Zoo Biology 18:247–260 (1999)

© 1999 Wiley-Liss, Inc.

A Comparison of Sex Steroid HormoneExcretion and Metabolism by PsittacineSpeciesJulian Lee, 1 Lisa Tell, 1* and Bill Lasley 2

1Department of Medicine and Epidemiology, School of Veterinary Medicine, University ofCalifornia, Davis, Davis, California

2Department of Population Health and Reproduction, School of Veterinary Medicine,University of California, Davis, Davis, California

The metabolism, excretory rates, and excretory patterns of carbon 14 (14C)radiolabeled estradiol (E2) and testosterone (T) were studied in female bud-gerigars (Melopsittacus undulatus) and orange-winged Amazon parrots (Ama-zona amazonica). Radiolabeled E2 and T were injected intramuscularly intosix budgerigars and two orange-winged Amazon parrots. Serial fecal/urinesamples were collected for 168 h post-radiolabel injection. Peak radiolabeledE2 excretion was observed at 4 h post-injection, and by 24 h, 93.3 ± 6.3 and65.9% (range, 59.1–72.7%) of the injected radiolabel was recovered in thefecal/urine matter of budgerigars and orange-winged Amazon parrots, respec-tively. Similarly, peak radiolabeled T excretion was observed at 4 h post-injection with 92.7 ± 3.6 and 66.2% (range, 57.5–75.2%) of the injectedradiolabel recovered in the fecal/urine matter by 24 h in the budgerigars andorange-winged Amazon parrots, respectively. High-performance liquid chro-matography (HPLC) analysis of the fecal/urine material revealed that bothparrot species excreted >80% of the radiolabel in the form of complex ste-roid conjugates. Immunoreactive E2 and T metabolites were detected usingestrone (E1) and C-21/C-19 conjugate enzyme immunoassays, respectively.Hydrolysis of the E2 metabolites and HPLC analysis of the ether extractsrevealed that E2 and E1 were the major steroid moieties. Hydrolysis of the Tmetabolites and HPLC analysis of the ether extracts revealed two and threemajor unconjugated peaks for the budgerigars and the orange-winged Ama-zon parrots, respectively. Zoo Biol 18:247–260, 1999. © 1999 Wiley-Liss, Inc.

Key words: fecal steroids; estradiol; testosterone; Amazon parrot; budgerigar

*Correspondence to: Dr. Lisa Tell, University of California, Davis, VM: Medicine and Epidemiology,Tupper Hall, Room 2108, Davis, CA 95616-8737. E-mail: latell@ucdavis.edu

Received for publication July 1997; revision accepted August 26, 1999.

248 Lee et al.

INTRODUCTION

In the past, gonadal activity in species of birds other than psittacines wascharacterized by measuring plasma hormone levels [Korenbrot et al., 1974;Temple, 1974; Wingfield and Farner, 1975; Cockrem and Seddon, 1994]. Re-cently, the use of serial fecal steroid analysis has been shown to provide an accu-rate assessment of gonadal function in domestic birds [Bishop and Hall, 1991;Cockrem and Rounce, 1994; Ishii et al., 1994]. This technique has also beenapplied to captive [Bercovitz, 1982; Ishii et al., 1994; Lee et al., 1995] and free-ranging [Kofuji et al., 1993; Cockrem and Rounce, 1995] non-domestic avianspecies. Furthermore, solubilization of avian feces has been shown to be a simpletechnique for providing material that can be analyzed by either radioimmunoas-says [Kanda et al., 1988; Cockrem and Rounce, 1994] or enzyme immunoassays[Lee et al., 1995; Tell, 1997]. These reports provide strong evidence that solubi-lization of avian feces is a simple sample-processing technique that is economi-cal (organic solvents for steroid extraction are not needed) and environmentallyfriendly and provides information regarding daily changes in ovarian functionfor a wide range of avian species.

The two major steroid hormones that are most commonly used for monitor-ing avian gonadal cycles have been estrogen and testerone (T). Increases in es-trogen metabolites reflect ovarian recrudescence [Cockrem and Rounce, 1994].Since T is a precursor of estradiol (E2), elevated T may reflect an overall in-crease in steroidogenesis in female birds. Increases in plasma [Wingfield andFarner, 1978, 1980] and fecal [Lee et al., 1995] T have been shown to coincidewith elevated plasma and fecal E2 levels in female white-crowned sparrows.

The order Psittaciformes is a diverse group of birds for which limited in-formation is available in terms of reproductive endocrinology. The majority ofavailable data is primarily in the form of fecal steroid metabolites that were mea-sured to identify gender [Czekala and Lasley, 1977; Bercovitz et al., 1979]. Onestudy characterized seasonal changes in the gonadal activity of a parrot; how-ever, this investigation was mostly informative for the male bird cycle [Cockremand Rounce, 1995]. Another report identified immunoreactive estrogens and an-drogens in hydrolzyed feces from four species of parrots [Erb and Bercovitz,1981]. Recently, a more comprehensive study was performed in which the timecourse of steroid excretion was identified, the hormonal metabolites were char-acterized, and aqueous solubilization of fecal/urine matter as a means for recov-ering the excreted metabolites of E2 and T was validated for one parrot species(the cockatiel) [Tell, 1997].

The primary objective of this study was to characterize the time course ofsex steroid excretion in female budgerigars (Melopsittacus undulatus) and or-ange-winged Amazon parrots (Amazona amazonica) to determine whether dailysex steroid production dynamics can be evaluated by daily fecal/urine monitor-ing. The second objective was to confirm that aqueous solubilization of fecal/urine matter recovers the excreted metabolites of E2 and T from fecal/urine mat-ter from these two species of parrots. The third goal of the study was to charac-terize the metabolites of E2 and T for these two parrot species so that applicabilityof specific enzyme immunoassays could be evaluated.

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MATERIALS AND METHODSAnimal Subjects

Six adult (age range, 1.25 ± 0.6 years) female budgerigars (Melopsittacusundulatus) and two adult female (older than 6 years) orange-winged Amazon parrots(Amazona amazonica) of proven fertility were used for this study. The birds wereheld in separate suspended cages in light-controlled rooms with a photoperiod of 10h of light:14 h of dark. A commercial pelleted diet (Roudybush maintenance crumbles;Roudybush, Sacramento, CA) and water were provided ad libitum. The birds weregiven complete physical examinations before initiating the study. The average bud-gerigar body weight was 44.2 ± 5.7 g and the orange-winged Amazon parrots weighed377 and 379 g, respectively. All procedures pertaining to this study were performedin accordance with a university-approved animal care and use protocol.

Injections

The injections for the budgerigars were prepared in 100 µL of vehicle (10%ethanol in 0.9% sterile saline) containing either 25 ng of unlabeled E2 or 50 ng ofunlabeled T as carrier. The injections for the orange-winged Amazon parrots werediluted in 600 µL of vehicle (10% ethanol in 0.9% sterile saline) containing either150 ng of unlabeled E2 or 300 ng of unlabeled T as carrier. Radiolabeled carbon 14(14C) E2 (45–60 mCi/mM, Dupont NEN Research Products, Boston, MA; 0.10 µCi)was injected intramuscularly into the pectoral musculature of budgerigars 1, 2, and3. Radiolabeled E2 (0.60 µCi) was injected intramuscularly into the pectoral muscu-lature of orange-winged Amazon parrot 1. Radiolabeled 14C-T (45–60 mCi/mM,Dupont NEN Research Products; 0.078 µCi) was injected intramuscularly into thepectoral musculature of budgerigars 4, 5, and 6. Radiolabeled 14C-T (0.45 µCi) wasinjected intramuscularly into the pectoral musculature of orange-winged Amazon par-rot 2. The same budgerigars and orange-winged Amazon parrots were re-injected onday 8 with the opposite hormone from the original injection (radiolabeled E2 plusunlabeled steroid for budgerigars 4, 5, and 6 and orange-winged Amazon parrot 2and radiolabeled T plus unlabeled steroid for budgerigars 1, 2, and 3 and orange-winged Amazon parrot 1). Before laboratory analysis, all collected samples werefrozen (–20°C) in borosilicate tubes without preservatives.

Sample Collection and Processing

Frozen fecal/urine samples were collected and prepared as previously described[Tell, 1997]. In brief, combined fecal/urine samples were collected from an aluminumfoil substrate for 24 h before injection with the radiolabeled hormones and every 4 hpost-injection for the first 72 h, then every 8 h for the next 96 h. The samples were driedat 58°C until a constant weight was achieved. Once constant weight was achieved, 0.1 Mphosphate buffer (pH = 7.0) with 0.1% bovine serum albumin was added to each sampleat a weight to volume ratio of 1:16 for the budgerigars and 1:8 for the orange-wingedAmazon parrots. The samples were vortexed, mixed vigorously for 24 h, then centri-fuged (828 g) at 5°C for 60 min. The buffer and pellet were separated and the radioactiv-ity measured using a liquid scintillation spectrometer (Tri-Carb 2000 Liquid ScintillationAnalyzer, Packard Instruments, Downers Grove, IL) with 15 mL of a scintillation cock-tail (Ultima gold, Packard Instruments). Samples were analyzed for radioactivity up to168 h in the buffer and up to 72 h in the pellet for both hormones.

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Ether Extraction

To determine the relative portion of conjugated versus free steroid in eachsample, 200 µL of supernatant from each phosphate buffer solubilized fecal/urinesample was combined with 100 µL of distilled water and extracted twice with fivevolumes of ethyl ether by vortexing for 1 min, snap freezing in a bath of methanol/dry ice, and decanting the unfrozen ether. The ether extract was air dried and recon-stituted with phosphate buffer. Aliquots of both the reconstituted ether extract andaqueous residual from the ether extraction were then analyzed by liquid scintillationspectrometry. The percentage of radioactivity within the ether extract or the residualaqueous phase was determined for both E2 and T.

Enzymatic Hydrolysis

To characterize further the conjugates found in the aqueous residual of the etherextracts, samples from each bird (n = 8) during the initial collection period (0–4 hpost-radiolabel injection) were subjected to enzymatic hydrolysis as described previ-ously [Tell, 1997]. In brief, 100 µL of the remaining aqueous residual from the etherextraction was transferred to a 12 × 75-mm test tube with 100 µL of citrate buffer atpH 5.3. Enzymatic hydrolysis was accomplished by addition of 20 µL of beta-glucu-ronidase/aryl sulfatase (Boehringer Mannheim, Mannheim, Germany) (5000 FishmanU/40,000 Roy U, respectively) and incubation of the mixture at 37°C for 24 h. Anestrone (E1) sulfate standard was included as a control. Hydrolyzed samples (0.2mL) were extracted twice with 1.0 mL of anhydrous ethyl ether each time. The etherand aqueous residual portions were collected into separate empty scintillation vials,scintillation fluid was added, and the counts obtained as described previously.

High-Performance Liquid Chromatography Analysis

Two different high-performance liquid chromatography (HPLC) techniques wereused to examine the steroid metabolites. A methanol/water mobile phase was used toseparate the steroid conjugates and an acetonitrile/water technique was used to sepa-rate the free steroids after the conjugates were hydrolyzed.

To verify that the radiolabel retained in the aqueous residue was an authenticsteroid or metabolic steroid by-products, samples from each bird (n = 8) from thefirst sampling interval (0–4 h post-radiolabel injection) were analyzed by reverse-phase HPLC as previously described [Tell, 1997]. In brief, 200 µL of the supernatantwas ether extracted as described previously. One hundred microliters of 20% metha-nol and 80% of the aqeous residual from the ether extracted sample was then sepa-rated by HPLC using a gradient reverse-phase system (methanol from 20 to 100%over 80 min, collecting separate 1.0-mL fractions each minute). Each of the 1.0-mLfractions was added to separate scintillation vials with 8 mL of scintillation fluid,vortexed, and counted. The resulting E2 profiles (n = 8) were compared to a sampleinjected with [3H]-E1 sulfate.

After the conjugated metabolites were analyzed, determination of the majorsteroid moieties of the E2 and T conjugates was performed by analyzing hydrolyzedaqueous residues from each bird by HPLC as previously described [Tell, 1997].Samples were obtained from tubes that demonstrated peak radioactivity after HPLCseparation of the aqueous residue after ether extraction. After HPLC separation, thesamples were dried and reconstituted in 100 µL of citrate buffer at pH 5.3, and 20

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µL of beta glucuronidase/aryl sulfatase (enzyme) was added to each sample. Themixture was capped, vortexed, and placed in a 37°C water bath for 24 h. The follow-ing day, the hydrolyzed samples (0.2 mL) were extracted twice with 1.0 mL of anhy-drous ethyl ether each time as described above. The ether and aqueous residualportions were separated by snap freezing, and the ether phase was dried overnightand reconstituted in 40% acetonitrile and 60% HPLC grade water. Column separa-tion was accomplished using an acetonitrile:water (40:60, vol/vol) solvent system.The ether phase was injected onto the column at a constant flow rate of 1 mL/min,and fractions at 0.3 mL/tube were collected and added to separate scintillation vialswith 8-mL scintillation fluid. The vials were vortexed and counted as previouslydescribed. Each of the resulting profiles was compared to those injected with [3H]-E1

and E2 or [3H]-androsterone or T.

Enzyme and Radioimmunoassay

Unprocessed solubilized fecal/urine aliquots from the first sampling period (0–4 h) were assayed by enzyme immunoassays for E1 conjugates (four budgerigars andtwo orange-winged Amazon parrots; E1C [Munro et al., 1991]) and androsterone-3-glucuronide (four budgerigars and two orange-winged Amazon parrots; A3G [Shideleret al., 1993]) immunoreactivity. The samples were serially diluted from 1:2 to 1:2,048 in distilled water and 20 µL of each dilution from each sample were taken tothe enzyme immunoassays. Serial dilutions of the same samples were also added toscintillation vials and disintegrations per minute counted as described above.

Since hydrolyzed aqueous phase samples from orange-winged Amazon parrotsexhibited radioactivity within the region of the T standard on HPLC, further analysiselucidating T immuno- or radioactivity was performed. One T sample from an or-ange-winged Amazon parrot was ether extracted, and the subsequent aqueous phasewas subjected to HPLC. The peak tubes from the HPLC analysis were analyzedusing a T-enzyme immunoassay [Lee et al., 1995]. Two T samples from orange-winged Amazon parrots were hydrolyzed and ether extracted, and the HPLC eluatesthat were within the region of the T standard were subjected to a T radioimmunoas-say (Diagnostic Products, Inc., Los Angeles, CA).

Data Analysis

The fecal/urine sample data and percentage of recoveries of the originally in-jected radiolabel for the budgerigars are presented as averages and standard devia-tions for the six birds studied. The percentage of recoveries of the originally injectedradiolabel for the orange-winged Amazon parrots are presented as averages and ranges.Standard deviations are not provided for the orange-winged Amazon parrots sinceonly two animal subjects were studied.

RESULTS

Animal Subjects

After injection of the radiolabeled hormone, all birds continued to eat and drinknormally. None of the birds displayed any evidence of health compromise as wasproven by a normal activity level, food consumption, fecal production, and stablebody weights. Fecal samples were reliably available for both the diurnal and noctur-nal collection periods.

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Radioactivity

Label Recovery and Time Course of Label Excretion

The average radiolabel distribution into the buffer and pellet (expressed as per-centage of recovery of the originally injected radiolabel) and time courses of radiola-beled hormone excretion after injection with radiolabeled E2 and T are displayed inFig. 1. In both species of birds, the radiolabel was found predominantly in the bufferfor both hormones. Radiolabeled E2 and T were first detected by 4-h post-radiolabelinjection, and that same sampling interval (0–4 h post-radiolabel injection) also rep-resented peak excretion. No radioactivity was detected by 16 h post-injection of ra-diolabeled E2. The T concentration started to decline by 8 h post-injection, and noradioactivity was detected by 20 h post-injection of radiolabeled T. In general, by 24h, the majority of the originally injected radiolabeled hormone was recovered fromboth species of birds for both hormones (average E2 recoveries were 93.3 ± 6.3 and65.9% [range, 59.1–72.7%] for the budgerigars and orange-winged Amazon parrots,respectively, and average T recoveries were 92.7 ± 3.6 and 66.2% [range, 27.5–75.2%] for the budgerigars and orange-winged Amazon parrots, respectively). After72 h, <0.5% of the injected radiolabel was detected in the pellet; therefore, no morepellet samples were analyzed. The average total recoveries (n = 6; 168 h of samplecollection) of the originally injected radiolabel from solubilized fecal/urine sampleswere 94.1 ± 5.4 and 93.9 ± 3.0% for radiolabeled E2 and T, respectively, for thebudgerigars. The average total recoveries (n = 2; 168 h of sample collection) of theoriginally injected radiolabel from solubilized fecal/urine samples were 67.6 (range,59.9–75.3%) and 66.5% (range, 57.9–75.2%) for radiolabeled E2 and T, respectivelyfor the orange-winged Amazon parrots.

Distribution of Free and Conjugated Metabolites/Enzymatic Hydrolysis

In the case of the budgerigars and the orange-winged Amazon parrots, the ex-creted radiolabel was found predominantly (>80%) in the aqueous residual (conju-gated forms) during peak periods of radioactivity excretion (0–4 h post-administrationof radiolabeled hormones) for both hormones.

Results from enzyme hydrolysis of samples collected during the first samplinginterval (0–4 h post-radiolabel injection) revealed an efficient conversion of the con-jugated metabolites to free steroid forms. The mean percentages, standard devia-tions, and ranges of radioactivity in the organic solvent or the aqueous residue beforeand after samples were hydrolyzed are reported in Table 1 for E2 and T treatmentsfor both parrot species. After enzymatic hydrolysis, the excreted label was foundpredominantly in the ether phase, indicating that the conjugated steroids were con-verted to free steroids by enzymatic hydrolysis (Table 1).

Identification of Fecal/Urine Sample Metabolites

High-Performance Liquid Chromatography

HPLC elution profiles of samples (0–4 h post-radiolabel injection) from bud-gerigars (n = 6) injected with radiolabeled E2 were consistent for all samples andshowed that the peak radioactivity was associated with a relatively polar steroid con-jugate moiety (Fig. 2A, fractions 21–30), which were consistent with samples spikedwith E1 sulfate (E1SO4, fractions 22–28). Subsequent HPLC analysis of hydrolyzedsamples (n = 6) revealed that E2 (Fig. 2B, fractions 14–23) and E1 (Fig. 2B, fractions

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Fig. 1. Average percentages ± standard deviations of originally injected radioactivity recovered from buffer (square) or pellet (triangle) of solubilizedfecal/urine samples collected every 4 h post-radiolabel hormone. The circle denotes total (pellet + buffer) percentage of radioactivity of originalinjectate recovered. A: 14C-E2 injection from six budgerigars. B: 14C-T injection from six budgerigars. C: 14C-E2 injection from two orange-wingedAmazon parrots. D: 14C-T injection from two orange-winged Amazon parrots.

254 Lee et al.

23–29 ) were the most likely free steroid moieties excreted by birds injected with E2.These fractions were consistent with samples spiked with E2 (fractions 18–22) andE1 (fractions 24–29).

HPLC elution profiles of samples (0–4 h post-radiolabel injection) from bud-gerigars (n = 6) injected with radiolabeled T were consistent for all samples andexhibited a series of three peaks that varied in magnitude and represented relativelypolar steroid conjugates (Fig. 3A, fractions 10–22, 22–29, 29–35). Subsequent HPLCanalysis of hydrolyzed samples from birds injected with T revealed two major peaks(Fig. 3B, fractions 6–12 and 12–22) and three to four minor peaks (Fig. 3B, fractions24–52). Samples spiked with radiolabeled T (fractions 23–28) or androsterone (frac-tions 56–62) had different elution patterns.

HPLC elution profiles of samples (0–4 h post-radiolabel injection) from theorange-winged Amazon parrots injected with radiolabeled E2 were variable for thetwo samples run. In general, peak radioactivity was associated with a relatively polarsteroid conjugate moiety that consisted of three peaks (Fig. 2C, fractions 4–14, 14–18, 18–30). The last peak was of greatest magnitude and was consistent with samplesspiked with E1 sulfate (fractions 22–28). Subsequent HPLC analysis of hydrolyzedsamples (n = 2) revealed that E2 (Fig. 2D, fractions 16–21) and E1 (Fig. 2D, fractions21–27) were the major free steroid moieties excreted by birds injected with E2. Thesefractions were consistent with samples spiked with E2 (fractions 18–22) and E1 (frac-tions 24–29).

HPLC elution profiles of samples (0–4 h post-radiolabel injection) from or-ange-winged Amazon parrots (n = 2) injected with radiolabeled T showed variabil-ity. In general, the birds had two major peaks (Fig. 3C, fractions 19–27 and 27–36)in addition to approximately 12 minor peaks (Fig. 3C). Subsequent HPLC analysisof hydrolyzed aqueous residue samples revealed three major peaks (Fig. 3D, frac-tions 7–18, 24–36, 33–47), of which the second was of greatest magnitude and wasconsistent with samples spiked with radiolabeled T (fractions 23–28). Samples spikedwith radiolabeled androsterone (fractions 56–62) had different elution patterns.

Enzyme and Radioimmunoassay

Serially diluted solubilized unprocessed fecal/urine samples (budgerigars andorange-winged Amazon parrots) from the first sampling interval (0–4 h post-radiola-

TABLE 1. Comparison of total percentage of radioactivity in non-hydrolyzed versus hydrolyzed(enzyme-treated) solubilized fecal/urine samples from six budgerigars and two orange-wingedAmazon parrots during the 0–4 post-radiolabel injection sampling period

Bird Non-hydrolyzed Hydrolyzed

species Hormone % Ether % Aqueous % Ether % Aqueous

Budgerigars E2 9.0 ± 7.2 91.0 ± 7.3 84.2 ± 4.7 15.8 ± 4.7Amazon parrots E2 6.7, 25.9 93.3, 74.1 51.3, 81.4 48.7, 18.6

Budgerigars T 10.3 ± 2.6 87.7 ± 2.5 65.28 ± 7.6 34.72 ± 7.6Amazon parrots T 14.4, 23.4 85.6, 76.6 41.9, 72.1 58.2, 27.9

Assay standard E1SO4 4 96 95 5

The budgerigar data are presented as average percentages ± standard deviations. The orange-wingedAmazon data are presented as individual values for the two birds treated.

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Fig. 2. HPLC (gradient system based on polarity) of the aqueous residue after ether extraction of a non-hydrolyzed solubilized fecal/urine samplecollected from a representative budgerigar (A) and orange-winged Amazon parrot (C) 0–4 h after 14C-E2 injection. The abscissa represents thefractions collected (1.0 mL/min). The E1SO4 standard eluted at fractions 22–28. HPLC (isocratic system) of the ether extract after ether extraction ofa hydrolyzed aqueous residue after HPLC analysis from a representative budgerigar (B) and orange-winged Amazon parrot (D) 0–4 h after 14C-E2

injection. The abscissa represents the fractions collected (0.3 mL/min). The E2 and E1 standards eluted at fractions 18–22 and 24–29, respectively.

256Lee et al.

Fig. 3. HPLC (gradient system based on polarity) of the aqueous residue aftger ether extraction of a non-hydrolyzed solubilized fecal/urine samplecollected from a representative budgerigar (A) and orange-winged Amazon parrot (C) 0–4 h after 14C-T injection. The abscissa represents thefractions collected (1.0 mL/min). HPLC (isocratic system) of the ether extract after ether extraction of a hydrolyzed aqueous residue after HPLCanalysis from a representative budgerigar (B) and orange-winged Amazon parrot (D) 0–4 h after 14C-T injection. The abscissa represents the fractionscollected (0.3 mL/min). The T and androsterone standards eluted at fractions 23–28 and 56–62, respectively.

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bel injection) exhibited parallelism between immunoreactivity, radioactivity, and stan-dards when assayed by an E1 conjugate enzyme immunoassay. In addition, serialdilutions of the crude solubilization, as well as eluates from at least four of the majorpeaks from the HPLC conjugated metabolites, exhibited immunoreactivity using anenzyme immunoassay that has been demonstrated to have cross-reactivity with bothC-21 and C-19 conjugates [Shideler et al., 1993]. This immunoreactivity was shownin samples from both budgerigars and orange-winged Amazon parrots. In the case ofthe samples from the orange-winged Amazon parrots, none of the multiple peaks ofconjugated T radiolabeled eluates from HPLC was immunoreactive as assessed byeither a T-enzyme immunoassay [Lee et al., 1995] or radioimmunoassay (DiagnosticProducts Inc., Los Angeles, CA), nor were the hydrolyzed ether extracted HPLCeluates that were within the region of the T standard immunoreactive in the T radio-immunoassay.

DISCUSSION

This study supports previous reports [Kanda et al., 1988; Cockrem and Rounce,1994; Lee et al., 1995; Tell, 1997] that aqueous extraction of hormones via solubili-zation of avian fecal/urine matter is a simple, economical, and environmentally su-perior laboratory technique for processing samples.

The results from this study demonstrated that steroid hormone excretion in par-rots is a very rapid and efficient process. Compared with the cockatiel [Tell, 1997]and other domestic avian species [Helton and Holmes, 1973; Holmes et al., 1974;Holmes and Slikker 1976], the budgerigars and orange-winged Amazon parrots ap-pear to be similar in metabolism and excretion of radiolabeled hormones. Both psit-tacine species appeared to metabolize the radiolabeled hormones as quickly as thecockatiel [Tell, 1997]. If the sampling periods had been more frequent, however,differences may have been distinguished between 0–4 hr post-radiolabel injection.The total radiolabel recovery efficiency appeared to be inversely proportional to thebody size of the birds (average percentages of 94.1 and 67.6% for budgerigars andAmazon parrots, respectively, for 0–168 h post-injection with radiolabeled E2, and93.9 and 66.5% for budgerigars and Amazon parrots, respectively, for radiolabeledT). This variability in radiolabel recovery efficiency is most likely attributable to thetype of feces that these birds produce and the difference in the overall body sizes forthe species studied. Budgerigars typically produce very small dry fecal samples,whereas the orange-winged Amazon parrots excrete fecal matter that has increasedviscosity and a higher moisture content. Therefore, when collecting the feces/urinefrom a smooth surface, complete collection is more efficient if the samples are drierand solid. The second variable that most likely affected the radiolablel recovery effi-ciency was the difference in the overall body sizes of the birds (budgerigar averageweight was 44 g and orange-winged Amazon parrot average weight 378 g). Sincethe orange-winged Amazon parrots had greater body mass and surface area, it wasassumed that the radiolabel distributed more widely into the body tissues, thus alarger percentage of the injected radiolabel remained unaccounted for.

Our HPLC data show that, similar to the cockatiel [Tell, 1997], the budgerigarand the orange-winged Amazon parrot excrete estrogen metabolites in the feces/urinethat are predominantly conjugated, appear to be E2 and E1 based, and exhibit immu-noreactivity when analyzed with an E1C enzyme immunoassay. Similarly, the T me-

258 Lee et al.

tabolites in the feces/urine of the budgerigar and orange-winged Amazon parrot werealso as described previously for the cockatiel [Tell, 1997] in which there were nu-merous metabolites before hydrolysis, and these metabolites showed immunoreac-tivity when analyzed with a C-21/C-19 conjugate enyme immunoassay. In the caseof the budgerigar, once these T metabolites were hydrolyzed, two polar peaks pre-dominated, as was found in the cockatiel [Tell, 1997]. The T metabolites from thefeces of the orange-winged Amazon parrots differed from the budgerigars and cock-atiels [Tell, 1997] in that once they were hydrolyzed, three major peaks were evidentand the second peak was of the greatest magnitude and consistent with the T stan-dard. Although this radioactive HPLC peak consistent with free T was found in theether extract of hydrolyzed fecal/urine samples, no authentic T could be identifiedby enzyme or radioimmunoassays in the aqueous phase of ether extracted samples orthe ether extracts from hydrolyzed samples. Therefore, in the case of the orange-winged Amazon parrots, it appears that the majority of the radiolabeled T was me-tabolized and conjugated, and neither the conjugates or the free steroids could bedetected by T enzyme or radioimmunoassays. Our data are consistent with the reportof Erb and Bercovitz [1981] in which multiple immunoreactive estrogens and andro-gens were identified in the hydrolyzed feces of four psittacine species using a HPLCmethod similar to that used in the present report. Although the immunoreactive com-pounds identified by Erb and Bercovitz [1981] were not known to be circulatingsteroidal estrogens or androgens, their work supported the concept that multiple ste-roid metabolites are formed before conjugation and excretion by psittacines.

In summary, the present data provide a characterization of the excretion patternof radiolabeled E2 and T in females of two psittacine species and provide a compari-son to data published for a third psittacine species, the cockatiel [Tell, 1997]. Theseresults show that steroid conjugates are the predominant metabolites for both sexsteroids and that the majority of the clearance occurs within 24 h. More important,this study supports the concept that aqueous extraction is an effective technique forprocessing avian fecal/urine samples and that conjugate assays are useful for mea-suring hormone levels. In addition, these findings identify the need to elucidate fur-ther the molecular structure of the sex steroid metabolites by providing newinformation regarding T metabolism in the orange-winged Amazon parrot. The presentdata suggest that some of the major metabolites of T in this species of bird are notdetected by some T immunoassays and that improvements could be made in moni-toring T with more specific assays.

CONCLUSIONS

1. E2 and T uptake, conjugation, and excretion are a rapid and efficient processin the budgerigar and the orange-winged Amazon parrot.

2. The rapid excretion of sex steroids in the budgerigar and orange-wingedAmazon parrot indicates that daily changes in E2 and T production are reflected inmeasurements from daily fecal/urine samples.

3. Solubilization of fecal/urine samples is a simple and effective way of pre-paring samples for subsequent measurement of steroid sex hormone concentrations.

4. In both the budgerigars and the orange-winged Amazon parrots, the majority

Steroid Excretion in Psittacines 259

of the radiolabeled metabolites of E2 and T is found within the aqueous componentof the solubilized fecal/urine samples, and most of the metabolites are in the conju-gated rather than the free form.

5. The E2 conjugates that are excreted by female budgerigars and orange-wingedAmazon parrots are monosulfates that are both E2 and E1 based.

6. The androgenic conjugates that are excreted by female budgerigars and or-ange-winged Amazon parrots are a complexity of metabolites. Some of the andro-genic conjugates that are excreted by the orange-winged Amazon parrots do not appearto have any T enzyme or radioimmunoreactivity.

ACKNOWLEDGMENTS

This work was partially funded by the University of California, Davis NewFaculty Research Grant and the EPA Center for Ecological Health Research, EPSR814709. Appreciation is extended to Dr. James Millam for his help in procuring thebirds and providing animal holding space for performing the study and to TomRoudybush for providing pelleted feed for the orange-winged Amazon parrots.

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