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.

Birth control vaccine targeting leukemia inhibitory factor.

The population explosion and unintended pregnancies resulting in elective abortions continue to impose major public health issues. This calls for a better method of contraception. Immunocontraception has been proposed as a valuable alternative that can fulfill most, if not all, of the properties of an ideal contraceptive. There are several targets that are being explored for contraceptive vaccine development. Leukemia inhibitory factor (LIF), a member of interleukin-6 family, is required for embryo development and successful blastocyst implantation in several mammalian species. The present study was conducted to examine if LIF can be a target for the development of a birth control vaccine. Three sequences from LIF and two sequences from LIF-receptor (LIF-R) that span the regions involved in ligand-receptor binding were delineated, and peptides were synthesized based upon these sequences. Antibodies raised against these five peptides reduced LIF bioactivity in an in vitro culture assay using BA/F3 mLIF-R-mpg130 cells. Vaccines were prepared by conjugating these peptides to various carrier proteins. Immunization of female mice with these peptide vaccines induced a long-lasting, circulating as well as local antibody response in various parts of the genital tract, and resulted in a significant (P ≤ 0.05) inhibition in fertility in all the three trials; the LIF-R peptide vaccines proved to be a better vaccine target. The data indicate that LIF/LIF-R is an excellent target for the development of a birth control vaccine. This is the first study, to our knowledge, that examined LIF/LIF-R as a target for immunocontraception. The findings of this study can be easily translated to humans since LIF/LIF-R is also important for implantation and pregnancy in women.

Immunocontraception in Eastern gray squirrels (Sciurus carolinensis): morphologic changes in reproductive organs.

Eastern gray squirrels (EGS) (Sciurus carolinensis) damage trees through bark stripping or gnawing due to territorial marking or agonistic gnawing behavior in concert with higher densities. This study was conducted to determine the effects of a contraceptive vaccine on EGS and its reproductive organ histology. Free-ranging urban EGS were vaccinated with the immunocontraceptive GonaCon. All EGS were > or = 6 mo of age as determined by a combination of pelage characteristics and body weights. The vaccine was administered by injection at a dosage rate of 0.4 ml containing 400 microg of GnRH-mollusk protein conjugate i.m. in the thigh to 33 EGS (17 male [m], 16 female [f]) in trapping session 1 (TS1), 23 (14 m, 9 f) in trapping session 2 (TS2), and 11 (8 m, 3 f) in trapping session 3 (TS3). A sham injection containing 0.4 ml saline-AdjuVac was given as control to 22 EGS (16 m, 6 f) in TS1, 20 (12 m, 8 f) in TS2, and 8 (4 m, 4 f) in TS3. In the last trapping session (TS4), 35 EGS (16 treated, 19 control) were killed for necropsy to evaluate histologic changes in testes and ovaries. Treated EGS males had testicular, prostatic, and epididymal atrophy compared with control EGS males. The tubuli seminiferi and prostatic glandular lumen of treated EGS males were atrophic, and the epididymal lumen contained no sperm cells. No histologic changes were observed in treated EGS females; however, females likely were not collected when changes due to GonaCon would have been observed. There were no observable histologic differences in the pituitary gland of treated and control EGS. There were no statistically significant differences in either testosterone or progesterone concentrations between control and treated EGS. Although there were no serious side effects to the vaccine, six EGS developed injection site abscesses. GonaCon may be a potential tool for EGS population control.

Does contraceptive treatment in wildlife result in side effects? A review of quantitative and anecdotal evidence.

The efficacy of contraceptive treatments has been extensively tested, and several formulations are effective at reducing fertility in a range of species. However, these formulations should minimally impact the behavior of individuals and populations before a contraceptive is used for population manipulation, but these effects have received less attention. Potential side effects have been identified theoretically and we reviewed published studies that have investigated side effects on behavior and physiology of individuals or population-level effects, which provided mixed results. Physiological side effects were most prevalent. Most studies reported a lack of secondary effects, but were usually based on qualitative data or anecdotes. A meta-analysis on quantitative studies of side effects showed that secondary effects consistently occur across all categories and all contraceptive types. This contrasts with the qualitative studies, suggesting that anecdotal reports are insufficient to investigate secondary impacts of contraceptive treatment. We conclude that more research is needed to address fundamental questions about secondary effects of contraceptive treatment and experiments are fundamental to conclusions. In addition, researchers are missing a vital opportunity to use contraceptives as an experimental tool to test the influence of reproduction, sex and fertility on the behavior of wildlife species.

Reimmunization increases contraceptive effectiveness of gonadotropin-releasing hormone vaccine (GonaCon-Equine) in free-ranging horses (Equus caballus): Limitations and side effects.

Wildlife and humans are increasingly competing for resources worldwide, and a diverse, innovative, and effective set of management tools is needed. Controlling abundance of wildlife species that are simultaneously protected, abundant, competitive for resources, and in conflict with some stakeholders but beloved by others, is a daunting challenge. Free-ranging horses (Equus caballus) present such a conundrum and managers struggle for effective tools for regulating their abundance. Controlling reproduction of female horses presents a potential alternative. During 2009-2017, we determined the long-term effectiveness of GnRH vaccine (GonaCon-Equine) both as a single immunization and subsequent reimmunization on reproduction and side effects in free-ranging horses. At a scheduled management roundup in 2009, we randomly assigned 57 adult mares to either a GonaCon-Equine treatment group (n = 29) or a saline control group (n = 28). In a second roundup in 2013, we administered a booster vaccination to these same mares. We used annual ground observations to estimate foaling proportions, social behaviors, body condition, and injection site reactions. We found this vaccine to be safe for pregnant females and neonates, with no overt deleterious behavioral side effects during the breeding season. The proportion of treated mares that foaled following a single vaccination was lower than that for control mares for the second (P = 0.03) and third (P = 0.08) post-treatment foaling seasons but was similar (P = 0.67) to untreated mares for the fourth season, demonstrating reversibility of the primary vaccine treatment. After two vaccinations, however, the proportion of females giving birth was lower (P <0.001) than that for control mares for three consecutive years and ranged from 0.0-0.16. The only detectable adverse side effect of vaccination was intramuscular swelling at the vaccination site. Regardless of vaccine treatment (primary/secondary), approximately 62% (34/55) of immunized mares revealed a visible reaction at the vaccine injection site. However, none of these mares displayed any evidence of lameness, altered gait or abnormal range of movement throughout the 8 years they were observed in this study. Our research suggests that practical application of this vaccine in feral horses will require an initial inoculation that may provide only modest suppression of fertility followed by reimmunization that together could result in greater reduction in population growth rates over time.

Immune-spaying as an alternative to surgical spaying in Iberian x Duroc females: Effect on the sensory traits and volatile organic compound profile of dry-cured shoulders and dry-cured loins.

The aim of this study was to assess the effect of immune-spaying on sensory characteristics and the volatile organic compound (VOC) profile of dry-cured shoulders and loins by comparing Iberian × Duroc surgically spayed females, immune-spayed females and entire females. VOC profile of dry-cured shoulders was not significantly affected by the reproductive status, probably due to the large heterogeneity of dry-cured shoulders as a product. Correspondingly, dry-cured shoulders showed little differences among treatment groups, with better scores for marbling, hardness and chewiness attributes in the immune-spayed females. Dry-cured loin sensory traits such as brightness, marbling, chewiness and juiciness, presented better scores in immune-spayed females. Moreover, dry-cured loins showed a higher homogeneity that allowed the effects of spaying to be observed, thus the Principal Component Analysis performed on VOC profile data indicated a better separation of samples among treatment groups. Consequently, immune-spaying could be a viable alternative to surgical spaying from the point of view of meat quality.

Effectiveness of Spayvac for reducing white-tailed deer fertility.

Overabundant white-tailed deer (Odocoileus virginianus) populations have been reported in many urban and suburban communities across the United States. Large populations of deer can potentially increase the risk of human-wildlife conflicts, such as deer-vehicle collisions, transmission of disease to humans, and vegetation damage. In 2003, efforts to control white-tailed deer numbers were initiated at the National Aeronautical and Space Agency's (NASA) Lyndon B. Johnson Space Center (JSC) in Houston, Texas, using the long-lasting, single-dose contraceptive SpayVac. Our objectives were to evaluate the effectiveness of SpayVac for reducing white-tailed deer fertility and determine the partial cost for treatment. Between 2003 and 2004, we monitored 45 adult female deer (34 treated with SpayVac, 11 controls treated with a placebo). Fawning rate over 2 yr for deer treated with SpayVac >30 days prior to the rut was 0% (n=31), whereas the fawning rate for control deer was 78% (n=11). Inoculation 1 mo prior to the breeding season was sufficient time to achieve fertility control. We conclude that SpayVac can effectively reduce the fertility of urban white-tailed deer.

Strategic use of anti-GnRH vaccine allowing selection of breeding boars without adverse effects on reproductive or production performances.

Boar stations raise only entire male pigs for selection as reproductive boars, but the majority of them will fail the selection process, ending at slaughter with a high risk of boar tainted meat. The aim of this study was to investigate whether a single dose of Improvac given to 16-week old boars had a negative effect on their subsequent sperm numbers and motility in 16 artificial insemination boars. We also aimed to generate more knowledge on incidence of boar taint at slaughter among Finnish pigs, compare production performances as average daily gain, feed conversion ratio, and carcass and meat quality (lean meat percentage, back fat, pH, color, androstenone, and skatole) of immunocastrated boars (n = 248) with those of entire boars (n = 268). Moreover, we aimed also to explore whether a fat biopsy taken at 16 weeks of age could already reveal the presence of boar taint compounds and be predictive of boar taint development at slaughter age. We found that 32% of entire boars (Figen Landrace, Figen Large White, and their crossbreed) slaughtered at an age of 25 weeks presented levels of androstenone and/or skatole above the threshold for boar taint in their meat. These boars (control) had higher androstenone and skatole levels in the back fat samples at slaughter (0.77 ± 0.55 and 0.09 ± 0.06 µg/g, respectively, mean ± standard deviation) than those in the immuno group (0.20 ± 0.25 and 0.06 ± 0.03 µg/g, respectively, P < 0.001). A single dose of anti-GnRH vaccine, given at 16 weeks of age, did not affect future sperm numbers and motility of boars selected for artificial insemination. We found no difference in the levels of testosterone, anti-GnRH antibodies titers, testicle morphology, and sperm numbers and motility between the boars vaccinated once, at 16 weeks of age, with anti-GnRH vaccine and the control boars (no vaccination). There were no differences in average daily gain, feed conversion ratio, lean meat percentage, and back fat between the immunocastrated boars and entire boars. Meat from immunocastrated boars had a higher pH and better color than meat from entire boars (P < 0.001), suggesting slightly improved meat quality.

Effects of time after a second dose of immunization against GnRF (Improvest) independent of age at slaughter on commercial bacon slicing characteristics of immunologically castrated barrows.

The objective was to determine the effects of time after a second dose of anti-GnRF immunization on fresh belly characteristics and slicing yields of immunologically castrated (IC) barrows, physically castrated (PC) barrows and gilts slaughtered at 24 weeks of age. The second dose was staggered so that IC barrows were slaughtered at 4, 6, 8, or 10 weeks after the second dose. Fresh belly characteristics (N=141) were collected at slaughter and bacon was manufactured commercially. The main effects in the model were treatment and the random effects of block and block within replication. Thickness, flop distance, and lipid content increased (L; P<0.04) and iodine value tended to decrease (L; P=0.08) with time after the second dose in IC barrows. Slicing yields increased with time after the second dose (L; P<0.01), but were similar (P=0.11) among sexes. Increasing time of slaughter after second anti-GnRF dose improves fresh belly and bacon slicing characteristics in IC barrows.

A comparison of the quality of fresh and frozen pork from immunologically castrated males versus gilts, physical castrates, and entire males.

The objective of this study was to compare pork quality and sensory characteristics of pork from gilts, physical castrates, entire males, and immunological castrates. Loins (Longissimus thoracis et lumborum) were collected at harvest, aged for 10 days, and subsequently cut into chops and roasts. Two chops and one roast were frozen for a minimum of 14 days to a maximum of 30 days and chops and roasts from the same loin were evaluated in fresh form. A trained sensory panel evaluated the samples and results showed that gilt pork, physical castrate pork, and immunological castrate pork were similar in terms of boar odor and pork flavor. The evaluated pork quality characteristics showed no differences among sexes except for marbling in the frozen samples (P<0.05). Results suggest that pork from immunologically castrated males is similar to pork from physical castrates in terms of sensory and pork quality characteristics and between fresh and frozen products.

Ham and belly processing characteristics of immunological castrated barrows (Improvest) fed ractopamine hydrochloride (Paylean).

Effects of sex class (physically castrated, PC or immunologically castrated, IC) and diet (0 or 5mg/kg ractopamine hydrochloride, RAC) on characteristics of ham and bellies were determined from pigs slaughtered in three groups with similar ending live weights. One carcass per pen per marketing group (n=8) was selected to evaluate further processing characteristics. Data were analyzed as a 2×2 factorial design with a split plot in time and fixed effects of sex, diet, marketing group, and their interactions. IC fresh bellies were thinner (P<0.01) and softer (P<0.01) than PC bellies. IC hams and bellies were leaner (P<0.05) than those from PC pigs. RAC feeding did not affect (P>0.05) fresh ham or belly characteristics but decreased (P<0.01) fat in cured PC bellies. Marketing group affected (P<0.05) fresh quality, processing characteristics, and composition of hams and bellies. Immunological castration and RAC produced leaner finished products but did not alter processing yield of hams or bacon.

Comparison of meat quality parameters in surgical castrated versus vaccinated against gonadotrophin-releasing factor male and female Iberian pigs reared in free-ranging conditions.

This study compared carcass and meat quality traits between 16 vaccinated (VF), 19 castrated (CF) and 8 entire (EF) female Iberian pigs, and between 21 vaccinated (VM) and 19 castrated (CM) male Iberian pigs reared in free ranging conditions. Vaccination consisted in the application of Improvac® at the age of 11, 12 and 14 months in VF and VM. Pigs were slaughtered at 16 months. In females, carcass and meat quality were found to be very similar regardless of the treatment. In males, VM had a leaner carcass, lower (P < 0.05) percentage of intramuscular fat, higher shear force and more rancidity than CM(P < 0.05 in all cases). It could be concluded that vaccination or suitable for free-range conditions in terms of product qualities. Vaccination in females did not alter carcass and meat quality, and specific interests should consider reproductive behavior in free-range conditions.

Comparison of entire male and immunocastrated pigs for dry-cured ham production under two salting regimes.

Due to the initiative to stop piglet castration, meat from entire male pigs is expected to take important share on the European market which can affect dry-cured ham industry. In the present study, hams of entire males (EM) and immunocastrates (IC) were submitted to dry-curing process. Sex category and salting regime were evaluated using standard (18 days; HS) and shortened salting (6 days; LS). At the end of processing, compared to HS, LS hams had lower (40%) salt content, were more proteolysed, were less salty, and had softer texture and different volatile profile. Sex effect was less evident; still, hams from EM exhibited higher processing losses and salt intake, and were drier, less marbled, harder, and more intensively coloured than IC hams. The panellists perceived higher off-flavours in hams with high boar taint compounds (correlation 0.67 and 0.53 for skatole and androstenone, respectively). The effect of sex category on volatile profile was negligible.

Effects of ractopamine administration and castration method on muscle fiber characteristics and sensory quality of the longissimus muscle in two Piétrain pig genotypes.

Single and combined effects of ractopamine supplementation (RAC, 7.5 vs. 0 ppm), castration method (surgical castration: SC vs. immuno-castration: IM) and genotype (genotype A: GA vs. GB containing 25% or 50% Piétrain) were determined on longissimus muscle (LM) fiber traits and quality of pork (n=512). RAC increased fiber IIX cross-sectional area (P=0.009) and decreased glycolytic potential (P=0.02) and pork tenderness (P<0.001). Fiber traits indicated that LM of IM pigs was more oxidative (P<0.05) and meat had slightly higher (P=0.04) off-flavor score and WBSF than SC. LM from GB pigs was paler (P<0.05) and had greater (P<0.05) glycolytic potential, IIX fiber cross sectional area and pork off-flavor than GA. RAC supplementation, castration method and genotype or their combination affected some fiber traits and some quality parameters but differences reported were small indicating these treatments or their combination could be used without major prejudice to meat quality.

The contraceptive potential of ZP3 and ZP3 peptides in a primate model.

It has been known for some time that antibodies raised against ZP3, the major component of the glycoprotein shell that surrounds all mammalian oocytes, can successfully inhibit sperm-egg interaction in vitro. In our own studies using the non-human primate Callithrix jacchus, active immunisation was successfully achieved when homologous or heterologous ZP3 was used as an immunogen. However this long-term suppression of fertility was at the expense of ovarian function. An ovarian pathology was observed which was characterised by a disruption of folliculogenesis and depletion of the primordial follicle pool. Adverse auto-immune reactions have also been observed in mice following induction of immunity to mouse ZP3. Following careful selection of B-cell epitopes on mouse ZP3, peptide vaccines were formulated which could circumvent these adverse side effects and induce reversible infertility in actively immunised mice. To identify similar epitopes on primate ZP3, epitope mapping studies were performed and several candidate regions of the molecule were identified. These were incorporated into chimeric peptide vaccines and administered as single or triple peptide vaccines. Active immunisation successfully induced antibodies that bound exclusively to the zona pellucida of marmoset and human ovarian sections. These antibodies were able to suppress human sperm-egg binding by up to 60% in vitro. Encouragingly, no adverse side effects on ovarian function were observed following long-term immunisation however, no loss of fertility was consistently observed in vivo. Thus considerable research is still required to identify a combination of ZP epitopes that will induce reversible infertility in the absence of any ovarian dysfunction.

Update on zona pellucida glycoproteins based contraceptive vaccine.

Zona pellucida (ZP) glycoproteins, due to their critical role in mammalian fertilization, have been proposed as candidate immunogens for development of a contraceptive vaccine. Active immunization studies in a variety of animal species, employing either native or recombinant zona proteins, has established their contraceptive potential. Hence, ZP glycoprotein-based contraceptive vaccines have a very good potential for controlling wild life population. To make it a realistic proposition, additional research inputs are required to develop new potent adjuvants and novel practical strategies for vaccine delivery. The observed ovarian dysfunction, often associated with immunization by ZP glycoproteins, is one of the major obstacles for their application in the control of human population. Ongoing studies to delineate epitopes of ZP glycoproteins that will generate an immune response capable of inhibiting fertility without any untoward effects on ovarian functions will help in determining their feasibility for human use.

Possible mechanisms of mammalian immunocontraception.

Ecological and conservation programs in ecosystems around the world have experienced varied success in population management. One of the greatest problems is that human expansion has led to the shrinking of wildlife habitat and, as a result, the overpopulation of many different species has occurred. The pressures exerted by the increased number of animals has caused environmental damage. The humane and practical control of these populations has solicited the scientific community to arrive at a safe, effective, and cost-efficient means of population control. Immunocontraception using zona pellucida antigens, specifically porcine zona pellucida (pZP), has become one of the most promising population control tools in the world today, with notable successes in horses and elephants. A conundrum has risen where pZP, a single vaccine, successfully induces an immunocontraceptive effect in multiple species of mammals. This review describes the most current data pertaining to the mammalian zona pellucida and immunocontraception, and from these studies, we suggest several potential mechanisms of immunocontraception.

Immune response of male baboons to testis-specific LDH-C(4).

Four sexually mature male baboons (Papio sp.) were immunized with a chimeric peptide containing a B-cell epitope of the testis-specific lactate dehydrogenase (LDH-C(4)) and a promiscuous T-cell epitope of tetanus toxin. LDH-C(4) is the testis-specific isozyme of lactate dehydrogenase, and antibodies to this protein reduce fertility significantly in female nonhuman primates. Animals were immunized on Day 0 and received booster injections at Days 29, 61, and 344 after priming. Serum specific antibodies were determined at regular intervals during the initial 6 months and after the last booster. Testis biopsies were taken at Days 61, 127, and 183 after the primary immunization. Sperm-zona binding was assessed prior to and three times after the last booster. The present study demonstrated that this epitope of LDH-C(4) did not cause autoimmune disease and that sperm from these immunized males had a diminished zona binding capacity. These results suggest that a safe male immunocontraceptive based on development of anti-sperm antibodies may be feasible.

Potential consequences and problems with wildlife contraceptives.

This presentation reviews multiple wildlife health considerations associated with contraception of wildlife, at the level of both the individual animal and wildlife population. Review of the literature demonstrates that many contraceptives studied in the past have had potential adverse effects characterized as follows: harmful effects on pregnant animals, inhibition of parturition or dystocia, changes in ovarian structure or function, changes in sex ratio, changes in lactation or mammary glands, impact on fertility of young, changes in testicular structure or function, changes in secondary sex characteristics, changes in bodyweight, changes in behaviour, changes in annual breeding season, other physiologic and pathologic changes, abscesses or inflammatory reactions, toxicity, interference with diagnostic tests and ecological alterations. Concern is expressed that the use of immunocontraception could create genetic changes in the target population that would influence disease resistance. The use of infectious agents as vectors to deliver immunocontraceptives was not considered wise animal health management because the product will be a new reproductive disease that could be difficult to contain within the target population. Criteria that need to be fulfilled for the safe use of contraceptives in wildlife are offered.

Sperm antigens and immunocontraception.

The development of novel forms of contraception is one way in which the world population crisis is being tackled. The concept of a contraceptive vaccine based on gamete-specific antigens is a particularly attractive approach. Much research has been carried out to identify sperm antigens which could be used as the immunogen. The most encouraging leads have come from groups using monoclonal antibodies to identify and characterize sperm antigens important for fertility (e.g. SP-10, PH-20 and PH-30). Identification of these molecules will also enable the development of specific tests for the diagnosis of immune infertility.