Vaccination against GnRH may suppress aggressive behaviour and musth in African elephant (Loxodonta africana) bulls--a pilot study.

Aggressive behaviour and musth are constant problems in captive and sometimes in free-ranging African elephant bulls. Aggressive bulls are difficult and musth bulls almost impossible to manage without severely restricting their movement either by leg-chaining or using tranquillisers. This study investigated the relationship between faecal androgen metabolites (FAM) and faecal cortisol metabolites (FCM) concentrations and aggressive behaviour and tested a GnRH vaccine as a means of down-regulating aggressive behaviour and musth in 1 free-ranging and 5 captive elephant bulls. The bulls were non-aggressive (n=3), aggressive (n=2) or in musth (n=1) at the onset of the study. The bulls were injected with a GnRH vaccine-adjuvant combination 3 or 4 times at 3- to 7-week intervals. Behaviour, FAM and FCM concentrations were measured during every week prior to vaccination until 4 months after the last vaccination. FAM concentrations were positively correlated with aggressive behaviour before the 1st vaccination. Androgen production, as reflected by FAM concentrations, was down-regulated in 3 of the 6 immunised bulls. At least 2 bulls and possibly a 3rd showed behavioural improvement following GnRH vaccination and in all 3 temporal gland secretion ceased. No further aggressive behaviour was observed until the end of the study in any of the bulls. The results of this 1st GnRH immunisation study suggest that it could be a useful method to control aggressive behaviour and musth in African elephant bulls.

Effects of GnRH immunization in sexually mature pony stallions.

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

Effects of active immunization against GnRH on serum LH, inhibin A, sexual development and growth rate in Chinese female pigs.

Surgical castration of young female pigs is common practice in Chinese pig farming today. The purpose of the present study is to investigate anti-GnRH immunization as a practical alternative to surgical castration for female pigs. Thirty-six Chinese female crossbred pigs (Chinese Yanan x Yorkshire) were selected from 12 litters, three pigs from each litter, at the age of 10-13 weeks. One pig from each litter was immunized with 62.5 microg D-Lys6-GnRH-tandem-dimer peptide conjugated to ovalbumin in Specol adjuvant at Week 0 (0 week post-vaccination, wpv), and a booster vaccination was given 8 weeks later (8 wpv). Its intact and castrate littermates (surgically castrated at the time of weaning, i.e. at 6 weeks of age) were administered the vehicle and served as controls. Antibody titers, serum LH and inhibin A were determined at the day of first vaccination, every 4 weeks thereafter and at the day of slaughter (18 wpv). At slaughter, ovaries were inspected for the presence of follicles and corpora lutea, and ovarian and uterine weights were recorded. Ten of twelve immunized pigs responded well to the immunization (immunocastrated animals), while the remaining two pigs responded poorly (nonresponders). Antibody titres in immunocastrated animals steadily increased after immunization, became maximal at 12 wpv and remained high until slaughter. Serum LH levels were reduced (P < 0.05) in immunocastrated pigs as compared to intact controls and surgical castrates. Serum inhibin A levels decreased after vaccination, and equaled surgical castrate levels from 8 wpv until the end of the experiment. Ovarian and uterine weights (1.3 +/- 0.2 and 43.9 +/- 11.4 g, respectively; mean +/- S.E.M.) were significantly lower (P < 0.05) in immunocastrates than in intact controls (9.4 +/- 1.1 and 390.9 +/- 67.2 g, respectively). Antibody titers were significantly lower (P < 0.05) in nonresponders than in immunocastrated pigs from 12 wpv to slaughter. Ovarian and uterine weights were similar in nonresponders and in intact controls. Macroscopically, no follicular structures were found in ovaries of immunocastrated pigs, while large follicles or corpora lutea were observed in the ovaries of both nonresponders and intact controls. Although not significant, immunocastrates had a numerically higher average daily gain than surgical castrates and intact controls (0.74 +/- 0.04 versus 0.66 +/- 0.04 versus 0.66 +/- 0.03 kg per day, respectively; mean +/- S.E.M., P = 0.09). Results obtained in the present study demonstrate that anti-GnRH immunization can be an attractive alternative to surgical castration for Chinese crossbred female pigs. Our results also question the beneficial effect of surgical castration on growth as compared to intact controls.

Performance of male pigs immunized against GnRH is related to the time of onset of biological response.

In this study, the performance of male pigs immunized against GnRH was determined in relation to the onset of their biological response to the immunization. Pigs were immunized at 9 and 17 wk of age and were housed in a pen together with both a surgically castrated and an intact boar littermate. Feed intake was restricted to 2.8 to 3.2 times maintenance requirement for energy. Animals were weighed weekly and slaughtered at 108 kg BW. Depending on the time of onset of the response after immunization in terms of biological effects, immunized pigs were retrospectively grouped into two categories. One category consisted of the immunized pigs, which had undetectable or low levels of LH and testosterone at the time of booster immunization-known as "early" responding immunocastrates (E-IM, n = 8), whereas the "late" responding immunocastrates (L-IM, n = 7) had substantial LH and testosterone levels at that time. This dichotomy of the response to immunization also was reflected in testis weight, with 17 g and 40 g for E-IM and L-IM pigs, respectively. At slaughter, testis size and weight were reduced (P < 0.001) in the immunocastrated pigs as compared to the intact boars. Androstenone concentrations in backfat of all immunocastrated pigs were undetectable. Growth performance (i.e., ADG and feed efficiency [FE, g gain/kg feed]), was better in boars and L-IM pigs than in surgical castrates and E-IM pigs (P < 0.05). Average daily gain and FE did not differ between E-IM pigs and the surgical castrates, but intact boars performed better than L-IM (P < 0.02). There were no significant differences in carcass quality (backfat thickness and meat percentage) between boars and surgical castrates at slaughter. However, for both characteristics L-IM pigs and intact boars performed better (P < 0.03) than E-IM pigs. Thus, growth performance in L-IM is better than in either E-IM or surgical castrates.

Pharmacological and toxicological assessment of a potential GnRH vaccine in young-adult male pigs.

Active immunization against gonadotrophin-releasing hormone (GnRH) is successfully applied to prevent boar taint in pork. In men, GnRH immunization could be an alternative to hormone therapy in patients with prostate cancer. In this study, a new GnRH vaccine formulation (a modified GnRH peptide conjugate formulated with CoVaccine adjuvant) was investigated for its pharmacological efficacy and safety in young-adult male pigs. Immunization resulted in castrate-like plasma testosterone levels in all treated pigs from week 8 until the end of the study, 30 weeks after the first immunization. Testosterone depletion retarded testes growth, reduced the relative weight of the testes and accessory sex organs, and reduced sperm counts and motility. There was no clinically relevant toxicity. Typical vaccination-related adverse reactions, such as swelling at the injection site and fever, were considered acceptable. We conclude that this GnRH vaccine efficiently and rapidly reduced serum testosterone levels, without inducing chronic toxic effects, and therefore could be applicable in both veterinary and human medicine.

GnRH tandem peptides for inducing an immunogenic response to GnRH-I without cross-reactivity to other GnRH isoforms.

Gonadotropin releasing hormone (GnRH) occurs in various isoforms in mammals, i.e. GnRH-I (mammalian GnRH), GnRH-II (chicken GnRH-II), GnRH-III (salmon GnRH) and two forms of lamprey GnRH. The function of the latter four molecules have only been partially investigated. Also not much is known about the physiological effects of GnRH-I immunization on the function of these GnRH isoforms. In order to avoid possible harmful side-effects due to undesired neutralization of GnRH isoforms, GnRH-I specificity of antibodies raised against a panel of alternative GnRH antigens was determined. The results show that GnRH antigens can be designed which generate antibodies that specifically bind GnRH-I, without cross-reacting with other GnRH isoforms.

Highly immunogenic and fully synthetic peptide-carrier constructs targetting GnRH.

To use peptides as synthetic vaccines, they have to be coupled to a carrier protein to make them more immunogenic. Coupling efficiency between a carrier protein and a peptide, however, is difficult to control with respect to loading density of the peptide. This makes these carrier proteins poorly suitable for practicle use. Attempts have been reported to find carrier molecules or delivery systems which allow easy coupling or incorporation of peptides, reproducible loading densities and well defined products. We compared several promising constructs or delivery systems by immunization of male pigs using a tandem GnRH peptide as a branched polylysine construct, a lipo-thioester, a lipo-amide or a KLH conjugate in CFA, and the lipoamide peptide in an immuno-stimulating complex (ISCOM). We found the lipo-thioester and the branched polylysine constructs to be the most effective carrier molecules for the induction of antibodies against GnRH and immunocastration of pigs.

The role of the individual amino acids of a GnRH-tandem-dimer peptide used as an antigen for immunocastration of male piglets determined with systematic alanine replacements.

Immunocastration targeting gonadotropin releasing hormone (GnRH) can be obtained in male piglets using native GnRH conjugates. However, due to insufficient efficacy of these conjugates, improved GnRH antigens, like peptides existing of repeats of the GnRH amino acid sequence, have been designed. We previously reported about a dimerised GnRH-tandem peptide with a D-Lys at position 6 of the native GnRH sequence (G6k-TD) being highly effective. To evaluate the contribution of each individual amino acid of the GnRH decapeptide to the efficacy of the G6k-TD peptide, each amino acid was replaced consecutively by alanine (Ala-scan). The G6k-TD peptides were conjugated to ovalbumin, used for immunisation and tested for their ability to elicit GnRH antibodies and to immunocastrate male piglets. The results show that four out of nine amino acids (pGlu-1, Ser-4, Arg-8 and Gly-10) can be replaced by alanine without negatively affecting immunocastration efficacy. Replacement of amino acids in other positions (Tyr-5, Leu-7 and Pro-9) gave partial decrease of efficacy, respectively, five, six and six out of seven piglets were immunocastrated. Replacements at two other positions (His-2 and Trp-3) completely negated immunocastration activity. Thus, seven out of nine amino acid positions in the basic unit of G6k-TD can be substituted by alanine without affecting immunocastration efficacy.

Efficient immunocastration of male piglets by immunoneutralization of GnRH using a new GnRH-like peptide.

Active immunization to immunomodulate regulatory processes suffers from the disadvantage that the antigen is usually 'self' and therefore poorly immunogenic. This has been well illustrated by the long-standing experience with immunocastration vaccines targeting GnRH, a ten amino acid peptide. Not all animals vaccinated with these vaccines are equally affected, even after multiple vaccinations. This is a severe handicap when immunocastration vaccines are applied to male piglets to circumvent surgical castration. Surgical castration is universally practised to prevent boar taint, produced in the testicles of mature boars. Alternative immunocastration is only acceptable if all animals are equally affected using a minimum of vaccinations. Vaccines based on the GnRH peptide itself cannot meet these goals. We showed that using a GnRH-like peptide, a 20 amino acid tandem repeat of the amino acid sequence of the GnRH peptide, these goals can be attained. Using the tandem GnRH peptide to vaccinate male piglets completely abolished the development and endocrinological functioning of the testicles, in contrast to monomer GnRH. These results show that superior antigens can be made for effective immunomodulation by appropriate alteration of the antigen.

New GnRH-like peptide construct to optimize efficient immunocastration of male pigs by immunoneutralization of GnRH.

Castration of male pigs is routinely performed in order to prevent the occurrence of boar taint in pig carcasses. However, boar taint can also be eliminated by immunological castration using a synthetic peptide vaccine against GnRH. For pig farming, to make immunocastration a feasible alternative method to surgical castration, the composition of the vaccine has to be not only reliable and effective but also cost-efficient and safe. Previously the authors have developed an effective immunocastration vaccine by replacing the monomer GnRH by a much more immunogenic tandem peptide. However, this tandem-GnRH vaccine preparation needs Complete Freund's adjuvant and to be applied at a relatively high dose. Therefore, alternative antigens were designed to cope with this problem and tested with different adjuvants and dosages. An effective new antigen was designed based on a GnRH-tandem peptide, which was dimerized and modified in one amino acid position of the decapeptide to allow conjugation of this tandem-dimer to ovalbumin. In mild adjuvants and in low dosage, this antigen was very effective in reducing testis weight, serum LH and androstenone level in backfat. Thus, an improved immunocastration vaccine has been designed that is relatively cost-efficient and highly efficacious in two vaccinations at low dose.

Active immunization against gonadotrophin-releasing hormone in Chinese male pigs.

We have investigated, under the normal conditions of local Chinese pig farming, castration of young male pigs by vaccination with a newly developed vaccine against gonadotrophin releasing hormone (GnRH). Because of the very early onset of puberty, long fattening period and relatively harsh circumstances in Chinese pig production, an investigation of the endocrine response of Chinese breeds to this type of vaccination was of particular interest. Fifteen crossbred boars (Yorkshire x Yanan) from three different litters were randomly assigned to three groups of five animals each. The first group was immunized at 13 weeks of age with a GnRH tandem dimer OVA-conjugate in Specol and received a booster immunization 8 weeks later. The second group was injected with Specol alone and served as untreated controls. The remaining group was surgically castrated at the time of weaning (at 6 weeks of age). Pigs were fed ad libitum from weaning onwards. All animals were slaughtered at 31 weeks of age. Immunized boars had undetectable or low serum testosterone (0.09 +/- 0.12 ng/ml), low fat androstenone (0.05 +/- 0.01 microg/g) levels and very low testes weights (19.1 +/- 4.3 g). Intact controls had much higher serum levels of testosterone (9.76 +/- 4.81 ng/ml), fat androstenone levels (2.26 +/- 0.87 microg/g) and testes weights (114.3 +/- 29.41 g) at slaughter. Both the immunized and castrated group grew significantly faster than intact boars (p < 0.01). Average daily gains in immunized, castrated and intact animals were 0.69 +/- 0.08, 0.63 +/- 0.05 and 0.42 +/- 0.07 kg (mean +/- SD), respectively. The present data demonstrate for the first time that the newly developed anti-GnRH vaccine works very well under practical Chinese pig farming conditions, and can be an attractive alternative to surgical castration.