Phytogenic additive and application thereof in promoting egg production and egg quality

Abstract

The present disclosure relates to Crassocephalum rabens (CR), an extract thereof or an active ingredient(s) contained therein and / or T. pratense (TP) and an extract thereof or an active ingredient(s) contained therein, and their applications in promoting egg production and quality.

Description

PATENTAttorney Docket No. G4590-20500PCTPHYTOGENIC ADDITIVE AND APPLICATION THEREOF IN PROMOTING EGG PRODUCTION AND EGG QUALITYCROSS REFERENCE TO RELATED APPLICATIONSThis application claims the priority benefit of U.S. Provisional Application No. 63 / 708,434, filed October 17, 2024, which is hereby incorporated by reference in its entiretyFIELD OF THE PRESENT DISCLOSURE

[0001] The present disclosure relates generally to a phytogenic field. Particularly, the present disclosure relates to Crassocephalum rabens (CR), an extract thereof or an active ingredient(s) contained therein and / or T. pratense (TP) and an extract thereof or an active ingredient(s) contained therein promoting egg production and quality.BACKGROUND OF THE INVENTION

[0002] Poultry farming plays a pivotal role in ensuring food security for the growing human population across the globe. Poultry meats and eggs are good nutrition and protein sources, and due to their bioavailability and affordability, eggs are important vehicles for ensuring all people have access to healthy food. It is estimated that egg production will rise by 50% by 2030 to keep up with demand. However, a wide range of environmental (e.g., heat stress, cold stress, stocking density, etc.) and biotic (e.g., virus, pathogen infection) stresses can adversely affect chicken growth and health, decreasing egg production and quality.

[0003] Egg laying in hens usually begins around 18-20 weeks of age. In commercial strains of layers, the laying performance peaks at around 27-28 weeks of age with hen-day egg production over 90%. This level of production is then maintained to 45 weeks of age. The egg production rate declines gradually to below 75% by 75 weeks of age. In some countries, the aged flocks are considered economically unviable and are slaughtered after 12 months of egg production. However, in some countries, the hens are force-molted to rejuvenate the second laying cycle.

[0004] All birds molt at least once during the year and during molting, feathers are shed and renewed in a sequence from the neck, chest, back, wing, and finally, tail. In domestic fowls under natural conditions, it usually takes 3-6 months to fully recover plumage. In the egg industry, layer chickens are forced to molt through food withdrawal for 7-14 days and water deprivation in the first 2 or 3 days. Forced molting can accelerate the natural process within 10 weeks, allowing chickens to grow a new set of feathers mainly in the autumn. This processPATENTAttorney Docket No. G4590-20500PCT occurs in tandem with decreasing day length and is generally accompanied by a sharp reduction or cessation of egg production. In the modern controlled-environment confinement housing system, hens are maintained at optimal temperatures with a photo schedule of day length of more than 14 h. After laying almost daily for nearly a year, hens develop photorefractoriness, and their egg production declines, as does the quality of the eggshell and the egg contents. In addition, the aged hens are overweight with some obesity-related morbidities, which exerts adverse effects on ovarian functions such as follicle maturation, atresia and ovulation (Liu Z.-C, Y.-L. Xie, C.-J. Chang, C.-M. Su, Y.-H. Chen, S.-Y. Huang, Walzem R. L„ and S.-E. Chen. 2014. Feed intake alters immune cell functions and ovarian infiltration in broiler hens -implications for reproductive performance. Biol. Reprod. 90:134, 1-8; Walzem, R.L. S.E. Chen. 2014. Obesity induced dysfunctions in female reproduction: lessons from birds and mammals. Adv. Nutr. 5:199-206). Feed withdrawal or nutritionally deficient diets accompanied by reduced photoperiods can decrease body weight and adiposity leading to ovarian regression and subsequent gonadal recrudescence after resumption of diet supply and photostimulation. However, the forced molting practice is not permitted in the European Union due to concern for animal welfare.SUMMARY OF THE INVENTION

[0005] In the present disclosure, it is surprisingly found that a phytogenic additive promoting egg yield, egg weight, egg quality, and / or lower yolk cholesterol contents. For example, Crassocephalum. rabens (CR), an extract thereof (CRE) or an active ingredient(s) contained therein (CR_API) and / or T. pratense (TP) and an extract thereof (TPE) or an active ingredient(s) contained therein (TP_API) can be used as a feed or feed additive or supplement in poultry to enhance egg production and quality.

[0006] In one aspect, the present disclosure provides a method of promoting egg yield, egg weight, egg quality, and / or lower yolk cholesterol contents and / or sustain ovarian function in a subject, comprising administering to the subject an effective amount of a composition comprising a CR or a CRE or a CR_API, or a combination of a CR and a CRE, or a CR and a CR_API, or a CRE and a CR_API.

[0007] In another aspect, the present disclosure provides a method of promoting egg yield, egg weight, egg quality, and / or lower yolk cholesterol contents and / or sustain ovarian function in a subject, comprising administering to the subject an effective amount of a composition comprising a TP or a TPE or a TP_API, or a combination of a TP and a TPE or a TP and a TP API or a TPE and a TP API.PATENTAttorney Docket No. G4590-20500PCT

[0008] In another aspect, the present disclosure provides a method of promoting egg yield, egg weight, egg quality, and / or lower yolk cholesterol contents and / or sustain ovarian function in a subject, comprising administering to the subject an effective amount of a preparation of a first composition comprising one or more component selected from a CR, a CRE and a CR_API and a second composition comprising one or more component selected from a TP, a TPE and a TP_API. In some embodiments, the preparation comprises a CR and a TP, a CRE and a TPE, or a CR_API and a TP_API.

[0009] In some embodiments, the components including the active ingredient(s) in CRE described herein comprises one, two, three, four, five or more of the following components: a- linolenic acid, mannitol, chlorogenic acid, glutamic acid, palmitic acid, and citric acid.

[0010] In some embodiments, the components including the active ingredient(s) in TPE described herein comprises one, two, three, four, five, six or more of the following components: formononetin, ononin, sissotrin, lactic acid, palmitic acid, citric acid and valine.

[0011] In at least one embodiment, the CRE has one or more the following characteristics: about 75% to about 95%, about 80% to about 95%, about 85% to about 95%, about 90% to about 95%, about 75% to about 95%, about 75% to about 90%, about 75% to about 85%, about 75% to about 80%, about 80% to about 95%, about 80% to about 90%, about 80% to about 85%, about 85% to about 95%, about 85% to about 90% or about 90% to about 95% of mannitol and about 25% to about 5%, about 25% to about 10%, about 25% to about 15%, about 25% to about 20%, about 20% to about 5%, about 20% to about 15%, about 20% to about 10%, about 15% to about 5%, about 15% to about 10% or about 10% to about 5% of a- linolenic acid in relative amount; about 0.5% to about 15%, about 0.5% to about 10%, about 0.5% to about 5%, about 0.5% to about 1%, about 1%, to about 15%, about 1% to about 10%, about 1% to about 5%, about 5% to about 15%, about 5% to about 10% or about 10% to about 15% of a-linolenic acid and about 99.5% to about 85%, about 99.5% to about 90%, about 99.5% to about 95%, about 95% to about 85%, about 95% to about 90% or about 90% to about 85% of chlorogenic acid in relative amount; about 80% to about 99%, about 80% to about 95%, about 80% to about 90%, about 85% to about 99%, about 85% to about 95%, about 85% to about 90%, about 90% to about 99% or about 90% to about 95% of glutamic acid and about 20% to about 1 %, about 20% to about 5%, about 20% to about 10%, about 20% to about 15%, about 15% to about 1%, aboutPATENTAttorney Docket No. G4590-20500PCT15% to about 5%, about 15% to about 10%, about 10% to about 1% or about 10% to about 5% of a-linolenic acid in relative amount; about 0.005% to about 5%, about 0.005% to about 1%, about 0.005% to about 0.5%, about 0.005% to about 0.1 %, about 0.005% to about 0.05%, about 0.005% to about 0.01 %, about 0.01% to about 5%, about 0.01% to about 1%, about 0.01% to about 0.5%, about 0.01% to about 0.1%, about 0.01% to about 0.05%, about 0.05% to about 5%, about 0.05% to about 1%, about 0.05% to about 0.5%, about 0.05% to about 0.1%, about 0.1% to about 5%, about 0.1% to about 1%, about 0.1% to about 0.05%, about 0.1% to about 0.001%, about 0.5% to about 5%, about 0.5% to about 1% or about 1% to about 5% of a-linolenic acid and about 99.995% to about 95%, about 99.995% to about 99%, about 99.995% to about 99%, about 99.995% to about 98%, about 99.995% to about 97%, about 99.995% to about 96%, about 99.99% to about 95%, about 99.99% to about 99.9%, about 99.99% to about 99%, about 99.99% to about 98%, about 99.99% to about 97%, about 99.99% to about 96%, about 99.95% to about 95%, about 99.95% to about 96%, about 99.95% to about 97%, about 99.95% to about 98%, about 99.95% to about 99%, about 99.9% to about 95%, about 99% to about 96%, about 99% to about 97% or about 99% to about 98% of palmitic acid in relative amount; and about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 15%, about 15% to about 30%, about 15% to about 25%, about 15% to about 20%, about 20% to about 30%, about 20% to about 25% of citric acid and about 90% to about 70%, about 90% to about 75%, about 90% to about 80%, about 90% to about 85%, about 85%, about 70%, about 85% to about 75%, about 80% to about 70% or about 75% to about 70% of chlorogenic acid in relative amount.

[0012] In a further embodiment, the CRE has one or more tire following characteristics: about 80% to about 90% of mannitol and about 20% to about 10% of a-linolenic acid in relative amount; about 1% to about 10% of a-linolenic acid and 99% to about 90% of chlorogenic acid in relative amount; about 85% to about 95% of glutamic acid and about 15% to about 5% of a-linolenic acid in relative amount; about 0.01% to about 0.1% of a-linolenic acid and about 99.99% to about 99.90% of palmitic acid in relative amount; andPATENTAttorney Docket No. G4590-20500PCT about 15% to about 25% of citric acid and about 85% to about 75% of chlorogenic acid in relative amount.

[0013] In a further embodiment, the CRE has one or more the following characteristics: a ratio of mannitol to a-linolenic acid of about 5.78 to about 1 or less a ratio of a-linolenic acid to chlorogenic acid of about 0.044 to about 1 or less a ratio of glutamic acid to a-linolenic acid of about 10. 13 to about 1 or less a ratio of a-linolenic acid to palmitic acid of about 0.00096 to about 1 or less; and a ratio of citric acid to chlorogenic acid of about 0.27 to about 1 or less.

[0014] In at least one embodiment, the TPE has one or more the following characteristics: about 80% to about 99%, about 80% to about 95%, about 80% to about 90%, about 80% to about 85%, about 85% to about 99%, about 85% to about 95%, about 85% to about 90%, about 90% to about 99%, about 90% to about 95% or about 95% to about 99% of lactic acid and about 20% to about 1%, about 20% to about 5%, about 20% to about 10%, about 20% to about 15%, about 15% to about 1%, about 15% to about 5%, about 15% to about 10%, about 10% to about 1%, about 10% to about 5%, about 5% to about 1% of formononetin in relative amount; about 0.005% to about 5%, about 0.005% to about 4.5%, about 0.005% to about 4.0%, about 0.005% to about 3.5%, about 0.005% to about 3.0%, about 0.005% to about 2.5%, about 0.005% to about 2.0%, about 0.005% to about 1.5%, about 0.005% to about 1.0%, about 0.005% to about 0.5%, about 0.005% to about 0.1%, about 0.005% to about 0.05%, about 0.005% to about 0.01%, about 0.001% to about 5%, about 0.001%, about 4.5%, about 0.001% to about 4.0%, about 0.001% to about 3.5%, about 0.001% to about 3.0%, about 0.001% to about 3.0%, about 0.001% to about 2.5%, about 0.001% to about 2.0%, about 0.001% to about 1.5%, about 0.001% to about 1.0%, about 0.001% to about 0.5%, about 0.001% to about 0.1%, about 0.001% to about 0.005%, about 0.05% to about 5%, about 0.05% to about 4.5%, about 0.05% to about 4.0%, about 0.05% to about 3.5%, about 0.05% to about 3.0%, about 0.05% to about 2.5%, about 0.05% to about 2.0%, about 0.05% to about 1.5%, about 0.05% to about 1.0%, about 0.05% to about 0.5%, about 0.05% to about 0.1%, about 0.1% to about 5%, about 0.1% to about 4.5%, about 0.1% to about 4.0%, about 0.1% to about 3.5%, about 0.1% to about 3.0%. about 0.1% to about 2.5%, about 0.1% to about 2.0%, about 0.1% to about 1.5%, about 0.1% to about 1.0%, about 0.1% to about 0.5%, about 0.5% to about 5%, about 0.5% to about 4.5%, about 0.5%PATENTAttorney Docket No. G4590-20500PCT to about 4.0%, about 0.5% to about 3.5%, about 0.5% to about 3.0%, about 0.5% to about 2.5%, about 0.5% to about 2.0%, about 0.5% to about 1.5%, about 0.5% to about 1.0% or about 1% to about 5% of formononetin and 99.995% to about 95%, about 99.995% to about 99%, about 99.995% to about 99%, about 99.995% to about 98%, about 99.995% to about 97%, about 99.995% to about 96%, about 99.99% to about 95%, about 99.99% to about 99.9%, about 99.99% to about 99%, about 99.99% to about 98%, about 99.99% to about 97%, about 99.99% to about 96%, about 99.95% to about 95%, about 99.95% to about 96%, about 99.95% to about 97%, about 99.95% to about 98%, about 99.95% to about 99%, about 99.9% to about 95%, about 99% to about 96%, about 99% to about 97% or about 99% to about 98% of palmitic acid in relative amount; about 0.5% to about 15%, about 0.5% to about 10%, about 0.5% to about 5%, about 0.5% to about 1%, about 1%, to about 15%, about 1% to about 10%, about 1% to about 5%, about 5% to about 15%, about 5% to about 10% or about 10% to about 15% of formononetin and about 99.5% to about 85%, about 99.5% to about 90%, about 95% to about 85% or about 90% to about 85%of citric acid in relative amount; about 80% to about 99%, about 80% to about 95%, about 80% to about 90%, about 80% to about 85%, about 85% to about 99%, about 85% to about 95%, about 85% to about 90%, about 75% to about 99%, about 75% to about 90%, about 75% to about 85%, about 75% to about 80%, about 80% to about 99%, about 80% to about 95%, about 80% to about 90%, about 85% to about 99%, about 85% to about 95%, about 85% to about 90%, about 90% to about 99% or about 90% to about 95% of valine and about 20% to about 1%, about 20% to about 5%, about 20% to about 10%, about 20% to about 15%, about 15% to about 1%, about 15% to about 5%, about 15% to about 10%, about 10% to about 1%, about 10% to about 5%, about 5% to about 1% of formononetin in relative amount; and about 1% to about 20%, about 20% to about 5%, about 20% to about 10%, about 20% to about 15%, about 15% to about 1%, about 15% to about 5%, about 15% to about 10%, about 10% to about 1%, about 10% to about 5%, about 5% to about 1% of formononetin and about 99% to about 80%, about 95% to about 80%, about 95% to about 85%, about 95% to about 90%, about 90% to about 80% or about 90% to about 85% of ononin in relative amount.

[0015] In a further embodiment, the TPE has one or more the following characteristics: about 85% to about 95% of lactic acid and about 15% to about 5% of formononetin in relative amount;PATENTAttorney Docket No. G4590-20500PCT about 0.01% to about 0.1% of formononetin and 99.99% to about 99.90% of palmitic acid in relative amount; about 1% to about 10% of formononetin and about 99% to about 90% of citric acid in relative amount; about 85% to about 95% of valine and about 15% to about 5% of formononetin in relative amount; and about 4% to about 15% of formononetin and about 96% to about 85% of ononin in relative amount.

[0016] In a further embodiment, the TPE has one or more the following characteristics: a ratio of lactic acid to formononetin of about 9.26 to about 1 or less a ratio of formononetin to palmitic acid of about 0.00052 to about 1 or less a ratio of formononetin to citric acid of about 0.061 to about 1 or less a ratio of valine to formononetin of about 9.46 to about 1 or less; and a ratio of formononetin to ononin of about 0.098 to about 1 or less.

[0017] In one embodiment, the subject described herein is poultry. In some embodiment, the poultry is a chicken, a duck, a turkey, a quail, an ostrich or a goose. In a further embodiment, the poultry is a chicken.

[0018] In one embodiment, the subject is with or without forced molting.

[0019] In one embodiment, the CR or CRE or CR_API or a composition comprising the CR or CRE or CR_API or a combination thereof, or the TP, TPE and TP_API or a composition comprising the TP or TPE or TP_API or a combination thereof promote estradiol secretion.

[0020] In some embodiments, the composition described herein is in a powder form (e.g., a dried powder) or a liquid form (e.g., aqueous solution, oil suspension), or a granule form.

[0021] In one embodiment, the composition described herein is used as an additive (such as a feed additive) or a supplement (such as a feed supplement).

[0022] In some embodiments, the composition described herein further comprises one or more essential oils prepared from M. spicata mint (MS), M. aquatica L. cv. Lime mint (MCL), M. arvensis mint (MA) and M. Kengting water mint (MKW).BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1. Ex vivo effects of herbal ingredients on estradiol production of isolated ovarian granulosa cells. Ovarian granulosa cells were isolated from F2-F5 follicles from 2 hens.PATENTAttorney Docket No. G4590-20500PCTCells were pooled and seeded in 6-well plates for 2 days, and then treated with various herbal ingredients overnight. The herbal ingredients included those from Crassocephalum rabens S. Moore (CR), Trifolium pratense (TP), Mentha spicata (MS), Mentha aquatica Lime (MCL), Mentha arvensis (MA), and Mentha Kengting Water Mint (MKW). Medium and cells were collected for 173 estradiol and viability analysis, respectively (n=3). *, significant differences vs. vehicle (P < 0.05, t-test). +, significant differences vs. 5 pg / mL dosage within the same treatment (P < 0.05, z-test).

[0024] Figure 2. Effects of phytogenic supplements on egg production of layers age 28-100 weeks (Trial 1). At 28 weeks of age, birds were randomly divided into 4 groups (120 hens per group) and fed with the standard layer diet (Control), or with additional phytogenic supplements: CR, TP, or CR+TP combination. At the beginning of 81 weeks of age, half the birds (n=60 hens of each group) were subjected to forced molting (FM) for 8 days, and the other half were maintained for natural molting (NM). Egg production was recorded every day.

[0025] Figure 3. Effects of phytogenic supplements on egg production of layers age 108-128 weeks (Trial 2). Birds at 108 weeks of age were randomly divided into 3 groups (n=100 hens per group) and fed with the standard layer diet (Control), or with additional phytogenic supplement CR or CR+TP combination. Egg production was recorded every day.

[0026] Figure 4. Effects of phytogenic supplements on egg quality of layers age 28-100 weeks (Trial 1). At age 28 weeks, birds were randomly divided into 4 groups (120 hens per group) and fed with the standard layer diet (Control), or with additional phytogenic supplements: CR, TP, or CR+TP combination. From 28 to 80 weeks of age, 6 eggs from each group (n=120 hens per group) were collected every 2 weeks for quality analysis. Eggs collected after 56 weeks of age were used for yolk cholesterol analysis.

[0027] Figure 5. Effects of phytogenic supplements on egg quality of layers age 108-128 weeks (Trial 2). At 108 weeks of age, birds were randomly divided into 3 groups (n=100 hens per group) and fed with the standard layer diet (Control), or with additional phytogenic supplement CR or CR+TP combination. From 114 to 128 weeks of age, 6 eggs from each group (n=100 hens per group) were collected every 2 weeks for quality analysis.

[0028] Figure 6. Effects of phytogenic supplements on serum estrogen and vitellogenin levels of layers age 28-100 weeks (Trial 1). At 28 weeks of age, birds were randomly divided into 4 groups (120 hens per group) and fed with the standard layer diet (Control), or with additional phytogenic supplements: CR, TP, or CR+TP combination. At the beginning of 81 weeks of age, half the birds (60 hens of each group) were subjected to forced molting (FM) for 8PATENTAttorney Docket No. G4590-20500PCT days and the other half were maintained for natural molting (NM). Serum collected at the indicated time points (n=6) was used for 170 estradiol and vitellogenin analysis. Means with different letters within the same time are significantly different (P < 0.05, by Tukey multiple comparison test).

[0029] Figure 7. Effects of phytogenic supplements on serum estrogen levels of layers age 108-128 weeks (Trial 2). At 108 weeks of age, birds were randomly divided into 3 groups (n=100 hens per group) and fed with the standard layer diet (Control), or with additional phytogenic supplement CR or CR+TP combination. Serum collected at the indicated time points (n=6) was used for 170 estradiol analysis. Means with different letters within the same time are significantly different (P < 0.05, by Tukey multiple comparison test).

[0030] Figure 8. Effects of TPE extract and TP_API (A), CRE extract and CR_API (B) on estrogen levels in chicken ovarian granulosa cells. Ovarian granulosa cells were collected from the F2 and F3 follicles of 2 hens and seeded in 6-well plates for 2 days in M-199 medium. Cells were treated with various herbal extracts or APIs for 24 h. Collected medium were used for estradiol analysis by ELISA (n=3). * Significant difference (vs. Vehicle): # Significant difference (vs. 5 ug / mL within the same treatment).DETAILED DESCRIPTION OF THE INVENTION

[0031] The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims. It is to be understood that both the foregoing general description and the following detailed description are examples and explanatory only and are not restrictive of the subject matter claimed in this application.

[0032] Terms such as "a", "an" and "the" are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration.

[0033] In this application, the use of "or" means "and / or" unless stated otherwise.

[0034] The term "feed" refers to food given to domestic livestock, and pet (companion animal) food.

[0035] The term "poultry" described herein may be used with all types of poultry, including other livestock, such as ducks, and turkeys. In one embodiment, the poultry of the present disclosure is a chicken.

[0036] As used herein, the term "about" when referring to the numerical value is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% from the numerical value. Such variations in the numerical value may occur by, e.g., the experimental error, calculation errors, routine minor adjustments, the typical error in measuring or handling procedures forPATENTAttorney Docket No. G4590-20500PCT making ingredients, supplements, compositions, or formulations, the differences in the source, manufacture, or purity of starting materials or ingredients used in the present disclosure, or like considerations.

[0037] As used herein, the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, which are included in the present disclosure, yet open to the inclusion of unspecified elements or steps, whether essential or not.

[0038] As used herein, the term "treatment" refers to the use of an effective agent to the poultry in need thereof with the purpose to cure, alleviate, relieve, remedy, ameliorate, reduce, or prevent the disease, the symptoms thereof, or the predispositions towards it.

[0039] As used herein, the term "preventing" or "prevention" refers to preventive or avoidance measures for a disease or symptoms or conditions of a disease, which include but are not limited to, applying or administering one or more active agents to the poultry which has not yet been diagnosed as the poultry suffering from the disease or the symptoms or conditions of the disease but may be susceptible or prone to the disease. The preventive measures of the present disclosure are provided to avoid, prevent, or postpone the occurrence of the disease or the symptoms or conditions of the disease.

[0040] As used herein, the phrase "an effective amount" refers to the amount of an active agent that is required to confer a desired preventive or therapeutic effect on the poultry in need thereof. Effective doses may vary, as recognized by those skilled in the art, depending on routes of administration, additional herbal ingredient usage, the possibility of co-usage with other phytogenic supplements, and the condition to be treated.

[0041] As used herein, the term "administering" or "administration" refers to the placement of an active agent into an animal by a method or route which results in at least partial localization of the active agent at the desired site to produce the desired effect. The active agent described herein may be administered by any appropriate route known in the art. For example, the feed composition of the present disclosure is administered to the animal by oral administration.

[0042] The term "relative amount" (or relative quantity or relative intensity) is used to compare values or express a quantity in proportion to another value.

[0043] The numeral ranges used herein are inclusive and combinable, any numeral value that falls within the numeral scope herein could be taken as a maximum or minimum value to derive the sub-ranges therefrom. For example, it should be understood that the numeral range "0.0001% to 99.9%" comprises any sub-ranges between the minimum value of 0.0001% to the maximum value of 99.9%, such as the sub-ranges from 10% to 90%, from 20% to 80%, from 30% to 70%PATENTAttorney Docket No. G4590-20500PCT and so on. In addition, a plurality of numeral values used herein can be optionally selected as maximum and minimum values to derive numerical ranges. For instance, the numerical ranges of 40% to 60%, 40% to 50%, and 50% to 60% can be derived from the numeral values of 40%, 50%, and 60%.

[0044] Medicinal plants contain diverse chemical ingredients that show biological or pharmacological properties, which can support layer chickens in combating stresses and improve their performance. Crassocephalum rabens (Asteraceae) has been demonstrated to have antiinflammatory effects on inhibiting endotoxin-induced nitric oxide production, the IKKOI / NF-KB signaling pathway, and COX-2 activity in macrophages (Hou, C.-C., Chen, Y. P., Wu, J.-H., Huang, C.-C., Wang, S.-Y., Yang, N.-S., and Shyur, L.-F. 2007. A galactolipid possesses novel cancer chemopreventive effects by suppressing inflammatory mediators and mouse Bl 6 melanoma. Cancer Res. 67, 6907-6915). Bioactive phytogalactolipids from the same plant ameliorated endotoxin-induced sepsis in animals by lowering the levels of systemic proinflammatory cytokines TNF-a and IL-6 and mitigating septic shock-induced multiple organ damage (Apaya, M. K., Lin, C.-Y., Chiou, C.-Y., Yang, C.-C., Ting, C.-Y., and Shyur, L.-F. 2015. Simvastatin and a plant galactolipid protect animals from septic shock by regulating oxylipin mediator dynamics through the MAPK-cPLA2 signaling pathway. Molecular Medicine 21, 988- 1001). On the other hand, Trifolium pratense (Fabaceae) (red clover) contains isoflavonoids, which show binding activity with either ERa or ERp. Red clover isoflavones have been suggested as an alternative to post-menopausal hormone replacement therapy. However, whether the estrogen-mimicking activity of red clover could be beneficial to layer chicken egg production or quality is unknown.

[0045] The present disclosure demonstrates that Crassocephalum rabens (CR) and Trifolium pratense (TP) are novel phytogenic supplements to promote egg production and quality in hens. In this disclosure, both CR and TP extracts and four mint essential oils can promote the chicken granulosa cells to secret estradiol. In some embodiments, the extracts are obtained by extracting CR or TP with 50% to 100% ethanol; for example, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95% or about 100% ethanol. The compounds in the CR and TP are further isolated and purified based on known methods. Such chemically defined phytogenic supplements CR and TP used alone or CR and TP used in combination as feed additives could significantly promote egg yield, egg weight, egg quality, and / or lower yolk cholesterol contents in hens with or without forced molting. CR, TP, and CR+TP supplementation differentially increased serum estradiol and vitellogenin levels in forced molting hens, suggesting anPATENTAttorney Docket No. G4590-20500PCT accelerated recovery of ovarian function to promote estrogen levels in aged hens, which in turn increased hen day egg production rate and total egg yield per hen. Therefore, supplemental herbs may accelerate the recovery process of the ovaries and reproductive tract after forced molting. The present disclosure demonstrates the mono-use or combinational use of the ingredients from six plants as novel phytogenic additives for the layer poultry industry.

[0046] The CR, CRT, CR_API or its composition described herein or the TP, TPE, TP_API, or its composition described herein can be made into a liquid or solid form. If it is a liquid, it can be made with a water, polyol (like glycerol, ethylene glycol, or propylene glycol), a salt (such as sodium chloride, sodium benzoate, potassium sorbate, or calcium carbonate), or a sugar or sugar derivative (like dextrin, glucose, sucrose, or sorbitol). For example, one version of the product is a liquid with the CR, CRE, CR_API or its composition described herein or the TP, TPE, TP_API, or its composition described herein and one or more formulating agents like water, glycerol, ethylene glycol, propylene glycol, sodium chloride, sodium benzoate, calcium carbonate, vitamins, minerals, potassium sorbate, starch, dextrin, glucose, sucrose, sorbitol, and oyster shell. This liquid can be sprayed onto animal feed after it has been made into pellets, or it can be added to the animals' drinking water.

[0047] For example, a solid formulation could take the form of granules, spray-dried powder, or agglomerates. The formulating agent might include a variety of salts (organic or inorganic zinc, sodium, potassium, or calcium salts like calcium acetate, calcium benzoate, calcium carbonate, calcium chloride, calcium citrate, calcium sorbate, calcium sulfate, potassium acetate, potassium benzoate, potassium carbonate, potassium chloride, potassium citrate, potassium sorbate, potassium sulfate, sodium acetate, sodium benzoate, sodium carbonate, sodium chloride, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate, zinc carbonate, zinc chloride, zinc citrate, zinc sorbate, zinc sulfate), starch, or a sugar or sugar derivative (such as sucrose, dextrin, glucose, lactose, sorbitol).

[0048] For example, the solid composition may be in granulated form in one embodiment. The granule may have a matrix structure with homogeneously mixed components. However, the granule typically consists of a core particle and one or more coatings, usually salt and / or wax coatings. Examples of waxes include polyethylene glycols, polypropylenes, Carnauba wax, Candelilla wax, beeswax, hydrogenated plant oil or animal tallow (such as hydrogenated ox tallow, hydrogenated palm oil, hydrogenated cotton seeds, and / or hydrogenated soybean oil), fatty acid alcohols, mono-glycerides and / or di-glycerides (such as glyceryl stearate, which is aPATENTAttorney Docket No. G4590-20500PCT mixture of stearic and palmitic acid), microcrystalline wax, paraffins, and fatty acids (such as hydrogenated linear long-chained fatty acids and their derivatives).

[0049] The CR, CRE, CR_API or its composition described herein or the TP, TPE, TP_API, or its composition described herein can be added into feed or drinking water as an additive or a supplement, but is not limited thereto. That is, the CR, CRE, CR_API or its composition described herein or the TP, TPE, TP_API, or its composition described herein can be prepared in any form suitable for the feed composition based on the actual needs. In at least one embodiment of the present disclosure, the feed composition can be formulated in the form of non-dried, dried, crushed, or granulated. For example but not limited thereto, the CR, CRE, CR_API or its composition described herein or the TP, TPE, TP_API, or its composition described herein can be mixed with a liquid carrier suitable for the feed composition, or the CR, CRE, CR_API or its composition described herein or the TP, TPE, TP_AP1, or its composition described herein can be absorbed into a carrier material suitable for the feed composition.

[0050] Without intent to limit the scope of the disclosure, exemplary instruments, methods and their related results according to the embodiments of the present disclosure are given below. It is noted that titles or subtitles may be used in the examples for convenience of a reader, which in no way should limit the scope of the disclosure. Moreover, certain theories are proposed and disclosed herein; however, in no way they, whether they are right or wrong, should limit the scope of the disclosure so long as the disclosure is practiced according to the disclosure without regard for any particular theory or scheme of action.EXAMPLES

[0051] Materials and Methods

[0052] Plant material and extract preparation

[0053] Crassocephalum rabens (Benth.) S. Moore (Asteraceae) (also known as C. crepidioides (Benth.) S. Moore) (CR) plants and Trifolium pratense L. (Fabaceae) (TP) plants grown in Nantou County, Taiwan were harvested and dried before experiments. The total extracts of medicinal plants were prepared by extraction with 50 to 99.5% ethanol at room temperature and the supernatants were collected and dried under vacuum. Four Mentha species (Lamiaceae), Mentha spicata (MS), M. aquatica L. cv. Lime (MCL), M. arvensis (MA) and M. Kengting Water Mint (MKW) were cultivated at the Taichung District Agricultural Research and Extension Station, Taichung, Taiwan. The above ground tissues of mint plants were harvested and dried, or subjected to water vapor distillation to collect essential oils. The mint essential oilsPATENTAttorney Docket No. G4590-20500PCT were stored at -20°C. Essential oils and plant extracts used in in vitro assay were diluted into different concentrations with dimethyl sulfoxide (DMSO). The chemical compositions of CR, TP, and Mentha plants were analyzed and determined by various spectral methods, including LC / mass spectrometry, GC / mass spectrometry, and / or NMR spectrometry.

[0054] Granulosa cell isolation and cultures

[0055] F2 to F3 follicles were collected from two laying hens for in vitro culture cell studies. In brief, follicles were quickly immersed in ice-cold Hanks’ Balanced Salt Solution. Granulosa cells were isolated as described (Xie Y.-L., Y.-E. Pan, C.-J. Chang, P.-C. Tang, Y.-F. Huang, R. L. Walzem, and S.-E. Chen. 2012. Palmitic acid in chicken granulosa cell death-lipotoxic mechanisms mediate reproductive inefficacy of broiler breeder hens. Theriogenology. 78: 1917-1928). Collected granulosa layers were dispersed in M199-HEPES medium containing 200 units / mL of type-2 collagenase and 0.3 mg / mL of trypsin inhibitor (Sigma-Aldrich, St. Louis, MO, USA) at 37°C for 30 min with gentle agitation. Cells collected from the F2 and F3 follicles were pooled and seeded in M-199 medium containing 10 mM HEPES, 18 mM NaHCOs, 1% antibiotic / antimycotic solution containing penicillin and streptomycin, and 10% fetal bovine serum (FBS), pH 7.4 and incubated at 37°C with 5% CO2. The next day, cells were replaced with new medium and allowed to culture for another 24 h. On the third day, cells were fed new medium containing various herb extracts (dissolved in DMSO) and cultured for another 24 h. Medium and cells were then collected for 17P-estradiol (E2) and viability analysis.

[0056] Estrogen, vitellogenin, and cell viability analyses

[0057] Blood samples were collected from hens with freshly laid eggs (eggshells were warm to the touch) on the sampling day. Collected serum and cell culture medium were used for estradiol and vitellogenin analysis using commercial ELISA kits (Cayman Chemical and Lifespan Biosciences, respectively). Cell viability was determined by MTT assay (Cayman Chemical).

[0058] Animal trial 1: Phytogenic supplement of hens aged 28-100 weeks to evaluate the effect on egg production

[0059] Six hundred HyLine Brown hens aged 24 weeks were purchased from a local farm in Ping Tung, Taiwan. Birds were fed a standard layer diet and allowed to adapt for 4 weeks. At 28 weeks of age, birds were randomly divided into 3 groups: (1) a control group, fed with the standard layer diet; (2) a CR group, fed with phytogenic supplement derived from C. rabens plant in the standard layer diet; (3) a TP group, fed with phytogenic supplement derived from T. pretense plant in the standard layer diet; and (4) a CR+TP group, fed with phytogenic supplement derived from C. rabens and T. pratense plants in the standard layer diet. The feedingPATENTAttorney Docket No. G4590-20500PCT trial lasted until the hens were 100 weeks of age. Hens were maintained on a photo schedule of 16 h light / 8 h dark with feed provided at 08:30 AM daily.

[0060] At the beginning of 81 weeks of age, half of the birds (60 hens from each group) were subjected to forced molting (FM), and the other half were maintained for natural molting (NM). The forced molting was achieved by feed withdrawal for 8 days and water deprivation in the first 3 days, followed by feed re -provision at 15, 30, and 60 grams on days 9, 10, and 11, respectively, and ad libitum feed intake after day 12. Hens that underwent forced molting were maintained on a photo schedule of 6 h light / 18 h dark for the first 4 weeks, and thereafter, the light period was increased by 1 h per week until reaching 16 h light / 8 h dark.

[0061] Animal trial 2: Phytogenic supplement of hens aged 108-128 weeks to evaluate the effect on egg production

[0062] Five hundred HyLine Brown hens aged 108 weeks were purchased from a local farm in Ping Tung, Taiwan. Birds were fed a standard layer diet and allowed to adapt for 8 weeks. At 108 weeks of age, birds were randomly divided into 3 groups: (1) a control group, fed with the standard layer diet; (2) a CR group, fed with phytogenic supplement derived from C. rabens plant in the standard layer diet; and (3) a CR+TP group, fed with phytogenic supplement derived from C. rabens and T. pratense plants in the standard layer diet. The feeding trial lasted to 128 weeks of age. Hens were maintained on a photo schedule of 16 h light / 8 h dark with feed provided at 08:30 AM daily.

[0063] Egg quality

[0064] From 28 to 80 weeks, 6 eggs per group were collected every 2 weeks and used for quality analysis within 24 h. Haugh unit (HU) was measured using the height of thick albumin and egg weight. The Haugh unit value ranged from 0 - 130 and was ranked as: AA, 72 or more; A, 71 - 60; B, 59 - 31; and C, 30 or less.HU = 100 * log (h - 1.70 37+ 7.6) h = observed height of the albumen in millimeters (mm) w = weight of egg in grams (g)

[0065] Vitelline Membrane Strength (VMS) was measured using the TA. HDPlus Texture Analyzer with a 5-kg load cell, and a sensitivity of 0.1 g (or texture analyzer trigger force). Deformation speed was 3.2 mm s1(Berardinelli et al., 2008). Yolk cholesterol was determined by the colorimetric method using a commercial kit (Sigma- Aldrich).

[0066] ResultsPATENTAttorney Docket No. G4590-20500PCT

[0067] An ex vivo assay was earned out using isolated chicken granulosa cells to validate the effect of various herbal extracts on estradiol secretion. The chemically defined extracts from CR and TP plants or essential oils prepared from M. spicata mint (MS), M. aquatica L. cv. Lime mint (MCE), M. arvensis mint (MA) and M. Kengting water mint (MKW) at 5 or 10 pg / mL treatment significantly increased estradiol secretion in the cells with little or no effect on cell viability (Figure 1). The results indicated that the six phytogenic candidates may have positive or improvement effects on egg production.

[0068] Dietary supplementation with phytogenic CR, TP, or CR+TP combination significantly increased hen-day-egg production (eggs / day / hen) and / or total egg yield (eggs / hen) in the laying stage from age 28 to 100 weeks (Trial 1, Table 1). The beneficial effect was more pronounced in hens after age 75 weeks (Table 1, Figure 2). Supplemental CR or TP also significantly promoted egg production in hens aged 85 to 100 weeks that were subjected to forced molting at age 80 weeks (Table 2, Figure 2). The beneficial effect on egg production by CR and CR+TP combination was further confirmed in aged hens from 108 to 128 weeks (Trial 2) (Table 3, Figure 3).

[0069] The chemically defined phytogenic supplements were found to improve egg quality in Trial 1, as shown by increased egg weight with CR, TP, and CR+TP, increased eggshell strength with CR+TP, increased egg freshness with TP, increased yolk membrane strength with CR+TP, and lowered yolk cholesterol levels with CR (Table 4, Figure 4). In Trial 2, supplemental CR significantly increased egg weight and suppressed yolk cholesterol levels, while CR+TP increased egg weight and egg freshness ('fable 5, Figure 5).

[0070] In accordance with improved egg production (Table 1), supplemental CR, TP, and CR+TP sustained high serum estrogen levels in hens under normal conditions from the age of 28 to 100 weeks (Figure 5). In hens that underwent forced molting (80 weeks), CR, TP, and CR+TP supplementation differentially increased serum estradiol and vitellogenin levels after 86 weeks, suggesting an accelerated recovery of ovarian function after cessation by feed withdrawal for forced molting (Figure 5). In Trial 2, CR and CR+TP also differentially promoted serum estrogen levels in aged hens from 108 to 128 weeks (Figures 6 and 7).

[0071] TPE and TP_API promote estradiol secretion in ovarian granulosa cells. Ovarian granulosa cells were isolated from F2-F5 follicles of 2 hens, cells were pooled and seeded in 6- well plates for 2 days, and then treated with various compounds for 24 hr. Collected medium were used for estradiol analysis by ELISA (n=3) (* Significant difference (vs. Vehicle); # Significant difference (vs. 5 ug / niL within tire same treatment). The results support the bioactivity of TP onPATENTAttorney Docket No. G4590-20500PCT maintaining ovarian function and promoting egg production yield in chickens (Figure 8A) (The treated cells were all with >90% viable using the indicated concentrations).L0072J CRE and CR_API promote estradiol secretion in ovarian granulosa cells. Ovarian granulosa cells were isolated from F2-F5 follicles of 2 hens, cells were pooled and seeded in 6-well plates for 2 days, and then treated with various compounds for 24 hr. Collected medium were used for estradiol analysis hy ELISA (n=3) (* Significant difference (vs. Vehicle); # Significant difference (vs. 5 ug / mL within the same treatment). The results support the bioactivity of CR on maintaining ovarian function and promoting egg production yield in chickens (Figure 8B) (The treated cells were all with >90% viable using tire indicated concentrations).

[0073] The chemical constituents in CRE includes a-linolenic acid, mannitol, chlorogenic acid, glutamic acid, palmitic acid, and citric acid, and they are in one or more of the following ratios:• a ratio of mannitol to a-linolenic acid of 5.78 to 1 or less• a ratio of a-linolenic acid to chlorogenic acid of 0.044 to 1 or less• a ratio of glutamic acid to a-linolenic acid of 10.13 to 1 or less• a ratio of a-linolenic acid to palmitic acid of 0.00096 to 1 or less• a ratio of citric acid to chlorogenic acid of 0.27 to 1 or less

[0074] The chemical constituents in TPE includes formononetin, ononin, lactic acid, palmitic acid, citric acid and valine, and they are in one or more of the following ratios:• a ratio of lactic acid to formononetin of 9.26 to 1 or less• a ratio of formononetin to palmitic acid of 0.00052 to 1 or less• a ratio of formononetin to citric acid of 0.061 to 1 or less• a ratio of valine to formononetin of 9.46 to 1 or less• a ratio of formononetin to ononin of 0.098 to 1 or lessTable 1. Effects of dietary supplementation of herbs on egg production of layers age 28-100 weeks under normal conditions (Trial 1).Control CR TP CR+TP28-100 weeksHen-day egg production 80.2±0.38 81.1±0.34* 80.8±0.35 81.3±0.35*(eggs / day / hen, %)Total egg yield 409.8±0.69 414.6±0.62* 412.8±0.64* * 415.5+0.65*PATENTAttorney Docket No. G4590-20500PCT(eggs / hen)75-100 weeksHen-day egg production 70.7±0.47 72.7±0.38* 72.12±0.41* 72.6±0.42*(eggs / day / hen, %)Total egg yield 128.6±0.52 132.3+0.42* 131.3+0.45* 132.0+0.46*(eggs / hen)At 28 weeks of age, birds were randomly divided into 4 groups (120 hens per group) and fed with the standard layer diet (Control), or with additional supplementation of herbs: CR (Crassocephalum rabens S. Moore), TP (Trifolium pratense), or CR+TP combination. At the beginning of 81 weeks of age, half of the birds (60 hens in each group) were subjected to forced molting (FM) for 8 days and the other half were maintained for natural molting (NM). *, significant difference vs. control (P < 0.05 by / -test). #, significant difference vs. CR+TP (P < 0.05 by / -test).Table 2. Effects of dietary supplementation of herbs on egg production and body weight of layers age 85-100 weeks under forced molting (Trial 1).Control CR TP CR+TP85-100 weeksHen-day egg production 70.9±0.55 82.0+0.58 * 83.9+0.62 * 71.5+0.52(eggs / day / hen, %)Total egg yield 79.4±0.48 91.9+0.50 * 93.9+0.53 * 80.1+0.44(eggs / hen)Body weight (kg / hen) before molting (81 weeks) 1.99+0.13 1.89+0.12 1.99+0.13 1.92+0.15 after molting (82 weeks) 1.46±0.10+1.45±0.13+1.47±0.15+1.48±0.16+Mortality (%) 6.67 5.0 3.33 5.0At the beginning of 81 weeks of age, half of the birds (n=60 hens of each group) were subjected to forced molting (FM) for 8 days and the other half were maintained for natural molting (NM). *, significant difference vs. control (P < 0.05 by / -test). +, significant difference vs. before molting (P < 0.05 by / -test).PATENTAttorney Docket No. G4590-20500PCTTable 3. Effects of dietary supplementation of herbs on egg production of layers age 108- 128 weeks under normal conditions (Trial 2).Control CR CR+TPHen-day egg 66.0+0.78c68.0+0.74* 68.3+1.01* production(eggs / day / hen, %)Total egg yield 97.1+0.61 100.0+0.36* 100.4+0.73*(eggs / hen)Birds aged 108 weeks were randomly divided into 3 groups (n= 100 hens per group) and fed with standard layer diet (Control), or with additional supplementation of herbs: CR (Crass ocephalum rabens S. Moore) or CR+TP (Trifolium pratense) combination. *, significant difference vs. control (P < 0.05 by t-test).Table 4. Effects of dietary supplementation of herbs on egg quality of layers age 28-80 weeks (Trial 1)Control CR TP CR+TPEgg weight (g / egg) 60.7+0.28 62.3+0.33* 62.7+0.34* 62.2+0.37*Yolk fraction 26.7+0.12 26.6+0.14 26.0+0.10 * 26.7+0.17(%of egg mass)Eggshell strength 3.52+0.044 3.56+0.045* 3.56+0.042* 3.73+0.056*(kg / cm2)Egg freshness 89.4+0.50 90.1+0.44 91.5+0.27** 90.1+0.40(Haugh unit)Yolk color 6.00+0.067 5.93+0.065 5.94+0.065 6.01+0.050Yolk membrane 3.14+0.077 3.27+0.070 3.26+0.077 3.34+0.091* strength (g)Yolk cholesterol 14.2+0.20 12.5+0.18** 13.6+0.31 13.7+0.16(mg / g yolk)Between 28 and 80 weeks of age, 6 eggs from each group (n=120 hens per group) were collected every 2 weeks for quality analysis. Eggs collected after 56 weeks of age were used for yolk cholesterol analysis. *, significant difference vs. control (P < 0.05 by t-test). #, significant difference vs. CR+TP (P < 0.05 by t-test).PATENTAttorney Docket No. G4590-20500PCTTable 5. Effects of dietary supplementation of herbs on egg quality of layers age 108-128 weeks (Trial 2)Control CR CR+TPEgg weight (g / egg) 66.4+1.05 68.6±0.43* 68.7±1.00*Yolk fraction 26.7+0.45 26.6+0.47 26.5±0.68(% of egg mass)Eggshell strength 3.11+0.07 3.15+0.09 3.25±0.07(kg / cm2)Egg freshness 71.4±3.1 71.5±4.2 76.1±1.9*(Haugh unit)Yolk color 6.19+0.18 6.20+0.21 5.80±0.27Yolk cholesterol 15.7+0.36 14.9±0.36* 15.0±0.52(mg / g yolk)From 114 to 128 weeks of age, 6 eggs from each group (n=100 hens per group) were collected every 2 weeks for quality analysis. *, significant difference vs. control (P < 0.05 by / - test )

[0075] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

PATENTAttorney Docket No. G4590-20500PCTCLAIMSWHAT IS CLAIMED IS:

1. A method of promoting egg yield, egg weight, egg quality, lower yolk cholesterol contents, and / or sustain ovarian function in a subject, comprising administering to the subject an effective amount of a composition comprising a Crassocephalum rabens (CR), an extract thereof (CRE) or an active ingredient(s) contained therein (CR_API), or a combination of a CR and a CRE, or a CR and a CR_API, or a CRE and a CR_API.

2. A method of promoting egg yield, egg weight, egg quality, lower yolk cholesterol contents, and / or sustain ovarian function in a subject, comprising administering to the subject an effective amount of a composition comprising a T. pratense (TP) and an extract thereof (TPE) or an active ingredient(s) contained therein (TP_API), or a combination of a TP and a TPE or a TP and a TP_API or a TPE and a TP_AP1.

3. A method of promoting egg yield, egg weight, egg quality, lower yolk cholesterol contents, and / or sustain ovarian function in a subject, comprising administering to the subject an effective amount of a preparation of a first composition comprising one or more component selected from a CR, a CRE and a CR_API and a second composition comprising one or more component selected from a TP, a TPE and a TP_API. In some embodiments, the preparation comprises a CR and a TP, a CRE and a TPE, or a CR_API and a TP_API.

4. The method of any one of claims 1 to 3, wherein the active ingredient(s) in CRE comprises one or more of the following components: a-linolenic acid, mannitol, chlorogenic acid, glutamic acid, palmitic acid, and citric acid.

5. The method of any one of claims 1 to 3, wherein the active ingredient(s) in TPE comprises one or more of the following components: formononetin, ononin, lactic acid, palmitic acid, citric acid and valine.

6. The method of any one of claims 1 to 3, wherein the CRE has one or more the following characteristics: about 75% to about 95% of mannitol and about 25% to about 5% of a-linolenic acid in relative amount; about 0.5% to about 15% of a-linolenic acid and 99.5% to about 85% of chlorogenic acid in relative amount; about 80% to about 99% of glutamic acid and about 20% to about 1% of a-linolenic acid in relative amount; about 0.005% to about 5% of a-linolenic acid and about 99.995% to about 95% of palmitic acid in relative amount; andPATENTAttorney Docket No. G4590-20500PCT about 10% to about 30% of citric acid and about 90% to about 70% of chlorogenic acid in relative amount.

7. The method of any one of claims 1 to 3, wherein the CRE has one or more the following characteristics: about 80% to about 90% of mannitol and about 20% to about 10% of a-linolenic acid in relative amount; about 1% to about 10% of a-linolenic acid and 99% to about 90% of chlorogenic acid in relative amount; about 85% to about 95% of glutamic acid and about 15% to about 5% of a-linolenic acid in relative amount; about 0.01 % to about 0.1 % of a-linolenic acid and about 99.99% to about 99.90% of palmitic acid in relative amount; and about 15% to about 25% of citric acid and about 85% to about 75% of chlorogenic acid in relative amount.

8. The method of any one of claims 1 to 3, wherein the CRE has one or more the following characteristics: a ratio of mannitol to a-linolenic acid of about 5.78 to about 1 or less a ratio of a-linolenic acid to chlorogenic acid of about 0.044 to about 1 or less a ratio of glutamic acid to a-linolenic acid of about 10.13 to about 1 or less a ratio of a-linolenic acid to palmitic acid of about 0.00096 to about 1 or less; and a ratio of citric acid to chlorogenic acid of about 0.27 to about 1 or less.

9. The method of any one of claims 1 to 3, wherein the TPE has one or more the following characteristics: about 80% to about 99% of lactic acid and about 20% to about 1% of formononetin in relative amount; about 0.005% to about 5% of formononetin and 99.995% to about 95% of palmitic acid in relative amount; about 0.5% to about 15% of formononetin and about 99.5% to about 85% of citric acid in relative amount; about 80% to about 99% of valine and about 20% to about 1% of formononetin in relative amount; and about 1% to about 20% of formononetin and about 99% to about 80% of ononin in relative amount.

10. The method of any one of claims 1 to 3, wherein the TPE has one or more the following characteristics:PATENTAttorney Docket No. G4590-20500PCT about 85% to about 95% of lactic acid and about 15% to about 5% of formononetin in relative amount; about 0.01% to about 0.1% of formononetin and 99.99% to about 99.90% of palmitic acid in relative amount; about 1% to about 10% of formononetin and about 99% to about 90% of citric acid in relative amount; about 85% to about 95% of valine and about 15% to about 5% of formononetin in relative amount; and about 4% to about 15% of formononetin and about 96% to about 85% of ononin in relative amount.

11. The method of any one of claims 1 to 3, wherein the TPE has one or more the following characteristics: a ratio of lactic acid to formononetin of about 9.26 to about 1 or less a ratio of formononetin to palmitic acid of about 0.00052 to about 1 or less a ratio of formononetin to citric acid of about 0.061 to about 1 or less a ratio of valine to fomiononetin of about 9.46 to about 1 or less; and a ratio of formononetin to ononin of about 0.098 to about 1 or less.

12. The method of any one of proceeding claims, wherein the subject is a female poultry.

13. The method of claim any one of proceeding claims, wherein the subject is a female chicken, duck, turkey, quail, ostrich or goose.

14. The method of claim any one of proceeding claims, wherein the subject is a hen.

15. The method of claim any one of proceeding claims, wherein the subject is with or without forced molting.

16. The method of claim any one of proceeding claims, wherein the composition is in a powder form (e.g., a dried powder) or a liquid form (e.g., aqueous solution, oil suspension), or a granule form.

17. The method of claim any one of proceeding claims, wherein the composition is used as an additive (such as a feed additive) or a supplement (such as a feed supplement).

18. The method of claim any one of proceeding claims, wherein the composition further comprises one or more essential oils prepared from M. spicata mint (MS), M. aquatica L. cv. Lime mint (MCL), M. arvensis mint (MA) and M. Kengting water mint (MKW).

19. The method of claim any one of proceeding claims, wherein the CR or CRE or CR_API or a composition comprising tire CR or CRE or CR_API or a combination thereof, or tire TP, TPE and TP_API or a composition comprising the TP or TPE or TP_API or a combination thereof promote estradiol secretion.

Images