handling and holding) on
P. multicolor. Cortisol levels also suggest no effect of repeating the
measurements over several days for the same animal, which is a major benefit of the holding water hormone method. It should be noted,
that our study assumes that holding water measurements of cortisol
are positively related to plasma measurements of cortisol in P. multicolor, based on documented correlations between waterborne and
plasma levels of cortisol in another species of cichlid [37]. Based on
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C.N. Friesen et al. / General and Comparative Endocrinology 179 (2012) 400–405
did not detect some females at the very early stages of brooding,
due to efforts to only detect brooders via visual observations,
which may have contributed to variance in hormone levels within
a stage.
The results of our experiment are only correlational and do not
provide any evidence concerning the causal relationships between
sex hormones and reproductive status, however, our findings support the hypothesis that sex hormones play a mediating role in
reproductive development. A correlation between reproductive
status and estradiol levels, characterized by a surge of estradiol
and testosterone as the individual approaches spawning followed
by a drop in concentrations post spawning, occurs in a number
of species of fish including: Oncorhynchus rhodurus [14], Salmo trutta [16], Cyprinus carpio [18], Salmo gairdneri [29], and Oreochromis
niloticus [34]. In female teleosts, estradiol has been shown to regulate the reproductive cycle by stimulating synthesis of the yolk
protein vitellogenin [20], which is critical for growing oocytes.
Our results measuring hormone levels during the reproductive cycle are in accordance with these observations. While most previous
research has been done using plasma samples, the increasing evidence that waterborne hormone levels provide indirect measures
of plasma hormone concentrations makes the holding water technique a powerful tool for continued research examining these complex relationships. Taken together, these results support the use of
the holding water technique to estimate plasma hormone concentrations in P. multicolor and open the door for careful studies of
hormone dynamics in individuals during reproductive cycling.
4.1. Conclusions
Fig. 3. Excreted (a) testosterone and (b) estradiol levels measured in female P.
multicolor (n = 14) across different reproductive stages. Hormone levels were log10transformed before analyses, means ± SEM are shown.
this assumption, our results suggest that the circulating levels of the
hormone associated with handling stress (cortisol) are not higher in
repeatedly handled fish. Our results also suggest, along with our
finding that waterborne and plasma levels of estradiol are positively
correlated in P. multicolor, that estradiol levels did not differ between
fish handled 1, 4, or 7 times prior to hormone sampling.
Our biological validation indicated a significant change in the
levels of estradiol across different reproductive stages of P. multicolor, providing additional support for the use of the holding water
technique and thus allowing us to study for the first time the
reproductive biology of female P. multicolor. In our observations
of P. multicolor throughout the reproductive cycle of multiple females, females brooded their eggs and hatched embryos for an
average of 17 days before releasing free swimming fry. The reproductive cycle was divided between three distinct periods: pre-,
mid-, and post-brooding based on the date that the female was
first observed with young in her mouth. Holding water measures
of testosterone and estradiol both exhibited peak levels of hormones during the pre-brooding phase, followed by a significant
drop in sex hormone levels during mid- and post-brooding. The
average levels of testosterone and estradiol measured from holding
water samples in our biological validation were comparable to
plasma concentrations reported in other studies [15] after using
the regression equation from our technique validation to calculate
holding water measures into plasma. Other studies have observed
a longer brooding period in P. multicolor [26]. It is possible that we
We validated a non-invasive technique of hormone measurement using commercial EIAs for use with the African cichlid, P. multicolor victoriae. We explored the effect of repeated holding water
sampling on stress and sex hormones (cortisol and estradiol,
respectively). We found no evidence that experimental manipulations during the holding water technique affect cortisol or estradiol
levels in P. multicolor, since fish experiencing repeated handling
events were characterized by similar excreted estradiol and cortisol levels to those measured in fish experiencing a single-handling
event prior to hormone sampling. Thus, our study suggests that the
repeated confinement of P. multicolor females across the reproductive cycle does not impair the collection of within-subject repeated
hormonal measures. We also found that the levels of sex hormones
(testosterone and estradiol) showed positive correlations between
water and plasma samples collected from the same individuals. Finally, we found a biologically relevant association between mean
excretion rates of estradiol and the brooding status of female P.
multicolor. Our results add to a growing body of literature supporting the use of the holding water technique as a non-invasive, reliable indicator of plasma hormone levels that enables repeated
measurement of hormones and aids in our understanding of the
complex interrelationship between reproductive physiology and
behavior.
Acknowledgments
Thank you to M.G. Abwooli, E. Amooti, C.A. Chapman, S. Gray,
S. Keller, S. Morgan, P. Omeja, and D. Twinomugisha for assistance
with field work. Care of experimental animals was aided by M. Bieber, J. Hunter, and L. Grigoryeva, and sample collection was assisted
by T. Molton, C. Walsh, and T. Tran. Thank you also to S. Cloutier and
M. Cuddy for their assistance modifying validation protocols and to
Cayman Chemicals for technical assistance with EIA plates. Funding
for this research was provided by a NSERC Discovery Grant and Canada Research Chair funds to L.J. Chapman, a NSERC Discovery Grant
C.N. Friesen et al. / General and Comparative Endocrinology 179 (2012) 400–405
to N. Aubin-Horth, as well as a NSERC CGS Grant and ALIS Grant to
C.N. Friesen.
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