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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.

The primary aim of the Makalali elephant immunocontraception programme is to test the efficacy of porcine zona pellucida (PZP) vaccine for practical population control of elephants in small, enclosed reserves, with the goal of stabilizing the current growth rate and reducing it to the 5-10% per annum displayed currently in the Kruger National Park. A secondary aim is to test the hypothesis that PZP treatment does not affect patterns of elephant social behaviour. Eighteen sexually mature cows (age > 12 years) were vaccinated in May 2000 using remote darts. Behavioural observations before, during and after vaccination included noting the activity of individual animals every minute for 15 min. No changes in general behaviour patterns have been noted to date although the animals' spatial use of the reserve was erratic during the period of vaccination, indicating irregular or disturbed patterns associated with vaccination. Normality was resumed on completion of the vaccinations. No aggressive or indifferent behaviour related to nursing, calf proximity or bull-cow interactions have been noted. Ten of the females were in various stages of pregnancy when treated. Subsequently, seven of them gave birth to healthy calves and the other three females are expected to calve shortly. It is too early in the study to draw conclusions about stabilization of growth rates.

Fawning rates and mating behaviour were compared between white-tailed deer (Odocoileus virginianus) treated with GnRH and porcine zona pellucida (PZP) immunocontraceptive vaccines from 1997 to 2000. Female deer from a herd of 102 deer at Seneca Army Depot, near Romulus, New York, were treated with prime and booster injections of PZP (n = 22) or GnRH vaccine (n = 32), or remained untreated as controls (n = 34). During the summers after booster treatment, observed fawning rates for adult female deer were similar for both PZP-treated (0.10-0.11 fawns per female) and GnRH-treated (0.13-0.22 fawns per female) female deer, and were significantly lower (t = -8.93 and t = -9.73; P < or = 0.0005, respectively) than those observed for control female deer (1.22-1.38 fawns per female). During the second (0.36 fawns per female) and third summers (0.61 fawns per female) after the last booster injection, GnRH-treated female deer still produced significantly fewer fawns than did the controls (1.38 and 1.31 fawns per female, respectively). In one breeding season after treatment, five of 18 (28%) females vaccinated with PZP produced fawns, similar to the rate for GnRH-treated females (29%). In addition, females treated with GnRH had fewer oestrous cycles per female (0.06, P < or = 0.05) than did either control (0.22 cycles per female) or PZP-treated deer (0.36 cycles per female). Initial PZP treatment followed by a booster dose 5-7 months later reduced fawn production and prolonged the breeding season as females repeatedly returned to oestrus, similar to results reported in other studies.

Two mouse-specific polyepitope protein antigens comprising different combinations of sequences chosen from the mouse fertility antigens zona pellucida proteins 1 and 3 (ZP1 and ZP3), prolactin, proliferin, granulocyte-macrophage colony-stimulating factor (GM-CSF), sperm protein SP56 and T-helper cell-stimulating epitopes were produced in bacterial protein expression systems. The recombinant proteins were fused to maltose binding protein (MBP) and used to immunize female mice; their effects on fertility were assessed. Controls were immunized with either MBP only or phosphate buffered saline (PBS). One antigen construct (MBP-polyepitope B), containing mouse-specific epitopes for ZP1, ZP3, SP56 and proliferin, significantly reduced the fertility of female BALB/c mice. Fertility in this group was decreased by > 40% compared with the MBP control and the number of viable embryos was decreased by > 60%. This construct will now be used to produce the antigen in a recombinant murine cytomegalovirus for assessment as a potential mouse-specific anti-fertility vaccine for use in the control of mice in the field.

Contraceptive management of publicly valued wildlife species requires safeguards to ensure that these populations are preserved in a healthy state. In addition, reversibility of contraceptive effects and safety in pregnant animals are major concerns. A population of wild horses has been immunized against porcine zona pellucida (PZP) over a 12 year period on Assateague Island National Seashore, MD (ASIS). Mares initially received one or two 65 microg inoculations and once a year 65 microg booster inoculations, all delivered by dart. All young mares aged > 2 years were treated with PZP for 3 consecutive years regardless of whether they have bred successfully and they were then removed from treatment until they had foaled. All mares vaccinated for 1 or 2 consecutive years became fertile again and 69% of mares treated for 3 consecutive years returned to fertility. All five mares treated for 4 or 5 consecutive years have also returned to fertility, but over longer periods of time. Mares treated for 7 consecutive years have not returned to fertility, but several, while still infertile, have started ovulating again. There was no difference in survival rates between foals born to treated and untreated mares, and PZP treatment of pregnant mares did not affect subsequent fertility of their female offspring.

Application of contraception for the control of suburban populations of white-tailed deer (Odocoileus virginianus) has been much debated, but few data are available on field applications and even fewer on population effects. Between 1993 and 1997, 74-164 individually known female deer living on Fire Island, New York, USA, were treated remotely with an initial shot of 65 microg porcine zona pellucida (PZP) in Freund's complete adjuvant followed by booster injections of 65 microg PZP in Freund's incomplete adjuvant. Starting in 1996, progressively increasing numbers of deer were treated with vaccinating/marking darts. Estimates of population density and composition, using distance sampling methods, began in 1995 in selected portions of the study area. Between 1993 and 1997, fawning rates among individually known, treated adult females decreased by 78.9% from pretreatment rates. Population density in the most heavily treated area increased by 11% per year from 1995 to March 1998 and then decreased at 23% per year to October 2000. In 1999-2000 surveys, fawns comprised 13-14% of the total population in the most heavily treated area, versus 16-33% in nearby untreated areas. These results show that PZP can be delivered effectively to sufficient deer to affect population density and composition in some environments, but that technical and logistical improvements are needed before contraception can be used widely to manage suburban deer populations.

It has long been established that there are major variations in both the immunogenicity and antigenicity of native zona pellucida (ZP) proteins. These differences appear to be more pronounced with respect to genetically engineered ZP proteins, which do not have native post-translational modifications (for example glycosylation and sulphation). As the number of animal species that are now included in population management programmes using native porcine zona pellucida (PZP) proteins expands, it is increasingly important to carry out studies to evaluate the immune response variations among different species as well as the individual variation within a species. In an attempt to compare these complex immune responses, we have evaluated antibodies from numerous species immunized with native, genetically engineered ZP and synthetic ZP peptides. Such an immunocontraceptive method could have great potential. These studies are critical not only for the development of predictable immune responses that result in permanent sterilization versus reversible contraceptive effects, but also for predicting which vaccinogens (native ZP protein versus genetically engineered ZP proteins) might have detrimental effects on animal and human populations.

Contraception is becoming a common approach for the management of captive and wild ungulates yet there are few data for contraceptive effects on entire populations. Management-level treatment of mares with porcine zona pellucida (PZP) vaccine resulted in zero population growth of the Assateague Island wild horse population within 1 year of initiation of treatment. Contraceptive efficacy was 90% for mares treated twice in the first year and annually thereafter. For mares given a single initial inoculation, contraceptive efficacy was 78%. The effort required to achieve zero population growth decreased, as 95, 83 and 84% of all adult mares were treated in each of the first 3 years, compared with 59 and 52% during the last 2 years. Mortality rates for mares and foals after the initiation of management-level treatments decreased below historic and pretreatment mortality rates of approximately 5%. Two new age classes have appeared among treated animals (21-25 years and > 25 years), indicating an increase in longevity among treated animals. Body condition scores for all horses, all adult mares and non-lactating mares increased significantly between summer 1989 and autumn 1999 but did not change significantly in lactating mares. These results provide reliable data for the construction of realistic models for contraceptive management of free-roaming or captive ungulate populations.

Porcine zona pellucida (PZP) immunocontraception was investigated for possible use in free-roaming wild horses in the western USA. A protocol of two injections (3-4 weeks apart) of vaccine lasting 1 year was first used and a single-injection controlled-release vaccine of 1 year duration was developed and tested in the field. Studies of a presumptive vaccine of 2 year duration were initiated. The parameters of anti-PZP antibody titre response, pregnancy testing and offspring production were used, and PZP vaccine was found to provide up to 94% infertility in free-roaming wild mares. In addition, a single-injection PZP vaccine of 1 year duration and containing a controlled-release component of PZP in a polymer matrix can provide infertility equivalent to the two-injection PZP vaccine. All the PZP vaccine preparations tested were associated with a return to normal fertility within 1 year. During the course of these studies, attention was given to practical aspects of management application of PZP contraception. Preparation of the controlled-release portion of the vaccine in pellets, which fit into the needle of a dart or syringe, has simplified vaccine handling and permitted long-term storage of the controlled-release component. Vaccine delivery is now performed using a jabstick on captured mares restrained in a field stock chute during routine horse gathers. Provision of a vaccine-training programme has maximized personnel safety during vaccine preparation and use.

Although there is some dissent, the animal protection community generally supports the concept of wildlife contraception. However, some contraceptive agents, delivery mechanisms and specific applications will be opposed by animal welfare advocates on environmental, humane or other ethical grounds, and some animal rights advocates may oppose wildlife contraception entirely. The Humane Society of the United States (HSUS) has supported and conducted wildlife contraception studies for more than 10 years. In general, we have invested in contraceptive agents (such as porcine zona pellucida) that we believe will prove environmentally, physiologically and behaviourally benign, and in delivery mechanisms that are narrowly targeted. As we consider contraception to be a major intervention into natural processes, we believe that wildlife contraception should be applied judiciously, locally and in a manner that is sensitive to the needs of animals, humans and ecosystem function.

Thirteen deer (Odocoileus virginianus) and three goats (Capra hircus) were given an initial immunization of 300 microg porcine zona pellucida (PZP) combined with 50 mg synthetic trehalose dicorynomycolate ml(-1) (STDCM) in drakeol i.m. in the semitendinosus and semimembranosus muscles. This immunization was followed by two booster injections of 300 microg PZP, 2 weeks apart. The vaccinations were made from the same purified batch of PZP and lot of adjuvant. All the doses were made at the same time and injected on the same day. The immune response was quantified by measuring serum anti-PZP IgG antibody concentration by ELISA. The results showed that the mean serum IgG concentration of the deer increased from 0.000 +/- 0.003 to 0.083 +/- 0.023 absorbance units, whereas in the goats the mean IgG concentration increase was from 0.044 +/- 0.019 to 1.245 +/- 0.774 absorbance units. Both the goats and deer showed a significant increase in IgG concentration (P < 0.05) after the final booster injection compared with preimmune serum. The final goat IgG concentration was significantly (P < 0.05) higher than that of the deer. If PZP and Freund's adjuvant are administered to deer, immunocontraception is achieved, but the IgG concentration in the deer in this study did not appear to be compatible with immunocontraception.