NZ196126A - Increasing ovulation rate in female cattle - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number 1 96126 Priority Date(s): .... .TP-. ^ Complete Specification Filed". • I ^ .

Publication Date: P.O. Journal, No: ll rlil SI i II li' NEW ZEALAND PATENTS ACT, 1953 No.: Date: COMPLETE SPECIFICATION ,s.

'CONTROL OF OVULATION' XI /We, COMMONWEALTH SCIEIITIFIC AND INDUSTRIAL RESEARCH OR.GAtOC2-AT(ON^ Oil CAIv I CAT 101?, a body corporate established under the Science and l°»V^ Industry Research Act 1941, as amended, of Limestone Avenue, Campbell, Australian Capital Territory, 2601, Australia hereby declare the invention for whicb£5i / we pray that a patent may be granted tcS^filfe/us, anc* the method by which it is to be performed, to be particularly described in and by the following statement:- I 196126 CONTROL OF OVULATION 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, e 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 speciies. 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 our copending -New Zealand patent, specification _Jl£^AT^NTOFFICE' V 14 JUL J9S3 \ 9 61> £ No. 196,125 it was also shown that by immunization of sheep 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 immunizations with good antibody production nor with changes in ovulation rate when the same immunogens that promote ovulation rate increases in sheep and goats have been injected into the cattle.

The present invention consists in a method for increasing the ovulation rate in female cattle 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) derivatives of the 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 loosely bonded steroid; and (iii) an immunoadjuvant, so as to produce a mean steroid binding antibody tit re in the female cattle of from 1 in 100 to 1 in 5,000 at the time of ovulation.

In another aspect the present invention consists in an immunogenic composition for increasing the ovulation rate in female cattle comprising (i) a conjugate of an acidic steroid androgen or oestrogen and an immunogenic protein (ii) 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 loosely bonded steroids; and (iii) an Immunoadjuvant. o u> > LU o LP cc As used in this specification, the following terms have ijieanings set out hereunder: Steroid Androgen: Any steroid substance that stimulates "the expression of secondary sex characteristics of the male. Androgenic activity can be assessed by measuring the regrowth of the involuted comb of a castrated cock following androgen administration or by measuring the growth response of the seminal vesicles in castrated male rates following androgen 196126 administration.

Steroid Oestrogen; Any steroid substance oestradiol- 1713 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- 17/5 have been found to render female animals anoestrus due to the neutralisation 10 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% by weight 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 30 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 N.Z. PATF-" '-rice 14 JUL 1983 RECEIVED other than 196126 N.Z. PATF'' r OcFICE " 5 " j 14 JUL 1983 I 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 invention 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 steroid-protein conjugate together with steroid loosely (non-convalently) hound to protein and a small proportion of unreacted steroid derivative. Surprisingly, the presence of the 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 to 90%, and preferably at least 50% and preferably up to 80% by weight, of the steroid moiety in the form of loosely bonded steroids. The compositions of this invention possess immunoge-nicity that is substantially greater than those skilled in the art would expect from a knowledge of the convalently 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 "i 9 612 6 adjuvant are often poorly effective which may be because it is difficult to prepare an emulsion at the concentrations of imnmnogen required that is stable and remains stable for sufficiently long to produce the immunological responses after injection; In preparation of the immunogenic compositions, substances already known in the art may be used. Thus, preferred androgen immunogens are prepared by the conjugation to protein of 4-androstene-3,17-dione derivatives functionalised with an acid ■P.&S. group at positions 1, 1( 11 or 15 of the steroid ring, for example^ -3, . 1 - dm**- -7«- "***£$ For immunization against testosterone, derivatives functional ized with an acid group at positions 3 or 17 may be used, for example, testosterone 3-carboxymethyloxime. The antibody titre range for androstenedione 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 antibody 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 unimmunized animals.

Suitable immunizations with the oestrogen, oestrone, can also promote an increase in the ovulation rate of cattle if titres lie in the range of 1 in 100 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, produce oestrone-specific antibody. Non-limiting examples of such oestrone derivatives already known in the art include oestrone-3-hemisuccinate, oestrone-3-carboxymethylether, ; oestroner-6-car boxymethyloxime and 15 car boxymethyles trone .

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

The invention places 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, y-globulin .and gelatin. - The invention recognises that the range of antibody titres may vary with the hormone used in the immunisation and N.Z. PATENT OFFICE 14 JUL 1983 ii; 196126 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 10 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 20 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, frcm 72 co 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 30 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 N.Z. PATENT OFFICE [ ' 1 14 JUL 1983 96 S1 26 N.Z. PATENT OFPtegroi 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 4% 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 d derivative and the immunogen it was found that 120 14.1 Ml moles steroid equivalent per mole protein were present in the immunogenic composition; 35 moles steroid/mole protein were covalently linked, the remainder being steroid loosely bound to protein.

In a similar preparation 7^-2-carboxyethylthio-4-androstene-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 steroid loosely bound to the-proteinV- In a similar preparation testosterone-^-carboxy-methyloxime 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 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 ompositions were pasted in 1 ml Freunds adjuvant and made to a suspension in a total volume of 30 ml in Freunds dj uvant. (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 about 10 1 96 1 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 10 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 20 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. 196126 - ii - TABLE 1 Enhanced Immunogenicity of Compositions Containing loosely Bound - steroid 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- 33 # 33 Below 1:50 3-carboxymethyl oxime 10 2 + 33 8 1:865 Androstenedione- 33 # 33 6 Below 1:50 loi-7. '-car boxy ethyl thioether 90 + 32 6 1:300 O estrone-3- 120 + 6 1:6420 carboxymethylether ★ + Following 2 treatments - primary and secondary • These preparations contain a high proportion of loosely bound steroid Preparations of steroid-protein conjugate conventionally dialysed to completely remove excess loosely bound steroid V.Z. PATENT OFF ICE 14 JUL 1983 RECEIVED r. / f; V (J / - 12 -TABLE 2 Ovarian changes in cattle 11 weeks after start of treatment Steroid Number of Animals with Animals with multiple Derivative used Animals enlarged corpora lutea or in making ovaries several large immunogenic follicles on the ovaries composition Oestrone-3 carboxy 9 5 5 Methylether Androstenedione- 6 4 2 VMr^Carboxy- ethylthioether Controls (Untreated) 10 1 0 1 96 1 EXAMPLE 3 Using immunogenic compositions and methods as described in Example 1 to maintain titres but including repeated treatments a further series of tests with immunizations against testosterone, androstenedione or oestrone was carried out. Palpation per rectum indicated ovarian responses and these were then confirmed and detailed by endoscopic examination of the cattle (Table 3) in which the ovaries were visualized and size, numbers of follicles and corpora lutea were recorded.

I ' Jf Q - 14 -TABLE 3 Endoscopic Examination of Cattle Following Immunization Against Steroids and Showing Resultant Increases in Ovulation Rate Immuni zation Number of Mean Number of Animals having Against (Steroid Animals in Groups Steroid Antibody Titre 2 or 3 Ovulations Testosterone 7 270 3 Androstenedione 6 300 2 Oestrone 8 1500 1 Total of Immuni zed Animals 21 — 6 (29%) Control (Reference) 2 0 0 Only a small control group was run in this series as it is well established that the multiple ovulation rate in untreated cattle of the Hereford or Hereford-Friesian breeds is very low and about 1% i.e. the untreated cattle have an ovulation rate of 1.01 as compared with the ovulation rate of the total of immunized animals in this example of 1.29. 1S6126

Claims (33)

WHAT WE CLAIM IS:

1. A method for increasing the ovulation rate in female cattle comprising administering to the cattle an immunogenic composition comprising (1) a conjugate of an acidic derivative of a steroid androgen (as herein defined) or steroid oestrogen (as herein defined) P &S an ^mmuno9en'I'c protein; (ii )^Teri vati ve^ of said steroid androgen ^4-3 S3 or oestrogen loosely bonded to said protein, the composition con- taining from 10 to 90% by weight of the steroid moiety in the form of said loosely bonded steroid; and (iii) an immunoadjuvant, so as to produce a mean steroid binding antibody titre (as herein defined) 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 loosely bonded steroids.

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 loosely bonded steroids.

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 • ^ S. steroid androgen derivative is 4-androstene-3 ,17-dione-"jioc-2 - • / ( ii^F*carboxyethyl thioether.

6. A method as claimed in claim 4 in which the acidic steroid androgen derivative is testosterone 3-carboxymethyloxime.

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-carboxy-methyl ether.

9. A method as claimed in any of claims 1-8 |n V?ta:.^h'ENTQFF)CS' f the immunogenic protein is human serum albumin, 14 JUL 1983 196126 - 16 -

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

». J. P. & S. 12. A method as claimed in any of claims 1-11 in which the immu- . a. 5 3 % - noadjuvant isAdiethylaminoethyldextran.

13. A method as claimed in any of claims 1-11 in which the immunoadjuvant is Freund's complete or incomplete adjuvant. . _ 14. A method as claimed in any of claims 1-3 in which the immunoge-

.. J. P. & 5. nic conjugate is formed between 4-androstene 3,17-dione ToC-2 -carboxyethylthioether and human serum albumin and the immunoadjuvant 4- isftdiethy1 aminoethyldextran.

15. 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 the cattle are artifically inseminated from 72 to 96 hours after the administration 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 tolin 5,000 at the time of ovulation.

18. An immunogenic composition for increasing the ovulation rate in „ J. P. & S. female cattle comprising (i ) a conjugate of an acidic derivative of a * 1'?* steroid androgen or oestrogen with an immunogenic p^otoiun; (ii).cf< Auen- vative^ of said steroid androgen or oestrogen loosely bpjided..-to—sai-4 Tci'JT OFFICE 14 JUL 1983 - >7 - protein, the composition containing from 10 to 90% by weight of the steroid moiety in the form of said loosely bonded steroid; and (iii) an immunoadjuvant.

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

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 loosely bonded steroids.

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 the acidic steroid androgen derivative is 4-androstene-3,17-dione-^<-carboxyethylthioether.

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

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

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

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 ^-globulin.

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

29. A composition as claimed in any of claims 18-28 2- ln which the immunoadjuvant is,, diethylaminoethyldextran A 196126 18 -

30. A composition as claimed in any of claims 18 to 29 in which the immunoadjuvant is Freund's complete or incomplete adjuvant.

31. A composition as claimed in any of claims 18 to 30 in which the immunogenic conjugate.is formed between 4-androstene-3,17-dione 7ec-a' -carboxyethylthioether and human serum albumin and the immunoadjuvant <2~ isAdi ethyl ami noethyldextran.

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

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