| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 United States Geological Survey-Biological Resources Discipline, Fort Collins Science Center, 2150 Centre Avenue, Building C, Fort Collins, Colorado 80526-8118, USA
2 Science and Conservation Center, 2100 S. Shiloh Road, Billings, Montana 59106, USA
3 Corresponding author (email: kate+schoenecker{at}usgs.gov)
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
-glucuronide (PdG) and iPdG followed a predictable curve over the course of the 180-day pregnancies. We conclude that estrone conjugates are not useful in diagnosing pregnancy; however, fecal steroid analysis of PdG and iPdG can be used to accurately determine pregnancy and reproductive function in bighorn sheep. This holds great potential as a noninvasive technique for understanding the role of reproductive disease in wild bighorn sheep. Key words: Animal reproduction, bighorn sheep, fertility, noninvasive, Ovis canadensis canadensis, pregnancy.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Pregnancy detection by fecal steroid analysis has been successfully applied to a host of ungulate species including moose (Alces alces; Monfort et al., 1992; Schwartz et al., 1995), muskoxen (Ovibos moschatus; Desaulniers et al., 1989), bison (Bison bison; Kirkpatrick et al., 1992, 1993, 1996), Equus spp. (Bamberg et al., 1991; Kirk-patrick et al., 1991; Barkuff et al., 1993), caribou (Rangifer tarandus; Messier et al., 1990), and black rhinoceros (Diceros bicornis; Berkeley et al., 1997). Safar-Hermann et al. (1987) reported using nonspecific radioimmunoassay in four species (red buffalo [Syncerus caffer nanus], yak [Bos mutus], Grevy’s zebra [Equus grevyi], and Nubian ibex [Capra ibex nubiana]) successfully. Fecal steroid metabolite analysis also has been used successfully to diagnose pregnancy in desert bighorn sheep and Rocky Mountain bighorn sheep (Ovis canadensis) using a nonspecific assay for progesterone metabolites (Borjesson et al., 1996) and multiple sampling. The assay used in Borjesson et al. (1996) was for metabolites related to pregnanediol-3-glucuronide (PdG), coupled with multiple samples (two samples collected 2 wk apart) from 60 days or later in gestation. The Borjesson et al. (1996) method proved to be 100% accurate. But not all wildlife managers and biologists have the time to conduct multiple sampling.
The current bighorn sheep (O. canadensis canadensis) population of Bighorn Canyon National Recreation Area (BICA) Wyoming and Montana (USA) is the product of several reintroductions to the surrounding areas (Coates and Schemnitz, 1989). Following a release in 1973 and growth rates near maximum potential of 19.8% per year, the population grew to an estimated peak population of about 211 animals in 1993 and 1994 (Kissell et al., 1996). The population began to decline rapidly in 1995 and 1996. Kissell et al. (1996) noted low ewe:lamb ratios during the decline phase. The population today is estimated at 100±18 (Schoenecker, un-publ. data). Understanding the reproductive dynamics of this herd could help managers and biologists better identify causes for the population decline and monitor current fertility and fecundity of ewes.
Our objectives were to 1) investigate the accuracy of single-sample fecal steroid analysis to predict pregnancy rates in free-ranging Rocky Mountain bighorn ewes, 2) compare the accuracy of three different fecal steroid metabolite assays for detection of pregnancy, and 3) quantify either embryonal loss, late fetal loss, or neonatal loss in free-ranging ewes by comparing pregnancy status with lambing status.
Study area
Free-ranging bighorn ewes were studied in BICA (45°05'00''N, 108°13'00''W). Bighorn Canyon is a National Park Service Unit that encircles a 114-km long reservoir in southeastern Montana and north-central Wyoming (Coates and Schemnitz, 1989). The sheep range also extends into portions of Custer National Forest, Bureau of Land Management lands (Pryor Mountain Wild Horse Range [PMWHR]), and some interspersed private lands in East and West Pryor Mountains. The park is dominated by Bighorn Canyon, a long canyon formed by the Bighorn River. A dam near Fort Smith, Montana, formed what is now Bighorn Lake. A strong precipitation gradient in the park provides 15 cm of rainfall annually at the south upstream end of the park and 45 cm at the north end (Knight et al., 1987). Vertical canyon walls where bighorn ewes lamb are up to 1,700 m high, containing limestone caves and talus slopes. Mountain slopes are forested, but alpine-like meadows, dry-land flats, and less vegetated canyons intersperse with forested areas (Gudorf et al., 1996). Elevations range from 900 to 2,500 m (Gudorf et al., 1996). Soils in the precipitous canyon areas originated from sandstone and limestone and dolomite in the nonprecipitous areas (Knight et al., 1987).
Vegetation communities in the park and surrounding lands have been categorized by Knight et al. (1987) and include desert shrubland, sagebrush steppe, basin grassland, juniper woodland, mountain mahogany-juniper woodland, riparian, and coniferous woodland.
Long cold winters and hot dry summers characterize the climate; however, diversity in geography creates locally variable weather conditions (Gudorf et al., 1996). Semiarid conditions along the Dry Head area of the park are contrasted with subalpine zones at higher elevations in the Pryor Mountains.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
In order to understand the serial changes in fecal steroid metabolites during the course of gestation, fecal samples from two pregnant captive ewes at ZooMontana were collected monthly from September to parturition in May. Approximate conception dates for the two captive ewes were calculated by counting back 180 days from parturition dates.
Fecal samples were kept frozen at –7.4 C until the time of extraction and assay. Thawed wet feces (0.5 g) were placed in scintillation vials with 5 ml of extraction buffer. The extraction buffer consisted of 450 ml enzyme immunoassay (EIA) buffer (5.42 g NaH2PO4·H2O, 8.66 g NaHPO4, 8.7 g NaCl, 1.0 g radioimmunoassay grade bovine serum albumin in 1.0 liter dH2O), 50 ml EIA wash solution (87.7 g NaCl, 0.5% Tween-20 in 1.0 liter dH2O), and 500 ml high performance liquid chromatography (HPLC) grade methanol. Samples were vortexed and shaken for 30 min and stored at –15 C overnight, vortexed again in the morning, and shaken for another 30 min. Samples were then centrifuged and the supernatant removed and frozen until assay.
Fecal extracts were assayed by EIA for progesterone metabolites related to PdG and estradiol metabolites, for estrone conjugates (E1C) as described by Shideler et al. (1991), and for immunoreactive pregnanediol-like progesterone metabolites (iPdG) as described by Kirkpatrick et al. (1991). Quantities of fecal extracts were 20 µl for PdG and iPdG and 40 µl for E1C. Assays were validated for bighorn sheep by means of testing halving dilutions for parallelism to the standard curve. Reproducibility of assays was calculated by determining inter-assay and intra-assay variation. The antibodies for PdG and E1C assays were P70 and R583, respectively (courtesy of C. Munro, University of California, Davis, California, USA). The antibody for iPdG was Ab 1284-1, raised against 20-hydroxy-4-pregnen-3-one-3 oxime (provided by R. Chatterton, Northwestern University School of Medicine, Evanston, Illinois, USA). Cross-reactivity for the estrone conjugate assay is estrone-3-glucuronide 100%; estrone-3-sulfate 66.6%; estrone 236%; estradiol 17ß 7.8%; estradiol-3-glucuronide 3.8%; estra-diol-3-sulfate 3.3%; all other steroid metabolites <0.01% (Munro et al., 1991). Cross-reactivity for the PdG assay is PdG 100%; 20-hydroxyprogesterone 60.7%; pregnanediol 7.3%; all other steroid metabolites <0.01% (Shideler et al., 1991). Cross-reactivity for the iPdG assay is 20-hydroxy-4-pregnen-3-one 100%; PdG 164%; 20-hydroxy-4-pregnen-3-one-3-oxime 41%; 20ß-hydroxy-5ß-pregnan-3-one 10%; 5-pregnane-3ß, 20ß-diol 4%; progesterone 2%; androsterone 0.2%; all other steroid metabolites <0.1% (Shideler et al., 1993).
Results are given in ng/g of wet feces. Differences in mean endocrine concentrations were tested for significance with the Mann-Whitney rank sum tests.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Approximate conception dates for the captive ewes at ZooMontana, "Princess" and "Jan" were 11 and 18 November, respectively. Fecal concentrations of iPdG rose steadily from conception until parturition with ranges of 3,000–18,000 ng/g, providing a clear picture of gestational progress in both captive ewes (Figs. 1
, 2). Fecal E1C concentrations did not increase rapidly until after days 67–72 of gestation and then declined from days 73–93 until parturition. However the range was small, from 2 to 15 ng/g feces, providing an accurate picture of pregnancy only for approximately days 70–90 of gestation. Fecal PdG concentrations showed little increase throughout pregnancy, until after days 79–93 of gestation, after which the range of increase was relatively small. Both PdG and iPdG concentrations provided an accurate indicator of pregnancy as early as 28 days postconception, using 1,800 ng/g and 8,000 ng/g, respectively.
|
|
). Pregnancy in the other nine animals was predicted accurately (Table 1). Of three free-ranging ewes sampled in 2002 that were predicted pregnant on the basis of iPdG concentrations (22,468, 18,168, and 32,848 ng/g wet feces), two were seen without lambs but with full mammary glands and extended teats, which suggests that they had both produced lambs that were lost soon after birth (Table 2). The third ewe was never observed leaving her group during the lambing season, as pregnant ewes do, which suggests she was either pregnant and lost the fetus somewhere between the time of fecal collection (4/22/02) and late May, or her pregnancy test was a false positive (Table 2).
|
|
Mean fecal concentrations of PdG were 3,143±254.29 ng/g wet feces in animals with lambs at their sides and 1,298±258.15 ng/g wet feces for ewes without lambs (P<0.0001). The range for individual pregnant ewe values was 1,498–4,913 ng/g wet feces and 358–3,432 ng/g wet feces for ewes without lambs. The two values that overlapped were those from the two ewes with full mammary glands and extended teats. The field data and observations from ground crews indicate that these latter values were in fact from pregnant animals.
Based on visual observations made by field crews that monitored lambs at the sides of ewes for over 2 yr, pregnancy was correctly predicted in 28 of 31 females (93%) using fecal iPdG or PdG concentrations. The remaining three samples (7%) include one false negative, one inconclusive test result, and one false positive, which we believe came from a ewe that terminated the pregnancy before parturition. In this case, the ewe did not leave the larger ewe group, did not go off on her own at any time, and was observed weekly during the time she would have been lambing. We interpret the data as indicating this ewe was pregnant at the time of the pregnancy test but lost the fetus before parturition, although her pregnancy test could have been a false positive. In the case of at least one ewe that was predicted not pregnant but lambed, we suspect field error since defecating ewes were often observed from a distance.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
It is important to note here that neither the iPdG nor the PdG assays are highly specific. The precise progesterone metabolites measured by the two assays is speculative. Cross-reactivities of the iPdG antibody have been reported previously (Kirkpatrick et al., 1991), but the three primary steroids include 20-ß-hydroxypro-gesterone, or 20-OHP (100%), PdG (164%), and 20-ß-hydroxy-5ß-pregnan-3-one (41%). Kirkpatrick et al. (1991), using this same assay for pregnancy diagnosis in equids, found that HPLC revealed at least three progesterone metabolites more polar than PdG and that, collectively, the immunoreactive metabolites measured by the iPdG assay positively correlated with blood progesterone.
The relatively low concentrations of metabolites cross-reacting in the PdG assay, compared to values derived from the iPdG assay, suggest both qualitative and quantitative differences in progesterone metabolites measured by the two assays. However, the collective metabolites cross-reacting in the PdG assay have been shown to positively correlate with blood progesterone in ewes (Borjesson et al., 1996).
The failure of E1C to produce an unambiguous diagnosis of pregnancy suggests that pregnancy results in very little estrone, estrone glucuruonide, and/or estrone sulfate production in wild ewes. In addition, all of these have significant cross-reactivity with the assay’s antibody (Shideler et al., 1991). Previous studies with domestic sheep have shown that, although this species produces increasing quantities of estrogens throughout the entire pregnancy, the primary metabolites are probably a nonreactive diconjugate of some sort rather than the collective E1C conjugates (Lasley, pers. comm.).
It is important to note that in various wildlife species, classic validations of assays for a specific steroid or its metabolite are not always possible or even advantageous. A whole spectrum of reproductive steroid metabolites is produced in mammals, and differences are significant. For example, PdG measurements in the Bovidae are extremely useful but E1C measurements are not, largely because the primary metabolite of estrogens in these taxa is 17ß-estradiol, which does not cross-react with the E1C antibody. Conversely, PdG measurements in the Equidae are of no value, while E1C measurements are extremely predictive of blood estrogen concentrations. Because the three assays used in this study cross-react with a significant number of metabolites, it is likely that some important steroids can be quantified. More importantly, for each assay some measurable and correlated reproductive event can be found, e.g., pregnancy, ovulation, blood steroid concentration, or birth of young. It is not vital that the assays be highly quantitative with respect to the specific metabolite because only working estimates are necessary to correlate to the specific reproductive event of interest (Lasley and Kirkpatrick, 1991). In the case of the current study, the confirming reproductive event was parturition and live young, and therefore pregnancy.
In previous studies with bighorn sheep, Borjesson et al. (1996) used multiple sampling and subsequent analysis of immunoreactive PdG metabolites provided to obtain 100% accuracy in diagnosing pregnancy when samples were collected after 60 days gestation. While multiple sampling over time ensures against sampling error or sample anomalies, such sampling presents new logistic challenges to field personnel. Single sampling used in our study yielded an accuracy in excess of 90%. Using the 1,800 ng/g cutoff for the PdG assay results, as reported by Borjesson et al. (1996), accuracy was exactly 90%, and using a cutoff value of 8,000 ng/g for the iPdG assay results, accuracy was also 90%.
The current study suggests that remote pregnancy evaluation of bighorn sheep may be valuable for quantifying fetal or possibly neonatal loss that might normally go undetected. There are diseases that can cause abortion in sheep. Campylobacter fetus fetus causes fetal loss in domestic sheep 4–6 wk prior to lambing (Collins and DeLisle, 1985; Salama et al., 1995), which in this study would have resulted in positive pregnancy diagnosis at the time of sampling using the tests described here. Enzootic abortion in ewes (caused by Chlamydia) also causes abortion shortly before parturition and could be the cause of fetal loss. Additionally, there is evidence of mountain lion (Felis concolor) and coyote (Canis latrans) predation on bighorn lambs on this particular range (Schoenecker, unpubl. data).
Our results offer a method for monitoring fertility of bighorn ewes that is noninvasive, safe, effective, and highly accurate. If field error can be kept to a minimum and ewes can be identified individually with preexisting radio collars or other marking devices to facilitate individual identification, much information can be gathered about the fertility and fecundity of bighorn sheep populations using fecal steroid analysis.
|
ACKNOWLEDGMENTS |
|---|
|
LITERATURE CITED |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
BAMBERG, E., E. MOSTL, M. PATZL, AND G. J. KING. 1991. Pregnancy diagnosis by enzyme immunoassay of estrogens in feces from nondomestic species. Journal of Zoo and Wildlife Medicine 22: 73–77.
BARKUFF, V., B. CARPENTER, AND J. F. KIRKPATRICK. 1993. Estrous cycle of the mare evaluated by fecal steroid metabolites. Journal of Equine Veterinary Science 13: 80–83.
BERKELEY, E. V., J. F. KIRKPATRICK, N. E. SCHAFFER, W. M. BRYANT, AND W. R. THRELFALL. 1997. Serum and fecal steroid analysis of ovulation, pregnancy and parturition in the black rhinoceros (Diceros bicornis). Zoo Biology 16: 121–132.
BORJESSON, D. L., W. M. BOYCE, I. A. GARDNER, J. DEFORGE, AND B. L. LASLEY. 1996. Pregnancy detection in bighorn sheep (Ovis canadensis) using a fecal-based enzyme immunoassay. Journal of Wildlife Diseases 38: 67–74.
COATES, K. P., AND S. D. SCHEMNITZ. 1989. The bighorn sheep of Bighorn Canyon National Recreation Area and Pryor Mountain Wild Horse Range: Ecological relationships and management recommendations. Completion report for the National Park Service and Bureau of Land Management, Department of Fishery and Wildlife Sciences, New Mexico State University, Las Cruces, New Mexico 88003-0003.
COLLINS, D. M., AND G. W. DELISLE. 1985. Typing of Campylobacter fetus fetus isolated from sheep abortions in New Zealand. New Zealand Veterinary Journal 33: 52–53.
DEFORGE, J. R. 1976. Stress: Is it limiting bighorns? Desert Bighorn Council Transactions 20: 30–31.
DESAULNIERS, D. M., A. K. GOFF, K. J. BETTERIDGE, J. E. ROWELL, AND P. F. FLOOD. 1989. Reproductive hormone concentrations in feces during the estrous cycle and pregnancy in cattle (Bos taurus) and muskoxen (Ovibos moschatus). Canadian Journal of Zoology 67: 1148–1154.
GEIST, V. 1971. Mountain sheep: A study in behavior and evolution. University of Chicago Press, Chicago, Illinois, 383 pp.
GUDORF, M., P. Y. SWEANOR, F. J. SINGER, A. BLANKENSHIP, V. BLEICH, T. EASTERLY, J. EMMERICH, C. EUSTACE, L. IRBY, D. JAYNES, B. JELLISON, R. KISSEL, J. LINDSAY, J. PARKS, T. PETERS, K. REID, S. STEWART, AND T. VOSS. 1996. Bighorn sheep habitat assessment of the Greater Bighorn Canyon National Recreation Area. National Park Service and National Biological Service cooperative report. Bighorn Canyon National Recreation Area, Lovell, Wyoming, 43 pp.
KIRKPATRICK, J. F., C. B. BAKER, J. W. TURNER, R. M. KENNEY, AND V. K. GANJAM. 1979. Plasma corticosteroids as an index of stress in captive feral horses. Journal of Wildlife Management 43: 801–804.
———, S. E. SHIDELER, B. L. LASLEY, AND J. W. TURNER. 1991. Pregnancy determination in un-captured feral horses by means of fecal steroid conjugates. Theriogenology 35: 753–760.
———, K. BANCROFT, AND V. KINCY. 1992. Pregnancy and ovulation detection in bison (Bison bison) assessed by means of urinary and fecal steroids. Journal of Wildlife Diseases 28: 590–597.[Abstract]
———, D. F. GUDERMUTH, F. L. FLAGAN, J. C. MCCARTHY, AND B. L. LASLEY. 1993. Remote monitoring of ovulation and pregnancy of Yellow-stone bison. Journal of Wildlife Management 57: 407–412.
———, J. C. MCCARTHY, D. F. GUDERMUTH, S. E. SHIDELER, AND B. L. LASLEY. 1996. An assessment of the reproductive biology of Yellowstone bison (Bison bison) subpopulations using non-capture methods. Canadian Journal of Zoology 74: 8–14.
KISSELL, R. E., JR., L. R. IRBY, AND R. J. MACKIE. 1996. Competitive interactions among bighorn sheep, feral horses, and mule deer in Bighorn Canyon National Recreation Area and Pryor Mountain wild horse range. Final report on Cooperative Agreement CA-1268-9017. Montana State University, Bozeman, Montana, 146 pp.
KNIGHT, D. H., G. P. JONES, Y. AKASHI, AND R. W. MYERS. 1987. Vegetation ecology in the Bighorn Canyon National Recreation Area. Final report submitted to the U.S. National Park Service and the University of Wyoming, National Park Service Research Center, 113 pp.
LARSEN, D. G., AND D. A. GAUTHIER. 1989. Effects of capturing pregnant moose and calves on calf survivorship. Journal of Wildlife Management 53: 564–567.
LASLEY, B. L., AND J. F. KIRKPATRICK. 1991. Monitoring ovarian function in captive and free-ranging wildlife by means of urinary and fecal steroids. Journal of Zoology and Wildlife Medicine 22: 23–31.
MESSIER, F., D. M. DESAULNIERS, A. K. GOFF, R. NAULT, R. PATENAUDE, AND M. CRETE. 1990. Caribou pregnancy diagnosis from immunoreactive progestins and estrogens excreted in feces. Journal of Wildlife Management 54: 279–283.
MONFORT, S. L., C. C. SCHWARTZ, AND S. K. WASSER. 1992. Monitoring reproduction in captive moose using urinary and fecal steroid metabolites. Journal of Wildlife Management 57: 400–407.
MUNRO, C. J., G. H. STABENFELDT, J. R. CRAGEN, L. A. ADDIEGO, J. W. OVERSTREET, AND B. L. LASLEY. 1991. Relationship of serum estradiol and progesterone concentrations to the excretion profiles of their major metabolites as measured by enzyme immunoassay and radioimmunoassay. Clinical Chemistry 37: 838–844.
SAFAR-HERMANN, N., M. N. ISMAIL, H. S. CHOI, E. MOSTL, AND E. BAMBERG. 1987. Pregnancy diagnosis in zoo animals by estrogen determination in feces. Zoo Biology 6: 189–193.
SALAMA, S. M., E. NEWNHAM, N. CHANG, AND D. E. TAYLOR. 1995. Genome map of Campylobacter fetus subspecies fetus ATCC 27374. FEMS Microbiology Letters 132: 239–245.[Medline]
SCHWARTZ, C. C., S. L. MONFORT, P. H. DENNNIS, AND K. J. HUNDERTMARK. 1995. Fecal progestagen concentration as an indicator of the estrous cycle and pregnancy in moose. Journal of Wildlife Management 59: 580–583.
SHIDELER, S. E., C. J. MUNRO, L. TELL, G. OWITI, L. LAUGHLIN, R. CHATTERTON, AND B. L. LASLEY. 1991. The relationship of serum estradiol and progesterone concentrations to the enzyme immunoassay measurements of urinary estrone conjugates and immunoreactive pregnanediol-3-glucuronide in Macaca mulatta. American Journal of Primatology 22: 122–133.
———, F. M. MORAN, P. N. LOHSTRAH, AND B. L. LASLEY. 1993. Enzyme immunoassays for ovarian steroid metabolites in urine of laboratory macaques. Journal of Medical Primatology 22: 301–312.[Medline]
STOOPS, M. A., G. B. ANDERSON, B. L. LASLEY, AND S. E. SHIDELER. 1999. Use of fecal steroid metabolites to estimate the pregnancy rate of a free-ranging herd of Tule elk. Journal of Wildlife Management 63: 561–569.
TURNER, J. C., AND C. G. HANSEN. 1980. Reproduction. In The desert bighorn, G. Monson and L. Sumner (eds.). University of Arizona Press, Tucson, Arizona, pp. 145–151.
Received for publication 8 May 2003.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
