1996 - black rhinoceros measurement of fecal - zoo biol

8/12/2019 1996 - Black Rhinoceros Measurement of Fecal - ZOO BIOL

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Zoo Biology 15359-171 1996)

TECHNICAL REPORTMeasu rement of Fecal Steroids in th eWack Rhinoceros Diceros bicornisUsing Group-Specific Enzym elmmuno assays for 20-Oxo -Preg nanesFranz Schwarzenberger, Krist ina TomaSova, Dana HoleCkova,Bernd Matern, and Erich MiSstllnst. f. Biochemie and L. Boltzmann-lnst. f. Vet. med. Endokrinologie, UniversityofVeterinary Medicine, Vienna, Austria F.S., E.M.); Zoo Dvdr Kralove, Czechia K.T.,D.H.); and Zoo Frankfurt, Germany B.M.

The m etabolism and ex cretion of progesterone in different animal species resultsin several fecal 5-reduced progesterone metabolites pregnanes), which in recentstudies were quantified using progesterone antibodies. To increase the accuracy offecal 20-oxo-pregnane evaluations in the black rhinoceros, enzyme immu noassaysEIA) using antibodies against 5a-pregnane-3P-ol-20-one3HS:BSA 5a-2O-oneEIA) and SP-pregnane-3a-ol-20-oneHS:BSA 5P-20 -one ETA) were developed.The assays showed high crossreactivities with pregnanes containing a 20-0x0group and are referred to a s group-specific; results of these assays were com paredwith an EIA using an antibody against 6HS-progesterone 4-ene-20-one EIA).Fecal samples of both subspecies of the black rhinoceros Diceros bicornismichaeli, n = 5 , and Dice ros bicornis minor, n = 1) during pregnancy werecollected 1-3 timesiweek. H PLC sep aration showed three major imm unoreactivefecal 20-oxo-pregnane peaks; their elution profiles and different crossreactivitiesin the three EIAs provided strong evidence that these peaks are Su-pregnane-3,20-dione, 5u-pregnane-3u-ol-20-one, and 5u-pregnane-3P-ol-20-one. Preg-nane values in the pregnant animals continuously increased between mon ths 3-7and were significantly P < 0.01) elevated above the levels of nonpregnantanimals 0.2 Fg/g) by week 11. During months 6-13 concentrations in the 5a-20-one and in the 5P-20-one EIA 5-11 Fglg) were significantly P < 0.01)higher than in the 4-ene-20-one EIA 1.5-3 Fglg . In conclusion, the immuno-reactive fecal 20-oxo-pregnane metabolites in the black rhinoceros are determinedmore accurately with antibodies against pregnane-20-one-C3 conjugates, a s com-pared with a progesterone antibody. 0 996 Wiley-Liss, Inc.Received for publication May 10, 1995; revision accepted July 27, 1995.Address reprint requests to Dr. Franz Schwarzenberger, Institut f. Biochemie, Vet. Med. Univ., LinkeBahngasse 11, A-1030 Vienna, Austria.

1996 Wiley-Liss, Inc.


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160 Schwarzenberger et al.Key words: noninvasive reproductive monitoring pregnancy diagnosis rhinocerosconservation Perrisodactyla

INTRODUCTIONThe two subspecies of African black rhinoceros, the eastern Diceros bicornismichaelij and the southern Diceros bicornis minor), are critically endangered due tocontinued poaching. The African population was reduced to less than 2,000 animalsby the end of 1992 [Klos and Frese, 19931. To save the remaining black rhinocerospopulation the current conservation strategies consist of conservation breeding in zoos[Klos and Frese, 19931 strengthening of antipoaching measures, and translocation tosanctuaries in Africa [Kock et al., 19951.

Successful breeding management may benefit from reliable reproductive mon-itoring. Reproductive data of rhinoceroses in recent studies were gained from theanalysis of steroids in blood [Kock et al., 1991; Berkeley, 19941; saliva [Kuckelkornand Dathe, 1990; Czekala et al., in press]; urine [Kassam and Lasley, 1981; Kasmanet al., 1986; Wagner, 1986; Ramsay et al., 1987, 1994; Hodges and Green, 1989;Hindle et al., 19921; and feces [Schwarzenberger et al., 1993a, 1994, 1995b; Berke-ley, 19941. Furthermore, the metabolism of radioactively labeled steroid hormoneswas studied in a white rhinoceros [Hindle and Hodges, 19901, and ultrasonographicimaging was used to investigate the reproductive tract of rhinoceroses [Adam et al.,1991; Schaffer et al., 19941.

The most appropriate techniques for reproductive monitoring in nondomesticanimals are the analysis of noninvasively collected fecal and urine samples. Recentinvestigations of fecal immunoreactive progesterone metabolites in several animalspecies used progesterone antibodies [Desaulniers et al., 1989; Messier et al. 1990;Wasser et al., 1991, 1994; Graham et al. 1993; Kirkpatrick et al. 1993; Monfort etal., 1993; Mostl et al., 1993; Schwarzenberger et al., 1993a, 1995a; Berkeley, 1994;Brown et al., 1994; Czekala et al., 1994; Latter et al., 19941. Resulting in usefulbiological data, most of these studies did not characterize the immunoreactive ma-terial by any method other than RIA. However, metabolism and fecal excretion ofradioactively labeled progesterone in feline, primate, and farm animal species re-sulted in several progesterone metabolites [Shideler et al., 1993; Brown et al., 1994;Wasser et al., 1994; Palme, personal communication]. In addition, HPLC separationwith subsequent immunoassay analysis displayed the presence of up to eight welldefined immunoreactive fecal progesterone metabolites [Kirkpatrick et al., 1991;Schwarzenberger et al., 1991, 1992, 1993a,b, 1995a; Heistennann et al., 1993;Mostl et al., 1993; Shideler et al. 1993; Brown et al., 1994; Wasser et al. 19941.These metabolites showed elution patterns similar to that of pregnanes, and authen-tic progesterone was either not detectable or detectable to a limited extent only.

Progesterone is metabolized to pregnanes before its fecal excretion. Some ofthese pregnanes (namely 5a and SP-pregnane-3,20-dione) still posses two 0x0groups in position C3 and C20 of the steroid molecule, and therefore these metabo-lites show crossreactivities with progesterone antibodies. However, depending oncrossreactivities, specific progesterone antibodies quantify only a small proportion ofthe actual amount of fecal pregnane compounds. A possibility for determining thesemetabolites more accurately is the application of antibodies against pregnane-C3conjugates. Since the functional group of the steroid in position C3 is masked, these


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ElAs of Fecal PO-0x0-Pregnanes 161antibodies show considerable crossreactivities with several progesterone metabolitessharing a similar C20 configuration (a 20-0x0, a 20a-OH, or a 20P-OH group) andtheir measurement is combined in one assay, which we refer to as group-specific.

In our recent study on the black rhinoceros we (1) determined the estrous cyclelength as 25 days; (2) diagnosed pregnancy by means of increasing fecal immuno-reactive progesterone metabolites after the first quarter of gestation; and (3) demon-strated the presence of immunoreactive 20-0x0- and 20a-OH-pregnane compounds[Schwarzenberger et al. 1993a1. However, lacking specific enzyme immunoassays(EIA) for 20-oxo-pregnanes, we relied on the crossreactivities of an antibody against6HS-progesterone, and assay specificity and accuracy were a serious concern. There-fore we raised antibodies against 5a- and 5 f.3-pregnanes containing a 20-0x0 groupand developed EIAs.

The aims of the present study were: (1) to develop specific assays for pregnanescontaining a 20-0x0 group (20-0x0-pregnanes); (2) to compare EIA measurements offecal 20-0x0-pregnanes in three EIAs with different degrees of specificity; (3) tocharacterize the possible structure of the fecal immunoreactive 20-0x0-pregnanes; and(4) to determine fecal 20-0x0-pregnane values in both subspecies of the black rhi-noceros.

METHODSAnim als and Sample Col lect ionFemale black rhinoceroses of the eastern Diceros bicornis michaeli) and the

southern Diceros bicornis minor) subspecies were studied. Animals of the easternsubspecies (n = 5 ) were housed at Dvh KralovC Zoo. At the times of parturitionsreported, the animals were 10, 15, 16, and 23 years old and had (including deliveriesreported in this study) 2, 3 ,4 , and 1 offspring, respectively. One 24-year-old animalhad a late pregnancy abortion after 14 months of gestation; this was her fifth preg-nancy and her previous pregnancies delivered viable calves. The rhino (n = 1) of thesouthern subspecies was housed at Frankfurt Zoo; she was seven years old and thereport in this study was her first pregnancy. Each female was allowed access to a maleduring the daytime and housed alone at night. Estrous behavior and mating wererecorded by the keeper staff. Animals were fed hay and grass, supplemented withleaves, branches, fruits, and vegetables, and had free access to water.

Morning fecal samples were collected off the ground and stored at -20C untilanalysis. Samples were collected 2-3 times/week for the first 4 months of pregnancyand the month before parturition. Samples during midgestation were collected once aweek or biweekly. Samples from nonpregnant animals were collected three times/week.Fecal Extraction for Analysis With ElAs

Fecal samples (0.5 g) were extracted with methanol as described previously[Schwarzenberger et al., 1993a1, except 1 O g powdered aluminum oxide was addedbefore extraction. Briefly, 0.5 g feces was mixed with 1.0 g powdered aluminiumoxide, extracted with 4.5 ml aqueous methanol (SO ), and defatted with petroleumbenzene. The methanol was diluted and analyzed with the EIAs. Aluminium oxidewas added to remove visible background pigment [Graham et al., 19931 and in an


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162 Schwarzenberger et al.TABLE 1. Enzyme immunoassays (EIA)EIA 4-ene-20-one 5a-20-one 5P-20-oneImmunogens 4-pregnene-6a-01- 5a-pregnane-3P-ol- SP-pregnane-3a-01-Labels 5a-pregnane-3P-ol- 4-pregnene-3,20-dione- 5a-pregnane-3P-ol-Antibody titers i:40 x 103 i : 15 x 103 1:15 x lo3

20-one-6HS:BSA 20-one-3HS:BSA 20-one-3HS:BSA20-one-3HS:DADOO-Bb 3CMO:DADOO-Bb 20-one-3HS:DADOO-Bb

Label titers 1:250 x lo3 i : m x 103 i:600 x 103Standard curve range 2-500 pg 2-500 pg 5-1,250 pgSensitivity (90% 1.5 k 0.12 pg 2.6 * 0.33 pg 5.7 k 0.37 pgIntrassay coefficient 10.4% 11.3% 9.6%Interassay coefficient 12.8% 16.2% 14.6%

binding)of variationof variation

~~

Assay described by Schwarzenberger et al. [1993].bDADOO-B = biotin-DADO0 = N-biotinyl-l,8-diamino-3,6-dioxaoctan.initial study was found to increase differences between low and high 20-oxo-pregnanevalues in all three assays used.Analys is With ElAs

Fecal extracts were analyzed with two newly developed EIAs (Tables I ,2) andthe data compared with that using the 4-ene-20-one EIA described in our previousstudy [Schwarzenbergeret al., 1993al. The antibodies for the three EIAs were raisedin rabbits. Progesterone was used as standard, and serial dilutions of fecal extractsgave displacement curves parallel to that of the standard curve in each assay.

The three assays followed the protocol described by Schwarzenberger et al.[1991, 1993a1, except that steroid-DADOO-biotin conjugates (DADOO-biotin =N-biotinyl-l,8-diamino-3,6-dioxaoctane ere used as labels [Palme and Mostl,1993; Pryce et al., 19941. Briefly, the three EIAs used a double-antibody techniqueand were performed on anti-rabbit-IgG-coated microtiter plates. After overnight in-cubation of standard or samples, steroid antibody, and biotinylated label, the plateswere emptied, washed, and blotted dry, before a streptavidin horseradish peroxidaseconjugate was added. After a 45 min incubation, plates were emptied, washed, andblotted dry. The substrate (tetramethylbenzidine) was added and incubated for 45 minat 4C before the enzymatic reaction was stopped with 2 mol/l sulfuric acid.

The synthesis of the steroid-DADOO-biotin conjugates followed the mixedanhydride reaction [Kellie et al. 19751. Steroid:HS or steroid:CMO derivatives(5-10 mg) were dissolved in 1 ml dimethylformamide, and cooled to - 8C underconstant stirring. Then 0.01 ml methylmorpholin and 0.01 ml isobutylchloroformiatewere added and incubated at - 18C for 30 min. Thereafter, 1.5 ml of a mixture of0.1 mol/l sodium phosphate buffer (pH 8.2) and dimethylformamide (1/2 v/v;- 18 C), containing 5 mg biotinyLDAD00, was slowly added under constant read-justment of the pH, using 1 moVl KOH. The mixture was incubated at room tem-perature for 3 hr, under stirring and light protection. Then 7.5 ml of water was addedand the solution was passed through a primed C18 cartridge. The cartridge waswashed with 5 ml water, and the steroid and steroid conjugates were eluted with 4 mlmethanol. The methanol was evaporated (40C) under nitrogen and the residue dis-


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ElAs of Fecal PO-0x0-Pregnanes 163TABLE 2. Crossreactivities a) f steroids in the differentenzyme imrnunoassaysSteroid 4-ene-20-one 5a-20-one 5P-20-one4 p r e g n e n

3,20 dione (progesterone) 100 100 1003P-ol-20-one 26 14 683a-ol-20-one 8 390 2020a-ol-3-one


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164 Schwarzenberger et al.fractions were compared with those of different crossreacting steroid standards 5a/P-pregnanes) and with 3[H]-progesteroneand 3[H]-20a-dihydroprogesterone. lutionpatterns of pregnane standards were determined by EIAs, and 3[H]-steroids weredetermined by liquid scintillation counting.Data Analysis

Data are presented as mean rt SEM. A total of 216 samples from nonpregnantanimals (n = 5 ) during the estrous cycle was used for comparison with those ofpregnant animals. The duration of pregnancy was calculated as the period betweenobserved mating and parturition. Samples from the first 13 months and the last 2months of pregnancy were standardized to the day of mating and to the day ofparturition, respectively. Results were grouped in weekly intervals during gestationalweeks 1-17 and 61-65, and in monthly intervals between these periods. Meanscalculated represent 5-1 8 samples per point. Statistical significance of the differencesbetween pregnant and nonpregnant animals was tested by the Students t test and theMann-Whitney U est. Differences among the EIAs during pregnancy were analyzedby a paired t test and the Wilcoxons signed-rank test.

RESULTSEnzyme lmmunoassays EIA)

Characteristics of the EIAs are summarized in Tables 1 and 2. Antibodies for the5a-20-one and 5P-20-one EIA were raised against steroid-C3-hemisuccinateproteinconjugates; thus group-specific antibodies directed against the C20 position of thesteroid were obtained. The assays quantify closely related 5-reduced progesteronemetabolites (pregnanes) containing an x group on the C20 atom, and the results areexpressed as 20-oxo-pregnanes . Crossreactivities with pregnanes were considerablyhigher in the 5a-20-one and 5P-20-one EIA, as compared with the 4-ene-20-one EIA.However, the former two EIAs did not completely differentiate between the 5a- andthe 5P-pregnane series. Either EIA could be used for fecal steroid monitoring, al-though HPLC separation provided strong evidence for the sole presence of 5a-pregnanesHPLC Separation of Fecal 20-oxo-pregnanes

HPLC separation and subsequent analysis of the fractions with the three EIAswere performed on fecal samples (n = 5 ) from both subspecies during differentstages of pregnancy. A representative midpregnancy profile is shown in Figure 1.Results did not differ between the two subspecies; three major immunoreactive 20-oxo-pregnane peaks were detected in each sample. Their relative proportions re-mained unchanged throughout pregnancy. Immunoreactive material coeluting with3[H]-progesterone was not detectable.

Comparison with corresponding reference steroids together with the differentcrossreactivities in the three respective EIAs provided good evidence that the fecalimmunoreactive peaks are ( 1 ) 5a-pregnane-3,20-dione7 (2) Sa-pregnane-3a-ol-20-one, and (3) 5a-pregnane-3P-ol-20-one.Further evidence for the identity of peaks 2and 3 was gained from the chromatography of fecal samples spiked with the authenticsteroid standards, and after ether extraction and rechromatography of the fractionscontaining peaks 2 and 3. These separations resulted in chromatograms similar to that


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1500

K

0.o 1200EE. 900

--.r

0 --v9r:m0 600 --

300 --9v

EIAs of Fecal PO-0x0-Pregnanes 165fcl q- 800

cross-react ions %)

6000.\yE400 2Q0

200

0 00 10 20 30 40 50 60 70

FractionsFig. 1 . HPLC separation of immunoreactive 20-0x0-pregnanes n a fecal sample of a 7-months-pregnantblack rhinoceros Diceors bicornis michaeli). Elution patterns of 1) 3[H]-progesterone, (2) 3[H]-20a-dihydroprogesterone were determined by liquid scintillation counting (A cpdfraction). Fractions wereanalyzed with EIAs for 20-0x0-pregnanes 0a-20-one EIA; 5p-20-one EIA;0 -ene-20-one EIA).Concentrations in ngifraction were calculated for 1 g of feces without correction for methodologicallosses. Crossreactivities of immunoreactive peaks a-c) in the three EIAs are given in the inset. Retentiontimes of peaks a-c were comparable to: (a) 5a-pregnane-3,20-dione, (b) 5a-pregnane-3a-ol-20-one, and(c) 5a-pregnane-3p-01-20-one.

obtained after fecal sample separation. The elution patterns of crossreacting 5P-pregnanes were different from the fecal immunoreactive metabolites; the steroidstandards SP-pregnane-3,20-dione,5P-pregnane-3a-ol-20-one, and 5P-pregnane-3P-01-20-one eluted in fractions 10, 39-42, and 42-46, respectively (Fig. 1) .Fecal 20-0x0-Pregnane Profiles

Estrous cycle profiles were comparable to those described previously[Schwarzenbergeret al., 1993al. Distinction between follicular and luteal phases waspossible in the three EIAs used, and mating corresponded with low fecal 20-0x0-pregnane concentrations. Therefore, mean values were calculated frorn samples col-lected during follicular and luteal phases, and used for comparison only with theresults obtained during pregnancy. Values of the nonpregnant animals were 117* 8 ,136 8 , and 140 ? 9 ng/g feces in the 4-ene-20-one, 5a-20-one, and 5P-20-oneEIA, respectively.

Gestation lengths for the animals of the Diceros bicornis m ichaeli subspecieswere 442, 455 67, and 469 days, respectively; one animal had a late pregnancyabortion on day 427 (Fig. 2). Values of this animal were used for mean SEMcalculation during the first 9 months of gestation only. Duration of pregnancy was464 days for the animal of the Diceros bicornis minor subspecies.


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166 Schwarzenberger et al.15.0 m

1 3 5 7 9 1 4Month of pregnancy

Fig. 2. Concentrations of immunoreactive 20-0x0-pregnanes in fecal samples of a black rhinocerosDiceors bicornis michaeli) during an estrous cycle, subsequent pregnancy, and after late pregnancyabortion. Samples were analyzed with EIAs for 20-0x0-pregnanes 0 a-20-one EIA; 5p-20-oneEIA). Note breaks of horizontal and vertical axes.

Pregnane concentrations during pregnancy were analyzed with the three EIAsand results of the Diceros bicurnis michaeli (mean SEM) and the Diceros bicurnisminor subspecies are shown in Figures 3 and 4, respectively; endocrine profiles of thetwo subspecies were comparable. Mean 20-0x0-pregnane values increased to lutealphase values within 2 weeks after mating and remained in that range during the first2 months of pregnancy. Pregnane concentrations continuously increased duringmonths 3-7, and values were significantly P < 0.01) elevated above mean values ofnon-pregnant animals from week 11 onwards. The increase in the 4-ene-20-oneETAvalues was moderate as compared with the pronounced increase in the 5a-20-one and5P-20-one EIA. During months 6-13 mean values in the 5a-20-one and the 5p-20-one EIA (5-11 pg/g) were significantly higher P < 0.01) than those in the 4-ene-20-one EIA (1.5-3 pg/g feces). The 20-0x0-pregnane values decreased during thelast 3 months of gestation and returned to basal levels within 2-3 days after partu-rition.DISCUSSION

The principal objective of this study was to compare measurements of fecal20-0x0-pregnane values in EIAs with different degrees of specificity. Results of ourrecent study on fecal immunoreactive progesterone metabolites in the black rhinoc-eros Diceros bicornis) demonstrated the presence of 20-0x0- and 2001-OH-preg-nanes; progesterone was not present, or present to a limited extent only [Schwarzen-berger et al., 1993al. However, lacking specific assays for 20-oxo-pregnanes, werelied on the crossreactivities of an antibody against 6HS-progesterone (the 4-ene-20-one EIA used in this study). Thus, assay specificity and accuracy of fecal 20-0x0-prenane determinations were a reasonable concern. Therefore, two antibodiesagainst 501- and 5P-pregnane-20-one 3HS:BSA derivatives (501-20-one EIA and 5p-


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EIAs of Fecal PO-0x0-Pregnanes 16715

mm9Cm--m 5m 32 Iag 2X

1 3 5 7 9 1 1 1 3 1 5Month o f pregnancyFig. 3. Concentrations (mean rt SEM; n = 5-18 per point) of immunoreactive 20-0x0-pregnanes infecal samples of black rhinoceroses Dice ors bicornis rnichaeli;n = 5 during pregnancy. Samples wereanalyzed with EIAs for 20-0x0-pregnanes 0a-20-one EIA; 5P-20-one EIA; Iz]4-ene-20-oneEIA).Note break of vertical axis.

15InPIumrn=L2 10

2 5m 3

?0 10

SmCE 2hl

1 3 5 7 9 1 1 1 3 1 5Month o f pregnancy

Fig. 4. Concentrations of immunoreactive 20-0x0-pregnanes in fecal samples of a black rhinocerosDiceors hicornis minor) during pregnancy. Samples were analyzed with EIAs for 20-0x0-pregnanes 05a-20-one EIA; 5P-20-one EIA; 4-ene-20-oneEIA). Note break of vertical axis.

20-one EIA ) w ere produced. In both assays considerably higher crossreactivities with20-0x0-pregnanes were obtained, as compared to the 4-ene-20-one EIA. However,the newly developed EIA s had crossreactions with 5a nd SP-pregnanes containinga 20-0x0-group and thus were not specific for the 5a and SP-pregnane series,respectively. Therefore, values obtained in these EIAs were comparable, but theywere considerably higher than those in the 4-ene-20-one EIA .In fecal samples of the black rhinoceros authentic progesterone, whichwould have coeluted with 3[H]-progesterone and which had crossreactivitiesof 100%


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168 Schwarzenberger et al.in all three EIAs used, was not detectable. HPLC separation showed three majorimmunoreactive peaks; their elution patterns and the different crossreactivities in thethree respective EIAs provided strong evidence that these peaks are Sa-pregnane-3,20-dione, 5a-pregnane-3a-ol-20-one, and Sa-pregnane-3P-ol-2O-one. lthoughrigorous identification was not achieved (GUMS was not available), our findings ofseveral 20-oxo-pregnanes are in accordance with other studies in different species.Using progesterone antibodies, the presence of 20-oxo-progesterone metabolites inthe feces of primates [Wasser et al., 1991, 19947, felids [Graham et al., 1993; Most1et al., 1993; Brown et al., 1994; Czekala et al., 19941, ruminants [Desaulniers et al.,1989; Messier et al., 1990; Monfort et al., 1993; Larter et al., 19941, white rhinoc-eroses [Schwarzenberger et al., 1994, 1995b1, and vicunas [Schwarzenberger et al.,1995al was demonstrated. In addition, radioinfusion studies [Shideler et al., 1993;Brown et al., 1994; Wasser et al., 1994; Palme, personal communication] and studiesusing HPLC separation with immunoassay analysis [Kirkpatrick et al., 1991;Schwarzenberger et al., 1991, 1992, 1993a,b, 1995a; Heistennann et al., 1993;Shideler et al., 1993; Brown et al., 1994; Wasser et al., 19941 showed that proges-terone metabolism and excretion results in several fecal steroid metabolites showingelution patterns similar to that of pregnanes.

The comparisons made in this manuscript deal with progesterone assays and notassays recognizing either a 2Oa-OH or a 20P-OH group. Pregnanediol(5 P-pregnane-3a,20a-diol) has a 2001-OH group in position C20 of the steroid molecule, and itscrossreactivities in our 20-oxo-assays are


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ElAs of Fecal 20-Oxo-Pregnanes 169tivities with 20-oxo-pregnanes in the present study were considerably higher thanthose of a progesterone antibody used in a comparable study [Berkeley, 19943. Ourresults were 3-5 times higher, although our calculations were based on wet feces andthose of Berkeley [1994] on dry feces. Similarly, a study on feline species using amonoclonal progesterone antibody with high crossreactivities to 5a-reduced preg-nanes reported 10-100-fold higher progesterone metabolite concentrations as com-pared to other studies [Brown et al., 19941.

The increase in assay specificity enabled us to determine fecal 20-oxo-pregnanevalues more accurately as compared with the 4-ene-20-one EIA [Schwarzenbergeretal., 1993a1. Thus, differences in the fecal pregnane values between nonpregnant and>2 months pregnant animals increased. The increase in accuracy causes an increasein reliabilityof pregnancy diagnosis. Although this is not of the utmost importance foranimals kept in zoos, where several samples can be collected over prolonged timeperiods, the high assay reliability is important for the study of free-ranging animals.Confirmation of pregnancy from one sample is possible by monitoring the high fecal20-oxo-pregnane values 3-4 months after mating. These high pregnane values mostprobably reflect placental steroid production. The increase in fecal steroid excretionin the black rhinoceros after the third month of pregnancy was also described byBerkeley [19941. Serum hormone levels of >1,000 ng/ml were obtained in a pro-gesterone RIA during the last third of pregnancy [Kock et al., 19911.

During the last quarter of gestation fecal 20-oxo-pregnane values declined. Thisis in contrast to the pattern of fecal progesterone metabolites containing a 2Oa-OHgroup which, in black rhinoceroses and mares, increased throughout gestation[Schwarzenberger et al., 1991, 1993al. HPLC separation of fecal immunoreactiveprogesterone metabolites in black rhinoceroses [Schwarzenberger et al., 1993al sug-gested that metabolites with higher polarity became more prominent as pregnancyprogressed. A shift in steroid productiodmetabolism and/or excretion to more hy-droxylated and thus more polar progesterone metabolites might explain this phenom-enon. The patterns of 5a-pregnanes containing a 20a-OH group using group-specificassays need to be elucidated in future investigations. These assays might improveestrous cycle monitoring, since the conversion of radioactively labeled progesteroneinto 20a-dihydroprogesterone in a rhinoceros was demonstrated [Hindle and Hodges,19901, and since urinary analysis of 20a-dihydroprogesterone was successfully usedfor estrous cycle monitoring [Hindle et al., 19921.

CONCLUSIONS1. Fecal 20-oxo-pregnanes in the black rhinoceros consist of closely related

immunoreactive metabolites. Their elution profiles on HPLC and different crossre-activities in the three EIAs used provided strong evidence that these metabolites are5a-pregnane-3,20-dione, 5a-pregnane-3a-ol-20-one, and Sa-pregnane-3P-ol-20-one.

2. Values of fecal 20-oxo-pregnanes are determined more accurately usinggroup-specific assays against 5-pregnane-20-one C3 conjugates, as compared to aprogesterone antibody.3. Using the newly developed assays, differences between values of nonpreg-nant and pregnant animals increased and thus reliability of pregnancy diagnosis wasimproved.


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170 Schwarzenberger et al.4.Application of group-specific assays for 20-0x0-pregnanes reaffirms that they

might be of general interest for future analysis of fecal progesterone metabolites inseveral animal species.ACKNOWLEDGMENTS

We thank the animal keepers Z. Vondra, P. Petriflek, M . Kober, J. &di;&ek,and V. Hrik (Zoo Dv8r Kralove); and K . Jahnel, W. Pittermann, S. Petermann, and N .Steinberg (Zoo Frankfurt) for the conscientious collection of the fecal samples; andA. Aichinger, E. Leitner, and A. Kuchar-Schulz for excellent technical assistance.REFERENCESAdams, G.P.; Plotka, E.D.; Asa, C.S.; Ginther,0 . Feasibility of characterizing reproductiveevents in large nondomestic species by transrec-tal ultrasonic imaging. ZOO B IOLOG Y 10247-259, 1991.Berkeley, E.V. FECAL STEROID HORMONENANCY IN THE BLACK RHINOCEROSDICEROSBICORNIS). M.S. Thesis, Ohio StateUniversity, 1994.Brown, J.L.; Wasser, S.K.; Wildt, D.E. ;GrahamL.H. Comparative aspects of steroid hormonemetabolism and ovarian activity in felids, mea-sured noninvasively in feces. BIOL OGY OF RE-

Czekala, N.M.; Durrant, B .S . ; Callison, L.;Williams, M.; Millard, S . Fecal steroid hormoneanalysis as an indicator of reproductive functionin the cheetah. ZOO BIOLOGY 13:119-128,1994.Czekala, N.M.; Callison, L. Pregnancy diagnosisin the black rhinoceros Diceros bicornis) by sal-ivary hormone analysis. ZOO BIOLOGY, inpress.Desaulniers, D.M.; Goff, A.K.; Betteridge, K.J.;Rnwell, J .E. ;Flood, P.F. Reproductive hormoneconcentrations in faeces during the oestrous cycleand pregnancy in cattle Bos taurus) and musk-oxen Ovibos moschatus). CANADIAN JOUR-

Graham, L.H. ; Raeside, J.I. ; Goodrowe, K.L. ;Liptrap, R.M. Measurements of faecal oestradioland progesterone in non-pregnant and pregnantdomestic and exotic cats. JOURNAL OF RE-

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