AU614496B2 - Improved female mammals - Google Patents

Description

AU-AI-78759/87 T WqD INELL NUAIOP O IZATION INTERNATIONAL APPLICATIO PUB ISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 4 (11) International Publication Number: WO 88/ 00827 A61K 31/56 Al (43) International Publication Date: 11 February 1988 (11.02.88) (21) International Application Number: PCT/US87/01787 (81) Designated States: AT (European patent), AU, BE (European patent), CH (European patent), DE (Euro- (22) International Filing Date: 22 July 1987 (22.07.87) pean patent), FR (European patent), GB (European patent), IT (European patent), JP, LU (European patent), NL (European patent), SE (European patent), (31) Priority Application Number: 891,158 (32) Priority Date: 28 July 1986 (28.07.86) Published With international search report.

(33) Priority Country: US Before the expiration of the time limit for amending the claims and to be republished in the event of the receipt of amendmnents.

(71) Applicant: UNIVERSITY OF ILLINOIS BOARD OF TRUSTEES [US/US]; 506 South Wright Street, Urbana, IL 61801 AQ.J P 24 MAR 1988 (72) Inventors: BERGER, Larry, L. 1806 Scottsdale, Champaign, IL 51820 KESLER, Darrel 22 Lark Park P.Rad, Champaign, IL 61821

AUSTRALIAN

(74) Agent: SHIMEI, Barbara, University Patents, Inc., 24 F 1988 P.O. Box 901, Westport, CT 06881 (US).988

SOFFICE

(54) Title: ADMINISTRATION OF ANDROGLN EMALE MAMMALS FOR INCREAsD E rCI.ENCY IN FOOD PRODUCTION f (57) Abstract Methods for producing improved female mammals intended for human or animal consumption, said methods comprising prenatally exposing said female mammals to androgen by administering to the mother an effectively constant amount of androgen sufficient to achieve and maintain a level of circulating androgen in the mother's bloodstream approximately equivalent to that of a normal adult male of the same species.

i, "WO 88/00827 PCT/US87/01787 DMINISTRATION OF .N DRONSN- -09GDM-BES?16 FEMALE MAMMALS FOR INCREASED EFFIn5- FRN This invention pertains to methods of producing improved female mammals of the type normally slaughtered at adulthood to obtain meat suitable for human or animal consumption, which method comprises exposing said female mammal solely during its prenatal period to androgen by administering to the mother during pregnancy an effectively constant amount of androgen sufficient to achieve and maintain a level of circulating androgen in the mother's bloodstream approximately equivalent to that of a normal adult male of the same species.

The key to improving the economic competitiveness of mammals as sources of proteins in the human and/or animal diet lies in improving the carcass merit (relative ratio of lean meat to fat), growth rate, and feed efficiency decreasing the amount of feed required per unit of weight gain).

Heifers grow approximately 12 to 15% slower and are to 12% less efficient in converting feed to weight gain than intact male herd-mates. Similarly, ewe lambs grow approximately 6 to 8% slower and require 4 to 6% more feed per unit of gain than intact ram lambs. Heifers routinely produce carcasses which are worth two to three dollars less per hundred pounds, compared to steer carcasses. This is due to the higher fat content, lower lean yield and lighter weight of heifer carcasses compared to steer carcasses of similar quality grade. The cause for these difference s between the sexes in growth rate, feed efficiency, carcass merit, and meat quality is not clearly understood.

The administration of estrogens to meat-producing animals to accelerate weight gain has been known since the 1950 See, U.S. Patents u I i I WO 88/00827 PCT/US87/01787 2 2,751,303 and 2,824,546. However, such treatment produced undesirable feminization which needed to be countered by simultaneous administration of estrogens and androgens. See, U.S. Patents 3*,009,857; 3,636,211 and 3,939,265. Androgens alone have been administered to induce hormonal castration of perinatal males. See, e.g. 4,252,799.

A great deal of work has been done on postnatal models, principally in the rat, to determine the effects of androgens and estrogens on secondary sexual characteristics, including growth rates, as the effects of growth hormone appear to be mediated by sex hormones. Bell, D.D. and I. Zucker, Physiol.

and Behavior 7:27-34 (1971) proposed that hormonal stimulation of the neonatal rat affects growth rate by altering hormonal sensitivity to neural weight regulating mechanisms. Tartellin, J.E. Shryne and R.A. Gorski, Acta Endocrinologica 79:177-191(1975) reported- that female rats given testosterone propionate on neonatal days 2 and 3 grew faster than control rats. Jansson, S. Ekberg, O. Isaksson, A. Mode, and J.A. Gustafsson, Endocrinology 117(5):1881-1888 (1985) suggested that neonatal androgenization of the rat (from endogenous steroids in the male and from exogenous sources in the female) determined the hormone secretory pattern which influenced body growth and hepatic steroid-metabolizing enzymes. It should be emphasized that high pharmacological doses of testosterone propionate were given to the rats neonatally; the more normal lower physiological dosages do not necessarily have the same effect as those described by the above researchers.

The applicability of the rat data to larger mammals must be interpreted very cautiously. For example, RalgroTM [zeranol, International Minerals ,WO 6/00827 PCT/US87/01787 3 and Chemical Corp., Northbrook, IL) is an anabolic implant which when given to the newborn calf increases growth rate, at least partially, by increasing circulating levels .of growth hormone.

However, the animal has to be reimplanted every 80 to 100 days to maintain the effect. The point is that stimulating growth hormone production at birth in both rats and cattle increases growth, however this effect is not sustained in cattle while it is in rats.

We have now discovered that carcass merit of female mammals can be improved by exposing the female mammal solely during its gestation to androgens.

This is conveniently done by administering to the mother during pregnancy an effectively constant amount of androgen sufficient to achieve and maintain a level of circulating androgen in the mother's bloodstream approximately equivalent to that of a normal adult male of the same species. This method avoids the time-consuming and costly prior art methods of repeatedly or continuously treating each female with androgens (and/or estrogens) for an extended period after birth, during growth and development. According to the methods of the present invention the levels of sex hormones in the female offspring are normal after birth, thus also avoiding having unnatural elevated levels of sex hormones present in the animal to be slaughtered and consumed.

There are several implants available which will increase the growth rate and/or feed efficiency of heifers. However, all of these products have to.be given directly to the animal at regular int:ervals (approximately every 80 days) after birth to maintain the improvements in growth rate and feed efficiency.

We have now discovered that the feed efficiency of female mammals can be improved by exposing the female WO 88/00827 PCT/US87/01787 4 mammal solely during its gestation to androgens by administering to the mother during pregnancy an effectively constant amount of androgen sufficient to achieve and maintain -a level of circulating androgen in the mother's bloodstream approximately equivalent to that of a normal adult male of the same species.

With this approach the animal to be consumed is itself never implanted; exposure during gestation via the mother is effective for the offspring's entire life.

Putney, W.E. Beal, and G.A. Good, Congr.

Proc. 10th Int. Congr. Anim. Reprod. Artif.

Insemin., Paper No. 5 (1984) showed that injection of 250 mg daily of 17-alpha-methyl-4-androsten-3-one to pregnant cows on days 40-60 resulted in similar mean birth weights for male, female, and androgenized .female .offspring.. Preweaning growth in the androgenized female was similar to that of bull calves and higher than that of female calves.

However, the external genitalia of the androgenized females were completely masculinized. We have surprisingly discovered that the growth rate in female mammals can be enhanced by exposing the female mammal solely during its gestation to androgens by administering to the mother during pregnancy an effectively constant amount of androgen sufficient to achieve and maintain a level of circulating androgen in the mother's bloodstream approximately equivalent to that of a normal adult male of the same species.

This represents a prenatal exposure to levels of androgens an average of 10-12 times lower than those taught by Putney et al.. Furthermore, this enhancement of growth rate occurs without the undesirable side effects associated with the complete masculinization of the external genitalia demonstrated by Putney et al.

WO 088/00827 PCT/LS81/O 1787 Although selecting for rapid growth rate has increased dystocia in both beef cattle and sheep, there are no products available which effectively reduce birth weight of calves or lambs. Bremner, and I.A. Cumming, Metabolism 27(3):253-255(1978) showed that injections of 250 mg testosterone ethanate every 2 weeks between days 72 and 147 of pregnancy resulted in increased fetal death and average lower birth weights for both male and female offspring. We have surprisingly discovered that the birth weight of female mammals can be reduced by exposing the female mammal solely during its gestation to androgens by administering to the mother during pregnancy an effectively constant amount of androgen sufficient to achieve and maintain a level of circulating androgen in the mother's bloodstream approximately equivalent to that of a normal adult male of the same species. This represents levels of androgens substantially less than those used by Bremner and Cumming. The method of the present invention avoids the intermittent high dosages of Bremner et al. and Cumming et al., leaves the male offspring unaffected, and avoids the high levels of fetal death reported by them. Lowered birth weight reduces dystocia, improves the efficiency of production by increasing the number of offspring weaned per mother and shortens the interval from delivery to first estrus in the mother.

Approximately 15% of the heifers entering the feedlot are pregnant. The average heifer that is in excess of 150 days pregnant when entering the feedlot costs the beef producer approximately $100 per head at present rates due to increased labor, lowered dressing percent, reduced meat quality and complications associated with calving in the feedlot.

Abortifacients are available and fairly effective WO 88/00827 PCT/US87/0 1787 6 within the first 150 days of pregnancy. After 150 days the producer has little option but to feed the pregnant heifer and hope she is ready to market before she calves, since slaughter soon after calving produces meat of reduced quality. Being able to guarantee that the heifers are not pregnant when entering the feedlot would be a real advantage to both the seller and the buyer.

Most feedlot heifers are fed melengesterol acetate (MGA), a synthetic progestogen to block estrus. The estrus activity of open heifers not fed MGA results in reduced feed intakes and rates of gain due to the extra activity associated with estrus.

Currently, MGA costs approximately $5 per head to feed for a typical finishing period.

We have discovered that the problems associated with fertility and estrus cycling can be eliminated by. partial masculinization of the' female. mammal, produced by exposing said female mammal solely during its gestation to androgens by administering to the mother during pregnancy an effectively constant amount of androgen sufficient to achieve and maintain a level of circulating androgen in the mother's bloodstream approximately equivalent to that of a normal adult male of the same species.

Clark, R.J. Scaramuzzi and R.V. Short, J.

Embryol. exp. Morph. 36:87-99(1976) implanted pregnant sheep with 1 g testosterone for 50 days at a time at various phases of pregnancy and studied the resultant effects on the female offspring. Results showed that the critical period for complete masculinization of the external genitalia is between days 40 and 50 of gestation. Wilson, P.R. and M.F.

Tartellin, Acta Endocrinologica 89:182-189(1978) showed that injection of pregnant ewes with 200 mg testosterone on each of days 20, 27, and 40 of -7pregnancy was capable of causing complete masculinization of the external genitalia of the resulting female offspring. However, both Clark et al.

and Wilson et al. were investigating the effects of prenatal hormonal imprinting on neural development; neither Clark et al. nor Wilson et al.

suggested any solution to the problems of unwanted fertility and cycling.

We have surprisingly found that sterility in female mammals can be achieved by exposure of the female mammals to active levels of androgens substantially lower than those demonstrated by the prior art, and furthermore without the drawbacks of complete masculinization reported by Clark et al. and Wilson et al.

According to a broad form of the invention there is provided a method for producing improved female mammals of the type normally slaughtered at adulthood to obtain meat suitable for human or animal consumption, by prenatally improving their eventual carcass merit, growth rate, feed efficiency and/or reducing their birth weight and producing stei ility without complete masculinization said method comprising the steps of: identifying a pregnant mammal of a particular species; o(b) identifying the normal circulating level of androgen in the normal adult male of the same species as said pregnant mammal, said level being within the range of from about 0.5 ng/ml up to about 20 ng/ml; administering androgen to said pregnant mammal throughout a predetermined period of her pregnancy extending from the approximate initiation of sexual differentiation through at least the first third of gestation; S maintaining through said period a level of circulating androgen in the bloodstream of said pregnant mammal within the range of from about ng/ml up to about 20 ng/ml, approximately equal to the normal androgen level of a normal adult male of the same species; and allowing offspring of said pregnant mammal to be born at the end of said pregnancy, whereby any male offspring are substantially unaffected by said treatment and any female offspring have normal levels of sex hormones, improved growth rate, and sterility without complete masculinization.

According to the method of the present invention, female mammals are exposed in utero to androgens by administering to the mother during pregnancy an effectively constant amount of androgen sufficient to achieve and maintain circulating levels of blood androgen approximately equivalent IL I 7a to those found in normal adult males of the same species.

The method of the present invention is suitable for use with any female mammal intended for human or animal consumption, including but not limited to cattle, sheep, pigs, horses, bison, deer, reindeer, goats, rabbits, etc.

Androgens suitable for use in this invention are male steroids capable of causing the biological changes that normally occur during inutero sexual differentiation of the developing male offspring, e.g. androstenediones, testosterone and derivatives of testosterone such as dihydrotestosterones, 19-nortestosterone, etc., apn their derivatives, especially their esters, such as testosterone proplonate, testosterone cypionate, and the like.

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*o*o *•go SWO 88/00827 PCT/'S87/01787 8 Testosterone and derivatives of testosterone, e.g.

testosterone propionate, are preferred.

The compositions of the present invention are administered to the mother during pregnancy in any convenient manner, for example by implant under the skin, e.g. in the ear lobe, in skin folds, or in the neck of the animal. Instead of implants, the compositions can be injected intravenously, intramuscularly, subcutaneously, or intraperitoneally in the form of a time-release preparation. The compositions of the present invention can also be deposited intraocularly as a gel or foam or intravaginally as described in U.S. Patent 4,449,980), intranasally as described in U.S.

Patent 4,331,651), or intrarectally in the form of suppositories or pessaries, as is known in the art.

The compositions may also be administered orally in *.formulations designed to survive the stomach fo.r absorption through the gut.* Compositions suitable for use in the present invention can also include other active ingredients, e.g. anti-inflammatory steroids such as natural or synthetic cortisones or derivatives thereof; antibiotics such as tetracyclines, penicillins, cephalosporins, or derivatives thereof; and other antibiotics which may prevent or ameliorate infection at the site of injection or implantation.

Formulations suitable for use in the present invention are made according to methods and procedures well known to those skilled in this art.

Preferably, the compositions of the present inventions are administered in the form of time-release implants according to methods and formulations known in the art for androgens and other steroids, e.g. U.S. Patents 2,824,546; 4,191,741; 3,737,521; 3,991,750; 4,096,239; Christensen, D.A.

WO 8/00827 PCT/LS87/01787 9 and D.J. Kesler, Animal Reproductive Science 7:531-536(1984); Kesler, R.R. Troxal, D.L.

Vincent, N.S. Scheffrahn, and R.C. Noble, Theriogenology 15(3):327-334(1981) all incorporated herein by reference. Alternatively, time-release implantable or injectable compositions comprising biodegradable capsules, spheres, or the like containing androgen according to methods known to those skilled in this art, e.g. Tice, R.M.

Gilley, R.J. Flores, J. Kirkpatrick, and R.H. Asch, Proc. Intl. Symp. Controlled Release Bioactive Materials, Vol. 12, pp. 108-109 (1985), may be used.

In addition to active ingredients, time-release formulations may also contain compounds useful in regulating release of androgen from the formulation.

The above compositions are administered to the mother during pregnancy in amounts sufficient to achieve and maintain circulating levels .of androgen in the bloodstream at levels approximately equivalent to those normally circulating in the bloodstream of a normal adult male of the same species. Methods of measuring circulating levels of androgens in normal adult males of each species are known to those in the art. Accordingly, for example, in cattle the mother would be administered a dosage of androgen calculated to maintain the circulating blood levels of androgen at approximately the level of a normal adult bull, e.g. at about 0.5 to about 15 ng/ml, preferably from about 2 to about 5 ng/ml. For the average cow weighing about 450 kg, this is equivalent to approximately from about 10 to about 150 mg (preferably about 20 mg) per head per day. For a sheep, the mother would be administered a dosage of androgen calculated to maintain the circulating blood levels at approximately the level of a normal adult ram, e.g. at about 1 to about 10 ng/ml, preferably WO 88/00827 PCT/US87/01787 about 2 to about 7 ng/ml. For the average ewe weighing about 50 kg, this is equivalent to approximately from about 7 to about 50 mg (preferably about 10 mg) per head per day. For a pig, the mother would be administered a dosage of androgen calculated to maintain the circulating blood levels of androgen at approximately the level of a normal adult boar, at about 1 to about 20 ng/ml, preferably from about 3 to about 8 ng/ml. For the average sow weighng about 180 kg, this is equivalent to approximately from about 10 to about 100 mg (preferably about 30 mg) per head per day.

The period during gestation when implantation must occur to get the desired effects is crucial.

For example, if it is desired to improve carcass merit, the female fetus must be exposed to an effectively constant amount of androgens from about the initiation of sexual differentiation through about the second third of gestation. If it is desired to improve feed efficiency, the androgens must be present from about the initiation of sexual differentiation through about mid-gestation; for improved growth rate without undesired complete masculinization, the female fetus should be treated from about the initiation of sexual differentiation through about the first third of gestation. To induce sterility without undesired complete masculinization, the preferred period of treatment is from about initiation of sexual differentiation 30 through about the first third of gestation; to reduce birth weight while still achieving subsequent improved growth, the female fetus should .be exposed from about mid-gestation through about 1 week prior to parturition. Methods of determining the time of onset of sexual differentiation and the various periods of gestation are readily determined by those 'WO 88/00827 PCT/US87/0 1787 11 skilled in the art. In all the above methods, since androgens are known to interfere with parturition, treatment of the pregnant mother with androgens is preferably halted before parturition, most preferably about 5 to about 30 days before delivery.

Examples of preferred embodiments are given in Table I. The indicated dosages (in mg/day) are administered to the mother during the appropriate period of pregnancy (equivalent to the periods of gestation given above) to achieve the desired effect: TABLE I carcass feed growth birth merit sterility efficiency rate weight cow 10-30 10-50 10-25 10-25 10-50 ewe 5-15 7-20 7-10 7-10 7-20 sow 10-60 10-75 10-35 10-35 10-75 A further understanding of this invention can be had from the following non-limiting examples. As used hereinabove and below unless expressly stated to the contrary, all temperatures and temperature ranges refer to the centigrade system and the terms ambient and room temperature refer to about 20-25 0 C. The term percent or refers to weight percent and the terms mole and moles refer to gram moles.

~Fg3 1~ i)llUI~ ICIX WO 88/00827 PCT/US87/01787 12 EXAMPLE I Pregnant crossbred ewes were implanted with testosterone propionate beginning at a point between days 40 and 60 of gestation; the implants were removed 82 days later. The implants were made of medical grade Silastic TM [Dow Corning, Midland, MI] tubing with an internal diameter of 6.4 mm and an outside diameter of 9.5 mm. A single 10 cm-long implant containing approximately 2.0 grams of testosterone propionate was given to each ewe just under the skin directly posterior to the carpus joint and perpendicular to the 6th and 7th ribs. The implants were designed to allow a controlled release of testosterone propionate resulting in a circulating level of 2 to 3 ng/ml in sheep (Christensen, D.A. and D.J. Kesler, Animal Reproductive" Science 7:531-536(1984)).

Twelve ewe and five ram lambs, born from ewes which were implanted, and ten ewe and nine ram lambs, born from nonimplanted ewes, were studied. Ram and ewe lambs were managed under different conditions.

Prenatally androgenized ewes had lower birth weights (P .05) than control ewes (3.73 vs 4.95 kg). Birth weights were similar for A rams and C rams (4.59 vs 4.64 kg). Daily gains were greater for A ewes compared to C ewes, but similar for A rams and C rams (.340 vs .344 kg/day).

Androgenized ewes did not exhibit regular estrus cycles while C ewes exhibited normal estrus cycles.

No A ewes became pregnant; however, 8 of 10 C ewes became pregnant. Although differences were not significant, fat thickness measurements were less and ribeye areas were greater for A ewes than C ewes.

Yield grades of A ewes were lower .10) than C ewes. The results are presented in Table II: I i WO 88/00827 PCT/US87/01787 TABLE II birth weight (ka) avg.

daily gain (ka) 12th rib fat (mm) ribeye area (mm2/kr) yield grade Control 4 95 a 0.1 79 a 7.62 53.08 4.01 c Treated 3 7 3 b 0 206 b 5.54 56.25 3 23 d 1 yield grade estimate of the closely retail cuts from designated portions of the (equivalent to carcass merit) trimmed carcass ab Means in same column with unlike superscripts differ c,d cd Means in 'same column with uhlike superscripts differ ;f WO 88/00827 PCT/US87/01787 14 Birth weights of female lambs were decreased approximately 17% by prenatal androgenization. From birth to 110 pounds (average slaughter weight) ewes which were androgenized in utero grew 13.5% faster than control ewes. It is interesting to note that improved growth continued past weaning, a situation not reported in postnatally treated animals without continuous treatment at regular intervals over their lifetimes. None of the androgenized ewes showed behavioral estrus or became pregnant, while 80% of the control ewes showed estrus and 70% became pregnant when exposed to a ram. At slaughter androgenized ewes produced carcasses which were leaner and contained more muscle per unit of body weight than control ewes.

EXAMPLE II In a procedure similar "to that of Example"I, cows were implanted with testosterone propionate/Silastic implants from approximately day 150 to day 240 of gestation and the implants were removed approximately 3 weeks prior to calving. The implants were 15 cm long and contained approximately 2.5 grams testosterone propionate per implant. Each cow was implanted subcutaneously with two implants, one behind each shoulder about 10 cm below the spine.

The implants were designed to allow a controlled release of testosterone propionate resulting in a circulating level of 1 to 2 ng/ml in cattle (Kesler,D.J., T.R. Troxel, D.L. Vincent, N.S.

Scheffrahn, and R.C. Noble, Theriogenology.

15(3) :327-334(1981)).

Results are shown in Table III: -I i i ii i 1 II-~ TABLE III birth weight (Ibs) weight per day of age to weaning bs) 12th rib fat (mm) ribeye area (mm2/kg) yield grade gain: feed ratio Control heifers 84.0 2.51 11.43 26.92 2.60 0 106 a Androgenized heifers 76.6 2.57 10.58 28.75 2.18 0.134 b ab Means with different superscripts are statistically different at P< i -I WO 98/00827 PCT/US87/01787 16 Birth weights of calves were decreased approximately 8% by prenatal androgenization.

Heifers which were androgenized prenatally had an weaning weight an average of 23 pounds heavier than control heifers. Rate and efficiency of gain were each improved approximately 8% for the first 72 .ays in the feedlot.

EXAMPLE III The procedure of Example I was repeated with a different group of control and treated ewes.

Thirty-seven ram lambs and 29 ewe lambs were obtained from untreated (control) mothers and 25 ram lambs and 28 ewe lambs were obtained from treated mothers. The results after 28 days in the feedlot are given in Table IV:

IL.

2 ,WC;88/00827 PCT/LSS7/O 1787 TABLE IV daily gain (lbs/dav) gain: feed ratio Control ewes 0.576 0.189 Treated ewes 0.608 0.210

I

WO 88/00827 PCT/'S87/01787 18 These data corroborate results of Example I in improved rate of gain and feed efficiency without undesired complete masculinization.

Further examples within the spirit and the scope of the invention will occur to those skilled in the art, and only those limitations appearing in the claims should be read thereon.

L.

Claims (4)

1. A method for producing improved female mammals of the type normally slaughtered at adulthood to obtain meat suitable for human or animal consumption, by prenatally improving their eventual carcass merit, growth rate, feed efficiency and/or reducing their birth weight and producing sterility without complete masculinization said method comprising the steps of: identifying a pregnant mammal of a particular species; identifying the normal circulating level of androgen in the normal adult male of the same species as said pregnant mammal, said level being within the range of from about 0.5 ng/ml up to about 20 ng/ml; administering androgen to said pregnant mammal throughout a predetermined period of her pregnancy extending from the approximate initiation of sexual differentiation through at least the first third of gestation; 15 releasing at a controlled rate through said period a level of circulating androgen in the bloodstream of said pregnant mammal within the range of from about 0.5 ng/ml up to about 20 ng/ml, approximately equal to the normal androgen level of a normal adult male of the same species; and allowing offspring of said pregnant mammal to be born at the end of 20 said pregnancy, whereby any male offspring are substantially unaffected by said treatment and any female offspring have normal levels of sex hormones, improved growth rate, and sterility without complete masculinization.

2. The method for producing improved female ovine offspring according to claim 1 wherein the amount of androgen administered to a pregnant parental ewe is an amount sufficient to achieve and maintain a level of circulating androgen, through said period, of between about 2 and 7 ng/ml.

3. The method for producing improved female bovine offspring according to claim 1 wherein the amount of androgen administered to a pregnant parental cow is sufficient to achieve and maintain a level of circulating androgen, through said period, of between about 2 and 5 ng/ml.

4. The method for producing improved female porcine offspring according to claim 1 wherein the amount of androgen administered to a pregnant parental sow is sufficient to achieve and maintain a level of circulating androgen, through said period, of between about 3 and 8 ng/ml. DATED this TWENTY-FIRST day of JUNE 1991 University of Illinois Board of Trustees Patent Attorneys for the Applicant SPRUSON FERGUSON INTERNATIONAL SEARCH REPORT International Aoplication No PCT/US87 0 1787 I. CLASSIFICATION OF SUBJECT MATTER (if several classification symools aopy, indicate all) 3 According to International Patent Classification (IPC) or to ooth National Classification ana IPC IPC(4): A61K 31/56 U.S.C1.: 514/177.178 II, FIELDS SEARCHED Minimum Documentation Searcnea 4 Classification System Classification Symools U.s. 514/177,178 Documentation' Searched other than Minimum Documentation to the Extent that such Documents are Included in the Fields Searched 6 III. DOCUMENTS CONSIDERED TO BE RELEVANT 14 Category Citation of Document. it with indication, .here appropriate, of the relevant oassages 1 Relevant Is Claim No. X N. Chemical Abstracts, Vol. 85, Abstract No. 172096g, (Unit Reprod. Biol., Med. Res. Counc., Edinburgh, Scot.), Clarke et al., "Effects of Testosterone Implants in Pregnant Ewes on Their Female Offspring", J. Embryol. Exp. Morphol., 1976, 36(1), pp 87-99. A N. Chemical Abstracts, Vol. 91, Abstract No. 53487z, (Inst. Tierz. Tierverhalten, Forschunganst. Lardwirtsch., Brunswick-Voelkenrode, Fed. Rep. Ger.), Elsaesser et al., "Estrogen Feedback in the Pig: Sexual Differentiation and the Effect Of Parental Testosterone Treatment", Biol. Reprod, 1979, 20(5), pp.

1187-93. Special categories of cited documents: is later document oublished after the international iling date or oriorlty date and not in conflict with the aoplication but document defining the general state of the art which is not cited to understand the princiole or theory underlying the considered to be of oarticular relevance invention earlier document but oublished on or after the international document of particular relevance; the claimed invention filing data cannot be considered novel or cannot be considered to document which may throw doubts on oriority claim(s) or involve an inventive step which is cited to estaolish the oublication date of another document of oarticular relevance: the claimed invention citation or other special reason (as specified) cannot be considered to involve an inventive steo when the document referring to an oral disclosure, use. exhibition or document is comoined with one or more other such docu- other means ments, such comoination being oovious to a person skilled document puoblished prior to the international filing date but n the art. later than me priority date claimed document member of the same patent family IV. CERTIFICATION Date of the Actual Completion of the International Search Date of Mailing of this International Search Report 0 1 DEC 13-" 03 NOVEMBER 1987 International Searcning Authority i Signature of Authorized Officer 1o ISA/US IJoseph A. Lipovsky Form PCTIISAI210 rsecond sheet) (October 1981) I r International Aoolication No. PCT/US87/01787 FURTHER INFORMATION CONTINUED FROM THE SECOND SHEET A N. Chemical Abstracts, Vol. 81, Abstract No. 21461p, (Inst. Exp. Endokrinol., Humboldt-Univ. Berlin, Berlin, E. Ger.), Hinz et al., "Prenatal Treatment of Sows WithTestosterone Propionate", Endokrinologie, 1974, 63(2), pp. 161-5. A US, A, 4,457,914 (COX ET AL.) 3 July 1984 (03.07.84), See entire document. A US, A, 4,331,657 (COX ET AL.) 25 May 1982 (25.05.82), See entire document. V.17 OBSERVATIONS WHERE CERTAIN CLAIMSWERE FOUND UNSEARCHABLE I This international searcn reoort nas not been established in respect of certain claims under Article 17(2) for the following reasons: Claim numoers because they relate to suoject matter tl not required to be searcned oy this Authority, namely: 2.1 Claim numoers because they relate to parts of the international aoplication that do not comely with the prescribed require- ments to such an extent that no meaningful international search can be carried out t. specifically: VI.l OBSERVATIONS WHERE UNITY OF INVENTION IS LACKING t1 This International Searching Authority found multiole inventions in this international aoplication as follows: As all required additional search fees were timely paid by the applicant, this international search report covers all searchable claims of the international appliation. 2.1.j As only some of the required additional search fees were timely paid by the applicant, this international search report covers only those claims of the international application for which fees were paid. specifically claims: 3.1[ No required additional search fees were timely paid by the applicant. Consequently, this international search report is restricted to the invention first mentioned in the claims: it is covered by claim numoers: 4.7 As all searchableclaims could be searched without effort lustifying an additional fee, the International Searching Authority did not invite payment of any additional fee. Remark on Protest 0 The additional search fees were accompanied by applicant's protest. E No protest accompanied the payment of additional search fees. Form PCT/ISA/210 (supplemental sheet (October 1981) 1