GB2115281A - The improvement of animal fecundity by treatment with antisera to gonadal steroids - Google Patents

Abstract

A composition for administration to breeding animals which comprises a mixture of an antiserum to an androgen steroid and an antiserum to an oestrogen steroid. There is also provided a method for the treatment of breeding animals to increase fecundity which includes administering to the animals prior to mating a composition comprising a mixture of an antiserum to an androgen steroid and an antiserum to an oestrogen steroid.

Description

SPECIFICATION The improvement of animal fecundity by treatment with antisera to gonadal steroids Prolificacy is a key component of the productivity of farm and other animals. It may be improved genetically in sheep by crossing or selection and environmentally by the provision of extra nutrients but to date no technique of improvement based on pharmacological intervention at or around the time of mating has found widespread acceptance in practice for this or indeed any domestic species.

Most of the variation in fecundity is the result of variation in the number of eggs shed and natural genetic variation in ovulation rate has been reported to arise from variation in the sensitivity of gonadotrophin release to the feedback effects of gonadal steroids. The artificial modifications of such feedback may be an effective way to raise fecundity.

In a series of sheep studies, active immunisation against steroids has been found to change the feedback balance in favour of additional ovarian activity; separate immunisation against androstenedione, oestradiol and oestrone, and testosterone all caused ovarian hyperplasia. The responses reported however have tended to be variable, excessive and in particular to be associated with reduced fertility.

An alternative approach would be to use a pharmacological antisteroid such as clomiphene which may be used in this way in women. A preliminary study confirmed the feasability of this but showed that clomiphene was only mildly anti-oestrogenic in the sheep in the circumstances studied.

The present invention utilises a different approach, namely the passive immunisation of the animals at the time of mating by the injection of sera from other immune animals. Such a treatment combines the advantages of known anti-steroidal activity of the antisera with dose control and short term effects of the antisteroidal compound; furthermore the sera can be mixed to give a combination of low activity against each of a range of steroids so that the best control of ovulation rate could be achieved without changes in the equilibria of any one steroid to such an extent that the essential roles were compromised.

The present invention therefore provides a composition for administration to female breeding animalps which comprises a mixture of an antiserum to an androgen steroid and an antiserum to an oestrogen steroid.

The present invention also provides a method for the treatment of female breeding animals to increase fecundity which includes administering to the animals prior to mating a composition comprising a mixture of an antiserum to an androgen steroid and an antiserum to an oestrogen steroid.

The invention may be applied to sheep, to cattle, to goats, or to horses and should have an even wider application.

The antiserum to an androgen steroid may be anti-testosterone, anti-androstenedione or anti - 5 dihidrostestosterone an antiserum to a synthetic androgen or mixtures thereof, and that antiserum to an oestrogen steroid may be anti-oestrone, anti-oestradiol or, an antiserum to a synthetic oestrogen, or mixtures thereof.

The antisera may be produced by immunisation of a small number of animals with the appropriate steroid derivative, which may be a derivative of the natural steroid or a synthetic steroid or a precursor thereof which will produce in the immunised sheep an antibody response yieiding an antiserum which will cross react with the natural steroids in the breeding ewes on passive immunisation with that antiserum. The antisera is collected by bleeding the immunised animal after a certain period of time and extracting it from the blood in known manner. The concentration of the antiserum and its cross-reactivits with the steroids may then be determined and quantities suitable for use in passive immunisation prepared.It would be possible to raise antisera to more than one steroid by immunising the donor animal with the appropriate mixture of steroids, but this would give rise to variations in proportions of antisera in the mixture.

As an alternative to the production of the antisera by conventional immunisation, the antisera may be prepared by monoclonal hybridoma technology.

A preferred composition is one containing a mixture of antisera to androstenedione (A), oestrone (eel), oestradiol (E2) and testosterone (T). The most suitable proportions of each antiserum in the mixture may vary with breed and would be determined by trials but40%T, 20%A, 20%E1, and 20%E2 is suggested as suitable where the percentage is the percentage of optimum dose of that steroid alone.

Particular examples of antiserum production and its use in sheep breeding will now be described.

As starting materials, 4-Androsten -3, 17-Dione (androstenedione), 1,3,5 (10) - oestratrien - 3 - ol - 17 - one (oestrone), 1,3,5 (10) - oestratrien - 3, 17(1-diol (17ss-oestradiol) and 4 - androsten - 17ss - ol - 3 - one (testosterone) were purchased from Steraloids Ltd.

(20a Chichester Road, Croydon, England). To prepare the immunogens, the following derivatives were synthesised: Androstenedione: Androsten - 4 - en - 3, 17 - dione - 7 a carboxyethyl thioether, by the method of Weinstein, Lindner, Friedlander and Bauminger (1972) (Steroids, 20,789-812).

Oestrone: Oestrone-6 carboxymethyloxime was prepared by oxidation of oestradiol - 6 - carboxymethyl oxime (Kohen, Bauminger and Lindner, 1975.

Steroid Immunoassay, p11-32. Cardiff: Alpha -Omega Publishing Ltd).

Oestradiol: (a) Oestradiol - 6 - carboxymethyloxime, by first synthesising the 6-oxo derivative (Longwell and Wintersteiner, 1940, J. biol. Chem. 133, 219-229) and converting this to the oxime (Dean, Exley and Johnson, 1971, Steroids, 18, 593-603).

(b) Oestradiol - 3 - carboxymethyloxime was prepared directly from oestradiol by the method of Rao and Moore (1977) (Steroids 29,461-469).

Testosterone: Testosterone - 3 - carboxymethyloxime was also synthesised by the method of Rao and Moore (1977).

These carboxy derivatives were conjugated to ova albumen (General Reagent Grade) (BDH, Poole, Dorset, England) by the mixed anhydride reaction of Erlanger, Borek, Beiserand Lieberman (1957) (J.

biol. Chem 228, 713-727). The only departure from the published method was that the conjugates were not precipitated with hydrochloric acid after dialysis.

The degree of derivatisation of each conjugate was determined by adding trace amounts of tritiated steroid to the reaction mixture in the preparation of the derivative and was found to be between 10 and 15 moles of steroid per mole of ovalbumen (MW 45,000 daltons).

Castrated male cross-bred sheep/cross-bred wetherswere used for antibody production and each was primed with 5 mg of conjugate. TheE1, E26CMO and T conjugates were each injected into two sheep, the E2-3CMO into a further three animals, and because of the low response to A initially, this particular conjugate was injected into six sheep.

An aqueous solution of each conjugate was emulsified with non-ulcerative complete Freund's Adjuvant (Morris) (Guildhay Antisera, Division of Clinical Biochemistry, University of Surrey, Guildford, Surrey, England) and injected intra-muscularly. After a suitable, but variable, interval of time, the animals were boosted with 2.0 to 2.5 mg of the same batch of conjugate in incomplete adjuvant via the same route. They were bled from the jugular vein 9 days after the booster injection. Approximately 500 ml of blood were collected from each animal in a sterile MRC bottle, using sterile needles and cannulae, and allowed to clot overnight at 40C. The following morning; the antiserum was harvested by aspiration and, after centrifugation at 40C and 1300 x g for 20 mins, was stored deep frozen prior to injection.

The titre of the antibodies present in each bleed was determined in the corresponding RIA, using tritiated labelled tracer and dextran-coated charcoal phase separation. Large bleeds were continued to be taken from the animals approximately every fortnight until the titres had fallen to low levels.

The anti A2 E and T2 sera finally produced were each a mixture of sera raised in two sheep, the anti-E2 serum a mixture from four of the five sheep (one injected with E2-6CMO conjugate and three with E2-3CMO conjugate).

Characterisation: Titre and specificity of the various antisera were measured by radioimmunoassay (Abraham 1975, Steroid Immunoassay, pp 67-78.

Cardiff: Alpha Omega Publishing Limited).

a) Titre. The titre was estimated by the addition of 0.1 ml twofold serial dilutions of each antiserum to 0.1 ml phosphate gelatin assay buffer (0.1% gelatin in 0.05hull phosphate buffered saline (PBS) pH 7.5) with 0.1 ml fixed mass of relevant radio-actively labelled steroid (Amersham & New England Nuclear, specific activities 53-108 Ci/mmol.).

The reaction volume was then incubated over night at 4"C, dextran-coated charcoal, 1 ml (25 mg: 250 mg: 100 ml assay buffer) was then added and incubated a further 40 mins at 4"C prior to centrifugation at 3200 rpm for 15 mins. The supernatant containing bound steroid was then decanted into 10 ml MINI RIA 20 scintillation fluid (koch-light) mixed and counted.

The final dilution of antiserum in the reaction volume that bound 50% of the labelled steroid was defined as the titre.

A, 1:144,000; E1, 1:480,000; E21:1,540,000;T, 1:276,000.

b) Specificity. The specificity of the steroid antisera was tested by performing cross reactivity studies with the four steroids under study and progesterone (P). The percentage cross reactivity was calculated by comparing dose response curves of each steroid with the four antisera and these are listed below.

Antiserum Nominal Steroid A E1 E2 T P Androstenedione (A) - 0 0 0.2 0 Oestrone (E,) 0 - 1.0 0 0.1 Oestradiol (E2) 0 6.8 - 0.1 0 Testosterone (T) 1.0 0 0.2 - 0 With only approximate information regarding the ultimate titre desired, the dose to be given to the breeding ewes was a compromise between that considered desirable on the basis of earlier informa tion and the number of animals available for treat ment. The doses chosen were A, 20 ml; Eel, 20 ml; E2, 7.5 ml; T, 23 ml, estimated to give final titres of approximately 1:300, 1:1,000, 1:1,300 and 1:650 respectively. (These calculations assume blood weight to be 8% of body weight, plasma to consti tute 55% of blood and blood plasma to constitute 40% of total intercellular fluid).One quarter of each of the four individual steroid doses was combined to make the 'mixture' (M).

Each of the animals to be treated was given a single injection (i.v.) of antiserum on the day of the start of mating with a fertile ram. Control animals were left untreated.

The sheep were treated in such a way that their ovulation rate could be recorded before and after mating and the duration of the oestrous cycle in which they were mated and the duration of any subsequent cycles, recorded. To measure the gener al effects of the stage of the oestrous cycle at the time of injection and to simulate commercial hus bandry conditions, animals were treated and mating started on a chosen day without regard to the oestrous cycle. To reduce the possibility that the stages of the oestrous cycle represented at the time of treatment might be reduced as a result of partial synchrony of the oestrous cycle by the introduction of the ram around the time of the start of the breeding season the flock was divided into two. Both were exposed to rams from the same day; one sub group was treated and became eligible for mating from that day, and in the other treatment and mating were postponed for 8 days.

Raddled vasectomised 'teaser' rams were intro duced on the first day and oestrus recorded in one sub group from the third day and in the other from the eleventh day. Within sub groups animals were allocated to be untreated controls or treated with A, E1, E2, T or mixed serum in rotation as they showed heat. The number of eggs shed at oestrus before treatment was recorded as the number of fresh (red) corpora lutea observed on the ovaries at laparos copy 3-13 days after oestrus. Treatment was given on the twentieth day and twenty-eighth day in the two subgroups respectively. From this time oestrus was recorded twice daily and ewes newly showing oestrus brought in for mating with fertile rams.

Oestrous ewes were penned singly with fertile rams and at least one mating recorded before being run with fertile rams for possible further mating at that oestrus. Returns to heat and remating were recorded through the use of keelmarks. If a ewe did not show oestrus during the 17 days prior to the designated start of mating, mating was postponed for 17 days so that its ovulation rate could be recorded pre-mating. This postponement was random with respect to treatment and was not considered in the analysis of the data. The number of eggs shed at the time of the first and any subsequent mating with a fertile ram was recorded at laparoscopy 3-13 days after mating and for a time thereafter.

The number of lambs per ewe was recorded at the time of lambing.

Statistical Methods. Differences among groups in the number of ewes with particular numbers of corpora lutea or lambs were tested by x2. Individual groups were compared to the control group by the use of Fishers Exact Test which gives the sum of the probabilities of the observed or any more extreme distribution arising by chance; a probability of 1 indicates that the observed allocation is the most likely; together with all other less likely allocations the collective probability is one.

Results Ovulation. The mean ovulation rate of ewes before and after treatment is given in Table 1 for each of the six groups. There were no statistically significant differences among the groups pre treatment when the number of single versus the number of multiple ovulations was compared for the various groups.

After treatment however the mean ovulation rate varied from 1.26 for the control group to 2.1 for those immunised against oestradiol or given the mixture of sera and the X2 test showed these differences to be statistically significant. The distribution of the ovulation rate is also shown in Table 1 where it can be seen that the principal effect of treatment was to increase the incidence of twin ovulations. The comparison of individual groups in turn with the control group using Fishers exact test showed that all treatments significantly increased the ovulation rate but that the statistical significance of the effect of the anti androstenedione serum was marginal. It must also be noted that the probability of obtaining one false significant result out of 5 with a threshold of Q.05 is 0.2.

The number of eggs shed at the time of return to oestrus by animals not conceiving to the first mating after injection is also given in Table 1 as the mean and the distribution for each group. There were relatively small numbers in this class but the mean ovulation rate of all treated groups was higher than that of the controls. In the oestrone and mixture group it was indistinguishable from that of the first ovulation after treatment while in the androstenedione and oestradiol groups it regressed 0.2 to 0.3 eggs towards the level of 1.25 of the control ewes, a mean indistinguishable from that at the first ovulation after the start of mating.

Conception. The number of ewes conceiving to a single mating is given in Table 1. The X2 test of the statistical significance of the differences among the groups indicated that they were real. The comparison of each group with the control by Fishers exact test gave a probability of 0.015 for the testosterone group. Possibly therefore the higher conception rate of the ewes of the T group was real. The removal of the T group from the comparison of all groups gives X2 of 6.4 for 4 d.F. (p > 0.1), compared to 13.8 for 5 d.F.

(p. < 0.05) when the T group was present; confirming the similarity of the remaining treated and control groups.

Number of Lambs. The total number of lambs present at the time of lambing was greater for treated than control ewes (Table 2). No ewe gave birth to triplets, and the X2 test of the number of ewes with single versus twin lambs indicated real differences among groups. The use of Fishers exact test, to compare individual groups to the controls indicated that the superiority was real for all but the androstenedione group.

Of the total of 216 lambs present 184 (85%) were born alive, but there was no indication of any difference in the incidence of mortality among the groups (Table 2). Four of the dead lambs resulted from 2 ewes which aborted shortly before lambing, 12 from 6 ewes which died with symptoms of pregnancy toxaemia shortly before the expected time of lambing. All 8 ewes were treated with sera but with 69 twin bearing treated ewes and only 5 untreated ones, 8 and 0 is the most likely allocation of a total of 8 failures (Fishers exact test), indicating no real difference.

The mean number of lambs born alive is given in Table 2 both per ewe lambing and per ewe treated.

The mortality of lambs (including that arising from the death of the mother) was the same in each treated group and the controls. Similarly there were no differences among the six groups in the number of ewes failing to give birth to any live lambs. The statistically significant differences among groups in live lambs born per ewe originally allocated for treatment were therefore similar to those for the number of lambs per pregnant ewe at lambing. The ewes given the mixture of antisera produced 1.5 lambs per ewe treated compared to an average of only 1 lamb for the controls.

Gestation Length. The mean gestation length for ewes in each group was either 148 or 149 days.

(Table 2). Neither the mean nor the variation (Table 2) was affected by the treatment.

Ovum Viability. The ovulation of viable ova, their fertilisation, implantation and gestation was estimated as the proportion of corpora lutea represented as lambs. This, together with the proportion of ewes showing oestrus and ovulating at the expected time after treatment gives an index of ewe fertility which is presented in Table 3. The index shows that overall, treatment with anti-oestradiol serum orthe mixture depressed ewe fertility while the anti-testosterone serum apparently raised it.

Statistical analysis of the two components of the index, the proportion of ewes returning to oestrus normally and the proportion of corpora lutea represented as lambs showed that the variation in the proportion of normal oestrous cycles among groups was statistically significant, p < 0.05. The proportion was lower for ewes in the oestradiol and mixture groups than for the controls (p = 0.05 and 0.005 respectively, Fisher exact test).

The differences in ovum viability, the other component of the index, were markedly different (X2 = 2.6, p < 0.001). The ewes of the testosterone group were significantly better than the control (p = 0.019, Fishers Exact Test) while the lower proportion surviving in the oestradiol group than in the controls approached statistical significance (0.05 < p < 0.1).

Effect of the Stage of the Oestrous Cycle. The number of eggs shed and the fertility of the return to oestrus following treatment is represented in the accompanying figure relative to the day of oestrous cycle when the individuals were treated. The number of corpora lutea (c.l.) observed on the ovaries of sheep ovulating after treatment with antisera to androstenedione, (A), oestrone (E1), oestradiol (E2), testosterone (T), a mixture of the four sera (M) and controls (C) according to the day of the oestrous cycle when treated. Each individual ewe is represented by a symbol.Those which conceived and where all c.l. observed are represented as lambs are represented *, ewes which conceived but gave birth to a different number of lambs by a sign and number; ewes failing to conceive R; ewes returning to oestrus less than 12 or more than 20 days after the previous oestrus, 0; not observed, N.

The incidence of abnormal oestrous cycles and the fertility of the return to oestrous but not the number of eggs shed were apparently greater in the anti-oestradiol and mixture groups when the serum was given late in the oestrus cycle. None of the three characteristics seemed to be affected by the time of treatment relative to mating in any of the other four groups. This was quantified by subdividing the data in Table 3 to give a separate index for the fertility of ewes treated during the early and luteal phases of the oestrous cycle (Day 1-12) and the follicular phase (Days 13-18). This index was only 0.2 for the oestradiol group and 0.4 for the mixture group when treated during the follicular phase compared to 0.6 for the controls.The number of animals was, in general too small for the present magnitude of the effects of the stage of the oestrous cycle observed to be demonstrated to be statistically significant on either component of the index of fertility for the oestradiol or mixture groups.

By contrast it can be seen from Table 3 that as it appears in Fig. 1, the number of eggs shed after treatment during the early and luteal phase is similar to that following treatment in the follicular phase; the largest difference between treatment early and late in the oestrous cycle was 0.29 corpora lutea. The long term nature of the effect on ovulation rate is confirmed by the high ovulation rate of animals returning to oestrus presented earlier.

The Linearityofthe Response to Mixed Sera. The response to the mixture of the four sera was compared to the average effects of the four indi vidual sera. The values of x2 for the comparison of all single sera (average) versus the mixture were computed for several traits to be as follows Trait Mixture Average x2 dF Number of corpora lutea 2.05 1.80 2.8 2p > 0.1 No. of lambs born alive 1.60 1.40 5.6 2p < 0.1 No. of lambs per ewe 1.68 1.46 3.2 2p > 0.1 No. of live lambs per 1.45 1.24 5.2 2p < 0.1 ewe allocated The mixture tended to lead to a higher response than that which would be predicted from the linear effects of its components. Although the superiority was not statistically significant, (0.1 > p > 0.05), the extra 0.2 lambs would be of very real practical significance.

The enhanced results from using a mixture of antisera to both androgen and oestrogen steroids compared with only one antiserum may be due to the effect on conception rates as well as ovulation rates although the mechanisms involved are complex and are inter-related.

As a result of these experiments, it has been shown that the use of antisera to steroids increased the prolificacy of sheep. Ewes given a mixture of antisera containing anti-androgen and anti-oestrogen sera gave birth to 50% more live lambs than did control ewes. Moreover, the improved prolificacy arose from an increase in the proportions of twin relative to single pregnancies not from an occasional ewe giving birth to many lambs. The schedule for treatment was the simplest possible - ewes were given a single injection on the day of the start of mating without any control of the oestrous cycle.

This would enable a farmer to treat his ewes when collected for mating without distinction between individuals.

The high conception rates achieved show that passive immunisation may be used to induce high prolificacy without any loss in fertility or indeed in the case of the anti-testosterone serum with a possible increase in fertility. Furthermore, passive immunisation has advantages over active immunisation in that the dose of antisera, and thus the final level of antibodies circulating in the breeding ewes, can be controlled and has an immediate effect.

In contrast, when active immunisation with a steroid is used, the variability in response between individuals is considerable both in the level of antibody produced and the time taken to produce a response and in some cases a hyperimmune state can result which effects a decline in fertility compared with untreated ewes. The increase in antibody levels in the passively immunised sheep is of short duration (about 4 weeks) after which the steroid levels return to normal, whereas with active immunisation the effect is prolonged.

Some of the differences between the treatment groups may have arisen from the physiological functions of the steroid to which the immune sera were prepared, others from the volume of antisera and hence the titre of antibodies given. Anti-oestradiol serum has been shown to block the oestrogen induced preovulatory discharge of LH and the response of ovariectomised ewes to the injections of oestrogen, the anti-oestradiol group in the present experiment showed the highest proportion of extended oestrous cycles. Further the index of the fertility of the ewes given anti-oestrodial serum was the lowest of all the six groups. The index of fertility in the other three groups given antisera to single steroids was similar to that of the control ewes but the ewes given the mixture of sera showed reduced fertility.It is therefore reasonable to argue that the reduction in the fertility of the mixture group arose from the anti-oestradiol component.

The small effect of androstenedione of the ovulation rate of the present sheep is by contrast likely to be the result of using too small a dose of serum. The results of both active immunisation with A and passive immunisation all show that antibodies to androstenedione may raise the number of eggs shed. The use of higher doses could well be profitable.

Ewes given antisera to testosterone showed higher fertility. All of the 38, fertile ewes conceived to mating after a single service, showing that anti-testosterone sera may increase fertility and by implication reduce embryo mortality. The combination of higher prolificacy and higher fertility would have considerable implications for the agricultural industry even though the mechanism of the action is not known. It has however recently been shown that even short periods of exposure to an asynchronous uterus may cause the death of embryos and a steroid induced lethal uterine product has been postulated.

The anti-testosterone serum may interfere with the production of such a protein. Alternatively it may lead to greater synchrony between the embryo and the uterus.

The stage of the oestruous cycle at the time of injection did not affect the response to anti A, E1 or T serum within the limits of the size of the experiment.

Intuitively however there would be expected to be a stage of the oestrous cycle, beyond which the favourable effects declined. For the anti-E2 and mixed sera the disadvantageous effects were much greater when the injection was given late in the oestrous cycle. The favourable effects of all sera however persisted through to the time of return to oestrus for those ewes which did not conceive to the first mating. The injection of sheep, say, three days before the introduction of the ram might therefore be appropriate.

The toxaemia at lambing was only observed in the present flock where particularly large rams were used. The absence of the disease in other treated flocks showed that it is not a necessary consequence of treatment; indeed the occurrence of twin lamb disease is an indirect measure of success.

On the basis of the present results it is possible to recommend suitable treatments, but only the Welsh mountain breed of sheep was used in the present experiments. These animals were used because they usually only produce a single lamb, since more than one could not be successfully raised by a ewe grazed on rough mountain pasture. When such mothers, are after a few years, brought to lower and more sheltered pasture it would then be profitable to breed increased numbers of lambs from them. The present dose of anti-testosterone and anti-oestrone serum might be useful in practice, but if anti-androstenedione serum were to be used a higher dose might be required. The general idea of a mixture has been vindicated, for that group gave birth to the highest average number of lambs.Further trials would be needed to describe the most appropriate mixture but, relative to the optimum dose of individual steroids, 40%T, 20%A, 20%E1, and 20%E2 might be a reasonable starting point. A mixture might be particularly appropriate if greater responses were required when the deleterious quadratic relationship of titre to mean ovulation rate might be specific to that antiserum while the favourable linear effects might be additive.

As mentioned above the invention could usefully be applied to hill breeds like the Welsh mountain sheep when an improvement in the ewes' situation would enable it to support more than one lamb. For other breeds which already produce a higher proportion of twins, the application could be used to increase that proportion and so enable the formerto match lamb numbers to expected food resources at the time of lambing. It would also present a cheaper alternative to the use of high planes of nutrition at the time of mating which are often used at present to obtain as high an ovulation rate as possible at the time of mating. For cattle however, particularly beef cattle, the use of this passive immunisation method for increasing the number of calves born to a herd would lead to a useful increase in productivity.

Similar remarks apply to horses, and particularly race horses where greater certainty of conception will avoid prolonged periods at stud and will result in births earlier in the season.

It will be apparent from the foregoing description that improved ovulation is achieved by compositions in accordance with the invention. It should be emphasised that another beneficial effect of the use of anti-testosterone antiserum and possibly anti-dihy.

drotestosterone antiserum is to improve conception rates. This beneficial effect is not necessarily achieved at the same time as increased ovulation.

This approach can result in increase success rate for artificial insemination of cattle, where normally the success rate is only 50% to 60%.

It is to be noted in this connection that tests over two seasons have resulted in 100% conception rate in sheep.

Claims (10)

1. A composition for administration to female breeding animals which comprises a mixture of an antiserum to an androgen steroid and an antiserum to an oestrogen steroid.

2. Acomposition according to claim 1 wherein the antiserum to an androgen serum is anti-testosterone, anti-androstenedione, or anti - 5 - dihydrotestosterone, an antiserum to a synthetic androgen, or a mixture thereof.

3. A composition according to claim 1 or claim 2 wherein the antiserum to an oestrogen steroid is anti-oestrone, anti-oestradiol, an antiserum to a synthetic oestrogen, or a mixture thereof.

4. A composition according to any one of the preceding claims which is a mixture of antisera to androstenedione (A), oestrone (E1), oestradiol (E2) and testosterone (T).

5. A composition according to claim 4 wherein the proportions of antisera in the mixture are about 40% T,20% A,20% Er, and 20% E2 where the percentage is the percentage of optimum dose of steroid alone.

6. A composition according to claim 1 substantially as herein described and exemplified.

7. A method of preparing a composition as claimed in any one of the preceding claims which includes immunising animals individually with the appropriate steroid derivative, collecting blood from the immunised animals after a certain period of time, extracting the antisera from the blood and mixing the resultant antisera in desired proportions.

8. A method as claimed in claim 7 wherein the steroid with which the animals are immunised is a derivative of a natural steroid or of a synthetic steroid or is a precursor thereof which will produce in the animal an antibody response yielding an antiserum which will cross-react with the natural steroids in the breeding animals on passive immunisation with that antiserum.

9. A method for the treatment of female breeding animals to increase fecundity which includes administering to the animals prior to mating a composition as claimed in any one of claims 1 to 6 or as prepared by a method as claimed in claim 7 or claim 8.

10. A method as claimed in any one of claims 7 to 9 substantially as herein described and exemplified.