WO2008064445A2

WO2008064445A2 - Oily parenteral formulation for the enhancement of the reproductive performance of production animals

Info

Publication number: WO2008064445A2
Application number: PCT/BR2007/000321
Authority: WO
Inventors: Celso Akio Maruta; Carolina Mieko Sadahira; Edson De Souza Moura; Márcio LIBONI
Original assignee: Vallée S.A.
Priority date: 2006-11-27
Filing date: 2007-11-23
Publication date: 2008-06-05
Also published as: AR063929A1; BRPI0604942A

Abstract

This invention refers to parenteral formulations comprising beta-carotene, vitamin E and oily substances proposed as a way of enhancing the biological effects of these compounds, taking into consideration that additional requirements are necessary for production animals when there is less availability of these substances in the ingested feed or a larger metabolic demand thereof. This invention will be widely used in the reproduction of production animals.

Description

"OILY PARENTERAL FORMULATION FOR THE ENHANCEMENT OF THE REPRODUCTIVE PERFORMANCE OF PRODUCTION ANIMALS" FIELD OF THE INVENTION

The present invention refers to, parenteral formulations comprising beta-carotene, vitamin E and oily substances provided as a way of enhancing the biological effects of these compounds, taking into consideration that additional requirements are necessary for production animals when there is less availability of these substances in the ingested feed or a larger metabolic demand thereof. This invention will be widely used in the reproduction of production animals.

BACKGROUND OF THE INVENTION

The concentrations of vitamin E and beta-carotene in feed are variable and influenced by several factors. The action of heat, moisture, rancid fat and transition metals increase the oxidization of vitamin E and, consequently, decrease its concentration in the feed. Moreover, the processes of trituration, pelletization, and mixing with other minerals in the concentrated commercial feed, also produce losses in the concentration. With respect to the beta-carotene, the riper the forage, the greater the losses of the compound. Furthermore, feed conservation processes, such as silage and haymaking, also reduce the amount of beta-carotene.

Another point that can generate losses of these actives is in the absorption phase. In ruminants, there are evidences of the destruction of vitamin E by the ruminal microbiota. With respect to beta-carotene, there is a lesser intake of this compound due to its adhesion to the cellular content of the ruminal fluid. Another important feature is the capability to absorb beta-carotene directly into the blood circulation, without converting beta-carotene into vitamin A. On average, relatively low quantities are absorbed by this manner, varying around 30% of the total present in the diet.

Several proposals have been presented to effectuate the supplementation of these substances but they require oral administration together with the feed. For example, there are Caroplus® (Daonechem) , beta-carotene compound, vitamin A, vitamin E, selenium and zinc; Rovimix® (DSM) , a 10% beta-carotene compound. The method of using these products cannot avoid the risk of the aforementioned losses. Moreover, said products need a daily administration over a relatively long period. Another product commercialized in the European market is Dalmavital® (Fatro Uriach) , an injected solution, indicated for intramuscular administration and composed of only a 4% concentration of beta-carotene .

US document No. 4,435,427 describes a process for the preparation of a parenteral aqueous solution containing beta-carotene . Nowadays, there is no product available in the market with the same concepts and features of the current invention. The composition of the product is formed with two potential antioxidants, beta-carotene and vitamin E, in an oily carrier, and with the indication for parenteral administration. This formulation will allow greater availability of these compounds for the animal, avoiding the previously mentioned losses of actives. The volume and number of administrations will be reduced in comparison with products that are orally administered. Furthermore, the experimental results showed an improvement in the quality of the embryos of super-ovulated cows and donor mares.

According to Fettman, 2001, the maintenance of an excellent sanitary status, and/or suitable conditions for sustaining the growth and the suitable productive and reproductive performance of production animals depends essentially on the presence in the diet of liposoluble vitamins, hydrosoluble vitamins, macrominerals, microminerals and other nutrients, such as essential fatty acids (linoleic and linolenic acid) and sulphurated amino acids, such as methionine, cystine and taurine, which perform significant metabolic functions. These substances have several structural and biochemical actions that sometimes operate in a complex of physiological functions of the animal organism. Most of these nutrients are supplied by the natural diets of the animals and are available in their natural environment. However, the extraordinary impositions of environmental conditions, or increased performance demands, can alter the physiological requirements for many of these substances. Moreover, the formulation of diets with alternative feeds can alter the bioavailability and interaction of these nutrients. Additionally, specific diseases can influence the absorption, metabolism and excretion of certain nutrients in such a way that deficiencies may occur. Veterinary medicine practice must have the objective of preventing nutritional deficiencies as well as the correction of imbalances. In view of the extraordinary conditions that unleash these deficiencies and imbalances, the pharmacological attributes of the nutrients must be considered, as a complement to their nutritional effects . On the other hand, Almeida (1999), states that the reproduction of mammals is a complex process that must occur in harmony with existing dietetic, physical and environmental conditions. Among the environmental factors that may influence the reproductive activity (photoperiod, temperature and behavior) , the availability of feed plays the most important role (Bronson, 1985, Bronson 1999) . In their natural habitat, adult mammals must look for their own feed, assimilating and distributing the energy obtained from this feed with other interactive and frequently competitive organic functions. The functions that need to be firstly supplied are: cellular maintenance, thermoregulation and the expenditure with locomotion to obtain the feed. Having supplied these primordial necessities, the remaining energy can be used for growth and reproduction or be stored as fat (Bronson 1985) . Therefore, when the feed availability is limited or when there is an increase in the energetic demand, without increasing the consumption of calories, the fertility of mammals can be reduced, including humans (Armstrong & Brett, 1987; Cameron & Nosbich, 1991; Berriman & Wade, 1991; Foster et al., 1995; Cuming et al., 1994; Loucks & Health, 1994; Zack et al., 1997).

US Patent No. 4,435,427 describes a parenteral aqueous solution containing injectable beta-carotene and its preparation process. For its administration, it is necessary that the formulations have a relatively high content of beta-carotene in a very fine form divided in such a way that the amount to be injected is a minimum. Nutritional infertility is commonly observed in female animals due to the high energetic needs of a complete reproductive cycle, comprising ovulation, conception, pregnancy and lactation. This is especially applied in the case of multiparous females. On the other hand, the reproduction can be re-established when there is an improvement in energetic conditions (Wade et al. 1996). Since the development of the reproductive capacity is essential for the survival of the species, a great deal of attention has been focused on the maintenance and improvement of the reproductive activity.

The interaction between nutrition and reproduction has been studied in several species of mammals, including rodents (Bronson & Marsteller, 1985; Bronson, 1986; Murahashi et al., 1996; Wade et al., 1997), pigs (Armstrong & Britt, 1987; Cox et al . , 1987; Booth, 1990a; Cosgrove et al., 1993b; Rozeboom et al., 1993; Zak et al., 1997), bovines (Zurek et al., 1995; Kinder et al., 1995; Schillo 1992), ovines (Schillo, 1992; Foster et al., 1995; Martin & Walkden-Brown, 1995) and primates (Cameron & Schreihofer, 1995) , as well as humans (Loucks, 1996) . In the commercial field particularly, reproductive efficiency is one of the most important factors to obtain profits. Consequently, several studies have been carried out with the objective of better understanding this interaction and the subsequent improvement in the fertility rates of the animals.

Among various factors that affect the reproductive performance of bovines, Santos, 1998, states that nutrition is the factor of greatest impact. Several researches have shown that the nutritional and metabolic state of the animal affect its reproductive functions. However, despite the vast literature found about this subject, very little is known with respect to the reproductive mechanisms influenced by nutrition, such as the hypothalamic-pituitary-ovary axis, and the establishment and maintenance of pregnancy. Among all the nutrients, energy appears as the main requirement for cows in the reproductive process, wherein an unsuitable supply in the diet has harmful effects on the reproductive efficiency of these animals. Cows that have a negative energy balance have lower plasmatic levels of glucose, insulin and a growth factor similar to insulin-I; they have fewer pulse frequencies of luteinizing hormone; they have lower plasmatic concentrations of progesterone; and they display irregularities in ovarian activity. The effects of the negative energy balance on bovine fertility appear to be divided by metabolic and endocrine irregularities, which result in changes in the ovarian activity, and also compromise not only the viability of the oocyte to be fertilized, but also the activity of the resulting corpus luteum. The manipulation of the composition of the diet and feed management, with the goal of increasing the energy supply for high production cows, will reflect in an improved reproductive performance of these animals. Among these actions, the supplementation with fat in the diet of cows in reproduction should be emphasized, wherein the increase in the energy ingestion alters the secretion of the prostaglandin F2α by the uterus, affecting the dynamic of the follicular growth and improving the luteal function and the fertility.

However, the ingestion of excessive quantities of raw protein or degradable protein in the rumen increases the concentration of uric nitrogen in the blood and milk, and alters some uterine secretory functions, which can compromise the conception rate of high production dairy cows. Recent data indicates that an excess of uric nitrogen in the uterine lumen can exacerbate the secretion of prostaglandins. Although the evidence indicates that the level of protein in the diet can interfere in the reproduction of high production cows, the data for herds of cattle with lower levels of production appear to be contradictory. In beef cattle, the low concentration of raw protein in the diet during the pre and postpartum periods influence the return to the cyclical activity, and reduce the pregnancy rate during the breeding season. The mineral and vitamin nutrition of cows during the transition period is very important for an excellent reproductive performance in the postpartum. Mineral and vitamin levels in the diet of similar dairy or beef cattle, or, in some cases, a little superior to the current recommendations, seem to be suitable for an excellent reproductive performance.

Prior art teachings demonstrated that the use of beta-carotene and vitamin E improve the reproductive efficiency of several animal species. However, the effects of these antioxidants have not been related to the quality of embryos in transference programs yet.

The formulation that will be described as follows supply this demand.

SUMMARY OF THE INVENTION

The present invention provides a parenteral supplement of beta-carotene and vitamin E, in oil. It is proposed as a way of enhancing the biological effects of these compounds, taking into consideration that additional requirements are necessary for production animals, in the following conditions; -when these substances are present in insufficient contents in the voluminous sources or in the concentrated feed offered to the animals, or present in forms with low biological availability;

-when low quality and deficient feed in these substances is offered to the animals (for example, animals kept in extremely dry pastures or in confinement) ;

-when are offered diets that contain unsaturated fats/nitrates or feed contaminated with heavy metals/aflatoxins, because they increase the usual requirements of vitamin E and selenium, respectively;

-when the production, conservation, and storage conditions of the feed result in the destruction of the initial contents of these compounds;

-when there is an increase in the metabolic demand, which often occurs in high performance animals;

-when there are conditions of stress, which frequently increase the susceptibility of various diseases (for example, the intensive production system) ;

-when the intake of these compounds offered in a normal diet is prejudiced, due to diseases, such as bad absorption syndrome, the presence of parasites in the gastro-intestinal tract and metabolic complications. The potential antioxidant agent beta-carotene acts in cellular tissues of low partial oxygen pressure and the potential antioxidant agent vitamin E acts in cellular tissues with high partial oxygen pressure. It is expected that the combined use of these substances will present synergy in the anti-oxidant effect. This antioxidant effect was demonstrated as being beneficial for the synthesis of steroids, of which we can highlight the progesterone sexual hormone. With respect to the vitamin, which is mainly recognized for its antioxidant effect, it also acts in several physiological processes, among them the synthesis of the cellular membrane and the prostaglandin, the blood coagulation, the reproductive function as a whole, and the immune response. In animal reproduction, specifically, it acts in the reduction of incidences of placenta retention, metritis, and ovarian cysts, as well as the reduction in time of post-metritis uterine degeneration. Other effects of vitamin E are related to the reduction of the incidence of clinical mastitis and the prevention of the milk oxidization.

Beta-carotene is a precursor of vitamin A, whose role in reproduction is widely recognized. However, there are several evidences that indicate its exclusive effect on reproduction, highlighted by the increase in the intensity of the estrous, the increase in the conception rate and the reduction in the number of services for conception, in the open days and in the incidence of ovarian cysts. After conception, the pregnancy is more efficiently maintained in animals that receive supplements with beta-carotene due mainly to the reduction in premature embryonic mortality and premature abortion rates.

DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION The parenteral supplementation of these compounds is recommended to maximize its availability, since there are several factors that interfere in their presence in the feed, absorption, distribution and metabolization. This aspect is particularly important for the case of beta- carotene, which has a high cost. The benefits of this supplementation can be expected when applied to the following animal species: bovines, buffalo, equines, pigs, ovines and goats. Therefore, it is provided the pharmaceutical formulation in question which comprises the solubilization of vitamin E in a lipophilic excipient, as well as a suspension of beta-carotene in this oily base. This preparation involves the concentration of vitamin E ranging from 0.01% to 15% (x = 5% w/v) , and concentrations of beta- carotene ranging from 0.01% to 30% (x = 8% w/v), from 0.01% to 30% w/v of beta-carotene, and 1 to 99% v/v of oily base. The beta-carotene is selected from a group consisting substantially of synthetic, semi- synthetic or naturally sourced beta-carotene. The vitamin E acetate is selected from a group consisting substantially of one of its 8 isomers. The oily substance is selected from a group consisting substantially of trygliceride of capric acid, caprylic acid, any vegetable oil such as sesame, sunflower, rice, soy, peanut, or a mixture thereof, being preferably trygliceride of capric acid or caprylic acid. The trygliceride of capric acid or caprylic acid is selected from a group consisting substantially of medium chain trygliceride, Captex, Estasan and Miglyol.

Furthermore, there is potential of association (in view of its practical applications) in this same preparation, other liposoluble vitamins such as vitamin A (such as retinyl palmitate, retinyl acetate or retinyl proprionate) and vitamin D (D2-ergocalciferol; D3- calciferol, calcitriol and colecalciferol) , wherein the liposoluble vitamin (s) can be present ranging from 0.01% to 15% w/v; and hydrosoluble vitamins such as vitamin C and B- complex vitamins (such as, for example, folic acid, thiamine-vitamin Bl, riboflavin-vitamin B2, pyridoxine- vitamin B6, cyanocobalamin-vitamin B12, biotin, pantothenic acid, etc) ; choline; minerals (such as selenium, zinc, copper, molibdenium, iodine, manganese, calcium and phosphorous) and essential fatty acids (such as linoleic acid and linolenic acid) , wherein the hydrosoluble vitamin (s) may be present ranging from 0.01% to 15% w/v.

The formulation can further comprise antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) ranging from 0.005% to 0.5% w/v.

In this same concept is disclosed the use of the formulation as described in the invention, for the reproduction of production animals. Due to the large margin of safety of use in vivo and the large variability of demand for the main constituents, as previously explained, the recommended posology for the preparation can vary between 5 and 50 micromol/kg of metabolic weight for the aforementioned species, as a single dose or in successive doses at intervals of between 3 and 90 days.

The parameters of reproductive efficiency are low inheritance features, in other words, they are little influenced by genetic effects. This allows the environmental components to have a greater impact on the reproductive performance than the genetic selection. Therefore, the reproductive efficiency of a herd of cattle specialized in dairy or beef production is highly influenced by the management and the environment. Among the environmental factors that affect the bovine reproduction, nutrition has the greatest impact. Conception and maintenance of pregnancy are highly influenced by any factor that can alter the metabolic and endocrine balance in bovines, and even in other species of production animals. Because of this, many of the impacts of deficiency, excess or imbalance of nutrients are reflected in the reproductive performance of heifers and dairy and beef cattle. The following table displays some reproductive parameters of bovine females and its relationship with the supply of nutrients.

Deficiency, excess or imbalance of nutrients and reproductive parameters

Parameter Deficiency Excess Imbalance

Abortion, stillborn and Energy, PB I, Se, debilitated calves Ca, P, Mn, Cu,

Vitamins A, D and E

Anestrous and reduction Energy, PB, P, I, Mn, F in the estrous cycle Co, Vitamin A

Low conception and Energy, PB I, Mn, PB, PDR PB/energy premature embryonic Vitamin A mortality

Dystocia and uterine Energy, Ca Energy, Catio-anionic complications P, Ca

Delayed puberty and Energy, PB, Se, I, P, Mo, S Cu/Mo-S sexual maturity Ca, Cu, Mn, Vitamins

A and E

Metabolic disorders that Energy, Se, I, Mg, P, Energy, Catio-anionic affect the reproductive Ca, Vitamins E, A and PB, Ca, P performance D This review has the objective of putting into focus the functions of vitamin E and beta-carotene in the performance of production animals, emphasizing their role in the reproductive processes and the advantages of the parenteral supplementation of these nutrients. VITAMIN E

Properties and metabolism

Eight forms of vitamin E are naturally found, four of them tocopherols and four of them tocotrienols . Not only the tocopherols but also the tocotrienols consist of a hydroquinone core and an isoprenoid side chain. Structurally, the tocopherols have a saturated side chain, while the tocotrienols have said unsaturated side chain containing three double bonds . Depending on the arrangement of the methyl groups in carbons 5, 7, and 8 of the side chain, alpha, beta, gamma or delta forms of the tocopherol or tocotrienol can be formed. Of these, the d-alpha- tocopherol, the main form that occurs naturally, has the largest biological activity of vitamin E. The form dl has the least potency. The form d-beta-tocopherol and d-alpha- tocotrienol have approximately half of this activity and the other forms have only one tenth or less of this potency (McDowell 1989c) . Alpha-tocopherol is a yellowish oil that is insoluble in water, but soluble in organic solvents. Because the stability of all tocopherols that occur naturally is low, there are substantial losses of vitamin E activity in processed and stored feed. The sources of vitamin E in feed deteriorate under conditions that promote their oxidation (heat, oxygen, moisture, oxidized fats and minerals) . For the concentrated feed used in animal food, the processes of grinding, mixing with minerals, addition of fats and pelletization, can increase the oxidation of this vitamin. In this last process, the inclusion of antioxidants can prevent the accelerated oxidation caused by the conditions of high temperatures and moisture adopted (McDowell 1989c) . The ester acetate is very stable to oxidation in vitro and does not have any activity as an antioxidant. Nevertheless, as it is hydrolyzed in the gastrointestinal tract to free tocopherol, it becomes a potential antioxidant in vivo.

The activity of vitamin E is expressed in international units (IU) or milligrams (mg) , the dl-alpha- tocopherol acetate is the standard one (1 mg = 1 IU) . The free, synthetic dl-alpha-tocopherol acetate has a potency of 1.1 IU / mg. The natural d-alpha-tocopherol has a potency of 1.49 IU / mg and the d-alpha-tocopherol acetate has a potency of 1.36 IU / mg. The absorption of vitamin E is dependent on the digestion of fats being facilitated, therefore, by biliary and pancreatic secretions. The biggest part of vitamin E is absorbed in the form of alcohol. The dietetics esters of vitamin E are hydrolyzed in the intestinal wall and the free alcohol is transported by lymphatic chain to the circulatory system. The vitamin E in the plasma is mainly attached to the lipoprotein in the globulin fraction, and within the cells, it is mainly concentrated in the mitochondria and microsomes. For ruminants, there is apparently little or no pre-intestinal absorption of tocopherol; on the contrary, there are reports of the destruction of this compound by the ruminal microbiota. The absorption efficiency of vitamin E is lesser than the absorption efficiency of vitamin A, because the alpha-tocopherol is more absorbed in other forms, generally in proportion to its biological activities.

Vitamin E is stored by all the body tissues and mainly in the fatty tissues. A large ratio is also found in the liver. However, unlike vitamin A, the liver cannot be considered as an organ that stockpiles vitamin E, because this hepatic reserve is rapidly exhausted in a nutritional deficiency. Vitamin E does not cross the placenta of ruminants in appreciable amounts, thus the newborn is very sensitive to the supplementation of this compound. Although the concentration of vitamin E in the colostrum is directly related to the quantity taken in by the female that delivered the newborn, the colostrum does not present great concentrations of this vitamin. Nevertheless, it is a natural source better than normal milk. The principal excretion route of absorbed vitamin E is the bile, wherein it appears in a free form. Functions

Vitamin E is required mainly for its antioxidant effect, which prevents the peroxidation of lipids in the cellular membranes, therefore preserving the structural integrity of cells in general. Several physiological processes are influenced by vitamin E, including the biosynthesis of the cellular membrane structure, the biosynthesis of the prostaglandins, blood coagulation, the reproductive function and the immune response (McDowell 1989c) . Some signs of vitamin E deficiency can be reversed by the nutritional supplementation of other antioxidants, of which selenium is the most important (a co-factor of the glutathione peroxide enzyme, which acts in the capture of free radicals) .

Integrity of the membrane and the antioxidant effect

The functions of vitamin E as an antioxidant refer to the neutralization of free radicals and the prevention of the lipid peroxidation of the membranes. At the ultrastructural level, it preserves the micro-architecture of the membrane, the enzymatic activity, and prevents the accumulation of oxidative reaction products that can perpetuate damage arising from the formation of free radicals. As an example of these actions there is the protection of the erythrocyte membrane, the maintenance of the integrity of the blood capillaries, the inhibition of the plaque aggregation and the prevention of diseases, such as muscular nutritional dystrophy and encephalomalacia. Reproduction

Vitamin E and selenium are required for the normal reproductive function. The supplementation of these substances increases the fertility and reduces the frequency of some reproductive diseases in various animal species. The incidence of retention of fetal membranes in cows can be related to the oxidative stress that occurs in the prepartum. Therefore, this situation is responsive to the supplementation of vitamin E (Brzezinska-Slebodzinska et al., 1994) .

The most likely mechanism whereby selenium and vitamin E can act in the reproductive tissues is through their antioxidant functions. Furthermore, selenium and vitamin E can be indirectly involved in the synthesis of prostaglandins, wherein the peroxide radicals are a normal part of the metabolic route (Diplock, 1981) .

The function of vitamin E in the metabolism of lipids and in the integrity of the membrane can be independent of its antioxidant action. Vitamin E is related to the control of the phospholipase A2 activity (Pappu et al., 1978), which is responsible for the cleavage of the arachidonic acid of the membrane phospholipids. The arachidonic acid is a common precursor of all prostaglandins and their correlated compounds. The cleavage of the phospholipids by the phospholipase A2 also produces lysolecithin in the membranes, which in excess can result in the fusion of the cellular membranes (Lucy, 1970). A relationship between the function of the phospholipase A2 and the function of the vitamin E in reproduction, however, is still not well defined. Immune function The deficiency of vitamin E is associated with the increase of the frequency and severity of many infectious diseases, due to alterations in the response of the immune system. It has already been disclosed how stress can reduce the tissue concentrations of vitamin E (Nockels et al., 1996) . Vitamin E deficiency causes a reduction in the lymphocyte differentiation, as well as its proliferation in response to various mitogens, which interfere in the phagocyte and bactericidal functions of the neutrophiles (Langweiler et al., 1983; Lessard et al . , 1991). The supplementation of vitamin E, whether in vivo or in vitro, results in the increase of the mitogen-induced lymphocyte proliferation and the immune cellular response (toxicity mediated by cells and natural killer activity) (Reddy et al, . 1987). By the same manner, mono-nuclear cells, stimulated with concanavalin A from young bulls supplemented with vitamin E, expressed (in vitro) 55% more messenger RNA of interleukin-1 than the cells of non-supplemented animals (Stabel et al. , 1992) . Despite some contradictory data, it is generally acceptable that the feed supplementation of vitamin E and/or selenium, besides nutritional requirements, can give some advantages of immunological defense against some diseases. Hematopoiesis The deficiency of vitamin E in newborn, suckling, and weaning pigs, is associated with the development of anemia, leucocytosis, multinucleation of erythrocyte precursor cells and the increase in the number of megacaryocytes in the bone marrow (Nyio, et al., 1980). In addition to the effects of the maturation of erythrocyte precursor cells, the vitamin E deficiency can result in abnormalities of the membrane of the erythrocytes which can lead to an abnormal fragility and hemolysis (Brady et al., 1982) . Experimental evidences of anemia in humans caused by deficiencies of glucose-6-phosphate dehydrogenase or glutathione synthetase also indicate a non-specific hematopoietic effect of the supplementation of vitamin E. Antioxidant activity and the prevention of cancer

Several functions of vitamin E can play a role in the chemo-prevention of cancer. The principal function is as an antioxidant, which interrupts the damage caused by the formation of the free radicals, initiated by the activated metabolites of various polycyclic hydrocarbons and aromatic amines (Gould et al., 1991; Ngah et al., 1991). The ability of vitamin E to increase the immune response can promote the anti-cancer "guard" activities of the immune system. Furthermore, vitamin E can act as a purifier of nitric compounds, inhibiting the conversion of carcinogenic nitrosamines. Epidemiological evidences in humans indicate an inverse relationship between the concentrations of serum antioxidants and the risk of the development of cancer. This can be particularly important to individuals who ingest high amounts of polyunsaturated fatty acids in their diet (Pacher, 1991) . The supplementation of vitamin E is associated, in humans, with up to a 60% reduction in the risk of death by cardiovascular diseases, but did not demonstrate any effect on the incidence risk of pulmonary- cancer. Despite this, effective and safe levels of dietetic vitamin E supplementation for the chemical prevention of cancer still remain theoretical, and await objective experimental verification.

Relationship with other oral antioxidants

Oral antioxidants, such as polyunsaturated fatty acids, can increase the requirements of vitamin E, whereas other oral antioxidants can have a reciprocal and compensatory effect. In further detail, the following actions are expected: 1. vitamin E prevents the formation of fatty acid hydroperoxides, 2. the sulphurated aminoacids are precursors of the glutathione, which can purify the free radicals, and be submitted to join with chemical oxidizing agents, and 3. selenium is a necessary component of glutathione peroxidase, which catalyzes the reduction of free radicals through the glutathione (Fettman, 1991) . Thus, many of the disorders associated with the deficiency of vitamin E can be prevented or treated with selenium or other antioxidants, and the vitamin E can improve the signs of the alimentary insufficiency of selenium.

Requirements

Plasmatic concentrations of tocopherol from 0.5 up to 1.0 mcg/mL are considered low in most species, and amounts lesser than 0.5 mcg/mL indicate a deficiency.

The following table lists some of the requirements defined by the NRC for the different species and categories of animals. However, when the supplementation is made above these levels, the following effects can be observed: maintenance of the membrane stability, the enhancement of the immune system, tendency to gain greater weight, as well as the better quality of the meat.

Intrinsic factors

The requirements for vitamin E are greater during pregnancy, lactation and during the phases of rapid growth. The potential risk of the development of a deficiency during these life phases is increased by the concomitant occurrence of extrinsic factors with respect to other oxidants or dietetic antioxidants, environmental stress, and infectious diseases .

Extrinsic factors Stressful environmental conditions and diseases increase the requirement for vitamin E. Gastrointestinal diseases can affect the absorption of vitamin E. Seasonal effects related to the status of vitamin E were observed in equines due to differences in the feed concentration, chronic effects in function of the stability of tocopherol under storage conditions, and alterations in the nutritional requirements associated with modifications in the environmental and reproductive status (Maenpaa et al . , 1988 a,b). Other feed constituents can have significant influence in the stability, in the absorption by the intestine, or in the availability of vitamin E by the organism. The requirements of vitamin E in dogs increases five times with the increase of ingestion of polyunsaturated fatty acids in the diet. As a function of the destruction of vitamin E by oxidation, the content of unsaturated fat in feed can affect its stability, particularly under adverse handling and storage conditions. When these fatty acids suffer oxidative rancidification, before the consumption of the feed by the animal, there is a consumption of the vitamin E originally present in the feed. When peroxidation occurs at the time of the consumption of said feed, the effects can be evidenced in the body stocks of vitamin E in the animal. Transition elements, including iron, zinc, copper and manganese, can act as catalysts in chemical reactions, and be consequently prejudicial against the stability of vitamin E (Dove and Ewan, 1991). The nutritional deficiency of selenium can adversely affect the capability to purify the free radicals by the reduction of glutathione peroxidase activity, thereby increasing the dietetic requirements of vitamin E and other antioxidants. Dietetic antioxidants such as vitamin C can protect the vitamin E from oxidative lysis and safeguard its use in the animal's defense against oxidative damages. Sources

Products of animal origin are generally poor sources of vitamin E, whereas vegetable products, in particular cereal grains and fresh and ripe forage are excellent sources. Wheat germ oil is the most concentrated natural source of vitamin E. Other oils such as soy, peanut and cotton are also rich sources. The stability of all natural tocopherols is low and substantial losses of vitamin E activity occur in food when it is processed or stored, as well as in manufactured and stored animal feed.

Losses of between 54 and 73% of vitamin E are reported in alfalfa stored at 33⁰C for twelve weeks and for losses of between 5 and 33% in dehydration processes of said feed.

The conservation of moist grains in silos can totally eliminate the vitamin E contents originally present. Signs of deficiency

The natural occurrence of vitamin E deficiencies is rare in adults and shortfall syndromes are only observed in young or growing animals that are fed with diets that lack vitamin E or selenium. Nutritional muscular dystrophy or white muscle disease^' occurs in young mice, rabbits, birds, dogs, pigs, bovines, ovines, goats and equines (McDowell, 1989c) . This disease is clinically characterized by generalized weakness, stiffness, locomotion disorders and cardiac insuffiency (Moore and Kohn, 1991) . Muscular injuries occur bilaterally and are evidenced by the pallid and spotted appearance of the tissue. Microscopic findings include hyaline degeneration, fragmentation and the lysis of muscular fibers, followed by coagulative necrosis and dystrophic mineralization (Dill and Rebhun, 1985) . In pigs, dietetic hepatosis and dietetic microangiopathy can be identified (Van Vleet and Kennedy, 1989; Rice and Kennedy, 1989) . The hepatosis is characterized by the edema and the musky nut appearance of the liver, which are microscopically evidenced lobular hemorrhage and coagulative necrosis. Microangiopathy, also known as mulberry heart disease, is characterized by the fibrinoid degeneration of the arterioles, thrombosis of the myocardial and capillary arteries, and subendocardial hemorrhages. Vitamin E deficiency in birds results in two syndromes besides muscular dystrophy: exudative diathesis, characterized by lethargy, anexoria and severe subcutaneous edema, and encephalomalacia, in which hens from 2 to 6 months old develop ataxia due to edema and cerebellar hemorrhages (McDowell, 1989c) . In equines, a particular and apparently genetic form of myelencephalonic degeneration seems to be deficient of vitamin E, although gastrointestinal absorption tests appeared normal (Blythe et al., 1991). Considerations for supplementation: uses and indications

The need for vitamin E supplementation (as well as other antioxidants) for ruminants must be dependent on the individual requirement of the species and the different production conditions of its available content in the diet. Factors of primary importance for this supplementation are:

-Shortage of vitamin E in the amounts or concentrations offered to the animals; -Extremely dry pastures for the field cattle;

-Diets lacking vitamin E for confined animals;

-Diets that contain ingredients with low contents of vitamin E or which are less bioavailable; -Diets that increase the requirements for vitamin

E or selenium, due to presenting unsaturated fats and nitrates or contamination with heavy metals and aflatoxins, respectively;

-Conditions of production, conservation and storage of feed that result in the destruction of the initial contents of vitamin E;

-Accelerated performance rates that can increase metabolic demands;

-Intensive production that can indirectly increase the demand for vitamin E due to stress, which frequently increases susceptibility to various diseases (McDowell, 1989c; McDowell, 1992; McDowell and Williams, 1991) .

Improvement of the reproductive performance

In male mice, the vitamin E deficiency causes the degeneration of the germinal epithelium (Scott 1978), and the selenium deficiency results in the inhibition of the spermatogenesis. In female mice, the vitamin E deficiency causes fetal death and reabsorption, degenerative alterations in the uterus, degeneration of the vascular system of the embryo and anemia of the embryo (Scott 1978) .

Additionally, the transfer of vitamin E through the placenta to the fetus is inefficient (Mason and Bryan 1940) , which can also contribute to the emergence of degenerative alterations in the fetus.

In some studies with cattle, the positive effect of vitamin E on reproduction was not confirmed (Gallickson et al. 1949, Schingoethe et al. 1978; Buchanan-Smith et al. 1969) . However, superovulated beef cows under good nutritional conditions, supplemented with selenium, presented 100% fertilization versus 41% for non-supplemented cows. Subsequent studies in cows that were not superovulated (Segerson and Libby 1982) did not demonstrate an increase in fertilization rates after the supplementation with vitamin E and selenium, but there was an implication of the increase of spermatic transport. The supplementation with vitamin E and selenium increased the uterine contractions in the direction of the oviduct in ovines (Segerson and Ganapathy 1981, Segerson et al. 1980). Animals deficient in selenium and vitamin E can present suppression of the immune response against infectious diseases. Leucocytes of animals deficient in selenium have low glutathione peroxidase activity and have a diminished microbicidal activity (Arthur and Boyne 1985) . Therefore, suitable levels of selenium and vitamin E can be very important for the prevention of metritis.

The deficiency of vitamin E in male mice does not impact upon the feedback processes of LH and testosterone, or FSH and inhibin, but causes testicular degeneration (Cooper et al. 1987) Vitamin E can affect the cellular germination by other mechanisms excepting the cellular antioxidant action. In a study performed with bulls, the supplementation with vitamin E did not affect the semen or sperm features and the fertility (Salisbury 1984). However, high doses of vitamins A, D, E and C were capable of altering the semen and sperm features (Kozicki et al. 1981) . In ovines, frequent administrations of injectable vitamin E and selenium did not result in any effect on the fertility or proliferation, but significantly increased the survival of the lambs in the pre-weaning period (Kott et al . 1983) . The quality of the semen of reproducer pigs was improved with the supplementation of vitamin E and selenium (Marin-Guzman et al. 1989) .

Inconsistent responses to the supplementation of vitamin E and selenium are not surprising, because we must take into consideration the large number of factors and the complexity of the interactions involved in the reproductive processes .

Prevention of the retention of the placenta The supplementation with selenium or with selenium and vitamin E reduces the incidence of the retention of the placenta in animals wherein its prevalence is high, or when these nutrients are deficient (Julien et al. 1976a, Julien et al. 1976b, Trinder et al. 1973, Trinder et al. 1969) . This prophylactic effect of improving the reproductive efficiency produced proposals of supplementation protocol for the pre-delivery period.

In bovines, the injection of vitamin E and selenium, and the oral supplementation with vitamin E reduced the incidence of placenta retention, metritis and ovarian cysts (Harrison et al. 1984). The intramuscular administration of 3000 IU / head of d-alpha-tocopherol, up to 14 days prepartum in dairy cows, reduced the incidence of placenta retention and metritis (Erskine et al . 1997). Additional studies demonstrated that the supplementation with vitamin E and selenium reduce the incidence of placenta retention (Eger et al. 1985, (Harrison et al. 1984), Hernken et al. 1978, Segerson et al. 1981b), metritis, ovarian cysts (Harrison et al. 1984), and the time necessary for the uterine involution in cows with metritis (Harrison et al. 1986) . The incidence of the placenta retention in one study (Segerson et al. 1981b), was reduced only in cows with a marginal deficiency of selenium and not in cows with suitable amounts or extreme deficient in selenium.

Improvement of the immune response

The dietetic supplementation of vitamin E increases the secondary humoral immune response of lambs affected by the Parainfluenza 3 virus, which was evidenced by the increase in the seric response of IgM to the viral challenge (Reffet et al. 1988). In pigs, the injectable supplementation of vitamin E and selenium in the prepartum period, in addition to the nutritional requirements, resulted in larger concentrations of IgM and IgG in female pigs, and larger levels of colostral IgM, seric IgM, and increased cellular immune response to phytohemagglutinin and to concavalin A in suckling pigs (Hayek et al. 1989, Nemec et al. 1994). The dietetic supplementation of young sows with alpha-tocopherol resulted in the increase in the humoral response to immunization with ovalbumin in its suckling pigs (Babinsky et al. 1991). In young heifers, the oral supplementation with dl-alpha-tocopherol acetate increased the concentration of the seric IgM, the lymphocytic blastogenesis in vitro stimulated by the phytohemagglutinin, and the seric inhibition in vitro of the viral replication of the infection by bovine rinotraqueitis (Reddy et al. 1986) . The feed supplementation of vitamin E in cows in peripartum resulted in the quicker migration of neutrophils to the milk, the increment of neutrophil chemotactic properties, and the increase in the intracellular bacterial death after an intramammary bacterial challenge (Smith et al. 1997, Politis et al. 1996, Politis et al. 1995). When cows in peripartum were supplemented with vitamin E and vaccinated against Escherichia coli J5, associated with Freund' s adjuvant, there was a significant increase in the amount of immunoglobulins in the serum and in the milk (Hogan et al. 1993) . The supplementation of vitamin E given to old age humans for four months increased the indicators of the immune function, including humoral and cellular responses to challenged antigens (Meydani et al. 1997). Safety Compared with vitamin A or with vitamin D, the majority of studies have demonstrated that vitamin E is relatively non-toxic. Upper safety limits are approximately 100 times higher than the nutritional requirements for most of the species studied up to this moment (NRC 1987) . In any case, extremely high doses are not completely deprived of adverse effects. Excessive doses of vitamin E in mice, chickens, dogs and humans can induce failures in coagulation or exacerbate other functions associated with vitamin K. High dietetic levels of vitamin E reduce the growth of mice and chickens and exacerbate the bone calcification abnormalities related to the deficiency of calcium and vitamin D. High doses of vitamin E in anemic children were correlated with the absence of a hematological response to the supplementation of iron. In some studies, the combined supplementation of vitamin A and E was found to provide less protection against bacterial infections than the isolated supplementation, and it was noted that there were antagonic effects in determined dietetic levels (Tengerdy and Nockels 1975) . In any case, studies of hypervitaminosis in mice, hens and humans indicate maximum tolerance levels ranging from 1000 to 2000 IU/kg of the diet (NRC 1987). BETA-CAROTENE Properties and metabolism

Beta-carotene is one of more than 600 different carotenoids that are found in vegetables, microorganisms and animals, and which can also be chemically synthesized. They are colorful and belong to the largest pigment group found in nature. The colors vary from yellow and orange to red and, thus, they are very noticeable in their natural state. In its pure form, beta-carotene appears as a crystalline powder, which is red-brown to violet in color. It is sensitive to light, oxygen and acids. It is insoluble in water, very slightly soluble in oils and fat (0.05 - 0.08%), and lightly soluble in organic solvents.

The carotenoids are chemically related to a more general class of compounds - the terpenes and terpenoids - which are characterized by the repetition of the isoprenoid units, containing five atoms of carbon. This same molecular structure of five carbons is found in several substances, such as: steroids, biliary acids, sexual hormones, ubiquinones, and in the side chains of vitamins E and K. The ubiquitous compound acetylCoA is the basic source of carbon. These isoprenoid chains consist of 3 and 8 units, and additionally vary by the curvature of the terminal groups in rings and by various modifications of hydrogenation and oxidation.

Beta-carotene has the chemical formula C40H56 and consists of 8 isoprenoid units whose terminations produce rings with specular images.

It is found in most green plants, generally intimately associated with other pigments such as chlorophyll. The isomer naturally found in nature is the all-trans form, although the acids - particularly in the presence of light - can convert the all-trans isomer into a range of cis-trans isomers. From those, the most common is the 13-cis isomer, although hundreds of different combinations are theoretically possible due to the number of double bonds in the long chain. This 13-cis form has much less potency as a source of vitamin A. Less than 10% of carotenoids can be metabolized to vitamin A, beta-carotene being the largest source of vitamin A in the natural diet of animals. Like most of the members of this chemical grouping, beta-carotene is liposoluble and found in association with lipid fractions of vegetal tissues, as much in its simple form as a complex with proteins. This protein-carotene complex can be lysated by proteolitic enzymes.

When carotene-containing feed passes through the stomach and the small intestine, the action of several proteases, lipases, and esterases liberate the carotene, which is, then, solubilized by the emulsifying action of the biliary salts. After being emulsified, it penetrates the plasmatic membrane of the intestinal wall. In the interior of the mucosa cells, the carotenoid dioxygenases degrade the beta-carotene molecule by successive oxidative cleavages and generate vitamin A. Although the examination of the molecular structures of beta-carotene and vitamin A can suggest that the cleavage of the carotene molecule on average can result in two molecules of vitamin A, research has demonstrated that this conversion in the intestinal lumen consists of successive removals of carbon groups from one of the molecular edges. To prove this fact, experiments have demonstrated the presence of the cleavage products of the long chain in the interior of the mucosa cells.

The retinol formed by this conversion process is transferred to the liver for sterification and sequentially for circulation or storage. The efficiency of the conversion depends on series of factors. Theoretically, a mole of beta-carotene should produce a mole of vitamin A, but this efficiency level is never achieved. There are great differences between the species and several factors that influence the efficiency of the conversion, for example: the diet, the physiological state and the genetic differences of the intestinal enzymatic activity. Another important control factor is the body reserves of vitamin A and other carotenoids. The larger these reserves, the smaller the absorption and the conversion rate of other carotenoids.

Some animals are capable of absorbing beat- carotene directly into the blood circulation without the conversion to retinol. Bovines, for example, can absorb considerable quantities of beta-carotene whereas ovines absorb little or no beta-carotene into the blood. In any case, the absorption of carotenoid is typically, relatively low (less than 30% of the diet concentration) , and this percentage reduces significantly with the increase of ingestion (Olson 1994) . For ruminants, it was initially proposed that the beta-carotene would be degraded in varied levels (20 to 40 % of the initial level) by the ruminal microbiota (Westendorf et al. 1990). However, it was ascertained that there is no direct destruction or microorganism attack, but an adhesion to the cellular content of the ruminal fluid that interferes in its utilization by the animal (Mora et al. 1999). The absorption of beta-carotene can be improved with the increase in the ingestion of fats, up to a determined level, probably by the stimulation of the secretion of biliary salts, which improve the manner of the dispersion of this compound in the gastrointestinal content and reduce the size of the micellae (Olson 1994). On the contrary, the presence of parasites in the intestine, diseases such as malabsorption syndrome, and the increase of the gastric pH prejudice this process.

Cats, pigs and buffalos do not absorb beta- carotene, due to the total biotransformation of this compound or by the absence of the dioxygenases that convert the carotenoids into retinol.

When the beta-carotene and other carotenoids pass without alteration through the systematic circulation they are transported to several parts of the body where they are deposited. Being lipophilic, they tent to migrate to the adipose tissue. In conjunction with the liver, they compose the largest store of beta-carotene in the animal organism. Beta-carotene has also been found in the lungs, kidneys, cervix, prostrate, skin and many other tissues (Schmitz et al. 1991). High concentrations of beta-carotene are found in tissues that contain many receptors for LDL, such as the corpus luteum, adrenal tissue and testicles, probably resulting of a non-specific flow of lipoproteins. Preliminary studies with stable radioactive tags and using mathematical models of physiological behavior, determined the presence of beta-carotene for 51 days in humans. This was a larger period than forecast in previous studies performed with non-isotope tags (Novotny et al. 1995). In bovines, 49 days were necessary to eliminate the circulating beta-carotene, after the supplementation of 0 to 352 mg of beta-carotene/kg DM/day. In this same study, the estimated digestibility of beta-carotene was from 66.25 to 88.14%. Animals appear to be incapable of synthesizing any type of carotenoid, except for a few, for example, the prawn and the lobster, which consume beta-carotene in their diets, produce alterations in its chemical structure and produce another carotenoid, the astaxanthin, which is deposited in the skin, flesh and external skeleton. In plants, there is evidence of a direct association between photosynthesis and the production of beta-carotene.

The excretion metabolites of carotenoids have not been well identified yet. It is assumed that the degradation process of carotenoids and their metabolites is probably similar to the process degradation of vitamin A and retinoids. It has been demonstrated that hepatic cytochrome P450 participates in the metabolism of the retinol and the retinoic acid in polar metabolites. Due to the low absorption of these compounds, most of the carotenoids that are ingested are excreted in the feces (Rock 1997).

Beta-carotene does not have any defined relationship with other micro-nutrients. It has a high affinity with lipoproteins (75% in LDL and the remaining in VLDL and HDL) , with which a stable complex is formed and is transported by the organism.

Functions (biological features) Recent researches suggest that beta-carotene has a special function in the reproductive cycle of bovines and possibly in other animals, such as equines, ovines, goats, pigs and rabbits. The original references for this hypothesis were revised by Hemkel & Bremel (Hemkel and Bremel 1982). Many experiments studied the relationship between the supplementation of beta-carotene and the reproductive function. Some evidenced negative results (Akordor et al. 1986, Bindas et al. 1984a, Bindas et al. 1984b, Bremer et al. 1982, Ducker et al. 1984, Folman et al. 1979, Larson et al. 1983, Lee et al. 1985, Wang et al. 1988, Wang and Larson 1983) , others positive results (Ahlswede and Lotthammer 1978, Ascarelli et al. 1985, Jackson 1981, Lotthammer 1979, Lotthammer and Ahlswede 1977, Lotthammer et al. 1976, Meyer et al. 1975, Rakes et al. 1985, Shams et al. 1977, Snyder and Stuart 1981, Wang et al. 1985, Wang et al. 1982), and there were also reports of adverse effects (Folman et al. 1987). The diversity of experimental conditions render difficult to reach a definitive conclusion with respect to the role of beta-carotene in reproduction, independent of its function as a precursor of vitamin A. These experimental differences include age, stage of lactation at the start of the supplementation, duration of the supplementation period, and the forms of the vitamin A supplementation and beta-carotene concentrations in the diet (Hemken and Bremel 1982) .

The base for the investigation of the specific role of beta-carotene in reproduction arose from the fact that the concentrations of this compound are high in the bovine blood plasma, in the follicular fluid and in the corpus luteum, whereas the hepatic stocks of beta-carotene are relatively low. Beta-carotene is an integral part of the microsomal membrane of the bovine corpus luteum, acting in the maintenance of the integrity of this structure. The beta-carotene present in cellular cytosol can be associated with the lipoproteins derived from plasma.

Retinol was isolated from the bovine corpeus luteum and studies in-vitro demonstrated the synthesis of this compound from beta-carotene (Gawienski et al. 1974). However, this production varies in accordance with the stage of the animal's estral cycle (Sklan 1983). Chew et al. 1984, identified low concentrations of retinol in the bovine corpeus luteum. These observations suggest that the beta- carotene in the corpeus luteum can act as a local storage of retinol, when the transport of the retinol-protein complex carriers does not supply sufficient quantity of the substance for the normal function of the corpeus luteum. The supplementation of beta-carotene in cows that were deficient in the compound significantly increased its concentration in the corpeus luteum. However, it did not alter the content of the vitamin A, weight, structure or function of the corpeus luteum (Georgevskii 1981) . The high concentration of beta-carotene in the corpus luteum can affect the production of ovarian steroids

(Gawienowski et al. 1974). Ovaries of mice deficient in vitamin A presented a low production of progesterone (Juneja et al. 1966) . Cows supplemented with beta-carotene and non- supplemented cows (Bindas et al.) presented differences in relation to the response to the synthesis of progesterone after the injection of human chorionic gonadotropin. In- vitro studies of bovine luteal cells suggest that different ratios of retinol, retinoic acid and beta-carotene in a culture medium can exercise different effects in the secretion of progesterone (Talavera and Chew 1987a) and that the retinoic acid and the beta-carotene can stimulate the use of low density lipoproteins to synthesize progesterone in said cells (Talavera and Chew 1987b) .

The quantity of retinol, retinyl esters, and beta- carotene found in the follicular fluid seems to be proportional to the blood concentration (Chew et al. 1984). The function of beta-carotene as an antioxidant is also ascertained (Burton and Ingold 1984), being an efficient purifier of oxygen and free radicals under conditions of low oxygen pressure (Burton and Ingold 1984, Freeman and Crapo 1982, Stahl et al. 1997) . It is known that these free radicals cause lipid peroxidation and the breakdown of the lipid membranes. Furthermore, these effects can reduce the lytic activity cytochrome P450 and the side chain of cholesterol, which are related to the steroidogenesis in adrenal and ovarian tissues (Hornsby, 1980; Young et al., 1995). Another action of beta-carotene is its capability to stimulate the formation of gap-type junctions in culture cells. It stimulates the transcription of the gene to connexin 43, which codifies the formation of this gap-type junction to a membrane protein; consequently, there is an increase in the communication between the cells in the human dermal fibroblasts (Zhang et al., 1991). This formation of gap-type junctions can be important for the coordination of the function of luteal cells (Redmer et al., 1991; Khan-Dawood et al.,' 1996; Grazul-Bilska et al., 1996) and can be important for the mechanism by which the beta- carotene stimulates the steroidogenesis. However, there is a hypothesis that this mechanism, when in the presence of high levels of LH and beta-carotene, leads to apoptosis and, consequently, luteolysis (Khan-Dawood et al., 1996). Arikan and Rodway, 2000, suggest that the increase in progesterone synthesis results in a greater use of beta-carotene as a function of the larger requirement of the antioxidant capacity of the luteal cells. This was confirmed by the fact that the ascorbic acid, also an antioxidant, was depleted in the ovary and in the adrenal, when hormonally stimulated (Parlow, 1972; Sayers et al., 1948). The vascular granular follicular layer can be included as an example of tissue that is exposed to low partial oxygen pressures. On the other hand, it is known that Vitamin E is more effective in conditions of high partial oxygen pressures and can complement the action of beta-carotene in the total antioxidant capacity of tissues (Burton and Ingold, 1984; Freeman and Crapo 1982) .

There are reports that beta-carotene is important for the maintenance of normal ovary function (Inaba et al., 1986) . Cows maintained on a diet deficient in beta-carotene with low plasmatic levels of this substance, in comparison with normal cows, had a greater number of ovarian cysts. Another study reported that cows supplemented with beta- carotene had a lower incidence of ovarian cysts than non- supplemented cows (Lotthammer and Ahlswede, 1977).

There are evidences that beta-carotene has influence on the bovine follicular development. Animals supplemented with beta-carotene ovulated one day after the beginning of the estrous, whereas in deficient cows two days were necessary. These results were confirmed by Schams et al. (1977), which demonstrated a time interval of 49 hours between the occurrence of the LH peak and the ovulation for the beta-carotene supplemented group, and a time interval of 72.5 hours for the deficient group. Requirements

As a source of vitamin A, beta-carotene is intimately correlated with the status of this vitamin in the organism. Thus, the supplementation level is evaluated by the corporal pool of vitamin A. However, the beta-carotene supplementation profile can also be specifically evaluated, which varies with the seasonality of the diet, ranging from 0 to 20 mcg/mL of blood plasma. With respect to cows that receive diets deficient in beta-carotene, the plasmatic levels may be below 1 mcg/mL. It is suggested that reproductive problems occur when plasmatic levels are below 3 mcg/mL or 1 mcg/mL. It is known that the ingestion of beta-carotene is reflected in the plasmatic concentrations of this compound (Akordor et al. 1986, Ascarelli et al. 1985, Bindas et al . 1984a, Ducker et al. 1984, Folman et al. 1979, Lotthammer 1979, Wang et al. 1982), and that the amount of tocopherol (vitamin E) can also affect this critical level.

The requirements for this compound are not well defined. The following oral supplement is suggested (except for cats) :

Dairy cows at the peak of lactation require beta- carotene for reproduction and release via milk, as much as a source of vitamin A. Thus, the required amount can achieve 200-300 mg/day, depending on the daily milk production level .

Sources

The content of beta-carotene in feed is very variable. Ripe forage is a good source of beta-carotene and presents concentrations higher than 200 mg/kg of DM (dry matter) . On the other hand, sensitive decreases in the beta- carotene were observed in mature forage (overripe) , brown forage (concentration of less than 20 mg/kg of DM) and in conserved feed after the processes of silage or haymaking. Signs of deficiency In case the diet is not suitably supplemented with vitamin A, the lack of beta-carotene can induce the deficiency of this vitamin. Chronologically, the observed symptoms are: less resistance to disease, growth alteration of the feathers, hair or integrity of the skin, reduction in the reproductive activity, and finally, reduction in visual capability.

Cows that are suitably supplemented with vitamin A can present reproductive problems if the supply of beta- carotene is unsuitable. These problems include: the manifestation of silent estrous, delayed ovulation, follicular cysts, reduced corpus luteum, as well as reduction in the levels of progesterone in the sorus. The general observed effect is non-conception or delayed conception (Kuhlman and Gallop 1942, Lotthammer et al. 1976) . When the beta-carotene deficiency is prolonged, there are harmful consequences for the hypophyseal, testicular and ovarian functions (Byers et al. 1956). Similar conditions were demonstrated in mares, pigs and rabbits. There are also risks of late abortion or the birth of a very weak animal.

Considerations for supplementation: uses and indications

The use of beta-carotene supplements as a source of vitamin A is generally not economically viable. However, for the reproduction animals of certain species, during determined critical phases, its use can be beneficial. The following oral levels were stated as supplementation proposals :

The expected effects of the beta-carotene supplementation on the reproductive functions are: the increase in the intensity of the estrous, the increase in the conception rate and the reduction in the number of services for conception, less open days and the reduction in the incidence of ovarian cysts (Ascarelli et al. 1985, Lotthammer 1979, Snyder and Stuart 1981) . After conception, the pregnancy is more efficiently maintained in groups of animals that received supplements with beta-carotene, which can be evidenced by the reduction in premature embryonic mortality and the decrease of premature abortion rates (Lotthammer 1979) . Safety The homeostatic mechanisms that control the absorption, circulation and storage of carotenoids normally prevent the absorption of excessive amounts of beta-carotene in the feed that pass through the gastrointestinal tract. Large amounts of beta-carotene can be absorbed if the diet is excessively supplemented for a long period of time. Beta- carotene deposits install themselves in the adipose tissues and have a yellowish color and the normal function of the liver can be reduced. However, the necessary amounts to generate this condition exceed the amounts of 10 g for each 100 kg of body weight for a period of 6 months or longer, which is impractical and economically not viable.

Examples : Example 1 - The effect of beta-carotene and FSH-P on the follicular dynamic and embryonic quality in Mangalarga Marchador mares. The objective was to evaluate the effect of beta-carotene and vitamin E supplementation on the production and embryonic quality of mares treated or non-treated with FSH-P, as well as evaluating the efficiency of the use of FSH-P (Folltropin®) to induce superovulation in mares.

Sixteen donor mares of the Mangalarga Marchador breed, between 4 to 16 years old and apt to reproduce, were used. Two doses of injectable beta-carotene (800 and 1200 mg) and vitamin E (500 and 750 IU) were intramuscularly used. They were administered on the 15^th day after ovulation to mares treated and non-treated with Folltropin-V®

(Vetrepham - Belleville, Canada) , with two daily intramuscular injections of 10 mg, administered at 8 am and 6 pm of the 15^th and 20^th day after ovulation or until the detection of one or more follicles greater or equal to 3.5 cm. The animals were randomly distributed into 6 treatments: treatment 1 (n = 8) - did not receive any medication; treatment 2 (n = 8) - FSH-P administration; treatment 3 (n = 4) - 800 mg of beta-carotene and 500 IU of vitamin E; treatment 4 (n = 4) - 1200 mg of beta-carotene and 750 IU of vitamin E; treatment 5 (n = 4) - FSH-P administration + 800 mg of beta-carotene and 500 IU of vitamin E; treatment 6 (n = 4) - FSH-P administration + 1200 mg of beta-carotene and 750 IU of vitamin E. The collection of the embryos was performed on the 6^th day after ovulation. Due to the fact that there was no difference between the treatments and the interactions, the principal effects of the treatment with FSH-P (n=16) versus non- treatment (n=16) and the supplementation with beta-carotene/ vitamin E (n = 16) versus non-supplementation (n = 16) were tested.

The embryonic quality was superior (p < 0.041) in the animals supplemented with beta-carotene/vitamin E (1.22 ± 0.22), versus non-supplemented (2.62 ± 0.21), but it was similar (p>0.20) between animals treated with FSH-P (1.47 ± 0.21), in comparison with non-treated animals (2.37 ± 0.22).

The average number of follicles increased (p <

0.0175) in animals treated with FSH-P (3.19 ± 0.34) versus non-treated (1.87 ± 0.34) and there was no effect (p>0.50) of supplementation (2.69±0.34) versus non-supplementation (2.37 ± 0.34) .

The number of ovulations did not vary (1.00 ± 0.0) between the principal effects.

There was no effect of the treatment with FSH-P (p > 0.80) or the supplementation (p > 0.90) on the duration of the estrous (8.00 ± 0.46 versus 8.23 ± 0.46 and 8.06 ± 0.46 versus 8.18 ± 0.46 days, for mares treated or non-treated with FSH-P and supplemented or non-supplemented with beta- carotene / vitamin E, respectively) and on the diameter of the ovulatory follicle (4.56 ± 0.18 versus 4.56 ± 0.18 and 4.50 ± 0.18 versus 4.62 ± 0.18 cm, for mares treated or non- treated with FSH-P (p > 0.99) and supplemented or non- supplemented with beta-carotene/vitamin E (p > 0.87), respectively) .

The follicular increase was not different to the main tested results (p > 0.05) being linear and crescent in relation to time (R2 = 0.95 and 0.92, treatment with FSH-P versus non-treatment; R2 = 0.94 and 0.95, supplementation with beta-carotene/vitamin E versus non-supplementation, respectively) .

The supplementation of beta-carotene/vitamin E improved the quality of the embryo. The treatment with FSH-P was associated with an increase in the number of palpable follicles; however, there was no change in the average number of ovulations.

Example 2 - The effect of beta-carotene and vitamin E on the quality of embryos collected from Dutch breed donors The objective of this research was to evaluate the effect of the (intramuscular) injection of beta-carotene and vitamin E on the quality of the embryos collected from Dutch breed donors.

Dutch breed cows (n=22) and heifers (n=50) were synchronized using Crestar® (Intervet International B. V. Boxmeer, Holland) which consists of the administration of a subcutaneous auricular implant of 3 mg of Norgetomet, and the intramuscular administration of 6 mg of Norgetomet and 10 mg of estradiol valerate (Dl). The superovulatory protocol began on the sixth day with 8 decreasing administrations of FSH/LHp (Pluset®, Calier S.A., Barcelona, Spain) in twelve-hour intervals. On the eighth day, an intramuscular injection of 0.5 mg of sodium cloprostenol (Sincrosin®, Vallee S.A., Montes Claros, Brazil) was administered and the removal of the implant was carried out on the ninth day. The animals were randomly allocated to one of three treatments. Treatment 1 consisted only of a carrier (n = 24), treatment 2 consisted of 800 mg of beta-carotene and 500 mg vitamin E (n = 20) , and treatment 3 consisted of 1200 mg of beta-carotene and 750 mg of vitamin E (n = 28) . The treatments were applied on the day of the implant (Dl) and at the beginning of the superovulatory protocol (D6) . The animals were inseminated 12 and 24 hours after the observation of the estrous and the collection was performed on the seventh day after the first insemination. After the collection, the embryos were evaluated according to the standard adopted by the IETS. An index of embryonic quality (IEQ) was proposed based on this classification (IEQ = (Excellent*l + Good*2 + Regular*3 + Poor*4 + Degenerate*5) / total of the collected embryos) . Absolute values closer to 1.00 indicate the better quality of the embryo. The IEQ was analyzed by PROCGLM and the total of the viable structures by PROCGENMOD of the Statistical Analysis System (SAS) .

With respect to the IEQ, there was an effect (P > 0.0058) of the interaction type of animal versus treatment, where the embryonic quality was improved in cows but not in heifers.

The IEQs for cows and heifers in treatments Tl, T2 and T3 were 3.13 ± 0.48, 2.32 ± 0.26; 2.57 ± 0.51 and 2.23 ± 0.27; 1.29 ± 0.51 and 2.50 ± 0.25, respectively.

There was no difference between the treatments in the total of collected structures and the viable embryos.

It was concluded that the intramuscular application of beta-carotene and tocopherol improved the quality of collected embryos of the Dutch breed cows and had no effect on the embryos of the heifers.

The number of viable embryos and the total of collected structures did not differ between the treatments.

Under the present experiment conditions, it was demonstrated that the application of antioxidants was an alternative to improve the quality of the embryos of superovulated cows.

It will be evident to a person skilled in the art that the present invention is not limited to preceding illustrative examples and it can be used in other embodiments without losing any of its essential features.

Therefore, it is desirable to consider the examples in all aspects, as illustrative and non-restrictive, with reference to the annexed claims and all modifications that can be found within the meaning and equivalent range of the claims and, therefore, intended to be included herein.

Claims

1. Parenteral formulation, CHARACTERIZED in that it comprises 0.01 to 30% w/v of beta-carotene, 0.01 to 15% w/v of vitamin E acetate 1000 IU/g and 1 to 99% v/v of an oily substance.

2. Formulation according to claim 1, CHARACTERIZED in that the beta-carotene is selected from a group consisting of a synthetic, semi-synthetic, or naturally originated beta-carotene.

3. Formulation according to claim 1, CHARACTERIZED in that the vitamin E acetate is selected from a group consisting of one of its 8 isomers.

4. Formulation according to claim 1, CHARACTERIZED in that the oily substance is selected from a group consisting of triglyceride of capric acid, caprylic acid, any vegetable oil, or a mixture thereof.

5. Formulation according to claim 4, CHARACTERIZED in that the oily substance is a triglyceride of capric or caprylic acid.

6. Formulation according to claim 5, CHARACTERIZED in that the triglyceride of capric or caprylic acid is selected from a group consisting of medium chain triglyceride, Captex, Estasan and Miglyol.

7. Formulation according to claim 1, CHARACTERIZED in that the formulation may further comprise other liposoluble vitamins, such as vitamin D (D2 ergocalciferol; D3 - calciferol, calcitriol or colecalciferol) and vitamin A (such as retinyl palmitate, retinyl acetate or retinyl propionate) .

8. Formulation according to claim 7, CHARACTERIZED in that the liposoluble vitamin (s) may be present in a range from 0.01% to 15% w/v.

9. Formulation according to claim 1, CHARACTERIZED in that the formulation may further comprise hydrosoluble vitamins, such as vitamin C, and B-complex vitamins (for example, folic acid, thiamine - vitamin Bl, riboflavin - vitamin B2, pyridoxine - vitamin B6, cyanocobalamin vitamin B12, biotin, pantothenic acid, etc) ; choline; minerals (such as selenium, zinc, copper, molibdenium, iodine, manganese, calcium and phosphorous) and essential fatty acids (such as linoleic acid and linolenic acid) which can be present in a range from 0.01% to 15% w/v.

10. Formulation according to claim 1, CHARACTERIZED in that the oily substance is selected from a group consisting of sesame, sunflower, rice, soy and peanut.

11. Formulation according to claim 1, CHARACTERIZED in that the formulation may further comprise antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) in a range from 0.005% to 0.5% w/v.

12. Use of the formulation as defined in claim 1, CHARACTERIZED in that it is for the reproduction of production animals.