IE50857B1

IE50857B1 - Control of ovulation

Info

Publication number: IE50857B1
Application number: IE158/81A
Authority: IE
Original assignee: Commw Scient Ind Res Org
Priority date: 1980-02-07
Filing date: 1981-01-28
Publication date: 1986-08-06
Also published as: IE810158L; DE3104190A1; BR8100722A; GB2068973A; GB2068973B; FR2475395B1; NZ196126A; AR230572A1; FR2475395A1

Abstract

A method and composition for increasing ovulation in female cattle wherein the cattle are administered with the composition so as to produce a mean steroid binding antibody titre in the female cattle of from 1 in 100 to 1 in 5000 at the time of ovulation. The composition comprises (i) a conjugate of a steroid androgen, such as androstenedione or testosterone, or of a steroid oestrogen, such as oestrone, and an immunogenic protein, such as human serum albumin, and (ii) acid anhydrides of the steroid derivative, the composition containing from 10 to 90% of the steroid moiety in the form of the anhydrides.

Description

This invention relates to a process whereby the natural genetically determined ovulation rate of cattle may be manipulated and increased. In particular, it is concerned with immunization of cattle against androgenic and oestrogenic steroids in such a way that ovulation may be increased to a degreee that significantly exceeds natural values. It is further concerned with the preparation and use of immunogenic compositions that can bring about the biological effects of the invention.

Because of their considerable economic importance, studies of the reproductive biology of domestic livestock have long been directed toward processes by which the individual fecundity of farm animals could be manipulated and in particular towards increasing the ovulation rate in females. The ovulation rate necessarily controls the maximum number of offspring that can be produced in a given pregnancy. Cattle have a very low natural ovulation rate, that is number of ovulations per animal occurring in the one oestrous cycle; for most breeds the ovulation rate lies between 1.00 and 1.01. Only in uncommon breeds are values such as 1.07 seen. Thus, cattle are very different in this aspect from many other domestic livestock species in which breeds often have ovulation rates of 2 and more. Clearly, for the natural fecundity of livestock to be increased, there must be at fertilization an increased ovulation rate.

It is already known in the prior art that, by artificially increasing the ovulation rate, in a domestic livestock species, by the administration of pregnant mare serum gonadotrophin (PMSG) for example, it is possible to increase the ovulation rate and ultimately the overall fecundity of the species. This process, however, suffers from the problem that the number of eggs shed cannot be controlled and the number of eggs shed by a cow treated with PMSG may vary from none up to 20.

In copending Australian Patent Application No. EE 2274/80 It was also shewn that hy immunization of sheep and and goats to produce antibodies directed against certain endogenous hormones, the ovulation rate of those animals could be increased.

The hormones were steroids of the oestrogen and androgen classes. However, surprisingly, cattle have not responded to such irmunizatiens with good antibody production nor with changes in ovulation rate when the same iawunogens that promote ovulation rate increases in sheep and goats have been injected into the cattle.

Ihe present invention consists in a method for increasing the ovulation rate in female cattle oatprlsing administering to the cattle an immunogenic ocepositicn carprising (i) a conjugate of an acidic derivative of a steroid androgen or oestrogen with an immunogenic protein; (ii) dialysable derivatives of the said androgen or oestrogen loosely bonded to said protein, the ccnposition containing fran 10 to 90% try weight of the steroid moiety in the form of said dialysable loosely banded steroid; and (iii) an inmunoadjuvant, so as to produce a mean steroid binding antibody titre in the female cattle of fran 1 to 100 to 1 in 5,000 at the time of ovulaticn.

In another aspect the present invention consists in an immunogenic ccrcpositicn for increasing the ovulation rate in female cattle oatprlsing (i) a conjugate of an acidic steroid androgen or oestrogen and an irnnunogenic protein; (ii) dialysable derivatives of said steroid androgen or oestrogen loosely bended to said protein, the oaipositicn captaining fran 10 to 90% by wei^it of the steroid moiety in the form of said dialysable loosely bended steroid; and (iii) an inmunoadjuvant.

As used in this Specification, the following terms have meanings set out hereunder: Steroid Androgen: Ary steroid substance that stimulates the expression of secondary sex characteristics of the male.

Androgenic activity can be assessed by measuring the regrewth of the involuted ccnfa of a castrated cock following androgen administration or hy measuring the growth response of the seminal vesicles in castrated male rats following androgen administration.

Steroid Oestrogen: Any steroid substance other than oestradiol- 17 [5 that stimulates the expression of secondary sex characteristics in the female. Oestrogenic activity can be assessed by measurement of uterine growth or cornification of the vaginal epithelium following oestrogen administration to spayed female rats or mice.

Immunisations against oestradiol- 17β have been found to render female animals anoestrus due to the neutralisation of its powerful hormonal effects and it is therefore not suitable for use in the present invention.

Antibody titre: Defined here as the dilution of the antiserum which binds 50% of the maximum amount of labelled steroid bound by the antiserum or by the standard reference quality control antiserum during incubation of about 50 picograms of steroid for about 18 hours at 4°C followed by the use of either dextran-coated charcoal or polyethyleneglycol to separate free from antibody-bound steroid .

The immunizations of the invention are carried out with substances and procedures which are individually known in the art but which in combination are novel. Thus, to render the androgens or oestrogens immunogenic, suitable acidic derivatives are synthesized and these are linked by chemical means, covalently, to an immunogenic protein such as human serum albumin, ovalbumin, gelatin or -globulins typically. To obtain effects on ovarian function and ovulation in cattle, it has been discovered, surprisingly, that the immunogens need to be prepared with particularly high levels of steroid incorporated into or associated with the carrier proteins. This is achieved by chemically reacting a large molar excess of steroid with the protein carrier. However, a high steroid incorporation alone is not a sufficient property of the immunogen to provide the effects of the invention. Commonly, the excess steroid used - 5 to facilitate a high molar steroid incorporation is deliberately removed from the conjugate by dialysis against aqueous, or aqueous organic, solvents or by column chromatography procedures. We have found, surprisingly, that the immunogenic compositions that achieve the effects of the invention are obtained if such dialysis is not pursued to completion to remove all the dialysable substances. It is part of its novelty that isolation of the immunogenic composition is achieved simply by incomplete dialysis of the reaction mixture against water alone for a period of 8-24 hours, optimally, followed by lyophilization.

Chemical analysis of the immunogenic composition so formed reveals that it is comprised of covalent steroidprotein conjugate together with dialysable steroid loosely (non-covalently) bound to protein. Surprisingly, the presence of the dialysable loosely bonded steroid particularly enhances the immunogenic response to the steroid protein complex (Table 1). We have found that to produce an enhanced response, the immunogen composition should contain 10-90%, preferably at least 50% and preferably up to 80% by weight, of the steroid moiety in the form of dialysable loosely bonded steroid. The compositions of this invention possess immunogenicity that is substantially greater than those skilled in the art would expect from a knowledge of the covalently linked hapten content.

The chemical procedure used to link the androgen or oestrogen to immunogenic protein can be any of the procedures known in the art which will at the same time form the necessary amount of steroid acid anhydride. The immunogenic compositions so formed are administered to animals and the immune response to them potentiated with adjuvants such as DEAE-Dextran, the immunogen being in solution or suspension, or with the immunogen suspended rather than emulsified, in Freunds complete or incomplete adjuvant. Surprisingly, it has been found that emulsions of the immunogen in Freunds 5085? adjuvant are often poorly effective ihich may be because it is difficult to prepare an emulsion at the concentrations of immunogen required that is stable and remains stable for sufficiently long to produoe the imnunological responses after injection, 4-androstene5 3,17-dicme-7a-caibc»yethylthioether.

In preparation of the imnunogenic coirpcsitions, substances already known in the art may be used. Thus, preferred androgen immunogens are prepared by the conjugation to protein of 4-androsteme3,17-dione derivatives functionalised with an acid group at positions 1, 7, 11 or 15 of the steroid ring, for exanple.

For innunization against testosterone, derivatives functionalized with an acid group at positions 3 or 17 may be used far exanple, testosterone 3-carbraymathyloxima. The antibody titre range for androstenedians or testosterone that should be attained to achieve an increase in ovulation rate in cattle should preferably lie in the range 1:100 to 1:5000 as a mean for the herd. With antibocy titres for these hormones which are significantly lower than this range, the incidence of multiple ovulations will tend to diminish and will approach that characteristic of unimnunized animals.

Suitable imnunizations with the oestrogen, oestrone, can also promote an increase in the ovulation rate of cattle if titres lie in the range of 1 in IDO to 1 in 5,000 as a mean for the herd.

The preferred steroid derivatives are those which, after conjugation with protein and administration to an animal, produoe oestrone25 specific antibody. Non-limiting exanples of such oestrone derivatives already known in the art include csstrons-3-hemisuccdnate, oestrone3-carbojymsthylether, oestrons-6-cszboxyinethyloxine and 15 carbosymethylestrone.

In general, the conjugate preferably contains 25 to 35 moles of covalently bonded androgen or oestrogen per mole of imnunogenic protein.

The invention plaoes no limitation on the protein used to form the immunizing antigen of the invention but human serum albumin has been found effective. Other useful proteins include oralbumin, γ-glcbulin and gelatin.

The invention recognises that the range of antibody titres may vary with the hormone used in the imuunization and - 7 with the breed of cattle being immunized. The selection of the most preferred antibody titre within the above range is therefore a matter of experimentation in the particular circumstances of any particular application of this method.

The immunogenic composition is preferably administered by injections spaced apart by a period of from 1 to 5 weeks. In the case of an animal which has been previously immunised only a single injection may be required. It is considered desirable to allow one full oestrus cycle to pass between the immunisation, or the second immunisation if that occurs, and mating of the animal. The immunogenic material should therefore be such that the desired antibody titre is obtained upon ovulation in an oestrus cycle separated from the time of immunisation by at least the length of one complete oestrus cycle.

In the examples given the biological effects achieved are a consequence of the development of antisteroid antibodies because immunizations of control animals against the protein carrier alone had no significant effect on the ovulation rate. In applying these effects to the breeding of cattle it will be recognised by those skilled in the art that they may conveniently be combined with synchronisation of the cycle, as with prostaglandins or progestagens, followed by artificial insemination from 72 to 96 hours later, in which case there is no requirement for the cattle to show oestrus and recording of oestrus is not essential.

Further, they may conveniently be combined with both synchronization of oestrus and the use of luteinizing hormone releasing hormone, typically 66-90 hr after the last synchronization treatment, followed by artificial insemination 6-12 hr later again, in order to improve fertility through timing of ovulation as well as ensuring optimum ovulation rate.

The scope of this invention is not limited to processes employing active immunization protocols, for passive immunizations using antisteroid antibodies raised in donor animals may be used to achieve the increases in ovulation rate encompassed by the invention.

The following example is given to illustrate preferred methods within the broad scope of this invention.

EXAMPLE 1 (a) Preparation of steroid-protein immunogenic compositions To an amount of 360 mg oestrone-3-carboxymethyl ether dissolved in 36 ml dioxane was added, dropwise and with stirring, a freshly prepared solution of 162 mg l-ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride (ECDI) dissolved in 10.8 ml distilled water. The reaction was allowed to proceed for 30 minutes at 25°C and then a solution of 360 mg human serum albumin (HSA) dissolved in 48 ml phosphate buffer pH 7.80, 0.05M was added dropwise with stirring. After 18 hr a further solution of 180 mg oestrone-3-carboxymethylether in 12 ml dioxane was mixed with 54 mg ECDI in 3.6 ml water, allowed to stand 30 minutes at 25° and added to the main reaction mixture. The reaction was allowed to continue for 4¾ hr and then a further 108 mg ECDI was added in solid form directly and with stirring to the mixture.

After a further 3 hr at 25°C the reaction mixture was transferred to dialysis tubing and dialysed against distilled water. The water was changed after about 1, 1%, 2 and 12 hr. The product (oestrone immunogenic composition) retained in the dialysis sack was then lyophilized and weighed. Yield 400 mg. The steroid content of the steroid-protein immunogen was calculated by incorporating a trace amount of ^H-labelled oestrone-3-carboxymethylether in the reaction.

By liquid scintillation counting of weighed amounts of the steroid derivative and the immunogen it was found that 120 moles steroid equivalent per mole protein were present in the immunogenic composition; 35 moles steroid/moles protein were covalently linked, the remainder being dialysable steroid loosely bound to protein.

In a similar preparation 7a-carboxyethylthio-4androstene-3,17-dione was linked to human serum albumin and yielded 380 mg of product (androstenedione immunogen composition) having 90 moles steroid equivalent per mole protein present of which 32 moles were covalently linked and the remainder being dialysable steroid loosely bound to the protein.

In a similar preparation testosterone-3-carboxymethyloxime was linked to human serum albumin and yielded 385 mg of product (testosterone immunogenic composition) having 102 moles steroid equivalent per mole of protein present, of which 33 moles were covalently linked, the remainder being dialysable steroid loosely bound to the protein. 100-250 mg of oestrone, androstenedione or testosterone immunogens were pasted in a 1 ml 0.9% by weight sterile saline and made into a total volume of 15 ml 0.9% by weight sterile saline. Then 15 ml DEAE-dextran solution was added. The DEAE dextran solution was prepared by dissolving 15 g in 100 ml water and adjusting the pH to 7.5-7.7 using saturated tri(hydroxy-methyl)-methylamine buffer (500 g/litre water).

The final volume of the DEAE dextran solution was then adjusted to 150 ml.

Alternatively, 100-250 mg of steroid immunogenic compositions were pasted in 1 ml Freunds adjuvant and made into a suspension in a total volume of 30 ml in Freunds adjuvant, (b) Immunization of cattle to test steroid immunogenic composition Cattle (of Hereford and Hereford-Friesan breeds) were injected each with 3 ml of the vaccine, containing 10 or 25 mg immunogen, each animal being given injections at S0857 - 10 about 1Ό sites subcutaneously over the neck region. An intramuscular injection of 1.5 ml pertussis vaccine was also administered. The injection treatment was repeated 4 weeks later.

Blood samples were taken by tail vein puncture one week after the second treatment. Blood was collected into heparinized tubes, stored on ice and centrifuged at 4°C.

The plasma so obtained was stored at -10°C until analysis of steroid antibody titre. The steroid-antibody titres obtained from the immunogenic compositions in (a) are shown in Table 1, together with the low values obtained with conventionally prepared steroid-protein conjugates.

EXAMPLE 2 Immunization of cattle against steroids and effect on ovarian size and activity Using immunogenic compositions and methods as described in Example 1 cattle were treated and examined. Ovarian changes were assessed by palpation per rectum in the period 8-17 weeks after commencing treatment and the number and size of follicles or corpora lutea on left and right ovaries were noted. Occurrence of oestrus in the cattle was determined using K-Mar heat mount detectors; cattle generally continued to show oestrus following treatment.

The results of immunization are given in Table 2, showing a marked increase in ovarian activity, follicle number and numbers of corpora lutea with immunization against oestrone and androstenedione compared to control animals.

S0857 - 11 TABLE 1 Enhanced Immunogenlclty of Compositions Containing Dialysable Loosely Bound Steroid in Addition to Steroid Protein Conjugates Steroid Derivative used in making Immunogen Composition Total Steroid Moiety Present Covalently Linked Hapten Content Number of of Animals Treated Mean Steroid Antibody Titre Produced* for Group Moles/Mole Protein Testosterone- 3-carboxymethyl oxime 33 102 # + 33 33 5 8 Below 1:50 1:865 Androstenedione- 33 # 33 6 Below 1:50 7

Claims

1. CLAIMS:1. A non-therapeutic method for increasing the ovulation rate in female cattle from the natural level to a higher level comprising administering to the cattle an immunogenic composition comprising (i) a conjugate of an acidic derivative of a steroid androgen or oestrogen with an immunogenic protein? (ii) dialysable derivatives of said steroid androgen or oestrogen loosely bonded to said protein, the caipositicn containing from 10 to 90% by weight of the steroid moiety in the form dialysable loosely bonded steroid? and (iii) an immunoadjuvant, so as to produce a mean steroid binding antibody titre in the female cattle of from 1 in 100 to 1 in 5,000 at the time of ovulation.

2. A method as claimed in claim 1 in which the immunogenic composition contains at least 50% by weight of the steroid moiety in the form of said dialysable loosely bonded steroid.

3. A method as claimed in claim 1 or claim 2 in which the immunogenic composition contains up to 80% of the steroid moiety in the form of said dialysable loosely bonded steroid.

4. A method as claimed in any of claims 1-3 in which the steroid androgen is 4-androstene-3,17-dione or testosterone.

5. A method as claimed in claim 4 in which the acidic steroid androgen derivative is 4-androstene-3,17-dione7a-carboxyethylthioether.

6. A method as claimed in claim 4 in which the acidic steroid androgen derivative is testosterone 3-carboxy— methyloxime. - 16

7. A method as claimed in any of claims 1-3 in which the steroid oestrogen is oestrone.

8. A method as claimed in claim 7 in which the acidic steroid oestrogen derivative is oestrone 3-carboxy5 methyl ether.

9. A method as claimed in any of claims 1-8 in which the immunogenic protein is human serum albumin.

10. A method as claimed in any of claims 1-8 in which the immunogenic protein is ovalbumin, gelatin or 10 γ-globulin.

11. A method as claimed in any of claims 1-10 in which the conjugate contains 25 to 35 moles of covalently bonded steroid androgen or oestrogen per mole of immunogenic protein. 15

12. A method as claimed in any of claims 1-11 in which the immunoadjuvant is diethylaminoethyldextran.

13. A method as claimed in any of claims 1-11 in which the immunoadjuvant is Freund’s complete or incomplete adj uvant. 20

14. A method as claimed in any of claims 1-3 in which the immunogenic conjugate is formed between 4-androstene3,17-dione 7a-carboxyethylthioether and human serum albumin and the immunoadjuvant is diethylaminoethyldextran.

15. A method as claimed in any of claims 1-14 in which 25 oestrus in the cattle is synchronised with prostaglandins or progestagens and in which the cattle are artifically inseminated from 72 to 96 hours after the administration - 17 of such prostaglandins or progestagens.

16. A method as claimed in any of claims 1-14 in which oestrus in the cattle is synchronised with prostaglandins or progestagens and in which luteinising hormone releasing hormone is administered to the cattle from 66 to 90 hours thereafter and the cattle are artificially inseminated from 6 to 12 hours after the administration of the luteinising hormone releasing hormone.

17. A method for increasing the ovulation rate in female cattle comprising passive administration to the cattle antibody raised in donor animals by a method as claimed in any of claims 1-16 so as to provide a mean steroid antibody titre in the female cattle of from 1 in 100 to to 1 in 5,000 at the time of ovulation

18. An immunogenic composition for increasing the ovulation rate in female cattle comprising (i) a conjugate of an acidic derivative of a steroid androgen or oestrogen with an immunogenic protein; (ii) dialysable derivatives of said steroid androgen or oestrogen loosely bonded to said protein, the composition containing from 10 to 90% by weight of the steroid moiety in the form of said dialysable loosely bonded steroid; and (iii) an immunoadj uvant.

19. A composition as claimed in claim 18 in which the Immunogenic composition contains at least 50% by weight of the steroid moiety in the form of said dialysable loosely bonded steroid.

20. A composition as claimed in claim 18 or claim 19 in which the immunogenic composition contains up to 80% by weight of the steroid moiety in the form of said dialysable loosely bonded steroid. - 18

21. A composition as claimed in any of claims 18-20 in which steroid androgen is 4-androstene-3,17-dione or testosterone.

22. A composition as claimed in claim 21 in which 5 the acidic steroid androgen derivative is 4-androstene3,17-dione-7a-carboxyethylthioether.

23. A composition as claimed in claim 21 in which the acidic steroid androgen derivative is testosterone 3-carboxymethyloxime. 10

24. A composition as claimed in any of claims 18-20 in which the steroid oestrogen is oestrone.

25. A composition as claimed in claim 24 in which the acidic steroid oestrogen derivative is oestrone 3-carboxymethylether. 15

26. A composition as claimed in any of claims 18-25 in which the immunogenic protein is human serum albumin.

27. A composition as claimed in any of claims 18-25 in which the immunogenic protein is ovalbumin, gelatin or γ-globulin. 20

28. A composition as claimed in any of claims 18-27 in which the conjugate contains 25 to 35 moles of steroid androgen or oestrogen per mole of immunogenic protein.

29. A composition as claimed in any of claims 18-28 in which the immunoadjuvant is diethylaminoethyldextran. 25

30. A composition as claimed in any of claims 18-28 in which the immunoadjuvant is Freund's complete or incomplete adjuvant. - 19

31. A conposition as claimed in any of claims 18-20 in which the immunogenic conjugate is formed between 4-androstene-3,17-dione-7o-carboxyethylthioether and human serum albumin and the immunoadjuvant is diethyl5 aminoethyldextran.

32. A method as claimed in claim 1 substantially as herein described with reference to any of Exanples 1-3.

33. A composition as claimed in claim 18 substantially as herein described with reference to any of Examples 10 1-3.