Fertility Control in Free-Ranging Elk Using Gonadotropin-Releasing Hormone Agonist Leuprolide:...

Fertility Control in Free-Ranging Elk Using Gonadotropin-Releasing Hormone AgonistLeuprolide: Effects on Reproduction, Behavior, and Body ConditionAuthor(s): Mary M. Conner, Dan L. Baker, Margaret A. Wild, Jenny G. Powers, Muhammad D.Hussain, Richard L. Dunn, Terry M. NettSource: The Journal of Wildlife Management, Vol. 71, No. 7 (Sep., 2007), pp. 2346-2356Published by: Allen PressStable URL: http://www.jstor.org/stable/4496348 .Accessed: 14/02/2011 17:52

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Research Article

Fertility Control in Free-Ranging Elk Using Gonadotropin-Releasing Hormone Agonist Leuprolide: Effects on Reproduction, Behavior, and Body Condition

MARY M. CONNER,I Department of Wildland Resources, 5230 Old Main Hill, Utah State University, Logan, UT 84322-5230, USA

DAN L. BAKER,2 Colorado Division of Wildlife, Research Center, 317 W. Prospect Road, Fort Collins, CO 80526, USA

MARGARET A. WILD, National Park Service, Biological Resource Management Division, 1201 Oak Ridge Drive, Suite 200, Fort Collins, CO 80525, USA

JENNY G. POWERS, National Park Service, Biological Resource Management Division, 1201 Oak Ridge Drive, Suite 200, Fort Collins, CO 80525, USA

MUHAMMAD D. HUSSAIN, School ofPharmacy, Department 3375, 1000 E. University Avenue, University of Wyoming, Laramie, WY 82071, USA

RICHARD L. DUNN, Atrix Laboratories, Incorporated, 2579 Midpoint Drive, Fort Collins, CO 80525, USA

TERRY M. NETT, Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA

ABSTRACT Overabundant elk (Cervus elapbhus) populations have become a significant problem in many areas of North America. This is particularly true for protected areas where high densities of elk can cause long-term ecological degradation. When lethal control is not acceptable in these environments, resource managers must often consider alternative methods for reducing the size of resident elk populations. A potential management alternative is controlling the fertility of female elk. A promising new approach to wildlife contraception involves the use of biodegradable implants containing the gonadotropin-releasing hormone (GnRH) agonist leuprolide. During fall 2002-spring 2004, we compared pregnancy rates, reproductive behavior, daily activity patterns, and body condition of 17 free-ranging female elk treated with a leuprolide formulation with 17 untreated females, in Rocky Mountain National Park, Colorado, USA. After treatment, the pregnancy rate of treated elk was 0%, whereas 79% of control elk became pregnant. The effects of treatment were reversed the subsequent year with the pregnancy rate of treated females 100% compared with 91% for controls. Reproductive behaviors were similar for treated and control elk during the breeding and postbreeding seasons; treated elk did not exhibit postrut reproductive behaviors. Moreover, we found no differences in daily activity patterns of experimental groups during the breeding or postbreeding seasons. Treated elk did not show improved body condition over pregnant females. Instead, treated females catabolized proportionately more body fat during winter after treatment and at a higher rate than pregnant control elk. However, this effect was reversed the next spring with no difference in body fat between treated and control elk. We conclude from this experiment that leuprolide, administered as a controlled release formulation, offers a safe and effective method of controlling fertility in free-ranging female elk. However, practical application is limited by treatment duration and the need to treat females before the breeding season. (JOURNAL OF WILDLIFE MANAGEMENT 71(7):2346-2356; 2007)

DOI: 10.2193/2006-463

KEY WORDS Activity budgets, body condition, Cervus elaphus, Colorado, contraception, elk, fertility control, gonadotropin- releasing hormone (GnRH) agonist, leuprolide, reproductive behavior.

Overabundant elk (Cervus elaphus) populations have become a significant problem for natural resource managers in many areas of North America (Jewell and Holt 1981, Garrott et al. 1993, Porter et al. 1994, Wagner et al. 1995, Porter and Underwood 1999). Fertility control offers a potential nonlethal method for reducing abundance of elk populations when lethal methods are not acceptable (Naugle et al. 2002, Shideler et al. 2002, Walter et al. 2002, Rutberg et al. 2004). Extensive research has been directed toward the development of different contraceptive technologies (Fagerstone et al. 2002), and simulation models have been constructed to offer general insight into the effects of fertility control agents on free-ranging ungulate population dynamics (Boone and Wiegert 1994; Seagle and Close 1996; Barlow et al. 1997; Hobbs et al. 2000; Merrill et al. 2003, 2006). However, despite these concerted efforts, minimal progress

has been attained in developing novel contraceptive agents that are efficacious without undesirable side effects, and that are economical and commercially available for application in free-ranging populations.

A promising new approach to reversible contraception for wild ungulates involves the use of biodegradable implants of gonadotropin-releasing hormone (GnRH) agonist (e.g., leuprolide, deslorelin). Continuous treatment with high doses of GnRH agonist has been shown to cause temporary suppression of pituitary responsiveness and gonadotropin secretion. The resulting decreases in serum concentrations of luteinizing hormone and follicle-stimulating hormone lead to suppression of ovulation, estrous cyclicity, and gonadal steroidogenesis (D'Occhio et al. 1989, 1996; Evans and Rawlings 1994; Gong et al. 1995). These conditions persist as long as the agonist is present, but once concentrations decline below therapeutic levels, normal ovarian function is restored (Gorospe and Conn 1988, Bergfeld et al. 1996). Recently, the development of long- acting biodegradable implants that can deliver a sustained

1 E-mail. [email protected] 2 Present address: Animal Reproduction and Biotechnology Labo- ratory, Department of Biomedical Sciences, Colorado State Uni- versity, Fort Collins, CO 80523, USA

2346 The Journal of Wildlife Management * 71(7)

release of GnRH agonist over a predetermined time have improved the practicality of this approach for wildlife applications (Ravivarapu et al. 2000, Trigg et al. 2001).

The use of GnRH agonist implants to suppress ovulation and reproduction is well documented for a number of domestic species, including dogs (Trigg et al. 2001), cattle (McLeod et al. 1991, D'Occhio et al. 2002), sheep (McNeilly and Fraser 1987), and horses (Montovan et al. 1990). Similar responses have been reported for a variety of captive wildlife species (Becker and Katz 1995; Baker et al. 2002, 2004; Brown and J6chle 2002; Herbert and Cooper 2006). However, to our knowledge, their evaluation in free- ranging wild ungulates has not been accomplished.

The present study is the second part of a 2-phase evaluation of the GnRH agonist leuprolide as a contraceptive agent in female elk. The goals of the initial phase of research were to develop an effective dose and delivery system in captive elk and to begin documenting any negative behavioral or physiological side effects of treatment. Results from this phase of research have been reported previously by Baker et al. (2002). Although captive investigations have provided fundamental knowledge on the potential utility and safety of leuprolide as an infertility drug, inferences to free-ranging elk are limited due, in part, to the artificial environment in which these measurements were taken. This is particularly true with regard to potential effects of the agent on body condition, reproductive behavior, and daily activity patterns. Thus, additional study is needed in free- ranging elk to evaluate whether this technology is truly safe, effective, and without undesirable side effects.

To address these questions, we conducted a field experiment with free-ranging elk in Rocky Mountain National Park (RMNP), Colorado, USA, to examine the efficacy of leuprolide as a contraceptive agent. Specifically, our objectives were to evaluate the effects of a leuprolide on pregnancy rates, reproductive behavior, daily activity patterns, and body condition of treated elk. We tested the following 3 a priori hypotheses: H/) Based on previous results from captive elk experiments (Baker et al. 2002), we predicted that leuprolide formulation would be 100% effective in suppressing reproduction in free-ranging elk and that the duration of infertility would extend for >1 breeding season. H2) In a prior experiment (Baker et al. 2002), we tested 2 hypotheses related to the effects of leuprolide on the reproductive behavior of captive female elk: 1) we predicted that leuprolide would suppress ovulation and estrous behavior during the breeding season, and as a result, sexual interactions would be reduced in treated elk compared with controls; and 2) we predicted that depletion of the leuprolide implant would occur before seasonal anestrus and as a result, ovulation, and estrous behaviors would increase in treated females compared with controls after the breeding season. Because we tested these hypotheses in an artificial setting with captive elk, we were uncertain whether previous findings would apply to free- ranging animals in a natural state. To gain additional insight into the effects of leuprolide on reproductive behaviors, we

retested these hypotheses with free-ranging elk in RMNP. H3) Because pregnancy has been shown to negatively affect body condition of female wild ungulates through increased energetic demands of gestation and lactation (Robbins 1983), we predicted that contracepted elk would show improved body condition over pregnant control elk (Kirkpatrick et al. 1997, McShea et al. 1997, Miller et al. 2001, Turner and Kirkpatrick 2002).

STUDY AREA We conducted this research primarily on elk winter ranges in RMNP. Elk wintered in the eastern portion of the park and adjacent Estes Valley, which includes the town of Estes Park, Colorado. Elk winter range encompassed approximately 8,000-10,000 ha between 2,500 m and 2,800 m in elevation. Glacial moraines extending eastward from the Continental Divide separate the winter range into 4 parallel valleys: Beaver Meadows, Horseshoe Park, Moraine Park, and Hollowell Park (Gysel 1959, Schoenecker et al. 2004). Vegetation was typical of the upper montane climax region (Marr 1967). During the study period, winter elk population estimates for the park and Estes Valley ranged from 1,700 animals to 2,200 animals, respectively, with >100 elk/km2 on some core winter ranges (Monello et al. 2005).

For elk in North America, the onset of the breeding season generally occurs in early September when dominant males begin to establish and defend a collection or harem of females. Breeding activities peak in early October, then gradually diminish by the end of the month. Most females conceive during the first estrous cycle; however, if conception fails, 5-8 recurrent estrous cycles of 18-21 days in length, extending through March, can occur. Once reproductive cycles cease, elk remain anestrus from April through August. For most females, parturition occurs in June after a gestation period of approximately 255 days (Morrison et al. 1959, Guinness et al. 1971, Bear 1989, Haigh and Hudson 1993).

METHODS Experimental Animals and Treatments Our study was conducted with the approval of the Colorado Division of Wildlife Animal Care and Use Committee

(study no. 5-2002) and in compliance with the United States Federal Animal Welfare Act Regulations. During 26 August 2002 to 23 March 2004, we evaluated the effects of leuprolide on pregnancy rates, reproductive behavior, daily activity patterns, and body condition of free-ranging female elk. We did not randomly select experimental animals. Instead, we attempted to choose only females of prime reproductive age (3-10 yr) and in moderate or better nutritional condition (Cook et al. 2001a). We assumed that if these criteria could be met, reproductive failure, if it occurred, would more likely be related to the effects of the contraceptive agent than diminished nutritional condition or ovulation failure of randomly selected animals (Albon et al. 1986, Fisher et al. 2000, Cook et al. 2004). We accomplished this in 2 ways. First, before chemical

Conner et al. * GnRH Agonist Contraception in Free-Ranging Elk 2347

immobilization, we made a visual assessment of the target animal's age (calf, yearling, ad, Smith and McDonald 2002) and relative fatness and body musculature (Riney 1960). Second, once the animal was anesthetized, we estimated age by tooth replacement and wear (Quimby and Gaab 1957), evaluated body condition using a body condition scoring system developed specifically for elk, and measured maximum rump fat depth with ultrasonography (Cook et al. 2001a, b). Then, using body condition indices and predictive equations developed by Cook et al. (2004), we determined the probability of pregnancy as a function of body fat (%) and selected as experimental animals only those females with

>90% probability of pregnancy (210% body fat). We captured elk by driving the roads in RMNP, locating

groups of elk, and then darting a selected individual from the vehicle or stalking the animal on foot. Most shots were at ranges of <35 m. We used a mixture of 2.7 mg of carfentanil (Wildlife Pharmaceuticals, Fort Collins, CO) and 10 mg ofxylazine (Tranquived; Vedco, St. Joseph, MO) delivered in a barbed, 1- or 2-cc self-injecting dart that we fired from a CO2 rifle (Dan-InjectTM; Dan-Inject of North America, Fort Collins, CO). We recorded capture locations (Universal Transverse Mercator) on a Garmin 12 hand-held Global Positioning System unit. Immediately after capture, we blindfolded the elk, monitored vital signs, estimated age, determined lactational status, palpated the reproductive tract for abnormalities, collected blood (20 ml), assessed body condition, and measured fat depth and muscle thickness

using ultrasonography. If the captured elk met the age and body condition criteria established previously, we randomly assigned it to either a treatment or control group.

We administered a subcutaneous injection, by hand- syringe, containing a polymeric matrix formulation of leuprolide acetate (D-Leu6-GnRH-Pro9-ethylamide) to treatment elk. This formulation was designed to deliver a 32.5-mg dose ofleuprolide at a controlled rate over a 90-day therapeutic period. A similar formulation was shown previously to suppress ovulation and pregnancy in captive elk for one breeding season (Baker et al. 2002). Control elk received the same polymer solution as the treatment group, but without leuprolide.

We fitted all experimental elk with very high-frequency radiocollars (Advanced Telemetry Systems, Isanti, MN). We sheathed radiocollars with a plastic identification sleeve marked with a unique alphanumeric code of 76-mm-high black characters on a colored background (white for control, yellow for treatment; Freddy 1993). We marked radiocollars with the words "Do Not Consume" and agency contact information. We reversed the effects of the immobilizing drugs with 300 mg of naltrexone (1/4 IV, 3/4 SQ; Wildlife Pharmaceuticals) and yohimbine (30 mg IV; Wildlife Pharmaceuticals). To minimize the possibility of infection associated with immobilization, we administered a subcutaneous dose (6,000,000 international units) of long-acting penicillin (Durapen; Vedco) to each elk. We removed radiocollars from as many experimental elk as possible after completion of final measurements.

Reproduction We evaluated the effects of leuprolide treatment on elk pregnancy rates during 24 March-15 April 2003 and the reversibility of these effects during 4-23 March 2004. We assessed contraceptive effectiveness and reversibility by recapturing as many of the original experimental elk as possible and comparing pregnancy rates of treated and control groups. We used radiotelemetry to locate animals, and we recaptured them using methods described previously. We confirmed pregnancy using 2 independent methods: rectal palpation of the fetus, fetal membranes or uterine cotyledons (Greer and Hawkins 1967), and pregnancy-specific protein B (PSPB) analysis (BioTracking, Moscow, ID; Willard et al. 1994, Noyes et al. 1997, Huang et al. 2000).

Behavior We tested behavioral hypotheses by examining the effects of leuprolide on reproductive interactions between male and female elk during 2 time periods. We defined these periods as the normal breeding period (11 Sep-31 Oct 2002) and postbreeding period (6 Jan-12 Mar 2003). We used a restricted randomized design to balance observations as much as possible among all experimental animals while also trying to observe the behavior of each female 2 times per week during the defined breeding periods (Heilmann et al. 1998). We located groups containing selected experimental elk by driving roads and using radiotelemetry. We generally made observations from a distance of 50-300 m with the aid of binoculars and spotting scopes. We sampled behaviors during the morning (sunrise-1100 hr), afternoon (1100- 1600 hr), and evening (sunset-1600 hr). We terminated an observation session if the focal elk or its associated group became disturbed by the observer or any other outside influence (e.g., dogs, vehicles, people). Each sampling period consisted of 1 hour of continuous observations.

We used focal animal sampling to record reproductive behaviors of experimental elk (Altmann 1952, Lehner 1996). To increase our sample size, we recorded behaviors not only on the focal animal but also for any experimental animal in the group with the focal animal. Based on previously reported elk breeding behaviors, we identified and recorded 19 reproductive interactions (Morrison et al. 1959, Geist 1982, Rapley 1985, Baker et al. 2002). However, because sample sizes were small, we grouped individual behaviors into 4 general categories: general breeding, male precopulatory, female precopulatory, and copulatory (Table 1). Thus, our experimental unit for analyses was the individual female elk. Sexual interactions were generally short duration (<30 sec) relative to sampling interval; therefore, we recorded the number of occurrences of each event rather than length of time and calculated reproductive behavior rates as behaviors per animal per hour.

To evaluate the effect of leuprolide on nonbreeding activities, we simultaneously sampled daily activity patterns of the same experimental elk. We made these measurements in conjunction with breeding behavior observations. Similar to breeding behavior sampling, we observed activity states of


Table 1. Description of reproductive and nonreproductive behaviors and associated behavior categories used during breeding (11 Sep-16 Oct 2002) and postbreeding (6 Jan-12 Mar 2003) seasons for female elk treated with a sustained release formulation of leuprolide in Rocky Mountain National Park, Colorado, USA.

Behavior category Description

Reproductive behaviors General breeding Male directed behavior related to establishing, maintaining, and defending a group or harem of female elk

(e.g., herding, guarding, tending) M precopulatory Male courtship behavior directed toward an individual female to induce or detect estrus or ovulation

(e.g., flehmen, tongue flick, smell, rub) F precopulatory Female courtship behavior directed toward the dominant male to arouse copulatory behavior

(e.g., lick and rub male, mount, lordosis, twitch hocks) Copulatory Male behavior directed toward a receptive female in estrus (e.g., intromission)

Activity budget behaviors

Feeding Standing or moving with head in vegetation (e.g., grazing, browsing) Moving Actively traveling with head up (e.g., walking, trotting) Idling Lying or standing but not feeding (e.g., bedded, ruminating, watching) Not seen Out of view of the observer

the focal animal, as well as any other experimental animal in the group with the focal animal. We observed the activity state of each elk at 2-min intervals and classified behaviors as feeding, idling, moving, or not visible (Table 1; Altmann 1952, Heilmann et al. 1998).

Body Condition We assessed the effects of leuprolide on elk body condition by comparing changes in body fat and muscle thickness of treated and control elk recaptured at 2 subsequent time periods: spring 2003 (6.5-7.5 months posttreatment) and spring 2004 (18.0-19.0 months posttreatment). We determined body fat (%) using a body condition scoring system developed for elk that was derived from palpation of the biceps femoris muscle (rump) combined with direct measurements of subcutaneous rump fat thickness using a portable Aloka, 500v ultrasound machine with a 5.0-MHz, linear array transducer (Corometrics Medical Systems, Wallingford, CT). We combined these 2 measurements to estimate body fat (%) using the equation body fat (%)=- -7.1527 + 7.3231x - 0.9898x2 + 0.0574x3, where x is rLIVINDEX, an arithmetic combination of 2 indices- subcutaneous rump fat thickness and rump body condition score (Cook et al. 2001a, b). We also used ultrasonography to measure thickness of the longissimus dorsi (loin) muscle between the 12th and 13th rib directly below the backbone. This measurement has been shown to provide a reasonably good measure of catabolism of lean muscle and is particularly useful for elk in poor or declining body condition (Stephenson et al. 1998; Cook et al. 2001a, b, 2004).

Statistical Analysis Reproduction.-To determine the sample size needed to

detect treatment differences for pregnancy rates, we performed a power-based sample size determination for a 1-sided Fisher's exact test (Krishnamoorthy and Thompson 2002, Kang and Kim 2004) using a software program, NCSS Pass 2000 (NCSS Statistical Software, Kaysville, UT). For this analysis, we assumed a 100% infertility rate for elk treated with leuprolide (Baker et al. 2002), 90%

recapture success for all experimental elk, and 95% accuracy of PSPB analysis for any elk >100 days gestation (Huang et al. 2000). To represent the worst-case scenario for control elk, we assumed the lowest pregnancy rate (63%) reported for elk in RMNP (Bender and Cook 2002). Our null

hypothesis was that the minimum difference in pregnancy rates would be 0.63, with pregnancy rates of untreated elk greater than treated elk. Based on this analysis, we estimated 25-30 female elk (equally divided between control and treatment groups or 13-15 elk per treatment group) to provide an adequate power (>80%) to detect expected differences in pregnancy rates. For year 1, we used a 1-sided Fisher's exact test to determine whether pregnancy rates of treated elk were lower than controls, and for year 2 we used a 2-sided Fisher's exact test to determine whether these rates were different between groups.

Behavior.-We conducted Monte-Carlo simulations

using bootstrapped data from a captive elk experiment (Baker et al. 2002) in a model based on our proposed experimental design. We used this approach to estimate sample sizes required to achieve acceptable statistical power (>80%) to detect fixed differences (>50%) in reproductive behavior rates during the breeding season. We constructed an analysis of variance (ANOVA) model (PROC MIXED; Littell et al. 1996, SAS Institute 1999), and we included treatment group and observation period (as a categorical variable) as fixed effects and individual elk as a random effect with a repeated measures structure by observation period. We used male precopulatory behavior rate as the behavioral response variable in our power simulations because it had the highest coefficient of variation compared with the other behavioral response variables in the captive elk experiment. We reasoned that if there was sufficient power to detect a difference in this behavior rate, the same effect size should be detected in the other behavior rates as well, due to their lower coefficients of variation.

We created a sample of behavior rates for each observation period by bootstrapping a given sample size (no. of control and treated elk) from captive elk data for each observation period. Behavior data from control elk were considered the

Conner et al. D GnRH Agonist Contraception in Free-Ranging Elk 2349

benchmark for comparison. That is, we only bootstrapped data from captive elk in the control group. To represent a 50% decrease in behavior rates of treated elk, we multiplied a behavior rate from a randomly selected control elk by 0.5. Once we selected data for a specified number of elk and observation periods, we analyzed the data set using the ANOVA model, and we recorded the number of times that behavior rates of treatment elk were statistically lower than behavior rates of control elk (i.e., P < 0.05; 1-sided test). We repeated this process 1,000 times. Thus, for each design (combination of no. of treatment and control elk and observation periods per elk), the power, or probability of detecting a difference in behavior rates, was the number of times we detected a difference divided by the number of simulations.

To estimate adequate sample sizes for behavior measurements, we used the initial minimum sample size criteria determined previously for detecting differences inpregnancy rates (13-15 elk/treatment group), and then we simulated the number of elk required for treatment and control groups with their attendant number of required observation periods (elk observed twice/week) to be able to detect a >50% difference in behavior rates at an acceptable power level (>80%). From these simulations, we determined that a

sample size of 34 elk (17/treatment group) and >16 observation periods per elk (e.g., 2 observations/week for 8 weeks) would be sufficient to meet this requirement.

We did not conduct power analyses for the postbreeding season because previous investigations with captive elk indicated that almost no reproductive behaviors occurred during this period (Baker et al. 2002). Thus, for the postbreeding season we used the same minimum number of observation periods as determined for the breeding season.

For analysis, we used an ANOVA model with a repeated measures structure (PROC MIXED; Littell et al. 1996, SAS Institute 1999), and we tested specific null reproductive behavior hypotheses that mean behavior rates would be lower for treated elk during the breeding season and higher during the postbreeding season compared with controls. Our model construction strategy for analyzing the reproductive behavior was as follows. 1) Construct a global model that included treatment as a fixed effect, observation period as a categorical variable, and female elk modeled as a random component. 2) Fit 4 variance structures to the global model and use Akaike's Information Criterion adjusted for small sample size (AICc) model selection to choose the best structure. The 4 variance structures we evaluated were 1) variance between females in their mean behavior rates was allowed to differ for treatment and control groups, 2) variance within female (via the repeated measurements) was allowed to differ for the treatment and control groups, 3) both forms of variance were heterogeneous between groups (1 and 2 above), and 4) both forms of variance were homogeneous between groups. 3) For the global model with the best variance structure, fit 3 covariance structures for each response variable and use AICc model selection to choose best structure. The covariance structures we

evaluated were appropriate for unequal time intervals and missing data: variance components (no covariance between measurements but accounts for repeated measurement and random effects), compound symmetry, and spatial power, which is an autoregressive covariance structure generalized for unequal time intervals. 4) Using the global model with the best variance and covariance structure for each response variable, construct a set of more parsimonious models and use AICc model selection to choose best model. We constructed 10 competing models (Table 2). 5) Using the model with the lowest AIC, estimate behavior rates for treated and control elk for each behavior group (Table 1).

We estimated activity budgets as the proportion of time each individual spent feeding, idling, or moving (Table 1) based on the total number of times we observed each animal. The model for analyzing activity budget observations was the same as the best model for reproductive behavior rates during the breeding season. For behavior rates and activity proportions, we estimated means and standard errors using least squares analysis, and we based hypothesis tests on type III generalized estimating equations to account for sample size imbalance.

Body condition.-Because the intervals between measurements varied from elk to elk, we standardized body fat (%) and loin depth (cm) measurements for spring 2003 and spring 2004 as follows: standardized measurement = non- standardized measurement + [(d between measurements - x d between measurements for group) x change in measurement/d]. We used standardized estimates for all

subsequent analyses. To evaluate the effect of leuprolide on body condition of

treated and control elk, we used an ANOVA model in PROC MIXED (Littell et al. 1996, SAS Institute 1999) with a repeated measures structure, and we tested for treatment, season, and treatment x season effects. We first modeled the variance-covariance structure for body fat (%) and loin depth (cm) via a model that included treatment and season as fixed (categorical) effects, with repeated measurements taken on experimental elk. Because we were unable to capture and sample every experimental animal during each season, we evaluated 3 covariance structures appropriate for unequal time intervals and missing values; variance components (no covariance between measurements but accounts for repeated measurement and random effects), compound symmetry, and spatial power, which is an autoregressive covariance structure generalized for unequal time intervals (Littell et al. 1996). We then selected the most appropriate variance-covariance structure using AICc model selection for subsequent estimates and tests. Additionally, we used this analysis structure to estimate the rate of change in body fat (%) and loin depth (cm) among seasons and tested the null hypothesis that rate of change of these indices were similar for treatment and control groups.

RESULTS Before the breeding season (26 Aug-5 Sep 2002), we successfully captured and radiocollared 34 female elk (17


Table 2. A priori models relating treatment group, maximum group size (group size), time of day (tod), and temporal influences (observation period) for estimating behavioral responses during breeding (11 Sep-16 Oct 2002) and postbreeding (6 Jan-12 Mar 2003) seasons for female elk treated with a sustained release formulation of leuprolide in Rocky Mountain National Park, Colorado, USA.

Model structure Ka Justification

Treatment group + observation period (as categorical variable) 128 Most global model that would converge Treatment group 3 More parsimonious additive effects Treatment group + observation period (as trend variable) 4 More parsimonious additive effects with time modeled as a rate Treatment group + tod 6 More parsimonious additive effects Treatment group + tod + observation period (as trend variable) 12 More parsimonious additive effects with time modeled as a rate Treatment group + group size (as rate) 4 More parsimonious additive effects Treatment group + group size (as categorical) 61 Evaluate whether group size is a better predictor than time Treatment group + group size + observation period 5 More parsimonious additive effects with time modeled as a rate

(as trend variable) Treatment group + group size + tod + observation period 8 All additive effects with time modeled as a rate

(as trend variable) From best structure above, add group size x time + treatment b Check for likely interactions, for example, we expected behavior rates

group x time, with time as a trend variable may decline faster for treated group compared with control group

a K= no. of parameters estimated in the model for fixed effects as estimated in PROC MIXED (SAS 1999) by the design matrix specification (X matrix). b The no. of parameters will vary depending on the top model.

treatment, 17 control) in RMNP. We captured 14 elk from the Kawuneeche Valley and surrounding alpine and subalpine areas on the west side of the park, and 20 from the general area of Beaver Meadows and Moraine Park on the east side of the park. The subsequent spring (Mar-Apr 2003), we recaptured and pregnancy-tested 14 treated females and all 17 control elk. We confirmed reversibility of the contraceptive treatment the next spring (Mar 2004) by recapturing 10 treated females and 12 controls (Table 3). During the course of the study, we captured experimental elk on 65 occasions without a capture-related mortality. Two elk were killed by hunters outside the park, 1 elk died from chronic wasting disease, and 6 elk died of unknown causes (2 treated elk, 4 control elk).

Reproduction Fertility measurements supported our prediction (H/) that leuprolide formulation would be 100% effective in prevent- ing pregnancy in free-ranging elk. Leuprolide suppressed pregnancy in all treated females, whereas pregnancy rate of control elk was 78.6% as determined by PSPB analysis and 82.3% when assessed by rectal palpation diagnosis (P < 0.001 for both tests, df = 1, 1-sided Fisher's exact test). Furthermore, the effects of leuprolide treatment were reversible, and they did not extend beyond one breeding season. Pregnancy rates were not significantly different for treated (100%) and control elk (90.9%; P= 1.000 for both

tests, df = 1, 2-sided Fisher's exact test) 1 year posttreatment, regardless of method of pregnancy detection.

Behavior

During the breeding season, we observed sexual interactions of female elk from 11 September to 2 November 2002. However, by 16 October daily reproductive behavior rates for all behavior categories had effectively declined to zero (Fig. 1). Therefore, to avoid diluting behavior rates by including uninformative zeros in our calculations, we restricted our analysis to 11 September-16 October. For this period, we collected data from 16 treated (153 hr) and 16 control (96 hr) elk during 125 observation periods (2 elk moved to locations that were inaccessible for observation). Total hours of observations exceeded the 125 observation periods because in many situations we observed >1 focal animal in a group. We evenly distributed observation periods throughout the day: 32% morning, 29% afternoon, and 39% evening. Group size associated with the focal animal averaged 35.6 (SE = 2.7) and 29.8 (SE = 2.6) animals per group for control and treatment elk, respectively. The number of male elk (yearlings and ad) associated with the focal animal(s) averaged 3.4 (SE = 0.2) males per group for control elk and 3.0 (SE = 0.2) for treated females.

Our best model (36 AAIC, units less than second best model) included treatment and observation period (time) as fixed categorical effects, and female elk as a random effect, and allowed within-individual variation to differ between female elk in control and treatment groups. Model residuals showed no major violation of assumptions. We followed the same procedure for the postbreeding season data analysis. Due to the sparse data, the best model (12 AAIC, units lower than second best model) contained only treatment as a

Table 3. Capture dates, sample size, and associated measurements taken September 2002 to March 2004 for female elk treated with a sustained release formulation of leuprolide in Rocky Mountain National Park, Colorado, USA.

Date Sample size Measurements

Fall 2002 17 treatment, 17 controls Capture elk and apply treatments: measure body condition and reproductive behavior Spring 2003 14 treatment, 17 controls Recapture elk: measure pregnancy rates and body condition Spring 2004 10 treatment, 12 controls Recapture elk: measure pregnancy rates and body condition


8.0 a. M precopulatory -Control

7.0 - - - Treatment

6.0- o r

5.0 .

Breeding period I Postbreeding period] 4.0 ,

3.0 :: S,0- T,

2.0 - .,

0.0 3.5

b. F precopulatory - Control

- -Treatment 3.0-

2.5-

S2.0 - Breeding period I Postbreeding period

L_(

. 1.s

• 1.o

s , 0.0 3.5

C. General breeding -Control -?-Treatment

3.0 -

2.5 -

2.0 - :: Breeding period I Postbreeding period

! ,,

1.5 -

1.0 - ,., 0.5 -

o n 0.0

0 CD 0 0 0 0 0 0 0 0 C 0 0 0 0 oL CL - o) > > o o o o u o o

C) W U Z0 Z 0 0 C) a -M -M C.) _

V--- , z'

, 0 LL.

- , LD '-- ,-

o4L C V O ,.-- 0,cc

C "--

0 LO 0 4 0

Date

Figure 1. Mean hourly behavior rates by day for (a) male precopulatory, (b) female precopulatory, and (c) general breeding behaviors during the breeding (11 Sep-16 Oct 2002) and postbreeding (6 Jan-12 Mar 2003) seasons for 16 control and 16 female elk treated with a sustained release formulation of

leuprolide in Rocky Mountain National Park, Colorado, USA. Note: y-axis scale is different for each graph.

fixed effect and did not support differences in within individual variation by treatment group.

Behavioral results did not support our prediction (H2a) that during the breeding season sexual interactions would be significantly reduced in treated elk compared with controls. Instead, we found no statistically significant effects of leuprolide on behavior rates of female elk for any behavioral category (P > 0.187; Fig. 2). Daily breeding activities were highly variable but they tended to begin during the first week of September, peak in early October, and abruptly end by the end of October (Fig. 1). Extended sexual interactions after October were rare, and we observed them only for female precopulatory behaviors (Fig. lb). We did not observe copulatory behavior during the study.

In the postbreeding season (6 Jan-12 Mar), we observed 13

1.2 *Control * Treatment

1.0 P= 0.709 P= 0.187 P= 0.498

0.8 -

0

o

S 0.6

CO

0.4

0.2

0.0, M precopulatory F precopulatory General breeding

Behavior category

Figure 2. Mean hourly reproductive behavior rates during the breeding (11 Sep-16 Oct 2002) season for 16 control and 16 female elk treated with a sustained release formulation of leuprolide in Rocky Mountain National Park, Colorado, USA. Error bars represent 95% confidence intervals.

treated (205 hr) and 15 control (299 hr) elk during 355 observation periods. Similar to the breeding season, observations were distributed relatively equally throughout the day; 31% morning, 41% afternoon, and 28% evening. Average group size was 207.1 (SE = 0.6) for controls and 198.7 (SE = 11.5) for treated elk. The number of male elk (yearlings and ad) per group averaged 9.7 (SE = 0.5) for the controls and 9.6 (SE = 0.6) for treated elk.

Behavioral results did not support our prediction (H2a) that sexual interactions would increase in treated elk after

depletion of the leuprolide implant compared with controls. Treated females apparently did not resume normal estrous cycles after implant diminution; reproductive behavior rates (no. of behaviors/hr) were low for both groups during the postrut period (Fig. 1). Male precopulatory rate was 0.02 (SE 0.01) for treated and 0.00 (SE = 0.01) for control elk (df 26, P = 0.099). General breeding behavior rate was 0.00 behaviors per hour (SE = 0.01) for both groups (df- 26, P - 0.238). We could not perform a statistical test for female precopulatory behaviors because we did not observe any during the postbreeding period.

Leuprolide treatments had no significant effect on the activity budgets of treated elk during the breeding (P > 0.204) or postbreeding seasons (P > 0.266; Fig. 3). With the exception of idling time, the proportion of time that we observed elk feeding and moving was similar between breeding and postbreeding seasons. Both groups of elk spent less time idling during the breeding season than the postbreeding period (2-sided test, P 0.020; Fig. 3).

Body Condition With the exception of lactational status, experimental groups were similar at the beginning of the study with regard to age, body fat (%; t = -1.54, df = 34, P = 0.133; Fig. 4a), and loin depth (cm; t = -0.58, df = 34, P= 0.567; Fig. 4b). There was a slightly higher proportion of lactating females in the treatment group compared with the controls (76.5% vs. 68.7%). Average values across all animals were


0.7 a. 0 Control

0.6 - P= 0.204 O Treatment

S0.5- E r 0.4 0 P= 0.674

0 0.3 CD.. 0 a 0.2 - P= 0.257

0.1 P= 0.338

0.0- Feeding Idling Moving Not seen

Behavior category

0.6- b. V% Control b.P= 0.266 cnol

05-P= 0.406 O0Treatment 0.5-

e E 0.4

CL

, 02-

0.1 - P= 0.639 P= 0.625 P = 0.625

0.0, Feeding Idling Moving Not seen

Behavior category

Figure 3. Mean proportion of time spent in nonreproductive activities during (a) breeding (11 Sep-16 Oct 2002) and (b) postbreeding (6 Jan-12 Mar 2003) seasons for 16 control and 16 female elk treated with a sustained release formulation of leuprolide in Rocky Mountain National Park, Colorado, USA. Note that the estimated proportions of time spent in nonreproductive activities does not sum to 1.0 for the breeding season because some proportion of time involved reproductive behaviors. Error bars represent 95% confidence intervals.

6.7 (SE = 0.5) years of age, 12.4% (SE = 0.3) body fat, and a loin thickness of 5.4 (SE = 0.07) cm.

Contrary to our hypothesis (H3), contracepted elk did not show improved body condition over pregnant controls. Instead, treated, nonpregnant females catabolized proportionately more body fat and lean body mass during winter than pregnant control elk (Fig. 4). Although we did not observe significant overall treatment effects of leuprolide on body fat (3 = 0.21, SE = 0.38, df = 26, P = 0.585) or loin depth (P = 0.03, SE = 0.10, df = 25, P = 0.615), we did detect a significant seasonal effect for both responses (body fat P < 0.001, loin depth P = 0.004). Note, there are >1 parameter estimates for season and treatment group x season effects; we do not present them here because their interpretation is not directly related to P-value or the estimates and 95% confidence intervals presented in Fig. 4. We expected the lack of treatment effect due to the observed interaction between treatment and season, which was significant for body fat but not for loin depth (body fat P= 0.008, loin depth P= 0.407). This interaction reflected the fact that body fat and loin depth were lower for treated

16 a. 0 Control

14 P= 0.133 OTreatment

12 - P= 0.609

10- -- P= 0.005

co~

co 6-

0 - Sep 2002 Mar 2003 Mar 2004

6.0 b. M Control

O Treatment P= 0.567

5.5 - P = 0.911 E P= 0.199

u

0-5.0-

0

4.5-

4.0- Sep 2002 Mar 2003 Mar 2004

Figure 4. Estimates of average total body fat (%), (a) calculated from LIVINDEX (Cook et al. 2001a, b) and loin muscle depth (cm), and (b) (Cook et al. 2001a, b, 2004) for female control elk and female elk treated with a sustained release formulation of leuprolide during September 2002 and March 2003-2004, in Rocky Mountain National Park, Colorado, USA. Error bars represent 95% confidence intervals.

elk in spring 2003 (Fig. 4). Between September 2002 and March 2003, treated elk tended to lose more body fat (57% vs. 38%) and lean body mass (12.6% vs. 6.3%) than controls such that by the end of winter the majority (93%) of treated animals retained less than 5% body fat.

For the same time period (Sep 2002-Mar 2003), we found rates of decrease in body fat (% change/d) was greater for treated elk (P = -0.036, SE = 0.002) than for controls (P =

-0.021, SE = 0.002, df = 14, P= 0.001). Similarly, rates of decrease in loin depth (% change/d) was greater for treated elk (P = -0.003, SE = 0.001) than for controls (P =

-0.0015, SE = 0.001, df= 14, P= 0.039). The rate of loss of body fat and loin depth was almost twice as much for treated elk compared with controls. However, once the effects of leuprolide were reversed and previously treated elk became pregnant (Mar 2004), we found no difference in body composition between groups (body fat P= 0.609, loin depth P= 0.911; Fig. 4).

DISCUSSION

Leuprolide, administered as a single injection, sustained release formulation, successfully (100%) suppressed reproduction in free-ranging female elk for one breeding season.

Conner et al. 0 GnRH Agonist Contraception in Free-Ranging Elk 2353

All treated elk regained normal reproductive function and conceived the next year. These findings are consistent with previous studies of captive elk (Baker et al. 2002, 2005), and they provide unambiguous evidence that leuprolide formulation can be a highly effective, short-term contraceptive agent in this species. Moreover, these results concur with those from previous studies in which long-acting GnRH agonist formulations effectively controlled reproduction in other mammalian species (Trigg et al. 2001, D'Occhio et al. 2002, Baker et al. 2004, Herbert et al. 2005, Herbert and

Cooper 2006). Leuprolide did not suppress reproductive behaviors of

female elk during the normal breeding season, nor did treated females display breeding behaviors during the postbreeding season. These results also parallel previous observations of captive elk (Baker et al. 2002), with one exception. In captivity, rates of male precopulatory behaviors during the breeding season were almost twice as high for treated females compared with controls. Increased behavior rates for treated females occurred because male precopulatory behaviors did not decrease during the breeding season, whereas for control elk, it dropped to zero once females became pregnant. Consequently, we found a significant interaction in behavior rates between time and treatment in the control group. In contrast, we did not observe these differences in behavior rates in free-ranging elk. Either these behaviors were exacerbated by confinement, did not occur in a natural setting, or they occurred at such a low rate that our sampling design was unable to detect them. Also, unlike captive experiments, where behavior rates could be modeled as an interaction between treatment group and time, here we were unable to observe every elk at each sampling period. Consequently, we did not have sufficient data to test for a small interactive effect.

We failed to detect any differences in the proportion of time spent, feeding, idling, or moving for treated elk compared with controls during the breeding or postbreeding seasons. Although similar measurements with GnRH agonist in other species are unreported, our results are similar to those reported previously for free-ranging elk (Heilman et al. 1998) and captive white-tailed deer (Odocoileus virginianus; McShea et al. 1997) treated with immunocontraceptive vaccines.

During late winter, treated elk were compromised in body fat composition (but not protein mass) compared with pregnant controls. Both groups of elk entered the breeding season in similar body condition, but by late winter (Mar 2003) most pregnant control elk (90%) >5% body fat reserves, whereas only 53% of treated, nonpregnant elk maintained this level of condition. Once the effects of treatment were reversed and treated females became

pregnant, differences in body condition seemed to converge. Improved body condition of pregnant females compared

with nonpregnant females during late winter was unex- pected, but it may be explained by the influence of gonadal hormones on food intake, body weight dynamics, and carcass adiposity. These nutritional indicators fluctuate

dramatically in response to differences in reproductive status. A delicate balance exists between progestins, estro- gens, and androgens that can influence the quantity and anatomical site of fat storage (Wallace et al. 1999, Pedersen et al. 2004, Rodriguez-Cuenca et al. 2006). Progesterone, the primary reproductive hormone during pregnancy, has been shown to promote subcutaneous fat deposition in primates (Wallace et al. 1999) and decreases lypolysis in rat adipocytes (Sutter-Dub and Cordoba 1997). Because reproductive hormones associated with pregnancy have also been shown to increase voluntary food intake and fat deposition (Wade 1975, Wade and Gray 1979, Schwartz and Wade 1981), pregnant elk could be predisposed to achieve seasonal target fat reserves that would ensure adequate milk production for neonatal survival. In contrast, these nutritional constraints may be relaxed in nonpregnant elk, and in the absence of a developing fetus, intake and body fat depots are without gonadal hormone stimulation.

MANAGEMENT IMPLICATIONS For management of some captive or free-ranging elk populations, leuprolide may be a preferable alternative to current immunological approaches because 1) only one capture and treatment is required for contraception the first year and any female in the population can be targeted for treatment in subsequent years; 2) leuprolide-based contraception is not dependent on an immunological response, which eliminates the need for an immunogenic adjuvant that can cause local tissue reactions (Stills and Bailey 1991); 3) behavioral side effects are minimal because recurrent estrous cycling does not occur in leuprolide-treated females; and 4) leuprolide formulation is currently approved by the United States Food and Drug Administration for therapeutic use in humans and is commercially available for extralabel use in female elk (Wildlife Pharmaceuticals). However, practical application of leuprolide is not without limitations. Leuprolide is relatively expensive ($150-200/ dose) and current technology requires annual treatment of females before the breeding season, a time when elk are widely dispersed and capture efficiency is low and un- predictable.

ACKNOWLEDGMENTS This research was supported by a grant from United States Department of the Interior, National Park Service, Rocky Mountain National Park, and the Colorado Division of Wildlife (Federal Aid to Wildlife Restoration, Project 153- R4). We offer special thanks to Atrix Laboratories, Incorporated, Fort Collins, Colorado, for generous donation of the leuprolide formulations used in this investigation. We sincerely thank B. Gill, R. Monello, M. Monello, D. Plattner, S. Ratchford, J. Ritchie, K. Griffin, M. Coffey, T. Johnson, T. Terrell, and M. K. Watry for technical assistance with animal capture, sample collections, and behavioral observations. The manuscript was significantly improved by comments from T. Shenk and L. Wolfe. In particular, we recognize and honor the late K. Zollers for


technical assistance, contagious enthusiasm, and devoted companionship during this study. We dedicate this manuscript to his memory.

LITERATURE CITED Albon, S. D., B. Mitchell, B. J. Huby, and D. Brown. 1986. Fertility in

female red deer (Cervus elaphus): the effects of body composition, age, and reproductive status. Journal of Zoology 209:447-460.

Altmann, M. 1952. Social behavior of elk Cervus canadensis nelsoni, in the Jackson Hole area of Wyoming. Behavior 4:116-143.

Baker, D. L., M. A. Wild, M. M. Conner, H. B. Ravivarapu, R. L. Dunn, and T. M. Nett. 2002. Effects of GnRH agonist (leuprolide) on reproduction and behavior in female wapiti (Cervus elaphus nelsoni). Reproduction (supplement) 60:155-167.

Baker, D. L., M. A. Wild, M. M. Conner, H. B. Ravivarapu, R. L. Dunn, and T. M. Nett. 2004. Gonadotropin-releasing hormone agonist: a new approach to reversible contraception in female deer. Journal of Wildlife Diseases 40:713-724.

Baker, D. L., M. A. Wild, M. D. Hussain, R. L. Dunn, and T. M. Nett. 2005. Evaluation of remotely delivered leuprolide acetate as a contraceptive agent in female elk (Cervus elaphus nelsoni). Journal of Wildlife Diseases 41:758-767.

Barlow, N. D., J. M. Kean, and C. J. Briggs. 1997. Modeling the relative efficacy of culling and sterilization for controlling populations. Wildlife Research 24:129-141.

Bear, G. D. 1989. Seasonal distribution and population characteristics of elk in the Estes Valley, Colorado. Colorado Division of Wildlife, Special Report No. 65, Fort Collins, USA.

Becker, S. E., and L. S. Katz. 1995. Effects of gonadotropin-releasing hormone agonist on serum LH concentrations in female white-tailed deer. Small Ruminant Research 18:145-150.

Bender, L. C., and J. G. Cook. 2002. Condition and health of elk (Cervus elaphus nelsoni) in Rocky Mountain National Park with reference to elk carrying-capacity relations. Final report for Rocky Mountain National Park from U.S. Geological Survey, New Mexico Cooperative Fish and Wildlife Research Unit, Las Cruces, USA.

Bergfeld, E. M., J. D'Occhio, and J. E. Kinder. 1996. Pituitary function, ovarian follicular growth, and plasma concentrations of 17 oz-oestradiol and progesterone in heifers during and after treatment with the luteinizing hormone-releasing hormone agonist deslorelin. Biology of Reproduction 54:776-782.

Boone, J. L., and R. G. Wiegert. 1994. Modeling deer herd management: sterilization is a viable option. Ecological Modeling 72:175-186.

Brown, J. L., and W. J6chle. 2002. Use of deslorelin to suppress estrous cyclicity in elephants: importance of endocrine monitoring to assess efficacy. Pages 20-21 in H. J. Bertschinger and J. F. Kirkpatrick, technical coordinators. Proceedings of 5th International Symposium on Fertility Control in Wildlife, Kruger National Park, South Africa.

Cook, R. C., J. G. Cook, D. L. Murray, P. Zager, B. K. Johnson, and M. W. Gratson. 2001a. Development of predictive models of nutritional condition for Rocky Mountain elk. Journal of Wildlife Management 65: 973-987.

Cook, R. C., J. G. Cook, D. L. Murray, P. Zager, B. K. Johnson, and M. W. Gratson. 2001b. Nutritional condition models for elk: which are the most sensitive, accurate, and precise. Journal of Wildlife Management 65: 988-997.

Cook, J. G., B. K. Johnson, R. C. Cook, R. A. Riggs, T. Delcurto, L. D. Bryant, and L. L. Irwin. 2004. Effects of summer-autumn nutrition and parturition date on reproduction and survival of elk. Wildlife Mono- graphs 155.

D'Occhio, M. J., W. J. Aspden, and T. R. Whyte. 1996. Controlled, reversible suppression of estrus cycles in beef heifers and cows using agonist of gonadotropin-releasing hormone. Journal of Animal Science 74:218-225.

D'Occhio, M. J., G. Fordyce, T. R. Whyte, L. A. Fitzpatrick, N. J. Cooper, W. J. Aspden, M. J. Bolam, and T. E. Trigg. 2002. Use of GnRH agonist implants for long-term suppression of fertility in extensively managed heifers and cows. Animal Reproductive Science 74:151-162.

D'Occhio, M. J., D. R. Gifford, C. R. Earl, T. Weathererly, and W. Von

Rechengerg. 1989. Pituitary and ovarian responses of post-partum acyclic beef cows to continuous long-term GnRH and GnRH agonist treatment. Journal of Reproduction and Fertility 85:495-502.

Evans, A. C. O., and N. C. Rawlings. 1994. Effects of long-acting gonadotropin-releasing hormone agonist (leuprolide) on ovarian follicular development in prepubertal heifer calves. Canadian Journal of Animal Science 74:649-656.

Fagerstone, K. A., M. A. Coffey, P. D. Curtis, R. A. Dolbeer, G. J. Killian, L. A. Miller, and L. Wilmot. 2002. Wildlife fertility control. Wildlife Society Technical Review 02-02. The Wildlife Society, Bethesda, Maryland, USA.

Fisher, M. W., B. J. McLeod, D. A. Heath, S. Lun, and P. R. Hurst. 2000. Role of ovarian failure in reproductive senescence in aged red deer (Cervus elaphus) hinds. Journal of Reproduction and Fertility 120:211-216.

Freddy, D. J. 1993. Estimating survival rates of elk and developing techniques to estimate population size. Job Progress Report, Colorado Division of Wildlife, Fort Collins, USA.

Garrott, R. A., P. J. White, and C. A. Vanderbil White. 1993. Overabundance: an issue for conservation biologists? Conservation Biology 7:946-949.

Geist, V. 1982. Adaptive behavior strategies. Pages 219-277 in J. W. Thomas and D. E. Toweill, editors. Elk of North America. Stackpole, Harrisburg, Pennsylvania, USA.

Gong, J. G., T. A. Bramley, C. G. Gutierrez, A. R. Peters, and R. Webb. 1995. Effects of chronic treatment with gonadotropin-releasing hormone agonist on peripheral concentrations of FSH, and LH, and ovarian function in heifers. Journal of Reproduction and Fertility 105:263-270.

Gorospe, W. C., and P. M. Conn. 1988. Restoration of the LH secretory response in desensitized gonadotrophs. Molecular and Cellular Endo- crinology 59:101-110.

Greer, K. R., and W. W. Hawkins, Jr. 1967. Determining pregnancy in elk by rectal palpation. Journal of Wildlife Management 31:145-149.

Guinness, F. E., G. A. Lincoln, and R. V. Short. 1971. The reproductive cycle of the female red deer, Cervus elaphus. Journal of Reproduction and Fertility 27:247-438.

Gysel, L. W. 1959. An ecological study of the winter range of elk and mule deer in Rocky Mountain National Park. Journal of Forestry 58:696-703.

Haigh, J. C., and R. J. Hudson. 1993. Farming wapiti and red deer. Mosby- Year Book, St. Louis, Missouri, USA.

Heilmann, T. J., R. A. Garrott, L. L. Cadwell, and B. L. Tiller. 1998. Behavior response of free-ranging elk treated with an immunocontraceptive vaccine. Journal of Wildlife Management 62:243-250.

Herbert, C. A., and D. W. Cooper. 2006. Fertility control in female eastern grey kangaroos using the GnRH agonist deslorelin. 1. Effects on reproduction. Wildlife Research 33:41-46.

Herbert, C. A., T. E. Trigg, M. B. Renfree, G. Shaw, D. C. Eckerly, and D. W. Cooper. 2005. Long-term effects of deslorelin implants on reproductive success and plasma progesterone, and LH concentrations in the female tammar wallaby. Reproduction 129:361-369.

Hobbs, N. T., D. C. Bowden, and D. L. Baker. 2000. Effects of fertility control on populations of ungulates: general, stage-structured models. Journal of Wildlife Management 64:473-491.

Huang, F., D. C. Cockrell, T. R. Stephenson, J. H. Noyes, and R. G. Sasser. 2000. A serum pregnancy test with a specific radioimmunoassay for moose and elk pregnancy specific protein B. Journal of Wildlife Management 64:492-499.

Jewell, P. A., and S. Holt. 1981. Problems in management of locally abundant wild mammals. Academic Press, New York, New York, USA.

Kang, S. H., and S. J. Kim. 2004. A comparison of the three conditional exact tests in two-way contingency tables using unconditional exact power. Biometrical Journal 46:320-330.

Kirkpatrick, J. F, J. W. Turner, Jr., I. K. M. Liu, R. Fayrer-Hosken, and A. T. Rutberg. 1997. Case studies in wildlife contraception: wild and feral equids and white-tailed deer. Reproduction Fertility Development 9:105- 110.

Krishnamoorthy, K., and J. Thompson. 2002. Hypothesis testing about proportions in two finite populations. American Statistician 56:215-222.

Lehner, P. N. 1996. Handbook of ethological methods. Second Edition. Cambridge University Press, Cambridge, United Kingdom.

Littell, R. C., G. A. Milliken, W. W. Stroup, and R. D. Wolfinger. 1996. SAS system for mixed models. SAS Institute, Cary, North Carolina, USA.


Marr, J. W. 1967. Ecosystems of the east slope of the Front Range in Colorado. Biological Series 8, University of Colorado, Boulder, USA.

McLeod, B. J., S. E. Dodson, A. R. Peters, and G. E. Lamming. 1991. Effects of a GnRH agonist (buserelin) on LH secretion in post-partum beef cows. Animal Reproduction Science 24:1-11.

McNeilly, A. S., and H. M. Fraser. 1987. Effect of gonadotropin-releasing hormone agonist-induced suppression of LH and FSH on follicle growth and corpus luteum function in the ewe. Journal of Endocrinology 115: 273-282.

McShea, W. J., S. L. Monfort, S. Hakim, J. Kirkpatrick, I. Liu, J. W. Turner, Jr., L. Chassy, and L. Munson. 1997. The effect of immunocontraception on the behavior and reproduction of white-tailed deer. Journal of Wildlife Management 61:560-569.

Merrill, J. A., E. G. Cooch, and P. D. Curtis. 2003. Time to reduction: factors influencing management efficacy in sterilizing overabundant white-tailed deer. Journal of Wildlife Management 67:269-281.

Merrill, J. A., E. G. Cooch, and P. D. Curtis. 2006. Managing an overabundant deer population by sterilization: effects of immigration, stochasticity and the capture process. Journal of Wildlife Management 70:268-277.

Miller, L. A., K. Crane, S. Graddis, and G. J. Killian. 2001. Porcine zona pellucida immunocontraception: long-term health effects on white-tailed deer. Journal of Wildlife Management 65:941-945.

Monello, R. J., T. L. Johnson, and R. G. Wright. 2005. The ecological role of elk in Rocky Mountain National Park. Report to the National Park Service, Estes Park, Colorado, USA.

Montovan, S. M., P. P. Daels, J. Rivier, J. P. Hughes, G. H. Stabenfeldt, and B. L. Lasley. 1990. The effect of potent GnRH agonist on gonadal and sexual activity in the horse. Theriogenology 33:1305-1321.

Morrison, J. A. 1959. Characteristics of estrus in captive elk. Behavior 16: 84-92.

Naugle, R. E., A. T. Rutberg, H. B. Underwood, J. W. Turner, Jr., and I. K. M. Liu. 2002. Field testing of immunocontraception on white-tailed deer (Odocoileus virginianus) on Fire Island National Seashore, New York, USA. Reproduction (supplement) 60:143-153.

Noyes, J. H., R. G. Sasser, B. K. Johnson, L. D. Bryant, and B. Alexander. 1997. Accuracy of pregnancy detection by serum protein (PSPB) in elk. Wildlife Society Bulletin 5:695-698.

Pedersen, S. B., K. Kristensen, P. A. Hermann, J. A. Katzenellenbogen, and B. Richelsen. 2004. Estrogen controls liplysis by up-regulating 02A- adrenergic receptors directly in human adipose tissue through the estrogen receptor o. Implications for the female fat distribution. Journal of Clinical Endocrinology and Metabolism 89:1869-1878.

Porter, W. F., M. A. Coffey, and J. Hadidian. 1994. In search of a litmus test: wildlife management in the U.S. National Parks. Wildlife Society Bulletin 22:301-306.

Porter, W. F., and H. B. Underwood. 1999. Of elephants and blind men: deer management in the U.S. National Parks. Ecological Applications 9: 3-9.

Quimby, D. C., and J. E. Gaab. 1957. Mandibular dentition as an age indicator in Rocky Mountain elk. Journal of Wildlife Management 21: 134-153.

Rapley, M. D. 1985. Behavior ofwapiti during the rut. Pages 357-361 in P. F. Fennessy and K. R. Drew, editors. Biology of deer production. Royal Society of New Zealand, Wellington, New Zealand.

Ravivarapu, H. B., K. L. Moyer, and R. L. Dunn. 2000. Sustained activity and release of leuprolide acetate from an in situ forming polymeric implant. American Association of Pharmaceutical Scientist 1:1-12.

Riney, T. 1960. A field technique for assessing physical condition of some ungulates. Journal of Wildlife Management 24:92-94.

Robbins, C. T. 1983. Wildlife feeding and nutrition. Academic Press, New York, New York, USA.

Rodriguez-Cuenca, S., M. Gianotti, P. Roca, and A. M. Proenze. 2006. Sex steroid receptor expression in different adipose depots is modified during midpregnancy. Molecular and Cellular Endocrinology 249:58-63.

Rutberg, A. T., R. E. Naugle, L. A. Thiele, and I. K. M. Liu. 2004. Effects of immunocontraception on a suburban population of white-tailed deer Odocoileus virginianus. Biological Conservation 116:243-250.

SAS Institute. 1999. SAS OnlineDoc?, Version 8. SAS Institute, Cary, North Carolina, USA.

Schoenecker, K. A., F. J. Singer, L. C. Zeigenfuss, D. B. Brinkley, and R. S. C. Menezes. 2004. Effects of elk herbivory on vegetation and nitrogen processes. Journal of Wildlife Management 68:837-849.

Schwartz, S. M., and G. N. Wade. 1981. Effects of estradiol and progesterone on food intake, body weight, and carcass adiposity in weanling rates. American Journal of Physiology 240:E499-E503.

Seagle, S. W., and J. D. Close. 1996. Modeling white-tailed deer (Odocoileus virginianus) population control by contraception. Biological Conservation 76:87-91.

Shideler, S. E., M. A. Stoops, N. A. Gee, J. A. Howell, and B. L. Lalsey. 2002. Use of porcine zona pellucida (PZP) vaccine as a contraceptive agent in free-ranging tule elk (Cervus elaphus nannodes). Reproduction (supplement) 60:169-176.

Smith, B. L., and T. L. McDonald. 2002. Criteria to improve age classification of antlerless elk. Wildlife Society Bulletin 30:200-2007.

Stephenson, T. R., K. J. Hundertmark, C. C. Schwartz, and V. V. Ballenberghe. 1998. Predicting body fat and body mass in moose with ultrasonography. Canadian Journal of Zoology 76:717-722.

Stills, H. F., Jr., and M. Q. Bailey. 1991. The use of Freund's complete adjuvant. Laboratory Animals 20:25-30.

Sutter-Dub, M. T., and P. Cordoba. 1997. Acute effects of progesterone on glucose metabolism in rat adipocytes: Are they modulated by endogenous adenosine? Metabolism 46:595-604.

Trigg, T. E., P. J. Wright, A. F. Armour, P. E. Willimason, A. Junaidi, G. B. Martin, A. G. Doyle, and J. Walsh. 2001. Use of GnRH analogue implant to produce reversible long-term suppression of reproductive function in male and female domestic dogs. Journal of Reproduction and Fertility (supplement) 57:255-261.

Turner, J. W., Jr., and J. F. Kirkpatrick. 2002. Effects of immunocontraception on population longevity and body condition in wild mares (Equus caballus). Reproduction (supplement) 60:187-195.

Wade, G. N. 1975. Some effects of ovarian hormones on food intake and body weight in female rats. Journal of Comparative Physiological Psychology 88:183-193.

Wade, G. N., and J. M. Gray. 1979. Gonadal effects on food intake and adiposity: a metabolic hypothesis. Physiological Behavior 22:583-593.

Wagner, F. H., R. Foresta, R. B. Gill, D. R. McCullough, M. R. Pelton, W. F. Porter, and H. Salwasser. 1995. Wildlife policies in the U.S. National Parks. Island Press, Washington, D.C., USA.

Wallace, J. M., C. A. Shively, and T. B. Clarkson. 1999. Effects of hormone replacement therapy and social stress on body fat distribution in surgically postmenopausal monkeys. International Journal of Obesity 23: 518-527.

Walter, W. D., P. J. Perkins, A. T. Rutberg, and H. J. Kilpatrick. 2002. Evaluation of immunocontraception in a free-ranging suburban white- tailed deer herd. Wildlife Society Bulletin 30:186-192.

Willard, S. T., R. G. Sasser, J. C. Gillespie, J. T. Jaques, T. H. Welsh, Jr., and R. D. Randel. 1994. Methods for pregnancy determination and the effects of body condition on pregnancy status in Rocky Mountain Elk (Cervus elaphus nelsoni). Theriogenology 42:1095-1102.

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