IE83361B1 - A method for improving the organoleptic qualities of meat - Google Patents

Description

A METHOD FOR IMPROVING THE ORGANOLEPTIC QUALITIES OF MEAT Ibe present invention relates to anwflmd »fOr producingi pig,cntt1e<1r sheep meat having inproved organoleptic qualities, particularly improved smell, juiciness and tenderness, from uncastrated male domestic animals.

The invention"also-relates to a vaccination kit associated therewith.

The advantages of using the intact male over the castrated male in the fattening of domestic animals intended for meat productioni have been stressed for several decades by‘ specialists in zootechnics. They relate to a higher growth rate, especially in cattle and sheep, to a better utilization of the feed ration and to a carcass which is leaner but better endowed with muscle mass in all domestic species (Sac, SEIDEMAN et ale, 3., of Animal Science, 1982, 55 (4) 826=840 and M: BONNEAU, i_L\‘H_fi‘: :r’rod, Anjhfno, 1988, l (2) i33~=l4~Ci} = The main drawbacks of this use of the intact male, pointed out in the reviews cited above, relate to ihe unpleasant smell and taste in the case or male pigs and sheep and the less tender‘ meat from intact Inale CgT1i@ and sheep, and justify the Current practices of surgical castration.

In effect, while androgenic steroids including androstenediol, androstenedione and testosterone are the decisive factors in the advantages expected in all domestic species for a faster growth and a better utilization of the feed ration, they are held responsible for the less tender meat from intact male cattle and sheep. The non—androgenic steroids or l6—androstene derivatives including 5a«androstenOne (5a—androst—16~en— 3—one), in the male pig, are partially responsible for the unpleasant smell and taste of the meat from a number of intact male pigs once they have reached puberty, these factors detracting from the quality of the meat and being an obstacle to its marketing in the fresh state.

Skatole, a product derived from tryptophan and produced by the intestinal microbial flora, is a compound partially responsible for the unpleasant smell and taste of the meat from the intact male pig. Its production depends on environmental, nutritional and breed factors._ Its accumulation in the adipose tissue is greater in the boar and is considered to be linked to the secretion of gonadal sex steroids.

For experimental purposes, an attempt has already been made to decrease or abolish the development of the male character in. young animals or the secretion. of dtesticular hormones, in particular testicular steroids, by active or passive immunoneutralization against these or against the hormones participating in their secretion, in particular luteinizing hormone or LH and the hormone gonadoliberin (GnRH), also known as luteinizing hormone releasing hormone (LHRH). Tests have also been conducted on pigs to lower the tissue level of 5a-androstenone, of the 16—androstene group, by active immunization directed towards this compound (E.D. WILLIAMSON et al., Livestock Production Science, 1985, 12, 251-264) or by’ passive CLAUS, Immunization with Hormones in Reproduction Research, ed.

Nieschlag, 1975). It is possible to seek to abolish or immunization against this same compound (R. decrease the secretion of testicular steroids by immuno- neutralization of the gonadotropic hormone LH specific to the species in question (R.E. FALVO et al., J. Anim.

Science, 1986, 63, 986-994), or" by anti-LHRH immuno- neutralization of endogenous LHRH. Only active anti-LHRH immunization has been recommended by various authors. In pigs, the lowering of a—androstenone has been obtained by this method (A. CARATY and M. BONNEAU, C.R. Acad. Sci.

Paris 1986, 303, Series III (16) 673-676; R.E. FALVO et al., J. Anim. Sci., 1986, 63, 986-994).

In sheep, B.D. SCHANBACHER (Am. J. 1982, 242, E201-E205) recomends anti—LHRH immunization to delay testicular development and produce a castration effect in male lambs. In cattle, P.S. ROBERTSON (Vet.

Rec., 1979, 105, 516-517) describes an anti—LHRH immuno- Physiol., logical castration.

The anti—LHRH immunoneutralization. tests " 833 described on laboratory animals (ARIMURA et al., Endocrinology, 1973, 93, 1092-1103; FRASER H.M. et al., J. Endocr. 1974, 63, 399-406; MAKINO T. et al., Contraception, 1973, 8 (2), 133-145; CARELLI C. et al., Proc. Natl. Acad. Sci., USA, 1982, 79, 5392-5395) and on species (JEFFCOATE et al., Theriogenology, 1978, 10(4), 323-335; ROBERTSON I.S. et al., Veterinary Record, 1979, 105, 556; SCHANBACHER B.D. Am. J. Physiol., 1982, 242, E201—E205) have shown that it is possible to obtain arrest of testosterone several domestic secretion, weight regression of the testicles and its appended glands, arrest of spermatogenesis and, at behavioral level, disappearance of libido.

This work has led to the suggestion of recourse to an early immunoneutralization, in particular anti-LHRH, to replace the traditional surgical castration for breeding purposes.

In Patent US No. 4,556,555, a nmthod is thus described for passive immunization of animals before puberty, using an antiserum containing antibodies directed towards gonadotropin.

International Patent Application WO 90/11,298 describes a method of anti—LHRH immunization at birth, using 2 LHRH sequences in tandem coupled to a carrier protein, to improve meat quality in pigs.

International Patent Application WO 88/00,056 describes a method of anti-LHRH immunological castration intended for improving the social and sexual behavior of male animals as a replacement for surgical castration which affects the growth rate. The bulls are_vaccinated at the age of 8 to 40 weeks and then receive several boosters.

An anti~LHRH vaccine sold under the brand name VAXSTRATE by the Australian company WEBSTERS is used in cows. _ R.E. Falvo et al., (J. Anim. Sci. 1986, 63 : 986- 994) have immunized several groups of boars using LHRH- human serum globulin. conjugates in Freund’s complete adjuvant or with muramyl peptide as adjuvant. After vaccination and several boosters, the authors observed high titers of anti—LHRH antibodies, but with the need to perform repeated boosters in order to maintain the high antibody titer.

I.S. Robertson describes a method of immunization with LHRH conjugated to tetanus anatoxin or to thyro- globulin, and suggests that the immunological approach would permit a late castration with the advantages which may be expected from the standpoint of weight gain. He concludes, however, that there are still efforts to be made in order to arrive at a castration method which is usable in practice, both in respect of the method itself and of the adjuvant, Freund’s adjuvant being prohibited in practice.

Lastly, A. Caraty and M. Bonneau (C.R. Acad. Sc.

Paris, vol. 303, Series III, No.16, 1986) have performed an anti~LHRH immunization. in male pigs. The authors suggest that the blockade of steroid production 2 to 3 weeks before slaughter would enable the high potential of this type of animal for meat production to be exploited while avoiding the problems created by the accumulation of androsterone in the adipose tissue. They conclude, however, that substantial progress remains to be made in the immunization. techniques before it is possible to propose active anti«LHRH immunization. as a technique which is usable in pig farming.

Moreover, late immunoneutralization creates in practice the considerable problem of the safety of the treatment, and in particular of the local reactions engendered by the vaccines, especially oily vaccines, with the risks of rejection or of downgrading of the meat resulting therefrom.

Improvement of the organoleptic qualities in cattle and sheep has not been suggested.

The Applicant has, in point of fact, found an industrially applicable method which enables the organo- leptic properties of the meat from animals to be improvedi .The present invention thus relates to arnmhod for producing pig,cfittk3Or sheep meat having improved organoleptic qualities, particularly improved smell, juiciness and tenderness, wherein uncastrated male domestic animals are fattened and the animals are given active immunoneutralisation using an anti~LHRH vaccine, characterised in that the advantages connected with the male nature of the animals are maintained virtually until they are slaughtered by the fact that, before or during the period of fattening of the animals, they are given a single anti—LHRH vaccine designed to induce a first low—intensity immune response, with no appreciable or even measurable effect on the secretion of sex steroids andgfimrtlybefore slaughtering, they are given an anti—LHRH vaccine to induce anti—LHRH immunoneutralisation which leads to suppression of or a significant reduction in the secretion of steroids.

We vaccine administered first and the VsCCin9 administered just before slaughtering are selected from emulsified vaccines and vaccines in an aqueous adjuvantl According to a first preferred embodiment of this method, administered. to the an anti~LHRH vaccine is animal, preferably in emulsion form, preferably during or before the phase of fattening of the animal, and then, shortly’ before slaughter’ of the animal, an anti~LHRH vaccine is administered again. The procedure may‘ be accomplished in two separate administrations, or by means of a controlled—release method.

In pigs, it» is especially advantageous to administer, before slaughter, the anti-LHRH vaccine with type hydroxide gel and/or saponin. an aqueous adjuvant, in particular aluminum This administration.is preferably performed 15 to 21 days before slaughter. ppreferably In contrast, in cattle and, where appropriate, in sheep, the administration preceding slaughter is prefer= ably carried out with an adjuvant in emulsion form, and preferably 1 to 2 months before slaughter. This adminia stration is preferably performed at least 4 weeks, and after the first . several months, ‘administration.

In any case, for the vaccine in emulsion form intended for the first administration and, in cattle, for the second administration, it is preferable for the vaccine to take the fort: of ex water-in-oil emulsion.

However other forms of emulsion may be envisaged.

This vaccine, preferably of the emulsion type, is designed, the invention, to induce a primary, according to low»intensity' immune response without a significant or even measurable effect on gonadal steroid secretion. The formulation as an emulsion is preferred, but other formulations are usable as long as they produce the same effect.

The administration which precedes slaughter is carried out with a vaccine formulated to produce, at this time, the abolition or a significant lowering of steroid secretion without an adverse local or general reaction capable of impairing the appearance or quality of the meat.

Preferably, in particular for pigs, the conjugate, in aqueous solution, is put into the following two formulations: the first, in the form ofia stable water—in—oil emulsion made from a mixture of highly ‘purified mineral, animal or vegetable oils and of non- ionic surfactants, for inducing a low—intensity immune response without a measurable effect on gonadal steroid secretion, and the second, not emulsified, with aluminum hydroxide gel and saponin, triggering a rapid and intense immune reaction resulting in sufficient production of neutralizing" anti—LHRH antibodies to bring" about the decrease in or abolition of gonadal steroids and the decrease in the associated transport of skatole of intestinal origin.

The emulsion used is, in distinction to that which is obtained using Freund’s complete or incomplete adjuvant, a stable emulsion enabling a ready=to=use vaccine to be prepared. The inflammatory skin reaction remains very weak and localized at the points of administration of the two vaccine formulations, and manifests itself in the form of well-circumscribed papules on external examination. Its internal development remains limited to the superficial dermis. It disappears without leaving an apparent granuloma at the time of slaughter of the animals.

According to another embodiment of this method. hyperimmune anti—LHRH serum or plasma or alternatively anti—LHRH monoclonal antibodies is/are administered to the animal a few days before slaughter, in particular 5 to 15 days beforehand.

The passive anti~LHRH imunization.bringing about the decrease in or even the abolition of androgenic and non—androgenic steroid secretion was obtained by intra~ muscular administration of hyperimmune equine plasma.

Brought to a sufficient level, measured. by‘ the LHRH _ 3- antibody titer of the serum of the recipient animal, this immunization. brings about a decrease in plasma testosterone from day 3 onwards; maintained at the same. level for the following 12 days, it is sufficient to bring about a lowering of the tissue androsterone to below 0.50 microgram/g, at which value the unpleasant smell and particular taste of male pig meat would no longer be perceived by the consumer. This passive immunization method has shown that maintenance of the significant ‘decrease in. testosterone for 12 days is sufficient to lower the tissue androsterone concentration to below the set threshold. This passive immunization can 'be envisaged by the use of anti—LHRH monoclonal anti- bodies secreted by porcine hybridomas or heterohybridomas.

The mode of administration of these formulations is preferably transcutaneous, in particular using a needleless injection apparatus, via a jet under pressure, in particular according to Patent Application FR=A~2,652,257.

The method according to the invention has the great advantage of being completely safe, in particular of not inducing local reactions liable to lead to down~ grading of the meat.

The inflammatory skin reaction remains localized at the points of administration of the two vaccine formulations, and manifests itself in the form of wellw circumscribed papules on external examination. Its internal development remains limited to the superficial dermis. It disappears without leaving an apparent granuloma at the time of slaughter of the animals. The inflammatory reaction, limited in time and to the points of administration, reflects the tolerance to ’the 'Uwo vaccine formulations and. is obtained by the trans~ cutaneous administration. of these, performed using a needleless injector. 4 The anti—LHRH immunization necessitates con— jugation of the LHRH peptide or a fragment of the LHRH non—immunogenic under economical peptide, which is _g;_ conditions for their use, to an immunogenic protein, termed carrier, via a covalent bond.

LHRH or GnRH, whether natural or synthetic, is composed of 10 amino acids, numbered from 1 to 10 proceeding from the amino—terminal terminus to the carboxy-terminal terminus, according’ to the following formula: pGlu-His—Trp—Ser—Tyr-Gly—Leu—Arg—Pro—Gly NH2 1 2 3 4 5 6 7 8 9 10 These symbols by convention. represent: pGlu, pyroglutamic acid; His, histidine; Trp, tryptophan; Ser, serine; Tyr, tyrosine; Gly, glycine; Leu, leucine; Arg, arginine; Pro, proline. .

Anti¥LHRH immunogenic conjugates, described by the various authors, may be produced, as regards the hapten, with: a) whole LHRH or LHRH modified in one or more of its parts to obtain the desired aminouterminal, carboxye terminal or intermediate conjugation, b) one of its peptide fragments composed of 5 to 7 amino acids, modified. or otherwise, to obtain the desired aminonterminal, carboxywterminal or intermediate conjugation, C) an agonist bearing a substituted amino acid, most commonly at position 6, to obtain an intermediate conjugation.

As regards the carrier protein, bovine serum albumin, human serum albumin, thyroglobulin, ovalbumin and human or equine globulins have been used.

Thus, European Patent Application EP—A—l8l,236 describes immunogenic conjugates comprising a nonapeptide or decapeptide including a sequence, corresponding to the last 8 amino acids of the LHRH molecule, to which a lysine or a cysteine—lysine sequence is added on the amino—terminal side.

Moreover, Patent Application WO 88/05,308 dis- closes conjugates, made using fragments of 5, 6 or 7 adjacent amino acids of the natural molecule, in which each fragment includes the N—terminal pyroglutamic acid _ H)- or the carboxy—terminal glycinamide and to which. an additional amino acid or amino acid sequence can.be added to the end linked to the immunogenic protein.

The conjugating agents used may be classified in three major categories: homo» activating agents, bifunctional agents and heterobifunctional agents.

Whereas, with.activating agents, the link between the two molecules is made between two functions already present, with the others, the link is made ‘Via a hydrocarbon residue termed ligand.

Among activating agents, there may be mentioned periodic acid, employed to oxidize the oligosaccharide residues of the glycoproteins to aldehydes, with which the amine groups of the other molecule participating in the conjugate will subsequently react.

Carbodiimides are activating agents widely employed for the coupling of antigens to proteins, and among these the most extensively’ used is certainly Neethyl—N’—(3—dimethylaminopropyl)carbodiimide (EDC) hydrochloride, which enables the reaction to be performed in an aqueous medium. Their action leads to the formation of an amide bond between a carboxyl group of one protein, activated in the form of an intermediate Owalkylisourea, and. an amine group borne by’ another' molecule. Their advantage lies in their simplicity of use.

Homobifunctional agents are molecules which possess two identical reactive groups separated by a hydrocarbon chain. Among these there may be mentioned glutaraldehyde, which reacts with two primary amine groups, alkyl or aryl diisothiocyanates, which react with primary amines and thiols, and bisdiazotized benzidine, which couples with the aromatic residues of tyrosine.

Bismaleimides and bisamidinates may be mentioned for the record. The major drawback of homobifunctional agents is that of poor control over the nature of the conjugates formed, since these agents can react with two molecules of the same nature and lead to the formation of oligomers or polymers. introduced To remedy" this, chemists have _ 11 _ heterobifunctional agents, in which the two groups have different specificities. In (flue general. case, one of these groups is an. N-hydroxysuccinimide ester which, under mild conditions, reacts with the free amine groups of proteins to give, on the one hand N—hydroxysuccinimide, and on the other hand the protein bearing via a covalent amide bond the coupling agent on which the 2nd function occurs. From a rather general standpoint, the latter can react with thiols supplied by the molecule to be coupled, these thiols being" either initially present in the molecule in the fornl of cysteine residues (it being possible for the latter to be natural constituents or, in the case rxf peptides, introduced intentionally during synthesis), or supplied by agents such as 2—iminothiolane orN—[3~(2—pyridyldithio)propanoyloxy]succinimide(SPDP), after reduction.

Among the possibilities stated above, it is preferable to use whole LHRH. In this case, natural LHRH is preferred to agonists such as (D~Lys5~LHRH by comm parison of the immunogenic activity of the conjugates prepared with these two peptides.

The carbodiimide is preferred to glutaraldehyde as an agent for conjugating natural=form LHRH with alpha~ globulin.

Human or equine alpha-globulin, fraction IV«l or IV»4, is preferred to human or bovine serum albumin.

Preferably, the vaccines comprise one and the same active principle, preferably comprising an alpha« globulin—LHRH conjugate; the LHRH is preferably in natural form and the alpha—g1obulin of human or equine origin, in particular fractions IV—l and/or IV—4. The conjugate is preferably obtained by adding from 0.5 to 2 volumes of a 2.5% solution of N—ethyl-N'—(3—dimethyl~ aminopropyl)carbodiimide (EDC) hydrochloride in 0.9% Nacl to 1 volume of alpha~globulin/LHRH mixture in solution con—taining 2 to 20 mg/ml in 0.9% NaCl. After stirring, the mixture is left overnight and then purified by gel permeation chromatography.

As regards the carrier protein, it is possible to use serum albumins, in.particular bovine or human, thyroi globulin, ovalbumin, and human or equine globulins, anatoxins, in particular tetanus anatoxin.

The predominance of the immune response of male pigs to the carboxy—terminal fraction.of the LHRH peptide conjugated. with the carbodiimide, (D~Lys%-LHRH conjugated with SPDP, which was observed, led to the definition of an anti—LHRH or of its agonist to alpha—globulin, immunogenic conjugate employing an advantageous peptide possessing the carboxy—terninal terminus of LHRH.

The Applicant found that it was very advantageous to use a conjugate of a peptide comprising the last 8 amino acids of LHRH, i.e. a decapeptide of formula: Trp — Set ~ Tyr ~ Gly — Len — Arg — Pro — Gly ~ NH, 4 5 6 7 8 9 10 which has considerable immunogenic activity without having the hormonal activity of natural LHRH.

The invention therefore envisages the use of conjugates incorporating this peptide (3~10) coupled to an immunogenic carrier protein from among those mentioned &bmVQ, ovalbumin and equine aloha Qiflbfllfll inparticular fractions IV=l and/or lV=4, being preferred, In the invention, the carbodiimide is preferred to glutaraldehyde and to heterobifunctional agents as an agent for conjugating the LHRH (3=lO) peptide, in pare ticular to equine alphaaglobulin or ovalbumin.

In the preferred preparation of conjugate, LHRH (3m10) ovalbumin. or alpha» globulin, are dissolved in the proportion of 2 to 40 mg per ml each in 0.1M NaCl/0.1M 2~(N—morpholino)ethane~ sulfonic acid buffer. 0.5 to 2 volumes of a 2.5% solution of N—ethyl—N’~(3—dimethylaminopropyl)carbodiimide in the same buffer are then added. The pH is adjusted by adding 1N sodium hydroxide. After stirring, the mixture is left overnight and is then purified by gel permeation chroma- tography, which removes uncoupled LHRH (3~10), residual carbodiimide and its hydrolysis products. and the carrier protein, -13 _ A subject of the invention is also the passive anti—LHRH. (3-10) immunization in accordance with the method described above.

The invention also relates to an anti—LHRH vaccination kit which can be used for the production of meat having improved organoleptic qualities from uncastrated male pigs, cattle or sheep, this vaccination kit comprising: — an anti—LHRH vaccine designed to be given in the first place before or during the stage of fattening the animals and designed to induce a first low intensity immune response with no appreciable or even measurable effect on the secretion of the sex steroids, to allow the male nature of the animals to develop, and — an anti—LHRH vaccine designed to be administered after the previous one and mumtly before slaughtering, this vaccine being designed to induce anti—LHRH immunoneutralisation causing the suppression or significant lowering of the secretion of steroids.

It also relates to kits combining, in a single pack, an equal number of doses of vaccine to be administered before slaughter and of vaccine to be administred jJ1 a primary injection. Preferbaly, these vaccines are packaged. in. a reduced volume and at an increased concentration for administration by trans~ cutaneous jet, for example according to the French patent application cited above.

The invention will now be described in greater detail by means, on the one hand of tests comparing several products and methods of vaccination according to the invention, and on "the other hand of tests which showed the predominance of the immune response of male pigs to the carboxy—terminal fraction of the LHRH peptide and of the test of anti—LHRH vaccination performed on male pigs according to the invention. -14 _ I — USE OF WHOLE LHRH A. Greater imunogenic activity of the conjugate maintaining intact the most extensive carboxy» terminal fraction of the LHRH peptide, and choice of the conjugate based on natural-formula LHRH in preference to that obtained using the agonist (D-Lys5)—LHRH.

Al. — Anti—LHRH immunization.of intact male pigs and of OFA male rats.

The comparison of activity of two. anti—LHRH vaccines consisting of conjugates between natural—form LHRH (B1 and B2) or (D—Lys°)~LHRH (A1 and A2) and human albumin, the conjugates being obtained with carbodiimide in the aqueous phase and SPDP, respectively, put into an oil—in-water emulsion and administered intramuscularly in pigs and subcutaneously in rats, leads to the following conclusions: « Greater activity’ of the vaccine based on naturalwform LHRH: mass of the conjugated LHRH peptide lower than that of the conjugated (D=Lys6)~LHRH peptide for a recruitment of a larger number of animals display" ing an immune response (Tables 1 and 3).

— Dose effect which manifests itself in a recruitment of a higher number of animals displaying an immune response with the same conjugate (Table 3).

Al.l — Preparation of (D—Lys5)~LHRH—albumin conjugates with SPDP.

The preparation of (D-Lys6)—LHRH—albumin conjugates is carried out in three steps: preparation of {N—[3—(2—pyridyldithio)propanoyl]—D—Lys6}—LHRH, prepara- tion of N—(3—mercaptopropanoyl)albumin, then coupling.

{N‘— [ 3 — ( 2 —Pyridyldithio ) propanoyl 1 —D—Lys" } -LHRH is prepared. by’ reacting an. excess of SPDP ‘with. LHRH in aqueous solution (6 mol of SPDP per mole of LHRH), and then, after one night at 4°C, centrifuging the product obtained. The latter is dissolved in 8M urea and the -pyridyldithio groups present are assayed. -15 _ N—(3—Mercaptopropanoyl)albumin.is obtained.by the action. of 0.2 mmol of SPDP on 1 g’ of human albumin dissolved in 100 ml of 0.1M phosphate buffer, and then, after one night of contact at 4°C and acidification to pH 6, by reduction with dithiothreitol. It is then purified by gel filtration chromatography. Assay of thiol and protein yields the average level of substitution.

Coupling is performed taking one 2—pyridyldithio group per 1.25 thiol groups. The pH is taken to 7-7.5 and then, after one hour, the yield is determined" by measurement of the 2—pyridinethione liberated.

The average level of substitution is deduced from this. Finally, the conjugate is purified by’ chroma- tography and concentrated by ultrafiltration.

A1.2 = Preparation of the LHRH-albumin conjugate with carbodiimide. mg of N»ethyl~N’=(3»dimethylaminopropyl)w carbodiimide hydrochloride, dissolved immediately before use in 40 ml of 0.9% NaCl, are added to 300 mg of LHRH and 300 mg of human albumin dissolved in 30 ml of 0.9% NaCl. After stirring, the mixture is left overnight at room temperature protected from light. It is then chromatographed. corresponding to the conjugate are collected, where appropriate concentrated and frozen.

From the fractions containing uncoupled LHRH, the amount of uncoupled LHRH and hence the average level of conjugation are determined. The latter is reproducible and varies from 8 to 10 mg of LHRH coupled per 100 mg of albumin.

From the UV spectra of the conjugate before and after chromatography, the chromatographic yields of conjugate and hence the amount (or concentration) of LHRH conjugated are deduced.

Al.3 - Assay techniques The antibody titer is determined according to the technique described by JEFFCOATE et al., Acta. Endocr., Copenh., 1974, 1;: 62S—635.

Testosterone is assayed directly on plasma by an RIA technique employing the radioligand testosterone Cl9~carboxymethyl ether—[ I]histamine. _ R3- Binding to the labelled peptide is determined after labeling the various peptides with iodine~125 according to COPPOLAND et al., Endocr., 1979 igg: 1504a 1506 described by JEFFCOATE et al., Acta Endocr., Copenh., 1974, 75, 625-635. ‘ T ' A1.4 - Illustrations and determination according to the technique — Tests on rats - Table no. 1: measured by the degree of binding of iodinelabelled LHRH anti-LHRH antibody response - Table no. 2: effect of anti-LHRH immunization on plasma testosterone concentration - Dosage Vaccines A1 : 50 pg of conjugated (D—Lys5)—LHRH Bl : 12 yg of conjugated LHRH W Tests on intact male pigs a Table no. 3: anti—LHRH antibody response measured by the degree of binding of iodine—l25=labelled LHRH ‘ Dosage Vaccines A1: 0g5 mg of conjugated (D=Lys6)=LHRH A2: 6 mg of conjugated (D~LysB)—LHRH B1: 0.15 mg of conjugated LHRH B2: 1.20 mg of conjugated LHRH _]7 1 Table l Anti—LHRH antibody response measured by the degree of binding of i0dine—125~labelled LHRH TESTING FOR ANTIBODIES (% Bo/T) IN THE SERUM (1/100) IN RATS INJEC— TIME GROUP A1 GROUP B1 TITER TIONS (WKS) 50 pg D1ys6—LHRH/HSA/AE1 12 pg LHRH/EDC/HSA/AE1 B/P{IM SC 0 - - _ SC A 0.0 15.4 <100 .8 7.6 <10O .0 35.3 .0 51.1 100 .3 91.1 1600 .1 85.3 980 .9 70.8 270 .8 94.9 210 .0 64.3 120 ; 11.3 67.9 220 ‘ 11.7 96.1 2éOO .0 68.3 280 / 14,5 79.5 £00 .0 92.4 210 .0 64.2 200 .5 67.2 240 .6 70.4 310 ' 0.0 98.8 3200 .2 68.7 2200 [ 0.0 38.8 310 _]3 _ Table 2 Effect of anti~LHRH immunization on plasma testosterone concentration ASSAY OF PLASMA TESTOSTERONE (NG/ML) IN RATS INJEG« TIME CONTROLS GROUP A1 GROUP B1 TIONS (wxs) 50 pg Dlys6-LHRH/PDP/HSA 12 pg LHRH/EDC/HSA SC 0 0.40 - - .26 - - .47 — - .00 - - sc 4 2.25 4.16 2.51 1.05 5.83 2.38 2.34 4.43 3.63 4 4.17 0.58 3.80 2.99 0.00 1.76 2.33 0.00 .05 2.85 0.00 6.67 1.54 0.00 6 2.01 3.26 0.00 .47 0.09 0.00 .69 1.10 0.00 .96 - 0.00 7 2.28 1.32 0.00 .92 0.89 0.00 .89 1.59 0.00 .65 2.17 0.00 .97 1.75 0.00 .91 1.48 0.00 2.71 1.91 0.00 1.22 2.33 0.00 ._]9 _ omq omm cum H.mm .m.Hm m.wo H.o¢ ¢.¢m m.qm m ¢.qN o o my oqo oqfl oma m.om m.¢m n.om @.wq H.qm m.mm m m.om o o my omm omm oma m.~m m.Nm m.om mnwi R.¢m H.m¢ m n.Hm o o ma omm oma oom a.om q.Hm N.m» N.om a.w¢ H.mN m N.Hw o o my o o o o o o o o o o .m:H saw me o o o o o o o o o o «H o o o o o o o o o o me .mcH uma ow mmm mmm mam mom H¢m mmw mam mom How mam mqw mam mam mwflm .oz mmfia .oz mwam .oz mmfim .02 mm Hm Ammv we N.H Hm<\ omv o m m.NN m.mm m o o o o o my omv o m m.wN H.¢¢ n.N o o o o c we omv o m m.®m m,m¢ m." o o o o o he mm o 0.0 H.Nq méwm m.qH Q Q o o o my om o o o q.mm o o o o o o .flaH mam my ooa o o o m.~@ o o o o o o qw N oma o o o w.Hw o o o o o o me .mafi "ma OH flaw Hmu nqm NNN Ham mow "mm m¢N Hmm mam Ham mwwm .oz mmfia .oz mwwm .oz H4 A~ Axomnexmv mmeHa mo OZHDZHQ Aom\H .QHQv zammm mma ZH mmHoomHaz4 ZMEQIHBZ4 mom ozHemma mama omqqmmmq1mmHsmzHooH MO mmmwmo WEB wm ammamamz mmzommmm WDOmHBZ¢ mmmq-Hez< m mqmma ,.2Q .

A2 — Comparative test of two anti-LHRH vaccines composed, respectively, of an LHRH-a—globulin conjugate with carbodiimide and a (D—-LYS5) -LHRH—a-globulin conjugate with SPDP, put into an oil—in—water emulsion and administered intramuscularly' (IM) or transcutaneously (ID) in pigs.

L A2.l - Preparation of (D—Lys5)—LHRH-a—globulin conjugate with SPDP.

The method described in Example Al is employed in exactly’ the same manner, but replacing albumin. at a mg/ml by concentration of 6 mg/ml. concentration of a—globulin at a The overall yield of coupled (D—Lys5)—LHRH is of the order of 45 to 50%.

Moreover, it is possible to modify at will the degree of substitution of the a—globulin, and hence the level of conjugationy by varying the SPDP and/or aeglobulin concentrations during the preparation of the MPaamglobulin.

A2.2 =Preparation of LHRHmhuman a=glObuliH conjugate with carbodiimide (EDC).

The method described in Example A1 is employed in exactly the same manner, but replacing human albumin by human amglobulin. The level of conjugation is from 24 to 28 mg of LHRH bound per 100 mg of human aaglobulin.

A2.3 = The efficacy of the vaccine based on the LHRH~a~globulin conjugate with carbodiimide is greater than the second one. The efficacy is expressed as the number of animals displaying a total disappearance of plasma testosterone (Table 4).

Table 4 LHRH—a—glob.EDC (1.2 mg) IM + ID IM + ID Abolition of testo— 5/10 2/10 sterone (D-Lyss)-LHRH-a~glob.SPDP (6 mg) _ mp- AJ3 — Predominance of the immune response of male pigs to the carboxy—terminal fraction of the LHRH peptide conjugated. with the carbodiimide, or of its agonist (D-Lys6)=-LHRH conjugated with SPDP, to human a—globulin.

This is determined by comparison of the per~ centages of binding of the anti~LHRH and anti—(D—Lys5)— LHRH sera by two labeled LHRH fragments, LHRH (3-10) deleted in respect of its amino—terminal fraction, and LHRH (1—l0) in free acid form and, as a result, deleted in respect of the amide fraction of its natural carboxy- terminal fraction, respectively. These two fractions recognize, respectively, more especially antibodies directed towards the carboxy—terminal fraction on the one hand, and amino~terminal fraction on the other hand" The predominance of the response to the carboxye terminal fraction of the peptide manifests itself in the number of animals displaying antibodies binding only the peptide LHRH (3010) to the exclusion of the binding of LHRH free acid (10/58 for the anti~LHRH serum and 3/10 for the anti»(D~Lys6)~~LHRH semmyo 0 No serum showed 100% binding of the LHRH free acid fraction, which would reflect an exclusive recognition of the aminowterminal fraction.

The most frequent mixed responses show a better recognition of the amino—terminal fraction by the anti-(D—Lys5)—LHRH sera than that of the anti~LHRH sera.

In the latter, only 3 sera out of 58 have a recognition of greater than 40% of the amino-terminal fraction, against 4 out of l0 for the anti—(D—Lys6)-LHRH serum.

B - Greater immunogenic activity of the LHRH—a—globnlin conjugate produced with the carbodiimide compared to that obtained with the conjugate prepared with glutaraldehyde.

B.1 m Preparation of the LHRH~a=globulin conjugate with glutaraldehyde. .5 ml of glutaraldehyde solution containing mg/ml are added dropwise and over a period of 30 min to 10 mg of LHRH and 50 mg of human a—globulin (Serva) ._22 _. dissolved in 5 ml of 0.1M phosphate buffer pH 7.5, stirring gently after each addition. After the mixture has been left for 2.5 h at room temperature, the reaction is stopped by adding 25 mg of sodium bisulfite dissolved in 0.5 ml of water. The conjugate is dialyzed at 4°C against 150 mM NaCl/10 mM phosphate buffer pH 7.5, and is concentrated by ultrafiltration.

B.2 — Comparative test on pigs of anti—LHRH vaccines formulated using identical amounts of conjugated LHRH. The efficacy is expressed as the number of animals displaying a total disappearance of plasma testosterone (Table 7).

Table 7 LHRH~a—glo. with LHRH—a—g1o. with glutaral— carbodiimide dehyde IM or ID adminiw IM or ID administration stration Abolition of plasma testo~ 5/10 0/10 sterone C Q Greater immunogenic activity of the conjugate employing human awglobulin compared to that obtained with the conjugate employing human serum ale bumin.

The efficacy is expressed as the number of animals displaying a total disappearance of plasma testosterone (Table 8).

Table 8 Tests on pigs — intramuscular injection LHRH—HSA with LHRH—a-glo. with carbodiimide carbodiimide Abolition of plasma 0/5 3/5 testosterone l\} UT _ Z3- D - Immunogenic activity of the conjugate employing equine a—globulin, fraction IV—l, equivalent to that obtained with the conjugate amglobulin.

D.l — Preparation of the LHRH—equine amglobulin conjugate with carbodiimide.

The method described in Example Al is employed in exactly the same manner, but replacing human albumin by equine a-globulin (fraction IV—1).

’ D.2 — Subcutaneous administration in rats, twice at an interval of 4 weeks, of a vaccine at a dose of 12 pg of LHRH conjugated to human or equine a-globulin.

Table 9 Tests on rats LHRH—human a-globulin fraction IV~l with carbodiimide LHRH«equine a—globulin fraction IV—1 with carbodiimide Abolition of plasma 12/12 12/12 testosterone E ~ Greater adjuvant activity of the wateroin~ oil emulsion of the invention over other emulsions {Table )" Tests on pigs employing the same conjugate composed of LHRH and human awglobulin with carbodiimide and administered at the same dose in the same volume transcutaneously at 5 points.

The emulsions examined are: a fluid oil—in—water emulsion (B), the emulsion of the invention (formula C in the table), a commercial emulsion to be diluted with the antigen (E) and an oily phase to be emulsified with the conjugate (F).

For all these formulae, the final amount of antigen per dose is the same. _ The emulsions are produced under the customary conditions used by those specializing in formulations of this type. employing human -24.. ai>_l_eilQ Emulsions B C E F Abolition of plasma 2/5 4/4 1/5 3/5 testosterone Number of animals displaying a tissue 2/5 4/4 3/5 3/5 androstenone con— ' centration below 0-5 pg/g F. Efficacy of passive anti—LHRH immunization for improving the organoleptic qualities of meat, measured by the lowering of tissue androstenone.

Table 11 Androstenone Content of the adipose tissue in Control animals and in those subjected to passive anti» LHRH immunoneutralization with a hyperimmune equine anti»(D~Lys5)~LHRH plasma administered in a ‘volume of ml on days 16; 13, 9 and 5 before slaughter.

,Controls Treated Number of animals displaying an androstenone concentration below 0.50 pg/g of adipose 2/5 5/5 tissue (significant difference at a~risk = 0.2) G. — Efficacy and tolerability of the formula» tions containing LHRH Conjugated to a-globulin with carbodiimide in the form of a water~in—oil emulsion (lst vaccine) and in aluminum hydroxide gel and saponin (2nd vaccine), administered transcutaneously at the same dose of conjugated LHRH, at the beginning of the fattening period and 18 to 21 days before slaughter, respectively, using a needleless injector known as Pigjet. —25— Two tests were performed in two stages, groups 1, 3 and 5 for the first and groups 2 and 4 for the second, respectively (Tables 12 and 13).

G.1 — The neutralization is of increased for an equal volume of of efficacy anti—LHRH immunow vaccine by multiplication of the points trans~ cutaneous administration.

Table 12 Groups 1 2 3 4 5 ml (5 points) ml (5 points) .4 ml (2 points) st vaccine 0.4 ml (10 points) ml (5 points) ml (5 points) ml (5 points) nd vaccine 0.4 ml (2 points) .4 ml (10 points) ,0.6 ml (2 points) Abolition of or marked decreasa in testosterone of animals) (no. /12 10/11 9/12 11/11 Tissue androstenone concentration below 645 pg/5 of animals) (no .

ND 10/23 ND ND ,. ._. \\ r- N ND: not determined G.2 Tolerance to the vaccines used is by the development of the judged inflammatory skin reactiony graded from O to 4 in an animal in accordance with the size of the papules appearing after administration; a papule appears at each administration point. The sum» mation of the scores in each of the groups is summarized as follows: mean score at the end of the first week following administration (Ad. 1) and mean score at the time of slaughter for each of the vaccines (S1) (Table 13). The best tolerance is observed with the use of the vaccines in group 4. _ g3_ Table 13 Tolerance on transcutaneous administration observed during the 2 tests performed (test 1 groups 1, and 5, test 2 groups 2 and 4).

Groups 1 2 3 /4 5 Vaccine 1 or 2 st. vac.Znd vac. 1st vac.2nd vac. 1st. vac.Znd vac. 1st. vac.2nd vac. lst. vac . Znd vac , animals 12 11 ll lNumbe): of 5 5 5 5 5 2 10 10 2 2 administra- tion points Ad. 1 1&2 11 31 11 41 16 33 11 30 10 S]. 2 4 0 0 5 3 U 0 2 0 Number of hi I _, USEL0Fi?HE_PEPElDBii3=1Ql AW Techniques of xneasurement, of the antimLHRH immune response and of the biological efficacy by the assay of plasma testosterone and tissue androsteroneu The anticLHRH immune response is measured by the antibody‘ titer which technique described by JEFFCOATE et al., , 1;, 625~635.

Bonding to the labelled peptides with is determinedo according‘ to the Actoa Endocrc (Copenha), is determined iodine~125 1979, l504~l506e The determination of the sera with respect to after labeling the various peptides according to COPPOLAND et al., Endocrinology, these peptides is performed according to the technique of JEFFCOATE et al. , The biological cited above. efficacy is measured by the lowering" or disappearance of plasma testosterone and tissue androstenone. The assay of plasma testosterone is performed directly on the plasma by an RIA technique employing the radioligand testosterone C19—carboxy methyl ether-[1"I]histamine (FURUYAMA S. et al., Steroids, 1972, lg, 415). The assay of tissue androstenone is performed adipose tissue by’ an RIA. technique on a sample of ,. _27__ employing the radiologand 5a-[3H]androstenone, desdribed by CLAUS, C.R. Acad. Sci., Paris, 1974. glg, 299»302.

B. Predominance of the immune response of male pigs to the carboxyuterminal fraction of the LHRH peptide conjugated. with the carbodiimide, or of its agonist (D—Lys6)-LHRH conjugated with SPDP, to human a—globulin.

B1. Preparation of the LHRH—human a—globulin conjugate with carbodiimide.

The conjugate is preferably obtained by adding from 0.5 toi2 volumes of a 2.5% solution of N—ethyl—N’—(3— dimethylaminopropyl)carbodiimide (EDC) hydrochloride in 0.9% NaCl to one volume of the a—g1obulin/LHRH mixture in solution containing 2 to 20 mg/ml in 0.9% NaCl. After stirring, the mixture is left overnight and then purified by gel permeation chromatography.

B2. Preparation of [(D—Lys6)=LHRH]=human amglobulin conjugates with SPDPO The preparation of [(DmLys5)=LHRH]»human a=globulin conjugates is carried out in 3 steps: preparau tion of {Na[3=(Zwpyridyldithio)propanoyl]=D=Lys5}=LHRH, preparation of N=(Bemercaptopropanoyl)=human a=globulin, then coupling" {N‘«[3m(2=Pyridyidithio)propanoyl]=D=Lys6}=LHRH is prepared by reacting an excess of SPDP with LHRH in aqueous solution (6 mol of SPDP per mole of (D—Lys6)~LHRH), and then, after one night at 4°C, centrifuging the product obtained. The latter is dissolved in 8M urea and the Zwpyridyldithio groups present are assayeda N»(3~Mercaptopropanoyl)-human a—globulin is obtained by the action of 0.2 mmol of SPDP on 0.6 g of human a~globulin dissolved in 100 ml of 0.1M phosphate buffer, and then, after one night of contact at 4°C and acidification to pH 6, by reduction with dithiothreitolo It is then purified by gel filtration chromatography.

Assay of thiol and protein yields the average level of substitution.

Coupling is performed taking one 2-pyridyldithio group per 1.25 thiol groups. The pH is taken to 7-7.5 and then, after one hour, the yield is determined by ~ fl3— measurement of the 2—pyridinethione liberated.

The average level of substitution is deduced from this. Finally, the conjugate is purified by chroma«_ tography and concentrated by ultrafiltration. The overall yield of coupled (D~Lys6)—LHRH is of the order of 45 to 50%.

B3. The predominance of the immune response of male pigs to the carboxy—terminal fraction of the LHRH peptide conjugated under the conditions described in A1 and A2 is determined by comparison of the binding-by the anti-LHRH and anti—(D-Lys5)—LHRH sera of two labeled LHRH fragments, LHRH (3-10) (LHRH deleted in respect of its amino—terminal fraction), and LHRH (1-10) in free acid form (LHRH deleted in respect of the amide fraction of its carboxy—terminal fraction), respectively. These two fractions recognize more especially antibodies directed, respectively, towards the carboxy=terminal fraction on the one hand, and amino=terminal fraction on the other hand, The response to the carboxy=terminal fraction of the peptide is general in all the animals immunized with one or other of the conjugates (68/68)" The sera of 3 out of 10 of the 10 animals immunized with conjugated (DwLys5)~LHRH and of l0 out of 68 of the animals immunized with conjugated LHRH showed exclusively a binding of the carboxy—terminal fraction. The other animals display a mixed response preferentially directed towards the carboxywterminal fraction.

The response to the aminouterminal fraction is not general (55/68). No serum showed binding exclusive to the LHRH free acid fraction.

C. Tests of active anti—LHRH immunoneutralization using LHRH (3—l0)—equine a—globulin IV»4 and LHRH (3«lO)o ovalbumin conjugates produced with carbodiimide.

Cl. Preparation of the LHRH a—globulin IV-4 conjugate with carbodiimide.

Eighty-five mg of LHRH (3-10) and 170 mg of equine a-globulin IV-4 are dissolved in 12.8 ml of 0.1M (3-l0)—equine NaCl/0.1M 2-(N-morpholino)ethanesulfonic acid buffers _29_ mg of N—ethyl—N’-(3~dimethylaminopropyl)Carbodiimide hydrochloride, dissolved in 17 ml of the above solution, are then added. The pH is immediately adjusted to 6.0 by adding 1.3 ml of 1N sodium hydroxide.

After stirring, the mixture is left for 16 h at room temperature, and the conjugate is then purified by gel permeation chromatography to separate the conjugate from unconjugated LHRH. Measurement of the amount of the latter enables the amount of LHRH coupled to be obtained by difference. It is possible to determine the coupling yield.

C2. Preparation of the LHRH (3-10)-ovalbumin conjugate with carbodiimide.

Sixty mg of LHRH (3-10) and 120 mg of ovalbumin are dissolved in 9 ml of 0.1M NaCl/0.1M 2=(N—morpholino)m ethanesulfonic acid. buffer. 150 mg" of N=ethyl=N’=(3e dimethylaminopropyl)carbodiimidehydrochloride,dissolved in l2 ml of the same buffer, are then addedg The pH is adjusted to 7.0 by adding 1N sodium hydroxide (approxi= mately l¢9 ml). The mixture is left overnight at room temperature and is then clarified by centrifugatlon. The supernatant is chromatographed on Sephadex gel to separate the conjugate from unreacted LHRH and products originating from the initial carbodiimide. By measurement of the amount of unbound LHRH (3=l0), it is possible to determine the coupling yield of LHRH (3=l0)a C3. Immune response, biological efficacy and tolerance to the anti~LHRH vaccine formulated from the conjugate obtained between the fragments LHRH (3»l0) and equine awglobulin IV—4 with Carbodiimide.

The formulations, consisting of conjugated LHRH (3-10) put into the form of a water—in—oil emulsion (lst vaccine) and into an aluminum hydroxide gel and saponin (2nd vaccine), were administered transcutaneously to'6 male pigs in a volume of 0.4 ml per dose, at the begin» ning of the fattening period and 17 ‘days before slaughter, respectively, using a needleless injector known as Pigjet delivering the dose volume in two applications of 0.2 ml distributed at 5 points at each ._ 3), application.

The immune response was maximal 10 days after administration of the 2nd vaccine. The individual antic body titers (reciprocal of the dilution at which iodinem 125 is 50% bound) were, respectively: Day 10 280 660 2,700 3,200 4,600 13,000 Day 16 290 400 2,000 2,400 3,100 8,600 The biological efficacy of this immune response itself in the testosterone from day 10 after administration of the 2nd manifests disappearance of plasma vaccine in all 6 animals. The disappearance of testosterone is accompanied, under the same conditions, by the disappearance of tissue androstenone.

Tolerance to the vaccine is judged by the development of the inflammatory skin reaction, graded in accordance with the size of the papules appearing at each point of delivery of the vaccine after administration‘ This local inflammation has completely disappeared from day 10 after administration of the 2nd vaccine,

Claims (7)

1.CLAIMS 1. AJndhmdfor producing pig,catth2or sheep meat having improved organoleptic qualities, particularly improved smell, juiciness and tenderness, wherein uncastrated male domestic animals are fattened and the animals are given active immunoneutralisation using an anti—LHRH vaccine, characterised in that the advantages connected with the male nature of the animals are maintained virtually until they are slaughtered by the fact that, before or during the period of fattening the animals, they are given a single anti—LHRH vaccine designed to induce a first low-intensity immune response, with no appreciable or even measurable effect on the secretion of sex steroids and,§untlybefore slaughtering, they are given an anti—LHRH vaccine to induce antiaLHRH immunoneutralisation which leads to suppression or a significant reduction in the secretion of steroids.

2. Amnahodaccording to claim 1, characterised in that the vaccine administered in the first place and the vaccine administered shortly before slaughtering are selected from emulsified vaccines and vaccines in an aqueous adjuvant.

3. A1nahmjaccording to either of claims 1 and 2, characterised in that the anti~LHRH vaccine administered in the first place is an emulsified vaccine.

4. Alnmhmdaccording to any one of claims l to 3, characterised in that, for‘ pigs, the anti—LHRH vaccine is administered with an aqueous adjuvant, shortly before slaughtering. S. Ainahmiaccording to claim 4, characterised in that aluminium hydroxide gel and/or saponin is used as the aqueous adjuvant. 6. Ainahodaccording to claims 4 or 5, characterised in that the vaccine is administered in an aqueous adjuvant 15 to 21 days before slaughtering. 7. Ajnmhodaccording to any one of claims 1 to 3, characterised in that, for cattle and sheep, an anti—LHRH vaccine is administered with an emulsified adjuvant before slaughtering. 8. Aindhod according to claim 7, characterised in that the emulsified vaccine is administered one to two months before slaughtering. 9» Ainmhod according to claims 7 or 8, characterised in that the emulsified vaccine is administered four weeks to several months after the initial administration» lo. zknwflmd according to any one of claims 2, 3 and 7 to 9, characterised in that the emulsified vaccine is a vaccine in a water~in—oil emulsion. ll. Ainmhod according to claim 10, characterised in that the water~in oil emulsion is made from a mixture of highly purified mineral oils and non—ionic surfactants. 12. Alnmhmjaccording to any one of claims 1 to 11, characterised in that an anti—LHRH immunogenic conjugate is administered comprising: ~ total or modified LHRH, — a peptide fragment of modified or unmodified LHRH, or — an LHRH agonist, coupled with an immunogenic carrier protein selected __33_ from among: — bovine or human serum albumin, — thyroglobulin, ~ ovalbumin, ~ the anatoxins, notably tetanus anatoxin, — human or equine globulins. 13. A1nahod according to claim 12, characterised in that the conjugate Comprises LHRH coupled with equine alpha qlobulin, hipmfimflm fractions IV—1 and/or IV—4. 14. Ainmhod according to claim 12, characterised in that the conjugate comprises LHRH (3-10) coupled with equine alpha globulin,inpankumrfractions IV—1 and/or TV—4, or with ovalbumin, 15» Arnmhod according to claims 13 or 14, characterised in that the LHRH/LHRH (3~10) and the immunogenic carrier protein are coupled by a carbodiimide. 16. A1nmh0daccording to claim 1, characterised in that anti IJHU1 seruu1<)r hypawuumnune plasma is tuhninistered to the animal, some days before slaughtering, 17. AJndh0daCCOrdiug to claim 1, characterised in that anti~LHRH monoclonal antibodies are administered to the animal several days before slaughtering. 18. Axnmhmiaccording to claims 16 or 17, characterised in that the substance is administered five to fifteen days before slaughtering, by subcutaneous or intramuscular route. 19. Arnmhmiaccording to any one of claims 1 to 18, characterised in that the substance is administered by transcutaneous route, preferably at several points, USiTK3 3 119€dJf31eSE37injection apparatus, via a jet under pressure. 20. Atndhod according to any one of claims 1 to 19, characterised in that the anti—LHRH vaccine administered in the first place is given before the stage of fattening the animal. 21; Anti—LHRH Vaccination kit which can be used for the production of meat having improved organoleptic qualities from uncastrated male pigs, cattle or sheep, this vaccination kit comprising: ~ an anti~LHRH vaccine designed to be given in the first place before or during the stage of fattening the animals and designed to induce a first low intensity immune response with no appreciable or even measurable effect on the secretion of the sex steroids, to allow the male nature of the animals to develop, and — an anti—LHRH vaccine designed to be administered after the previous one and shonjy before slaughtering, this vaccine being designed to induce anti—LHRH immunoneutralisation causing the suppression or significant lowering of the secretion of steroidsi 24, Vaccination kit according to claim 21, characterised in that the vaccines contain an active principle which is a conjugate of an immunogenic carrier protein and an LHRH peptide. 2

5. Vaccination kit according to claim 22, characterised in that the LHRH peptide is selected from the group consisting of: ~ total or modified LHRH, ~ peptide fragment of LHRH, which may or may not be modified, — LHRH agonist. 24. Vaccination kit according to claim 22 or 23, characterised in that the immunogenic carrier protein is selected from the group comprising: ..35_ — serum albumin, — thyroglobulin, — ovalbumin, — anatoxins, ~ tetanus anatoxin, — equine globulins, — human globulins, — alpha globulins. 25. Vaccination kit according to one of claims 21 to 24, characterised in that the anti—LHRH vaccine to be administered by the first injection is a Vaccine in the form of an emulsion. 2

6. Vaccination kit according to one of claims 21 to .L. 25, characterised in that the anti—LHRH vaccine no be administered shortly betore slaughtering is a vaccine in the form of an emulsion. 27» Vaccination kit according to claim 25 or 26, characterised in that the Vaccine is the form of an emulsion is a vaccine in the form of a watcr—in oil emulsion. 28. Vaccination kit according to one of claims 25 to 27, characterised in Lhat the Vaccine in the form of an emulsion is made up of a mixture of mineral oils and non—ionic surfiactants. 29. Vaccination kit according to one of claims 21 to 25, characterised in that the anti—LHRH vaccine intended to be administered shortly before slaughtering is a vaccine with an aqueous—type adjuvant. 30. Vaccination kit according to claim 29, characterised in that the aqueous~type adjuvant is selected from the group consisting of aluminium 25 _ 36 _ hydroxide gel, saponin and mixtures thereof. 31. Vaccination kit according to one of claims 21 to 25 and 29, 30, anti—LHRH vaccine to be administered shortly before intended for pigs, characterised in that the slanghtering is a vaccine with an aqueous—type adjuvant_ 32. Vaccination kit according to one of claims 31 to 28 for use in cattle and sheep, characterised in that the anti~LHRH vaccine to be administered shortly before slaughtering is a vaccine in the form of an emulsion. 33. Vaccination kit according to any one of claims 21 to 32, characterised in that the vaccination kit consists of an identical number of doses of each of the vaccines in a single package. 34. AJnfih0d according to any one of claims 1 to 11, characterised in that a vaccine is administered containing an immunogenic active principle which comprises the LHRH peptide. 35. Vaccination kit according to claim 21 or any one of claims 25 to 33 together with c1aim.21, characterised in that the anti—LHRH Vaccines comprise an immunogenic active principle containing the peptide LHRHl 36. A method according to any one of claims 1 to 20 or claim 34 substantially as hereinbefore described with reference to the examples. 3

7. An anti—LHRH vaccination kit according to any one of claims 21 to 33 or claim 35 substantially as hereinbefore described with reference to the examples. Tomkins & Co.