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PubMed2002

In search of the active PZP epitope in white-tailed deer immunocontraception.

Native porcine zona pellucida (PZP) has been shown to be highly effective as an immunocontraceptive in white-tailed deer. However, the immunogenicity of PZP extracted from pig ovaries may vary from lot to lot and the extract has the potential of containing either viral or pathogenic material. Determination of the immunocontraceptive epitopes of PZP would allow portions of the molecule to be synthesized or inserted into a recombinant system for production of a consistent and safe vaccine. In this study, epitopes of PZP were selected and tested by in vitro binding, immunogenicity in rabbits, immunogenicity and immunocontraception in deer. Sera from PZP immunocontracepted deer were tested on ELISA plates containing immobilized peptides from ZP1 and ZP3alpha. Peptides with which sera from infertile deer reacted (six peptides from ZP1 and six peptides from ZP3alpha) were selected, synthesized and tested for immunogenicity in rabbits. Deer were then immunized with combinations of peptides from either the ZP1 or ZP3alpha groups. ZP3alpha peptides induced high immune titers against native PZP, but did not induce infertility in the deer. Although ZP1 peptides induced lower titers, deer immunized with two ZP1 peptides exhibited multiple estrus events and infertility, typical of that for deer immunized with native PZP vaccine. Competitive inhibition assays using the ZP1 peptides demonstrated that the peptide comprising pins 10-16 was most effective in blocking binding by the serum antibody of native PZP immunized deer. This peptide was used to immunocontracept deer, resulting in a significant reduction in fawning for 1 year.

Amino Acid Sequence↗
PubMed2003

Absence of effects from immunocontraception on seasonal birth patterns and foal survival among barrier island wild horses.

Despite a large body of safety data, concern exists that porcine zonae pellucidae (PZP) immunocontraception--used to manage wild horse populations--may cause out-of-season births with resulting foal mortality. Our study at Assateague, Maryland indicated the effects of immunocontraception on season of birth and foal survival between 1990 and 2002 on wild horses from Assateague Island. Among 91 mares never treated, 69 (75.8%) of foals were born in April, May, and June (in season). Among 77 treated mares, 50 (64.9%) were born in season. Of 29 mares foaling within 1 year after treatment (contraceptive failures), 20 (68.9%) were born in season. Of 48 mares treated for greater than 2 years then withdrawn from treatment, 30 (62.5%) of 48 foals were born in season. There were no significant differences (p <.05) between either treatment group or untreated mares. Survival did not differ significantly among foals born in or out of season or among foals born to treated or untreated mares. Data indicate a lack of effect of PZP contraception on season of birth or foal survival on barrier island habitats.

Animals↗
PubMed2002

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.

Animals↗
PubMed2002

Effects of immunocontraception on population, longevity and body condition in wild mares (Equus caballus).

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.

Animals↗
PubMed1988

Sperm antibodies induced by anti-idiotype antibodies: a strategy in development of immunocontraceptive vaccines.

The potential of using anti-idiotype antibodies as immunocontraceptive vaccines is evaluated in this study. Two sperm monoclonal antibodies, HS 63 and MS 204, which have significant inhibitory effect on the in vitro and in vivo fertilization of mouse were selected to elicit heterologous anti-idiotype antibodies. Rabbit antisera against HS 63 or MS 204 were collected after the third immunization. After the removal of anti-Fc fragments with an irrelevant mouse IgG immunoadsorbent column, anti-idiotype antibodies (anti-Id HS 63 or anti-Id MS 204) were purified from the antisera with affinity chromatography. HS 63 or MS 204 was used as respective ligand. The purified anti-idiotype antibodies conjugated with hemocyanin were used to immunize female CD-1 mice as the experimental group. Another group of female CD-1 mice were immunized with adjuvant only as the control group. Each group had three mice. The immune responses varied significantly among individual mice. The antisera could stain the acrosomal region of sperm in the indirect immunofluorescent assay in a way which is similar to the original monoclonal antibodies (HS 63 and MS 204). The inhibitory effect of antisera on the sperm fertilization of mouse oocytes in vitro was significant in both cases. In the case of antisera against anti-Id HS 63, the control group showed 81.8% and 49.3% of fertilization rates, whereas the corresponding rates of the experimental group were only 35.7% and 20.5%, respectively. Similarly, for antisera against anti-Id MS 204, the experimental group also revealed lower fertilization rates as compared with those of the controls (50.0% vs. 95.8% and 36.8% and 81.3%). The results of this study suggest that anti-idiotype antibodies against HS 63 and MS 204 contain the internal image of the sperm antigen and they could elicit an immune response with a significant antifertility effect. Therefore, they might not only be contributory to further study and understanding of the original antigens in structure and function but also be used as an alternative in the development of immunocontraceptive vaccines.

Animals↗
PubMed2002

Antifertility vaccines.

The possibility of using immunization as a method of birth control has been explored actively since the 1930s, with several different sperm, egg or hormonal antigens having been studied as suitable targets for intervention. However, it is only in the past decade that the efficacy of vaccination against fertility has become established firmly in both humans and free-roaming animal populations. We will review recent progress in the continuing development of antifertility vaccines, with an emphasis on vaccines intended ultimately for use in humans, whilst highlighting also some of the notable successes achieved with vaccines produced for use in other species.

Animals↗
PubMed1998

Testis-specific proteins and their role in contraceptive vaccine development.

Development of a vaccine(s) based on sperm antigens represents a promising approach for contraception. The utility of an antigen in immunocontraception is contingent upon its testis/sperm specificity and involvement in spermatogenesis and/or fertilization. The aim of the present article is to review the information regarding the proteins that have been reported to be testis/sperm-specific and may have an important function in spermatogenesis and/or fertilization. The potential role of these proteins in the development an antisperm contraceptive vaccine(s) is discussed.

Contraception, Immunologic↗
PubMed1988

Identification of sperm antigens that regulate fertility.

Polyclonal antisera were generated in rabbits against human sperm extracts. Out of many such antisera, one was selected (antiserum I) because it recognized relatively few antigens on Western blots. Antiserum I identified immunostainable material on the acrosome of human, monkey, rabbit, hamster, rat and mouse sperm. A detailed histochemical study using rat testes showed that the antigen was detectable on early spermatids and sperm, but not on less mature germ cells. Immunohistochemistry at the electron microscope level localized the antigen on the plasma membrane of rat sperm. In Western blots using human sperm extracts, the antibody recognized a major band with an apparent molecular weight (MW) of 40,000 and minor bands at 43,000 and 72,000. With extracts of monkey sperm and rat testicular cytosol, antiserum I recognized antigens of 69,000 and 23-24,000, respectively. Functionally, antiserum I produced strong agglutination of human sperm. It also prevented attachment of mouse sperm to mouse oocytes in vitro and reduced fertility when administered to female mice. These results suggest that antiserum I can be used as a possible reagent for selecting sperm antigens as components of a contraceptive vaccine.

Animals↗
PubMed2002

Ecological basis for fertility control in the house mouse (Mus domesticus) using immunocontraceptive vaccines.

Laboratory studies confirm the potential for fertility control in the house mouse Mus domesticus using mouse cytomegalovirus (MCMV) as a vector for an immunocontraceptive vaccine. This article presents an overview of key results from research in Australia on enclosed and field populations of mice and the associated epidemiology of MCMV. The virus is geographically widespread in Australia. It also persists in low population densities of mice, although if population densities are low for at least a year, transmission of the virus is sporadic until a population threshold of approximately 40 mice ha(-1) is reached. The serological prevalence of MCMV was high early in the breeding season of four field populations. Enclosure studies confirm that MCMV has minimal impact on the survival and breeding performance of mice and that it can be transmitted to most adults within 10-12 weeks. Other enclosure studies indicate that about two-thirds of females would need to be sterilized to provide effective control of the rate of growth of mouse populations. If this level is not maintained for 20-25 weeks after the commencement of breeding, the mouse population can compensate through increased recruitment per breeding female. The findings from this series of descriptive and manipulative population studies of mice support the contention that MCMV would be a good carrier for an immunocontraceptive vaccine required to sustain female sterility levels at or above 65%.

Animals↗
PubMed2002

Evaluation of a porcine zona pellucida vaccine for the immunocontraception of domestic kittens (Felis catus).

With a seasonally polyestrus breeding structure, the unwanted domestic cat population has proven difficult to control. Various lethal methods have been used in an attempt to lower this population of cats. Recently, humane attempts to control "pest species," such as the feral cat, have focused on immunocontraception. SpayVacTM is a vaccine that uses antibodies raised against porcine (ZP) antigens to prevent fertilization of the ovum. SpayVac, delivered in a single dose, has been evaluated in fallow deer and several species of seals with >90% reduction in fertility and no adverse reactions. This study evaluated the effectiveness of SpayVac in reducing fertility in domestic kittens. Thirty female kittens were treated with SpayVac containing either Freund's complete adjuvant (FCA) or alum, or with a control vehicle. Kittens were monitored for side effects, estrus cycling at maturity, and fecundity. Anti-porcine ZP antibodies were quantified by ELISA. Immunohistochemical assays measured the species specificity of the antibodies produced and IgG binding in vivo. Despite high anti-porcine ZP antibody titers, neither formulation of SpayVac prevented estrus cycling at maturity or reduced fecundity. Immunohistochemical assays indicated that antibodies produced by cats treated with SpayVac recognized porcine ZP, but not feline ZP.

Animals↗
PubMed1981

The influence of anti-zona and anti-sperm antibodies on sperm--egg interactions.

Anti-zona antibodies are effective inhibitors of fertilization in vitro and, regardless of whether passive or active immunization techniques are used, in vivo. Antibodies raised against unfractionated zona pellucida antigens are chiefly directed against a group of carbohydrate-rich components localized on the outer surface of the zona. The interaction of anti-zona antibodies with these sites induces the formation of a surface precipitate which occludes the sperm binding sites by a process of steric hindrance, and stabilizes the zona structure against digestion by the proteolytic enzymes of the sperm head. Active immunization studies indicate that the long-term induction of infertility without adverse side effects is feasible in both laboratory rodents and primates when the zona pellucida is used as a target. Anti-sperm antibodies also exhibit a capacity for inhibiting fertilization in vivo and in vitro. To determine the most appropriate detection method to screen patients for anti-sperm antibodies several homologous and heterologous antisera were analysed by 5 different agglutination and immobilization techniques and then compared for their ability to inhibit the fertilizing capacity of human spermatozoa using the zona-free hamster egg penetration test. The results obtained with the Franklin--Dukes tube--slide test exhibited the closest correlation with the anti-fertility activity of a given antiserum; this activity could be amplified by the addition of complement to the medium. It is concluded that antibodies directed against the sperm head are responsible for limiting the fertilizing capacity of human spermatozoa in vitro and that it is these antibodies of which attention should be focused to unravel the role that immunological factors play in the aetiology of infertility in vivo.

Animals↗
PubMed2002

Field testing of immunocontraception on white-tailed deer (Odocoileus virginianus) on Fire Island National Seashore, New York, USA.

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.

Animals↗
PubMed2002

Porcine zona pellucida (PZP) immunocontraception of wild horses (Equus caballus) in Nevada: a 10 year study.

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.

Animals↗
PubMed2002

Reversibility of action and safety during pregnancy of immunization against porcine zona pellucida in wild mares (Equus caballus).

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.

Animals↗
PubMed2005

Effects of GnRH immunization in sexually mature pony stallions.

Immunization against gonadotrophin releasing hormone (GnRH) was studied as an alternative for the commonly used surgical castration in stallions. Two GnRH vaccines comprising non-mineral oil adjuvants were evaluated for their potential to induce high antibody titers directed against GnRH and subsequent effects on reproductive characteristics. Twelve sexually mature male hemicastrated Shetland ponies were assigned to three groups. Group 1 and 2 were injected with 1mg peptide equivalent of G6k-GnRH-tandem-dimer conjugated to ovalbumin (OVA) in CoVaccine HT adjuvant (GnRH/CoVaccine) and in Carbopol (GnRH/Carbopol), respectively, and group 3 was injected with CoVaccine HT adjuvant without antigen (controls). After immunization no adverse effects were observed with respect to the injections sites or general health. Two weeks after the second vaccination antibody titers against GnRH increased rapidly in all animals of the GnRH/CoVaccine group, at the same time reducing serum testosterone levels maximally for the further duration of the experiment. In the GnRH/Carbopol group antibody responses and effects on testosterone levels were intermediate in two stallions and not apparent in the remaining stallions of this group. Semen evaluation showed that from 2 weeks after the second immunization onwards, sperm motility was affected in all stallions treated with GnRH/CoVaccine and one stallion treated with GnRH/Carbopol. Seven weeks after the second immunization, no semen could be collected from two stallions, one of each group, due to suppressed libido. Histological examination of the testes, 15 weeks after the initial immunization, demonstrated reduction in seminiferous tubuli diameters in all stallions of the GnRH/CoVaccine group and one stallion of the GnRH/Carbopol group. Furthermore, spermatogenesis was extremely disorganized in these stallions, as indicated by absence of the lumen in the seminiferous tubules, the absence of spermatozoa and spermatids in the tubular cross-sections and the impossibility to determine the stage of the tubular cross-sections. Testis size was also substantially reduced in three out of four stallions treated with GnRH/CoVaccine. The results demonstrate that two immunizations with G6k-GnRH-tandem-dimer-OVA conjugate in a suitable adjuvant such as CoVaccine HT caused a rapid and complete reduction of serum testosterone levels in sexually mature stallions, subsequently leading to reduced sperm motility and affected testis function, while no adverse reactions were observed after immunizations.

Animals↗
PubMed2006

Molecular cloning and assessment of the immunocontraceptive potential of the zona pellucida subunit 3 from Brandt's vole (Microtus brandti).

A full-length cDNA encoding Brandt's vole (Microtus brandti) zona pellucida glycoprotein subunit 3 (vZP3) was isolated using rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The cDNA contains an open reading frame of 1254 nucleotides encoding a polypeptide of 418 amino acid residues. The deduced amino acid sequence of vZP3 revealed high overall homology with hamster (82.1%), mouse (81.3%) and rat (80.6%). A synthetic vZP3 peptide corresponding to amino acid residues 328-343 was conjugated to keyhole limpet hemocyanin (KLH-vZP3(328-343)) and used to immunise female Brandt's voles in order to test the efficacy of this peptide as a contraceptive antigen. High IgG antibody levels to the vZP3(328-343) peptide were present in the sera of female voles immunised with KLH-vZP3(328-343) and these also cross-reacted to the zona pellucida in ovaries of Brandt's vole. The fertility of the KLH-vZP3(328-343) -immunised voles was reduced by 50% compared with controls without evidence of significant ovarian pathology.

Amino Acid Sequence↗
PubMed2006

Evaluation of the contraceptive potential of brZPCp vaccine in BALB/c mice: their safety and efficacy.

In this study we have examined the potential ability of Microtus branditi partial ZPC (brZPCp) cDNA sequence (436-1150 nt) as a target for immunocontraception. Immunogenicity studies and fertility trials were performed in BALB/c mice using recombinant construction pCR3.1-brZPC(p). ELISA outcome indicated that antibodies could be generated by immunized mice, and IgG titer was increased compared to the control. Immunohistochemistry outcome indicated that antibodies could recognize native ZP in vivo, which in turn, prevented the binding of sperm to ovulated eggs. Antibodies could also recognize recombinant protein expressed by BL21 in vitro, which was confirmed by SDS-PAGE and Western blot. Fertility rate was reduced by 45% compared to the control immunized with pCR3.1. Meanwhile, there was no incidence of significant ovarian pathology in treated mice. This experiment indicates that this vaccine can elicit the specific antibody which binds exactly to the corresponding ZPC. This construction is proved to be immunogenic, and can reduce fertility without obvious oophoritis. The result in this study suggests a potentially important method for controlling population for its safety and easy production.

Animals↗