Major locus on ECA18 influences effectiveness of GonaCon vaccine in feral horses.

Contraceptive vaccines are used to reduce birth rates in wild and feral animal populations. While the immunocontraceptive GonaCon-Equine has proven effective in reducing fertility among female feral horses, there is individual variation in the duration of infertility following treatment. To identify genetic factors influencing the effectiveness of GonaCon-Equine, we conducted a genome-wide association study of 88 mares from a feral population genotyped using the Illumina GGP Equine 70k SNP array. Contraceptive treatment schedules and long-term foaling rates have been recorded for each individual. We used mixed linear models to control for relatedness among mares. We found a significant association (p < 5 ×10-8) with a locus on equine chromosome 18. The most likely candidate genes in this region are STAT1 and STAT4, which are both involved in immune system function. Variation in STAT function could affect the immune response to the vaccine, leading to variation in contraceptive efficacy. Additional SNPs reaching a less stringent threshold of significance (p < 5 ×10-6) were located on other chromosomes near known immune system genes, supporting the hypothesis that variation in immunocontraceptive efficacy can be attributed to genetic variation in immune response rather than fertility genes.

Zona pellucida glycoproteins: Relevance in fertility and development of contraceptive vaccines.

Mammalian zona pellucida (ZP) is composed of three to four glycoproteins, which plays an important role during fertilization. Mutations in the genes encoding zona proteins are reported in women with empty follicle syndrome, degenerated oocytes and those with an abnormal or no ZP further emphasizing their relevance during fertility. Immunization with either native or recombinant ZP glycoproteins/proteins leads to curtailment of fertility in various animal species. Observed infertility is frequently associated with ovarian pathology characterized by follicular atresia and degenerative changes in ZP, which may be due to oophoritogenic T cell epitope(s) within ZP glycoproteins. To avoid ovarian dystrophy, B cell epitopes of ZP glycoproteins have been mapped by using bio-effective monoclonal antibodies. Immunization with the immunogens encompassing the mapped B cell epitopes by and large led to amelioration of follicular atresia. However, their use for human application will require more rigorous research to establish their safety and reversibility of the contraceptive effect. Nonetheless, to minimize human-animal conflicts, ZP-based contraceptive vaccines have been used successfully in the population management of free-ranging animal species such as feral horses, white-tailed deer and elephants. To control zoonotic diseases, attempts are also underway to control the population of other animal species including stray dogs, which acts as one of the major vectors for the rabies virus.

Contraceptive efficacy of recombinant porcine zona proteins and fusion protein encompassing canine ZP3 fragment and GnRH in female beagle dogs.

PROBLEM: To manage population of dogs (Canis familiaris), the efficacy of recombinant proteins-based contraceptive vaccines to inhibit fertility has been evaluated in female beagle dogs. METHOD OF STUDY: Female beagle dogs (n = 4) were immunized with physical mixture of Escherichia coli-expressed recombinant porcine ZP3 with promiscuous T cell epitope of tetanus toxoid (TT-KK-pZP3) and porcine ZP4 with promiscuous T cell epitope of bovine RNase (bRNase-KK-pZP4), or with a fusion protein encompassing dog ZP3 fragment and two copies of GnRH with appropriate promiscuous T cell epitopes (dZP3-GnRH2 ); control animals received only alum, the adjuvant. The immunized animals were followed-up for antibody titres by ELISA as well as for fertility status subsequent to mating with male dogs. RESULTS: Active immunization of female dogs following a three injections schedule at 4-week intervals with a physical mixture of TT-KK-pZP3 + bRNase-KK-pZP4 as well as dZP3-GnRH2 , led to generation of significant antibody titres against respective recombinant proteins. Active immunization with dZP3-GnRH2 also led to generation of antibodies reactive with both dZP3 and GnRH. A booster dose on day 383 led to an increase in antibody titres and circulating antibodies against respective recombinant proteins could be observed on day 528. Antibodies in immune serum samples from dogs immunized with TT-KK-pZP3 + bRNase-KK-pZP4 or dZP3-GnRH2 reacted with native canine ZP as assessed by an indirect immunofluorescence assay. Mating studies revealed a reduced number of pregnancies as well as a significant reduction in the number of pups born in the female dogs immunized with dZP3-GnRH2 as compared to the adjuvanted control. Curtailment of pregnancy in dZP3-GnRH2 immunized group was associated with antibody titres against dZP3-GnRH2 . However, immunization with recombinant TT-KK-pZP3 + bRNase-KK-pZP4 did not significantly decrease the number of pups born as compared to the adjuvanted control. CONCLUSION: These studies revealed the potential of recombinant dZP3-GnRH2 -based contraceptive vaccine to curtail fertility in female dogs. Large scale studies to establish the efficacy and safety of this recombinant protein for the management of community dog population are thus warranted.

Production and characterization of a human antisperm monoclonal antibody against CD52g for topical contraception in women.

BACKGROUND: Approximately 40% of human pregnancies are unintended, indicating a need for more acceptable effective contraception methods. New antibody production systems make it possible to manufacture reagent-grade human monoclonal antibodies (mAbs) for clinical use. We used the Nicotiana platform to produce a human antisperm mAb and tested its efficacy for on-demand topical contraception. METHODS: Heavy and light chain variable region DNA sequences of a human IgM antisperm antibody derived from an infertile woman were inserted with human IgG1 constant region sequences into an agrobacterium and transfected into Nicotiana benthamiana. The product, an IgG1 mAb ["Human Contraception Antibody" (HCA)], was purified on Protein A columns, and QC was performed using the LabChip GXII Touch protein characterization system and SEC-HPLC. HCA was tested for antigen specificity by immunofluorescence and western blot assays, antisperm activity by sperm agglutination and complement dependent sperm immobilization assays, and safety in a human vaginal tissue (EpiVaginal™) model. FINDINGS: HCA was obtained at concentrations ranging from 0.4 to 4 mg/ml and consisted of > 90% IgG monomers. The mAb specifically reacted with a glycan epitope on CD52g, a glycoprotein produced in the male reproductive tract and found in abundance on sperm. HCA potently agglutinated sperm under a variety of relevant physiological conditions at concentrations ≥ 6.25 µg/ml, and mediated complement-dependent sperm immobilization at concentrations ≥ 1 µg/ml. HCA and its immune complexes did not induce inflammation in EpiVaginal™ tissue. INTERPRETATION: HCA, an IgG1 mAb with potent sperm agglutination and immobilization activity and a good safety profile, is a promising candidate for female contraception. FUNDING: This research was supported by grants R01 HD095630 and P50HD096957 from the National Institutes of Health.

Evaluation of multi-epitope recombinant protein as a candidate for a contraceptive vaccine.

Contraceptive vaccine (CV) is a valuable, non-invasive, and alternative method for purposeful contraception. Sperm antigens are useful targets for producing CVs due to their specialized expression in sperm. In this study, a recombinant protein containing three main sperm epitopes (IZUMO1, SACA3, and PH-20) was designed and evaluated as CV to control fertility in male mice. The chimeric recombinant protein was expressed and purified in E. coli. Male mice were immunized by 100 µg purified protein and sera were collected to assess IgG antibodies. Evaluating the reproductive performance, immunized male mice mated with normal-fertile female mice and mating rate and the number of newborns was studied. Immunized mice were sacrificed and necropsy and histopathology studies were conducted. The results revealed that the designed chimeric protein stimulated the immune system of the mice effectively. The level of IgG antibody was significantly higher in vaccinated mouse rather than control mouse. Eighty percent of the vaccinated mice became infertile and in the remaining ones, the number of children decreased to 4-6 offspring instead of 10-12 in normal mice. Histopathological studies showed that no organs including heart, brain, lung, liver, kidney and intestine were damaged. However, Normal spermatogenesis has been disrupted and necrotic spermatogonia cells were reported in Seminiferous tubules. We concluded that the designed chimeric protein containing IZUMO1, SACA3, and PH-20 epitopes can stimulate the immune system and cause male contraception without any side effects.

Promises and pitfalls of male contraceptive vaccines.

I have not failed. I've just found 10,000 ways that won't work. -Thomas A. Edison.

Immuno-castration of female and male pigs with anti-gonadotrophin releasing hormone vaccine: Morphometric, histopathological and functional studies of the reproductive system.

Immuno-castration is increasingly recommended in pigs due to welfare reasons; however, there are few studies in females compared to males. This aim of this study was to investigate the effects of immuno-castration in female and male pigs. The weight, the morphometric and microscopic characteristics of the reproductive organs, and the hormone concentrations were studied in 12 immunocastrated females (IF) and 12 immunocastrated males (IM) and compared with control animals (C). At slaughter, IF tended to have greater body weights than CF (P =  0.051), whereas in IM and CM pigs there were not body weight differences (P =  0.140). The weight of the reproductive tract and size of all individual organs were less in IF compared with CF. Results from histological assessments indicated IF had more atretic follicles and a thinner endometrial mucosa than control females. Hormone concentrations were not different between CF and IF (P >  0.050). As a result of immuno-castration, there was impaired spermatogenesis in most males. Results from microscopic evaluations indicated there was a marked decrease of spermatogonial cells and size of Leydig cells in the testicles. Accessory gland structures were affected in CM and IM with there being differences in gross and microscopic characteristics. Testosterone concentrations, unlike estradiol, were different in IM compared to CM (P <  0.001). These results provide evidence that immuno-castration with the anti-gonadotrophin releasing hormone vaccine is effective in female and male pigs and induces morphological and endocrine changes incompatible with fertility.

Contraceptive and molecular function of a novel recombinant vaccine based human leukemia inhibitory factor on Balb/c mice: An experimental in vivo study.

The functional competence of leukemia inhibitory factor (LIF), as immunocontraceptive vaccine in mice, was investigated. Balb/c mice were divided into two groups of vaccinated and controls. The recombinant human LIF (rhLIF) protein and phosphate buffer saline was emulsified with Freund's adjuvant and injected into vaccinated and control groups, respectively. Theinhibition of implantation was evaluated in mice uterine. The concentration of secreted interferon-γ (IFN-γ) and interleukin (IL)-4 were measured in cultured splenocyte of mice stimulated by rhLIF. The expressions of immune responsive gene 1 (IRG-1), cochlin (COCH), amphiregulin(Ar), and heparin-binding EGF-like growth factor (HB-EGF) genes were determined. Mice were assessed for inhibition of fertility after delivery, reversibility of immune response against rhLIF, and survival rate. Active immunization of mice with rhLIF resulted in reduction of the implantation and fertility rate up to 80.49% and 75%, respectively. All mice produced a high titer of anti-rhLIF antibodies in serums and vaginal fluids washes after 16 weeks; however, these antibodies were cleared from vaginal fluid washes after six months. A significant down-regulation in mRNA levels of IRG-1, Ar and HB-EGF was observed in vaccinated group compared to controls; however, no significant change in the expression profile of cochlin gene was detected. The results showed that rhLIF prevented pregnancy in a high percentage of female mice. Although the immunization of female Balb/c mice with rhLIF inhibited fertility and expression of genes associated with this molecule, further studies are needed to support this protein as a suitable candidate for contraceptive vaccine in mammals.

[Oestrus suppression in a dairy herd by means of anti-GnRH vaccination Improvac®: A prospective field study]. / Brunstunterdrückung mittels Anti-GnRH-Impfung Improvac® in einem Milchviehbestand: Eine prospektive Feldstudie.

INTRODUCTION: The aim of this study was to evaluate the duration of estrus suppression after a double administration of the anti-GnRH vaccine Improvac® (Zoetis Schweiz GmbH, 2800 Delémont) in cows. Furthermore, it should be investigated, if a third administration could prolong the effect of the cycle suppression. A total of 21 cows (more than four weeks post partum) were vaccinated twice, at least 35 days apart, with 2 ml Improvac® (0.4 mg of a GnRH-analogon) subcutaneously on one side of the neck. Over a period of 368 days and in the course of 18 farm visits these cows were examined gynecologically and re-vaccinated if they showed signs of estrus behaviour or ovarian activity. After the second vaccination the cycle of the cows was suppressed for an average period of 114 days (59-175 days) and the effect could be prolonged by a booster of the vaccine for another 127 days in three cows. Estrus behaviour was absent for a longer period than ovarian activity was. The vaccine was tolerated well: apart from slight swelling at the injection site, no side effects were observed. Our results demonstrate that two immunizations with Improvac® are an easily applicable method for the suppression of cyclic activity in cows for a mean period of 114 days. The duration of cycle suppression was prolonged by a booster of the vaccination.

INTRODUCTION: Le but de cette étude était d'évaluer la durée de la suppression des chaleurs chez les vaches après une double administration du vaccin anti-GnRH Improvac® (Zoetis Schweiz GmbH, 2800 Delémont). En outre, on a recherché si une troisième administration pourrait prolonger l'effet de la suppression du cycle. Au total, 21 vaches (plus de quatre semaines après vèlage) ont été vaccinées deux fois, à au moins 35 jours d'intervalle, avec 2 ml d'Improvac® (0,4 mg d'analogue de la GnRH) par voie sous-cutanée d'un côté du cou. Sur une période de 368 jours et au cours de 18 visites à la ferme, ces vaches ont été examinées gynécologiquement et revaccinées si elles présentaient des signes de comportement œstral ou d'activité ovarienne. Après la deuxième vaccination, le cycle des vaches a été supprimé pendant une période moyenne de 114 jours (59 - 175 jours) et l'effet a pu être prolongé par un rappel du vaccin pendant 127 jours supplémentaires chez trois vaches. Le comportement d'œstrus était absent pendant une période plus longue que l'inactivité ovarienne. Le vaccin a été bien toléré: à part une légère enflure au site d'injection, aucun effet secondaire n'a été observé. Nos résultats démontrent que deux immunisations avec Improvac® sont une méthode facilement applicable pour la suppression de l'activité cyclique chez les vaches pendant une période moyenne de 114 jours. La durée de la suppression du cycle a été prolongée par un rappel de vaccination.

Phage constructs targeting gonadotropin-releasing hormone for fertility control: evaluation in cats.

OBJECTIVES: Phage-gonadotropin-releasing hormone (GnRH) constructs with potential contraceptive properties were generated in our previous study via selection from a phage display library using neutralizing GnRH antibodies as selection targets. In mice, these constructs invoked the production of antibodies against GnRH and suppressed serum testosterone. The goal of this study was to evaluate this vaccine against GnRH for its potential to suppress reproductive characteristics in cats. METHODS: Sexually mature male cats were injected with a phage-GnRH vaccine using the following treatment groups: (1) single phage-GnRH vaccine with adjuvant; (2) phage-GnRH vaccine without adjuvant and half-dose booster 1 month later; or (3) phage-GnRH vaccine with adjuvant and two half-dose boosters with adjuvant 3 and 6 months later. Anti-GnRH antibodies and serum testosterone, testicular volume and sperm characteristics were evaluated monthly for 7-9 months. RESULTS: All cats developed anti-GnRH antibodies following immunization. Serum antibody titers increased significantly after booster immunizations. In group 3, serum testosterone was suppressed 8 months after primary immunization. Total testicular volume decreased in group 1 by 24-42% and in group 3 by 15-36% at 7 months after immunization, indicating potential gonadal atrophy. Vacuolation of epididymides was observed histologically. Although all cats produced sperm at the conclusion of the study, normal morphology was decreased as much as 38%. Phage alone produced no local or systemic reactions. Immunization of phage with AdjuVac produced unacceptable injection site reactions. CONCLUSIONS AND RELEVANCE: Our phage-based vaccine against GnRH demonstrated a potential for fertility impairment in cats. Future research is required to optimize vaccine regimens and identify animal age groups most responsive to the vaccine. If permanent contraception (highly desirable in feral and shelter cats) cannot be achieved, the vaccine has a potential use in zoo animals or pets where multiple administrations are more practical and/or reversible infertility is desirable.

Effects of an anti-gonadotrophin releasing hormone vaccine on the morphology, structure and function of bull testes.

Castration reduces aggressive and sexual behaviour and provides better carcass quality in bull calves. Vaccination against gonadotrophin-releasing hormone (GnRH) is used as an alternative to surgical castration for the purposes of reducing pain and distress in the animals. Currently, no anti-GnRH vaccine has been authorized for use in cattle in the European Union. The aim of the present study was to assess the effect of an anti-GnRH swine-specific vaccine (Improvac®, Zoetis, USA) on the morphology, structure and function of bull testes. Animals were vaccinated at days 1, 21 and 104 of the experimental period and were classified based on their live weight into the following two groups: LIGHT (172.9 ±â€¯30.00 kg) and HEAVY (323.8 ±â€¯37.79 kg). The scrotal circumference was measured on day 1 and prior to slaughter (day 164). At slaughter, the sperm motility and concentration in the caudae epididymis were assessed. Testes were weighed, measured and examined using ultrasound, and then tissue samples were collected and fixed in formalin. Histological and immunohistochemical studies were performed on the testes to measure the diameter of the seminiferous tubules and assess the testicular cell populations. The results revealed that suppression of testicular development was associated with the use of the Improvac® vaccine, which resulted in a smaller size of the testes and impaired spermatid production. However, the effect of Improvac® was more pronounced and consistent in calves vaccinated at a low live weight than at a heavy live weight, which suggested that vaccination is more effective when calves are vaccinated before or early during puberty. However, testes from calves vaccinated at a low live weight were more prone to the development of intraluminal concretions in the seminiferous tubules.

Immunogenicity and contraceptive efficacy of recombinant fusion protein encompassing Sp17 spermatozoa-specific protein and GnRH: Relevance of adjuvants and microparticles based delivery to minimize number of injections.

PROBLEM: Requirement of multiple injections of contraceptive vaccines to achieve infertility is one of the important impediments for their application. In the present study, attempts have been made to reduce the number of injections of contraceptive vaccine. METHOD OF STUDY: Fusion protein encompassing C-terminus fragment of sperm protein Sp17 (aa residues 76-126) and two copies of gonadotropin-releasing hormone along with T-cell epitopes and dilysine linkers (abbreviated as Sp17C -GnRH2 ) was expressed in Escherichia coli. Its immunogenicity and contraceptive efficacy have been evaluated in female FVB/J mice using different adjuvants and delivery platforms. RESULTS: Immunization of female mice with recombinant Sp17C -GnRH2 (25 µg/injection/mouse) emulsified with squalene-arlacel A following two injections schedule led to failure of 88.8% immunized animals to conceive, which was not significantly different from mice immunized with same protein along with alum following three injections schedule. To make single-dose vaccine, poly d,l-lactic acid-based microparticles (PLA-MPs) entrapping Sp17C -GnRH2 were prepared. Immunization of female mice with a combination of soluble Sp17C -GnRH2 (12.5 µg/injection/mouse) along with Sp17C -GnRH2 entrapped in PLA-MPs (12.5 µg/injection/mouse) in alum showed higher antibody titres and contraceptive efficacy as compared to mice immunized with Sp17C -GnRH2 entrapped in PLA-MPs alone in alum. Immunization with recombinant Sp17C -GnRH2 led to long-term infertility as second mating (150 days after immunization) of various groups of immunized mice showed similar infertility as observed during first mating. CONCLUSION: Single-dose immunization with PLA-MPs entrapping Sp17C -GnRH2 along with soluble recombinant protein in alum generated long-lasting infertility in female mice.

Chronic use of a GnRH agonist (deslorelin) or immunization against GnRH: effects on testicular function and sperm quality of bucks.

Chronic use of GnRH agonists and immunization against GnRH have been used as reversible contraceptive methods. The aim of this study was to compare the effectiveness of both treatments to inhibit reproductive function of adult bucks, in terms of strength and duration of the effects. We used 9 control untreated bucks (CON), 7 bucks treated chronically with a GnRH agonist (subcutaneous implants with 7.4 mg of deslorelin, Suprelorin, Virbac) (AGO), and another 7 bucks were immunized against GnRH (dose of 2 mL of Improvac-Zoetis with 300 µg of a synthetic incomplete analog of natural GnRH; 300 mg of diethylaminoethyl-dextran; and 2.0 mg of chlorocresol) (IMM). Testicular and sperm evaluations, testosterone concentrations, and male odor were determined from 4 wk before applying the treatments until 17 mo of their application. Scrotal circumference of CON (21.0 ± 0.1 cm) and IMM (21.2 ± 0.2 cm) was greater than that of AGO bucks (19.9 ± 0.2 cm) (P < 0.05 for each), without difference between CON and IMM bucks. Pixels' color intensity of testicular ultrasound images was not affected by treatment (general mean ± SEM: 116.0 ± 1.8). Testosterone concentration was greater in CON than AGO and IMM in months 3 and 4, greater in CON and IMM than AGO bucks in months 15 and 16, and greater in IMM than CON and AGO bucks in month 17 (P < 0.05 for all comparisons). Male odor was greater in CON (1.5 ± 0.0) than IMM bucks (1.3 ± 0.0) and greater in IMM than AGO (1.1 ± 0.0) bucks (P < 0.05 for each). Treatment negatively affected all the sperm variables: the total number of sperm in the ejaculate, sperm motility, sperm with normal morphology and sperm with integral membrane function. It was concluded that both treatments were effective in inhibiting the reproductive axis; however, neither of them produced azoospermia or decreased testosterone concentrations to undetectable levels. With both treatments, there were individual males exhibiting characteristics of fertility in all periods of the study. However, chronic use of a GnRH agonist seemed to be the most effective treatment in terms of duration and strength.

Brazilian Spotted Fever Prevention through a Nonlethal Capybara Population Control Strategy.

INTRODUCTION: Brazilian spotted fever (BSF), a lethal tick-borne Rickettsioses (2000 - 2018 >600 human deaths) involving synanthropic capybara as host. METHODS: We introduced an alternative to mitigate human-capybara conflicts and epidemiologic concerns of BSF. Complex aspects like transmission dynamics, risk areas, host mobility, and birth rate control, were considered to develop a prevention strategy using an anti-GnRH vaccine. RESULTS: The propositioned immunocontraceptive potentially remove and prevent the spread of BSF from endemic areas. CONCLUSIONS: We propose the anti-GnRH vaccine as a BSF prevention strategy based on these favorable results.

Field-testing a single-dose immunocontraceptive in free-ranging male capybara (Hydrochoerus hydrochaeris): Evaluation of effects on reproductive physiology, secondary sexual characteristics, and agonistic behavior.

Controlling wildlife populations to mitigate human-wildlife conflicts and the spread of zoonotic diseases is an ever-growing necessity. The objective of this study was to evaluate a single-dose anti-gonadotropin-releasing hormone vaccine (GonaCon, USDA/NWRC, Fort Collins, CO, USA) as a non-lethal alternative for population control in free-ranging, synanthropic male capybara. In addition to infertility efficacy of this treatment, potential effects on the alpha male's secondary sexual characteristics and agonist behavior need to be assessed because any alterations in these factors could lead to population management failure. The treatment group (n = 3) received 1 mL of the anti-GnRH vaccine, intramuscularly, and the control group (n = 2) a 1 mL sham vaccine. Reproductive behavior and social group dynamics were monitored for 30 days prior to inoculation (June 2017) with continuous observations occurring during the study period. Antifertility effects were assessed by conducting exams of testicular morphology, semen characteristics, and histological analysis (after 270 days via hemi-gonadectomy). Compared to the control group, the testicles of the treated males had severe atrophy (P <  0.05), oligozoospermia and greater numbers of sperm cells in a static developmental phase. Courtship and agonistic alpha male behavior were not altered, and the group's social integrity was maintained. Results indicate there was 100% infertility in capybara males, observed throughout the study period of 18 months, and equally important, the male's alpha characteristics were not affected by the treatment, which is imperative for successful capybara population control efforts.

Researching immunocontraceptive vaccines with mares (Equus caballus) as both a target and model for African elephant (Loxodonta africana) cows: A review.

A sequence of studies is reviewed that reported the domestic horse (Equus caballus) mare as an appropriate and accessible research platform for recording clinical and laboratory data post-immunisation with anti- GnRH and -zona pellucida (ZP) immunocontraceptive vaccines. Experience with a native porcine ZP (pZP) vaccine in African elephant (Loxodonta africana) cows highlighted needs for improving vaccine formulations and more clearly defining associated ovarian effects and safety profiles. Initially, the efficacy, reversibility and safety of the GnRH vaccine Improvac® in mares was demonstrated using reproductive tract ultrasonography and concurrently measuring serum antibody titres and progesterone concentrations. Results informed the study design and minimally invasive monitoring of post-treatment ovarian steroid responses of this vaccine in free-ranging African elephant cows. A subsequent sequence of studies reported reversible contraceptive and immunological efficacy in pony mares immunised with pZP formulated with Freund's adjuvants. By comparison, mares treated with a recombinant ZP3 and ZP4 (reZP) vaccine showed disappointing responses. Unexpectedly, most pZP-treated mares showed ovarian inactivity. In attempting to understand this response, results showed the involvement of cytotoxic (CD8+) T-cells negatively correlated to serum ovarian steroid and anti-Müllerian hormone (AMH) levels. Of concern was the prevalence of injection-site lesions ascribable to Freund's adjuvants. Following this, mares treated with both pZP and a novel reZP vaccine formulated with non-Freund's adjuvants showed comparable immunological responses and ovarian inactivity, notably without adverse treatment reactions. In addition, measuring AMH showed promise for monitoring ovarian function in anti-ZP-treated animals.

Evaluation of infertility efficacy of the E. coli expressed STF2-GnRH vaccine in male cats.

Gonadotropin-releasing hormone (GnRH) is secreted from the hypothalamus and anti-GnRH antibodies are not formed under normal conditions. However, administration an excess of recombinant GnRH protein results in the formation of anti-GnRH. We evaluated the efficacy of the recombinant Salmonella typhimurium flagellin fljB (STF2)-GnRH vaccine in inducing infertility in 17 intact male cats. The first vaccination and a boosting vaccine was injected for examination. Serum was obtained from blood collected at monthly intervals and anti-GnRH antibodies and testosterone concentrations were determined. Six months after the vaccination, testicular samples are obtained and used for histological examination. Compared with sham control group, the injection groups showed an increase in anti-GnRH antibody titers and testosterone concentrations tended to be reduced in the injection groups and increased in the control group. Histological evaluations and Johnsen's testicular biopsy scores revealed testicular hypoplasia in the 2 injection groups. Consequently, normal sexual maturation with sperm production was observed in the control group. In contrast, the cats that received the GnRH vaccine showed weak (2 of 7 cats) or moderate (4 out of 7 cats) dose-dependent infertility effects. On the basis of the results, the STF2-GnRH vaccine was identified to be effective in inducing infertility in male cats. The results of this study thus indicate the possibility of immunological castration targeting feral cats.

BOARD INVITED REVIEW: Immunocontraception as a possible tool to reduce feral pig populations: recent and future perspectives.

The feral pig populations of many countries continue to increase. Scientific studies on population size are scarce, while the numbers of reported observations on presence of and damage caused by feral pigs are increasing. Feral pigs can carry and spread several diseases (including zoonotic), but African Swine Fever (ASF) is of most concern. It is a highly transmissible viral disease associated with an extremely high mortality rate. Since 2009 ASF has appeared in several European countries, with cases being identified first among local feral pigs and consequently in domestic pig production units, indicating a clear linkage with the movement of the feral pig population and the spread of the disease across national boundaries. Control of feral pig populations is currently under discussion. Because massive culling raises questions of animal welfare and ethics, fertility control could represent an important and effective means to control feral pig populations. Contraceptive vaccines have been used with some degree of success in many wild species because they are able to provide a long-term effect without any consequent health problems. However, extensive and efficacious use of vaccines to control feral pig populations is not simple. The aim of this article was to review the progress in immunocontraception use in feral pigs, providing an account of the current status and future perspectives.

Serum antibody immunoreactivity and safety of native porcine and recombinant zona pellucida vaccines formulated with a non-Freund's adjuvant in horses.

Commercial and regulatory limitations associated with native porcine zona pellucida (pZP) vaccines formulated with Freund's adjuvants may be overcome by developing effective recombinant ZP vaccines (reZP) and identifying alternative adjuvant formulations. A two-part preparatory study used 15 geldings and identified potentially effective alternative adjuvant formulations based on anti-pZP antibody response following treatment with pZP formulated with Addavax (AddaVax ™, Invivogen), Quil A (Quil-A® Adjuvant, Invivogen), Quil A and Poly (I:C) (HMW VacciGrade™, Invivogen), Pet Gel A (Montanide™ Pet Gel A, Seppic) and Pet Gel A and Poly (I:C). Injection site reactions, rectal temperature and respiratory and heart rates were also monitored for three days post-treatment. Suitable anti-pZP antibody titres were seen in response to Pet Gel A and Pet Gel A and Poly (I:C). Subsequently in 31 mares, following administration of pZP, reZP and a combination of pZP and reZP proteins prepared in Pet Gel A and Poly (I:C), both serum anti-pZP and -reZP antibody responses were monitored. In addition, safety was assessed for up to seven days post-treatment by inspection and palpation of gluteal intramuscular injection sites and measurement of rectal temperature. The measured antibody titres in all treatment groups differed significantly to an adjuvant control group (P < 0.001). Temporal changes in both anti-pZP and -reZP antibody titres in all ZP treatment groups were similar to patterns reported previously in various species vaccinated with pZP formulated with Freund's adjuvants. There were no differences in anti-pZP antibody titres between the pZP and reZP treated groups (P > 0.05). Side effects were mild and transient in nature. This represents the first application of a reZP vaccine formulated with non-Freund's adjuvants evoking a similar antibody titre response to native pZP vaccination in mares.