Application of Enzyme Immunoassay to Fecal Steroid Analysis in Sika Deer (Cervus nippon)

Journal of Reproduction and Development, Vol. 45, No. 6, 1999 Technical Note Application of Enzyme Immunoassay to Fecal Steroid Analysis in Sika Deer (Cervus nippon) Kiyoshi YAMAUCHI, Shin-ichiro HAMASAKI 1), Yukari TAKEUCHI and Yuji MORI Laboratory of Veterinary Ethology, The University of Tokyo, 1 1 1 Yayoi, Bunkyo-ku, Tokyo 113-8657, and 1) Wildlife Management Office, 5-8 Fuda Tama-ku, Kawasaki, Kanagawa 214-0011, Japan Abstract. We have previously demonstrated that the gonadal function of sika deer (Cervus nippon) can be successfully monitored by using fecal steroid analysis. In this study we examined some technical aspects of applying this method to the estimation of the reproductive status of wild sika deer. Enzymeimmunoassays (EIA) for fecal progesterone and testosterone were established, and the fecal concentrations of these steroids obtained were compared with those measured by conventional radioimmunoassays (RIA). The patterns of fecal progesterone during the estrous cycle and the annual fecal testosterone profile in a male deer measured by EIA were virtually identical with those measured by RIA. Then the effect of environmental temperature on the fecal progesterone concentration was examined by monitoring the change in fecal progesterone at 4 C and 20 C up to 144 h after defecation. Fecal progesterone levels increased markedly at 20 C (but not at 4 C) with the degree of increase being variable in each sample. This increase was inhibited by adding ethanol, antibiotics or silica gel. It therefore appears that the increase was caused by the action of intestinal microorganisms, which converted conjugated steroids to unconjugated forms. These results suggest that wild sika deer fecal samples should be collected in winter time when the environmental temperature is low for steroid analysis in field conditions. The EIAs appeared more feasible means for field-endocrinological studies than RIA considering their simpler and less timeconsuming procedures. Key words: Sika deer, Feces, Enzymeimmunoassay, Pregnancy diagnosis, Progesterone, Testosterone. (J. Reprod. Dev. 45: 429 434, 1999) M easurement of steroid hormone levels in the blood has been the most common technique for assessing the reproductive status in laboratory as well as domesticated species. Nevertheless, blood sampling is sometimes extremely difficult in the wild species such as cervidae, because it often requires capturing and immobilizing of the target animal. In order to develop a feasible method which enables monitoring the gonadal function in Accepted for publication: October 5, 1999 Correspondence: Y. Mori captivity and assessing the reproductive population dynamics in field conditions, we have applied the technique of fecal steroid analysis [1 4] to sika deer and demonstrated cyclic fluctuations during the estrous cycle and a pregnancy-related increase in fecal progesterone in the doe as well as annual changes in fecal testosterone in the buck [5]. Despite the obvious potential advantages of fecal steroid analysis, there have been technical problems to be solved before applying it to wildlife studies. First, the radioimmunoassay (RIA) used in our previous study has a number of limitations due to

430 YAMAUCHI et al. the strict regulation of the use of radioisotopes, which forced us to develop an alternative assay method. Second, since it was suggested that steroid concentrations in feces increased after defecation [6, 7], it appeared necessary to evaluate the postdefecation change in fecal steroid under possible environmental conditions for collecting feces in a study in which the pregnancy rate for a certain wild population was to be estimated. In the present study we therefore examined the feasibility of applying enzymeimmunoassays (EIA) of fecal progesterone and testosterone to fieldendocrinological studies in wild sika deer. Materials and Methods Animals and sampling Fecal samples were collected from three adult female and one male adult sika deer kept at the Children s zoo in the Osaki Park, Urawa city (35 1 N, 139 9 E), Saitama Prefecture. They were allowed free access to fresh or dried hay supplemented with a pelleted diet. Immediately after defecation, fecal samples were picked up with tweezers from the ground surface and put in a vinyl bag, and used in the subsequent experiments. Sample processing The feces processing methods for the extraction of steroid hormone have been described elsewhere [5]. Briefly, feces were dried at 100 C for 1.5 h in an oven and thoroughly crushed. A portion (0.25 g) of the powdered feces was put in an extraction tube with a teflon-sealed cap. Then 1.5 ml of distilled water and 5 ml of diethyl-ether were added, and steroids were extracted for 10 min with a vortex mixer. The ether layer was recovered in another tube by decantation after snap-freezing, and evaporated at 42 C in a water bath. The residue was redissolved in 2 ml of ethanol by vortex mixing for 10 min. Assay procedures The RIA procedures for fecal progesterone and testosterone were the same as those described in our previous report [5]. The anti-progesterone (#337) and anti-testosterone (#250) antisera were provided by Dr. G. D. Niswender (Animal Reproduction and Biotechnology Laboratory, Department of Physiology, Colorado State University, Colorado, U.S.A.). The labeled hormones were 125 I-progesterone (Amersham, IM140) and 125 I-testosterone (Amersham, IM128). The recovery rates of progesterone and testosterone added to the dried feces of sika deer were 76% and 93%, respectively. Intra-assay coefficients of variation for progesterone and testosterone were 2.4% (n=6) and 3.2% (n=6), respectively. Inter-assay coefficients of variation of progesterone and testosterone were 6.8% (n=7) and 10.2% (n=7), and the minimum detectable levels for progesterone and testosterone were 1.95 pg/tube and 1.25 pg/ tube, respectively. The EIA procedures for progesterone and testosterone have been described elsewhere [8]. The antisera used were OK-1 for progesterone which was provided by Dr. K. Okuda (Division of Animal Science and Technology, Faculty of Agriculture, Okayama University, Okayama, Japan) and FKA 102 for testosterone (COSMO Co.,Tokyo, Japan). The recovery rates for progesterone and testosterone added to dried feces of sika deer were 86% and 70%, respectively. Intra-assay coefficients of variation of progesterone and testosterone were 4.2% (n=4) and 4.5% (n=4), respectively. Inter-assay coefficients of variation of progesterone and testosterone were 7.1 % (n=7) and 8.8% (n=3), and the minimum detectable levels of progesterone and testosterone were 2.8 pg/well and 4.9 pg/well, respectively. Trials for choosing suitable conditions for fecal sampling Effect of ambient temperature: In order to examine the change in the fecal progesterone concentration after defecation, 2 4 grains of feces were collected from each of 3 female deer (one pregnant and two non-pregnant females) and frozen either immediately after defecation (0 h) or at 6, 12, 24, 48, 96 and 144 h after being kept at 20 C. All the fecal samples were thawed and assayed for progesterone concentrations at once. Then the effect of temperature on the pattern of fecal progesterone change after defecation was examined. Portions of the same feces were frozen either immediately after defecation or after 48 h incubation at 20 C or 4 C. All the samples were thawed and assayed at once for comparison of fecal progesterone concentrations.

EIA FOR FECAL STEROID ANALYSIS 431 Effect of controlling intestinal microorganisms: In order to examine the effect of inactivating intestinal bacteria, 2 4 grains of each feces sample were frozen either immediately after defecation (0 h) or 48 h after being kept at 20 C or 4 C. To the feces kept at 20 C were also added silica gel (20 g per one grain), 70% ethanol or one of three antibiotics, namely, streptomycin benzylpenicillin (Mycillin: Meiji-seika Co., Tokyo, Japan), ampicillin sodium (Viccillin: Meiji-seika Co., Tokyo, Japan), and oxytetracyclin (Terramycin: Pfizer Co., Tokyo, Japan) 300 400 µl per one grain of feces. All the samples were thawed and assayed at once, and the fecal progesterone concentrations obtained were compared. Results The displacement curves for the reference standard and the serial dilution of fecal extracts from sika deer obtained by EIA were confirmed to be parallel for both progesterone and testosterone as shown in Fig. 1. There was a good correlation between values obtained by RIA and EIA for both progesterone (r=0.94) and testosterone (r=0.99) as shown in Fig. 2. The patterns of fecal progesterone during the estrous cycle and the annual fecal testosterone profile in a male deer measured by EIA were virtually identical with those measured by RIA previously [5] (Fig. 3), but fecal testosterone concentrations measured by EIA were lower than those measured by RIA. Progesterone concentrations increased abruptly in feces kept at 20C, and reached stable levels by 24 48 h after defecation (Fig. 4; a). And by comparing the amount of increase in the progesterone concentration in feces kept at 20 C with that kept at 4 C, the increase in progesterone concentrations was more remarkable in feces kept at 20 C than at 4 C, with greater variation among individual samples (Fig. 4; b, c). By adding silica gel, ethanol or antibiotics to fresh feces, an increase in the fecal progesterone concentration was prevented (p<0.05, Mann- Whitney s U-test) as compared with feces kept at 20 C for 48 h without any treatment (Fig. 5). Fig. 1. Parallelism between displacement curves for the reference standard ( ) and serial dilution of extracts ( ) from sika deer feces obtained by EIA for progesterone (top) and testosterone (bottom). Pregnant female and male deer fecal samples were used for progesterone and testosterone assays, respectively. 25 µl fecal extracts were assayed in duplicate. Discussion Fecal steroid analyses have been reported in several species so far [9], by mostly using RIAs. In this study it has been shown that EIAs also have sufficient sensitivity for analysing progesterone and testosterone concentrations in feces of the deer. Fecal progesterone concentrations measured by EIA were similar to those obtained by RIA, whereas fecal testosterone concentrations were considerably lower when assayed by EIA. It has been reported that some antibodies to particular steroids show signs of considerable cross-reactivity with several other steroid metabolites in the feces [9, 10]. It therefore seems plausible that the difference between the fecal testosterone concentrations obtained with RIA and EIA was due to the different

432 YAMAUCHI et al. Fig. 2. Correlation between the assay results obtained by RIA and EIA for fecal progesterone (top) and testosterone (bottom). Fecal samples from three female deer (one pregnant and two non-pregnant females) and one male deer were used for progesterone and testosterone assays, respectively. characteristics of the antibodies. Nevertheless, it was shown that sequential changes in fecal steroid concentrations could be monitored by EIA, because there were good correlations between the values obtained with the RIA and EIA systems. Actually the patterns of fecal progesterone and testosterone profiles obtained by EIA and by RIA closely corresponded (Fig. 3). Based on these results, the EIAs were considered to be applicable to fecal steroid hormone measurement in sika deer under wild as well as captive conditions. And it seems that this method is also useful for monitoring the reproductive status of nontractable zoo animals besides the application to wildlife. Fig. 3. Correspondence between RIA ( ) and EIA ( ) assay results. Fecal progesterone profiles for two non-pregnant female deer during the estrous cycle (top) and the annual fecal testosterone change in one male deer (bottom) are shown. It has been reported that fecal concentrations of steroid hormones tend to increase if the feces are left unfrozen after defecation [6, 7]. Since increased storage time prior to freezing results in higher estrogen levels in feces and then the levels will remain constant, Holtz [7] suggested that the feces should be kept unfrozen for some time in order to obtain standardized results. Regarding this we failed to confirm that fecal steroid levels can be standardized after being left unfrozen for certain time. The addition of desiccant silica gel and the administrations of various antibiotics or ethanol elution resulted in an inhibition of the increase in the fecal progesterone concentrations. Similar results to those for fecal testosterone in our preliminary experiment were obtained (data not shown). These results suggest that intestinal

EIA FOR FECAL STEROID ANALYSIS 433 Fig. 5. Effects on the increase in fecal progesterone concentrations after 48 h incubation at 20 C of adding 1, silica gel; 2, streptomycin and benzylpenicillin; 3, ampicillin sodium; 4, oxytetracyclin and 5, 70% ethanol to feces (mean ± SEM, n=4; p<0.05, Mann-Whitney s U-test). Solid bars indicate the control results without any of these treatments. Data are presented as the relative ratio to the fecal progesterone concentration frozen immediately after defecation (0 h). Feces of three females deer (one pregnant and two non-pregnant females) and one male deer were used for their assays. Fig. 4. Changes in progesterone concentrations in feces kept at 20 C after defecation (a: mean ± SEM, n=3), and changes in progesterone concentrations in individual feces before and after 48 h incubation at 20 C (b) or 4 C (c). by the ambient temperature and other environmental factors such as humidity. The present results suggest that feces need to be collected in winter (e.g. February or March), when fecal progesterone concentrations in pregnant deer are high [5] and environmental temperature and humidity are both low, for successful application of this technique to the assessment of the population dynamics in wild sika deer. Acknowledgments microorganisms would be involved in the gradual post-defecation increase in fecal steroid concentrations. It was reported that in pregnant women large quantities of conjugated steroid appeared in the feces during ampicillin administration due to a reduction of the intestinal flora [11]. It is therefore supposed that the increase in the fecal progesterone concentration observed in this study was caused by intestinal microorganisms which would have decomposed the conjugated progesterone into the unconjugated form, with the degree of its increase being affected We would like to thank Mr. A. Nagumo, Mr. K. Harayama, Mr. K. Izumisawa and the other staff members of the Children s zoo for their assistance in collecting the samples. We also thank Dr. G. D. Niswender for providing antisera for progesterone and testosterone. We are grateful to Dr. K. Okuda of Okayama University and Dr. A. Miyamoto of Obihiro University of Agriculture and Veterinary Medicine for providing antisera and peroxidase-labeled hormone for progesterone and testosterone as well as for helpful advice.

434 YAMAUCHI et al. References 1. Kirkpatrick JF, McCarthy DF, Gudermuth SE, Shideler SE, Lasley BL. An assessment of the reproductive biology of Yellowstone bison (Bison bison) subpopulations using noncapture methods. Can J Zool 1996; 74: 8 14. 2. Monfort SL, Schwart CC, Wasser SK. Monitoring reproduction in captive moose using urinary and fecal steroid metabolites. J Wildl Manage 1993; 57: 400 407. 3. Borjesson DL, Boyce WM, Garder IA, DeForge J, Lasley B. Pregnancy detection in bighorn sheep (Ovis canadensis) using a fecal enzyme immunoassay. J Wildl Dis 1996; 32: 67 74. 4. Lasley BL, Kirkpatrick JF. Monitoring ovarian function in captive and free-ranging wildlife by means of urinary and fecal steroids. J Zoo Wildl Med 1991; 22: 23 31. 5. Yamauchi K, Hamasaki S, Takeuchi Y, Mori Y. Assessment of reproductive status of deer by fecal steroid analysis. J Reprod Dev 1997; 43: 221 226. 6. Hirata S, Mori Y. Monitoring reproductive status by fecal progesterone analysis in ruminants. J Vet Med Sci 1995; 57: 845 850. 7. Holtz W. Pregnancy diagnosis through faecal estrogens in goats. In: Proceedings of the First International Symposium on Faecal Steroid Monitoring in Zoo Animals; 1992; Royal Rotterdam Zoological and Botanical Gardens, Rotterdam. 62 67. 8. Miyamoto A, Okuda K, Schweigert FJ, Schams D. Effect of basic fibroblast growth factor, transforming growth factor-b and nerve growth factor on the secretory function of the bovine corpus luteum in vitro. J Endocrinol 1992; 135: 103 114. 9. Schwarzenberger F, Mostle E, Palme R, Bamberg E. Faecal steroid analysis for non-invasive monitoring of reproductive status in farm, wild and zoo animals. Anim Reprod Sci 1996; 42: 515 526. 10. Schwarzenberger F, Tomasova K, Holeckova D, Matern B, Mostle E. Measurement of fecal steroids in the black rhinoceros (Diceros bicornis) using group-specific enzyme immunoassays for 20-oxopregnanes. Zoo Biol 1996; 15: 159 171. 11. Martin F, Peltonen J, Laatikainen T, Pulkkinen M. Excretion of progesterone metabolites and estriol in faeces from pregnant women during ampicillin administration. J Steroid Biochem 1975; 6: 1336 1346.