MXPA97009579A - Inhibit compositions and methods to improve producc's performance - Google Patents

Abstract

The present invention relates, in general, to a method for improving the yield of poultry production, by administering to a bird a heterologous protein comprised of inhibin protein, or a fragment thereof and a carrier protein. The present invention also relates to a method for improving the production performance of poultry, by administering to a bird a product of fusion gene comprising a gene encoded for expression of avia-inhibin protein subunit -alpha, or a fragment thereof, and a gene encoded for the expression of a carrier protein. An effective amount of the heterologous protein or fusion gene product is administered to an animal such that the immune response against the heterologous protein occurs in the animal. The present invention further relates to the above heterologous protein and the fusion gene product and to the production methods of the same.

Description

I HIBINA COMPOSITIONS AND METHODS TO IMPROVE PRODUCTION PERFORMANCE FIELD OF THE INVENTION The present invention relates in general to a method for improving the yield of poultry production by administering to a bird a heterologous protein comprised of an inhibin protein, or a fragment thereof, and a carrier protein. The present invention also relates to a method for improving the yield of poultry production by administering to a bird a fusion gene product comprising a gene encoding the expression of an alpha subunit bird inhibin protein, or a fragment thereof, and a gene that codes for the expression of a carrier protein. The present invention further relates to the above heterologous protein and the fusion gene product, and to methods for producing same.

BACKGROUND OF THE INVENTION The ratites are birds that run, usually large, without flight, which include several orders including the species of Ostrich, Emu, Rhea, Cassowaries and Kiwis. An emu (Dromiceius novaehollandiae) is an Australian ratite bird, which is characterized by its rudimentary wings and a feathered head and neck. An average adult emu has a height of approximately 1.82 m (6 feet) and weighs approximately 68 kg (150 pounds). An ostrich (Struthio ca elius) is a large runner bird with small wings and powerful thin legs. A normal adult ostrich has a height of approximately 2.43 m (8 feet) and weighs approximately 147.5 to 170.2 kg (325 to 375 pounds). The term "rhea" is the common name for members of birds of the order of Rheiformes. The Rheiformes are an order of South American running birds, called American ostriches, which differ from the true ostrich in its smaller size, head and neck plumed, and three-toed feet, among other aspects. Ostrich and emu have had commercial value in their natural environments of South Africa and Australia, respectively. Ostrich products have been in demand for over 100 years, and there is a substantial global market for their skins, meat and feathers, for example, ostrich skin is used in boots, bags, jackets, suitcases, purses and many other articles. Ostrich feathers are used in fashion, clothing, and feather dusters.

In contrast, emu is a newcomer relative to the common market. It is valued for the same products with the addition of an essential oil that is used in the cosmetics industry. Emu oil, which makes a thin layer of subcutaneous fat, has deep penetration properties, which make the oil useful in cosmetic creams, such as wrinkle-retarding emollients. Also, medicinal uses for emu oil, such as arthritis treatment, are currently being investigated. A typical fully developed emu can have a height of 1.6 to 1.9 m or more, and a weight of 30 to 45 kg or more. The emus mature to about 1 year, and the pre and post-pubescent emus never show specific phenotypic differences in the genus. Similar to ostriches, the Emu population in the United States has also experienced explosive growth in recent years. In 1994, there were approximately 150,000 emus total including 15,000 pairs of offspring in the United States. It has been predicted that emus numbers in 1995 will increase from 500,000 to 750,000 birds, of which 45,000 are expected to be pairs of offspring. There is a growing demand for ratite products in several countries including Australia, Belgium, Israel, Canada, Holland, Namibia, South Africa and Zimbabwe. Therefore, during the last years there has been an explosive growth in the domestic market for ostrich and emu, and to a much lesser degree the rhea. In the last 5 years, the number of ostrich breeding pairs and the total number of birds in the United States has increased from 7.5 to 20 times, respectively. It is estimated that in 1995, 200,000 ostriches, including 20,000 breeding pairs, existed in the United States. The tremendous interest in raising these animals is due to the significant value of adults, as well as of immature animals, and especially to test ostrich breeding pairs, which are evaluated as high as $ 75,000, with a pair of emus valued at 30,000 dollars or more. Immature ostriches from 3 to 4 months of age are valued at approximately $ 7,500, and immature emus are valued at approximately $ 5,000. A majority of animals are purchased between 3 and 6 months of age. In addition, there is tremendous interest in ratites as an alternative to more traditional forms of animal husbandry. Several factors in relation to the ratites make them a superior alternative to the more traditional forms of animal husbandry (ie cattle farms, pigs and sheep). These factors include: higher feed conversion ratios, a greater ability to be raised on farms intensively, large animal size, improved reproductive capacity, and exceptional nutritional value of their meat. For example with ostrich meat, which is a red meat similar to beef, contains significantly less fat, calories and cholesterol than chicken or turkey meat. More particularly, an 85 gram serving of ostrich meat contains 2 grams of fat, 58 mg of cholesterol and 97 calories. In contrast, a 85-gram serving of turkey meat contains 3 grams of fat, 59 mg of cholesterol and 135 calories. A serving of 86 grams of chicken meat contains 3 grams of fat, 73 mg of cholesterol and 140 calories. A serving of 85 grams of beef (fillet) contains 15 grams of fat, 77 mg of cholesterol and 240 calories. And finally, a serving of 85 grams of pork contains 19 grams of fat, 84 mg of cholesterol and 275 calories. (The values for the ostrich meat were derived from AMSI Quality Laboratory Report # 0800100. The values for the other meats were derived from USDA Handbook No. 8, "Nutritive Valué of Foods.") Similar to the ostrich, emu meat is a red meat with a low fat content. More particularly, a 100 gram serving of emu meat contains 1.7 g of fat, 57.5 mg of cholesterol and 109 calories. (The values for emu meat were derived from Siliker Laboratories of Texas, Inc.) Also, ratites such as ostriches provide approximately 45.4 kg (100 pounds) of meat at the age of 12 months and then produce a substantial amount of meat. meat in a relatively short period. An illustration of how the ratites are a superior alternative to the more traditional forms of animal agriculture is the following comparison of an ostrich and a cow. First, an ostrich has a gestation or incubation period of 42 days, where a cow requires 280 days. Second, an average ostrich produces more than 20 progenies per year, while a cow produces one progeny per year. Third, the feed conversion ratio of an ostrich is less than 2: 1, while the feed conversion ratio of a cow is 5: 1. Fourth, the days from conception to slaughter are approximately 407 days for an ostrich in contrast to 645 for a cow. Finally, ostriches produce feathers in addition to their flesh and skin, while cows do not produce other products than meat and skin. Considering these attributes, and the increased needs for the world population for meat, which is nutritious and still low in fat and cholesterol, and which can be efficiently produced with minimal negative impact on the environment, the ratite industry has a high potential for future growth. Currently, the demand for ratites exceeds supply more. Nevertheless, ratite producers are limited by suboptimal egg production in most females of breeding age. Depending on the species, most captive ratites currently produce an average of 10-20 eggs per year, while their genetic potential for egg production is believed to exceed more than 60 eggs per year. For example, an ostrich with high production in wild conditions can produce an egg approximately every 48 hours during the breeding season, and a high production emu in wild conditions can produce an egg approximately every 72 hours in the breeding season. In contrast, an ostrich in captivity usually takes 5 to 10 days to lay an egg, and an emu usually takes 4 to 8 days to lay an egg. A market for slaughter of ratites does not occur until sufficient numbers of progeny are produced annually. According to some values, in order for a slaughter market to be maintained, there must be at least 250,000 animals available annually. Therefore, a method to improve production performance will greatly accelerate market growth. Accordingly, there is a need for a composition and method for improving the yield of production in birds, and particularly in ratites such as ostriches and emus. In addition to the recently emerging ratite industry, there is a huge industry encompassing established poultry species, mainly egg-type chickens (Single Comb White Leghorns), meat-type chickens (broilers), turkeys, ducks, geese and quail. The global demand for poultry meat and egg products is huge and has increased steadily over the last decade. The following is an attempt to characterize the size and complexity of the poultry meat industry in the United States, exclusive of ratite production. For the purposes of this discussion, it should be understood that the current population figures (United States census, 1990 data), suggest that there are approximately 250 million inhabitants in this country. In addition, the population of America for the year 2000 is projected to be approximately 275 million people, which represent an increase of 25 million people. From 1990 to 1995 (using figures projected by USDA for 1995), per per capita consumption of chickens and average turkey meat 34.8 and 8.2 kg (66.7 and 18.1 pounds), respectively (See Anonymous, Poultry Processing Sourcebook, Meat Processing, Vol 33 (9): 22-25, (1994) and Bowman, M. Beef and Pork: Competing for the Food Dollar, Meat Processing, Vol. 33 (12): 16-25 (1994)). This translates to a total average per capita poultry meat consumption of 43 kg (94.8 pounds) during the last 5 years. The stable trend of increased consumption of poultry meat is also documented. For example, a per capita consumption of young chickens has increased approximately 12.7 kg (28 pounds) (from 25.2 kg (55.6 pounds) in 1985 to 37.9 kg (83.5 pounds) in 1995). Likewise, turkey consumption per capita has increased approximately 1.13 kg (2.5 pounds) (from 7.2 kg (15.9 pounds) in 1989 to 8.35 kg (18.4 pounds) in 1995). This trend of increased consumption of chicken and turkey meat products is expected to continue in the future parallel with the anticipated growth in the human population (see values cited above). If both predictions are correct, then by the year 2000 almost 16.6 million tons of chicken and turkey meat will be consumed annually in this country only. The trend of increased consumption of poultry is directly related to the fact that poultry meats are considered to be "heart healthy" foods (they have a low content of animal fat) and have a good price., compete favorably with the most expensive red meats (such as steak, pork and lamb). Improving the production yield of chickens and chicken breeding hens could therefore be a significant economic value to an industry that currently enjoys high growth due to a demand for production, still increasing. To satisfy consumer demand and maintain their competitive edge in the price of meat, breeders of young turkeys and turkeys will continue in the business of producing as many hatched eggs as possible. Therefore, any method capable of increasing egg production even in small quantities could generate significant economic benefits. As an example, the scenario of a single chicken broiler hen (with a stored chicken base) that produces 15 additional eggs during a production cycle (approximately one year duration) is as follows. This case could result in the current prices of chicks in the market (0.16 / chick), in the generation of an extra income of 2 dollars for the sale of birds newly hatched and 10-12 dollars preceded by sales added chicken meat generated from the growth of these chicks (after considering the appropriate deductions for food costs and fixed costs). Since the population of estimated chick broiler hens is believed to be an excess of 60 million hens in the United States, the economic gain could be approximately 750+ million dollars. Including estimation money that could be anticipated by the improvement of production performance in turkey breeders (as well as in hens comprising the specialized poultry business of ducks, quail geese, and including chickens discussed above), is estimated conservatively that is between 1-2 trillion dollars for all the economic gain that could be obtained from the meat side of the poultry industry, including all the poultry raised for the consumption of meat, excluding ratites. It should be noted that the poultry industry first developed in this country as a mainly table egg industry. When the poultry were subsequently selected for their improved body weight (meat-type chickens), such genetic selection had deleterious effects on the egg production regimes. In other words, egg production proved to be negatively correlated with the genetic change found after selection for increased body weight. Therefore, the potential for improving the production yield of broiler chickens and turkeys (meat type birds) through endocrine manipulation as contemplated in the present invention) should be greater in birds of type of meat than in Single Comb Ehite Leghorns birds, bred by intensity of egg production since the late 1920s. On the one hand, there are 3 to 4 times more egg table layers as discussed below, than breeder birds of tender chickens of America. Therefore, even very small improvements in the yield of production in egg-type birds are greatly increased when considering the size of bird populations that may be affected. The following is a discussion of the size and importance of the table egg industry in this country. The per capita consumption of eggs has remained reasonably stable from 1989 to 1992 and ranged from 13.6 to 13.8 kg (30.0 to 30.4 pounds) (See Table 653: Per capita of major food commodities, Unites States, 1984-92, Acrricultural Statistics, 1993, USDA Nati. Agri. Stat. Ser., US Govt. Printing Office, Washington, DC p 457; 1993 and 1994 dates unavailable). The stagnant table egg consumption regimes in America are probably directly associated with the public interest in the consumption of egg yolks, which are perceived to have a high cholesterol content. Despite stable per capita egg consumption regimes, table egg table numbers have steadily increased from 228.8 million hens in 1990 to 240.7 million hens in 1994 (See Bell, DD, University of California Monthly Statistical Report, Table 28: Table egg layers; Number on farms during the month, 1980-93, UC Riverside). This trend of increasing numbers of production hens may simply reflect the need for more eggs to meet increasing population numbers, increased export of eggs and egg products, or even other unidentified factors. Whatever the reason, since almost 80% of the world's eggs are produced outside of America, this suggests that any positive effect can be obtained within the United States table egg market that can be multiplied by a factor of 5 to obtain global economic importance. However, if the calculations are restricted to the United States, and if a stable per capita consumption is maintained at approximately 13.7 kg (30.2 pounds), and the projection that the population will increase to 275 million persons in the following is accepted 5 years, then by the year 2000 almost 4.2 million tons of chicken eggs will be produced annually in this country, only. A dollar value for the improvement of egg production in table egg layers is difficult to derive due to the existence of many complex aspects of a Leghorn production cycle that affect the decision making (for example, the cost of small chickens of replacement against the use of molting, harmful effects of the molting on the yield after the molt, loss of egg sales during the molt, etc.). However, it could be unreasonable to conclude that an increase in egg production in egg-type chickens in a stored chicken base of only half the magnitude of those discussed above for young chicken breeders could at least be translated an economic benefit of the type of billions of dollars. The phrase "improve production yield" is understood by those of ordinary skill in the art to denote an increase in one or more of the following female birds: accelerate the onset of egg production; accelerate the start of maximum egg production; prolonged persistence of egg production; increased intensity of egg production; or increased total egg production. The phase also includes improved feed conversion ratios; Improved egg shell quality or improved resistance to adverse production conditions such as heat stress, overcrowding, poor nutrition and noise. The phrase means an increase in one or more of the following in males: accelerated onset of puberty or sperm production; accelerated start of maximum sperm production; increased persistence of sperm production; increased intensity of sperm production (sperm count), - volume of ejaculation increasing; improved sperm viability; increased testosterone production; or increased libido. Recently, the hormone inhibin has been studied as a potential means to increase ovulation in mammals. Inhibin is a peptide hormone mainly produced by the gonads, and more particularly through the growth of follicles and testes. In mammals, it functions as a feedback regulator inhibiting the secretion of the pituitary follicle stimulating hormone ("FSH"). Since the existence of inhibin was first postulated 60 years ago, its chemical isolation was only recently achieved.

Mammalian inhibin is a dimeric protein hormone, which is composed of a subunit a (molecular weight 18,000) and a subunit β (molecular weight 14,000). The subunit is unique to inhibin like the dimers of the activin form of the β subunit, a hormone that releases FSH from the pituitary gland. The ß subunit exists in two forms (&A and ßß), which are distinct but absolutely similar. Therefore, depending on the β subunit involved, inhibin exists as an inhibin-A or inhibin-B. Both subunits a and β, when bound through disulfide bonds, are required for biological activity to suppress the secretion of follicle stimulating hormone ("FSH") from the pituitary gland. The amino acid sequence of the a subunit of inhibin exhibits approximately 80-90% similarity between porcine, bovine, human, murine and domestic chickens. Excellent reviews on the isolation, production, analysis and biological actions of inhibin are available from Risbridger et al., Current Perspectives of inhibition Biology, Endocrinological Act (Copenh), 122: 673-682, (1990); and Rivier, C., et al., Studies of the Inhibin Family of Hormones: A Review, Hormone Research, 28: 104-118 (1987), which is incorporated herein by reference.

In mammals and birds, FHS plays an important role in follicular growth and development, while luteinizing hormone ("LH") is thought to induce ovulation. Several factors of the brain and gonadal (peptide and steroidal hormones) interact to control the release of gonadotropin hormone. Of these factors, gonadotropin releasing hormone ("GnRH") and inhibin exert opposite controls on the secretion of FSH from the pituitary in mammals. Gonadotropin releasing hormone is a decapeptide in the brain which acts to stimulate FSH and LH secretion, while inhibin is a gonadal protein, which apparently acts to selectively inhibit secretion of FSH in mammals. A basic knowledge of the ovulatory procedure in poultry is necessary to understand the role of inhibin in the endocrine control of ovulation in birds. The growth of follicles in the functionally mature ovary of the domestic hen exists in an inheritance of different size. A typical ovary contains yolk-filled follicles with a length of 4 to 6 centimeters and a diameter of 2 to 4 centimeters (F, to F4, Fg), accompanied by a larger number of smaller yellow follicles, from 2 to 10 mm, and many much smaller clear follicle. The major preovulatory follicle (F,) is intended to ovulate the next day, the second largest (F2) on the next day (approximately 26 hours later) and so on. The control of follicular isolation and development within this hierarchy is poorly understood. The implication of pituitary gonadotropin has been proven, even the role of inhibin in the control of gonadotropin secretion in poultry and the ovulation control remains unclear. A recent strategy to induce hyperovulation in mammalian species has been the development of methods which involve the neutralization of endogenous inhibin activity. For example, active immunization of mammals against various compounds containing inhibin has been studied. The immunoneutralization of inhibin has been associated with increased ovulation regimes in heifers, sheep, sows and rats. Accelerated ovulation regimens found in mammals vaccinated with preparations of antigenic inhibin is thought to be a consequence of elevated FSH levels in the plasma, which lead to improved follicular development of the ovary. A variety of antigens have been used as vaccines in studies demonstrating an elevation in the ovulation regimen of mammals. Some of the antigens tested in mammals include: Recombinant DNA derived from fragments of the inhibin a subunit (Wrathall et al., Effects of active immunization against a synthetic peptide sequence of the inhibin a-subunit on plasma gonadotrophin concentrations, ovulation rate and lambing rate in ewes, J. Reprod. Fert ., 95: 175-182, 1992; and Meyer et al., Antiserum to inhibition of Alpha-Chain Peptide Neutralizes Inhibin Bioactivity and Increases Ovulation Rate in Sheep, Scientific Journal Series of the Minnesota Agrie. Exp. Sta., Paper No. 17,103, 1991) replicates of synthetic peptides from the N-terminal sequence of bovine inhibin-a-subunit coupled with ovalbumin (Glencross et al., Effect of active immunization of heifers against inhibin on plasma FHS concentractions, ovarian follicular development and ovulation rate, Journal of Endocrinolog ?, 134, 11-18, 1992), synthetic peptide sequences of the bovine inhibin subunit conjugated to human serum albumins (Morris, et al Effect of immunization against synthetic peptide sequences of bovine inhibin a-subunit on ovulation rate and twin-calving rate in heifers, Journal of Reproduction and Fertility, 97: 255-261, 1993), and partially purified inhibin of bovine follicular fluid (Morris, et al Effect of immunizing Prepuberal Lambs of Low and High Ovulation Rate Genotypes with Partially Purified Inhibin from Bovine Follicular Fluid, Theriogenology, Vol 35 No. 2, 1991). Despite the conflicting data on how FSH levels fluctuate during the ovulation cycle, in all cyclization mammals studied, immunoneutralization of endogenous inhibin consistently increased ovarian follicular development and ovulation regimen, without considering the antigen used or the species of mammal attacked. As stated previously, the involvement of inhibin in the regulation of reproductive function in poultry species remains uncertain. In this way, published reports have been restricted to the reproductive function of inhibin in domestic poultry. The volume of this literature supports the theory that inhibin probably exerts parallel physiological roles in poultry to those documented in mammals: in chickens, inhibin can serve as a regulator of follicular isolation and / or development. However, in birds, the involvement of inhibin in the control of the ovulation regimen may or may not be through the suppression of FSH secretion from the pituitary. For example, although chickens with low egg production have been found to have higher levels of inhibin in plasma and cell layers. granulose of the preovulatory follicles that in hens with high egg production, no difference in the plasma FHS levels associated with the egg production regime has been found. Wang et al., Increase in Ovarian a- Inhibition Gene Expression and Plasma Immunoreactive inhibin Level is Correlated with a Decrease in Ovulation Rate in the Domestic Hen. General and Comparative Endocrinology, 91, 52-58 (1993). Therefore, this reference suggests that in chickens, the changes related to the ovulation regimen in the expression of the inhibin a subunit gene and the levels of immunoreactive inhibin in the plasma do not directly affect the ovulation regimen through a modulation of FSH levels in the plasma. In addition, in Johnson, PA, Inhibin in the Hen, Poultrv Science, 72: 955-958, (1993), an RIA system of bovine inhibin was used to successfully determine immunoreactive inhibin in hen plasma, however, no significant peak of immunoreactive inhibin was detected through the ovulatory cycle despite a preovulatory LH surge. Therefore, the role of inhibin in folliculogenesis in birds remains unclear. Recently, the a subunit of chicken inhibin was successfully cloned and sequenced. Wang and Johnson, Complementary Deoxyribonucleic Acid Cloning and Sequence Analysis of the a-Subunit of Inhibin from Chicken Ovarían Granulosa Cells, Biology of Reproduction, 49, 1-6 (1993), which is incorporated herein for reference in its entirety. The comparison of the inhibin sequence of poultry to know the mammalian inhibin a subunit sequences showed a homology of 86-89%. Northern staining analysis using two isolated probes (cINAg and cINA12) revealed that the a subunit of inhibin is expressed in ovarian granulosa cells of chickens, but not in the brain, kidney, liver or spleen tissues of chickens. Therefore, the biology of inhibin in birds remains poorly understood, and the responses of birds for exchange with antigenic inhibin have not yet been attempted or verified. Therefore, as the ratite market is substantially limited by the suboptimal production yield of many ostriches, emus and ñandus, what is needed is a composition and method to improve the production yield of these birds. In addition, the improvement of the production performance of all poultry is necessary to increase the amount of poultry produced for consumption and to improve the efficiency of such production, or feed conversion ratio. Accordingly, there remains a need for a composition and method for improving or increasing the yield of production for all poultry, including chickens, turkeys, ducks, quails and geese, among others. There is also a need for a composition and a method to improve production performance in exotic birds, such as Psitaciformes. Psittaciformes include parrots, and are a monofamilial order of birds that exhibit zigodactilism and have a strong hook beak. A parrot is defined as any member of the poultry family of Psittacidae (the individual family of psittaciformes), distinguished by a short, strong, strongly hooked beak. The need for a composition and method for improving production yield is not limited to birds. There remains a need for effective composition and methods to improve production performance in many animals. For example, there is a continuing need to improve the yield of production in most of the animals that arise agriculturally, such as pigs, cows and sheep. There is also a continuing need to improve production performance in hair-bearing animals such as mink, fox, otter, urones, raccoons and in rodents such as rats, mice, gerbils and hamsters used as pets and as subjects for laboratory research, and there is an increased need for other animals whose skins are used for decorative purposes. Also, a composition and a method is needed to improve the yield of production to increase the population of many animals such as exotic or endangered species to prevent their extinction. There is a continuing need to improve production performance on animals used for racing, training or exhibitions (competitions) such as horses, dogs, cats, zoo animals and circus animals. As shown by the increased demands for treatment of infertility in humans, there is also a need to improve production performance in humans. Accordingly, there is a need for a composition and method for improving the yield of production in many animals.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates, in general, to a method for improving the yield of animal production, by administering to the animal a heterologous protein composed of inhibin protein, or a fragment thereof, and a carrier protein. The present invention also relates to a method for improving the production performance of animals, by administering to the animal a fusion gene product comprising a gene that encodes the expression of the inhibin protein of subunit a, or a fragment thereof, and a gene that codes for the expression of a carrier protein. An effective amount of the heterologous protein or fusion gene product is administered to an animal, so that an immunological response occurs in the animal against the heterologous protein. It is understood that the method of the present invention improves the production yield of animals that produce inhibin. Preferably, the animal is a bird. More preferably the bird is a chicken. Another preferred bird is a ratite, such as an emu, ostrich, ñandu or cassowary. The present invention further relates to the above heterologous protein and the fusion gene product and methods for producing same. More particularly, the present invention is directed to a composition and method for making a heterologous protein composed of inhibin, or a fragment thereof, and a carrier protein. The inhibin protein or fragment thereof can be inhibin of poultry, inhibin of mammals, inhibin of fish or inhibin of reptiles. The carrier protein includes, but is not limited to, maltose binding protein, globulin shot, key lime hemocyanin, or bovine serum albumin, among others. The preferred carrier protein is maltose binding protein. The heterologous protein can be either an inhibin conjugated to the carrier protein or an inhibin fused to the carrier protein. The method for producing the fused heterologous protein comprises inserting cDNA, which is encoded to express inhibin, or a fragment thereof, to a vector containing coding information for the production of a carrier protein. After inserting the vector into an expression system, the heterologous fused protein is expressed by the system. Preferably, the heterologous protein is composed of ratin inhibin, such as ostrich inhibin, emu inhibin, and rhamnin inhibin. Another preferred heterologous protein is composed of chicken inhibin. The present invention is also directed to a method for improving the production performance in animals through the administration of the heterologous protein of the present invention, comprising inhibin protein or a fragment thereof, and a carrier protein. In one embodiment, the method comprises administering an effective amount of the protein to a female animal. In another embodiment, the method comprises administering an effective amount of the protein to a male animal. Preferably, an immunological response occurs in the animal directed against the heterologous protein. More preferably the immune response, which occurs in the animal, is also directed against the inhibin protein produced by the animal (endogenous inhibin). The present invention is also directed to a fusion gene product comprising a gene encoding the expression of the subunit inhibin protein or a fragment thereof, and a gene encoding the expression of a carrier protein. The gene encoding inhibin protein expression, or fragment thereof, can be encoded to express poultry inhibin, mammalian inhibin, fish inhibin or reptin inhibin. The gene encoding the expression of a carrier protein can be encoded to express maltose binding protein or bovine serum albumin, among others. The preferred gene encoded to express a carrier protein is encoded to express maltose binding protein.

The present invention also relates to a method for improving the yield of animal production, by administering to the animal a fusion gene product comprising a gene encoded for the expression of the inhibin protein of subunit a, or a fragment thereof. , and a gene encoded for the expression of a carrier protein. More particularly, the present invention also encompasses gene therapy methods, wherein the DNA sequences encoding inhibin or fragments thereof and a carrier protein, are introduced into an animal. The fusion gene product of the present invention can be administered directly to the animal, or it can be administered in a vector, or in a cell containing a vector having the fusion gene product therein. The method of the present invention improves the yield of production in female animals, which produce inhibin, such as mammals, reptiles, fish and birds. More particularly, this method improves the yield of production in galliforms and ratites. More particularly, this method improves the yield of production in chickens, turkeys, ostriches, emus and ñandus. This method also improves the yield of turtle production, including endangered turtle species. Unexpectedly, the method of the present invention increases the onset of puberty or egg production in animals. Also, the method of the present invention unexpectedly accelerates the onset of maximum egg production in an animal. In addition, the method of the present invention increases the intensity of egg production in an animal. In addition, the method of the present invention surprisingly extends the persistence of maximum egg production in animals. Furthermore, the method unexpectedly increases the total egg production of an animal's life span. In poultry, the method of the present invention also improves the feed conversion ratio of a bird. Also, the method of the present invention unexpectedly reduces or eliminates the effect of adverse production conditions on egg production regimes of animals exposed to such conditions. Such adverse conditions include high temperatures, overcrowding, poor nutrition and noise. Surprisingly, the method of the present invention also improves the yield of production in male animals, which produce inhibin, such as mammals, reptiles and birds. More particularly, the method of the present invention increases testosterone levels in male animals. Similarly, the method of the present increases the onset of puberty or sperm production in male animals. Also, the method of the present invention accelerates the onset of maximum sperm production in a male animal. In addition, the method of the present invention unexpectedly increases the intensity of sperm production (sperm count) by a male animal. Still further, the method of the present invention prolongs the persistence of maximal sperm production in animals. Also, the method improves the viability of sperm in animals. Moreover, the method unexpectedly reduces or eliminates the effect of adverse conditions on the production of sperm from animals exposed to such conditions. Such adverse conditions include high temperatures, overcrowding, poor nutrition and noise. The method of the present invention also surprisingly increases the libido and therefore the reproductive potential of a male bird. As stated above, the method of the present invention is used to improve the production yield of any animal that produces inhibin, including, but not limited to, most agriculturally arising animals, such as pigs, cows, sheep, turkeys. , quail, ducks, geese, chickens and fish in animals that wear hair such as mink, fox, otter, urones, rabbits and raccoons; laboratory animals, such as rats, mice, gerbils, and guinea pigs; for animals whose skins are used for decorative purposes such as lizards and snakes, - exotic or endangered species; animals used for racing, training or exhibitions (competitions) such as horses, dogs, cats, zoo animals and circus animals; and human beings. Additional poultry, wherein the method of the present invention improves its production yield, include ratites, psittaciformes, falconiformes, piciformes, strigiformes, paseriformes, coraciformes, raliformes, cuculiformes, columbiformes, galliformes, anseriformes and herdionas. More particularly, the method of the present invention can be used to improve the production yield of an ostrich, emu, ñandu, chicken, turkey, ducks, geese, quail, partridge, kiwi, cassowaries, parrots, parakeet, hawk, eagle, dove, cockatoo, singing bird, talking bird, blackbird, finch, singer, canary, toucan, mainato or sparrow. Accordingly, it is an object of the present invention to provide an inhibin composition that induces an immune response in an animal after its administration to an animal. It is a further object of the present invention to provide a heterologous protein comprising inhibin protein, or a fragment thereof, and a carrier protein.

It is a further object of the present invention to provide a composition comprising a fused heterologous protein composed of inhibin or a fragment thereof. It is a further object of the present invention to provide a method for producing a fused heterologous protein comprising inhibin protein or a fragment thereof and a carrier protein. It is yet another object to provide a fusion gene product comprising a gene encoded for the expression of the subunit inhibin protein or a fragment thereof, and a gene encoding the expression of a carrier protein. It is another object of the present invention to produce an immunological response directed against the heterologous protein of the present invention through direct injection of the fused gene product of the present invention to an animal. Still another object of the invention is to provide compositions and methods useful for gene therapy for demodulation of inhibin levels. It is another object of the present invention to provide a method for improving production performance in animals.

It is an object of the present invention to provide a method for improving the yield of production in birds. It is another object of the present invention to provide a method for improving yield performance in ratites. It is also an object of the present invention to provide a method for improving the production performance in chickens. It is another object to provide a method for improving the yield of production in reptiles. It is another object of the present invention to provide a method for improving the yield of production in mammals. It is another object of the present invention to provide a method for improving fish production performance. It is a further object of the present invention to provide a method for improving production performance in humans. These and other objects, aspects and advantages of the present invention will be apparent from the review of the following detailed description of the described embodiments and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a SDS-PAGE gel wherein A is anti-ostrich antibodies (chicken inhibin maltose binding protein), b is the normal vector of pMALMR-c, C is a normal protein molecular weight, D is a real pMAL -c vector used in the preparation of the fused heterologous protein, E is the purified fused chicken inhibin-maltose binding protein (heterologous protein) of the present invention and F is an eluent of a purification that does not It was loaded with the heterologous protein. Figure 2 is an illustration of the effect of immunoneutralization of the inhibin α subunit on day-old egg production ("HDEP") in Japanese quail using maltose binding protein fused to the protein encoded by cINA521. More particularly, Figure 2 is a graphic representation of the data in Example 8.

DETAILED DESCRIPTION The present invention relates generally to a method for improving the yield of animal production, by administering to the animal a heterologous protein composed of inhibin protein, or a fragment thereof and a carrier protein. The present invention also relates to a method for improving the production performance of animals by administering to the animal a fusion gene product comprising a gene encoded for expression of the alpha subunit inhibin protein, or a fragment thereof, and a gene that codes for the expression of a carrier protein. An effective amount of the heterologous protein or fusion gene product is administered to an animal so that an immunological response occurs in the animal against the heterologous protein. It is understood that the method of the present invention improves the production yield of animals that produce inhibin. Preferably, the animal is a bird. More preferably the bird is a chicken. Another favorite bird is a turkey. Yet another preferred bird is a ratite, such as an emu, an ostrich, a rhea or a cassowary. The present invention further relates to the above heterologous protein and the fusion gene product and to a method for producing the same. After the following definitions, the composition of the present invention is described in detail, followed by a detailed description of the methods of the present invention.

Definitions The term "bird" or "bird", as used herein, is defined as a member of the class of birds of animals that are characterized as vertebrates producing warm-blooded eggs, mainly adapted to fly. The term "ratites" as used herein, is defined as a group of mostly large, non-flying brooding birds, comprising several orders and including emus, ostriches, kiwis and cassowaries. The term "psitaciformes" as used herein, includes parrots, and are of a monofamilial order of birds that exhibit zigodactilism and have a strong hook beak. A "parrot" is defined as any member of the psitacidae family of birds (individual family of Psitaciformes), distinguished by the short, strong, strongly hooked beak. The term "chicken" as used herein, denotes both chickens used for egg production, such as Single Comb White Leghorns, as chickens that emerge for consumption, or young chickens. The term "egg" is defined herein as a large female sexual cell enclosed in a porous, calcareous or skin shell produced by birds and reptiles. "Egg production through a bird or reptile", as used in the present is the act of a bird that lays an egg, or "egg-laying". The term "ovules" is defined as a female gamete, and is also known as an egg. Therefore, egg production in all animals other than birds and reptiles, as used herein is defined as the production and discharge of an egg from an ovary, or "ovulation". Accordingly, it is understood that the term "egg" as used herein, is defined as a large female sex cell enclosed in a porous calcareous or skin shell, when it is produced through a bird or reptile, or is a ovule when it is produced by other animals. The terms "egg production start", "first egg production" and "puberty" in relation to birds are used interchangeably in the present, and denote when a bird lays its first egg. Accordingly, "accelerating the onset" of egg production or puberty in poultry, as used herein, denotes inducing an early date of the first egg production that a bird could normally have. Similarly, "puberty" and "sperm production initiation" in males are used interchangeably. The phrases "improve production performance", "increase production yield" and "increase production yield" are used interchangeably to detect an improvement in one or more of the following areas: accelerated onset of puberty (egg production or ovulation in females; sperm production in males); accelerated start of maximum egg production or ovulation in females or accelerated start of maximum sperm production in males; increased intensity of egg production in females or sperm in males; prolonged persistence of egg production in females or sperm production in males; production of increased total life egg or ovulation in females; improved feed conversion ratios; improved shell quality of the egg; improved resistance to adverse conditions such as high temperatures, overcrowding, poor nutrition, and noise; improved sperm viability in males; improved testosterone production in males, - improved volume of ejaculation; and increased libido in males. The phrase "intensity of egg production" is known to those skilled in the art and denotes the frequency of egg production. The phrase "total egg production of life" of a bird is defined as the total number of eggs produced by a bird throughout its life. The phrase "daily egg production of a hen" or "HDEP" as used herein is defined as the number of eggs produced by a particular group of hens per day. The phrase "accelerated onset of maximum egg production" or "accelerated onset of maximum egg production" as used herein denotes the period from birth to when the animal produces eggs or ovulates at 50% of its diet. peak production or ovulation regimen, which is shorter than the normal period from inception to maximum egg production. It should be understood that a method "reducing cholesterol levels" of an egg, as used herein, denotes a method to induce a bird to produce one or more eggs having a lower cholesterol content than the average cholesterol content. of eggs produced by birds of the same species. In contrast to the term bird or bird, the term "mammal", as used herein, is defined as a member of the Ma malia class, which is a large class of warm-blooded vertebrates containing animals, characterized by mammary glands, a body covered with hair, three oscicles in the middle ear, and a muscular diaphragm that separates the thoracic and abdominal cavities, red blood cells without nucleus and embryonic development in allantois and amnion.

The term "reptile", as used herein, is defined as any member of the class Reptilia which is a class of terrestrial vertebrates that characteristically lack hair, feathers and mammary glands, their skin is covered with scales, they have a heart of three chambers, and their pleural and peritoneal cavities are continuous. A heterologous protein, as used herein, is defined as a protein composed of inhibin protein or a fragment thereof, and a carrier protein. It should be understood that the terms "inhibin" and "inhibin fragment" are used interchangeably in the heterologous protein composition, the method for making the heterologous protein, and the method for using the heterologous protein of the present invention. It should also be understood that "cINA521", as used herein, denotes a sequence of 521 base pairs (SEQUENCE OF IDENTIFICATION NO: 1). The cINA521 codes for a portion of the inhibin alpha subunit of an ostrich, represented by the SEQUENCE OF IDENTIFICATION NO: 2. As used herein "MBP-cINA521 is the heterologous protein that is expressed from a recombinant host cell , after the cloning of cINA521 to a recombinant host cell and expressing a fused heterologous protein comprising maltose binding protein ("MPB") and the inhibin protein subunit of the inhibin protein encoded by cINA521.Preferably, MBP-cINA521 is produced in host E. coli cells after the expression of cINA521 cloned using the commercially available vector pMAL RM-c. Thus, "cINA521" denotes a nucleotide sequence, and "MPB-CINA521" denotes a fused heterologous protein. A fused heterologous protein, as used herein, is defined as two different proteins fused together, for example, a protein It is composed of inhibin protein or a fragment thereof, fused to a carrier protein. The fused heterologous protein is expressed from an expression system comprising a fused gene product containing a gene encoded for inhibin protein expression, or a fragment thereof, fused to a gene encoding the expression of a carrier protein. "Fused gene product" as used herein, is defined as the product resulting from the fusion of the gene encoded for the expression of the inhibin protein, or a fragment thereof, and the gene encoded for the expression of a carrier protein A heterologous conjugated protein as used herein, is defined as a protein composed of inhibin protein, or a fragment thereof, conjugated to a carrier protein. The conjugated heterologous protein is produced through a chemical reaction which binds the inhibin protein to the carrier protein with a covalent bond. An immunological response of an animal to a substance that has been administered to the animal, as used herein, is defined as the cell-mediated and / or humoral response of an animal that is specifically directed against the substance. The term "selectively interacting", as used herein, is defined as when two objects are associated with each other through a covalent bond, a non-covalent bond, a hydrogen bond, electrostatically, a receptor-ligand interaction, a enzyme-substrate interaction or through another binding or binding means. The association is selective since the two objects interact in a specific way, in a specific position or only with each other.

Compositions of Inhibin The present invention relates generally to a composition used in the method for improving production performance in animals, including birds. The composition is composed of a heterologous protein comprising inhibin protein or a fragment thereof and a carrier protein. Inhibin can be inhibin of any species of animal that produces inhibin. Inhibin includes, but it is not limited to inhibin of birds, inhibin of mammals, inhibin of reptiles, inhibin of amphibians or fish inhibin, among others. More specifically, mammalian inhibin includes, but is not limited to, cow inhibin, human inhibin, horse inhibin, cat inhibin, dog inhibin, rabbit inhibin, sheep inhibin, minin inhibin, inhibin fox, otter inhibin, ferret inhibin, raccoon inhibin, donkey inhibin, rat inhibin, mouse inhibin, hamster inhibin and pig inhibin. Bird inhibin includes, but is not limited to, ostrich inhibin, emu inhibin, rhea inhibin, cassowary inhibin, kiwi inhibin, turkey inhibin, quail inhibin, chicken inhibin, duck inhibin, goose inhibin and inhibin of members of the order of psitaciformes. A preferred inhibin is poultry or bird inhibin. A more preferred inhibin is ratin inhibin such as ostrich inhibin, emu or ñandu. A particularly preferred inhibin is ostrich inhibin. Another preferred inhibin is chicken inhibin.

More preferably, the heterologous protein of the present invention comprises a subunit inhibin protein, or a fragment thereof, and a carrier protein. Inhibin, or fragment thereof, can be isolated from animal fluids, expressed from genetically engineered cells in an expression system or synthetically produced from a series of chemical reactions. More particularly, the inhibin fragment includes, but is not limited to the following compositions: subunit inhibin α, β subunit inhibin, - 'recombinant DNA derived from fragments of subunit inhibin α or β subunit inhibin; replicates of synthetic peptide or inhibin fragments of subunit α or β subunit inhibin; synthetic peptide replications of the N-terminal sequence of the subunit inhibin, or β subunit inhibin; fragments of partially purified inhibin of follicular fluid; fragments of inhibin from subunit to endogenous or inhibin of subunit β and fragments of inhibin from subunit to exogenous or inhibin of subunit β. As stated above, it is more preferable that the inhibin fragment be a subunit inhibin (a), or a fragment thereof. By inhibin it is understood by one skilled in the art that it encompasses inhibin with amino acid substitutions that can make it more immunogenic or more active in a receptor. The inhibin in the heterologous protein is either fused to or conjugated to the carrier protein as described below. When inhibin is fused to the carrier protein, the heterologous protein is a "fused heterologous protein". Where inhibin is conjugated to the carrier protein, the heterologous protein is a "conjugated heterologous protein". A preferred heterologous protein is a heterologous fused protein. The identity of the carrier protein in the heterologous protein is not a critical aspect of the present invention. Any carrier protein known in the art can be used in the heterologous protein. Carrier proteins that can be used in the present invention include, but are not limited to the following group: maltose binding protein "MBP"; bovine serum albumin "BSA"; lime key hemocyanin "KLH" ovalbumin; flagellin; thyroglobulin; serum albumin of any species; gamma globulin of any species; syngeneic cells; syngeneic cells carrying la antigens; and polymers of amino acids D- and / or L-. A preferred carrier protein is MBP. Another preferred carrier protein is BSA if the heterologous protein will not be administered to a cow or horse. Yet another preferred carrier protein is ovalbumin if the heterologous protein will not be administered to a bird. The most preferred carrier protein is MBP. It is preferred that the carrier protein be immunogenic to the animal to be administered. The present invention also relates to a method for producing the heterologous conjugated protein of the present invention. Methods for producing conjugated proteins are known in the art. Methods for conjugating proteins to proteins are fully described in Antibodies, a Loboratory Manual, edited by Ed Harlow & David Lane, Coldspring Harbor Lab (1988), which is incorporated here for reference. Additional methods for producing conjugated heterologous proteins including conjugation reagents, such as dialdehydes, carbodiimides, bisdiazotized benzidine and others, carrier proteins, and immunization schedules, are described in detail in Chapter 38, pp. 605-618 and Chapter 42, pp. 665-678, in Section VI, "Preparation of Antibodies" in Neuroendocrine Peptide Methodology, edited by P. Michael Conn Academic Press, New York, 1989, which is incorporated herein by reference. Although conjugated proteins can be used in the methods of the present invention, fusion proteins are preferred. More particularly, the heterologous proteins that are fused produce a homogeneous product, where the different segments of the proteins are always fused in the same position, and the same number of segments of the proteins are fused. Also, the fused heterologous proteins can be produced uniformly, inexpensively, and in large amounts. In contrast, conjugated heterologous proteins are not as uniform as fused proteins. For example, depending on which proteins are to be conjugated, the conjugation reaction can produce a mixture of proteins having one or more conjugations, proteins that have conjugations in different locations or proteins that remain unconjugated. In addition, some of the conjugations can make the heterologous protein sterically hindered for its intended use (for example the immunogenic portion of the protein is sterically hindered). Also, the conjugation reaction conditions and reagents can degrade the proteins produced therein. For example, glutaraldehyde is commonly used in conjugation reactions, and this modifies the conformation of proteins. In addition, conjugated proteins are more expensive to produce in large quantities than fused proteins. The present invention is also directed to a fusion gene product comprising a gene encoding the expression of the alpha subunit inhibin protein or fragment thereof, in a gene encoded for the expression of a carrier protein. The gene encoding the expression of the inhibin protein, or fragment thereof, can be encoded to express poultry inhibin, mammalian inhibin, fish inhibin or reptin inhibin. The gene encoding a carrier protein can be encoded to express maltose binding protein or bovine serum albumin, among others. The preferred gene encoded to express a carrier protein is encoded to express the maltose binding protein. The fusion gene product and the method for making the fusion gene product are described in more detail below. Briefly described, the method for producing the fused heterologous protein of the present invention is comprised of the steps of inserting a fusion gene product into a coding region of a plasmid, transforming it into a host cell with the plasmid and expressing the protein heterologous fused from the host cell from methods well known in the art. More particularly, the method for producing the fused heterologous protein comprises inserting cDNA which is encoded to express inhibin or a fragment thereof, into a vector containing coding information for the production of a carrier protein. After inserting the vector into an expression system, the fused heterologous protein is expressed through the system. Many methods for making fused heterologous proteins are known in the art. Therefore, any method known in the art can be used to produce the fused heterologous protein of the present invention. Many commercially available expression systems and equipment can be used to prepare the heterologous fused protein of the present invention. An example of such a commercially available vector expression system and equipment is pMALMR-c from New England Biolabs, Beverly Massachusetts. The cytoplasmic expression of the fused heterologous protein occurs in the pMAL-c system. The method for producing the fused heterologous protein of the present invention from a pMAL MR-c kit is fully described below in Examples 1 and 2. Other sources of vector equipment and expression systems that can be used to produce the heterologous fused protein of the present invention include, but are not limited to: Pharmacia Biotech of Piscataway, New Jersey; and Clonetech, of Palo Alto, California.

The present invention further relates to a fusion gene product comprising a gene encoding the expression of the inhibin protein or a fragment thereof, and a gene encoding the expression of a carrier protein. The inhibin gene can be from any animal species that produces inhibin. The inhibin gene can be a bird inhibin gene, a mammalian inhibitory gene, a reptile inhibin gene, an amphibian gene, or a fish gene among others. More specifically, the mammalian inhibin gene includes, but is not limited to, bovine inhibin gene, human inhibin gene, equine inhibin gene, cat inhibin gene, dog inhibin gene, inhibin gene of sheep, gene of inhibin of mink, gene of inhibin of fox, gene of inhibin of nutria, gene of inhibin of ferret, gene of inhibin of raccoon, gene of inhibin of rat, gene of inhibin of mouse, gene of inhibin of hamster , donkey inhibin gene, and pig inhibin gene. The bird inhibin gene includes, but is not limited to, an ostrich inhibin gene, an emu inhibin gene, a rhea inhibin gene, or a casuarin inhibin gene, a kinase inhibin gene, a turkey inhibin gene, a quail inhibin gene, a chicken inhibin gene, an inhibin gene of any member of the psittacine order, an inhibin gene of any falconiform, an inhibin gene of any piciform, a inhibin gene of any straigiform, an inhibin gene of any coraciform, an inhibin gene of any raliform, or an inhibin gene of any passeriform, an inhibin gene of any cuculiform, an inhibin gene of any columbiform, a gene of inhibin of any galliform (domestic bird), an inhibin gene of any anseriform (goose, duck, other waterfowl), an inhibin gene of any herodion, and an inhibin gene of any of the following birds: hawk, eagle ila, dove, parakeet, cockatoo, macao, parrot, canary, maniato, toucan and perch bird (such as songbird, talking bird, blackbird, finch, singer, and sparrow). A preferred inhibin gene is a bird inhibin gene. A more preferred inhibin gene is a ratin inhibin gene. An especially preferred inhibin gene is an ostrich inhibin gene. Still another preferred inhibin gene is an emu inhibin gene. A more preferred inhibin gene is a rhamn inhibin gene. Another preferred inhibin gene is a chicken inhibin gene. The chicken inhibin alpha subunit cDNA clone (cINA6; Wang and Johnson, Complementary Deoxyribonucleic Acid Cloning and Sequence Analysis of the a-Subunit of Inhibin from Chicken Ovarian Granulosa Cells, Biologv Of Reproduction, 49: 453-458, 1993) , which is incorporated herein by reference in its entirety, inserted into the Bluescript EcoR 1 site (Stratagene, La Jolla, CA), was obtained as a donation from PA Johnson (Cornell University). The cINA6 clone specifically hybridized to ostrich genomic DNA in Southern assays indicating significant DNA homology between these two species (Chouljenko et al., Expression and purification of chicken a-inhibin as a protein fusion with E. coli maltose binding protein , Poultry Science, 73 (Suppl 1): 84, 1994). A DNA fragment ("cINA521") was divided from clone cINA6 using Pst I digestion. The cINA521 DNA fragment comprised the majority of the a subunit of mature chicken inhibin. Although A521 was separated from clone cINA6 reported in Wang and Johnson, the sequence obtained, mainly SEQUENCE OF IDENTIFICATION NO: 1, differs from the DNA sequence published by Wang and Johnson. The ostrich inhibin a subunit sequence was obtained through polymerase chain reaction (PCR) methods that are well known in the art. More particularly, the following primers were constructed based on the sequence reported by Wang, and were used in the PCR reaction with the ostrich genomic DNA: 5 '-CTCAGCCTGCTGCAGCGCCC-3'; and 5 '-GTGTCGACCGCGCGACGCCGAC-3'. More particularly, the above primers correspond to base pairs 778 to 798 and 1348 to 1326, respectively, of the chick inhibin α subunit cDNA clone, CINA6 reported by Wang and Johnson. The PCR product was digested with endonuclease Pst 1 and subcloned into the commercially available vector PUC19 (New England Biolabs). The inhibin gene sequence of the ostrich fragment Pst 1 is identical to the corresponding portion of chicken inhibin a. As stated above, it is understood that the carrier protein is not a critical aspect of the present invention, therefore, a gene encoded to express any carrier protein can be used in the present invention. The carrier protein gene includes, but is not limited to, genes that code for expressing the following proteins: maltose binding protein "MBP"; bovine serum albumin "BSA"; key lime haemocyanin "KLH"; ovalbumin; flegelin; thyroglobulin; serum albumin of any species, gamma globulin of any species; syngeneic cells; syngeneic cells that carry antigens la; and polymers of amino acids D- and / or L-. The preferred carrier protein gene is a gene that codes for expressing MPB. Another preferred carrier protein gene is a BSA gene if the resulting heterologous protein will not be administered to a cow or horse. Yet another preferred protein gene is an ovalbumin gene if the resulting heterologous protein will not be administered to a bird. The most preferred carrier protein gene is a gene that codes for expressing MBP or its derivatives. Preferred carrier protein genes encode proteins that will increase both the intensity and duration of the host immune response to the inhibin protein. The present invention further relates to a method for making a fusion gene product comprising the step of fusing a gene encoding inhibin protein expression, or a fragment thereof, to a gene encoding the expression of a carrier protein. Briefly described, the method for making the fusion gene of the present invention comprises the steps of isolating the preferred inhibin complementary DNA (cDNA), which produces double chain structure inhibin DNA, to obtain a carrier protein double chain, and fusing the double stranded structure inhibin DNA to the double stranded structure carrying protein DNA in such a way that the fused DNA allows the expression of a fused heterologous protein comprising the inhibin protein, or a fragment of it, and the carrier protein.

Many methods for isolating genes and making fusion gene products are known in the art. See, for example Sambrook, Fritsch & Maniatis, Molecular Cloning, A Laboratory Manual, 2nd? D. Cold Spring Harbor Laboratory Press, 1989, Vols. I, II, III. Therefore, any method known in the art can be used to produce the fusion gene product of the present invention. Commercially available vector equipment can be used to prepare the gene product of the present invention. An example of such commercially available vector equipment is pMAL MR-c from New England Biolabs, Beverly, Massachusetts. The method for producing the fusion gene product of the present invention from a pMAL -c kit is fully described below in Example 1. Other sources of vector equipment that can be used to produce the fused gene product of the present invention include, but are not limited to: Pharmacia Biotech of Piscataway, New Jersey; and Clonetech, of Palo Alto California. As stated above, the cDNA clone subunit of chicken inhibin (cINA6) inserted into the Bluescrip EcoR 1 site was obtained as a donation from P. A. Johnson (Cornell University). A DNA fragment ("cINA521") was separated from clone cINA6 using Pst I digestion. The fragment (cINA52?) Sf2 cloned into the plasmid p-MALMR-c in frame with the maltose binding protein ("MBP" ) and a fusion protein of appropriate size (Line E; Figure 1) was detected after IPTG (isopropyl β-D-thiogalactopyranoside) induction and SDS-PAGE. The resulting protein conjugate ("MBP-cINA521") was used as an antigen to immunize Japanese prepubescent female quail (Coturnix coturnix japonica) against circulating inhibin levels as fully described in Example 8.

Methods for Improving Production Yield It has been unexpectedly discovered that the composition of the present invention improves the yield of animal production, and in particular the yield of bird production. Accordingly, the present invention is also directed to a method for improving the production performance in animals through the administration of the heterologous protein of the present invention. In one embodiment, the method comprises administering an effective amount of the protein to a female animal so that the production yield of the animal is increased. In another embodiment, the method comprises administering an effective amount of the protein to a male animal so that the production yield of the animal is increased. Preferably, an immune response occurs in the animal directed against the protein. More preferably, the immune response that occurs in the animal is also directed against the inhibin protein produced by the animal (endogenous inhibin). More particularly, the method of the present invention comprises administering an effective amount of the heterologous protein of the present invention (comprising inhibin or a fragment thereof, and a carrier protein) to an animal, so that performance is improved of animal production. Preferably, the animal is a bird. It should be understood that a "treated" bird is a bird to which the heterologous protein of the present invention has been administered. The method of the present invention can be used to improve the yield of production in any species of female bird that produces inhibin. The female bird includes, bolt is not limited to, a ratite, psitaciformes, falconiformes, piciformes, estrigiformes, pseriformes, coraciformes, raliformes and cuculiformes, columbiformes, galliformes (domestic bird), an anseriforme (goose, ducks, and other aquatic birds) , and herodionas. More particularly, the female bird includes, but is not limited to, an ostrich, emu, ñandu, kiwi, cassowary, turkey, quail, chicken, hawk, eagle, pigeon, parrot, cockatoo, macao, parrot, bird perch. (such as songbird, talking bird, blackbird, finch, singer, and sparrow) and any number of other psitaciformes. A favorite bird is a ratite. A more preferred bird is an ostrich. Another favorite rattle is an emu. Still another favorite ratite is a rhea. Another preferred bird is any member of the order of psitaciformes. Still another favorite bird is a chicken. Still another favorite bird is a quail. The method of the present invention can also be used to accelerate the onset of egg production in endangered bird species. Such endangered birds include, but are not limited to eagles, hawks, condors, and owls. The inhibin and the carrier protein in the heterologous protein composition of the present invention vary according to which species of bird, the composition will be administered. It is preferred that the poultry inhibin and the maltose binding protein be used when the composition is to be administered to a bird. A preferred inhibin is a domestic chicken or ratite inhibin, when the composition is to be administered to a ratite. More preferably, the preferred inhibin is inhibin of domestic chicken or ostrich, when the composition is to be administered to an ostrich or ratite. Another preferred inhibin is an inhibin of domestic chicken or ostrich, when the composition is to be administered to a chicken. It should be understood that inhibin in the heterologous protein need not be of the same species to which the heterologous protein will be administered. For example, a heterologous protein that is administered to an ostrich may be composed of chicken inhibin and a carrier protein. It should also be understood that the composition may further comprise auxiliaries, preservatives, diluents, emulsifiers, stabilizers and other known components that are known and used in vaccines of the prior art. Any auxiliary system known in the art can be used in the composition of the present invention. Such auxiliaries include, but are not limited to, Freund's incomplete adjuvant, Freund's complete adjuvant, acetylated maleate bonded to polyspersed β- (1,4) polyesters ("Acemannan"), Titermax® (polyoxyethylene-polyoxypropylene copolymer auxiliaries from CytRx Corporation ), modified lipid auxiliaries from Chiron Corporation, Cambridge saponin-derived auxiliaries, Biotech, Killed Bordetella pertussis, lipopolysaccharide (LPS) of gram negative bacteria, large polymer anions such as dextran sulfate and inorganic gels such as alum, aluminum hydroxide, or aluminum phosphate. A preferred auxiliary system is Freund's incomplete auxiliary. Another preferred auxiliary system is the complete Freund auxiliary. The heterologous protein composition of the present invention can be administered to a bird by any means known in the art. For example, the composition can be administered subcutaneously, intraperitoneally, intradermally, or intramuscularly. Preferably, the composition is injected subcutaneously. The composition can be administered to the bird in one or more doses. Preferably, the composition is administered to the bird in multiple doses, wherein an initial immunization is followed by booster immunizations. The composition can be administered to an animal at any time before the animal stops ovulating or producing sperm due to disease or age. The preferred age at which the composition of the present invention is administered to an animal depends on the species of the animal involved, the mating season (if any) of an animal and the purpose of administration of the composition. For example, when the composition is administered to accelerate the onset of egg production or sperm production, the composition of the present invention is to be administered to a bird before the bird reaches egg production or puberty. As stated above, the preferred age at which the composition of the present invention administered to an animal first depends on the species of the animal involved, the mating season (if any of an animal), the size of the bird, and the identity of the components (inhibin and carrier protein) in the composition. As another example, when the composition is administered to improve the yield of production of agricultural animals, which have breeding seasons, the preferred time to administer the composition is before the start of the breeding season. In contrast, when the composition is to be administered to a mature animal, which has a suppressed egg production regime or a suppressed sperm production regime, then the composition could be administered at the time the suppression is recognized as problem. With respect to an animal having a breeding season, although the heterologous protein of the present invention can be administered to a bird such as a ratite of any age, the bird immunization during the six months before the first breeding season of the Bird is preferable. It should be understood by those skilled in the art that average female birds initiate egg production during the first breeding season. It is still more preferable to immunize the bird approximately six months before the first breeding season of the bird, and then to administer booster immunization at intervals of one month before the first breeding season of the bird. It is more preferable to immunize the bird approximately six months before the first breeding season of the bird, and then administer booster immunizations at one-month intervals for six months. For example, for best results to increase the egg production of a female ostrich, a primary immunization is administered to the ostrich approximately six months before its first breeding season, and then reinforcement immunizations are administered at one month intervals for six months. months The primary immunization comprises from about 0.5 to 4.5. mg of the heterologous protein of the present invention. Booster immunizations comprise from about 0.30 to 3.0 mg of the heterologous protein of the present invention. Preferably, the primary immunization comprises between about 1.5 to 3.0 mg of the heterologous protein of the present invention. Booster immunizations comprise between about 0.75 to 1.5 mg of the heterologous protein of the present invention. It is also preferable that the heterologous protein be emulsified in complete Freund's assistant (Sigma Chemical Co., St. Louis, MO) in the primary immunization, and that the heterologous protein be emulsified in an incomplete Freund's assistant (Sigma) in the immunizations of reinforcement. Even more preferably, the heterologous protein composition is injected subcutaneously. More preferably, the heterologous protein composition is injected subcutaneously at three sites along the region of the ostrich's upper thigh. The amount administered to a bird of the heterologous protein of the present invention varies according to the species of the bird, the age and weight of the bird, when the protein is administered in relation to the breeding season (if the bird has a breeding season). breeding), and how many times the protein is going to be administered. Also, the beginning of the administration schedule, or treatment schedule, varies according to the species of the bird, the average age of puberty of that species of bird, the family history of the bird (with respect to the family history of puberty age), the time of year in which the bird swelled, the nutritional plane of the bird (highly fed birds enter puberty before those that are poorly fed), the general health of the bird at the time of commencement, immunological competence of the bird, the long-term health history of the bird, the presence of extreme weather conditions (excessive inclement weather prolonged, such as rain, heat or winter to which the bird does not It is customary, accommodation conditions (overpopulation) and lack of exercise.An expert in the art, in view of the teachings of the present invention, may be able to determine by routine tests the amount of heterologous protein that will be administered to produce an immunological response to the protein by the bird Another example of the method to improve production performance is as follows: The immunologically effective conjugated heterologous protein composition is administered to a mammal such that an immunological response occurs in the mammal, which is directed against the heterologous protein. The heterologous protein is preferably composed of mammalian inhibin conjugated to the maltose binding protein. Another preferred heterologous conjugated protein is composed of inhibin from poultry or reptiles; and maltose binding protein. For example, the following is a brief summary of the method of the present invention for improving production performance in Japanese quail, as described in Example 8. The average age at puberty for an untreated quail is approximately 6 to 8. weeks The following may be a treatment schedule for Japanese quail having an approximate body weight of between 0.454 to 0.1135 kg (0.1 to 0.25 pounds): primary (first) injection of 0.75 mg of the heterologous protein of the present invention on its 25th day of age; and reinforcements of 0.375 mg on days 32, 39, 46, 53, 60 and 90 of age, followed by reinforcements every 35 days afterwards for three additional attacks (ie at 95, 130 and 165 days of age). More particularly, at the age of 25 days, 50 female quail were randomized and equally assigned to one of the two injection groups (25 birds per group), as follows: (1) MPB-cINA521 in Freund's auxiliary ("MBP- cINA521 / FRN "), or (2) Freund's auxiliary control; "FRN"). Birds immunized against inhibin (Group 1) were given approximately 0.75 mg of MBP-cINA? 2, per bird in the appropriate control vehicle. Equivalent vehicle injection volumes (0.2 ml) of FRN were used in Group 2. All injections were administered subcutaneously using tuberculin syringes equipped with 25-gauge needles. As discussed above, booster inhibin immunizations of approximately 0.375 mg of MBP-cINA521 per bird or appropriate control attacks were subsequently administered and the birds were observed for a total of 20 weeks. Starting on day 41 of age, which is considered to be day 1 of the egg production cycle, daily measures of egg production per day in the hen ("HDEP") and mortality ("MORT") were recorded daily. for 20 consecutive weeks. In addition, an average age at the first egg production ("FIRST") and an age at which the hens reach 50% of egg production ("FIFTY") were calculated for each of the treatment groups. As discussed more fully in Example 8, the HDEP, MORT, FIRST, and FIFTY data were subjected to variability analysis. The immunoneutralization of inhibin clearly accelerated puberty in quail hens. As shown in Table 2, the average age of FIRST egg production was reduced (P <.0088) by approximately six days in the hens treated with inhibin. Also, as shown in Table 3, the age of FIFTY egg production was markedly reduced (12 days; P < .01) in hens treated with inhibin. A positive effect of inhibin treatment of egg production intensity also existed, most notably at the start and end of the production cycle, as shown in Figure 2. For example, average HDEP regimens were observed (P < 05) significantly higher in hens treated with MPB-cINA521 / FRN when compared with the FRN controls during Weeks 1 (16.5 vs 2.6%), 2 (50.0 vs 28.6%), and 4 (96.6 vs 79.7%) and other time during Weeks 15 (98.8 vs. 86.9%), 16 (69.9 vs. 86.3%), 18 (85.7 vs. 66.1%) and 20 (96.8% vs. 73.8%). The total HDEP regimen, including the 20 weeks of production, for hens treated with inhibin was 83.5% compared to 75.4% for controls (P <.14). In addition to accelerating puberty, prolonging egg production and improving total production intensity, inhibin treatment reduced the time required to reach peak egg production by approximately 3 weeks. Referring now to Figure 2, compare MBP-cINA521 / FRN, which is at 96.6% HDEP in Week 4 against FRN which is at 96.6% HDEP in Week 7. Although the differences in the peak values of HDEP were not statistically evaluated, the treatment differences in the mean age at which the hens reached levels of 50% HDEP, reflected the peak yield (FIFTY). Mortality was not a factor in this study, since only eight birds died (three controls, and five of treatment). The mortality of 16% is within the expected limits for quail that reached 180 days of age. The most calculated biological responses to the treatments were studied for the effects of onset, magnitude and duration of response. In the present, the data represent that a complete Japanese quail production cycle could be considered (ie 20 weeks post-pubertal or egg production), therefore, the following comments on the effects of inhibin immunoneutralization at the start , magnitude and duration of egg production in this species are justified.The data support the conclusion that the onset of puberty was accelerated in the immunoneutralized group with inhibin.This was evidenced in the marked differences in treatment observed in the variables FIRST and FIFTY and in the differences observed during the initial weeks of the HDEP data The acceleration of puberty coupled with the increased persistence of egg production in birds attacked with inhibin contributed to an increase in total HDEP that was marked ( 8.1%) For example, on a per-hen basis the inhibin treatment essentially resulted in a daily gain of approximately 0.081 eggs for each day of the production cycle that a hen remained viable (that is, capable of producing an egg). This means that approximately 11 more eggs were obtained for each chicken housed during 20 weeks in the form of examination (0.081 eggs / hen for 140 days of production = to 11.34 eggs per hen per production cycle). Similar results in chickens and turkeys, as found in Coturnix, will have substantial strategic relevance in the poultry industry. It should be noted that Japanese quail have been selected by intensity of egg production, and that the egg production potential is considered to be the highest in Coturnix than in the single comb (Single Comb White Leghorns) commercially bred for the sole purpose of table egg production. Therefore, the intensification of egg production through the inhibin vaccine in chickens that have not been selected for egg production but for meat production, for example breeding of young chickens, the increase for the consumption of their meat, it can be even bigger. Accordingly, the above data shows that the inhibin composition of the present invention improves the yield of production as it accelerates the onset of puberty., increases the intensity of egg production and accelerates the start of maximum egg production in Japanese quail. Since Japanese quail are an acceptable animal model for chickens with respect to their reproductive systems, the above data indicates that the method of the present invention will also accelerate the initiation of egg production in chickens. Accordingly, the method of the present invention will result in an egg producer that is capable of producing more eggs with lower food costs. The above data also show that the inhibin composition of the present invention improved the production yield as it minimized the adverse effects of high temperatures of the egg production regime of Japanese quail. More particularly, in Week 18 of the study described in Example 8, the quails were inadvertently exposed to elevated temperatures. As can be seen in Figure 2, birds in Group 1, (treated with MBP-cINA521 / FRN) sustained a drop in the egg production rate of approximately 5%. In contrast, the birds in Group 2 control: FRN) sustained a drop in the egg production rate of approximately 26%. Accordingly, the method of the present invention for improving the yield of production decreases the negative impact on the egg production regimes of poultry exposed to adverse egg production conditions. This aspect of the invention is important since poultry usually grows in open, uncontrolled environments. Consequently, poultry supplies are usually exposed to adverse conditions such as high temperatures, and other extreme weather conditions that are not acclimatized, thus reducing egg production regimes in the poultry industry . The following is a brief summary of the method of the present invention for improving production performance in ostriches as discussed in Example 9. The average age at puberty for untreated ostriches is approximately 28 and 32 months. The following could be the treatment schedule for ostriches that have a body weight scale of approximately 68.1 to 136.2 kg. (10 to 300 pounds): injection (primary) of 5.0 mg of the heterologous protein of the present invention at its 26th month of age; and reinforcements of 2.5 mg at 27, 28, 30, 32, 34 and 36 months of age. The following is a brief summary of the method of the present invention for improving emu production performance as discussed in Example 10. The average age at puberty for an untreated emu is approximately 20 months. The following could be the treatment schedule for an emu on an approximate body weight scale of 22.7 to 40.8 kg (50 to 90 .lbs); primary (first) 3.0 mg of the heterologous protein of the present invention at its 18th month of age; and reinforcements of 1.5 mg in the 19, 20, 22, 24, 26 and 30 months of age. The following is a brief summary of the method of the present invention for improving the production yield in chickens as discussed in Example 11. The average age at puberty for a chicken is approximately 20 weeks. The following could be the treatment schedule for a chicken that has a body weight scale of approximately 0.908 to 6.58 kg (2.0 to 3.5 pounds). Primary (first) injection of 1.5 mg of the heterologous protein of the present invention at week 15 of age; and reinforcements of 0.75 mg in the 17, 20, 24, 30, 40 and 50 weeks of age. The following is a brief summary of the method of the present invention for improving turkeys production performance as discussed in Example 12. The average age at puberty for an untreated turkey is approximately 30 weeks. The following could be the treatment schedule for a turkey that has an approximate body weight scale of 4.08 to 5.4 kg (9.0 to 12 pounds); primary (first) injection of 2.0 mg of the heterologous protein of the present invention at its 28th week of age; and 1.0 mg boosters at 29, 30, 34, 38, 46 and 54 weeks of age. The following is a brief summary of the method of the present invention for improving parrot production performance as discussed in Example 13. The average age of puberty in an untreated parrot is approximately 30 months. The following could be the treatment schedule for a parrot having an approximate body weight scale of 0.22 to 0.56 kg (0.5 to 1.25 pounds): primary (first) injection of 0.75 mg of the heterologous protein of the present invention in its month 28 years old; and reinforcements of 0.375 mg in the 29, 30, 32, 34, 36 and 38 months of age. As discussed above, the method of the present invention improved the yield of production by accelerating the onset of puberty in the animal to which the composition of the present invention was administered. The term "accelerate" with respect to the start of egg production denotes that the egg production of a treated bird begins at least about 3% earlier than the egg production that ordinarily begins in an untreated bird. Preferably, the production begins at least about 5% before, and more preferably begins at least about 7% before. Even more preferably, egg production begins at least about 10% before, and more preferably begins at least 13% before egg production could ordinarily begin on an untreated bird. Also, as discussed above, the method of the present invention improved the production yield by increasing egg or sperm production in the animals. The term "increase" with respect to egg production, denotes that the egg production of a treated bird increased approximately at least 3% with respect to the amount of egg production in an untreated bird. Preferably, egg production increased by at least about 7% and more preferably increased by at least about 12%. In addition, as discussed above, the method of the present invention improves production yield by accelerating the onset of maximum egg production in an animal. The term "accelerate" with respect to the start of maximum egg production, denotes that the maximum egg production of a treated bird begins at least about 3% before the egg production could ordinarily start in an untreated bird. Preferably, maximum egg production begins at least about 5% earlier, and most preferably begins at least 7% earlier. Still most preferably, maximum egg production begins at least about 10% earlier, and more preferably begins at least 13% earlier than maximum egg production could ordinarily begin in an untreated bird. Surprisingly, the composition of the present invention can also be used to increase the total life of bird egg production. The term "increase" with respect to the life of egg production denotes that the total life of the egg production of the treated bird is increased by at least about 3% with respect to the total egg production life of a bird not treaty. Preferably, the total life of egg production is increased by at least about 7%, and more preferably by at least about 12%. More preferably, the total life of the egg production is increased by at least about 15%. Unexpectedly, the composition of the present invention can also be used to reduce or eliminate the need for feather fashion of a female bird, for example, to prolong the persistence of egg production, by providing a second production cycle. More particularly, if the composition described above, is continuously administered to a female bird, as described in the above method, the rate of egg production of the bird, compared to the bird that was not treated with the composition of the present invention. , it could remain high enough that the bird could not need to change feathers to improve its egg production regime. In a common practice in the art, changing feathers of a female bird, such as chickens (Single Comb White Leghorns, egg producers), when their egg production is reduced, so that the economic cost to keep the bird saturates the economic benefit produced by the eggs produced. To "move from feathers" to a chicken, the bird undergoes a fasting period of about four to fourteen days until it begins to move, for example, to lose its feathers. During the molting period, the bird stops producing eggs. After the bird is returned to normal feeding levels, egg production begins again after a period. The entire molting period is approximately two months from the beginning of the fasting period to the start of the next egg production cycle. In reality, the egg production regime of the bird is rejuvenated. However, after the molting of a chicken, its egg production regime for the next cycle is not the same as egg production during the first cycle of egg production (before moulting) M. North and D. Bell, Commercial Chicken Production Manual, fourth edition, Chapter 19, Published by Van Norstrand Reinhold of New York. For example, chickens reach egg production at approximately 20 weeks, and produce an economically significant number of eggs of approximately 40 to 50 weeks. At the peak of egg production, chickens produce eight to nine eggs every ten days. However, after approximately 50 weeks of egg production, the egg production rate decreases from approximately 60% of the peak egg production. At this point, the cost of feeding the chicken is greater than the value of the eggs it produces. It is an important practice to move the chicken at this time, so that when the egg production of the chicken begins again, its egg production regime increases. By "prolonging the persistence of egg production" with reference to chickens and quail, among other birds, it is understood that egg production will be prolonged for approximately one to four weeks. Therefore, the composition of the present invention, as it maintains the egg production rate at a higher level than the bird was not treated with the composition, reduces or eliminates the need to move the bird. Reducing or eliminating the need to move feathers to a bird results in significant savings. More particularly, during the period in which the bird is moving, and before that time, the bird is unproductive with respect to its feeding cost before it moves from feathers, and then it is unproductive for a period after feeding begins . Maintaining the egg feeding regime, at an improved level, therefore eliminates or reduces these unproductive phases of the bird, thus reducing producer costs and increasing the producer's benefits. Maintaining the egg production regime at an improved level, egg producer levels are also improved as the egg production regime after moulting, which is not equal to the egg production regime in the first production cycle of egg. egg as discussed above. Briefly described, the egg egg production regime could be increased, thus avoiding the need for bird feather moulting, by administering an effective amount of the heterologous protein of the present invention to induce an immunological response thereof, and then administer an effective amount of heterologous protein (boosters) to maintain a higher than normal egg production regimen. Accordingly, the method of the present invention improves the yield of production in female animals that produce inhibin, such as mammals, reptiles, and birds such as ratites. More particularly, this method improves production performance in ratites such as ostriches, emus and ñandus and in chickens. Unexpectedly, the method of the present invention increases the onset of puberty or primary egg production in animals. Also, the method of the present invention accelerates the onset of maximum egg production. In addition, the method of the present invention increases the number of eggs laid by an animal. In addition, the method of the present invention prolongs the persistence of maximum egg production in animals. Even more preferably, the method increases the total life of an animal's total egg production. In poultry, the method of the present invention also improves the conversion ratio of the bird. Also, the method of the present invention reduces or eliminates the effect of adverse production conditions on egg production regimes in animals exposed to such conditions. Such adverse conditions include high temperatures, saturation, poor nutrition and noise. Although not intended to be limited to the following, a theory is applied that the method of the present invention for improving the yield of production in animals provides a greater increase in egg production in species that have not been genetically selected for the Prophylactic egg production treatment. This is particularly true for certain poultry. For example, egg-type chickens have been genetically selected for maximum production yield since the late 1920s (See, for example, Jull, M.A., 1932, Poul try Breeding, John Wiley &; Sons). In terms of a chicken's short life span, a large selection target for this occurred during the period from about 1928 to the present. In contrast, the ratites and psittaciformes, the other exotic birds, and to a lesser degree meat-type chickens (young chickens) have not been genetically selected for the prophylactic egg production treatment. Also, the birds that are in danger have not been genetically selected for the prophylactic egg production treatment either. Therefore, since egg-type chickens are already genetically excellent egg producers, the amount of improvement that can be seen with the method of the invention is limited in comparison to the birds that genetically are egg producers of poor to medium. . Therefore, a much greater amount of improvement in production yield is seen with the method of the present invention with birds that have not been genetically selected for prophylactic egg production, such as ratites, psittaciformes, and other exotic birds, Endangered birds, turkeys and meat-type chickens. Immunization of an animal with the heterologous protein of the present invention induces the animal to produce antibodies selectively directed against the heterologous protein. Preferably, the immunization also induces the animal to produce antibodies selectively directed against endogenous inhibin. The production of such antibodies reduces the time of onset of puberty or egg production. The production of such antibodies from the animal also improves the animal's egg production capacity or sperm production, since the antibodies neutralize the biological activity of inhibin in the animal's bloodstream. Not wishing that it is bound by theory, it is believed that the a subunit of inhibin binds to FSH receptors and therefore, competitively inhibits the binding of FSH to such receptor sites. By reducing inhibin levels that can bind with receptor sites, therefore the biological effect of FSH in the animal is increased, as competition for FSH receptor sites is reduced. It is believed that the antibodies neutralize inhibin by interacting with inhibin with circulation, thereby interfering steinarically with the binding of the inhibin-interacted to the FSH receptor sites. Unexpectedly, the method of the present invention also improves the yield of production in male animals, which produce inhibin, such as mammals, reptiles and birds. More particularly, the method of the present invention increases testosterone levels in male animals. Similarly, the method of the present invention increases the onset of puberty or sperm production in male animals. Also, the method of the present invention accelerates the onset of maximum sperm production in a male animal. In addition, the method of the present invention increases the intensity of sperm production (sperm count) by a male animal. Still further, the method of the present invention prolongs the persistence of maximal sperm production in animals. Also, the method of the present invention increases the volume of ejaculation in male animals. In addition, the method improves the viability of sperm in animals. Furthermore, the method unexpectedly reduces or eliminates the effect of adverse conditions of sperm production in animals exposed to such conditions. Such adverse conditions include high temperatures, saturation, poor nutrition and noise. The method of the present invention also unexpectedly increases the livid, hence the reproductive potential of a male bird. Another unexpected and surprising aspect of the present invention is that the composition herein also produces more eggs having a reduced cholesterol content compared to eggs laid by untreated animals. More particularly, if the composition described in the above is administered to a female bird, as discussed in the above method, the cholesterol content of the eggs laid by the bird will be reduced by a longer period compared to the bird that was not treated with the composition of the present invention. Therefore, the composition of the present invention increases or produces a greater number of eggs with a low cholesterol content. The terms "increase" or "number greater than" denotes that the number of eggs with a low cholesterol content produced by a treated bird increases by at least approximately 2% with respect to the number of eggs on a low cholesterol content produced for an untreated bird. Preferably, the number of eggs with a low cholesterol content produced increases by at least about 5%, and more preferably increases by at least about 10%. It should be understood that a "treated" bird is a bird to which the heterologous protein of the present invention was administered. The term "low cholesterol content" or "lower cholesterol content" denotes that the cholesterol content of an egg is lower than the average content of egg cholesterol produced by bird species during the life of such bird by at least approximately 10%. Preferably, the cholesterol content of an egg with a low cholesterol content is less than average by at least about 20%. More preferably, the content of egg cholesterol with a low cholesterol content is less than average by at least about 30%. It is known that in chickens, the first five to six eggs that they put for a female bird (chicken) after they reach puberty, have a lower cholesterol content than the eggs they produce later. The composition of the present invention induces the female bird to lay eggs that contain a lower cholesterol content for a longer period. Due to the health consequences associated with high levels of cholesterol in the blood, there is a need for egg products with a low cholesterol content. Accordingly, the composition of the present invention provides the egg producer with a higher number of a highly sought-after type of egg.

Gene Therapy Using the Fusion Gene Product The present invention also relates to improving the production performance of animals, by administering to the animal a fusion gene product, comprising a gene encoding the expression of the inhibitory protein of the gene. alpha subunit or fragment thereof, and a gene that codes for the expression of the carrier protein. The fusion gene product of the present invention can be administered directly to the animal or can be administered in a vector, or in a cell containing a vector having a fusion gene product therein. Various methods to transfer or supply the DNA to cells for the expression of the protein to gene product, otherwise referred to as gene therapy is described in Gene Transfer into Mammalian Somatic Cells in vivo. N. Yang, Crit. Rev. Biotechn. 12 (4): 335-346 (1992), which is incorporated herein by reference. Gene therapy encompasses the incorporation of DNA sequences into somatic cells or germline cells for use in a therapy either ex vivo or in vivo. Gene therapy works to replace genes, or increase normal and abnormal gene function. Strategies for gene therapy include therapeutic strategies such as identification of a defective gene and then adding a functional gene either to replace the function of the defective gene or to increase a slightly functional gene; prophylactic strategies, such as adding a gene for the product protein. As an example of prophylactic strategy, a fused gene product that encodes inhibin or fragment thereof, and a carrier protein can be placed in an animal, thereby secondarily reducing inhibin levels in the animal due to the immune response. Any protocol for the transfer of the fused gene product of the present invention is contemplated as part of it. Transfection of promoter sequences, instead of one normally found specifically associated with inhibin or other sequences that could reduce inhibin protein production are also encompassed as gene therapy methods. An example of this technology is found in Transkaryotic Therapies, Inc., of Cambridge, Massachusetts, using homologous recombination to insert a "genetic switch" that turns on an erythropoietin gene in cells. See GENESIS Engineering News, April 15, 1994. Gene transfer methods for gene therapy fall into three broad physical categories (eg, electroporation, direct gene transfer and particle bombardment), chemical (carriers based on lipids, or other non-viral vectors) and biological vectors (vector derived from virus and receptor consumption) For example, non-viral vectors can be used, which include liposomes coated with DNA Such liposome / DNA complexes can be directly injected in the form intravenously to the animal It is believed that the liposome / DNA complexes are concentrated in the liver where they deliver the DNA to macrophages and Kupffer cells.These cells are long-lived and thus provide long-term expression of the DNA delivered. In addition, the vectors or "naked" DNA of the gene can be directly injected into the desired organ, tissue or tumor for the target delivery of AD N therapeutic The methodologies of gene therapy can also be described through the delivery site. Fundamental ways of supplying genes include ex vivo gene transfer, gene transfer in vivo, and in vitro gene transfer. In the ex vivo gene transfer, the cells are taken from the animal and developed in the culture medium. The DNA is transfected into the cells, and the cells transfected in number and then reimplanted in the animal. In in vitro gene transfer, transformed cells are cells that grow in culture, such as tissue culture cells, and not particular cells of a particular animal. These "laboratory cells" are transfected, the transfected cells are selected and expanded either for implantation to the animal or for other uses. In vivo gene transfer involves the introduction of DNA into the cells of the animal, when the cells are inside the animal. The methods include using virally mediated gene transfer using a non-infectious virus to deliver the gene in the animal or injecting the naked DNA to a site in the animal and the DNA is taken through a percentage of cells in which the protein Gene product is expressed. In addition, the other methods described herein, such as the use of a "gene gun", can be used for the in vitro insertion of inhibin DNA or inhibin regulatory sequences.

Chemical methods of gene therapy may involve a lipid-based compound, not necessarily a liposome, to transport DNA through the cell membrane. Lipofectins or cytofectins, positive lipid-based ions, which bind to negatively charged DNA, form a complex club that can cross the cell membrane and deliver DNA into the cell. Another chemical method uses receptor-based endocytosis, which involves the binding of a specific ligand to a cell surface receptor and envelops and transports through the cell membrane. The ligand binds to the DNA and the entire complex is transported to the cell. The ligand gene complex is injected into the bloodstream and then into target cells that have the receptor that will specifically bind the ligand and transport the ligand-DNA complex to the cell. Many gene therapy methodologies employ viral vectors to insert genes into cells. For example, altered retrovirus vectors have been used in ex vivo methods to introduce genes to tumor infiltration lymphocytes, and peripherals, hepatocytes, epidermal cells, myocytes and other somatic cells. These cells are then introduced to the animal to provide the gene product of the inserted DNA.

Viral vectors have also been used to insert genes into cells using in vivo protocols. To direct the tissue-specific expression of foreign genes, cis-acting regulatory promoter elements that are known to be tissue specific can be used. Alternatively, this can be achieved by using the in situ delivery of DNA or viral vectors to specific anatomical sites in vivo. For example, gene transfer to blood vessels in vivo was achieved by implanting in vitro, transduced endothelial cells at selected sites on arterial walls. The virus infected the surrounding cells, which also expressed the gene product. A viral vector can be delivered directly to the site in vivo, through a catheter, for example, thus allowing only certain areas to be infected by the virus and providing a specific gene expression at the long-term site. Gene transfer in vivo using retrovirus vectors has also been demonstrated in breast tissue and liver tissue by injecting altered virus into the blood vessels leading to the organs. Viral vectors that have been used for gene therapy protocols include, but are not limited to retroviruses, other RNA viruses, such as poliovirus or Sindbis virus, adenovirus, adeno associated virus, herpes virus, SV 40, vaccinia and other DNA viruses. Replicating defective murine retroviral vectors are the most widely used as gene transfer vectors. The murine leukemia retroviruses are composed of an RNA of single chain structure that complexes with a nuclear core protein and polymerase (pol) enzymes, enclosed by a protein core (gag) and surrounded by a glycoprotein envelope ( env) that determines host scale. The genomic structure of the retroviruses includes the gag, pol and env genes enclosed by the 5 'and 3' long terminal repeats (LTR). Retroviral vector systems exploit the fact that the minimum vector contained in the 5 'and 3' LTRs and the packaging signal are sufficient to allow vector packaging, infection and integration into the target cells provided that the viral structural proteins are supplied in trans in the packaging of the cell line. The fundamental advantages of retroviral vectors for gene transfer include efficient infection and gene expression in most cell types, integration of precise individual copy vectors to target cell chromosomal DNA, and ease of manipulation of the retroviral genome.

The adenovirus is composed of DNA of single chain, linear structure, which complexes with core proteins and is surrounded by capsid proteins. Advances in molecular virology have led to the ability to exploit these organisms in order to create vectors capable of transducing novel genetic sequences to target cells in vivo. Adenovirus-based vectors will express the peptides of gene products at high levels. Adenoviral vectors have high efficiencies of ineffectiveness, even with low virus titers. In addition, the virus is completely infectious since a cell-free virion thus injected from the producer cell lines is not necessary. Another potential advantage of adenoviral vectors is the ability to achieve long-term heterologous genes in vivo. Mechanical methods of DNA delivery include fusogenic lipid vesicles such as liposomes or other vesicles for membrane fusion, lipid particles, cationic lipid incorporating DNA, such as lipofectin, polylysine-mediated DNA transfer, direct injection of DNA, such as microinjection of DNA into germ or somatic cells, particles coated with pneumatically-supplied DNA, such as the gold particles, used in a "gene gun", and inorganic chemical aspects, such as calcium phosphate transfection. Another method, ligand-mediated gene therapy, involves the formation of the DNA complex with specific ligands to form ligand-DNA conjugates to direct the DNA to a specific cell or tissue. It has been found that injecting plasmid DNA into muscle cells produces a high percentage of cells, which are transfected and have sustained expression of marker genes. The plasmid DNA may or may not be integrated into the genome of the cells. The non-integration of the transfected DNA could allow the transfection and expression of the gene product proteins in terminally differentiated, non-proliferative tissues, over a prolonged period without the fear of insertions, deletions or mutational alterations in the mitochondrial cell or genome. The long-term, but not necessarily permanent, transfer of therapeutic genes to specific cells can provide treatments for genetic diseases, or for prophylactic use. The DNA can be periodically reinjected to maintain the level of the gene product without mutations occurring in the genomes of the recipient cells. The non-integration of exogenous DNAs may allow the presence of several different exogenous DNA constructs within a cell with all constructs expressing several gene products. The gene transfer methods mediated by particles were first used to transform plant tissue. With a particle bombardment device or "gene gun", a driving force is generated to accelerate the high density particles coated with DNA (such as gold or tungsten) at a high speed that allows the penetration of organs, tissues or target cells. Bombardment of particles can be used in in vitro systems, or with ex vivo or in vivo techniques to introduce DNA to cells, tissues or organs. Electroporation for gene transfer uses an electrical current to make the cells or tissues susceptible to gene transfer mediated by electroporation. A short electrical impulse with a given field strength is used to increase the permeability of a membrane, so that DNA molecules can penetrate the cells. This technique can be used in in vitro systems or with ex vivo or in vivo techniques to introduce DNA into cells, tissues or organs. Carrier-mediated gene transfer in vivo can be used to transfect foreign DNA to cells. The DNA-carrier complex can be conveniently introduced into the body fluids or into the blood stream afterwards to the site specifically directed towards the organ or tissue in the body. Both liposomes and polycations, such as polylysine, lipofectins or cytofectins can be used. The liposomes can be used, which are cell-specific or organ-specific and in this way the foreign DNA carried by the liposome will be taken up by the target cells. The injection of immunoliposomes that are targeted to a specific receptor in certain cells can be used as a convenient method to insert the DNA that carries the receptor. Another carrier system that has been used is the conjugate system of asialoglycoprotein / polylysine to carry DNA to hepatocytes for gene transfer in vivo. The transfected DNA can also form complexes with other types of carriers, so that the DNA is taken to the recipient cell and then resides in the cytoplasm or nucleoplasm. DNA can be coupled to nuclear carrier proteins in vesicular complexes specifically engineered and directly carried to the nucleus. Inhibin gene regulation can be achieved by administering compounds that bind to the inhibin gene, or control regions associated with the inhibin gene, or its corresponding RNA transcriber to modify the transcription or translation regime. In addition, cells transfected with a DNA sequence encoding inhibin or fragment thereof, and a protein can be administered to an animal to provide an in vivo source of the heterologous protein of the present invention. For example, the cells can be transfected with a vector containing the fusion gene product of the present invention, which encodes inhibin, or a fragment thereof, and a carrier protein. The term "vector" as used herein, means a carrier that may contain or be associated with specific nucleic acid sequences that function to transport the specific nucleic acid sequences to the cells. Examples of the vectors include plasmids and infectious microorganisms, such as viruses, or non-viral vectors, such as ligand-DNA conjugates, liposomes, lipid-DNA complexes. It may be necessary that the recombinant DNA molecule comprising the fused gene product of the present invention be operably linked to an expression control sequence to form an expression vector capable of expressing the heterologous protein of the present invention. The transfected cells can be derived from normal animal tissue, the diseased tissue of the animal, or they can be non-animal cells. For example, cells from an animal can be transfected with a vector capable of expressing the heterologous protein of the present invention, and re-introduced into the animal. Then, the transfected cells produce the heterologous protein of the present invention, thus inducing an immunological response to inhibin. The cells can also be transfected through non-vector, or physical or chemical methods known in the art, such as electroporation, incorporation or through a "gene gun". Additionally, the fused gene product of the present invention can be directly injected, without the aid of a carrier, into the animal. In particular, the fused gene product of the present invention can be injected into the skin, muscle or blood. The gene therapy protocol for transfecting inhibin, or a fragment thereof, to an animal, can be either through the integration of the gene product fused into the genome of the cells, into micromosomes, or as a separate replication or construction of DNA without replication or in the cytoplasm or nucleoplasm of the cell. The heterologous protein expression may continue for a prolonged period, or the fused gene product of the present invention may be periodically reinjected to maintain the desired level of the heterologous protein of the cell, tissue, or organ, or a given blood level. The fused gene product of the present invention can be administered to a bird through any means known in the art., for example, the composition can be administered subcutaneously, intraperitoneally, intradermally, intramuscularly or intravascularly. Preferably, the composition is injected subcutaneously. Another preferred administration is intravascular infusion near the preferred site of therapy. The composition can be administered to a bird in one or more doses. Preferably, the composition is administered in birds in multiple doses, wherein an initial immunization is followed by booster immunizations. The preferred amount of the fused gene product to be administered is between 50 and 300 micrograms per kilogram of body weight. Preferably, the fused gene product is administered in a vehicle, such as a pH regulator or Freund's adjuvant. The methods of the present invention to improve the yield of production in birds, will greatly accelerate the population growth and therefore the market for ratites, such as ostriches and emu as their suboptimal egg production regimes will be increased through the method of the present. The method of the present invention will also satisfy the need for expansion for poultry such as domestic chickens and their eggs. The utility of the method of the present invention for improving the production yield is not limited to improving the yield of production in birds. The method of the present invention for improving production yield can be used in many animals. As stated above, the method of the present invention is used to improve the production yield of any animal that produces inhibin, including, but not limited to, most of the animals that are grown agriculturally, such as pigs, cows, sheep , turkeys, quail, ducks, geese, turtles, fish and chickens; in animals that wear hair such as mink, fox, otter, ferrets, rabbits and raccoons; rodents for laboratory tests such as mice, rats, hamsters, guinea pigs and gerbils; for animals whose skins are used for decorative purposes such as lizards and snakes; exotic or endangered species-animals used for racing, entertainment or exhibition (competition) such as horses, dogs, cats, zoo animals, and circus animals; and human beings. Additional poultry that the method of the present invention improves its production yield include ratites, psittaciformes, falconiformes, piciformes, estrigiformes, paseriformes, coraciformes, cuculiform raliforms, columbiformes, galliformes, anseriformes, and herodiones. More particularly, the method of the present invention can be used to improve the production yield of an ostrich, emu, ñandu, kiwi, cassowaries, parrot, parakeet, hawk, iguana, pigeon, cockatoo, songbird, talking bird, blackbird , finch, singer, canary, toucan, maniato or sparrow.

Qualitative or Quantitative Methods of the Present Invention Another aspect of the present invention is a method for producing antibodies directed against the heterologous protein of the present invention. Generally, the method for producing antibodies directed against the heterologous protein comprises the steps of: administering an effective amount of a heterologous protein comprising inhibin protein or a fragment thereof, and a carrier protein, to an animal so that a immune response in the animal against the heterologous protein; remove a blood sample from the animal; and then isolate any antibody directed against inhibin from the serum of the blood sample. Preferably, the antibodies are isolated from the serum of the blood sample by passing the serum through a column containing effective amounts of a carrier protein to separate the antibodies from the serum. Alternatively, the column will contain the heterologous protein of the present invention. In another technique the antibodies are isolated by first passing them through a column containing the carrier protein and then passing them through a column containing the heterologous protein of the present invention. The techniques used to produce and purify antibodies directed against the heterologous protein of the present invention are well known to those skilled in the art. It is also understood that the heterologous protein of the present invention can be administered to any animal depending on the type of antibodies desired. In addition, it should be understood that inhibin may be exogenous or endogenous. Therefore, the type of inhibin in the heterologous protein of the present invention and the animal species in which the composition is administered, are determined by the type of the antibody that is desired. For example, the heterologous protein comprising chicken inhibin and maltose binding protein can be administered to an ostrich to produce inhibin antibodies against ostrich chicken. Also, the heterologous protein comprising ostrich inhibin and maltose binding protein can be administered to an ostrich to produce anti ostrich inhibin antibodies for ostrich. The present invention is also directed to a cost-effective, reliable, simple, rapid method to determine whether an animal is hormonally predisposed to be a high-level or low-level egg producer. More particularly, the present invention also relates to a method for determining the amount of inhibin produced by an animal, which, therefore, allows the determination of the egg production capacity of the animal. Briefly described, the method for determining the amount of inhibin in the blood of an animal comprises the steps of: removing a blood sample from the animal; contacting the blood sample with anti-inhibin antibodies that are specific directed against the endogenous inhibin of the animal under conditions that allow the antibodies to selectively interact with any inhibin if present in the sample. Remove any non-interacting antibodies from any of the interacting antibodies, and determine the amount of antibodies that are interacted. One aspect in the art will understand that the immunoassay techniques that can be used in the above method are well known in the art. Therefore, any immunoassay technique, known branding and visualization method, can be used in the above method, including ELISA and radioimmunoassay (RIA). A preferred immunoassay is ELISA ("enzyme-linked immunosorbent assay"), and a preferred brand is horseradish peroxidase. Another preferred brand is a colored latex bead. The colored latex bead can be any desired color for viewing purposes. Preferably, the red yellow latex bead, blue or green. The colored latex bead may be hollow or solid, but is preferably hollow to minimize its weight. The size of the latex bead varies according to its intended use in immunoassay. One skilled in the art may be able to ascertain through routine testing the largest test size that is visible that does not interfere stereally with the immunoassay reactions. Preferably, the latex beads have a number less than 0.5 μ in diameter and more preferably less than 0.2 μ in diameter.

For example, circulating inhibin concentrations in a bird's blood can be determined using normal sandwich ELISA techniques. First, anti-bird inhibin antibodies, which were directed against a portion of * - inhibin *** or fragment thereof, towards the cavities of a microtiter plate. After washing and blocking the plate, then an amount of blood plasma that was obtained from the bird being tested is added. After allowing any inhibin in the sample, if present, to interact selectively against the immobilized anti-inhibin antibody, the sample is washed with water from the cavity of the plate. Then, labeled anti-inhibin antibodies are added to the cavities which are directed with a different portion of the inhibin or fragments thereof, than the antibody immobilized in the cavity. The antibody can be labeled with any brand known in the art, such as horseradish peroxidase. After allowing the labeled anti-inhibin antibody to act selectively with any immobilized inhibin, anti-inhibin anti-inhibin antibodies, labeled by washing, are removed. The amount of inhibin present in the plasma sample is determined using the appropriate visualization media for the label using ELISA to quantify the amount of labeled anti-inhibin antibody immobilized in the cavity. The normal positive and negative controls, which are going to be operated simultaneously near the cavities of the plate. It should be understood that the reproductive potential of any animal that produces inhibin can be determined by the above method. The method of the present invention can be used to determine the amount produced by a female animal of any species that produces inhibin. The animal can be bird, mammal, fish, or reptile, among others. More specifically, the animal includes, but is not limited to, a cow, human being, horse, cat, dog, sheep, mink, fox, otter, ferret, raccoon and pig. The bird includes, but is not limited to, an ostrich, rhea and chicken. A favorite animal is a bird. A more preferred animal is a ratite. A more preferred animal especially is an ostrich. Another preferred animal is an emu. Still another preferred animal is a chicken. Animals that have high levels of inhibin have a low reproductive potential, and depend on the species involved and if they are developed for agricultural purposes, such as an animal with low egg production can be taken for slaughter instead of being kept for breeding purposes. breeding. In contrast, those animals that develop agriculturally that have lower amounts of inhibin are high egg producers and are generally used for breeding purposes, and are not taken for slaughter. The level of inhibin in the animal varies according to the age, species and time in years in relation to the maid season (if any). Therefore, the determination of the potential egg production of an animal is relative to these factors, and a measure of one of the animal's inhibin levels are very valuable when compared to the average inhibin levels of the animal species. , of approximately the same age, the same time of the year if the animal has a breeding season. Since the amount of inhibin produced by a bird varies according to the above factors, relative amounts of inhibin are established below different categories of birds of ages. Table 1 illustrates the variation of ratites such as emus and ostriches in their inhibin production depending on whether they are poor or good egg producers, and dependent on the age of the ratite. Table 1 Age Reproductive Potential Level (Months) Inhibin 6-12 > 5 Poor 6-12 -25 Moderate 6-12 0 - 1.5 Good 24+ > 7 Poor 24+ 3 - 7 Moderate 24+ 0 - 2.5 Good In the previous table, the inhibin level is relative to a normal poor value of 1 for female ratites that vary from 50 to 60 eggs per breeding season, which is good and a value of 7 for a female ratite deposit functionally production, which produce less than five eggs per season, which is poor. Yet another aspect of the present invention is a method for producing animal antibodies directed against a class of antibodies from another animal, such as IgG. Briefly described, the method for producing antibodies in an animal, which are directed against the IgG of another animal, comprises the steps of: administering an effective amount of a class of antibodies of a first animal, such as those produced by the method described previously, to a second animal, so that an immunological response occurs in the second animal against the antibodies of the first animal; withdraw a blood sample from the second animal; and isolating the antibodies of the second animal from the serum of the blood sample as described above. Preferably, the second animal is a species different from the first animal. The present invention is also directed to a rapid, simple, reliable, cost-effective method for determining whether an animal has responded immunologically to an attack with the inhibin composition. The method uses antibodies from a second animal directed against a class of antibodies of a first animal as described above. Briefly described, the method comprises binding inhibin or the heterologous protein of the present invention to a solid phase and contacting the immobilized inhibin with a blood sample of the animal to be tested. The sample is contacted with immobilized inhibin under conditions wherein inhibin will interact selectively with any anti-inhibin antibody in the sample. After removing the non-interacting antibodies from the sample and washing, a quantity of antibodies labeled from the second animal, which are directed against a class of the antibodies of the first animal, are added. The labeled antibodies that are directed against the animal's antibodies will then selectively interact against the antibodies that are bound with the immobilized inhibin. After the removal of the labeled non-interacting antibodies, the presence or amount of labeled antibodies interacted is determined by visualizing the label. In this way, the method detects the presence of antibodies directed against inhibin in the animal, and therefore determines whether the animal has responded immunologically from the administration of a composition comprising inhibin. It should be understood that the method for determining whether an animal has responded immunologically to the administration of a composition comprising inhibin can be performed on a female animal of any species. The animal can be a bird, mammal, fish or reptile, among others. More specifically the mammal includes, but is not limited to, a cow, human being, horse, cat, dog, sheep, mink, fox, otter, ferret, raccoon and pig. The bird includes, but is not limited to an ostrich, emu, ñandu, and chicken. A favorite animal is a bird. A very preferred animal is a ratite. A more preferred animal especially is an ostrich. Another preferred animal is an emu. Still another preferred animal is a rhea. One skilled in the art will understand that immunoassay techniques can be used in the above method are well known in the art. Therefore, any immunoassay, branding and visualization technique can be used in the above method. A preferred immunoassay is ELISA and a preferred brand is horseradish peroxidase. Another preferred brand is a colored latex bead. The colored latex bead can be any desired color for viewing purposes. Preferably, the latex bead is yellow, red, blue or green. The colored latex bead may be hollow or solid, but is preferably hollow to minimize its weight. The size of the latex bead varies according to its intended use in the immunoassays. One skilled in the art may be able to ascertain the size of the bead that is visible that it does not interfere stereically with the immunoassay reactions. Preferably, the latex bead has a diameter less than 0.5μ, and more preferably has a diameter less than 0.2μ. Another embodiment of the present invention is directed to the above method to determine whether an animal has responded immunologically to the administration of a composition comprising inhibin wherein the immunoassay method is modified as follows. Briefly described, the method comprises obtaining a blood sample from an animal, and contacting it with a labeled animal inhibin, or fragment thereof. The sample is contacted with labeled animal inhibin under conditions where animal inhibin will interact selectively with any of the inhibin bodies in the sample. After removing the labeled non-interacting inhibin from the sample, the presence or amount of interacting labeled inhibin is determined by visualizing the label. The inhibin used in this method is selected from, but is not limited to the heterologous fused inhibin protein of the present invention; endogenous inhibin, or fragments thereof; and exogenous inhibin or fragments thereof. Preferably, the labeled inhibin is endogenous inhibin. This invention is further illustrated through the following examples, which are not constructed in a way that imposes limitations on the scope thereof. On the contrary, it should be clearly understood that reclassifications can be made to various other modalities, modifications and equivalents thereof, which after reading the present description, can suggest by themselves to those skilled in the art, without departing from the spirit of the present invention and / or the scope of the appended claims.

EXAMPLE 1 Production of a fused gene product comprising a gene coding for the expression of chicken inhibin, and a gene coding for the expression of maltose binding protein. The following is a method for producing a fused gene product comprising a gene (cINA52-I) expressed to encode a fragment of the alpha subunit chicken inhibin (SEQUENCE OF IDENTIFICATION NO: 2) and a gene encoded to express the protein of maltose binding. The fused gene product of the present invention is made from the vector equipment pMAL-c from New England Biolabs, Beverly, Massachusetts. The pMAL MR vectors provide a method for producing an expressed protein of a cloned gene in a reading frame. The cloned gene is inserted downstream of the male gene, which encodes the maltose binding protein ("MBP"), and results in the expression of the MBP fusion protein ("MBP-cINA521)." The method produces an expression of high-level cloned sequences and a one-step purification for the fusion protein, MBP-cINA521, using affinities of MBP for maltose.The following is a method for ligating the INhibin, cINA521 to a pMAL -c vector: 1. Digest 0.5 μg of the plasmid DNA pMALMR "-c in 20 μl with the restriction endonuclease Pstl 2. Digest 5 μg of plasmid DNA cINA6 containing the gene of chicken inhibin with the same enzyme, Pstl. 3. Verify the complete digestion by operating 4 μl of the reaction of pMALMR- and 4 μl of the reaction of CINA6 on 0.8% of agarose gel. Then, operate the preparative agarose gel and purify the Pstl cINA521 fragment through the pre-A-Gene preparation kit. 4. Add 0.05 units of calf intestinal alkaline phosphatase (NEB # 290) to the DNA digestion vector. Incubate at 37 ° C for one now. 5. Add to an equal volume of a 1: 1 mixture of phenol / chloroform to the vector restriction digestion, mix and then centrifuge, remove the aqueous phase (top) and color in fresh tube. Repeat with chloroform alone. 6. Add 10 μg of glycogen or tRNA to the vector digestion, such as adding 1/10 volumes of 3 M sodium acetate, mixing and then adding two volumes of ethanol. Incubate at -20 ° C for 30 minutes. 7. Microcentrifuge for 15 minutes. Empty the supernatant, rinse the pellet with 70% ethanol, and allow to dry. 8. Resuspend each sample in 20 μl of water. 9. Mix: 0.2 μg of vector digestion; 0.5 μg of insert digestion; add water, up to 18 μg; then add 2 μl of 10 times ligase pH regulator; 0.5 μl of NEB T4 ligase (# 202, "200 units), and incubate at 16 ° C for 2 hours at night 10. Heat at 65 ° C for 5 minutes, cool on ice. 11. Mix 5 μl of the ligation mixture with 100 μl competent DH5a or any complementing strain JJaaZC a) and incubate on ice for 15-30 minutes.

Heat at 42 ° C for 2 minutes. 12. Add 1 ml of L and incubate at 37 ° C for 60 minutes. Extend on a LB plate containing 100 μg / ml ampicillin. They incubated overnight at 37 ° C.

Collect the colonies with a sterile stick on a master LB plate and an LB ampicillin plate containing 80 μg / ml Xgal and 0.1 mM IPTG. Incubate at 37 ° C for 8 a 16 hours. Classify the Lac phenotype on the Xgal plate and retrieve the "white" clones from the master. 13. Classify the presence of inserts in one or both of the following ways: A. Prepare minipreparation DNA. Digest with an appropriate restriction endonuclease to determine the presence and orientation of the insert. B i) Develop a 5 ml culture in an ampicillin broth of LB at approximately 2 x 108 / ml. ii) Take a sample of 1 ml. Microcentrifuge for 2 minutes, discard the supernatant and resuspend the cells in a 50 μl protein gel SDS-PAGE sample buffer. iii) Add IPTG to the rest of the culture at 0.3 mM, for example, 15 μl of a 0.1 M solution. Incubate at 37 ° C with good aeration for 2 hours. iv) Take a sample of 0.5 ml. Microcentrifuge for 2 minutes, discard the supernatant and resuspend the cells in the sample pH regulator SDS-PAGE in 100 μl. v) Boil the samples for 5 minutes. Electrofarm 5 μl of each sample with a 10% SDS-PAGE gel together with a set of protein PM standards and 15 μl of the supplied MBP and pH regulator of SDS-PAGE. Dye the gel with brilliant blue of Cumasie. An induced band is easily visible at a position corresponding to the molecular weight of the fusion protein. The molecular weight of MBP alone is 42,000 daltons.

EXAMPLE 2 Production of a heterologous fusion protein, "MBP-cINA52?", Comprising chicken inhibin and maltose binding protein. The following is a method for producing a fused heterologous protein comprising chicken inhibin and maltose binding protein, "MBP-cINA521". The fused gene product of Example 1 expresses the fused maltose binding protein inhibin protein, "MBP-cINA521", as follows: 1. Inoculate 80 ml of enriched broth + glucose and ampicillin (see Media and Solutions below) with 0.8 ml of an overnight culture of cells containing the fusion plasmid of Example 1. 2. Develop at 37 ° C with good aeration at 2 x 10 cells / ml (Ag00 of ~ 0.5). Take a 1 ml sample and microcentrifuge for 2 minutes (non-induced cells). Discard the supernatant and re-suspend the cells in a 50 μl SDS-PAGE sample buffer. Swirl and place on ice. 3. Add IPTG (isopropylthiogalactoside) to the remaining culture to give a final concentration of 0.3 M, for example 0.24 ml of a 0.1 M water supply solution (see Media and Solutions). Continue incubation at 37 ° C for 2 hours. Take a sample of 0.5 ml and microcentrifuge induced cells for two minutes). Discard the supernatant and re-suspend the cells in a sample pH regulator SDS-PAGE 100 μl. Shake the resuspended cells and place on ice. 4. Divide the crop into two aliquots. Harvest the cells by centrifugation at 4000 x g for 10 minutes. Discard the supernatant and re-suspend a pellet (sample A) in 5 ml of regulator lysis (see Media and Solutions). Resuspend the other pellet (sample B) in 10 ml of 30 mM Tris-Cl, 20% sucrose, pH 8.0 (8 ml of each 0.1 g of wet cells by weight). 5. Freeze the samples in a dry ice-ethanol bath (or overnight at -28 ° C). Thawing in cold water (20 ° C is more effective than 70 ° C, but takes more time). 6. Apply sound, verify cell breakdown and measure release of the protein by applying a Bradford assay or releasing the nucleic acid at A2g0 ° until it reaches its maximum. Add 0.6 ml of 5M NaCl. 7. Centrifuge at 9,000 x g for 20 minutes. Decant the supernatant (crude extract 1) and store on ice. Resuspend the pellet in 5 ml of lysis buffer. This is a suspension of the insoluble matter (crude extract 2). Column Purification of the protein-inhibin heterologous fused maltose-binding protein, "MBP-cINA52?", As previously produced, is as follows: 1. Inflate amylose resin (1.5 g) for 30 minutes, 50 ml of column pH (see Media and Solutions) in a 250 ml filter flask. Degassing with a vacuum cleaner. Empty in a 2.5 x 10 cm column. Wash the column with 3 column volumes of the same pH regulator + 0.25% Tween 20.

The amount of resin required depends on the amount of fusion protein produced. The resin binds to approximately 3 mg / ml bed volume, so that a column of approximately 15 ml to produce up to 45 mg of the protein fusion / culture per liter. A 50 ml syringe capped with silanized glass fiber can be replaced by a 2.5 cm column. The ratio of height to column diameter should be less than or equal to 4. 2. Dilute the crude extract 1: 5 with column buffer + 0.25% Tween 20. Load the diluted crude extract at a flow rate of [ lOx (diameter of the column in cm)] ml / hour. This is approximately 1 ml / min for a 2.5 cm column. The dilution of the crude extract aims to reduce the protein concentration by approximately 2.5 mg / ml. If the raw extract is less concentrated, do not dilute it that much. A good rule of thumb is that 1 g of wet weight of cells gives approximately 120 mg of protein. 3. Wash with 2 column volumes of column pH regulator + 0.25% Tween 20. 4. Wash with 3 column volumes with pH regulator without Tween 20. 5. Elute the fusion protein, "MBP-cINAc2? " with column pH regulator + 10 mM maltose + 0.1% SDS (optional 10 mM ß-mercaptoethanol, 1 mM EGTA), Collect 10-20 fractions of 3 ml. Analyze the fractions for the protein, for example, through Bradford or A2600 analysis; the fractions contain the fusion protein has easily detected the protein. The fusion protein elutes faster after the hollow volume of the column.

Means and Solutions * Enriched medium + glucose and ampicillin = per liter; 10 g of triplone, 5 g of yeast extract, 5 g of NaCl, 2 g of glucose. Autoclave; add sterile ampicillin at 100 μg / ml. * Supply solution 0.1 M IPTG = 1.41 g of ITG (isopropyl-β-o-thiogalactoside (add H20 to 50 ml.

Filter and sterilize. * 0.5 M sodium phosphate buffer, pH 7.2, (supply material) = (A) 69.0 g NaH2P04H20 at 1 liter with H20. (B) 70.9 g Na2HP04 at 1 liter with H20. Mix 117 ml (A) with 383 ml (B). The pH of this supply solution should be approximately 7.2. Dilute to 10 mM in a column pH regulator, the pH should be 7.0.

Lysis pH regulator Per liter Concentration Final 20 ml of Na2HP04 0.5 M phosphate 10 mM 1. 75 g of NaCl NaCl 30 mM 10 ml of Tween 20 25% Tween 20 0.35% 0. 7 ml of 10 mM ß-ME ß-mercaptoethanol ("ß-ME") (optional) 20 ml of 0.5 M EDTA (pH 8) 10 mM EDTA 10 ml of 1 M EGTA (pH 7) 10 mM EGTA Adjust to pH 7.0 with HCL or NaOH Column pH regulator Per Liter Final concentration 20 ml of sodium phosphate, 10 mM phosphate pH 7.2 29.2 g NaCl 0.5 M NaCl 1 ml of sodium azide I M azide 1 mM 0.7 mM ß-ME (optional) ß-ME 10 mM 1 ml EGTA IM (pH 7) EGTA 1 M (optional) Adjust to pH 7.0 if necessary Low Salt Column Regulator Per Liter Final concentration ml of 0.5 M sodium phosphate 10 mM phosphate pH 7.2 1.75 g of 30 mM NaCl NaCl 1 ml of 1 mM ÍM azide sodium azide 0.7 ml of 10 mM β-Meß-mercaptoethanol (optional) 1 ml of EGTA ÍM ( pH 7) 1 mM EGTA (optional) Adjust to pH 7.0, if necessary The purity of the heterologous chicken MBP protein fused "MBP-cINA521", after passing through the column is illustrated in Figure 1, columns "E" The column marked "F" is the eluent from the column when no heterologous protein has been loaded in the column (negative control). The columns marked "B" represent the vector normals of pMALMR - c. The columns marked "C" are normal molecular weight. The columns marked with "D" are the real vector of pMALMR * -c used in the preparation of the heterologous MBP-inhibin-fused chicken protein, "MBP-cINA521", before insertion of the inhibin gene as described in FIG. Example 2. The above proteins were electrophoresed on an SDS-PAGE gel in a pH regulator of SDS-PAGE, and stained with a bright blue Cumasie stain.

EXAMPLE 3 Immunization of an Ostrich against Inhibin The following is a method to immunize an ostrich against inhibin. A primary immunization is administered to the ostrich approximately six months before the first breeding season of the bird and then reinforcement immunizations were administered at one-month intervals for six months. Accordingly, it is preferred to administer the primary immunization to an ostrich when it is approximately between about 18 and 24 months of age. The primary immunization comprises from about 1.5 to 3.0 mg of a fused heterologous protein comprising chicken inhibin (a fragment of the alpha subunit) and maltose binding protein produced by the methods described in Examples 1 and 2. Immunizations of reinforcement comprise between about 0.75 to 1.5 mg of the fused heterologous protein. The heterologous protein is emulsified in Freund's Complete Auxiliary (Sigma Chemical Co., St. Louis, MO) in the primary immunization, and the fused heterologous protein is emulsified in Freund's Incomplete Auxiliary (Sigma) in the booster immunizations. The fused heterologous protein composition is injected subcutaneously at three sites along the upper region of the ostrich's thigh.

EXAMPLE 4 Production of Selectively Targeted Ostrich Antibodies Against Inhibin The following is a method for producing ostrich antibodies ("ostrich inhibin inhibitor antibodies"), which are selectively directed against a heterologous protein of the present invention comprising chicken inhibin. and maltose binding protein. More particularly, the ostrich antibodies are IgG antibodies. The heterologous protein used is a fused protein comprising chicken inhibin and maltose binding protein produced by the methods described in Examples 1 and 2. To produce the antibodies, immunize an ostrich with inhibin of chicken-heterologous protein MPB according to the method described in Example 3. The amount administered to the ostrich should be sufficient to produce an immunological response in the ostrich against the heterologous protein. Remove approximately 5 ml of blood from the ostrich and then isolate the antibodies directed against the inhibin from the rest of the blood sample. Any separation method known in the art can be used to isolate antibodies. Preferably, normal ELISA techniques are used, along with affinity and CLAP columns.

EXAMPLE 5 Production of Goat Antibodies Targeted Against Ostrich Antibodies The following is a method to produce goat antibodies that are selectively directed against a class of ostrich antibodies, including the antibodies produced in Example 4, more particularly the ostrich antibodies. of IgG. In this method, a goat was immunized with 0.5 to 3.0 mg of ostrich with IgG, so that an immunological response occurs in the goat against the ostrich IgG. The ostrich IgG was obtained in a deposit of sera from different ostriches. The blood was obtained from the goat and the goat antiavestrus IgG was then isolated from the sample using normal techniques that are well known in the art. The serum deposit was purified using normal methods involving precipitation using 50% ammonium sulfate, and subsequent fractionation using a protein-A sepharose column. Preferably, the precipitation of IgG is conducted as follows. Twelve milliliters of serum containing IgG were diluted 1: 1 with 50 mM Tris (pH 8.0). Then, 24 ml of saturated ammonium sulfate was added slowly with stirring (all at 4 ° C), and the mixture was stirred for about 2 hours. The mixture is centrifuged at 10,000 rpm for 10 minutes to collect the precipitate. Resuspend the precipitate in 50 mM-Tris / Saline (to 12 ml) and dialyze overnight against 2 L of 50 mM-Tris at 4 ° C, the final volume takes 20 ml. Preferably, the subsequent fractionation using a Sepharose protein-A column is as follows. The column contains protein-A sepharose CL-4B (Pharmacia Biotech, Inc., Piscataway, NJ). The column was loaded with approximately 5 ml of ammonium sulfate / serum precipitate. The sample was allowed to bind protein A sepharose for about 30 minutes. Then, the column was washed with a 0.1 M phosphate buffer (pH 7.5). and the absorbed IgG was eluted with 0.1 M glycine (pH 2.8). Finally, the eluted fractions were neutralized by adding a few drops of 1 M Tris-HCl (pH 9). The quality of the purified ostrich IgG was tested by visualization after SDS-polyacrylamide gel electrophoresis, before being administered to a goat. The method for immunizing and the auxiliaries used are not critical to the invention, thus any method known in the art can be used, and any system known in the art can be used. Preferably, the purified ostrich IgG will be injected into a goat subcutaneously. Preferably, booster injections can also be administered at four-week intervals, using Freund's incomplete adjuvant. Preferably, the IgG will be administered with Freund's Complete Assist or Titermax Hunter (Sigma Chemical Co., St. Louis, Missouri). A goat develops a satisfactory immune response after three to four injections. Then, 5-10 ml of goat blood was removed, and goat antibodies directed against IgG directed against ostrich, were isolated from the blood sample. Any method known in the art can be used to separate the goat antibodies from the blood sample. A preferred method to separate lo-; Goat antibodies from the blood sample is to pass the blood sample through a column of ostrich IgG. The goat antibodies were then collected from the column by washing the column with glycine buffer pH-8.0.

EXAMPLE 6 Verification of the Immunological Response of a Truce Bird After Its Vaccination With the Heterologous Protein Fused A The following is a method to verify the immunological response of an ostrich after it has been vaccinated with a fused heterologous protein composed of chicken inhibin. and maltose binding protein produced by the method of Examples 1 and 2, wherein the immunological response was verified using the goat antibodies produced in Example 5. To determine if an ostrich has reacted immunologically to the fused inhibin-heterologous protein MBP, the heterologous protein is first bound to a solid phase. Then, remove 5-10 ml of blood from an ostrich that has been immunized with a heterologous protein, and isolate the serum from the blood. Contacting the immobilized heterologous protein with the serum under conditions, wherein the heterologous protein will interact selectively with any of the anti-inhibin antibodies in the serum. After washing, add the goat antibodies produced in Example 5, which have been labeled with HRP. The labeled goat antibodies will then selectively interact with the ostrich antibodies, which bind to the immobilized heterologous protein. After the removal of the labeled non-interacting antibodies, the presence or amount of goat antibodies labeled with HRP interacted was determined, visualizing the mark. Preferably, the tag was visualized by adding Nitro Tetrazolium Blue ("NBT"), a substrate to HRP. One skilled in the art will understand that the immunoassay techniques that are used in the above method are well known in the art. Therefore, any immunoassay technique, brand and visualization method can be used in the above method. An immunoassay is ELISA and a preferred brand is horseradish peroxidase. Another preferred brand is a colored latex bead.

EXAMPLE 7 Determination of the Reproductive Potential of an Ostrich The following is a method to determine the reproductive potential of an ostrich by quantifying the amount of inhibin in the blood of the ostrich. The inhibin concentrations of blood circulation of an ostrich can be determined, using radioimmunoassay (RIA) or normal sandwich ELISA techniques. First, bind the anti-inhibin antibodies produced in Example 4 to the cavities of a microtiter plate. After washing and blocking the plate, after adding a quantity of blood or serum that was obtained from an ostrich to a quantity of microtiter plate. After leaving any inhibin in the sample, if present, selectively interacting with the immobilized anti-inhibin antibodies, the sample was washed from the cavity of the plate. Then, add a different anti-inhibin antibody to the plate than that produced by Example 4, which is conjugated to horseradish peroxidase. The anti-inhibin antibody conjugated with HRP differs from the immobilized anti-inhibin antibody in that they are selectively directed against different portions of inhibin. After allowing the labeled antiinhibin antibody to interact selectively with any immobilized inhibin, any non-interacting labeled anti-inhibin antibodies are washed out. The amount of inhibin present in the plasma sample was determined by adding NBT to the well and visualizing the amount of labeled anti-inhibin antibody immobilized in the well. The normal positive and negative controls are operated simultaneously near the plate cavities. Many immunoassay techniques, brands and visualization methods are well known in the art. Accordingly, any method of immunoassay, branding and visualization technique can be used in the present invention.

EXAMPLE 8 Improvement of Egg Production in Codorni cs As stated above, the chicken inhibin a subunit cDNA clone (cINA6) inserted into the Bluescript EcoR 1 site was obtained as a donation from P. A. Johnson (ornell University). A DNA fragment ("cINAc i") was separated from clone cINA6 using Pst I digestion. The cINA521 DNA fragment comprised the majority of the a subunit of mature chicken inhibin. This fragment (cINA521) was cloned into the plasmid p-MAL MR '-c in frame with the maltose binding protein ("MBP") and a fusion protein of the appropriate size (Lane E; Figure 1) was detected after of the induction of IPTG (isopropyl-β-D-thiogalactopyranoside) and SDS-PAGE. The resulting protein conjugate ("MBP-cINA521") was used as an antigen to immunize Japanese, pre-pubescent female quail (Coturnix coturnix japonica) against circulating inhibin levels as described below. Freshly hatched quail were incubated in an incubator battery Model 2S-D were modified for quail. The initial incubation temperature was about 37.8 ° C with a weekly reduction of about 2.8 ° C until room temperature was obtained. During the growing period (that is, up to approximately 6 weeks of age), a quail starter ration (28% CP, 2,800 kcal ME / Kg of feed) and water for consumption ad libitum, and continuous subdued light ( 22 lx) with a light of 14 hours (280 to 300 lx): 10 hours dark operation was used. At 25 days of age, 50 quail were randomized and equally assigned to one of the two injection groups (25 birds per group) as follows; (1) MPB-cINA521 in Freund's auxiliary ("MBP-cINA521 / FRN"), or (2) Freund (auxiliary control, "FRN"). Birds immunized against inhibin (Group 1) was given approximately 0.75 mg MBP-cINA521 per bird in the appropriate control vehicle. The equivalent injection volumes (0.2 ml) of FRN were administered to the group. All injections were submitted subcutaneously using tuberculin syringes equipped with 25 ga needles. After the injections, to the wings of the quails ee put some bands to identify them by the treatment before lodging them (individually) in production cages. Immunizations of weekly booster inhibin of approximately 0.375 mg of MBP-cINA521 per bird, or appropriate control attacks, were subsequently administered for five consecutive weeks (ie, at 32, 39, 46, 53 and 60 days of age) and then every 35 days of age, after the three additional attacks (ie, at 95, 130 and 165 days of age). Beginning at 6 weeks of age, a food and water relationship was provided to the quails (21% CP, 2,750 kcal ME / kg of food) for ad libitum consumption. Beginning at 41 days of age (considering Day 1 of the egg production cycle) egg production measurements were recorded per day in the hen ("HDEP") and mortality ("MORT") daily for 20 consecutive weeks. In addition, the average age in the first egg production ("FIRST") and the age at which the hens reached 50% of egg production ("FIRST") and the maximum egg production as defined above, were calculated. for one of the treatment groups. The egg production data per day of the hens were subjected to variation analysis ("ANOVA") of which a completely randomized design with a treatment division graph layout was incorporated. The main graph consisted of two injection treatments (MBP-cINA521 / FRN or FRN) and the 20 production periods of 7 days each comprised the division. The immunoneutralization of inhibin clearly accelerated puberty in quail hens. The average age of FIRST egg production was reduced (P <.0088) by approximately six days in the hens treated with inhibin (Table 2). Likewise, the age of the FIFTY egg production was markedly reduced (12 days: P <01) in the hens treated with inhibin (Table 3). A positive effect of inhibin treatment on egg production per day of hens (HDEP) also existed, very remarkably at the beginning and end of the production cycle (Figure 2). For example, mean HDEP regimens (P <.05) were observed significantly in hens treated with MBP-cINA521 / FRN when compared with FRN controls during weeks 1 (16.5 versus 2.6%), 2 (50.0 against 28.6%) and 4 (96.6 against 79.7%) and again during weeks 15 (98.8 against 86.9%), 16 (96.9 against 86.3%), 18 (85.7 against 66.1%) and 20 (96.8% against 73.8%). The total HDEP ratio (including all 20 weeks of production) for hens treated with inhibin was 83.5% compared to 75.4% for controls. In addition to accelerating puberty, prolonging egg production and improving total production intensity, treatment with inhibin reduced the time needed to reach peak egg production by approximately 3 weeks (Figure 2; Compare MBP-cINA521 / FRN = 96.6% HDEP in the fourth week centers FRN = 96.6% HDEP by week 7). Although the differences in HDEP peak values were not statistically evaluated, treatment differences in the mean age at which hens reached 50% HDEP levels (FIFTY) reflected peak yield. Mortality was not a factor in this study since only eight birds died (three from the control, five from the treated). Such MORT (16%) could be within the expected limits for the quail that reached 180 days old.

Table 2 Effect of Inhibone Immunoneutralization of Inhibin in Middle Ages (± SE) in the first egg production in Japanese quail Treatment Age in the first egg production (days) FRN1 56.15 = 1.82a MBP-cINA521FRN2 50.38 = 1.08b 1 = Freund 2 auxiliary control = MBP-cINA521 / FRN = Maltose Binding Protein-GEV- ^ chicken inhibin fusion protein in Freund's auxiliary. a'b (P <0088).

Table 3 Effect of Inhibitone neutralization in the mean age (± SE) to 50% of egg production in Japanese quail Treatment Age in the first egg production 50% (days) FRN1 73.04 = 3.78a MBP-cINA521FRN2 61.00 = 2.70b 1 = Freund 2 auxiliary control = MBP-cINA521 / FRN = Maltose binding protein-chicken inhibin fusion protein 515 in Freund's auxiliary. a'b (P <01). Signs of eggs with eggshells and thin shells (without eggshells) occurred at higher frequencies in control birds, particularly in the final stages of the production cycle, than in birds immunized with inhibin. This suggests that higher numbers of defective egg levels (ie, eggs that were either not set for consumption or similarly broke before consumption or not established as incubation / brooding eggs) were associated with the control treatment.

EXAMPLE 9 Improvement of Production Performance in Ostriches The protein conjugate (MBP-cINA521) was used as an antigen to immunize prepubescent female ostriches, against the circulation of inhibin levels and to accelerate afterwards the start of egg production in the treated ostriches . The method described in Example 8 was followed with the following exceptions. The average age at puberty for an untreated ostrich is approximately 28 and 32 months. The following is the treatment schedule for an ostrich having an approximate body weight scale of 68.10 to 136.20 kg (150 to 300 pounds): 5.0 mg primary injection (first) of the heterologous protein of the present invention in the 26 months old; and reinforcements of 2.5 mg at 27, 28, 30, 32, 34 and 36 months of age.

EXAMPLE 10 Improve Production Performance in Emu The protein conjugate (MBP-cINA521) was used as an antigen to immunize preusbescent female emus against the circulating levels of inhibin, and thus accelerate the onset of egg production in the emus treated. The method described in Example 8 was followed with the following exceptions. The average age at puberty for an untreated emu is approximately 20 months. The following is the treatment schedule for a parrot having an average body weight scale of 22.7 to 40.8 kg (50 to 90 pounds): primary (first) injection of 3.0 mg of the heterologous protein of the present invention in its month 18 old; and reinforcements of 1.5 mg in the months 19, 20, 22, 24, 26 and 30 of age.

EXAMPLE 11 Improve Production Performance in Chickens The protein conjugate (MBP-cINA, - ^,) was used as an antigen to immunize prepubescent female chickens against circulating levels of inhibin, defectin circulating inhibin, and thus accelerate the start of egg production in treated chickens. The method described in Example 8 is followed with the following exceptions. The average age at puberty for an untreated chicken is approximately 20 weeks. The treatment schedule for a chicken having a body weight scale of approximately 0.908 to 1.58 kg (2.0 to 3.5 pounds) primary (first) injection of 1.5 mg heterologous protein of the present invention at its 15 week age, and boosters of 0.75 mg in the 17, , 24, 30, 40 and 50 weeks of age.

EXAMPLE 12 Improvement of Turkey Production Performance The protein conjugate (MBP-cINA521) was used as an antigen to immunize prepubescent female turkeys, against the circulation of inhibin levels and to accelerate afterwards the start of egg production in the treated turkeys . The method described in Example 8 was followed with the following exceptions. The average age at puberty for an untreated turkey is approximately 30 weeks. The following is the treatment schedule for a turkey having an approximate body weight scale of 4.08 to 5.44 kg (9.0 to 12 pounds): primary (first) injection of 2.0 mg of the heterologous protein of the present invention in its week 28 old; and 1.0 mg boosters at 29, 30, 34, 38, 46 and 54 weeks of age.

EXAMPLE 13 Improve Production Performance in Parrots The protein conjugate (MBP-cINA521) was used as an antigen to immunize prepubescent female parrots against the circulating levels of inhibin, and thus accelerate the onset of egg production in the parrots treated. The method described in Example 8 was followed with the following exceptions. The average age at puberty for an untreated parrot is approximately 30 months. The following is the treatment schedule for a parrot having an average body weight scale of 0.22 to 0.56 kg (0.5 to 1.25 pounds): primary (first) injection of 0.75 mg of the heterologous protein of the present invention in its week 28 old; and reinforcements of 0.75 mg in weeks 28, 30, 32, 34, 36 and 38 of age. It should be understood, of course, that the above refers only to preferred embodiments of the present invention and numerous modifications and alterations thereof may be made, without departing from the spirit and scope of the invention as set forth in the appended claims.

SEQUENCE LIST (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Agritech Technologies, Ltd. (B) STREET: Jones & Askew, 191 Peachtree Street, Ste. 3700 (C) CITY: Atlanta (D) STATE: Georgia (E) COUNTRY: United States of America (F) ZIP CODE (ZIP): 30303 (G) TELEPHONE: (404) 818-3700 (H) TELEFAX: (404) 818-3799 (A) NAME: The Board of Supervisors of Louisiana State University and Agricultural and M (B) STREET: Jones & Askew, 191 Peachtree Street, Ste. 3700 (C) CITY: Atlanta (D) STATE: Georgia (E) COUNTRY: United States of America (F) ZIP CODE: 30303 (G) TELEPHONE: (404) 818- 3700 (H) TELEFAX: (404) 818-3799 (ii) TITLE OF THE INVENTION: Compositions of Inhibin and Methods for Improving Production Yield (iii) NUMBER OF SEQUENCES: 2 (iv) LEADABLE FORM OF THE COMPUTER: (A) ) TYPE OF MEDIUM: Flexible disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.30 (EPO) (2) INFORMATION FOR SEQUENCE IDENTIFICATION NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 521 base pairs (B) TYPE: nucleic acid (C) SHAPE FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) ASPECT: (A) NAME / KEY: CDS (B) LOCATION: 1.303 (xi) DESCRIPTION OF SEQUENCE: IDENTIFICATION SEQUENCE NO: 1: CTG CAG CGC CCA TCG GAG GAC GTG GCC GCC CAC ACC AAC TGC CGC CGG 48 Leu Gin Arg Prc Ser Glu Asp Val Aid Wing Kis Thr Asn Cys Arg Arg 5 10 15 GCG TCC CTC AAC ATC TCT TTC GAG GAG CTG GGC TGG GAC AAT TGG ATC 96 Wing Ser Leu Asp lie Ser Phe Glu Giu Leu Gly Trp Asp Asn Trp He 20 30 GTG CAC CCC AGC AGC TTC GTT TTC CAC TAC TGC CAC GGG AAC TGT GCC 144 Val His Pro Ser Ser Phe Val Phe His Tyr Cys His Gly Asn Cys Ala 35 40 45 GAA GGC CAC GGG CTG AGC CAC CGG CTG GGG GTG CAG CTG TGC TGC GCC 192 Glu Gly His Gly Leu Ser His Arg Leu Gly Val Gln Leu Cys Cys Ala 50 55 60 GCG CTG CCC GGC ACC ATG CGC TCA CTG CGT GTC CGC ACC ACC TCT GAT 240 Wing Leu Pro Gly Thr Met Arg Ser Leu Arg Val Arg Thr Thr Ser Asp 65 70 75 80 GGT GGC TAC TCC TTC AAG TAC GAG ACG GTG CCC AAC ATC CTG GCG CAG 288 Gly Gly Tyr Ser Phe Lys Tyr Glu Thr Val Pro Asn He Leu Wing Gln 85 90 95 GAC TGC ACC TGT GTC TAGCAGCTGG CATGCACGGC CAGACCCGCG TGGATCTCCC 343 Asp Cys Thr Cys Val 100 CGTTGCCTCT GGACTGCCCC AGTGCCAGAT GATGAGCCCA TCCCAGGGAT GGAGGAGTCA 403 CTCACACGGG CACTGCGCAG CCCGGAGCAG GGAGAGGGAC CCAGGTGGAA GTTTTGGTGG 463 TGCCACCCTC CCTTTGACTG CCAGGGTTTC ATGGTTTCAG GTTGCGTGGG TGCTGCAG 521 (2) INFORMATION FOR IDENTIFICATION SEQUENCE NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 101 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear iii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: IDENTIFICATION SEQUENCE OR: 2: Leu Gln Arg Pro Ser Glu Asp Val Ala Ala His Thr Asr. Cys Arg Arg 1 5 10 15 Wing Ser Leu Asn lie Ser Phe Glu Glu Leu Gly Trp Asp Asn Trp He 20 25 30 Val His Pro Ser Phe Val Phe His Tyr Cys His Gly Asn Cys Ala 35 40 45 Glu Gly His Gly Leu Ser His Arg Leu Gly Val Gln Leu Cys Cys Wing 50 55 60 Wing Leu Pro Gly Thr Mee Arg Ser Leu Arg Val Arg Thr Thr Ser Asp 65 70 75 80 Gly Gly Tyr Ser Phe Lys Tyr Glu Thr Val Pro Asn He Leu Wing Gln 85 90 95 Asp Cys Thr Cys Val 100

Claims (26)

1. A method for improving a bird's production performance, characterized in that it comprises administering an effective amount of a heterologous protein comprising a protein of inhibin of alpha subunit poultry, a fragment thereof, and a carrier protein, to a bird so that the production performance of the bird is improved.

2. The method in accordance with the claim 1, further characterized in that the bird is selected from the group consisting of ratites, psittaciformes, falconiformes, piciformes, estrigiformes, paseriformes, coraciformes, raliformes, cuculiformes, columbiformes, galliformes, anseriformes, and herodionas.

3. The method in accordance with the claim 2, characterized because the ratite is an ostrich, emu, ñandu, kiwi or cassowary.

4. The method according to claim 2, characterized in that the bird is a chicken, a turkey, a parrot, parrot, hawk, eagle, quail, dove, cockatoo, bird singer, talking bird, blackbird, chaffinch, cantor, goose, duck , canary, maniato, toucan or sparrow.

5. The method according to claim 1, characterized in that the inhibin protein has or contains substantially the same sequence of SEQUENCE OF IDENTIFICATION NO: 2.

6. The method according to claim 1, characterized in that the carrier protein is maltose binding protein, bovine serum albumin, ovalbumin, flagellin, key lime hemocyanin, thyroglobulin, serum albumin, gamma globulin, syngeneic cells, cells syngeneics that carry antigens or amino acid polymers.

7. The method according to claim 1, characterized in that the bird is a female bird.

8. The method according to claim 1, characterized in that the bird is a male bird.

9. A fused heterologous protein, characterized in that it comprises an alpha subunit inhibin protein, a fragment thereof, fused to the carrier protein.

10. The heterologous protein fused in accordance with claim 9, further characterized in that the inhibin protein has or contains substantially the same SEQUENCE sequence. IDENTIFICATION NO: 2.

11. The fused heterologous protein according to claim 9, characterized in that the carrier protein is maltose binding protein, bovine serum albumin, ovalbumin, flagellin, key limed hemocyanin, thyroglobulin, serum albumin, gamma globulin, syngeneic cells, syngeneic cells that carry antigens or amino acid polymers.

12. A method for producing a heterologous fused protein, characterized in that it comprises the steps of: (a) providing a double-stranded chain cDNA, which encodes alpha subunit inhibin or fragment thereof; (b) providing a vector which has coding information for the production of a carrier protein; (c) ligating the inhibin cDNA to the vector; (d) inserting the vector linked to an expression system; and (e) expressing a fused heterologous protein comprising an inhibin protein, or a fragment thereof, and the carrier protein.

13. The method according to claim 12, characterized in that the inhibin cDNA has or substantially contains the same sequence as the SEQUENCE OF IDENTIFICATION NO: 1.

14. The method according to claim 12, characterized in that the inhibin protein or fragment thereof in the fused heterologous protein, has or contains substantially the same sequence of SEQUENCE OF IDENTIFICATION NO: 2.

15. The method according to claim 12, characterized in that the carrier protein is maltose binding protein, bovine serum albumin, ovalbumin, flagellin, key limed hemocyanin, thyroglobulin, serum albumin, gamma globulin, syngeneic cells, syngeneic cells that carry antigens or amino acid polymers.

16. A fusion gene product, characterized in that it comprises a gene coding for the expression of the inhibin protein of alpha subunit poultry or a fragment thereof, fused to a gene encoded for the expression of a carrier protein.

17. The fusion gene product according to claim 16, characterized in that the gene encoded for the expression of the alpha subunit inhibin poultry protein is encoded for the expression of a protein having or substantially containing the same sequence as IDENTIFICATION SEQUENCE NO: 2.

18. The fusion gene product according to claim 16, characterized in that the gene encoded for the expression of the poultry inhibin protein of the alpha subunit is substantially the same as the SEQUENCE OF IDENTIFICATION NO: 1.

19. The fusion gene product according to claim 16, characterized in that the gene encoded for the expression of a carrier protein is encoded for the production of maltose binding protein, bovine serum albumin, ovalbumin, flagellin, lipid hemocyanin. of key, albumin serum, thyroglobulin, gamma globulin, syngeneic cells, syngeneic cells bearing the antigen, or amino acid polymers.

20. A composition comprising a cell containing a vector, wherein the vector contains a DNA sequence encoding inhibin, a fragment thereof, and a carrier protein, and wherein the vector is capable of expressing inhibin or a fragment of the same, fused to the carrier protein, when it is present in the cell.

21. The composition according to claim 20, characterized in that the DNA sequence encoding inhibin or fragment thereof is SEQUENCE OF IDENTIFICATION NO: 1.

22. A composition comprising a vector, characterized in that the vector contains a DNA sequence encoding the inhibin or a fragment thereof or a carrier protein wherein the vector is capable of expressing inhibin or fragment thereof, fused to the carrier protein , when it is present in the cell.

23. The composition according to claim 22, characterized in that the DNA sequence encoding inhibin or fragment thereof is SEQUENCE OF IDENTIFICATION NO: 1.

24. A method for improving the production performance of a bird, characterized in that it comprises administering an effective amount of a fusion gene product, comprising a gene encoded for the expression of alpha subunit inhibin poultry protein, a fragment of the same, and a gene encoded for the expression of the carrier protein, to a bird such that the production yield of the bird is improved.

25. The method comprising implanting in an animal, a cell containing a vector, wherein the vector contains a DNA sequence encoding inhibin or a fragment thereof, and a carrier protein, and wherein the vector is capable of expressing inhibin or a fragmentary of it, fused to the carrier protein, when it is present in the cell.

26. The method according to claim 25, characterized in that the DNA sequence encoding inhibin, or a fragment thereof, is SEQUENCE OF IDENTIFICATION NO: 1.