DESCRIPTION

Field of application

The present patent relates to a feed composition or a food supplement for improving female fertility for use in both breeding animal species such as rabbit, pigs, etc., and in pet animals, such as dogs and cats, etc. and in the human species.

In mammals, the normal reproductive function of the female is dependent on a complex hormonal interaction between the endocrine system and target organs. The hypothalamus produces the gonadotropin releasing hormone (GnRH) that promotes the luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion from adenohypophysis. LH and FSH promote the maturation of the egg cell and the secretion of estrogen and progesterone by ovaries.

Estrogens and progesterone in turn stimulate target organs of the reproductive system (ie, mammary glands, uterus and vagina) and exert positive and negative feedback actions on the ovarian axis by inhibiting or stimulating the secretion of the gonadotropins.

The menstrual cycle can be divided into three phases: follicular, ovarian and luteal. In the follicular phase (preovulatory), FSH secretion stimulates the growth of a group of 3 -30 follicles up to the selection of a dominant follicle that will grow to maturity, while the others degenerates trough follicular atresia. When the circulating LH levels increase compared to FSH, it begins to increase the production of estradiol by the selected follicle, which establishes positive feedback with the hypothalamus-pituitary axis leading LH to reach the peak.

Once LH reaches its peak, the ovulatory phase begins, and usually lasts 36-48 hours.

This phase is characterised by multiple releases of large amount of hormone. The peak of LH which determines the full maturation of the follicle, is necessary for the ovulation and for the release of the egg cell by the mature Graaf follicle This typically occurs from 16 to 32 hours after the beginning of the peak of LH.

The oocyte remains in the meiotic prophase up to the peak of LH. Within 36 hours of the LH peak, the oocyte completes the first meiotic division, in which each cell receives half of the original chromosomes number and the first polar body is eliminated. The second meiotic division, in which each chromosome divides longitudinally into two identical copies, is not completed and the second polar body is not eliminated until the spermatozoa penetrates the egg cell.

In the luteal phase (postovulatory), granulosa and teca cells form the corpus luteum. The length of this phase is the most constant phases, with an average period of 14 days in non- pregnant women and ending on the first day of the subsequent menstruation. The corpus luteum supports the fertilized egg nidation by secreting increasing amounts of progesterone up to a maximum of about 25 mg / day at 6-8 days after the LH peak.

Although the mechanisms of ovulation are quite conserved in mammals, there are some differences among various species. For example, in the rabbit, the induction of the ovulation does not occur spontaneously as in humans, but after external stimulation, mainly by coitus, sexual behavior among females in the follicular phase, electrical stimulus, vulvar mechanical stimulus, abrupt environmental variations or hormonal treatments with LH, LH-RH or gonadotropins. These external or pharmacological stimulus support in a more or less direct way the nervous reflex that comes to the hypothalamus, and triggers the ovulation. Ovulation occurs 10-14h after stimulation.

Various plant species proved to have an effect on fertility. For example, Suzuki and

Ukyo (1968) showed that from 41 different plants, the fruit and the lemon peel, the horseradish root, the stalks of Aralia cordata, the hops leaves, the young rice plants and Lycium chinense Miller induce ovulation in female rabbits. Lycium chinense Miller was subjected to further experimentation in order to identify the substances that induce ovulation (Suzuki et al., 1972).

Suzuki M, Ukyo T. Induction of ovulation by plant extracts in the rabbit. Acta Med

Biol (Niigata). 1968 Jan; 15(3): 169-72.

Suzuki M, Osawa S, Hirano M. A Lycium chinense Miller component inducing ovulation in adult female rabbits. Tohoku J Exp Med. 1972 Mar; 106(3):219-31.

Lycium barbarum is a distinct species of the Lycium chinense Miller. Its berry (commonly called Goji berry) is used in the traditional Chinese medicine and is recognized to possess antioxidant properties. It has been reported that the administration of Goji berry in animal models reduces glucose and lipid levels in serum and has antioxidant and immunomodulating properties, useful in age-related diseases, including atherosclerosis, neurodegeneration and diabetes.

Chang R C C, So K F. Use of anti-aging herbal medicine, Lycium barbarum, against aging-associated diseases. What do we know so far?. Cell Mol Neurobiol. 2008; 28 643-652

Potterat O. Goji {Lycium barbarum and L. chinense): Phytochemistry, pharmacology and safety in the perspective of traditional uses and recent popularity. Planta Med. 2010 Jan;76(l):7- 19. doi: 10.1055/S-0029-1 186218.

The antioxidant activity of Lycium barbarum was mainly associated with the presence of polysaccharides (LBPs) and flavonoids.

Li X M, Li X L, Zhou A G. Evaluation of antioxidant activity of the polysaccharides extracted from Lycium barbarum fruits in vitro. Eur Polymer J. 2007; 43 488-497

Li X L, Zhou A G. Evaluation of the antioxidant effects of polysaccharides extracted from Lycium barbarum. Med Chem Res. 2007; 15 471 -482

Lycium barbarum polysaccharides have been proved to exhibit beneficial effects on male reproductive systems in many aspects: 1) the increase of sexual performance in rats (Luo et al., 2006)

Luo Q, Li Z, Huang X, Yan J, Zhang S, Cai YZ. Lycium barbarum polysaccharides: Protective effects against heat-induced damage of rat testes and H202-induced DNA damage in mouse testicular cells and beneficial effect on sexual behavior and reproductive function of hemicastrated rats. Life Sci. 2006 Jul 10;79(7):613-21.

2) the enhance of the quality, quantity and motility of the sperm (Luo et al., 201 1);

Luo Q, Cui X, Yan J, Yang M, Liu J, Jiang Y, Li J, Zhou Y. Antagonistic effects of Lycium barbarum polysaccharides on the impaired reproductive system of male rats induced by local subchronic exposure to 6OC0-Y irradiation. Phytother Res. 201 1 May;25(5):694-701. doi: 10.1002/ptr.3314. 3) the prevention of the testicular and seminiferous tubules damage induced by hyperthermia, hydrogen peroxide and radiation (Luo et al., 2006 and 201 1) and 4) the counteract the reduction of the testosterone levels in hemicastrated rats(Luo et al., 2006 and 201 1).

There are several patents that describe methods for the purification of lipopolysaccharides from Lycium barbarum berry, for example: Method for preparing lycium barbarum polysaccharides CN1743341A, Method for producing Matrimony wine polysaccharide CN101029088A.

In the US Patent No. 2008/0075799 Al Use of Medicinal Plants And Extras Thereof With Growth Promoting Activity In Veterinary Medicine And Zootechnics is claimed the use of Goji berries in livestock animals and its aqueous extracts in order to improve animal welfare. This patent claims the administration of a quantity of fruit / extract berries in the range of 0.1 to 10 grams/day for animal. Moreover, it claims the use of Goji berry to increase the body weight by avoiding the use of antibiotics.

The practice of artificial insemination has been a turning point in the organization of livestock breeding. For example, in intensive rabbit breeding, artificial insemination allows to obtain a higher production standard and a greater health surveillance on female rabbits.

In order to obtain a bigger probability of succes using the artificial insemination it should be taken into account several factors related to the reproductive performance, such as: receptivity, fertility, prolificity and the hormonal antagonism that is created between reproduction and lactation, to which is added the negative energy deficit found in young female rabbits which have not reached the adult body weight at the time of the first pregnancy worsened by a lower capacity of ingestion due to the growth of the fetuses in the uterus. Prolactin, hormone necessary for milk production and progesterone, necessary for the maintenance of pregnancy, have an inhibitory crosstalk effect.

Sexual receptivity is another factor that it should not be underestimated. A receptive animal has an increased frequency of ovulation, fertility and prolificity compared to a non-receptive animal. Such receptivity is greater in the immediate post-partum, then it decreases for a few days (less than 1 week) and finally increases again to acceptable values as a consequence of the maturation of the follicles and then by the production of estrogens.

Surprisingly, the present patent application found that Goji berry supplements in a precise quantitative range does not improve body weight as reported in the US patent application 2008/0075799, but increases the fertility and the milk production when administered in food.

SHORT DESCRIPTION OF FIGURES

Figure 1. The effect of the supplementation with two concentrations of Goji berries in rabbit does feed intake at different physiological phases (A), on the body weight of the does at AI (B), on the lactation curves (C), on the mean weight of the litter at the moment of birth and at 20 days, in all three groups (D) and on the productive performance of the rabbits at 60 and 72 days of age (E). The bars that do not share the same letter have significant differences with P <0.05.

Table 1. Cross-tabulation of the results regarding fertility.

Table 2. Mortality and productive pre -weaning performance.

Table 3. Hormone profile of the rabbit does during pregnancy and lactation.

Table 4. Hormonal profile of the rabbit does during pregnancy and lactation.

Summary of the Patent

The Applicant has now found that a composition or formulation comprising Goji berries of the species Lycium barbarum is able to satisfy the requirements in:

a) improving female fertility;

b) enhancement of lactation;

c) improvement of litter welfare.

Indeed, as demonstrated in the experimental part reported below, the Applicant has found, for example, that surprisingly the administration of Goji berry in the diet of rabbit does results in an improvement of the fertility and improvement of litter walfare as a consequence of the enhancement of the milk production.

According to the invention, the feed composition, known as "feed formulation " is based on a mixture comprising: Raw protein, Raw oils and fats, Crude cellulose, Crude ash, Calcium, Phosphorus, Sodium, Lysine and Additives such as Vitamins, Pro -Vitamins and Substances with similar effect and other Oligo -Elements.

Among vitamins, provitamins, antioxidants and substances with similar effect, there are: Vitamin A (3a672a-retinal acetate), Vitamin E (3a700), Vitamin B l (3a821), Vitamin B2 (roboflavin), Vitamin B6 (3a831 pyridoxine HCL), Vitamin B12 (cyanocobalamin), Vitamin H, Niacin (3a314), Calcium D-Pantothenate (3a841), Choline Chloride (3a890), Vitamin K, Folic Acid (3a316) and hydroxytyrosol.

Among oligo-elements are enumerated, but not exhaustive: Anhydrous calcium iodate E2, Zinc oxide E6, manganese oxide E15, sodium selenite S8, Ferrous sulphate monohydrate, Copper sulphate pentahydrate.

Preferably, according to the invention, the percentage of raw protein included in the feed formulation ranges between 0 and 30.

Preferably, according to the invention, the percentage of Oils and Raw Fats included in the feed formulation ranges between 0 and 10.

Preferably, according to the invention, the percentage of crude cellulose included in the feed formulation ranges between 10 and 20.

Preferably, according to the invention, the percentage of crude ash included in the feed formulation ranges between 0 and 20.

Preferably, according to the invention, the percentage of calcium included in the feed formulation ranges between 0 and 5.

Preferably, according to the invention, the percentage of phosphorus included in the feed formulation ranges between 0 and 3.

Preferably, according to the invention, the percentage of sodium included in the feed formulation ranges between 0 and 3.

Preferably, according to the invention, the percentage of lysine included in the feed formulation ranges between 0 and 3. The animal feed formulation made of Raw Protein, Raw Oils and Fats, Raw Cellulose, Raw Ash, Calcium, Phosphorus, Sodium, Lysine can result from the mixing of raw materials of mineral, vegetable and animal origin such as goji berries (Lycium barbarum ), corn wheat, chicken meat wheat, meat greaves, chicken fat, beef fat, lamb meat wheat, fish wheat, rice wheat, hydrolyzed liver, whole powder eggs, beetroot molasses, apple pomaces, sodium chloride, yeast (dried), potassium chloride, barley (fermented), seaweed (dried), flax seeds, yeast (extract), artichoke, dandelion, ginger, birch leaves, nettle, sage, coriander, rosemary, thyme, licorice root, dried shellfish pulp, chamomile, olivaria spirea, garlic, alfalfa wheat, Wheat bran, sunflower seed wheat, Barley wheat, soy wheat (seeds), rice bran, cane molasses (sugar), vegetable soybean fat, calcium carbonate (limestone).

Preferably, in the composition according to the patent, the content of Goji Berries (Lycium barbarum) is within the percentage range of not more than 2%, preferably higher than 0 and less than 1 % for fertility-based nutrition formulations or with a percentage superior or equal to 3% in feed formulation.

Preferably, in the composition according to the patent, the content of Lycium barbarum polysaccharides is within the range of not more than 0.2% preferably higher 0 and not greater than 0.1 % for the feed formulation useful for fertility or with a percentage equal to or greater than 0.3%> in the feed formulation.

Preferably, in the composition according to the patent, the content of Vitamin A (3a672a-retinal acetate) is within the range of 0 to 20000 U.I. / Kg of feed formulation.

Preferably, in the composition according to the patent, the content of Vitamin E (3a700) is within the range of 0 to 100 mg / Kg of feed formulation.

Preferably, in the composition according to the patent, the content of Vitamin Bl (3a821) is within the range of 0 to 10 mg / Kg of feed formulation.

Preferably, in the composition according to the patent, the content of Vitamin B2

(roboflavin) is within the range of 0 to 10 mg / Kg of feed formulation. Preferably, in the composition according to the patent, the content of Vitamin B6 (3a831 -pyridoxine HCL) is within the range of 0 to 10 mg / Kg of feed formulation.

Preferably, in the composition according to the patent, the content of Vitamin B12 (cyanocobalamin) is within the range of 0 to 1 mg / Kg of feed formulation.

Preferably, in the composition according to the patent, the vitamin H content is within the range of 0 to 1 mg 7 Kg of feed formulation.

Preferably, in the composition according to the patent, the content of Niacin (3a314) is within the range of 0 to 100 mg / Kg of feed formulation.

Preferably, in the composition according to the patent, the calcium content of D- Pantothenate (3a841) is within the range of 0 to 100 mg / Kg of feed formulation.

Preferably, in the composition according to the patent, the content of choline chloride (3a890) is within the range of 0 to 800 mg / Kg of feed formulation.

Preferably, in the composition according to the patent, the Vitamin K content is within the range of 0 to 10 mg / Kg of feed formulation.

Preferably, in the composition according to the patent, the folic acid content (3a316) is within the range of 0 to 10 mg / Kg of feed formulation.

Preferably, in the composition according to the patent, the content of anhydrous calcium iodate E2 is within the range of 0 to 10 mg / Kg of feed formulation.

Preferably, in the composition according to the patent, the content of zinc oxide E6 is within the range of 0 to 100 mg / Kg of feed formulation.

Preferably, in the composition according to the patent, the manganese oxide content El 5 is within the range of 0 to 100 mg / Kg of feed formulation.

Preferably, in the composition according to the patent, the selenium content of sodium E8 is within the range of 0 to 10 mg / Kg of feed formulation.

Preferably, in the composition according to the patent, the ferrous monohydrate sulphate content is in the range of 0 to 800 mg / Kg of feed formulation. Preferably, in the composition according to the patent, the content of copper pentaidrate sulphate is within the range of 0 to 100 mg / Kg of feed formulation.

Further features and advantages of the present patent will become apparent from the following description of some preferred embodiment examples of data given by way of non-limiting example.

Preferably, in the composition according to the patent, the hydroxytyrosol content is comprised within the range of 0 to 100 mg / Kg of feed formulation.

Example 1

Examples of feed formulation according to the invention are described below

Example 2

Effects on in vivo fertility in rabbit does

Thirthy three New Zealand White rabbits does were randomly assigned to 3 groups (n = 1 1) according to the feed diet: control group (C) fed with feed formulation(I), Goji Low group (LG) fed with feed formulation(II) and Goji High group (HG) fed with feed formulation (III). After a 30-day food adaptation period, the rabbits does were sumitted to artificial insemination (AI) and subsequently pregnancy was diagnosed by abdominal palpitation. After parturition, rabbit does were monitored daily during lactation for a period of 20 days ("controlled lactation") in order to evaluate the amount of milk produced. The experimental trial in the rabbit does finished 35 days after parturition.

Feed intake was higher in the control group than in the groups fed with feed supplemented of Goji berry (P <0.01, Figure 1A). The body weight and the BCS of the animals were higher in the HG group at the time of the AI compared to the control group (P <0.01, Figure IB). Similarly, during pregnancy the highest body weight was found in the HG group (P <0.05). Fertility rate was higher in the LG group than in the control group (P <0.05), while the HG group showed intermediate values (Table 1).

LG group had a higher milk production than the control and HG groups(P <0.001, Figure 1 C). This result, as shown in the following paragraphs, is further confirmed by the increased weight of litters at 20-days (Figure ID) and by the reduction of pre -weaning mortality recorded in the LG group.

The LG group of rabbit does showed a greater number of young rabbits at 20 (P <0.05) and 35 days (P <0.001). In addition, the little rabbits of this group showed a lower pre -weaning mortality than the control and HG groups (P <0.01). At weaning, the young rabbits of the group HG reached the highest body weight (P <0.001) and this trend was maintained until the end of the experiment (P <0.05). The Data is summarized in the Table 2 and Figure IE.

Regarding plasma concentrations of T3, T4, insulin and NEFA, no differences were found between the groups. The LG rabbits does had lower Cortisol levels than control and HG groups (P <0.01). In LG rabbit does, glucose blood levels decreased, despite the fact that glycaemia is a highly controlled homeostatic parameter, and it might be due to the increased milk production of these animals or to a hypoglycemic effect of Goji at this dosage. Leptin is higher in the HG group, especially at the time of the AI, compared to the other groups (P <0.01). This result is in agreement with a higher body weight and value of BCS of these animals. These evidence could explain the impaired rate of fertility shown by these animals. Data is summarized in Tables 3 and 4.

Table 1. Crossed-tabulation of the results regarding fertility obtained with the two consecutive inseminations (significant results are in red).

Table 3. Hormonal profile of the rabbit does during pregnancy and lactation (significant results are in red non significant results not showed).