US2824546A

US2824546A - Treating animals with hormone preparation

Info

Publication number: US2824546A
Application number: US193042A
Authority: US
Inventors: Klette Hermann
Original assignee: Individual
Current assignee: Individual
Priority date: 1950-10-20
Filing date: 1950-10-20
Publication date: 1958-02-25
Anticipated expiration: 1975-02-25

Description

United States Patent TREATING ANIMALS WITH HORMONE PREPARATION Hermann Klette, Frankfurt am Main, Germany No Drawing. Application October 30, 1950 Serial No. 193,042

Claims. (c1.119--1 This invention relates to hormone preparations and more particularly to hormone preparations having the activity of the steroid hormones, and a method of making same.

One object of this invention is to provide a hormone preparation which, although it is applied by hypodermic injection, possesses a prolonged activity similar to that of implanted pellets and thelike.

Another object of this invention consists in providing an injectable hormone preparation which exhibits a shock effect as well as a prolonged effect on application to the organism, said effects being controllably adjustable.

A further object of this invention consists in provida hormone preparation which, on account of its compo sition, is especially suitable in the veterinary field and may be applied, for instance, for fattening cattle, hogs and other domestic animals, for hormonal castration of domestic animals, especially of male animals, such as boars, steers, cockerels and the like, for initiating milk secretion in heifers, sterile cows, goats, sheep and others, for sex modification in the chick embryo, for increasing egg production, for combatting Trichomonas infections of cattle, and for various other veterinary purposes.

Still another object of this invention consists in providing a hormone preparation suitable in human therapy in cases in which an initially strong and also a prolonged effect of the hormone upon the body is required.

Other objects of this invention will appear hereinafter.

it is known to use in veterinary as well as human therapy solutions of steroid hormones in vegetable oils or in the form of implants. But these ways of application are connected with a number of disadvantage. Solutions of the hormones in oil are very quickly absorbed. Hence, their effect lasts only for a comparatively short time. In order to obtain a more lasting effect, the injection has to be repeated quite frequently. Implantation of pellets on the other hand requires skilled personnel and is usually done by the physician himself. Although they exert a prolonged effect, one has no way to determine the exact duration of said effect. Quite often the implanted pellets become covered with connective tissue by which they areso so sayencysted. This results in their losing their activity completely because no hormone can be dissolved and absorbed by the body fluids. Sometimes they even are discharged from the body under suppuration. Furthermore the initial effect of said implanted pellets is only very slight and sometimes insufficient to produce the desired therapeutic or other result. Aqueous suspensions of hormones have also been suggested, but the effect of the known suspensions is also rather uncertain.

It has been found, however, that uniform, certain and prolonged effects are achieved when applying suspen- 2,824,546 Patented Feb. 25, 1958 sions of a composition according to this invention. For this purpose suspensions of said hormones are produced in which the particle size of each hormone particle in said suspension is chosen in such a manner that it will produce the maximum effect. In order to achieve a strong initial effect particles of a comparatively small particle size are employed while the prolonged effect is caused by particles of a comparatively larger particle size. For producing the initial strong or shock effect, the particle size of the hormone should be not larger than 0.01 mm. in diameter while the prolonged effect is produced by particles of more than 0.01 mm. diameter. By suitable variations of the content of particles of different size suspensions are obtained which may be used for various purposes with maximum results. The upper limit of the particle size is determined by the width of the needle to be employed. Instead of using suspensions of particles of small particle size, one may also employ solutions of the hormones in suitable solvents in which the hormone particles of larger particle size are suspended.

Suspensions according to this invention are preferably produced with the natural follicle hormones such as estradiol, estrone, estriol, and their derivatives, especially their esters, such as estradiol benzoate, estradioldipropionate and the like, and with synthetic compounds having the acivtity of said follicle hormone, for instance, compounds of the stilbene series, such as diethyl stilbestrol, di-(p-hydroxyphenyl)-hexadiene, di (p hydroxyphenyl) hexane and their derivatives, especially their esters and ethers, but also compounds as ethinyl estradiol, doisynolic acid, equilenic acid and the like, di- (p-methoxyphenyl)-phenyl ethylenebromide and others more. Di-(p-acetoxyphenyl)-hexadiene has proved of special value because, on account of its specific crystal structure, it is capable of forming especially suitable suspensions.

Although compounds having the activity of the natural follicle hormones are especially suitable for this purpose, other hormones may also be used, such as the corpus luteum hormones progesterone and ethinyltestosterone, the male-sex hormones testosterone and methyl testosterone and their derivatives, especially their esters, such as testosterone propionate and the like, the adrenocortical hormones, such as desoxy corticosterone and its acetate, corticosterone and even cortisone and other ll-substituted compounds of this type. In general, this invention is applicable to all steroid hormones and their synthetic analogues of which an initial shock effect and at the same time a prolonged effect on application is desired.

An especially suitable field of application of the suspensions according to this invention is the veterinary field. It is known that, when fattening female animals, the highest fattening effect to be achieved with any animal in accordance with its race and age, takes place only after a longer starting period during which the animals do not gain considerably. This is due to the fact that the sexual functions are only gradually reduced whereby finally so-called self-castration takes place the reason for which is, among others, luteinisation and fatty degeneration of the ovaries. With male animals the sexual instinct disturbs also the fattening effect and diminishes the quality of their meat. Therefore they usually are subjected to bloody castration in order to achieve full fattening effect.

In all these cases the application of suspensions of cornpounds having the activity of the follicle hormone according to this invention has proved to be of great advantage. But it was found that for effecting complete and hormonal castration in the male as well as the female animal definite minimum amounts of the hormonal substance have to be applied to the animal, part of which must be very rapidly absorbable so as to cause a shock-like effect while the remainder must be absorbed gradually, i. e. in a prolonged manner. Under these circumstances hormonal castration takes place already after a much shorter starting period, namely after a period of 3 to 4 weeks at the most, while when applying the previously used amounts of hormones said changing period was much longer and was about 7 to 10 weeks.

Furthermore it has been found that with said minimum effective dosis a modification in the metabolism of the treated animals takes place causing fattening, said fattening effect being directly proportional to the amount of hormone applied. Gains in weight are obtained thereby which are 50-l00% higher than with control animals kept under the same conditions. Due to the prolonged effect of the hormone suspension according to this invention, the state of castration and of fattening is maintained with female animals for any length of time and with male animals for about four months. Besides the extraordinary fattening result, the meat quality is improved because its structure becomes less dense and fat is interspersed therein, and it acquires a lighter color. The disadvantage of the meat of male animals, especially of that of boars, having a disagreeable specific odor and taste so that it is unfit for human consumption, is almost completely eliminated by the treatment with a suspension of follicle hormones according to this invention. Thereafter the meat can be used as food.

The following minimum effective doses sufficient to cause castration and fattening in a short period of transformation were determinedi Suspensions according to this invention are obtained, for instance, by suspending previously prepared hormone particles of the desired size and in the required amounts and proportions in an aqueous medium which preferably contains suitable emulsifying and/or dispersing agents and, if necessary, protective colloids and the like. Especially suitable are emulsifying and dispersing agents and protective coloids which prevent or at least considerably retard agglomeration of the hormone particles and increase in size caused by growing of the crystals. Furthermore, on shaking up the suspensions before use in order to uniformly distribute the hormone particles therein, said agents must be capable of keeping the hormone in uniform suspension for a period of time sufiicient for injecting the same.

It is, of course, also possible to produce suspensions of the hormone in other liquids than water. Especially advantageous have proved suspensions of coarser horrnone particles in oil solutions of the same or a different hormone. The dissolved hormone exerts the shock-like effect because it is readily absorbed while the undissolved hormone particles produce the prolonged effect. When using water-soluble hormone derivatives, such as the estradiol glucoside phosphate compound, it is possible to employ aqueous solutions of the same in which, for instance, estradiol benzoate which is insoluble in water, is suspended. Such a solution-suspension is also capable of exerting a powerful initial eflect upon the body whereafter the slowly absorbed estradiol benzoate particles are maintaining the condition created by said initial action.

In order to produce hormone particles of a larger diameter than 0.01 mm., the active compound is, for instance, dissolved in suitable organic solvents, especially in ethyl alcohol or ether, so as to obtain a hot-saturated solution. By cooling while stirring slowly, the hormone is allowed to crystallize whereby the speed of cooling down the solution and the manner of stirring are adjusted in such a manner that substantially uniform crystals of a particle size of more than 0.01 mm. are obtained. The crystallized particles are separated from the mother liquor, for instance, by filtering by suction or by centrifuging or the like, and are carefully dried under conditions whereby conglomeration of the particles is avoided. Thereafter the dry crystal particles are first freed, for instance, by sieving, wind sifting, vibration classification or the like from particles of 0.01 mm. diameter and less and are then separated, if this is required, into particles of various particle size. One may also produce particles of specific and desired size by grinding dry hormone crystals in suitable mills and by subsequently sieving, wind sifting and the like of the obtained powder. In order to obtain very fine hormone particles of uniform size it is advisable to treat suspensions of the hormones, for instance, in an aqueous medium, preferably in the presence of emulsifying and/ or dispersing agents and/ or protective colloids, in a swinging mill, a colloidal mill or the like until all the hormone particles have been reduced, as can be ascertained by microscopic investigation, to substantially the same size less than 0.01 mm. in diameter. A suspension in which the hormone particles possess to a large extent such small size, is obtained, for instance, by dissolving the hormone in a suitable solvent watermiscible solvent, for instance, in ethanol, and adding said solution while stirring vigorously to an aqueous solution of a suitable emulsifying and/or dispersing agent and/or protective colloid. Thereby the hormone precipitates in a very finely divided form because said emulsifying agents etc. prevent agglomeration of the crystals. Said suspension is then. equalized in size as stated above. In order to produce a preparation according to this invention, hormone particles of more than 0.01 mm. diameter are then worked into said suspension in an amount necessary for the desired use of said preparation. It is, of course, also possible, by suitably selecting the working conditions when precipitating the hormone by pouring its alcoholic solution into the aqueous solution of an emulsifying agent etc., to obtain a preparation containing smaller particles as well as larger ones in the desired proportion.

Another process of producing preparations according to this invention consists in first preparing saturated solutions of the hormone at elevated temperatures in a suitable solvent, for instance, in a vegetable oil, such as olive oil, sesame oil and others, and then cooling said solution and crystallizing the hormone which at lower temperatures does not remain in solution, under conditions whereby crystal particles are obtained of a size corresponding to the requirements. The suspension obtained contains then part of the hormone in solution and, if necessary, part of it in the form of finer crystals of less than 0.01 mm. diameter for shock action while part of it is present in the form of coarser crystals of more than 0.01 mm. diameter for prolonged action. One may, of course, add suitable emulsifying and/or dispersing agent's and/or protective colloids to said solutions in order to improve the suspending power of said suspensions and to prevent conglomeration of the hormone particles.

As emulsifying and/or dispersing agents and/or protective colloids there may be used with great advantage water-soluble cellulose ether compounds, such as methyl cellulose or the sodium salt of carboxymethyl celluabout 1.0 to 1.5% of the same. trated solutions are too viscous for normal use and therelose. They are used as base for the hormone particles.

Especially suitable have proved solutions containing More highly concenfore, tend to clog the hypodermic needle. Less concentrated solutions, on the other hand, have not the emulsifying and dispersing power necessary for keeping the hormone particles in suspension for a sufliciently long period of time. Lower and higher concentrations, however, may also find proper application in specific cases.

Other agents of this type may also be used provided that they are substantially non-toxic and non-irritating, compatible with the body, and resorbable by the body fluids. Among them may be mentioned certain starch fractions, like amylopectin, the polyoxyethylene sorbitan monolaurates, monopalmitates, monostearates, monooleates which are known by the tradenames Tween 20, 40, 60, 80 respectively, sodium cellulose sulfate, certain types of purified carrageen, which may also be added to cellulose ether solutions to increase their suspending properties, hydroxyethyl cellulose and others more.

Another way of providing a preparation which exerts a strong initial and also a prolonged hormone effect consists in preparing pellets, tablets, small cylinders, pills and other structures which can be implanted easily into the body, with hormone particles of different particle size and/or different absorbability. Such pellets etc. are composed, for instance, of an inner core containing particles of larger size crystals, for instance, of n crystals of more than 0.01 mm. diameter, while their outer layer covering the core partly or completely, con sists of particles of a size less than 0.01 mm. diameter or of crystals which are readily soluble in the body fluids. To obtain such two part pellets etc., one may moisten the coarser hormone particles with a concentrated solution of a binding agent, preferably soluble in the body fluids, and may form therefrom a core around which then may be placed in a suitable manner a coating consisting of finer hormone particles moistened likewise with a concentrated solution of a binding agent which is readily soluble in the body fluids. The outer layer may also consist of a water-soluble hormone with a suitable binding agent. As binding agents there may be used cellulose ether compounds such as methyl cellulose and others, gum acacia, sugar syrup, blood serum, lymph, and the like. After drying said two part pellets under suitable conditions an implant is obtained which on application to the body will first release very rapidly the hormone in the outer layer due to the large surface of the small particles or the solubility in body fluids of the water-soluble hormone, thus, causing a shock effect, while the core, on account of the larger particle size of the hormone therein, will be absorbed more slowly, thus, effecting a more prolonged action. In principle, the composition of a bipartite implant according to this invention may be such, that, after implanting the same, it must be capable to separate into a crystal mixture consisting of single particles of different size and, hence, of ditferent resorbability. Implants of this type do not possess the disadvantages of the known implants which consist of a solid, coherent mass of the hormone in question, because shortly after implantation they disintegrate into a crystal powder which does not tend to become covered with connective tissue as readily as a compact mass.

One very interesting application of an aqueous suspension according to this invention is its use for modifying the sex in the embryo of chicks and other domestic birds, whereby when employing suspensions of follicle hormone, substantially only female chickens are hatched. This process of sex modification consists in injecting an aqueous suspension of a compound having the activity of the natural follicle hormone, into the air space of the egg at an early stage in development, preferably not later than 6 days after starting incubation. Amounts of about 20 I. U. of estrogenic activity are already sufficient to cause modification of the sex of the embryo in the sense of a feminisation of the hatched chicken. With higher amounts this effect is more pronounced, but if too high amounts are applied the hatched animals show considerable changes of the cloaca which return to normal the older the animals become. The best results were obtained with hormone amounts between about 20 I. U. and 80 I. U. per egg.

It is already known to cause sex modification in the chick embryo by injecting oily solutions of follicle hormone into the allantois part of the egg a few days after incubation has started. But this method cannot be used for practical purposes; for, it is very difficult to find the rather small allanteis in the egg, while injection into the air space of the egg according to the present invention can be carried out even by an unskilled person. In order to introduce the hypodermic needle into the egg, a small hole is first drilled into the shell, for instance, by means of a tooth drill whereby, however, the skin underneath the shell is not penetrated. Thereafter, the hypodermic needle is introduced through said hole in the shell and the skin into the air space and the hormone is injected. Preferably, not more than 0.3 cc. of suspension are applied in which the necessary amount of hormone is suspended. After injection and removal of the needle, the hole is closed by wax, paraffin or other indifferent, substantially non-toxic and non-irritating material which does not melt at incubation temperature. The eggs are then incubated in the customary manner without further treatment. The hatched chickens are almost all of female sex. Of course, the process is not only applicable to hens eggs but also to eggs of other fowl, such as geese, turkeys, ducks, and the like.

Another economically very valuable application of hormone suspensions according to this inventions consists in increasing the egg production of fowl. There are already known a number of agents which are said to increase the egg production of fowl. In general, they comprise highly proteinaceous fodder to which often lime or other substances capable of promoting the formation of the egg shell, are added. But the results obtained thereby are not certain but subjected to variations; A treatment of the laying hen with a hormone preparation according to this invention, however, increases the egg production considerably, especially during the off-season when the egg production decreases. Furthermore it was found that the molting period is shortened considerably and that egg production starts earlier in the year. This effect of the hormone upon the egg production is of great importance for two reasons. First, eggs in larger quan tities are available in months with reduced egg production, and second, since each hen is capable to lay only a certain number of eggs (about 700900 eggs) during its life-time, it is much younger at the time its egg production stops when it has been treated with the hormone, than an untreated hen. For, the egg laying period is reduced from 3 to 4 years to 2 to 3 years. This means that the meat of such a treated younger hen which is slaughtered after egg production stops, is more tender.

An especially suitable preparation according to this invention is obtained, for instance, by impregnating and intimately mixing preferably grain-fodder for fowls with an aqueous suspension of a follicle hormone compound or a compound having the activity of a follicle hormone containing an emulsifying and/ or dispersing agent and/ or protective colloid and an agent capable of increasing ad herencc of the hormone particles to the grain-fodder, said hormone particles being of very small particle size. Suitable adhesive agents are the above mentioned cellulose ether compounds and the other emulsifying and/or dispersing agents, especially those which are somewhat nygroscopic and therefore quite sticky. But other substances may be used likewise, such as gum acacia, algiru'c 7 acldgand other mucilaginous extracts and the like, pro vided they are non-toxic and non-irritating to the animal. Of course, it is also possible to impregnate the fodder with a solution of the hormone in a suitabl solvent, thereby adding preferably an agent capable of increasing adherence of the hormone particles to the fodder, whereafter the impregnated fodder is intimately mixed, if necessary, with further amounts of fodder so as to guarantee uniform distribution of the hormone throughout the fodder.

One may also produce aqueous solutions of watersoluble hormone preparations and add the same in the required amounts to the drinking water. furthermore, one may include the hormone into fodder as it is used for cramming poultry. This manner of application has the advantage that each bird receives a predetermined amount of hormone. One may also apply hormone suspensions according to this invention by injection, whereby the advantage is attained that considerably smaller amounts of the hormone are required. Any other suitable way of applying the hormone may also be employed.

The amounts of hormone to be given daily depend, of course, upon the kind of fowl and the various races to be treated. Furthermore, the resorbability and the solubility of the hormone used play also an important role. The diacetate of di-(p-hydroxyphenyl)-hexadien has been proved of special advantage. Of this compound an amount of about 4000-6000 I. U. daily has given very satisfactory results. Of course, other hormone compounds may be used likewise.

Suitable local anesthetics as well as preserving agents may be added to the suspensions and other preparations provided these additions do not irritate the body and do not react or in any way affect the properties of the active hormone ingredient. As preserving agents there may be used, for instance, chlorobutanol, phenol, cresol, thimerosal [sodium-(ethylmercuri)-thiosalicylate], n-butyl-phy'droxybenzoate and others while benzocaine, butacaine sulfate, tetracaine hydrochloride and the like may be used as local anesthetics.

Instead of using water as base for preparing the hormone suspensions according to this invention, isotonic sodium chloride solution, Ringers solution, blood serum and others may be used likewise.

The term hormone as used throughout the specification and the claims annexed hereto indicates not only the actual natural hormones but also synthetic chemical compounds and their derivatives having the activity of said natural hormones.

The following examples serve to illustrate the invention without, however, limiting the same to them.

Example 1 0.5 g. of amylopectin are dissolved in 100 cc. of water, 10,000,000 I. U. of estradiolglucosido phosphate are added thereto and are dissolved therein by heating while stirring. After cooling the solution to room temperature, 10,000,000 I. U. of the dimethyl ether of di-(p-hydroxyphenyl)-hexadiene crystals having a particle size of between about 0.05 and 1.00 mm. diameter are added slowly while stirring vigorously to said solution. A suspension of hormones is obtained which contains part of the hormones in dissolved and, thus, readily and rapidly absorbable form while the remainder of the hormones is present in coarsely crystalline form which is absorbed only gradually and very slowly.

Hormone crystals of between about 0.05 and 1.0 mm. diameter are obtained by dissolving the above mentioned methyl ether in methanol so that a hot-saturated solution is formed, and allowing the solution to cool while stirring slowly whereby part of the ether crystallizes. The crystals are then filtered, dried, carefully ground, and separated by sieving into crystals of about 0.05 to 1.0 mm. diameter.

Example 2 10,000,000 1. U. of di-(p-hydroxyphenyl)-hexadien are dissolved in cc. of olive oil (U. 5. Ph. )GV) while heating to about C. and stirring continuously. The solution is then cooled within 5 hours to about 0 C. while stirring slowly, is kept at this temperature for about 3-4 hours and is then slowly allowed to attain room Example 3 4 g. of diethyl stilbestrol (40,000,000 I. U.) are dissolved in 10 cc. of boiling ethanol and are gradually added to 40 cc. of a solution of 1% of methyl cellulose in water whereby the mcthylcellulose solution is stirred by means of a very effective stirring apparatus. The hormone is thereby precipitated in finely divided form. The suspension obtained is homogenized in a swinging mill until most of the particles show under the microscope a particle size of about 0.001 mm. to about 0.005 mm. diameter.

6 g. of diethyl stilbestrol crystals having a particle size of between about 0.05 mm. to about 0.15 mm. diameter, as they are obtained by allowing a hot-saturated alcoholic solution of said hormone to crystallize, filtering off the crystals by suction, drying them, and separating crystals of said size by sieving, are slowly and gradually added to 60 cc. of a 1.5% solution of a mixture of equal parts of methyl cellulose, known by the tradename Tylose S 400, and of the sodium salt of carboxymethyl cellulose, known by the tradename Tylose K 2000, in water while stirring thoroughly until a uniform suspension of the hormone is obtained.

Both suspensions are mixed with each other while stirring thoroughly. A suspension is obtained containing about 40% of the hormone having a particle size of about 0001-0005 mm. diameter and about 60% of the hormone having a particle size of about 0.05-0.15 mm. diameter.

By mixing such previously prepared hormone suspension in any desired proportion, suspensions of various composition are obtained which may be employed for many purposes. They are readily applicable by hypodermic injection and allow the application of very small as well as very large hormone doses of a composition adapted to any desired use.

With suspensions obtained according to the above given examples or in a similar manner the following results were obtained.

A hormone suspension containing at least 50% of very finely divided particles is subcutaneously injected behind the ear. Experiments were carried out, for instance, with non-pregnant sows, 7 months to 2 years old. The animals did not grow properly and did not respond well to fattening fodder. They were frequently and strongly in heat. Within l-2 days after the injection ruttish animals calmed down and did not exhibit any heat during the following fattening period while with animals which were free of heat at the time of injection, ruttishness appeared which, however, disappeared after 4-5 days. When applying at least 60,000 I. U. hormone per kg. live weight, about l8-21 days were required to effect the necessary change in the endocrine system. Thereafter the fattening effect proper sets in and the animals gain considerably more in weight than untreated animals under like conditions. The average increase in gain amounted to 50-.100% more than with the controls. Sows of 70 kg. weight, for instance, were injected with about 5,000,000 I. U. of a suspension of the diacetate of di-.(p-hydroxyphenyl)-hexadiene, while sows with about kg. live weight received about 10,000,000 I. U. of the same suspension and sows with about- 200 kg. live weight about 15,000,000 1. U. The

following table shows the fattening effect of these animals in comparison with untreated controls:

This table shows furthermore that, in order to obtain about the same final weight, the fattening duration can be reduced 30-50%. A further great advantage of the treatment is as stated above, the considerable improvement of the meat quality due to the interspersion of fat in the muscle. The meat-fat-ratio, however, remains in general normal. Bloody castrated boars respond to the treatment in about the same manner as sows, while with not castrated bears the results were less uniform. A higher increase in weight of -100% over that of untreated boars was achieved by the injection of a hormone suspension according to this invention. With these animals the great advantage is achieved that, as stated above, their meat becomes fit for human consumption. It loses its peculiar odor and taste about 4-6 weeks after the injection. The castration effect of the hormone, however, lasts only for about 3-4 months. Hence, the treatment should be carried out about 3 months before the animal will be slaughtered.

With wethers, especially young animals, a gain of about 30% more than with untreated animals is observed, but only half the amount of hormone per kg. live weight is required than with sows and boars.

With oxen and cows an additional gain of about 30-70% over untreated animals is observed. Compare, for instance, the following table, whereby the animals of 200 kg. of weight received about 5,000,0007,000,000 I. U. of the hormone while those of 400 kg. of weight were injected with about 10,000,00014,000,000 I. U. of the hormone:

With cows similar results were obtained. Other experiments showed the following results:

Average daily increase in weight with animals treated with a hormone suspension according to this invention:

Treated. Controls, More inanimals, g. crease,

percent Oxen 897 660 36. O Steers 1, 141 710 60. 8 Cows and Heifo 983 750 31. 1

Hence, the treated animals could be slaughtered about one month earlier than the controls.

Another field of application of hormone suspensions is the hormonal caponizing of fowls. This process has considerable advantages over the bloody castration. First of all, no losses due to the castration operation occur. And the speed of hormonal castration is much higher than that by surgical means. The following table gives the comparative results of bloody castrated animals and of hormonal castrated ones:

Weight at Weight after beginning of experiment, g. 2 weeks, 4 weeks, 6 weeks,

g. g. g.

Bloody castration 795 1, 032 1, 246 1, 418 Hormonal oastration 795 866 1, 416 1, 754

This table shows that, although in the beginning the gain in weight is not as high with hormonal castration than with bloody castration, the picture has entirely changed after 6 weeks, when the hormonal capons show a 23.7% higher weight than the bloody castrated ones. The overall increase in weight is with the former about with the latter about 78%.

A further possibility of utilizing the hormone suspensions exists for promoting milksecretion in heifers, sterile cows, goats, and sheep. With goats positive results could be achieved in almost all cases. Sterile goats gave by one injection with 2,000,0002,500,000 I. U. hormone after about 14 days to 3 weeks about 1.5-2.5 liters of milk daily. The milk production remained almost the same for about 7-8 months. With cows in general the results are not as certain; but in some cases the'milk production amounted to 12 liters daily. Amounts of 17 liters daily were also reported.

Hormone suspensions containing a large percentage of very small particles of a size of 0.001-001 mm. diameter may be successfully used instead of oil solutions of the hormone, for instance, for the treatment of trichomonas infection of cattle and in other cases where a very strong initial effect and a comparatively short prolonged eifect is desired.

Example 4 A mixture of roughly ground corn germs and wheat bran 1:1 is spread in a thin layer upon a plate and is then sprayed with a suspension of di-(acetoxyphenyl)- hexadien in an aqueous 0.5% methyl cellulose solution so that 5 g. of the impregnated material contains about 5,000 I. U. of the hormone. The sprayed mass is then furthermore ground and intimately mixed so as to obtain a preparation throughout which the hormone is uniformly and evenly distributed. 5 g. of the preparation added daily, preferably in the morning, to the fodder of each laying hen increases the egg production of said hen in the off-season to about 50%.

Example 5 19.0 g. of finely ground oats, 15.0 g. of wheat middlings, 10.0 g. of wheat bran, and 5.0 g. of soybean meal are thoroughly mixed with each other. This mixture is sprayed with a concentrated alcoholic solution of 100,000 I. U. of di-(p-hydroxyphenyl)-hexadien in a mixer which can be heated to 50 C. while an air stream is blown through it. The alcohol is evaporated thereby and recovered in the customary manner. The impregnated mass is then intimately mixed with 10.0 g. of dried skim milk, 10.0'g. of meat scrap containing 55% of protein, 15.0 g. of alfalfa-leaf meal, 4.0 g. of linseed meal, 5.6g. of ground limestone, 2.4 g. of streamed bonemeal, 1.2 g. of a salt mixture containing 100 parts of common salt and 1.7 parts of anhydrous manganous sulfate, and 2.8 g. of cod-liver oil, until the hormone is uniformly and evenly distributed therein. 5 g. of this laying mash is fed daily to each laying hen.

Example 6 A paste-like mass containing only very little water is made by moistening estradiol benzoate crystals having a particle size of about 0.1-0.3 mm; diameter, with a concentrated aqueous solution of gum acacia whereby the amount of binding agent is just high enough to cause the single hormone crystals to stick to each other without filling out all the interstices betweensaid crystals. When preparing this paste care has to be taken that the particles are not substantially broken up into smaller particles.

Likewise another paste-like mass is produced by binding estradiol crystals having a particle size of about 0.001-0.005 mm. diameter with a concentrated aqueous solution of methyl cellulose.

The pastes are then brought into extrusion machines the head of which is provided with two orifices, one in the midle forming a core and the other surrounding concentrically the former, thus, forming a tube which, on

' account of the adhesiveness of the paste sticks to the inner core and forms a cover around it. The mass containing the coarse estradiol benzoate crystals is forced through the inner orifice while the mass containing the fine estradiol crystals is forced through the outer con- Example 7 A tablet of 1.5 mm. diameter and 1.5 mm. height is pressed from di-(p-aeetoxyphenyl)-hexadien crystals having a particle size of about 0.2-0.3 mm. diameter and a suitable binding agent, such as carboxy methyl cellulose sodium, whereby care is taken that the pressure is not too high but that the crystals substantially maintain their size and shape and are not reduced in size to powder. This tablet is then used as a core around which in a manner known per se a coating is applied consisting of a concentrated sugar syrup containing di- (p-acetoxyphenyl)-hexadien crystals having a particle size of about 0.001-0.005 mm. diameter. Thereby pellets are obtained consisting of a core of coarse crystals and a coating of fine crystals which can be implanted quite easily.

Of course, many changes and variations may be made by those skilled in the art in the reaction conditions, the

hormones, binding, emulsifying, dispersing, adhesive agents, solvents employed, the particle sizes of the hormone, the preparation of hormone crystals of specific particle size, and the like, provided these changes and variations are made in accordance with the principles set forth herein and the claims annexed hereto.

What I claim is:

1. In a method of increasing body growth and improving meat quality of cattle and other domestic animals, the step comprising administering to said animals, by a single non-intravenous injection, a suspension of an estrogen compound in an injectable vehicle, said suspension containing estrogen particles of a size substantially between about 0.001 mm. and about 0.005 mm. diameter and estrogen particles of a size substantially between about 0.05 mm. diameter and about 0.50 mm. diameter.

2. In a method according to claim 1, wherein the finer estrogen particles and the coarser estrogen particles are present in the suspension in about equal amounts by weight.

12 3. In a dosage procedure for increasing body growth and improving meat quality of cattle and other domestic animals, the steps comprising administering to said animals, by a single non-intravenous injection, :1 suspension of an estrogen compound in an injectable vehicle,

said suspension containing estrogen particles of a size between about 0.001 mm. and about 0.005 mm. diameter and estrogen particles of a size between about 0.05 mm. and about 0.50 mm. diameter, the finer and the coarser particles being present in said suspension in about equal amounts, the total amount of estrogenic compound being injected corresponding to at least the following estrogenic international units per kg. of live weight of said animal:

I. U. per kg. ltvc Bulls Cows, steers, heifers" Boa ams 20,000 Sheep 15,000. Goats, male 25,000. Goats, female. 18,000. Old cocks, hen 600,000 per animal. Coekerels.- 350,000 per animal.

700,000 per animal.

and keeping said treated animals on ordinary feed until slaughtering time.

4. In a method according to claim 3, wherein the estrogenic compound is di-(p-acetoxy phenyl) hexadiene.

5. In a method according to claim 1, wherein the estrogen compound is di-(p-acetoxy phenyl) hexadiene.

References Cited in the file of this patent UNITED STATES PATENTS 1,928,346 Axelrod Sept. 26, 1933 2,142,537 Tisza Jan. 3, 1939 2,207,990 Miller July 16, 1940 2,343,625 Abramson et al. Mar. 7, 1944 2,385,117 Turner et a1 Sept. 18, 1945 2,394,628 Meyer Feb. 12, 1946 2,413,419 Saunders Dec. 31, 1946 2,486,426 McGaha Nov. 1, 1949 2,541,447 Turner Feb. 13, 1951 2,557,052 Himelick June 12, 1951 2,589,898 Turner Mar. 18, 1952 FOREIGN PATENTS 417,715 Great Britain Oct. 1, 1934 515,566 Great Britain Dec. 8, 1939 543,897 Great Britain September 1941 OTHER REFERENCES Forbes: Article in Science, Apr. 21, 1941, pp. 404-405.

Greene: Abstracts of Surg. Gym. and Obst, June 1942, pp. 595-599, p. 596 is especially pertinent.

J. of Endocrinology, vol. 4, 1944-1945, pp. 51 and 77.

Koref et al.: Article in Endocrinology, Mar. 1946, pp. 214 and 215.

Bowling et al.: Article in J. A. M. A., Nov. 1, 1947, pp. 567-569.

Squibb Abst. Bulletin, July 19, 1950.

Claims

1. IN A METHOD OF INCREASING BODY GROWTH AND IMPROVING MEAT QUALITY OF CATTLE AND OTHER DOMESTIC ANIMALS, THE STEP COMPRISING ADMINISTERING TO SAID ANIMALS, BY A SINGLE NON-INTRAVENOUS INJECTION, A SUSPENSION OF AN ESTROGEN COMPOUND IN AN INJECTABLE VEHICLE, SAID SUSPENSION CONTAINING ESTROGEN PARTICLES OF A SIZE SUBSTANTIALLY BETWEEN ABOUT 0.001 MM. AND ABOUT 0.005 MM. DIAMETER AND ESTROGEN PARTICLES OF A SIZE SUBSTANTIALLY BETWEEN ABOUT 0.05 MM. DIAMETER AND ABOUT 0.50 MM. DIAMETER