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This review summarizes the advancements over a decade of research on antigens of anti-sperm antibodies (ASAs), which are key to male immune infertility. Despite the progress in assisted reproductive technologies, understanding the roles and mechanisms of ASAs and their antigens remains vital for immune infertility management. We conducted a comprehensive literature search on PubMed from January 2013 to December 2023 using the following keywords: "anti-sperm antibody," "sperm antigen," and "immune infertility." In this review, we focus on the discoveries in sperm antigen identification and characterization through proteomics, gene disruption technology, and immunoinformatics, along with the development of fertility biomarkers. Here, we discuss the clinical applications of improved ASA detection methods and the progress in the development of immunocontraceptive vaccines. The intersection of advanced diagnostic techniques and vaccine development represents a promising frontier in reproductive health. The findings also highlight the need for standardized ASA detection methods and a comprehensive molecular-level approach to understanding ASA-related infertility. These insights underscore the significance of ongoing reproductive immunology research in enhancing clinical fertility outcomes and contraceptive vaccine development., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objective: Human Chorionic Gonadotropin (hCG) plays a crucial role in embryo implantation and in maintenance of pregnancy. An immuno-contraceptive approach involves the use of a recombinant hCGβ-LTB vaccine formulated with adjuvant Mycobacterium indicus pranii (MIP), to prevent pregnancy without disturbing ovulation, hormonal profiles, and menstrual cycles in women. The present work in mice was designed to address issues encountered in clinical trials conducted with hCGβ-LTB vaccine, with focus on two primary concerns. Firstly, it aimed to determine the optimal vaccine dosage required to induce a high level of anti-hCG antibodies. Secondly, it aimed to assess the safety profile of the vaccine, specifically injection site reactions in the form of nodules, observed in some of the subjects., Methods and Results: Studies undertaken indicate that a 2 µg dose of the protein version of the vaccine, administered in mice through the intramuscular route, can induce high anti-hCG titres. Furthermore, administering a booster dose enhances the antibody response. Our findings suggest that the concentration and frequency of administration of the adjuvant MIP can also be reduced without compromising vaccine efficacy., Conclusion: The issue of nodule formation at the injection site can be mitigated either by administering the vaccine along with MIP intramuscularly or injecting hCG vaccine and MIP at separate intradermal sites. Thus, protein vaccine administered at a 2µg dose via the intramuscular route addresses both efficacy and safety concerns.

CatSper is a voltage dependent calcium ion channel present in the principal piece of sperm tail. It plays a crucial role in sperm hyperactivated motility and so in fertilization. Extracellular loops of mouse sperm CatSper were used to develop a vaccine to achieve protection from pregnancy. These loops were inserted at one of the three hypervariable regions of Human Papilloma Virus (HPV) capsid protein (L1). Recombinant vaccines were expressed in E.coli as inclusion body (IB), purified, refolded and assembled into virus-like particles (VLP) in vitro, and adsorbed on alum. Four vaccine candidates were tested in Balb/C mice. All the constructs proved immunogenic, one showed contraceptive efficacy. This recombinant contraceptive vaccine is a non-hormonal intervention and is expected to give long-acting protection from undesired pregnancies., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Chris Bystroff reports financial support was provided by Grantham Foundation for the Protection of the Environment. Chris Bystroff reports a relationship with Grantham Foundation for the Protection of the Environment that includes: funding grants. Chris Bystroff has patent #US20220152166A1 issued to Rensselaer Polytechnic Institute.]., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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 IgG 1 constant region sequences into an agrobacterium and transfected into Nicotiana benthamiana. The product, an IgG 1 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., Competing Interests: Declaration of Competing Interest LZ and KJW own Mapp Biopharmaceutical Inc., a company interested in commercializing contraceptive antibodies. TRM, MB, and MP are employees of Mapp Biopharmaceutical Inc. The other authors (GBV, JGM, JAP, EM, JP, JD, EN, DJA) have no conflict of interest., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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 Sp17 C -GnRH 2 ) 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 Sp17 C -GnRH 2 (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 Sp17 C -GnRH 2 were prepared. Immunization of female mice with a combination of soluble Sp17 C -GnRH 2 (12.5 μg/injection/mouse) along with Sp17 C -GnRH 2 entrapped in PLA-MPs (12.5 μg/injection/mouse) in alum showed higher antibody titres and contraceptive efficacy as compared to mice immunized with Sp17 C -GnRH 2 entrapped in PLA-MPs alone in alum. Immunization with recombinant Sp17 C -GnRH 2 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 Sp17 C -GnRH 2 along with soluble recombinant protein in alum generated long-lasting infertility in female mice., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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 ± 30.00 kg) and HEAVY (323.8 ± 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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., (© The Author(s) 2019. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Feral swine cost around $1.5 billion each year in agricultural, environmental, and personal property damages. They are also the most widespread carriers of the zoonotic disease brucellosis, which threatens both livestock bio-security and public health. Currently, there is no approved vaccine against brucellosis in pigs. This is a preliminary report on the development of a live-attenuated B. suis vaccine that could be employed to deliver heterologous antigens to control swine populations. An attenuated vaccine strain provided significant protection against B. suis challenge in mice. Leucine auxotrophy in the vaccine strain allowed the over-expression of heterologous antigens without the use of antibiotic resistant markers. Vaccinated mice showed the development of antibodies against expressed antigen. Further evaluation is required to assess its ability to cause infertility using the mouse model prior to further testing for use as a tool for feral swine population and disease control., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Zona pellucida 3 (ZP3) is a potential antigen for the development of contraceptive vaccines to control animal population. In this study, we designed a canine ZP3 (CZP3) DNA vaccine through targeting DEC-205 (named as pcD-scFv-CZP3c) and investigated its contraceptive effect in mice. Female BALB/c mice were intramuscularly immunized 3 times at 2 weeks intervals. After immunization, humoral and cellular immune responses were detected by ELISA and flow cytometry. The results showed that pcD-CZP3 and pcD-scFv-CZP3c induced CZP3-specific antibody (Ab) responses both in serum and vaginal secretions compared to pcDNA3.1. Additionally, compared to pcD-CZP3, pcD-scFv-CZP3c increased the levels of CZP3-specific Abs after a third immunization. Abs induced by these two DNA vaccines could bind with mice and dogs oocytes. Moreover, pcD-scFv-CZP3c enhanced the activation of CD4 + T cells characterized by the increased frequencies of CD4 + CD44 + T cells. Finally, the contraceptive effect was evaluated in the immunized mice. These two DNA vaccines significantly decreased a mean litter size of mice compared to pcDNA3.1, but pcD-scFv-CZP3c group showed the smallest mean litter size. The mean litter size of pcD-scFv-CZP3 were 3.2 ± 0.742 and 4.6 ± 1.118 in two mating tests, which were significantly lower than pcDNA3.1(P < 0.001 and P < 0.05). Our results suggest that the CZP3 DNA vaccine targeted with DEC-205 may be a potential strategy for developing a contraceptive DNA vaccine., (Copyright © 2018 Elsevier Inc. All rights reserved.)

uPA, a trypsin-like serine protease, was found to take active part in male reproduction. Our previous work had demonstrated the antifertility effects of its full length protein immunization, but with immune tolerance and other latent side effects. Here we discovered two effective B-cell epitopes of uPA for male contraception in growth factor-like domain and kringle domain respectively. Together with carrier protein, immunization of these two epitope peptides could induce high titers of specific antibodies in male mice. Significant reduction of fertility was observed in these two groups in mating trial without evident systemic illness or abnormal mating behavior. Epididymal sperms of immunized males exhibited impaired progressive motility and ability to fertilize eggs in vitro. The immunization of another predicted epitope in serine protease domain and the control groups showed no similar positive results. Importantly, T cells were not activated after the challenge of these B-cell epitopes itself, which suggests that these vaccines do not induce cell-mediated autoimmunity. Taken together, our study discovered two uPA B-cell epitopes as novel targets for male immunocontraception with minimum side effects. Considering their high identity with human uPA protein, these two epitope vaccines hold great promise to be developed for man use in the future., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Currently there is no contraceptive vaccine that can cause permanent sterility in mares. This study investigates the effect of vaccination against oocyte-specific growth factors, Bone Morphogenetic Protein 15 (BMP-15) and Growth Differentiation Factor 9 (GDF-9), on ovarian function of mares. It was hypothesized that immunization against these growth factors would prevent ovulation and/or accelerate depletion of the oocyte reserve. For this study, 30 mares were randomly assigned to three groups (n = 10/group) and vaccinated with BMP-15 or GDF-9 peptides conjugated to KLH and adjuvant, or a control of phosphate buffered saline and adjuvant. Horses received vaccinations at weeks 0, 6, 12, and 18. Ovarian activity and estrous behavior were evaluated 3 days a week via ultrasonography and interaction with a stallion. The study was initiated on March1, 2016. Upon evaluation of ovulation rate, the GDF-9 group did not have a difference (P = 0.66) in ovulation rate when compared to controls (10.8 and 10.0 ovulations, respectively), but the number of ovulations in the BMP-15 group was less (P = 0.02; 4.9 ovulations). Average follicle size prior to ovulation was less (P < 0.0001) in both treatment groups compared to controls. Estrous behavior was altered in both the BMP-15 and GDF-9 groups compared to controls after the second vaccination (P = 0.05 and 0.03, respectively). Although further research is required to determine the continued effects of vaccination against GDF-9 on ovulation rates, these results indicate that vaccination against BMP-15 and GDF-9 could serve as a contraceptive in wild horse populations., (Copyright © 2018 Elsevier B.V. All rights reserved.)

To investigate the effect of gonadotropin-releasing hormone 2-multiple antigen peptide (GnRH2-MAP) on reproductive function. In our study, 20-day-old male rats (n = 90) were randomly allocated to one of three treatment groups: GnRH2-MAP immunization, GnRH2 immunization, and non-immunized control groups. The immunized animals were administered three doses of GnRH2-MAP or GnRH2 vaccines from 0 to 6 weeks at 2-week intervals. The control group only received oil adjuvant. Blood and right testis samples were collected, and the left testis was weighed and its volume was measured at 0, 2, 4, 6, 8, 10 and 12 weeks after the first immunization. The serum antibody titer and testosterone concentration were determined by ELISA, and the right testis samples were collected for histological analysis. The results revealed that the serum of vaccinated rats elicited a significantly higher antibody titer and a lower T concentration compared with the control group two weeks after the first immunization (P < 0.05), but the highest antibody titer and lowest T concentration were found in animals treated with GnRH2-MAP (P < 0.05). The second immunization resulted in a significant decrease in testicular weight and volume (P < 0.05) in both immunized groups compared to the control, but these values were significantly lower in the GnRH2-MAP group than in the GnRH2 group. Furthermore, seminiferous tubules revealed more significant atrophy in the GnRH2-MAP group than in the GnRH2 group, and no sperm were observed in rats of the GnRH2-MAP group. Thus, GnRH2-MAP may be an effective antigen and a potential immunocastration vaccine with higher effectiveness., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Effective tools for male contraception are important in the control of reproduction in animal populations. The aim of the present study was to evaluate the effects of active immunization against gonadotropin-releasing hormone (GnRH) on male reproductive function assessing testicular morphological changes and serum-gonadotropin levels in pre-pubertal rabbits, guinea pigs and ram lambs. An anti-GnRH vaccine was developed by linking a GnRH-homologous molecule to a tetanus clostridial toxoid (Al(OH)3 coadjuvant). After vaccination protocols testicular morphometry, histopathological alterations and endocrine responses (FSH, LH, testosterone and cortisol serum levels) were evaluated. Testicular volume was significantly reduced in vaccinated animals with respect to the control group in rabbits, guinea pigs and ram lambs (P<0.05 to P<0.001). The anti-GnRH vaccine generated a reduction in testicular volume of 15-, 27- and 11-fold, respectively. Tubule diameters decreased in the vaccinated group with respect to the control ~2.0-, 1.2- and 3.5-fold, respectively (P<0.001). Tubule, intertubular and lumen volumes significantly decreased in vaccinated rabbits (P<0.05), guinea pigs and ram lambs (P<0.01). Vaccinated animals of the three species showed significant reductions in spermatogonial numbers (10- to 40-fold; P<0.01). Sperm was absent in all seminiferous tubules of all rabbits, and most individuals of guinea pigs (80%) and ram lambs (60%). No significant differences were observed between vaccinated and control groups regarding FSH and LH during the experiments in the three experimental species/models used. Testosterone, however, was only significantly lower (~22-fold, P<0.01) in vaccinated rabbits. In conclusion, the present study demonstrated that pre-pubertal active immunization against GnRH leads to endocrine disruption and marked differences on testicular morphometry, development and activity among lagomorphs, hystricomorphs and ovine species with species-specific sensitivity regarding the anti-GnRH immune response.

Circulating anti-Müllerian hormone concentration (AMH) is positively correlated to the number of small growing follicles in the mare and may reflect ovarian function. Dynamics of AMH during immunocontraception have not previously been investigated. This study aimed to compare serum AMH in mares following treatment with native porcine zona pellucida (pZP), recombinant pZP3 and pZP4 (reZP) or gonadotrophin releasing hormone (GnRH) vaccines, and saline-treated controls. Stored sera collected during two previous studies examining ovarian activity in mares during zona pellucida (ZP) or GnRH immunocontraception were analysed for serum AMH. Data were compared among treatment groups using mixed-effects linear regression and one-way ANOVA with post hoc testing. Correlations between AMH and previously reported clinical variables were estimated using Spearman's rho. Mares immunized against GnRH showed variable but detectable AMH throughout successive breeding and non-breeding seasons that were not significantly different to unvaccinated control mares. Mares treated with pZP demonstrated marked, reversible suppression of AMH. Mares immunized using reZP showed an intermediate effect. In the ZP study, AMH was positively correlated to serum progesterone concentrations, mean ovarian volumes and antral follicle counts, whereas no correlations between AMH and serum progesterone concentrations, mean ovarian volumes, or the presence of one or more follicles ≥20 mm in diameter were detected in the GnRH study. In conclusion, marked suppression of AMH during pZP immunocontraception, but not during GnRH immunocontraception, suggested enhanced suppression of ovarian follicular development and, or follicular function during pZP immunocontraception. Serum AMH concentrations may provide a novel tool for the assessment of ovarian function during ZP-based immunocontraception., (Copyright © 2017 Elsevier Inc. All rights reserved.)

An experimental contraceptive vaccine was evaluated in Atlantic salmon (Salmo salar). A peptide derived from the beta subunit of luteinizing hormone (LH) was conjugated to two different carrier proteins, bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH), and formulated with one of four immunostimulants in a water-in-oil emulsion. Specific antibody responses to the peptide and each carrier protein were evaluated. While the antibody response to KLH was stronger than the response to BSA, both carrier proteins stimulated comparable antibody responses to the LH peptide. The immunostimulant proved to be more important for enhancing the LH peptide antibody response than the carrier protein selection; vaccines containing a combination of Aeromonas salmonicida and Vibrio anguillarum stimulated significantly greater LH peptide antibody production than any of the other three immunostimulants evaluated at 12 weeks post-vaccination. This study provides proof-of-concept for specific antibody production against a hapten-carrier protein antigen in Atlantic salmon and reinforces the importance of vaccine immunostimulant selection., (Copyright © 2017. Published by Elsevier Ltd.)

Background: Population control of domestic, wild, invasive, and captive animal species is a global issue of importance to public health, animal welfare and the economy. There is pressing need for effective, safe, and inexpensive contraceptive technologies to address this problem. Contraceptive vaccines, designed to stimulate the immune system in order to block critical reproductive events and suppress fertility, may provide a solution. Filamentous bacteriophages can be used as platforms for development of such vaccines., Objective: In this review authors highlight structural and immunogenic properties of filamentous phages, and discuss applications of phage-peptide vaccines for advancement of immunocontraception technology in animals., Results: Phages can be engineered to display fusion (non-phage) peptides as coat proteins. Such modifications can be accomplished via genetic manipulation of phage DNA, or by chemical conjugation of synthetic peptides to phage surface proteins. Phage fusions with antigenic determinants induce humoral as well as cell-mediated immune responses in animals, making them attractive as vaccines. Additional advantages of the phage platform include environmental stability, low cost, and safety for immunized animals and those administering the vaccines., Conclusion: Filamentous phages are viable platforms for vaccine development that can be engineered with molecular and organismal specificity. Phage-based vaccines can be produced in abundance at low cost, are environmentally stable, and are immunogenic when administered via multiple routes. These features are essential for a contraceptive vaccine to be operationally practical in animal applications. Adaptability of the phage platform also makes it attractive for design of human immunocontraceptive agents., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

The objective was to compare effects of anti-GnRH immunization (immunocastration) versus surgical castration on hypothalamic-pituitary function in boars. Thirty-six boars were randomly divided into three groups (n = 12/group): control, surgically castrated, or immunized against GnRH at 10 wk of age (boostered 8 wk later). Compared to intact boars, immunocastration reduced (P < 0.05) serum concentrations of LH, FSH, testosterone and inhibin B and caused severe testicular atrophy, whereas surgical castration increased (P < 0.05) serum concentrations of LH and FSH. Both immunocastration and surgical castration consistently reduced hypothalamic GnRH synthesis, with decreased (P < 0.05) mRNA expressions of GnRH, GnRH up-stream gatekeeper genes kiss1 and its receptor (GPR54), and androgen receptor in the hypothalamic arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV), as well as GnRH content in the median eminence. Inconsistently, mRNA expressions of gonadotropin-inhibitory hormone (GnIH) in ARC and AVPV as well as its receptor (GPR147) in pituitary were selectively reduced (P < 0.05), but mRNA expressions of estrogen receptor alpha and aromatase (CPY17A1) in pituitary were selectively increased (P < 0.05) in surgical castrates. In response to selectively attenuated suppressive signaling from GnIH and testosterone, mRNA expressions of GnRH receptor (GnRHR), LH-β and FSH-β in pituitary were increased (P < 0.05) in surgical castrates, whereas these pituitary gene expressions were decreased (P < 0.05) in immunocastrates, due to loss of hypothalamic GnRH signaling. We concluded that immunocastration and surgical castration consistently reduced hypothalamic GnRH synthesis due to a testosterone deficiency disrupting testosterone-Kisspeptin-GPR54-GnRH signaling pathways. Furthermore, selectively attenuated GnIH and testosterone signaling in the pituitary increased gonadotropin production in surgical castrates., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Suppression of cyclic activity in cattle is often desired in alpine farming and for feedlot cattle, not intended for breeding. A cattle specific anti-GnRF vaccine (Bopriva™) is registered for use in heifers and bulls in different countries. In adult cows vaccinated with Bopriva™, the median period until recurrence of class III follicles was 78 days from the day of the 2nd vaccination and reversibility could be proven, as out of 11 experimental cows 10 cows became pregnant at first, and one cow at second insemination. In the present study, 76 healthy, cyclic Eringer heifers and cows were vaccinated twice with Bopriva™ 3-7 weeks apart, to prevent estrus during alpine pasturing. Blood samples were taken for progesterone and GnRF antibody titer analysis on the day of inclusion (7-9 d before the first vaccination) and at the first vaccination. At the same time, gynaecological examinations were performed. When estrus occurred in the course of the alpine pasturing season, a gynaecological examination was done including analysis of a blood sample (progesterone, anti-GnRF antibody titer). Cows were followed for fertility out to 26 months post second vaccination., Results: Median duration of estrus suppression was 191 days after the second vaccination (when the 2 vaccinations were given 28-35 days apart). From n = 13 cows showing signs of estrus on the alpine pasture, n = 7 could not be confirmed in estrus (serum progesterone value >2 ng/ml, no class III follicles seen using ultrasonography). Median duration between second vaccination and next calving was 496 days (25%/75% quartiles: 478/532 days)., Conclusion: Bopriva™ induced a reliable and reversible suppression of estrus for more than 3 months in over 90% of the cows.

HLA-G is a nonclassical HLA immunotolerogenic molecule expressed in different human cell types. Successful embryo implantation is a consequence of information exchange between the uterus and the blastocyst. It is widely accepted that HLA-G expression by the fetus promotes the establishment of several mechanisms that, ultimately, would protect the developing embryo from maternal immune rejection and seems to be essential to both an adequate implantation and a healthy pregnancy. MicroRNAs miR-148a and miR-152 down-regulate HLA-G expression. The levels of both microRNAs in the placenta are very low. Although various contraceptive methods are available in the market, several of the most popular are based on hormone administration, an approach that have been causing concerns regarding their adverse effects. This scenario has led the research and development of new contraceptive methods meant to induce low disturbances in women body. Based on this context, we hypothesize that the delivery of miR-148a and miR-152 microRNAs, carried by liposomes, into the uterus, would locally induce a down-regulation of the immunotolerogenic HLA-G molecule. In this sense, a local concentration increase of both miR-148a and miR-152 would counteract HLA-G expression and therefore prevent pregnancy development, being a potential tool for the development of a new contraceptive therapy., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The aim of this study was to detect the effect of immunization against gonadotropin-releasing hormone (GnRH) on cell-meditated immunity. Three-week-old male Sprague-Dawley rats (n=32) were randomly and equally assigned to two groups: 1) GnRH-tandem-ovalbumin immunized group; and 2) the control group (injected with an equivalent Al(OH) 3 adjuvant). Blood samples were collected at two-week intervals to assess the level of GnRH-specific antibodies and testosterone. Moreover, blood and thymus samples were also collected to analyze the T lymphocyte subpopulations one and two months after the last booster immunization. T lymphocyte immunity against GnRH was activated during the first month post-immunization as exhibited by increased numbers of CD3+ (P<0.05) and CD4+ (P<0.05)T lymphocytes following testosterone suppression (P<0.01), which was then restored and maintained at appropriate levels in the second month. In contrast, the differentiation of T lymphocytes in the thymus was reduced during the first month after immunization as exhibited by the significant decreased number of CD3+ (P<0.05) cells, followed by the restoration and heightened numbers at later time points for both the number of CD3+ (P<0.05) and CD4+ (P<0.01)T lymphocytes. These results suggest that immunization against GnRH interferes with the number of lymphocytes during the early time points following immunization. The number of T lymphocytes initially decreased in the peripheral blood following immunization, but was replenished by newly exported cells from the thymus which eventually restored the T lymphocytes to normal levels., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Sterilization is a key strategy to reduce the number of domestic cats entering and killed in shelters each year. However, surgical sterilization is expensive and labour-intensive and cannot fully address the 70 million free-roaming cats estimated to exist in the United States. GonaCon™ is a gonadotropin-releasing hormone vaccine originally developed for use as a wildlife immunocontraceptive. An earlier formulation was tested in domestic cats and found to be safe and effective for long-term contraception. However, the current Environmental Protection Agency (EPA)-registered formulation consists of a different antigen-carrier protein and increased antigen concentration and has never been tested in cats. A pilot study was undertaken to evaluate the short-term safety of a single GonaCon immunization, assess the consequences of vaccinated cats receiving an accidental second GonaCon injection and determine the humoral immune response to immunization. During Phase 1, cats in Group A (n = 3) received a single intramuscular injection of GonaCon and Group B (n = 3) received a single intramuscular injection of saline. During Phase 2, Group A received a second GonaCon injection and Group B received their initial GonaCon injection. All cats developed GnRH antibodies within 30 days of vaccine administration. The endpoint titre (1:1,024,000) was similar among all cats, and levels remained high throughout the duration of the study. Four cats developed a sterile, painless, self-limiting mass at the site of injection. The mean number of days to mass development was 110.3 (range, 18-249 days). In conclusion, this preliminary study suggests that the EPA-registered GonaCon formulation is safe for continued testing in domestic cats, an accidental revaccination should not increase the risk of a vaccine reaction and the EPA-registered formulation effectively elicits a strong humoral immune response., (© 2016 Blackwell Verlag GmbH.)

Reasons for Performing Study: Few studies have investigated ovarian function in the mare undergoing porcine zona pellucida (pZP) immunocontraception despite reported ovarian dysfunction in other species., Objectives: This study aimed to describe ovarian function and oestrous cyclicity in pony mares following treatment with either the conventional pZP vaccine or a novel recombinant form of the vaccine derived from porcine ZP3 and ZP4 (reZP). In addition, the contraceptive efficacy of pZP vs. reZP was assessed., Study Design: Blinded, randomised, prospective clinical trial., Methods: Mares (n = 21) were randomised into 3 groups of 7: Group I received the pZP vaccine, with a booster 5 weeks later; Group II received the reZP vaccine, with a booster 5 weeks later; and Group III (controls) received 2 treatments, 5 weeks apart, of saline and adjuvant alone. Mares underwent weekly monitoring via transrectal palpation and ultrasound examination of the reproductive tract, with daily monitoring during oestrus. Data were collected over a 24 week period coinciding with the physiological breeding season; treatments commenced in Week 4. Serum samples were obtained for antibody titres and ovarian steroid level analyses at 7 day intervals. Cycling mares were bred via fresh semen artificial inseminations over a maximum of 2 consecutive oestrous cycles, commencing 5 weeks post booster vaccination., Results: Control mares cycled throughout the trial. After treatment, 6 of 7 pZP mares (86%) and one reZP mare (14%) had an extended anoestrus that correlated with basal serum oestradiol and progesterone levels. All mares resumed cyclicity by 10 months post treatment. Pregnancies were diagnosed in all controls, 4 reZP- (57%) and none of the pZP-immunised mares., Conclusions: The current study demonstrates the reversible suppression of ovarian function in pony mares following treatment with pZP. The effect of the reZP vaccine on pregnancy outcome requires further investigation., (© 2015 EVJ Ltd.)

Objective To construct a DNA contraception vaccine targeting canine zona pellucida 3 (CZP3) antigen and assess the immunological efficacy and contraceptive effect of the vaccine. Methods The CZP3 gene was amplified from total RNA of canine ovary by reverse transcription PCR and analyzed by bioinformatics, such as ProtScale, TMHMM, Signal P, InterProScan, PREDICT PROTEIN, homology modeling, etc. The constructed DNA vaccine pcDNA-CZP3 was used to vaccinate mice, and then its immune effect and contraceptive effect were evaluated in the mice. Results The CZP3 gene had 426 amino acids with two hydrophobic regions at its N-terminal and C-terminal, respectively. The top 22 amino acids at the N-terminal of the CZP3 was the signal peptide and there was a transmembranous helix from extracellular to intracellular at the C-terminal. CZP3 also had 8 B cell epitopes. The DNA contraception vaccine pcDNA3-CZP3 induced high levels of antibody and lower average litter size of mouse compared with the blank and negative control groups significantly. Conclusion The canine contraception DNA vaccine pcDNA-CZP3 has been successfully constructed and it can reduce the mouse fertility remarkably.

Pigs immunized against gonadotropin-releasing factor (GnRF) have increased carcass fatness compared to entire males; however, the timing of this increase in fatness after the second immunization against GnRF has not been determined. An experiment was conducted to identify and compare the growth performance, body composition, and physiological changes in immunocastrated males (IC males) at different BW and feeding levels. A total of 64 pigs were used in a 2 × 2 × 2 factorial experiment with the treatments being 1) sex (entire males or IC males), 2) initial BW (45.9 kg [light] or 78.3 kg [heavy]), and 3) feeding regime (2.5 times maintenance [restricted] or ad libitum). The pigs were individually housed, and the diets were fed for 4 wk after the second immunization against GnRF until slaughter at either 68.4 kg BW (light) or 105.8 kg BW (heavy). Immunocastrated males on a restricted feed intake had a lower ADG compared to entire males from d 15 to 28 and d 0 to 28 ( 0.011 and 0.011, respectively). Fat deposition was not affected by sex from d 0 to 14, but from d 15 to 28 IC males deposited 45 g/d more fat than entire males ( = 0.025). Immunocastrated male pigs fed ad libitum deposited 87 g/d more fat from d 15 to 28 than entire males fed ad libitum ( = 0.036). However, there was no difference in fat deposition between IC males and entire males when feed intake was restricted from d 15 to 28. Plasma urea nitrogen levels were greater in IC males compared to entire males from d 7 after the second immunization against GnRF ( 0.05 for d 7, 10, 14, 21, and 28). Plasma concentrations of IGF-1 were lower for IC males compared to entire males on d 3, 7, 10, and 28 ( 0.05 for all days). The following conclusions were made: 1) when pigs are immunized at a light BW (50 kg) and/or are on a restricted feed intake, they have a reduced propensity to deposit fat; however, the restriction in feed intake adversely affects growth rate. 2) The majority of fat deposition for males immunized at heavy BW (80 kg) occurs from d 15 to 28 after the second immunization against GnRF.

Problem: To evaluate the effectiveness and security of a contraceptive vaccine using plasmid DNA encoding mouse CRISP1 as antigen and chitosan nanoparticles as the carrier., Method of Study: Chitosan-pcDNA3.1-mCRISP1 Nanoparticles (CS-DNA NPs) were prepared and characterized in terms of morphology, zeta potential, polydispersity index, and binding capacity of pDNA. The cytotoxicity and gene transfer capability of CS-DNA NPs were assessed in COS-7 cells compared to Lipofectamine 2000(™) . Four groups of mice received three injections of 0.9% normal saline, pcDNA3.1 vector, pcDNA3.1-CRISP1, or CS-DNA NPs, respectively. ELISA was used to examine the immune responses. Fertility and mean litter size were analyzed by natural mating., Results: CS-DNA NPs have a spherical or elliptical shape with a mean diameter of 189.3 nm, positive zeta potential, and good DNA condensation. It also showed high DNAse resistance and good transfection efficiency without cell toxicity. The titers of anti-mCRISP1 antibodies from CS-DNA NP-immunized mice were significantly higher than that of pcDNA3.1-CRISP1 group. Male and female CS-DNA NP-immunized animals were recognized with a statistically significant reduction in their fertility compared with pcDNA3.1-CRISP1-immunized mice., Conclusion: This study demonstrated that using chitosan-DNA nanoparticles as the carrier can improve the immunogenicity of mCRISP1 DNA contraceptive vaccine with good security., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

An experiment was conducted to determine the standardized ileal digestible (SID) Lys requirement of entire male and male pigs immunized against gonadotrophin releasing factor (GnRF; immunocastrates). A total of 420 entire male and immunocastrated (IC) male pigs weighing 60.1 kg BW (SEM 0.49) were used in a 2 × 5 factorial experiment with the main effects being gender (entire males or IC males) and 5 concentrations of SID Lys:DE ratio (0.32, 0.43, 0.54, 0.64, or 0.75 g SID Lys/MJ DE). The diets were fed for 6 wk until slaughter at 107.5 kg BW (SEM 5.72). Over the entire period, IC males had a greater ADG ( < 0.001), greater ADFI ( < 0.001), and lower G:F ( < 0.001) compared with entire males. Immunocastrated males had increased plasma urea nitrogen (PUN) concentrations compared with entire males from d 10 to 42 ( < 0.001 for all days). Plasma urea nitrogen concentration also increased as Lys concentrations increased from d 3 to 42 ( < 0.001 for all days). Using the linear-plateau model, the optimal ADG for entire males was achieved at SID Lys concentrations of 0.68, 0.62, 0.54, and 0.58 g/MJ DE whereas optimal G:F was achieved at SID Lys concentrations of 0.72, 0.60, 0.54, and 0.51 g/MJ DE for the time periods d 0 to 14, d 15 to 28, d 29 to 42, and d 0 to 42, respectively. For IC males, optimal ADG was achieved at SID Lys concentrations of 0.64, 0.43, 0.38, and 0.40 g/MJ DE whereas optimal G:F was achieved at SID Lys concentrations of 0.64, 0.43, 0.36, and 0.42 g/MJ DE for the same respective time periods. Using the quadratic polynomial model, maximum ADG for entire males was achieved at SID Lys concentrations of 0.62 and 0.58 g/MJ DE whereas maximum G:F was achieved at SID Lys concentrations of 0.59 and 0.68 g/MJ DE for d 29 to 42 and d 0 to 42, respectively. For IC pigs, maximum ADG was achieved at SID Lys concentrations of 0.69, 0.54, and 0.64 g/MJ DE whereas maximum G:F was achieved at SID Lys concentrations of 0.81, 0.54, and 0.64 g/MJ DE for d 0 to 14, d 29 to 42, and d 0 to 42, respectively. A solution could not be found using the quadratic polynomial model for entire males for d 0 to 14 for both ADG and G:F and for both entire males and IC males for d 15 to 28 within the range of Lys values tested. When both the growth performance and PUN values are considered, the results suggest that IC males show a response to dietary SID Lys similar to that of entire males for 2 wk after the second immunization against GnRF. After this, IC males have a lower requirement for SID Lys than entire males.

An alternative to surgical sterilization for fertility control of animals (wild, zoo, farm, and domestic) is needed to prevent problems related to overpopulation, including culling and relocation. A PubMed and Google Scholar database search was conducted using the keywords 'contraceptive vaccine animals,' 'immunocontraception animals,' 'non-surgical sterilization animals,' 'PZP vaccine,' and 'GnRH vaccine.' The searches from 1972 to 2015 yielded over 1500 publications. These articles were read, and 375 were selected for detailed analysis. Articles referenced in these publications were also thoroughly examined. PZP and GnRH contraceptive vaccines (CVs) have been extensively investigated for fertility control of wild, zoo, farm, and domestic animal populations. Both vaccines have shown tremendous success with PZP vaccines taking the lead. Novel technologies and targets are being developed to improve existing vaccines and generate second-generation CVs. Single-shot vaccines, which can be delivered remotely, will greatly advance the field of immunocontraception for animal use with potential human application., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Problem: The development of a new and suitable contraceptive methods, as well as in-depth and systematic research into underlying contraceptive mechanisms, is crucial. IZUMO1 plays an important role in the fusion of the sperm and ovum during fertilization. Izumo(-/-) mice are infertile. Therefore, IZUMO1 may be a potential target for the development of a contraceptive vaccine., Method of Study: Linear B-cell epitopes (BCE) were identified in IZUMO using biosynthetic peptides and used to immunize female mice., Results: Five IZUMO BCE were identified: DLVLDCL177-183, YSFYRV196-201 (named BCE-2), YLT217-219, SMVGPED221-227, and DAGNY228-232. Active immunization with the BCE-2 vaccine sharply decreased the fertility rate in female mice in a safe and reversible manner. In vitro fertilization showed that the BCE-2 vaccine interferes with and blocks the fusion of the sperm and the ovum., Conclusions: B-cell epitopes-2 may be a new candidate for the development of contraceptive vaccine due to its effectiveness, safety, and reversibility., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Contraceptive vaccines have been used for the management of wildlife population. In the present study, we have examined the contraceptive potential of Escherichia coli-expressed recombinant fusion protein comprising of 'promiscuous' T cell epitope of tetanus toxoid [TT; amino acid (aa) residues 830-844] followed by dilysine linker (KK), dog ZP3 fragment (aa residues 307-346), triglycine spacer (GGG), T cell epitope of bovine RNase (bRNase; aa residues 94-104), GnRH, T cell epitope of circumsporozoite protein of Plasmodium falciparum (CSP; aa residues 362-383), and GnRH. SDS-PAGE analysis of the purified refolded protein revealed a dominant ∼12 kDa band, which in Western blot reacted with mouse polyclonal antibodies against dog ZP3 fragment and mouse monoclonal antibodies against GnRH. Immunization of female FvB/J mice following two booster schedule with the above recombinant protein supplemented with alum led to high antibody titres against the immunogen as well as ZP3 and GnRH as determined by ELISA. The immune sera reacted with zona pellucida of mouse oocyte and also inhibited in-vitro fertilization. The qRT-PCR studies showed decrease in the ovarian GnRH receptor in mice immunized with the recombinant fusion protein. Mating studies revealed high contraceptive efficacy of the recombinant protein as in two independent experiments, 90% of the immunized female mice failed to conceive. Following one booster immunization schedule, 50% of the immunized female mice failed to conceive. However, in adjuvanted controls, all the female mice became pregnant. To conclude, the recombinant protein described herein has a good potential to be developed as candidate contraceptive vaccine., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Immunocastration in pigs has been proposed as a cruelty-free alternative to surgical castration. In this work the effect of immune-spaying of female pigs on the volatile compound profile of Iberian dry-cured products was evaluated. The head-space volatile compound of dry-cured shoulders and loins from surgically spayed, immune-spayed and entire Iberian × Duroc females was characterized by proton transfer reaction-time of flight-mass spectrometry. It was not possible to identify a significant effect of the castration modality on dry-cured shoulders, probably because of the heterogeneity of samples. Contrarily, Principal Component Analysis of dry-cured loins indicates a better homogeneity of samples and the separation of loins from surgically spayed and immune-spayed females. Some mass peaks tentatively identified as important flavor compounds in dry-cured products, 3-methylbutanal, 2,3-butanedione and 3-methylbutanoic acid, were significantly higher in the immune-spayed females. Therefore, immune-spaying seems to have a negligible effect on the volatile compound profile of dry-cured shoulders, whereas it could affect the VOC profile in the case of dry-cured loins., (Copyright © 2015. Published by Elsevier Ltd.)

The possibility of a contraceptive vaccine targeting human chorionic gonadotropin has long been recognized, but never fully realized. Here we describe an epitope-specific approach based on immunogenic display of hCG-derived peptides on virus-like particles of RNA bacteriophage. A number of recombinant VLPs were constructed, each displaying a different hCG-derived peptide. Some were taken from the disordered C-terminal tail of the hormone, another came from an internal loop, and yet another was an epitope mimic produced by affinity-selection on an hCG-neutralizing antibody target. Immunization of mice with some VLPs yielded antisera that bound the hormone and neutralized hCG biological activity.

Problem: Necessity to elicit antibody response above the protective threshold titres by sexually active women immunized to prevent pregnancy., Method of Study: Recombinant hCGβ-LTB vaccine expressed as both DNA and protein. Balb C mice employed for testing immunogenicity., Results: Necessity to give three primary injections of the vaccine to elicit proper antibody response. Immunization twice with DNA form of the vaccine at fortnightly interval followed by the protein elicits a distinctly higher antibody response than proteinic vaccine alone. Antibodies generated are bio-effective against hCG., Conclusion: Immunization with the DNA form of the recombinant hCGβ-LTB vaccine twice at fortnightly interval followed by the proteinic form of the vaccine induces distinctly higher antibody response., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The objective of this study was to determine the effects of increasing balanced protein, with and without ractopamine hydrochloride (RAC), in the finisher diets of immunocastrates on their carcass cutting yields and respective muscle, fat, and bone proportions. Carcass traits and yields of 120 individually housed entire male pigs and immunocastrates were evaluated following a 2 × 2 × 3 factorial design. Vaccination occurred at 16 and 20 wk of age and slaughtering at 24 wk. Up until 20 wk of age, all pigs received a commercial grower diet. Low-, medium-, and high-protein (7.50, 9.79, and 12.07 g standardized ileal digestible [SID] lysine/kg, respectively) diets were then fed for the last 28 d with RAC supplemented at 0 or 10 mg/kg. Immunocastration and RAC supplementation increased ( = 0.003 and = 0.017, respectively) the live weight at slaughter. The interaction between sex and protein ( = 0.039) for caliper backfat depths showed greater backfat depths in immunocastrates fed medium protein whereas RAC decreased ( = 0.027) the caliper backfat depth. Feeding RAC increased the trotters ( = 0.040), tenderloins (fillets; < 0.001), shoulder ( = 0.002), hindquarter ( = 0.026), loin ( = 0.028), and belly ( = 0.044) percentages. The shoulder ( < 0.001), hindquarter ( < 0.001), and loin ( = 0.005) muscle percentages were increased and the hindquarter fat percentages were decreased ( = 0.032) with RAC supplementation. Immunocastration increased the belly cut ( = 0.006), loin fat ( < 0.001), and belly fat ( < 0.001) percentages. Therefore, carcass cutting and lean yields can be improved by RAC supplementation while decreasing the backfat depth, and the increase in backfat due to immunocastration could be avoided with the correct dietary protein level.

Immunocontraception with porcine ZP (pZP) can be an effective means of fertility control in feral horses. Previous studies suggest that antibodies produced after pZP vaccination may both inhibit fertilization and cause follicular dysgenesis. Zonastat-H, PZP-22, and SpayVac are three pZP vaccines proposed for use in horses. Although all these vaccines contain the pZP antigen, variations in antigen preparation and vaccine formulation lead to differences in antigenic properties among them. Likewise, despite numerous efficacy and safety studies of Zonastat-H and PZP-22, the contraceptive mechanisms of SpayVac remain unclear. The preparation of pZP for SpayVac is thought to include more nonzona proteins, making it less pure than the other two vaccines. This may result in increased antigenicity of the vaccine. We therefore investigated the immunoreactivity of serum antibodies from SpayVac-vaccinated mares to equine zona protein. Western blot analyses revealed an immunoreactivity of these antibodies to protein isolated from mature equine oocytes, ZP, follicular tissues, and ovarian tissues. Immunohistochemical analyses were used to locate the binding of serum antibodies to the ZP of immature oocytes in ovarian stromal tissue. We also found serum antibodies from SpayVac-treated mares to be predominantly specific for zona protein 3. Collectively, our results suggest a model where serum antibodies produced in response to SpayVac vaccination are immunoreactive to equine zona protein in vitro. Our study lends insight into the contraceptive mechanisms underlying the infertility observed after SpayVac vaccination., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The objective was to determine effects of active immunization against GnRH on reproductive function in Tibetan rams. Peripubertal Tibetan rams (n = 30) were randomly and equally allocated into three groups: control (no treatment); surgically castrated; or immunized against 100-μg d-Lys6-GnRH-tandem-dimer peptide conjugated to ovalbumin in Specol adjuvant at 24 weeks of age (with a booster 8 weeks later). Blood samples (for antibody titers and hormone concentrations) were collected at 4-week intervals until rams were killed (40 weeks). Immunization triggered a good antibody response in all immunized rams (P < 0.01). Compared with intact controls, anti-GnRH immunization reduced (P < 0.01) serum concentrations of testosterone, inhibin A, LH, and FSH, and it induced testicular atrophy (suppression of spermatogenesis). Androstenone concentrations in fat tissues of GnRH-immunized rams were also rendered nondetectable (P < 0.001). Furthermore, mRNA expressions of GnRH receptor, LH-β, and FSH-β in the pituitary and of LH receptor, FSH receptor, and inhibin α and βA subunits in the testes were decreased in immunized rams compared with intact controls (P < 0.05). This was apparently the first report that active immunization against GnRH-tandem-dimer-ovalbumin conjugate in Specol adjuvant was an effective alternative to surgical castration for Tibetan rams under practical Tibetan plateau conditions., (Copyright © 2015 Elsevier Inc. All rights reserved.)

KISS1 gene-encoding kisspeptins are critical for the onset of puberty and control of adult fertility. This study investigated whether KISS1 can be used as a novel target for immunocastration. Human KISS1 was fused with the HBsAg-S gene for constructing an antibiotic-free recombinant plasmid pKS-asd that coded for 31.168 kDa target fusion protein. Six male Hu sheep lambs were divided into two equal groups, treatment and control. The vaccine (1mg/ram lamb) prepared in saline solution was injected into lambs at weeks 0, 3 and 6 of the experiment, respectively. Vaccine efficacy was evaluated in terms of KISS1-specific IgG antibody response, serum testosterone levels, scrotal circumference, testicular weight, length and breadth, extent of testicular tissue damage, and sexual behaviour changes. The specific anti-KISS1 antibody titre in vaccinated animals was significantly higher than that in controls (p<0.05). In addition, vaccinated animals showed lower serum testosterone level, testicular weight and length and smaller scrotal circumference than those in controls (p<0.05). Spermatogenesis of seminiferous tubules in vaccinated animals was suppressed; sexual behaviours in vaccinated animals were significantly lower (p<0.05) than those in controls. In conclusion, the immunization against KISS1 in this DNA vaccine induced a strong antibody response and resulted in the suppression of gonadal function and sexual behaviour in animals, demonstrating that KISS1 can be used as a novel target for developing a DNA immunocastration vaccine., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Introduction: There is continuing need for contraceptives. According to World Health Organization, 210 million pregnancies occur each year, out of which some 80 million are unintended. A vaccine offering privacy and periodic intake would be an attractive proposition., Areas Covered: The article is a brief review of three vaccines developed against human chorionic gonadotropin (hCG) with progressively better attributes. Clinical trials have proven in more than one country the complete safety and reversibility of the anti-hCG vaccine(s) in women. Vaccination does not entail any disturbance in levels of reproductive tract hormones of the woman or any disturbance in menstrual regularity and bleeding profiles. Phase II clinical trials show the effective prevention of pregnancy in sexually active women of proven fertility. A recombinant vaccine amenable to industrial production has been developed; it induces substantially higher antibody titers in mice of four different genetic strains than those required to prevent pregnancy in women. Rigorous toxicology studies have been completed on this vaccine in rodents and marmosets., Expert Opinion: This unique vaccine, requiring periodic intake and demonstrating no impairment of ovulation, hormonal profiles and menstrual regularity, is on the verge of final clinical trials under the aegis of the Indian Council of Medical Research and should be a valuable addition to the available contraceptives.

Objectives: To study the anti-fertility effect of a DNA vaccine using Bin1b as the target antigen in male mice., Methods: A novel recombinant eukaryotic vector containing a fusion gene sequence of mouse Bin1b in tandem with three copies of C3d fragment (C3d3) was used to construct pSG.SS.C3d3.YL.Bin1b. The correct expression of the Bin1b-C3d3 protein was confirmed in transfected HEK293 cells by indirect immunofluorescence and western blot analysis. The fertility of immunised mice was determined by a mating experiment and sperm motility test. Anti-Bin1b antibody titres in sera were examined by ELISA assays. Binding activity of C3d3 fragment of the fusion protein was verified in C3d receptor-expressing Raji cells and flow cytometric analysis., Results: Immunisation of pSG.SS.C3d3.YL.Bin1b recombinant DNA vaccine significantly decreased sperm motility and compromised fertility in male mice. ELISA results showed that the titres of anti-Bin1b IgG in sera of immunised mice increased markedly with the immunisation process. Further, the anti-fertility effect of pSG.SS.C3d3.YL.Bin1b was significantly better than that of pSG.SS.YL.Bin1b DNA vaccine and generated higher titres of anti-Bin1b antibody., Conclusions: Our results show that recombinant DNA vaccine targeting Bin1b can markedly reduce fertility in male mice, providing an alternative approach for birth control.

Zona pellucida (ZP) glycoproteins based contraceptive vaccines have been proposed for the management of wildlife population. In the present study, a fusion protein encompassing promiscuous T cell epitope of tetanus toxoid [TT; amino acid (aa) residues 830-844] followed by a dilysine linker and an ectodomain of dog ZP3 (ZP3; aa residues 23-348) without any affinity tag (TT-KK-ZP3) has been expressed in Escherichia coli. The recombinant protein was successfully produced in fed-batch fermentor and purified. The average yield of purified refolded protein was 12.20 ± 0.61 mg/2g wet cell pellet. Female FvB/J mice immunized with the varying doses of recombinant TT-KK-ZP3 supplemented with alum/PetGel A as adjuvants following a three injection schedule, showed dose dependent increase in serum IgG titer. Antibodies against TT-KK-ZP3 recognized native mouse/dog ZP and significantly inhibited mouse in-vitro fertilization (p=0.012). Immunized mice showed significant reduction in fertility (p<0.05). Higher antibody titers were associated with a decrease in the number of pups born to the immunized female mice. To reduce the number of injections, two injection schedule using various dose combinations of TT-KK-ZP3 supplemented with alum revealed lower immunogenicity and contraceptive efficacy as compared to the three injection schedule. To overcome this, CpG motif was included in addition to alum and both intraperitoneal and intranasal route of immunization following the two injection schedule was investigated. Inclusion of CpG significantly enhanced the antibody titer and improved contraceptive efficacy. In the mice immunized following intraperitoneal route, serum/vaginal IgG and in the mice immunized through intranasal route, vaginal IgA seemed to be important for curtailment in fertility. To conclude, the recombinant protein described herein may be a good candidate for developing contraceptive vaccine for the wildlife population management, in particular street dogs., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Two gonadotrophin releasing hormone (GnRH) constructs prepared by either chemical conjugation to keyhole limpet hemocyanin (GnRH-KLH) or as an expressed recombinant fusion protein (Multimer) were evaluated with or without adjuvants (immunostimulating complexes, ISCOMs, or cytosine-phosphate-guanosine oligodeoxynucleotides, CpG ODNs). After subcutaneous administration to Balb/c male mice at Weeks 0, 2 and 4, these preparations were assessed for induction of immune responses and effects on reproductive organs. GnRH-KLH plus ISCOMs formulation induced strong IgG immune responses from Week 4 through Week 12 resulting in consistent reproductive organ atrophy by Week 12 after subcutaneous administration. GnRH-KLH plus CpG ODNs generated immune responses but no atrophy of reproductive tissues by Week 12. Multimer plus ISCOMs induced poor immune responses and no effects on reproductive tissues by Week 12. In the absence of additional adjuvant, none of the GnRH constructs induced reproductive organ atrophy. GnRH-KLH induced stronger immune responses when formulated with ISCOMs or CpG ODN compared to Multimer. GnRH-KLH with ISCOMs could be an effective colloidal alternative for emulsion GnRH vaccine formulations., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Growth performance and carcass characteristics of physically castrated (PC) and immunologically castrated (IC) pigs fed ractopamine hydrochloride (RAC; 5 mg/kg) were evaluated in 64 pens of 22 pigs each. Male pigs were randomly assigned to castration method at birth. Pigs in the PC group were physically castrated at 5 d of age while IC pigs were administered Improvest at 11 and 18 wk of age. Pigs entered the grow-finish barn at approximately 9 wk of age (d 0). Dietary treatments (control or RAC) were initiated on d 87. Final treatment arrangement was a 2 × 2 factorial of castration method and diet. Data were analyzed using a mixed model with fixed effects of castration method, diet, market group, and all 2- and 3-way interactions. Pen was the experimental unit. From d 0 to 65, IC pigs had 11.2% greater (P < 0.01) G:F and 11.6% less (P < 0.01) ADFI than PC pigs, but ADG was increased 1.0% in PC pigs compared with IC pigs (P < 0.01). From d 65 to 87, IC pigs had 7.9% greater (P < 0.01) ADG and 12.1% greater (P < 0.01) G:F than PC pigs while having similar (P = 0.16) ADFI. At the initiation of diet (RAC) treatments, BW of all treatments were similar (P ≥ 0.32). From d 87 to 120 (RAC feeding period), IC pigs had 10.0% greater (P < 0.01) ADG and 10.5% greater (P < 0.01) ADFI than PC pigs while having similar (P = 0.64) G:F. Feeding RAC increased (P < 0.01) ADG by 16.9% and G:F by 17.9% while having no effect (P = 0.42) on ADFI from d 87 to 120. There were no significant interactions between castration method and diet on growth performance from d 87 to 120. For the entire study (d 0-120), IC pigs had 2.6% greater (P < 0.01) ADG, 4.6% less (P < 0.01) ADFI, and 7.3% greater (P < 0.01) G:F than PC pigs. Averaged over market groups, IC pigs were 2.5 kg heavier (P < 0.01) and had similar (P = 0.10) carcass weights and 1.8 percentage units less (P < 0.01) dressing yields than PC pigs. Additionally, IC pigs had 1.3 mm less (P < 0.01) fat and 1.7 mm less (P < 0.01) loin depth than PC pigs. Pigs fed RAC were 2.9 kg heavier (P < 0.01) and had 2.3 kg heavier (P < 0.01) carcasses and 2.2 mm deeper (P < 0.01) loins but similar (P = 0.21) dressing yields and tended (P < 0.10) to have 0.4 mm less fat than control-fed pigs when averaged over market groups. Group 3 pigs were the heaviest (P < 0.01) at slaughter and had the heaviest (P < 0.01) carcasses, greatest (P < 0.01) dressing yields, and the most (P < 0.01) carcass fat of all market groups. Overall, immunological castration and RAC were additive in terms of improving growth performance and carcass characteristics.

The effects of immunocastration and time after second Improvest dose on proximate composition and fatty acid profile of adipose tissue from jowl and belly of finishing barrows were determined. Physically castrated (PC) and immunologically castrated (IC) barrows were assigned to treatments at birth. Within 5 d of age, PC barrows were physically castrated and IC barrows were administered Improvest (gonadotropin-releasing factor analog; Zoetis, Kalamazoo, MI) at 16 and 20 wk of age. Diets were formulated with corn and soybean meal and did not contain ethanol coproducts. Subsequently, PC (n = 23) and IC (n = 24) barrows were slaughtered biweekly from 22 to 28 wk of age, 2 to 8 wk following second Improvest dose. Adipose tissue samples were collected from the jowl and belly. Main effects of castration method and time after second Improvest dose and their interaction were analyzed with the MIXED procedure of SAS. Overall, IC barrows were heavier at slaughter (136.1 vs. 100.6 kg; P < 0.01), had similar HCW (P = 0.13), and were leaner (50.9 vs. 48.9%; P < 0.01) than PC barrows. In jowl adipose tissue, IC barrows had less lipid content (64.7 vs. 80.2%; P < 0.01) than PC barrows at 2 wk. However, there were no differences (P ≥ 0.75) in lipid content between IC and PC barrows from 4 to 8 wk. Total MUFA content was reduced (42.5 vs. 44.8%; P = 0.04) and PUFA content was greater (16.9 vs. 14.8%; P < 0.01) in IC compared to PC barrows. Increasing time after second dose reduced (P = 0.04) PUFA content from 17.5% at 2 wk to 15.4% at 8 wk. In IC barrows, iodine value (IV) was increased by 6.9 and 3.3 g/100 g compared with PC barrows at 2 and 4 wk, but IV was not different between castration methods (P ≥ 0.95) at both 6 and 8 wk. Meanwhile, in belly adipose tissue, IC barrows had less lipid content than PC barrows (75.3 vs. 82.7%; P < 0.01). Lipid content was unchanged over time (P = 0.34) in either PC or IC barrows. Total SFA and MUFA content of the belly were similar for both IC and PC barrows. Belly adipose tissue from IC barrows had greater concentrations of PUFA than PC barrows (16.2 vs. 14.7%; P < 0.01). In IC barrows, IV was increased by 5.3 g/100 g compared with PC barrows at 2 wk, but IV was not different between castration methods (P ≥ 0.91) from 4 to 8 wk. Results indicate that as time after second Improvest dose progressed, IV of jowl and belly was reduced. This was accompanied by an increase in lipid deposition in jowl, while lipid content remained constant in belly.

Effects of feeding ractopamine (RAC; 5 mg/kg) to physically castrated (PC) and immunologically castrated (IC) pigs on carcass characteristics, cutting yields, and loin quality were evaluated using 285 carcasses. Male pigs were randomly assigned to sex treatments (PC and IC) at birth and fed the same nursery diets before allotment into 32 pens with 22 pigs per pen in a grow-finish barn. Pigs in the PC group were physically castrated at approximately 5 d of age, and pigs in the IC group were administered Improvest at 11 and 18 wk of age. Diet treatments (control or RAC) were initiated on study d 87. Pigs were marketed at 12 d (4.5 wk post-second Improvest dose), 19 d (5.5 wk post-second Improvest dose), and 33 d (7.5 wk post-second Improvest dose) following the start of final diet treatments. Three carcasses per pen were selected for evaluation of cutting yields and loin quality. Data were analyzed using PROC MIXED in SAS with fixed effects of sex, diet, market group, and their interaction; carcass (N = 285) was the experimental unit. Carcasses from RAC-fed pigs were heavier (P < 0.01) and had deeper (P = 0.02) loins than control-fed carcasses. Carcasses from IC pigs were similar (P = 0.22) in weight but had less (P < 0.01) fat and shallower (P = 0.02) loins when compared to PC carcasses. There were differences (P < 0.05) among market groups for carcass weights, fat depths, loin depths, and estimated carcass leanness. For cutting yields, RAC-fed carcasses had greater (P ≤ 0.03) bone-in lean and total carcass cutting yields than control-fed carcasses while there were no differences (P > 0.05) between RAC-fed and control-fed carcasses when evaluating LM color, marbling, firmness, pH, drip loss, and tenderness. Carcasses from IC pigs had greater (P < 0.05) boneless lean yields, bone-in lean yields, and total carcass cutting yields than PC carcasses. There were minimal differences (P < 0.05) in LM marbling, firmness, composition, and tenderness between PC and IC pigs. There was an interaction (P = 0.03) between sex and diet for LM composition. Control-fed PC loins had more (P < 0.01) lipid than all other treatment combinations. Market group had effects (P < 0.05) on carcass cutting yields, LM color, marbling and firmness scores, pH, purge loss, composition, and tenderness. The results from this study indicated RAC and immunological castration were additive in terms of improving carcass cutting yields while having minimal effects on pork quality.