Effect of porcine zonae pellucidae immunisation on ovarian follicular development and endocrine function in domestic ewes (Ovis aries).

Domestic ewes (Ovis aries) were immunised with porcine zonae pellucidae (pZP) or pZP conjugated to keyhole limpet haemocyanin (KLH) in adjuvant(s) to examine the feasibility of the species to serve as a model for further development of pZP-based vaccines in ungulates. Two immunisation groups were employed, with a third group receiving only adjuvant (n = 5 per group). Early in the study, oestrous activity was monitored by the use of a vasectomised ram fitted with a marking harness. Eventually, ewes were exposed to an intact ram for breeding. In addition, weekly serum and every-other-day faecal samples were collected to measure pZP antibodies and progesterone metabolite concentrations respectively. At the conclusion of the study, fecundity was established, and ovarian tissue was examined. Ewes immunised against pZP : KLH with adjuvant produced minimal antibody absorbance levels, displayed normal oestrous cycles, became pregnant upon introduction of the intact ram and exhibited normal ovarian histopathology. Ewes immunised against pZP with adjuvant produced high antibody absorbance levels, were acyclic following primary immunisation and were infertile. Examination of the ovarian tissue revealed atrophic changes that included: (1) the absence of growing follicles; (2) significant reduction in the number of primordial follicles; and (3) the presence of abnormal granulosa cell clusters lacking oocytes. Antisera displayed immunoreactivity to the major components of pZP, and immunohistochemical labelling of ovarian tissue showed specificity to the ZP. These data are the first generated in an ungulate species showing deleterious effects of pZP immunisation on folliculogenesis and oestrous cyclicity.

Monitoring gonadotropin-releasing hormone administration by measurement of urinary steroid conjugates.

The use of measuring urinary steroid conjugates in nontimed, randomly collected morning urine samples expressed as a function of creatinine concentration was assessed to monitor ovarian response to pulsatile administration of gonadotropin-releasing hormone in ambulatory patients. This method of evaluating ovarian steroid production provided a convenient, inexpensive, and noninvasive means of monitoring responses to gonadotropin-releasing hormone treatments and documents that clomiphene-resistant amenorrheic patients can be induced to ovulate with appropriate gonadotropin-releasing hormone therapy. Different ovarian responses in the same woman to similar doses and frequencies of gonadotropin-releasing hormone indicate that discrete adjustments of individual doses may be required to facilitate consistent ovulatory responses. The strategy presented here allows for subsequent gonadotropin-releasing hormone therapy in the individual patient to be determined by an objective and quantifiable ovarian response to an initial treatment.

Ovarian-pituitary hormone interactions during the perimenopause.

To describe the hormone changes that occur at the onset of the perimenopause, daily urine and random blood samples were collected from 5 peri-menopausal women for 3 or 4 consecutive cycles. Estrone conjugate and pregnanediol-3-glucoronide concentrations were determined for urine samples. Circulating luteinizing hormone, follicle stimulating hormone, progesterone, estradiol and estrone concentrations were determined in serum samples. Two of the 5 women experienced irregular menstrual intervals during the study period. One of these subjects experienced a prolonged intermenstrual interval. Three other women exhibited apparently regular ovulatory menstrual cycles. The prolonged intermenstrual interval of one women exhibiting irregular menstrual intervals was associated with low urinary estrogen levels in the early follicular phase of the affected cycle, followed by increased gonadotrophin levels and increased estrogen levels that rose to exceed normal cycle concentrations by 2- or 3-fold. Increased estrogen levels were followed by declining gonadotrophin levels, minimal progesterone production, and, ultimately, vaginal bleeding. These data suggest that there are some forms of menstrual variability at the time of the perimenopause associated with lowered early follicular phase estrogen levels. Reduced negative feedback and subsequently increased gonadotrophin levels may have stimulated estrogen production which may have suppressed gonadotrophin secretion and lowered estrogen excretion, resulting in the observed oscillations between episodes of hypo- and hyperestrogenism.

Urine and fecal sample collection on filter paper for ovarian hormone evaluations.

A practical method for collecting, storing, and transporting liquid biological samples in a dry state for subsequent hormone metabolite analyses is presented. This method employs the use of ordinary filter paper strips that imbibe liquid samples. Samples taken up by the filter paper were allowed to dry and were retained at ambient conditions in capped vials for up to 5 years prior to analysis. Examples presented in the present report include urine samples from human and nonhuman primates as well as solubilized fecal samples from nonhuman primates. Hormone metabolite analysis of the paper-stored samples provided data that were comparable to the results obtained from analyses of the original liquid samples. One year of storage had no effect on hormone concentration. Five years of storage resulted in concentrations that were quantitatively less but qualitatively similar to the concentrations obtained by direct analysis of the initial samples. These data demonstrate the versatility and reliability of paper as a matrix for biological samples that may provide a more convenient approach for collecting and transporting samples collected in the field. © 1995 Wiley-Liss, Inc.

Monitoring female reproductive function by measurement of fecal estrogen and progesterone metabolites in the white-faced saki (Pithecia pithecia).

A simple method for extracting and measuring ovarian steroids in feces is applied to the ovarian cycle, pregnancy, parturition, and period of lactational amenorrhea in Pithecia pithecia. Small amounts of wet, unmixed feces were combined with a modified phosphate buffer, shaken, centrifuged, and decanted, and the supernatant was directly measured for estrogen and progesterone metabolites by enzyme immunoassays. Urinary estrogen and progesterone metabolite measurements were compared to paired fecal measurements to determine the degree to which fecal hormone levels detected the same ovarian events as urinary measurements. The correlation coefficients for the relationship between urinary and fecal hormones for individual animals studied (n = 5) were found to be statistically significant in every case except one sexually immature animal. The application of the method presented here demonstrates that simple solubilization and non-radiometric measurement of ovarian steroids excreted in feces reliably reflect reproductive events in Pithecia pithecia. © 1994 Wiley-Liss, Inc.

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.

Paired urine and serum samples from four conceptive and six nonconceptive ovarian cycles of seven adult Macaca mullatta were analyzed by radioimmunoassay (RIA) for circulating estradiol (E2) and progesterone (Po), and urinary estrone conjugates (E1C) and immunoreactive preganediol-3-glucuronide (iPDG) using enzyme immunoassay (EIA). Nonconceptive cycles exhibited a fivefold increase in urinary E1C and serum E2 levels from follicular phase levels to the preovulatory peak. Linear correlation between urinary E1C and serum E2 nonconceptive cycle hormone levels was significant (P <0.01, r = 0.69). Luteal phase levels of iPDG and serum Po levels were approximately parallel in nonconceptive cycles. Similarly, conceptive cycle urinary E1C levels and serum E2 measurements had a correlation coefficient that was significant (P<0.01, r = 0.45). Nonconceptive and conceptive cycle iPDG and Po levels were significantly correlated (P = 0.05, r = 0.63, and P<0.01, r = 0.66, respectively). These data demonstrate that EIA measurements of ovarian hormones in daily urine samples can be used to accurately monitor ovarian function and early pregnancy in Macaca mulatta.

Estrogen-behavior correlates in the reproductive physiology and behavior of the ruffed lemur (Lemur variegatus).

The animal subjects of this study consisted of seven male-female pairs, living an open-air, off-exhibit area of the San Diego Zoo. Daily urinary estrogen levels in each of five females were measured and daily behavioral observations of the seven pairs were made. Behavioral patterns aligned by objectively determined, discrete physiologic events were analyzed to determine their temporal correlation to changes in estrogen excretion. The data indicate that approximately one-sixth of the female behaviors and one-third of the male behaviors sampled were significantly correlated to urinary estrogen levels in the females (P less than 0.05-0.005). In addition, both attractivity and receptivity were distinguishable and their component behaviors in males and females were found to be related to the estrogen profile. Proceptivity, however, was only weakly identified and its description in this sample population was ambiguous. Mating was observed to occur exclusively on 1 day, presumably the day of ovulation.

A prototype for ovulation detection: pros and cons.

A noninstrumented enzyme immunoassay for urinary estrone conjugates was adapted from an instrumented microtiter plate enzyme immunoassay assay. The end point of the assay was a color change from green to clear, which was visible to the unaided eye. The visible color change was adjusted to allow 80 ng/ml estrone conjugates (on the basis of a sample size of 6.5 microliters urine) to be distinguished from an infinite dilution without instrumentation. The evaluation of human urine collected from ovulatory ovarian cycles demonstrated that early follicular phase concentrations (35.9 +/- 6.8 to 79.4 +/- 14.7 ng/ml, n = 10) produced a dark-green color, whereas late follicular phase concentrations (162.9 +/- 20.1 ng/ml, n = 10) produced no color. Daily urine samples throughout 10 ovulatory ovarian cycles produced parallel profiles when compared to measurements of estradiol in paired blood samples. Complete analysis of the data indicated that ovarian follicular dynamics can be accurately monitored through the noninstrumented analysis of daily estrone conjugates in urine samples.

PIP: The purpose of this study of the timing of ovulation was to evaluate and compare the results of estrogen concentrations in serum using radioimmunoassay (RIA) with urine samples assessed by instrumented (EIA) and noninstrumented enzyme immunoassays (NEIA). This was done to determine the timing before ovulation of the 1st enzyme rise. The advantage of urinary analysis over serum analysis is that subjects can collect and freeze samples at home, toxic regulated substances are eliminated, time and cost is reduced, and the design allows for a larger population sample size. The study population was a group of women 23-40 years with normal menstrual cycles. Early morning urine samples and midmorning blood samples were obtained for 1 complete menstrual cycle. Reanalysis of 6 of the refrozen urine samples was made using NEIA. Diagnostic Product kits were used to establish ovulatory cycles, and Munro et al laboratory methods were used to analyze urinary estrone conjugated (EIC) and progesterone (PdG). The methods of Taussky was used to measure creatinine. The noninstrumented EIA was similar to the EIC EIA format of Czekala et al and the materials reported by Munro et al. However, there were 3 changes in the microtiter plate EIC EIA format: 1) high binding star tubes were substituted for the microtiter plate, 2) the EIC enzyme label was altered by eliminating the glucuronide moiety, and 3) small Whatman No. 1 filter paper pads (7.5 mm diameter) were used to measure and transfer urine samples (storage at 4 degrees Centigrade and dried completely). Reliability was comparable to micropipettors. The process is described. The results were that an increase of serum estradiol (E2) concentrations accurately and consistently predict the occurrence of future ovulation. It was also demonstrated that changes in urinary estrogen excretion can be used to predict ovulation, and can be detected with either EIA or NEIA. The rise in urinary EIC was detected by EIA (35.9 + or - 6.8 to 70.4 + or - 14.7 ng/ml and dark green color in the early follicular phase) and NEIA (70-80 ng/ml) between 6-2 days prior to the midcycle LH peak and comparable to estradiol rises in paired blood samples (r=0.88, p.01). E2 measurements are better predictors but eliminate self evaluation. The detection of the EIC rise (EIC values plus PdG and LH values) provide a complete and comprehensive monitor of all ovarian events of a complete menstrual cycle. The development of a noninstrumented test or urine osmolality will contribute to ending false positives or negatives.

Oestrous cycle of the North American bison (Bison bison) characterized by urinary pregnanediol-3-glucuronide.

An enzyme immunoassay for urinary pregnanediol-3 alpha-glucuronide (PdG) was evaluated for the indirect measurement of progesterone metabolites during the oestrous cycle and early pregnancy of uncaptured North American bison. Comparisons between plasma progesterone and urinary PdG, dose-response parallelism between the standard curve and diluted urine samples and high-performance liquid cochromatography revealed that PdG was a primary immunoreactive urinary metabolite of progesterone in bison. Urine samples were collected directly from the soil from 29 bison cows during the August rutting season and analysed for PdG. Eight bison cows demonstrated complete oestrous cycles ranging from 19 to 26 days (mean cycle length = 23.12 +/- 0.76 days) and behavioural oestrus among four of these cows correlated with PdG nadirs. Mean PdG nadirs were 63.62 +/- 21.61 ng/mg urinary creatinine (Cr) and mean peak midluteal values were 546.01 +/- 130.73 ng/mg Cr. Seven of eight became pregnant, indicating that bison exhibit a second seasonal oestrus. Eighteen other bison cows were pregnant prior to the beginning of the study and demonstrated non-cyclic increased PdG concentrations (greater than 200 ng/mg Cr) during the 30-day course of collection. Three cows ovulated and became pregnant during the 30-day collection period and then exhibited increasing urinary PdG concentrations. This report demonstrates that ovarian function in uncaptured bison can be monitored by means of urinary PdG and that both ovulatory cycles and early pregnancy can be detected.

Monitoring ovulation and implantation in the cynomolgus macaque (Macaca fascicularis) through evaluations of urinary estrone conjugates and progesterone metabolites: a technique for the routine evaluation of reproductive parameters.

Investigations were undertaken to determine the applicability of recently reported specific radioimmunoassays for urinary estrone conjugates and progesterone metabolites for monitoring ovarian function in the cynomolgus macaque (Macaca fasciularis) and other macaque species. Mean estrone conjugate measurements appear to accurately reflect the preovulatory estrogen peak in both conceptive (n = 5) and nonconceptive (n = 6) cycles, as well as to indicate early pregnancy through increases which are significantly elevated by Day + 15 (p less than 0.049) post estrone conjugates peak. The mean luteal phase levels of these progesterone metabolites are significantly elevated by Day + 14 (p less than 0.012) in conceptive cycles when compared to the mean values for nonconceptive cycles.

Urinary estrogens during pregnancy of the ruffed lemur (Lemur variegatus).

Pregnancy in the ruffed lemur (Lemur variegatus) was monitored by analyses of urinary estrogens. Urine samples were collected weekly throughout pregnancy (gestation: 100 +/- 1.6 days) from ten females analyzed for total immunoreactive estrogen (Et) and indexed by creatinine (Cr). Maternal urinary total estrogen excretion remained low until the last half of gestation at which time excretion increased steadily, reaching values 1000 times greater than those observed at estrus. Chromatographic separation after enzyme hydrolysis of lemur urine indicates that estrone is the major estrogenic component of pregnancy in the ruffed lemur, with the remaining components consisting of equal amounts of estradiol-17 alpha and -17 beta.

Excretion and measurement of estradiol and progesterone metabolites in the feces and urine of female squirrel monkeys (Saimiri sciureus).

The first objective of the present study was to determine the metabolic form and rate of excretion of ovarian hormone metabolites in the urine and feces of female squirrel monkeys injected with radiolabeled progesterone (Po) and estradiol. The major portion of the urinary metabolites of both hormones was excreted within 16-24 hr post-injection. Estrogen and Po isotopes in feces exhibited an excretion peak at 16 hr post-injection. The majority of recovered radiolabel of both hormones was excreted in feces. Chromatographic separation of fecal extractions indicated that the major estrogen metabolites in feces are in the free as opposed to the conjugated form. The radioactivity and immunoreactivity for estrone and estradiol (E(1) and E(2), respectively) in eluates of fecal samples subjected to celite co-chromatography indicated that both free E(1) and E(2) exist as excretion products in the feces of female squirrel monkeys. The major radioactive peaks for Po metabolites showed peaks in the elution profile at or very near the Po standard, and corresponded with the celite co-chromatography elution profile of Po standard when subjected to enzyme immunoassay (EIA). The second objective was to validate the application of EIA systems to measure fecal metabolites. Reproductive events of one female squirrel monkey across one annual reproductive cycle are described using the endocrine profile generated from fecal steroid assays. Examination of this profile confirmed that longitudinal fecal sampling and steroid hormone metabolite measurement in feces was not only feasible and practical, but accurately detected known reproductive events as well.

Estrogen and progesterone metabolites and follicle-stimulating hormone in the aged macaque female.

The study presented characterizes the ovarian and pituitary function of the aged female macaque through a complete annual reproductive cycle to compare hormone dynamics during the human and nonhuman primate menopausal transition. Data collected over an entire year from aged macaque females indicated that urinary FSHbeta subunit baseline levels statistically significantly increased in females after age-related abnormal menstrual cycles occurred. These abnormal cycles were followed by anovulation and complete cessation of follicular activity. No statistically significant difference in urinary FSHbeta subunit levels was seen between females that exhibited year-round normal ovarian cycles and those that exhibited seasonal ovarian cycles followed by an interval of anovulation during the nonbreeding season. Basal urinary estrogen metabolite levels were not observed to decrease until ovarian cycles became abnormal and FSHbeta subunit levels began to rise. Early follicular phase circulating inhibin beta levels were statistically significantly reduced only when ovariectomized females were compared to the year-round normally cycling females. A statistically nonsignificant trend toward decreased inhibin secretion, however, was apparent in aged females with normal cycles, aged females with abnormal cycles, anovulatory aged females, and finally, ovariectomized females. Whereas decreased circulating levels of dehydroepiandrosterone sulfate showed a general decline over the 1-yr study period in all groups, they were lowest in the year-round normally cycling group, progressively higher in the normal-to-anovulatory group and abnormal-to-anovulatory group, and highest in the anovulatory group. Finally, the nonbreeding season was associated with the highest number of abnormal cycles, suggesting that onset of complete ovarian senescence in these study macaques was more likely to occur during that time (i.e., females were less likely to return to normal ovarian cycles the following breeding season and more likely to exhibit permanent ovarian quiescence).

Fecal analysis of ovarian cycles in female black-handed spider monkeys (Ateles geoffroyi).

An enzyme immunoassay (EIA) was applied to characterize the reproductive endocrinology of adult female black-handed spider monkeys (Ateles geoffroyi). Analysis of paired urine and fecal samples, collected from two females housed at San Diego Zoo, confirmed that the EIAs employed provided quantitative measurements of ovarian sex steroid hormones. Fecal metabolite levels were significantly correlated with those in urine, confirming that feces are a valid source of steroid metabolites in this species. The excretion of these metabolites in feces lagged urinary excretion by 1-2 days. The ovarian cycle profiles of the two captive females and five free-ranging females are comparable, with an average length of approximately 20-23 days. Cyclical bleeding, as previously reported, was observed in one of the two captive females. Pregnancy was detected in four free-ranging females, and early fetal loss for one female was indicated by hormonal data.

Enzyme immunoassays for ovarian steroid metabolites in the urine of Macaca fascicularis.

In vivo studies using carbon 14 labeled estradiol (E2) and progesterone (Po) were performed to characterize the time course and metabolic fate of circulating E2 and Po. Co-chromatography of human, orangutan, and macaque luteal phase urine samples demonstrated the presence of a steroid conjugate peak in all three species that was identified as being androsterone and etiocholanolone glucuronides. An enzyme immunoassay for urinary metabolites of Po was developed subsequently for Macaca spp. using a monoclonal antibody that cross-reacted with both C-19 and C-21 metabolites.

Comparison of serum estradiol to urinary estrone conjugates in the rhesus macaque (Macaca mulatta).

Paired urine and serum samples were collected daily during fourteen nonconceptive (7 females) and ten conceptive (9 females) ovarian cycles from a total of 12 female rhesus monkeys (Macaca mulatta). Daily urine samples were analyzed for concentrations of estrone conjugates (Ei Conj). Serum samples were evaluated for concentrations of estradiol (E2) and progesterone (P) by radioimmunoassay (RIA), and bioactive luteinizing hormone (bLH) and monkey chorionic gonadotropin (mCG) were analyzed by a mouse Leydig cell bioassay. Linear correlation (r) between urinary E1 Conj and serum E2 (r range = -0.176-0.948) during nonconceptive cycles aligned by the preovulatory E1 Conj peak (Day 0) improved when daily hormone values were realigned to account for an approximately 24-h delay in the excretion of hormonal metabolites in urine (r range = 0.465-0.967). Similarly, correlation between urinary E1 Conj and serum E2 during conceptive cycles aligned by Day 0 (r range = 0.300-0.824) improved when values were offset by 24 h (r range = 0.408-0.876). When conceptive cycles were compared to nonconceptive cycles, serum P levels were significantly elevated over nonconceptive levels by Day +12 (p less than 0.001), and urinary E1 Conj levels by Day +13 (p less than 0.02), whereas serum E2 and bLH were both significantly elevated by Day +14 (p less than 0.0006 and p less than 0.01, respectively). In both nonconceptive and conceptive cycles, urinary E1 Conj paralleled serum E2 and demonstrated incremental increases above baseline levels, which were greater than for serum E2.

Simple extraction and enzyme immunoassays for estrogen and progesterone metabolites in the feces of Macaca fascicularis during non-conceptive and conceptive ovarian cycles.

A simple method for extracting ovarian steroids from feces is presented, together with enzyme immunoassay systems for measuring estrogen and progesterone metabolites. Small amounts of feces were combined in a 1:10 proportion with a modified phosphate buffer, shaken for 24 h, centrifuged, and decanted; the supernatant was directly measured for estrogen and progesterone metabolites by enzyme immunoassays. Serum estradiol and progesterone profiles were compared to urinary and fecal profiles in the same animals to determine the degree to which each reflected the ovarian events detectable in serum. The correlation coefficients for the relationship between serum, urinary, and fecal hormones for individual animal cycles were found to be statistically significant in every case but one, where the relationship between serum estradiol and urinary estrone conjugates was not significant. Urinary and fecal measurements were used to determine whether estrogen and progesterone metabolism and excretion varied within and between animals. Variation in unconjugated estrogen and progesterone metabolites was observed in the follicular phase, the luteal phase, and early pregnancy.

Characterization of the onset of menopause in the rhesus macaque.

The objective of this study was to assess ovarian activity in a cohort of aged female rhesus macaques. Menstrual records for 26 rhesus macaques ages 20-29 yr were evaluated over a 1-yr period, and daily urinary estrone conjugate (E1C) and pregnanediol-3-glucuronide (Hygeia [Hy]-PdG) levels were determined for 12 wk. Each animal was categorized as either pre-, peri-, or postmenopausal based on menstrual and hormonal data. Eleven animals (mean age 22.5 yr) were premenopausal, thirteen (mean age 24 yr) were perimenopausal, and two (mean age 29.5 yr) were postmenopausal. Hormone profiles for perimenopausal animals reveal prolonged follicular phases and/or a lack of patterned Hy-PdG dynamics. Breakthrough bleeding occurred in four of these perimenopausal animals. The postmenopausal animals were amenorrheic and exhibited low E1C levels (less than 10 ng/mg creatinine). The results of this study illustrate that the decline of ovarian function in female macaques during the third decade of life parallels the menstrual and hormonal events associated with the climacteric in women, and that menopause does occur in rhesus macaques.

Monitoring ovulation and implantation in the lion-tailed macaque (Macaca silenus) through urinary estrone conjugate evaluations.

Urine samples were collected daily during ten nonfertile and four fertile ovarian cycles of four adult female lion-tailed macaques (Macaca silenus). Urine was analyzed for concentrations of total immunoreactive estrogen (Et), estrone conjugates, and bioactive luteinizing hormones (LH). The estrone conjugates of selected samples were separated by high-performance liquid chromatography (HPLC) to evaluate the relative proportions of estrone glucuronide (E1 G) to estrone sulfate (E1 S) contributing to the sum total of the conjugate measured in the samples. The estrone conjugate profile was found to accurately reflect the preovulatory estrogen peak in both nonfertile and fertile cycles as well as the early pregnancy increase which was found to be statistically significant on Day + 14 postovulation (P = 0.003). Estrone conjugate levels rose in the early follicular phase from 126.00 +/- 24.07 (SEM) ng/mg creatinine to a preovulatory peak of 471.90 +/- 62.95 ng/mg creatinine. Fertile cycles exhibited a postovulatory climb to a peak of 515.00 +/- 38.00 ng/mg creatinine on Day + 19, in contrast to the secondary rise observed in nonfertile cycles that peaked at 148.11 +/- 13.80 ng/mg creatinine on Day + 10. Bioactive LH evaluations confirmed ovulation and, in the fertile cycles, reflected the subsequent elevation of chorionic gonadotropin on Day + 18. The estrone conjugate profile of fertile cycles and early pregnancy compared favorably to the Et profile: both showed the same time course and increases in estrogen excretion.

Use of porcine zona pellucida (PZP) vaccine as a contraceptive agent in free-ranging tule elk (Cervus elaphus nannodes).

The potential for the application of porcine zona pellucida (PZP) immunocontraception in wildlife population management has been tested over a 15 year period and promises to provide a useful wildlife management tool. These studies have provided evidence indicating that the use of PZP immunocontraception in wildlife: (i) is effective at both the physiological and population level (Liu et al., 1989; Kirkpatrick et al., 1996; Turner et al., this supplement); (ii) is deliverable by remote means (Kirkpatrick et al., 1990; Shideler, 2000); (iii) is safe in pregnant animals (Kirkpatrick and Turner, this supplement); (iv) is reversible (Kirkpatrick et al., 1991; Kirkpatrick and Turner, this supplement); (v) results in no long-term debilitating health problems (Kirkpatrick et al., 1995; Turner and Kirkpatrick, this supplement); (vi) has no implications for passage through the food chain (Harlow and Lane, 1988); and (vii) is reasonably inexpensive (J. F. Kirkpatrick, personal communication). This report presents the results of a 5 year study in tule elk (Cervus elaphus nannodes), 3 years of which were on the application of PZP immunocontraception to an expanding elk population living in a wilderness area of Point Reyes National Seashore in Marin County, CA, where hunting is not allowed and culling is not publicly acceptable.