Evaluation of antibody response to an adjuvanted hapten-protein vaccine as a potential inhibitor of sexual maturation for farmed Atlantic salmon.

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.

Immunization against GnRF in adult cattle: a prospective field study.

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.

Safety and effectiveness of a single and repeat intramuscular injection of a GnRH vaccine (GonaCon™) in adult female domestic cats.

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.

Reduction of fertility in male mice immunised with pSG.SS.C3d3.YL.Bin1b recombinant vaccine.

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.

Single-treatment porcine zona pellucida immunocontraception associated with reduction of a population of white-tailed deer (Odocoileus virginianus).

Previous reports have demonstrated gradual reductions of white-tailed deer (Odocoileus virginianus) populations through immunocontraception, with stabilization occurring after 2-4 yr of treatment, and subsequent reductions of 6-10% annually. These studies employed porcine zona pellucida (PZP) vaccines that required two initial treatments and annual retreatments. From 2005 to 2010, 258 adult and yearling female deer on Fripp Island, South Carolina, were treated with one of several PZP preparations designed to produce 2+ yr of effective contraception with a single treatment. These included several preparations of SpayVac and of native PZP-adjuvant emulsion plus PZP and QA-21 in timed-release pellets. Deer were chemically immobilized, ear-tagged, and administered initial treatments by hand in February-March. Some treated deer were boosted remotely with PZP-adjuvant emulsion 1.5 - 4.5 yr after initial treatments. Ground-based distance sampling was used to estimate deer population density at Fripp Island, a resort community, and at a relatively undeveloped neighboring control site, Hunting Island. Most vaccine preparations tested reduced fawning rates by 75% to 95% for at least 1 yr. From 2005 to 2011, deer density on Fripp Island declined by 50%, from 72 deer/km(2) to 36 deer/km(2), an average annual reduction of 11%. In contrast, population density on the Hunting Island control site fluctuated between 2005 and 2011, averaging 23 deer/km(2) (range, 19-28 deer/km(2)). Population declines on Fripp Island were associated with an increase in the proportion of treated females and with a progressive decrease in winter fawn:doe ratios, from 1.21 fawns/doe in 2005 to 0.19 fawns/doe in 2010. Winter fawn:doe ratios averaged 1.36 fawns/doe (range, 0.84 - 1.62 fawns/doe) at the Hunting Island control site. Annual survivorship averaged approximately 79% among ear-tagged females. The rate at which deer populations diminished in association with PZP treatments on Fripp Island was higher than that seen at other study sites, although the reasons for the more rapid decline on Fripp Island are not well understood.

Twenty years of immunocontraceptive research: lessons learned.

The National Wildlife Research Center (NWRC) began immunocontraception vaccine research by testing porcine zona pellucida (PZP) on white-tailed deer (Odocoileus virginianus). Early PZP research demonstrated that PZP induced infertility; however, increased length of the rut was observed in PZP-treated deer. An alternative vaccine using a keyhole limpet hemocyanin-gonadotropin-releasing hormone (KLH-GnRH) conjugate formulated with modified Freund's adjuvant was developed at NWRC. Suppression of GnRH has reduced reproduction in both sexes but is most effective in females. This vaccine was effective in preventing contraception in female deer for several years after a prime and boost. Due to adverse side effects of Freund's adjuvant, NWRC developed a new adjuvant called AdjuVac, a mineral oil/surfactant adjuvant with the addition of Mycobacterium avium as an immunostimulant. The price of KLH prompted a search for a more economical hemocyanin carrier protein for the GnRH peptide. Blue protein, derived from the mollusk Concholepas concholepas, proved to be a successful option. Formulation improvements resulted in a vaccine that can be effective as a single injection for multiple years, now called GonaCon. GonaCon is registered with the Environmental Protection Agency (EPA) for use in white-tailed deer in urban/suburban areas and for wild horses (Equus caballus) and burros (Equus asinus). Future GonaCon applications may include reducing reproduction to manage populations of other wildlife species, such as prairie dogs (Cynomys ludovicianus) in urban areas and suppressing reproduction to reduce the spread of venereal diseases such as brucellosis. Research is being conducted to develop a GnRH vaccine used in combination with the rabies vaccine to control population growth in free-roaming dogs, with the secondary effect of managing the spread of rabies. The EPA would regulate all these uses. Research is also ongoing on a GnRH vaccine to delay the onset of adrenocortical disease in pet ferrets (Mustela putorius), a use regulated by the United States Department of Agriculture.

Immunocontraception of captive exotic species: V. Prolonged antibody titers in Dall sheep (Ovis dalli dalli) and domestic goats (Capra hircus) immunized with porcine zona pellucida.

Native porcine zona pellucida (PZP) immunocontraception has been used to inhibit fertility in more than 80 species of ungulates, although the duration of contraception efficacy varies among species in both Perissodactyla and Artiodactyla. This study examined anti-PZP antibody titers in Dall sheep and domestic goats at the Milwaukee County Zoo, and also Himalayan tahr and Armenian Mouflon sheep at the San Diego Zoo Safari Park, and, for comparison, Altai wapiti, lowland wisent, Javan banteng, and southern pudu at the San Diego Zoo Safari Park, all were given a primer dose and booster dose of PZP. Of the San Diego Zoo Safari Park animals, the 4 comparison species demonstrated the typical 1-yr pattern of anti-PZP antibodies, whereas the Armenian sheep and Himalayan tahr showed prolonged (2-3 yr) antibody responses after a single primer and booster dose. The Dall sheep and domestic goats had significantly longer durations of antibody titers (3 yr) from a single year's treatment (primer plus booster). Analysis of the data indicates that Armenian sheep, Himalayan tahr, Dall sheep, and domestic goats have prolonged responses, and are more sensitive to PZP in that they produce a protracted antibody response.

Romance without responsibilities: the use of the immunocontraceptive porcine zona pellucida to manage free-ranging bison (Bison bison) on Catalina Island, California, USA.

Prior to 2010, the introduced population of American bison (Bison bison) on Santa Catalina Island, California, was managed through the shipment of surplus bison to private ranches, Native American reservations, and livestock auctions on the mainland. In response to escalating costs, transport-induced stress to the animals, and ecologic impacts associated with high bison numbers on-island between shipments, the use of the immunocontraceptive vaccine porcine zona pellucida (PZP) as a fertility control option for managing the population was investigated. Between 2009 and 2012, a total of 64 bison cows (> or =1 yr old) received primer inoculations of 100 microg PZP emulsified with 0.5 ml Freund's modified adjuvant (FMA) delivered through a combination of intramuscular injections by hand (50 bison cows) during roundups and via field darting (14 bison cows). Pre-rut booster inoculations of 100 microg PZP emulsified with 0.5 ml Freund's incomplete adjuvant (FIA) were administered exclusively via field darting in 2010, 2011, and 2012 to 45, 48, and 61 bison cows (> or =1 yr old), respectively. During the present study, 38 adult cows (marked and unmarked) received one or more PZP inoculations during their first, second, or third trimesters of pregnancy, and of these individuals, 35 successfully produced calves. Low pregnancy values detected in the remaining three cows have been attributed to residual progesterone associated with unsuccessful fertilization. The 2010 pretreatment calving rate (calves born per cow) determined via direct observation was 67.4% (29 calves from 43 cows). Through the use of PZP, the calving rate was reduced to 10.4% by 2011 and to 3.3% by 2012. Considering the annual mortality rate of 2-5% documented during this study, the results demonstrate the potential of PZP use as an effective nonlethal tool for controlling population growth in free-ranging bison.

Lack of spatial and behavioral responses to immunocontraception application in African elephants (Loxodonta africana).

Opinions are divided as to whether human intervention to control elephant (Loxodonta africana) population growth is desirable, partly because of elephant welfare concerns. Female contraception through immunization with porcine zona pellucida (PZP) proteins is viable. The effects of sustained use and application of the PZP vaccine on elephant behavioral and spatial responses were examined by evaluating herd ranging, fission-fusion dynamics, association patterns, and reproductive and sexual behaviors. Minimal change was anticipated as a result of long calf dependence on and association with cows, a reduced but not indefinite 0% growth rate and the known mechanism of action of PZP vaccines, and minimal expected change in resource requirements necessitating behavioral or spatial use adaptations. Although behavioral effects identified in previous hormonal contraceptive trials were evident, it was demonstrated that immunocontraception caused no prolonged behavioral, social, or spatial changes over the 11-yr study period. Individually identified elephants were monitored from 1999 to 2011. Minimal, short-term social disruption, with temporary changes to the herds' core ranges, was observed during the annual treatment events, particularly in the first three treatment years, when vaccinations were conducted exclusively from the ground. Thereafter, when vaccinations were conducted aerially, minor disruptions were confined to the morning of administration only. Despite sustained treatments resulting in demographic changes of fewer calves being born, treatments did not alter spatial range use, and no adverse interherd-intraherd relations were observed. Similarly, resource requirements did not change as calving still occurred, although in fewer numbers. It was concluded that PZP immunocontraception has no detectable behavioral or social consequences in elephants over the course of 11 yr, providing a convincing argument for the use of sustained immunocontraception in the medium to long term as an important tool for elephant management. Behavioral consequences of alternative management approaches should all receive similar scrutiny to enable managers to make informed decisions when weighing management interventions.

Active immunization against the proregions of GDF9 or BMP15 alters ovulation rate and litter size in mice.

The transforming growth factor ß (TGFB) superfamily proteins bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9), are essential for mammalian fertility. Recent in vitro evidence suggests that the proregions of mouse BMP15 and GDF9 interact with their mature proteins after secretion. In this study, we have actively immunized mice against these proregions to test the potential in vivo roles on fertility. Mice were immunized with either N- or C-terminus proregion peptides of BMP15 or GDF9, or a full-length GDF9 proregion protein, each conjugated to keyhole limpet hemocyanin (KLH). For each immunization group, ovaries were collected from ten mice for histology after immunization, while a further 20 mice were allowed to breed and litter sizes were counted. To link the ovulation and fertility data of these two experimental end points, mice were joined during the time period identified by histology as being the ovulatory period resulting in to the corpora lutea (CL) counted. Antibody titers in sera increased throughout the study period, with no cross-reactivity observed between BMP15 and GDF9 sera and antigens. Compared with KLH controls, mice immunized with the N-terminus BMP15 proregion peptide had ovaries with fewer CL (P<0.05) and produced smaller litters (P<0.05). In contrast, mice immunized with the full-length GDF9 proregion not only had more CL (P<0.01) but also had significantly smaller litter sizes (P<0.01). None of the treatments affected the number of antral follicles per ovary. These findings are consistent with the hypothesis that the proregions of BMP15 and GDF9, after secretion by the oocyte, have physiologically important roles in regulating ovulation rate and litter size in mice.

Physiologic responses following gonadotropin-releasing hormone immunization in intact male dogs.

We investigated the use of a commercial gonadotropin-releasing hormone (GnRH) vaccine as a method of temporary and reversible immunocastration in intact male dogs. Four privately owned dogs were vaccinated twice at 4-week intervals. Blood samples were collected at 0, 4, 12 and 20 weeks following the initial vaccination. These samples were analysed for GnRH antibody titres, luteinizing hormone (LH) and testosterone concentrations. Scrotal measurements were made at the time of sample collection, and testicular volume was calculated using the formula of an ellipsoid. As a result of vaccination, dogs displayed an elevated GnRH antibody titre, decreased LH and testosterone concentrations and decreased testicular volume, which reversed by the end of the study period. Therefore, these results suggest that immunizing against GnRH may be a possible choice for temporary and reversible immunocastration.

Generation and characterization of phage-GnRH chemical conjugates for potential use in cat and dog immunocontraception.

Overpopulation of cats and dogs is a serious worldwide problem that demands novel, safe and cost-effective solutions. The objective of this study was to generate and characterize phage-peptide conjugates with gonadotropin-releasing hormone (GnRH) for potential use as an immunocontraceptive. A filamentous phage vector f5-8 with wild-type phage coat proteins was used as a carrier for construction of chemical conjugates with GnRH, a peptide that acts as a master reproductive hormone. In such conjugates, the phage body plays the role of a carrier protein, while multiple copies of GnRH peptide stimulate production of neutralizing anti-GnRH antibodies potentially leading to contraceptive effects. To generate the constructs, four different GnRH-based peptides were synthesized and conjugated to phage particles in a two-step procedure: (i) peptides were reacted with phage to form a conjugate using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride chemistry (EDC) and (ii) the conjugates were separated from remaining free peptides by dialysis. Formation and specificity of phage-GnRH conjugates were confirmed by three independent methods: spectrophotometry, electron microscopy and ELISA. When the conjugates were tested for interaction with sera collected from cats and dogs immunized with GnRH-based vaccines in independent studies, strong specific ELISA signals were obtained, suggesting the potential use of the conjugates for cat and dog immunosterilization. The ability of the conjugates to stimulate production of anti-GnRH antibodies in vivo was evaluated in mice. While optimization of dose, immunization route and adjuvant still requires investigation, our preliminary results demonstrated the presence of anti-GnRH antibodies in sera of mice immunized with such conjugates. Fertility trials in cats and dogs will be needed to evaluate contraceptive potentials of the phage-GnRH peptide chemical conjugates.

Induction of specific immune response and suppression of fertility by B-cell-epitope-based mimovirus vaccine.

SPINLW1 (previously known as eppin (epididymal protease inhibitor)) is a target under intense scrutiny in the study of male contraceptive vaccines. B-cell-dominant epitopes are now recognized as key parts of the induction of humoral immune responses against target antigens. The generation of robust humoral responses in vivo has become a crucial problem in the development of modern vaccines. In this study, we developed a completely novel B-cell-dominant-epitope-based mimovirus vaccine, which is a kind of virus-size particulate antigen delivery system. The mimovirus successfully self-assembled from a cationic peptide containing a cell-penetrating peptide of TAT49-57 and a plasmid DNA encoding both three SPINLW1 (103-115) copies and adjuvant C3d3. The male mice were immunized with the epitope-based mimovirus vaccine, which resulted in a gradual elevation of specific serum IgG antibody levels. These reached a peak at week 4. Mating for the fertility assay showed that the mimovirus vaccine had accomplished a moderate fertility inhibition effect and investigation into the mechanism of action showed that it did so by interfering with the reproductive function of the sperm but that it did not damage the structures of the testes or cause serum testosterone to decline. Our results suggest an ideal protocol for suppressing fertility in mice by an engineered mimovirus vaccine.

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.

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.

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.

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.