COMPANION ANIMAL FEED WITH COMBINATION OF 25-HYDROXYVITAMIN D AND ANTIOXIDANTS/ANTI-INFLAMMATORIES

Abstract

This invention relates to the use of the combination of 25-hydroxyvitamin D3 (“25-OH D3”) and antioxidants/anti-inflammatories (ascorbic acid vitamin E and canthaxanthin) to make a premix or feed which can ameliorate various problems observed in companion animals which have been subject to overfeeding. Foods containing the 25-OH D3 and antioxidants/anti-inflammatories and premixes are also provided.

Description

BRIEF DESCRIPTION OF THE INVENTION

This invention relates to the combination of 25-hydroxyvitamin D (“25-OH D3” and/or “25-OH D2”) and antioxidants/anti-inflammatories (ascorbic acid, Vitamin E and a carotenoid) for use in food for companion animals, especially cats and dogs. This combination of nutritional supplements protects against various adverse effects associated with hyperphagia and related obesity. It also relates to feed and feed premixes for companion animals containing the combination of 25-hydroxyvitamin D and the antioxidants/anti-inflammatories.

BACKGROUND OF THE INVENTION

Hyperphagia and therefore adiposity can occur in companion animals including dogs and cats. Overfeeding-induced diseases which can result include metabolic disorders including diabetes and cardiovascular problems. Pet owners do not always regulate the volume of food which is offered to the animal, and some animals will eat past the point of satiation.

In poultry which is used as a model, elevated concentrations of plasma glucose, non-esterified fatty acids, very low density lipoprotein, triacylglycerol, phospholipids, ceramide and sphingomyelin were observed during hyperphagia. Furthermore, hepatic and circulating ceramide and sphingomyelin accumulation, and up-regulation of pro-inflammatory IL-1β expression in liver and adipose tissues systemically manifested the development of lipotoxicity in feed-satiated hens. Ceramide is a key intermediate linking certain nutrients (i.e. saturated fats) and inflammatory cytokines (e.g. tumor necrosis factor-α, TNFα) to the regulation of cell function and antagonizing insulin signaling and mitochondrial function. Moreover, as a result of its toxic effects on particularly susceptible cell types, ceramide has the capacity to damage the heart, pancreas, and vasculature. Lipotoxicity leading to impaired ovarian dysfunctions, including follicle atresia, ovarian regression, and a decline of circulating estradiol levels in feed-satiated hens, was further exemplified by ceramide accumulation and up-regulation of IL-1β, serine palmitoyltransferase, and sphingomyelinase transcript abundance, but suppressed protein kinase Akt activation within the hierarchical follicles. In vivo evidence has thus delineated the actions of ceramide and IL-1β in mediating overfeeding-induced follicle atresia and progression of ovarian involution in broiler hens.

Hy⋅D® (registered trademark for 25-OH-D3; available from DSM Nutritional Products, Switzerland has been used to promote bone and joint health in companion animals, particularly large breed dogs.

The combination of 25-OH D3 and canthaxanthin has also been used in poultry. WO2010/057811 (DSM IP ASSETS, BV) describes this combination for use in improving hatchability, fertility, and lower embryo mortality in poultry. The combination is commercially available under the trademark MAXICHICK. There is no mention in the patent publication of the inclusion of ascorbic acid and high vitamin E levels, nor the uses to ameliorate the adverse effects of hyperphagia-related obesity.

WO14/191153 (DSM IP ASSETS B.V) teaches the combination of canthaxanthin and at least one of Vitamin C, Vitamin E, selenium, and optionally at least one of thymol, eugenol, vanillin and gamma-terpinene can improve immune statues, bone health, skeletal development and growth and feed conversion, particularly when flocks are subject to stress associated with vaccination.

Vitamin C (ascorbic acid) is often not included as a supplement in some animal diets, as the many animals can produce sufficient Vitamin C under normal rearing conditions. However, it has been used in some specific conditions, such as in heat stress situations.

Vitamin E is generally added to companion animal food. For dogs the recommended minimum amounts range from 36-50 IU/kg on a dry matter basis. For cats, the minimum is from 38-50.7 IU/kg, also on a dry matter basis. Many premium pet foods have a much higher amount of Vitamin E, and it can be as high as 500 IU/kg.

There is a need to reduce or ameliorate the metabolic and hormonal dysregulations as a result of hyperphagia in companion animals in order to ameliorate, prevent or treat impaired glucose regulation, insulin actions, cardiomyopathies, and ultimately ascites and sudden death.

DETAILED DESCRIPTION OF THE INVENTION

It has been found, accordance with this invention that the combination of 25-hydroxyvitamin D (25-OH D3 and/or 25-OH D2) and antioxidants/anti-inflammatories ameliorates adverse metabolic conditions observed when hens are fed ad libitum. The hen was uses as a model for ad libitum fed companion animals such as a cat or dog at they also can experience hyperphagia-related obesity. It has also been found in accordance with this invention, that the bio-actives traditionally included in the food as antioxidants also possess an anti-inflammatory activity which mitigates the adverse effects of hyperphagia-related obesity.

It has also been found that the combination of 25-OH D and antioxidants/anti-inflammatories can ameliorate, reduce the severity of, or prevent cardiovascular problems related to hyperphagia.

As 25-OH D2 and 25-OH D3 may act in a similar fashion after administration, it is envisioned that either may be used separately in combination with antioxidants/anti-inflammatories, or a mixture of both 25-OH D3 and 25-OH D2 may be used in combination with antioxidants/anti-inflammatories. If used together, the ratio of 25-OH D3: 25-OH D2 is not a critical part of the invention. 25-OH D3 used alone is preferred.

The antioxidants/anti-inflammatories of this invention comprise the combination of ascorbic acid, vitamin E and a carotenoid. Thus one aspect of this invention is the combination of 25-OH D3, a carotenoid, vitamin E and ascorbic acid. Another embodiment is the combination of 25-OHD2 a carotenoid, Vitamin E and ascorbic acid. Another embodiment is the combination of 25-OHD3, 25-OHD2, a carotenoid, Vitamin E and ascorbic acid. The carotenoid is at least one selected from the group consisting of: lycopene, astaxanthin, cryptoxanthin, beta-carotene, lutein, zeaxanthin and canthaxanthin. Most preferred is beta carotene.

Another aspect of this invention is the combination of

• • a) 25-OH D,
  • b) a carotenoid selected from the group consisting of: lycopene, astaxanthin, cryptoxanthin, beta-carotene, lutein, zeaxanthin and canthaxanthin,
  • c) vitamin E
  • d) ascorbic acid, and
  • e) optionally further comprises at least one further bio-active ingredient selected from the group consisting of:

Vitamin D, Vitamin B2, Vitamin B6, Niacin, Zinc, Copper, Manganese, and Selenium. Preferably the 25-OH D is 25-OH D3. Preferably at least Vitamin D is a further bio-active ingredient. Sometimes the further bio-active ingredients include at least Vitamin D and Selenium. In some cases, all the further bio-active ingredients are added.

Another aspect of this invention is a companion animal food comprising the combination of 25-OH D2 or 25-OH D3 or mixtures thereof, ascorbic acid, Vitamin E and a carotenoid, preferably beta carotene.

Yet another embodiment is a companion animal food comprising the combination of 25-OH D, a carotenoid, preferably beta carotene, vitamin E and ascorbic acid, which optionally further comprises at least one further bio-active ingredient selected from the group consisting of: Vitamin D, Vitamin B2, Vitamin B6, Niacin, Zinc, Copper, Manganese, Selenium and combinations thereof. Preferably the 25-OH D is 25-OH D3. Sometimes the further bio-active ingredients include at least Vitamin D and Selenium. In some cases, all the further bio-active ingredients are added.

Another aspect of this invention are premixes for a companion animal food which comprise the combination of 25-OH D, vitamin E, ascorbic acid and a carotenoid, preferably beta carotene. Preferably, the 25-OH D is 25-OH D3. The premixes and subsequent feed, when fed to an animal prone to hyperphagia or obesity improves/ameliorates adverse metabolic conditions associated with hyperphagia. In some embodiments the feed and premix also comprises at least one further bio-active ingredient selected from the group consisting of:

• • Vitamin D, Vitamin B2, Vitamin B6, Niacin, Zinc, Copper, Manganese, Selenium and combinations thereof. Sometimes the further bio-active ingredients include at least Vitamin D and Selenium. In some cases, all the further bio-active ingredients are added.

Another aspect of this invention is a method of ameliorating the amount of weight gained by a companion animal which is fed ad libitum comprising: administering the combination of 25-OH D, a carotenoid, preferably beta-carotene, Vitamin C and Vitamin E to the animal. Optionally the animal may also be fed the aforementioned combination and at least one further bio-active ingredient selected from the group consisting of:

• • Vitamin D, Vitamin B2, Vitamin B6, Niacin, Zinc, Copper, Manganese, Selenium and combinations thereof. Sometimes the further bio-active ingredients include at least Vitamin D and Selenium. In some cases, all the further bio-active ingredients are added.

When using the feed of this invention, the animals may be fed ad libitum and the ill effects normally experienced will be experienced to a lesser degree or not at all. This eliminates the problems encountered where the household has multiple pets and the owner is trying to manage weight of some of the animals.

In preferred embodiments, the combination 25-OH D, carotenoid, vitamin E and ascorbic acid are added to a basal diet which contains all the necessary ingredients for complete nutrition, i.e. the combination provides a supra-physiological amount of the components. Thus, this can be distinguished from prior use of one or several of the ingredients which are provided in order to merely meet nutritional requirements so that the animal is not vitamin or nutrient deficient.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows glucose clearance and insulin secretion of broiler breeder hens in response to ad libitum feed intake in combination with 25-OH D3+antioxidants/anti-inflammatories. Hens were injected with a single dose of glucose (0.5 g/kg BW) through wing vein after 3 weeks of feeding. Blood samples were collected through cannulation of wing vein at indicated time points after glucose infusion, n=3.

FIG. 2 shows tissue interleukin-1β contents and plasma IL-6 levels of broiler breeder hens in response to ad libitum feed intake in combination with 25-OH D3 and antioxidants/anti-inflammatories. Tissues and blood samples were collected after 10 weeks of the feeding trial. Means with different superscript letters are significantly different (P<0.05), n=3.

FIG. 3. shows tissue STAT-3 activation of broiler breeder hens in response to ad libitum feed intake in combination with 25-OH D3 and antioxidants/anti-inflammatories inclusion. Tissues and blood samples were collected after 10 weeks of the feeding trial. Means with different superscript letters are significantly different (P<0.05), n=3.

FIGS. 4-6 show pictures taken during the necropsy of broiler hens in response to ad libitum feed intake in combination with 25-OH D3 and antioxidants/anti-inflammatories inclusion. Hens were necropsied after 10 weeks of the feeding trial.

FIG. 7 shows the effect of dietary supplementation of 25-OH-D3+antioxidants/anti-inflammatories on egg production of broiler hens with restricted or ad libitum feed intake.

FIG. 8 shows the effect of dietary supplementation of 25-OH-D3+antioxidants/anti-inflammatories on the survival rate of broiler hens with restricted or ad libitum feed intake.

FIG. 9 shows the effect of dietary supplementation of 25-OH-D3+antioxidants/anti-inflammatories on body weight of broiler hens with restricted or ad libitum feed intake.

FIG. 10 is a series of photos showing the gross morphology of the heart of dead hens with dietary supplementation of 25-OH-D3+antioxidants/anti-inflammatories under restricted or ad libitum feed intake.

FIG. 11 is a series of photographs showing the gross morphology of the heart of dead hens with dietary supplementation of 25-OH-D3+antioxidants/anti-inflammatories under restricted or ad libitum feed intake

FIG. 12 is a series of electrocardiograms (EGCs)demonstrating the effect of dietary supplementation of 25-OH-D3+antioxidants/anti-inflammatories on EGC of broiler hens with restricted or ad libitum feed intake. The arrows point to irregularities in the patterns.

FIG. 13 is a series of EGSs showing the effect of dietary supplementation of 25-OH-D3+antioxidants/anti-inflammatories on arrhythmic ECG pattern of broiler hens with restricted or ad libitum feed intake.

FIG. 14 shows photos and a graph demonstrating the effect of dietary supplementation of 25-OH-D3+antioxidants/anti-inflammatories on cardiac fibrosis of broiler hens with restricted or ad libitum feed intake. (at age of 35 weeks)

FIG. 15 are graphs showing the effect of dietary supplementation of 25-OH-D3+antioxidants/anti-inflammatories on plasma IL-6 and IL-1β concentration of broiler hens with restricted or ad libitum feed intake.

FIG. 16 are photos showing the effect of dietary supplementation of 25-OH-D3+antioxidants/anti-inflammatories on cardiac cell apoptosis of broiler hens with restricted or ad libitum feed intake. (at age of 35 weeks)

As used throughout this specification and claims, the following definitions apply: “25-OH D” refers to any form of 25-hydroxyvitamin D (i.e. either 25-OH D2 or 25-OH D3, or mixes thereof). 25-OH D3 specifically refers to 25-hydroxyvitamin D3; 25-OH D2 specifically refers to 25-hydroxyvitamin D2.

“Vitamin D” means either Vitamin D2, Vitamin D3 or a combination. Vitamin D3 used alone is preferred.

“Companion animal” means dogs, cats, rats, rabbits, ornamental birds, ornamental fish and reptiles.

“Hyperphagia” is excessive eating; the animal does not voluntarily limit its feeding.

“Ameliorating weight gain” means that there is a significant lower amount of weight the poultry would gain when feeding ad libitum with ingesting the combination of 25-OH D3 and antioxidants/anti-inflammatories described herein. The animals ingesting the combination may gain more than those fed a restricted diet.

“Ascorbic Acid” and “Vitamin C” are used interchangeably throughout the specification and claims.

“Basal diet” means that the food used supplies the animal with sufficient vitamins and minerals so that the companion animal is vitamin and mineral replete.

“25-OH-D3+antioxidants/anti-inflammatories” means the combination of 25-OH D3, vitamin E, carotenoid and ascorbic acid, administered in feed as an addition to a basal diet, at a dosage range as set forth in the specification. Optionally, and preferably, additional bio-active ingredients, selected from the group consisting of: Vitamin D, Vitamin B2, Vitamin B6, Niacin, Pantothenic Acid, Folic Acid, Biotin, Zinc, Copper, Manganese, Selenium and combinations thereof are added to the 25-OHD3, vitamin E, canthaxanthin and ascorbic acid combination.

“Sudden Death” means that the individual bird died without showing previous signs or illness or trauma. Upon necropsy, the bird usually had unrecognized cardiovascular problems.

1. Metabolic Problems

Metabolic problems associated with hyperphagia and which can be lessened, reduced or eliminated through use of the feed/premix of this invention include:

• • a) decreased weight gain and/or no further weight gain;
  • b) clearance of non-esterified fatty acids
  • c) amelioration of plasma dyslipidemia (triglycerides, sphingomyelin, and ceramide)
  • d) amelioration of triglyceride and ceraminde accumulation in the liver, leg, breast muscle, and heart
  • e) suppression the tissue pro-inflammatory IL-1β production and plasma IL-6 concentration
  • f) cardiac protection and enhanced cardiac function through the up-regulation of the phosphorylation of STAT-3 (signal transducer and activator of transcription 3) in the heart.
  • g) suppression of the infiltration of immune cells into the heart
  • h) decreasing the incidence of ascites.

These above-mentioned observed improved conditions result in a lowering of mortality rate, improved insulin signaling, reduced lipotoxic development and systemic inflammation, and activation of cardio-protective mechanisms against fuel-overload induced cardiac pathogenesis.

Importantly it has been found in accordance with this invention that the combination of 25-OH D3, a carotenoid, Vitamin C and Vitamin E can lessen the amount of weight gained when an animal is allowed to eat ad libitum. Thus, this combination can be added to the companion animal's normal food, and weight management can be easier for the owner.

Along with the lesser weight gain, other benefits associated with decreasing obesity have also been observed.

2. Cardiovascular Problems

Birds in a flock can experience a sudden death, i.e. the cause of death is not readily apparent. We investigated this further, as is detailed in EXAMPLE 4. We investigated this phenomenon in flocks fed ad libitum and a restricted diet, with or without the combination of 25-OH D3 and antioxidants/anti-inflammatories. Some of our results are set forth below.

It has been found, in accordance with this invention, that the combination of 25-OH D3, and the antioxidants/anti-inflammatories that in In birds fed ad libitum, sudden death birds (i.e, birds which have undergone sudden death) which had been fed with 25-OH-D3+antioxidants/anti-inflammatories had higher body weight, but lower relative liver, abdominal fat, and heart weight.

In the birds with sudden death, ad libitum feeding caused cardiac adaptive hypertrophy; and some of the hypertrophic growth may develop pathologically into ventricle dilation. As a result, the heart requires a higher contractility to maintain pumping function to meet the need of blood supply for oxygen delivery to the peripheral tissues. This condition may have caused heart failure.

Importantly, we found that 25-OH-D3+antioxidants/anti-inflammatories decreased cardiac pathogenic progression and thereby the incidence of heart failure in birds fed ad libitum. Thus another aspect of this invention is the use of 25-OH-D3+antioxidants/inflammatories to reduce the amount of cardiac problems leading to sudden death.

In both restricted and ad libitum fed birds, birds with 25-OH-D3+antioxidants/anti-inflammatories exhibited less adaptive hypertrophic growth, supporting the hypothesis that most excessive fuels may be partitioned to the muscle, and thereby, hypertrophic growth of the heart for increased pumping function cannot meet the need of oxygen supply for higher growth rate (muscle) and thus may provoke cardiac arrhythmia and failure.

25-OH-D3+antioxidants/anti-inflammatories were seen to have the following effects:

• • decreased the incidence of cardiac morbidities (dilation, pericardial effusion, rupture) observed in necropsies of in the dead birds.
  • Decreased the amount of irregular incidence of ECG patterns
  • Decreased arrhythmia of broiler hens fed ad libitum
  • ameliorated sudden death induced by cardiac morbidities
  • ameliorated cardiac fibrosis in hens fed ad libitum.
  • ameliorated chronic systemic inflammation in hens fed ad libitum.
  • ameliorated cardiac cell apoptosis in hens with restricted or ad libitum feed intake.

As these symptoms are expected to involve the same pathways in the companion animals as in the observed birds, it can be concluded that the same pathologies are occurring in the obese companion animals as well. Thus, the 250H D3+antioxidants can protect the cardiovascular system in companion animals as well.

3. Doses

In one aspect of this invention the combination of 25-OH D3 and the antioxidants/anti-inflammatories are given to animals which are vitamin replete rather than vitamin deficient. The vitamin replete status is preferably due to the use of a basal feed which supplies at least the minimum amount of vitamins and minerals for the cat or dog. The combination of this invention is thus preferably used in addition to the basal diet. The lower doses given below are suitable for companion animals who have reached an ideal weight, as it will assist them from gaining extra unwanted weight. The medium dosages are suitable for animals which are overweight or are tending to obesity. The high dosages are suitable for those animals which have become obese. All these dosages may be given in combination with a reduced calorie companion animal food.

25-OH D3:

The amount of 25-OH D3 can range from 15-200 μg/kg food. Preferably, the amount of 25-OH D3 is from 35-150 μg/kg food. For food with a low dose of the combination of the invention, 35 μg per kg 25-OH D3/food is preferred; for food with a medium dose of the combination, 69 μg per kg food is preferred; and for food with a high dose of 150 μg feed is preferred.

Vitamin E:

The amount of Vitamin E can range from 40-400 mg/kg food. Preferably the amount is 80-300 mg/kg food. For food with a low dose of the combination of the invention, 80 mg/kg Vitamin E is preferred. For food with a medium dose, 150 mg Vitamin E is preferred; for food with a high dose, 300 mg/kg Vitamin E is preferred.

Beta Carotene:

The amount of canthaxanthin can range from 1-15 mg/kg feed. Preferably the amount is 3-12 mg/kg feed. For feed with a low dose of the combination of the invention, 3 mg/kg canthaxanthin is preferred. For feed with a medium dose, 6 mg canthaxanthin is preferred; for feed with a high dose, 12 mg/kg canthaxanthin is preferred.

Ascorbic Acid:

The amount of ascorbic acid can range from 40-400 mg/kg feed. Preferably the amount is 100-300 mg feed. For feed with a low dose of the combination of the invention, 100 mg/kg ascorbic acid is preferred. For feed with a medium dose, 150 mg ascorbic acid is preferred; for feed with a high dose, 300 mg/kg ascorbic acid is preferred.

Thus specific preferred feeds of this invention comprise the following dosages (all amounts are per kg/feed):

Preferred Feed #1:

• • 25-OH D3: 15-200 μg,
  • Vitamin E: 40-400 mg,
  • Beta Carotene: 1-15 mg; and
  • Ascorbic acid: 40-400 mg.

Preferred Feed #2:

• • 25-OH D3: 35-150 μg,
  • Vitamin E: 80-300 mg,
  • Beta carotene: 3-12 mg, and
  • Ascorbic acid: 80-300 mg

Preferred Feed #3: (Low dose feed) This feed is preferred for animals who are at their ideal weight, and is used to help maintain it.

• • 25-OH D3: 35 μg
  • Vitamin E: 80 mg
  • Beta Carotene: 3 mg
  • Ascorbic acid: 80 mg.

Preferred Feed #4 (medium dose feed) This feed is preferred for moderately to severely obese companion animals:

• • 25-OH D3: 69 μg
  • Vitamin E: 150 mg
  • Beta carotene: 6 mg
  • Ascorbic acid: 150 mg.

Preferred Feed #5 (high dose feed) This feed is preferred for severely obese companion animals:

• • 25-OH D3: 150 μg
  • Vitamin E: 300 mg
  • Beta carotene: 12 mg
  • Ascorbic acid: 300 mg.

The ratios of the above antioxidants/anti-inflammatories relative to each other may range as follows:

• • Vitamin E to Vitamin C may range from 1-10:10-1; preferably 1-5:5-1; and more preferably 1.5:1 to 1:1.5, and most preferably 1:1.
  • Vitamin E or Vitamin C to Beta Carotene may range from 40:1 to 1:1; preferably from 20:1 to 1:1; and more preferably from 10:1 to 1:1

Preferred ratios include the following

Vit E	Vit C	Beta Carotene
40	40	1
20	20	1
10	10	1

Optional Additional Ingredients:

To each of the feeds listed above, at least one of the additional ingredients may be added. Preferably at least one, and more preferably more than one of the following ingredients are added. In other embodiments, all the following ingredients are added:

• • Vitamin D3—generally this is present in poultry diets at approximately 2500 IU per kg feed. In accordance with this invention, if desired, the amount of Vitamin D is increased to at least 3000 IU per kg.
  • Vitamin B2: this can be added at 3-25 mg per kg; preferably from 6-20 mg/kg. For low dose feed, 6 mg/kg is preferred. For medium dose feed, 14 mg/kg is preferred; and for high dose feed 20 mg/kg is preferred.
  • Niacin: this can be added at 25-300 mg per kg feed. Preferably it ranges from 60-200 mg/kg. For low dose feed, 60 mg/kg is preferred. For medium dose feed, 120 mg/kg is preferred; and for high dose feed 200 mg/kg is preferred.
  • Pantothenic acid: this can be added at 10-120 mg per kg feed. Preferably it ranges from 15-80 mg/kg. For low dose feed, 15 mg/kg is preferred. For medium dose feed, 30 mg/kg is preferred; and for high dose feed 80 mg/kg is preferred.
  • Folic acid: this can be added at 1-8 mg per kg feed. Preferably it ranges from 2-6 mg/kg. For low dose feed, 2 mg/kg is preferred. For medium dose feed, 4 mg/kg is preferred; and for high dose feed 6 mg/kg is preferred.
  • Biotin: this can be added at 0.05-1.0 mg/kg feed. Preferably it ranges from 0.2-0.8 mg/kg. For low dose feed, 0.2 mg/kg is preferred. For medium dose feed, 0.4 mg/kg is preferred; and for high dose feed 0.8 mg/kg is preferred.
  • Zinc: this can be added at 50-300 mg/kg feed. Preferably it ranges from 70-250 mg/kg. For low dose feed, 70 mg/kg is preferred. For medium dose feed, 125 mg/kg is preferred; and for high dose feed 250 mg/kg is preferred.
  • Copper: this can be added at 5-50 mg/kg feed. Preferably it ranges from 10-30 mg/kg. For low dose feed, 10 mg/kg is preferred. For medium dose feed, 20 mg/kg is preferred; and for high dose feed 30 mg/kg is preferred.
  • Manganese: this can be added at 50-300 mg/feed. Preferably it ranges from 80-270 mg/kg. For low dose feed, 80 mg/kg is preferred. For medium dose feed, 150 mg/kg is preferred; and for high dose feed 270 mg/kg is preferred.
  • Selenium: this can be added at 0.05-0.6 mg/kg feed. Preferably it ranges from 0.1-0.4 mg/kg. For low dose feed, 0.1 mg/kg is preferred. For medium dose feed, 0.3 mg/kg is preferred; and for high dose feed 0.5 mg/kg is preferred.

Premixes can be made to give the above-mentioned doses and preferred doses. One premix which forms part of this invention is formulated so that 1 gram of premix is added to one kilogram food, and that the resulting food contains the dosages described in any of the given dosages above. The amounts of the individual ingredients can, of course be varied so that one kilogram of premix is added to one metric ton of feed, and that the resulting food contains the dosages described in any of the given dosages above. There are specific illustrations of this in the Examples, below.

Further, it is envisioned that the combinations specified herein may be added to any commercially available dog or cat food, and thus the total amount of 25-OHD3 and antioxidants/anti-inflammatories present may be equal to the amount originally present in the food plus the addition as specified herein. It is also envisioned that the 25-OHD3 and antioxidants/anti-inflammatories as specified herein are the sole additions to a basal diet which contains at least the minimum required nutrients for cat or dog nutrition.

The following non-limiting Examples are presented to better illustrate the invention

EXAMPLES

Example 1

A total of thirty 45-week-old broiler breeder hens (ROSS 308) were obtained from a commercial flock for the study. A basal broiler breeder laying diet was formulated as shown in Table 1. The calculated nutrient composition is shown in Table 2.

TABLE 1
Ingredient composition of the basal broiler breeder laying diets.
Composition          %, w/w
Corn                 66.9
Soybean meal         22.2
Oil fat              1.67
Ca Carbonate (ground 6.36
oyster shell)
Dicalcium phosphate  1.8
Choline-Cl (70%)     0.1
Mineral Premix1      0.1
Copper sulfate       0.05
Vitamin Premix2      0.1
1Mineral premix provided (per kg of diet for treatment groups 1, 2 and 3): Cu 18 mg; I 1.1 mg; Fe 80 mg; Mn 150 mg; Zn 125 mg; and Se 0.25 mg.
2Refer to Table 2 for further detail.

TABLE 2
Vitamin premix composition (provided per kg of diet)
		Treatment 3:
	Treatments 1 and 2:	3 = ad libitum feeding + 25-
	1 = restricted feeding	OH-D3 + antioxidants/anti-
Vitamin	2 = ad libitum feeding	inflammatories
A (IU)	10000	12000
D3 (IU)	2500	3000
E (mg)	100	150
K3 (mg)	3	5
B1 (mg)	3	5
B2 (mg)	8	14
B6 (mg)	6	8
B12 (mg)	0.03	0.03
Niacin (mg)	60	120
Pantothenic acid	18	30
(mg)
Folic acid (mg)	1	4
Biotin (mg)	0.2	0.4
C (ascorbic acid)	0	150
(mg)
25-OH-D3 (mcg)	0	69
Canthaxanthin	0	6
(mg)

TABLE 3
Calculated nutrient composition (%) of the
basal broiler breeder laying diets.
Composition      % w/w
Crude protein    16
Crude fat        4.2
Calcium          3.1
Sodium           0.16
Total Phosphorus 0.64
Total ME         2910 kcal/kg

Diet was supplemented with or without 25-OH D3 at 69 mcg/kg diet in combination with antioxidants/anti-inflammatories (vitamin E, ascorbic acid, canthaxanthin) and enriched levels of selected vitamins. Hens were randomly allocated to 3 treatment groups according to feeding regimen (restricted and ad libitum) as follows:

Basal diet—restricted feeding (140 g/day)
Basal diet—ad libitum feeding
Basal diet—ad libitum feeding+25-OH-D3 at 69 mcg/kg diet+antioxidants/anti-inflammatories

They were individually-housed in wire cages placed in a controlled room with 14 h:10 h light:dark period and at a temperature of 25±3° C. Water was available ad libitum. The experimental period was lasted for 10 weeks. Three weeks after the feeding trial, some birds were used for relevant plasma parameter analyses. At the end of experiment, hens were euthanized and sacrificed for tissue sample collection for further studies: Necropsy of tissue morphology

• • Determination of lipid and sphingolipid profile—serum and tissues
  • Determination of tissue pro-inflammatory cytokines
  • Determination of insulin resistance
  • Collection of tibia for bone strength analysis
  • Harvesting heart (cardiomyopathy) and skeletal muscle (breast and thigh) for myopathy analyses.

Example 2

Results and Discussion

25-OH D3 and Antioxidants/Anti-Inflammatories Suppressed Adiposity and Abdominal Fat in Overfed Broiler Hens

Breeder hens are capable of storing large quantities of excess energy (in the form of triglycerides) in the liver, adipose tissue and yolk of developing oocytes. Lipogenesis (i.e., the conversion of glucose to triglycerides) takes place primarily in the liver of birds and involves a series of linked, enzyme catalyzed reactions including glycolysis, the citric acid cycle and fatty acid synthesis. Hepatic lipogenesis is subject to both nutritional and hormonal control and is highly responsive to changes in the diet. Adipose tissue serves primarily as a storage site for lipid with little lipogenic activity. Differential lipogenic capacity of liver vs. adipose tissue in birds is a function of the expression of a key transcription factor, sterol regulatory element binding protein-1 (SREBP-1). The gene for SREBP-1 is highly expressed in the liver, but to a much lesser extent in adipose tissue. Moreover, the expression of a number of lipogenic enzyme genes such as fatty acid synthase, malic enzyme, acetyl CoA carboxylase, ATP citrate lyase and steroyl CoA desaturase 1 is directly influenced by SREBP-1.

Breeder hens fed ad libitum accreted more abdominal fat than those restricted fed. Dietary supplementation of 25-OHD3 and antioxidants/anti-inflammatories ameliorated the deleterious effect of ad libitum feeding on body and tissue weight, particularly on relative adipose tissue weight (adiposity) (Table 4). Tibial strength of ad libitum-fed hens was enhanced by 25-OH D3 and antioxidants/anti-inflammatories.

TABLE 4
25-OH D3 and antioxidants/anti-inflammatories on body
weight, liver weight, abdominal fat weight and tibial
strength of ad libitum-fed broiler breeder hens
			Ad libitum
			feeding +
			25-OH-D3 +
		Ad	Antioxidants +
	Restricted	libitum	anti-inflam-	Pooled
	feeding	feeding	matories	SEM1
Body weight (kg)	3.67c	4.60a	4.04b	0.26
Liver weight (g)	41.4b	48.5a	49.6a	5.6
Liver/body	1.14	1.06	1.23	0.18
weight (%)
Abdominal fat	40.9c	185.9a	110.8b	28.9
weight (g)
Abdominal fat	1.10c	4.01a	2.74b	0.70
weight/body
weight (%)
Tibial strength	32.3b	38.3ab	44.3a	3.57
(kg/cm2)
a-cWithin a row, means without a common superscript differ (P < 0.05).
1Pooled standard error of the mean.

25-OH D3 and Antioxidants/Anti-Inflammatories Lowered Mortality and Improved Egg Production, Ovarian Morphology and Plasma 17B Estradiol Level

Secretion of estradiol is the hallmark of successful ovulatory follicles. In addition to its role in triggering the preovulatory surge of gonadotropins, estradiol is an important intra-ovarian growth, differentiation, and survival factor. Inclusion of 25-OH D3 and antioxidants/anti-inflammatories reduced mortality and incidence of ovarian degeneration and ovarian-tumor-like morphology, increased egg production and sustained plasma estradiol levels in birds under ad libitum feed intake.

25-OH D3 and Antioxidants/Anti-Inflammatories Ameliorated Impaired Glucose Clearance and Insulin Sensitivity

Dietary inclusion of 25-OH D3 and antioxidants/anti-inflammatories improves insulin resistance as evidenced by ameliorating fasting plasma glucose and non-esterifed fatty acid level in overfed hens for 10 weeks (Table 6). In glucose clearance test, lean hens showed a very sharp clearance rate between 30-60 min after glucose infusion, and conversely obese hens had a very sluggish clearance rate between 30-90 min (FIG. 1). In insulin secretion, obese hens showed a higher plasma insulin level under fasting status and after glucose infusion when compared to lean hens (FIG. 1). Both glucose clearance and glucose-induced insulin secretion were corrected by 25-OH D3 and antioxidants/anti-inflammatories inclusion in overfed hens for 3 weeks (FIG. 1).

TABLE 5
25-OH D3 and antioxidants/anti-inflammatories on
plasma glucose, non-esterified fatty acid (NEFA)
and insulin of ad libitum-fed broiler breeder hens
			Ad libitum
			feeding +
			25-OH-D3 ++
		Ad	antioxidants/
	Restricted	libitum	anti-inflam-	Pooled
	feeding	feeding	matories	SEM1
After 3 weeks
of feeding
Plasma glucose	181.5	202.5	188.5	11.9
(mg/dL)
Plasma NEFA	0.21b	0.35a	0.25b	0.05
(μmole/mL)
After 10 weeks
of feeding
Plasma glucose	180.6b	212.6a	195.6b	12.7
(mg/dL)
Plasma NEFA	0.35b	0.44a	0.33b	0.05
(μmole/mL)
Fasting plasma	1.38	1.15	1.59	0.21
insulin
Glucose-induced	2.66a	1.97b	2.46a	0.36
insulin
a-bWithin a row, means without a common superscript differ (P < 0.05).
1Pooled standard error of the mean.

25-OH D3 and Antioxidants/Anti-Inflammatories Ameliorated Dyslipidemia

Ad libitum-fed hens elevated plasma triglyceride, ceramide and sphingomyelin levels. However, supplementation of combined 25-OH D3 and antioxidants/anti-inflammatories lowered the level of these lipid metabolites in the plasma of ad libitum-fed hens (Table 6).

TABLE 6
25-OH D3 and antioxidants/anti-inflammatories
on plasma triacyglycerol, ceramide and sphingomyelin
of ad libitum-fed broiler breeder hens
			Ad libitum
			feeding +
			25-OH-D3 ++
		Ad	antioxidants/
	Restricted	libitum	anti-inflam-	Pooled
	feeding	feeding	matories	SEM1
After 3 weeks
of feeding
Plasma	15.6	17.4	14.5	2.9
triacyglycerol
(mg/mL)
Plasma ceramide	11.5b	18.2a	13.5b	2.83
(nmole/mL)
Plasma	0.14b	0.28a	0.16b	0.05
sphingomyelin
(μmole/mL)
After 10 weeks
of feeding
Plasma	12.75b	15.2a	11.5b	2.2
triacyglycerol
(mg/mL)
Plasma ceramide	8.1b	12.3a	8.8b	1.65
(nmole/mL)
Plasma	0.15b	0.22a	0.12b	0.05
sphingomyelin
(μmole/mL)
a-bWithin a row, means without a common superscript differ (P < 0.05).
1Pooled standard error of the mean.

25-OH D3 and Antioxidants/Anti-Inflammatories Reduced Accumulation of Tissue Triglyceride and Ceramide Content

Accumulation of triglyceride and ceramide in the liver, heart and leg muscles was lower in hens fed supplemental 25-OH D3 and antioxidants/anti-inflammatories than in those fed ad libitum (Table 7).

TABLE 7
25-OH D3 and antioxidants/anti-inflammatories on tissue triacyglycerol
and ceramide content of ad libitum-fed broiler breeder hens
			Ad libitum
			feeding +
			25-OH-D3 +
		Ad	Antioxidants +
	Restricted	libitum	anti-inflam-	Pooled
	feeding	feeding	matories	SEM1
Triacyglycerol
(mg/g tissue)
Liver	69.0b	94.8a	79.8a	10.6
Heart	33.1b	55.6a	45.6a	6.8
Breast muscle	15.6	18.0	17.4	0.05
Leg muscle	30.2c	52.3a	41.2b	6.6
Ceramide
(mg/g tissue)
Liver	174.5b	287.9a	235.0a	52.7
Heart	17.5c	30.2a	23.5b	2.4
Breast muscle	2.25	2.91	2.52	0.54
Leg muscle	4.12b	7.12a	6.01a	0.85
a-bWithin a row, means without a common superscript differ (P < 0.05).
1Pooled standard error of the mean.

25-OH D3 and Antioxidants/Anti-Inflammatories Depressed Tissue Proinflammatory IL-1β Production and Plasma IL-6 Concentrations in Overfed Broiler Hens

Obesity-associated inflammation was ameliorated by dietary 25-OH D3 and antioxidants/anti-inflammatories supplementation as evidenced by suppressed circulating IL-6 levels and IL-1β production in adipose tissue, liver, leg and breast muscle, and heart (FIG. 2).

25-OH D3 and Antioxidants/Anti-Inflammatories Ameliorated Lipotoxicity in Broiler Breeder Hens Fed Ad Libitum

A central complication of obesity is the development of insulin resistance, which is when insulin is incapable of eliciting postprandial nutrient storage in its primary target tissues, skeletal muscle and liver. Without wishing to be bound by theory, it appears that two probable mechanisms may explain how increased adipose stores affect overall insulin sensitivity throughout the body, contributing to the down regulation of insulin signaling in peripheral tissues. Firstly, the delivery of nutrients to cells or tissues is in excess of their storage capacities and thus this leads to the generation of metabolites that inhibit insulin action. Of particular importance, lipid derivatives, such as triacylglycerol and ceramide, have been shown to inhibit specific insulin signaling intermediates, thus blocking postprandial glucose uptake and/or glycogen synthesis. In the case of broiler breeder females being fed ad libitum, the persistent accumulation of these metabolites in peripheral tissues likely contributes to a sustained state of insulin resistance throughout the hen and of lipotoxic development. Secondly, increased adiposity induces a chronic inflammatory state characterized by elevated circulating levels of proinflammatory cytokines produced from adipocytes or from macrophages infiltrating the fat pad. These inflammatory mediators have been shown to antagonize insulin signaling directly, and also to induce catabolic processes, thus further increasing the delivery of nutrient metabolites to insulin-responsive organs.

Overall, excess supply of glucose leading to the formation of excess saturated fatty acids and therefore accumulation of lipids in non-adipose tissues elevates the cellular levels of—active lipids (sphingolipids) that inhibit the signaling pathways implicated in metabolic regulation together with activated inflammatory responses and lipotoxic development. In particular, ceramide is a putative intermediate linking both excess nutrients (i.e., saturated fatty acids) and inflammatory cytokines to the induction of insulin resistance. Moreover, ceramide is toxic in a variety of different cell types and is capable of damaging the heart, pancreas and vasculature. Moreover, 25-hydroxy D3 and antioxidants/anti-inflammatories were effective in ameliorating the deleterious effect of metabolic and endocrine dysregulations and pro-inflammatory responses resulting from increased adiposity occurring in broiler breeder hens fed to satiation.

25-OH D3 and Antioxidants/Anti-Inflammatories Ameliorate Cardiac Morbidities, Ascites, and Inflammation in Overfed Broiler Hens

The heart may become dysfunctional due to excess lipid accumulation. That ad libitum feeding promoted triglyceride accumulation in the heart suggested that increased cardiac fatty acid availability is adaptively esterified into triglyceride. In addition, ceramide content of the heart was also increased as a result of ad libitum feeding. Ceramide is a cardiotoxin in lipotoxic cardiomyopathy, which elicited inflammatory responses as evidenced by more cardiac infiltration of immune cells. (Table 9).

TABLE 8
25-OH D3 and antioxidants/anti-inflammatories on cardiac
responses of ad libitum-fed broiler breeder hens
			Ad libitum
			feeding +
			25-OH-D3 +
		Ad	antioxidants +
	Restricted	libitum	anti-inflam-	Pooled
	feeding	feeding	matories	SEM1
Heart weight (g)	14.5b	19.2a	17.3a	1.8
Heart/body weight	0.40	0.47	0.43	0.17
(%)
Heart septum (HS)	2.73	3.01	2.83	0.57
weight (g)
HS weight/heart	18.7	14.6	15.7	3.9
weight (%)
Right atrium (RA)	1.20b	1.97a	1.72ab	0.38
wall weight (g)
RA wall weight/	8.2	9.6	9.6	1.9
heart weight (%)
Right ventricle	0.95b	1.51a	1.73a	0.27
(RV) wall weight
(g)
RV wall weight/	6.3b	7.5ab	9.4a	1.5
heart weight (%)
Left atrium (LA)	1.17b	2.26a	2.02a	0.43
wall weight (g)
LA wall weight/	12.2	11.1	10.9	3.1
heart weight (%)
Left ventricle (LV)	3.78b	4.45a	4.65a	0.34
wall weight (g)
LV wall weight/	25.5a	21.7b	25.8ab	2.4
heart weight (%)
Incidence of	1/7	5/10	3/10
transudate within
pericardium
(heart/total)
Incidence of heart	1/7	6/10	3/10
ventricle dilation
(heart/total)
Incidence of	0/7	3/10	1/10
ascites (hen/total)
Cardiac immune	97.9a	127.7a	57.7b	32.4
cell count
a-bWithin a row, means without a common superscript differ (P < 0.05).
1Pooled standard error of the mean.

Cardiac hypertrophy represents clinically an adaptive response to increased workload on the heart. However, cardiac responses to neural and hormonal factors can also incite hypertrophic changes independent of increases in afterload or vascular resistance. Fuel overloading-induced cardiac compensatory growth occurred in broiler breeder hens (Table 8). Cardiac hypertrophy may become maladaptive and eventually develop into pathological conditions, leading to heart failure. These results supported the fact that lipotoxic development and hypertrophic growth in the heart tend to elicit inflammatory responses.

The cardioprotective role of phosphorylated STAT-3 (signal transducer and activator of transcription 3) is becoming increasingly clear in recent years. Interestingly, combined 25-OH D3 and antioxidants/anti-inflammatories induced greater activation of STAT-3 (i.e., phosphorylation of STAT-3) in the heart than restricted-fed breeder hens (FIG. 3), with the lowest activation being observed in ad libitum-fed broiler breeder hens. The incidence of transudate fluid within pericardium, heart ventricle dilation and ascites was alleviated in ad-libitum-fed breeder hens when supplemented with combined 25-hydroxy D3 and antioxidants/anti-inflammatories.

Example 3

Premixes

TABLE 9, below, presents some of the final dose ranges of the composition of this invention to be added to the feed.

		Ranges	Low	Medium	High
Vitamin	Unit	per kg feed	per kg feed	per kg feed	per kg feed
25-OH-D3	mcg	15-200	35	69	150
Vit E	mg	40-400	80	150	300
Canthaxanthin	mg	1-15	3	6	12
VIt C	mg	40-400	100	150	300

TABLE 10. below presents the quantity of premix dosed per 1 kg feed:

		0.3 g	0.6 g	1 g	1.8 g	2 g
Vitamin	Unit	premix	premix	premix	premix	premix
25-OH-D3	mg	0.0207	0.0414	0.069	0.1242	0.138
Vit E	mg	45	90	150	270	300
Canthaxanthin	mg	1.8	3.6	6	10.8	12
Vit C	mg	45	90	150	270	300

TABLE 11
1 gram premix can be made with the following ingredients:
	Vitamin	Unit	1 g premix
	25-OH-D3	mg	0.069
	Vit E	mg	150
	Canthaxanthin	mg	6
	Vit C	mg	150
	Carrier and others	mg	to 1000 mg or 1 g

Conclusions

Supplemental 25-OH D3 and antioxidants/anti-inflammatories ameliorated deleterious effects associated with overfeeding of broiler breeder females by:

lowering mortality rate and improving ovary function and therefore reproductive performance of overfed broiler breeder hens.
improving endocrine (insulin) signaling
reducing lipotoxic development and systemic inflammation
activating cardio-protective mechanisms against fuel-overload induced cardiac pathogenesis

Example 4

Foods

A dry pet food formula

Ingredients               %
Chicken By-Product Meal   35.40
Corn                      30.00
Rice, Brewers             10.00
Chicken Fat               6.00
Wheat                     5.00
Beet Pulp                 4.00
Corn Gluten Meal, 60%     3.00
Fish Meal                 2.00
Digest (palatants)        1.00
Fish Oil                  1.00
Dicalcium Phosphate       1.00
Potassium Chloride        0.40
Salt                      0.30
Choline Chloride, 60% dry 0.30
Calcium Carbonate         0.20
Vitamin Premix            0.20
Mineral Premix            0.20

To this formula, 25-OHD3, beta carotene, Vitamin C and Vitamin E are added.

A wet pet food formula

Ingredients        %
Water              55
Chicken            25
Rice               5
Chicken liver      2
Chicken heart      2
Natural flavor     2
Flaxseed           2
Peas (dried)       1.3
Carrots (dried)    1
Apple (dried)      1
Guar Gum           1
Carrageenan        1
Soybean Oil        1
Potassium Chloride 0.5
Vitamin premix     0.1
Mineral Premix     0.1

To this formula, 25-OHD3, beta carotene, Vit C and Vitamin E are added.

Example 4

Cardio-Myopathy Trial

Example 5

Cardio-Myopathy Trial

Materials and Methods

A total of thirty 45-week-old broiler breeder hens (ROSS 308) were obtained from a commercial flock for the study. A basal broiler breeder laying diet was formulated as shown in Table 12. The calculated nutrient composition is shown in Table 13.

TABLE 12
Ingredient composition of the basal broiler breeder laying diets.
Composition         %, w/w
Corn                66.9
Soybean meal        22.2
Oil fat             1.67
Calcium carbonate   6.36
(ground oyster shell)
Dicalcium phosphate 1.8
Salt                0.08
Choline-Cl (70%)    0.1
Mineral premix1     0.1
Cooper sulfate      0.05
Vitamin premix2     0.1
1Mineral premix provided (per kg of diet for treatment groups 1, 2 and 3): Cu, 18 mg; I, 1.1 mg; Fe, 80 mg; Mn, 150 mg; Zn, 125 mg; and Se, 0.25 mg.
2Refer to Table 13, below for further detail.

TABLE 13
Vitamin premix composition (provided per kg of diet)
		Treatments 2 and 4
		2 = restricted feeding +
		25-OH-D3 +
	Treatments 1	antioxidants/
	and 3	anti-inflammatories
	1 = restricted	4 = ad libitum feeding +
	feeding	25-OH-D3 +
	3 = ad libitum	antioxidants/
Vitamin	feeding	anti-inflammatories
A (IU)	10000	12000
D3 (IU)	2500	3000
E (mg)	100	150
K3 (mg)	3	5
B1 (mg)	3	5
B2 (mg)	8	14
B6 (mg)	6	8
B12 (mg)	0.03	0.03
Niacin (mg)	60	120
Pantothenic acid (mg)	18	30
Folic acid (mg)	1	4
Biotin (mg)	0.2	0.4
C (mg)	0	150
25-OH-D3 (mcg)	0	69
Canthaxanthin (mg)	0	6

TABLE 14
Calculated nutrient composition (%) of
the basal broiler breeder laying diets.
Composition      %, w/w
Crude protein    16
Crude fat        4.2
Calcium          3.1
Potassium        0.44
Sodium           0.16
Total phosphorus 0.64
Total ME         2910 kcal/kg

Diet was supplemented with or without Hy⋅D® at 69 mcg 25-OH-D3/kg diet in combination with antioxidants (ascorbic acid, canthaxanthin) and enriched levels of selected vitamins. Hens were randomly allocated to treatment groups according to feeding regimen (restricted and ad libitum) as follows:

• • 1. Basal diet—restricted feeding (140 g/day)
  • 2. Basal diet—restricted feeding+Hy⋅D® (25-OH-D3 at 69 mcg/kg diet)+antioxidants/anti-inflammatories
  • 3. Basal diet—ad libitum feeding
  • 4. Basal diet—ad libitum feeding+Hy⋅D® (25-OH-D3 at 69 mcg/kg diet)+antioxidants/anti-inflammatories

Results:

TABLE 15
Effect of dietary supplementation of 25-OH-D3+antioxidants/anti-
inflammatories on egg production of broiler hens with restricted or ad libitum feed
intake.
		Restriction + 25-		Ad libitum +
		OH-D3+		25-OH-D3+
		antioxidant/		antioxidant/
		anti-		anti-
	Restriction	inflammatories	Ad libitum	inflammatories
		Dead		Dead		Dead		Dead
	Whole	bird	Whole	bird	Whole	bird	Whole	bird
	flock	flock	flock	flock	flock	flock	flock	flock
	(n = 68)	(n = 19)	(n = 70)	(n = 11)	(n = 80)	(n = 58)	(n = 79)	(n = 47)
PROD	51.4 ± 2.5a	47.2 ± 1.9a	51.3 ± 2.7a	40.2 ± 1.8b	32.0 ± 2.9b	43.4 ± 1.7ab	34.8 ± 2.7b	45.8 ± 2.1ab
YIELD	129.0 ± 5.5b	47.9 ± 2.5b	143.2 ± 5.7a	33.3 ± 1.9b	41.1 ± 5.3d	41.5 ± 2.1b	54.7 ± 5.9c	41.1 ± 2.0b
PROD = Egg production rate (eggs/day/hen, %);
YIELD = Egg yield (eggs/hen)
Results were expressed with mean ± SEM.
Means with different superscript letters are significantly different within the same flock (P < 0.05).

FIG. 7 shows the effect of dietary supplementation of 25-OH-D3+antioxidants/anti-inflammatories on egg production of broiler hens with restricted or ad libitum feed intake. From TABLE 15 and FIG. 7, it can be concluded that:

• 1. 25-OH-D3+antioxidants/anti-inflammatories improved total egg yield by promoting survival.
• 2. In dead birds, 25-OH-D3+antioxidants/anti-inflammatories had no effect on egg yield and egg production rate in bird fed ad libitum, but decreased egg yield and egg production rate in restricted birds. In the whole flock, however, 25-OH-D3+antioxidants/anti-inflammatories increased egg yield but not egg production rate.
• 3. These results suggested that 25-OH-D3+antioxidants/anti-inflammatories accelerated the progression into death in restricted birds that are susceptible to sudden death, and thus acting as a flock culler to exclude the sudden death-susceptible birds for longer survival and thereby reduced the flock maintenance cost.

TABLE 16
Effect of dietary supplementation of 25-OH-D3 + antioxidants/anti-
inflammatories on mortality of broiler hens with restricted or ad
libitum feed intake and body characteristics of the dead hens
		Restriction +		Ad libitum ++
		25-OH-D3 +		25-OH-D3 +
		antioxidant/		antioxidant/
		anti-inflam-	Ad	anti-inflam-
	Restriction	matories	libitum	matories
	(n = 19)	(n = 11)	(n = 58)	(n = 47)
Mortality	19/68	11/70	58/80	47/79
(dead birds	(26.47%)	(15.71%)	(72.5%)	(59.49%)
of the total)
Body weight	3.91 ± 0.090 c	4.01 ± 0.084bc	4.07 ± 0.083 b	4.37 ± 0.082 a
of the dead
birds (kg)
Liver weight	89.10 ± 6.79 b	99.27 ± 7.92 ab	105.52 ± 5.48a	106.70 ± 4.68 a
of the dead
birds (g)
Relative	2.28 ± 0.0016 d	2.47 ± 0.0020 b	2.59 ± 0.0011a	2.44 ± 0.0009 c
liver weight
of the dead
birds (g/100
g BW)
Abdominal	60.45 ± 4.56 b	68.54 ± 6.05 b	148.64 ± 8.45c	133.54 ± 9.44a
fat weight
of the dead
birds (g)
Relative	1.55 ± 0.0013 d	1.71 ± 0.0016 c	3.65 ± 0.0014 a	3.06 ± 0.0009 b
abdominal
fat weight
of the dead
birds (g/100
g BW)
Heart	18.12 ± 0.81 c	19.93 ± 0.87b	23.66 ± 0.96a	22.29 ± 0.86a
weight of
the dead
birds (g)
Relative	0.46 ± 0.00023 c	0.50 ± 0.00021 b	0.58 ± 0.00024a	0.51 ± 0.00018b
heart
weight of
the dead
birds (g/100
g BW)
Results were expressed with mean ± SEM.
Means with different superscript letters are significantly different (P < 0.05)

FIG. 8 shows the effect of dietary supplementation of 25-OH-D3+antioxidants/anti-inflammatories on the survival rate of broiler hens with restricted or ad libitum feed intake

From Table 16 and FIG. 8 it can be concluded that:

• • 1. 25-OH-D3+antioxidants/anti-inflammatories improved bird survival rate.
  • 2. In birds fed ad libitum, dead birds with 25-OH-D3+antioxidants/anti-inflammatories had higher body weight, but lower relative liver, abdominal fat, and heart weight, suggesting that most excessive fuels may be partitioned to the muscle, and thereby, adaptive hypertrophic growth of the heart for increased pumping function cannot meet the need of oxygen supply for higher growth rate (muscle) and thus may provoke cardiac arrhythmia and failure.

TABLE 17
Effect of dietary supplementation of 25-OH-D3 + antioxidants/anti-
inflammatories on carcass characteristics at age of 35 and 50 weeks
of broiler hens with restricted or ad libitum feed intake
		Restriction +		Ad libitum +
		25-OH-D3 +		25-OH-D3 +
		antioxidant/		antioxidant/
		anti-inflam-	Ad	anti-inflam-
	Restriction	matories	libitum	matories
	(n = 6)	(n = 6)	(n = 6)	(n = 6)
Body wt (kg):
at 35 wks	3.57 ± 0.19b	3.66 ± 0.15 b	4.32 ± 0.27 a	4.36 ± 0.33 a
at 50 wks	3.78 ± 0.21b	3.83 ± 0.23 b	4.62 ± 0.34 a	4.71 ± 0.38 a
Liver wt(g):
at 35 wks	55.35 ± 4.42 b	57.37 ± 2.27 b	98.45 ± 4.76a	83.37 ± 4.17 b
at 50 wks	58.67 ± 3.84 b	59.75 ± 4.67 b	72.56 ± 5.14 a*	68.45 ± 3.42 b*
Relative liver wt
(g/100 g BW, %):
at 35 wks	1.55 ± 0.06 d	1.57 ± 0.11 b	2.28 ± 0.10 a	1.91 ± 0.09 b
at 50 wks	1.55 ± 0.05	1.56 ± 0.14	1.57 ± 0.13*	1.45 ± 0.07*
Abdominal fat wt
of the dead birds
(g):
at 35 wks	47.63 ± 2.16 c	50.57 ± 3.64 c	123.84 ± 9.84a	95.47 ± 8.12 b
at 50 wks	51.24 ± 2.38 c	53.12 ± 2.57 c	147.62 ± 8.55c*	112.47 ± 9.01 c
Relative
abdominal fat wt
(g/100 g BW, %):
at 35 wks	1.35 ± 0.11 c	1.38 ± 0.06 c	2.86 ± 0.0 a	2.19 ± 0.03 b
at 50 wks	1.36 ± 0.12 c	1.39 ± 0.50 c	3.20 ± 0.06 a*	2.39 ± 0.08 b*
Heart wt (g):
at 35 wks	12.69 ± 0.38 b	12.09 ± 0.31 b	17.33 ± 0.65a	17.76 ± 0.70 a
at 50 wks	14.12 ± 0.41 b	13.88 ± 0.0.36 b	22.21 ± 0.71a*	20.88 ± 0.73a*
Relative heart wt
(g/100 g BW, %):
at 35 wks	0.357 ± 0.024b	0.331 ± 0.011 b	0.401 ± 0.016a	0.414 ± 0.046ab
at 50 wks	0.374 ± 0.025b	0.362 ± 0.024 b	0.481 ± 0.015a*	0.443 ± 0.024 ab
Results were expressed with mean ± SEM.
Means with different superscript letters are significantly different (P < 0.05)
*significant difference vs. age at 35 wks.

The low relative liver weight in ad libitum birds at age of 50 weeks appears to be due to ovarian regression developed and thus decreased estrogen secretion leading to decreased lipid synthesis in the liver for yolk deposition.

FIG. 9 shows the effect of dietary supplementation of 25-OH-D3+antioxidants/anti-inflammatories on body weight of broiler hens with restricted or ad libitum feed intake.

Results were expressed as a ratio.

FIGS. 10-11 illustrate the gross morphology of the heart of dead hens with dietary supplementation of 25-OH-D3+antioxidants/anti-inflammatories under restricted or ad libitum feed intake.

Conclusions and annotations from Tables 17 and 18 and FIGS. 9-11:

• 1. Ad libitum feeding caused cardiac adaptive hypertrophy, and some of the hypertrophic growth may develop pathologically into ventricle dilation. As a result, the heart requires a higher contractility to maintain pumping function to meet the need of blood supply for oxygen delivery to the peripheral tissues and thus may provoke heart failure.
• 2. 25-OH-D3+antioxidants/anti-inflammatories decreased the incidence of cardiac morbidities (dilation, pericardial effusion, rupture) in the dead birds.
• 3. In both restricted and ad libitum fed birds, birds with 25-OH-D3+antioxidants/anti-inflammatories exhibited less adaptive hypertrophic growth. This supports the hypothesis that most excessive fuels may be partitioned to the muscle, and thereby, hypertrophic growth of the heart for increased pumping function cannot meet the need of oxygen supply for higher growth rate (muscle) and thus may provoke cardiac arrhythmia and failure.

TABLE 19
Effect of dietary supplementation of 25-OH-D3 + antioxidants/anti-
inflammatories on electrocardiogram (ECG) pattern and arrhythmia
of broiler hens with restricted or ad libitum feed intake. See
FIGS. 12 and 13 for examples of the EGC patterns.
		Restriction +		Ad libitum +
		25-OH-D3 +		25-OH-D3 +
		antioxidant/		antioxidant/
		anti-inflam-	Ad	anti-inflam-
	Restriction	matories	libitum	matories
	(n = 8)	(n = 8)	(n = 8)	(n = 8)
ECG pattern	4/8	4/8	1/8	3/8
A at age of
35 wks
ECG pattern	2/8	3/8	2/8	3/8
B or C at
age of 35
wks
ECG pattern	2/8	1/8	5/8	2/8
D, E, F, or
G at age of
35 weeks
ECG pattern	3/8	4/8	0/8	2/8
A at age of
50 wks
ECG pattern	3/8	2/8	1/8	3/8
B or C at
age of
50 wks
ECG pattern	2/8	2/8	7/8	3/8
D, E, F, or
G at age of
50 weeks
Arrhythmic	0/8	0/8	2/8	1/8
ECG pattern
at age of
35 wks
Arrhythmic	1/8	1/8	4/8	2/8
ECG pattern
at age of
50 wks

Conclusions and annotations from Table 19 and FIGS. 12 and 13:

• • 25-OH-D3+antioxidants/anti-inflammatories decreased irregular incidence of ECG pattern (pattern D to G) and arrhythmia of broiler hens fed ad libitum and ameliorated sudden death induced by cardiac morbidities.

FIG. 14 shows the effect of dietary supplementation of 25-OH-D3+antioxidants/anti-inflammatories on cardiac fibrosis of broiler hens with restricted or ad libitum feed intake. (at age of 35 weeks) Means with letters over the bars are significantly different (P<0.05)

Conclusions and annotations from FIG. 14:

• 1. 25-OH-D3+antioxidants/anti-inflammatories ameliorated cardiac fibrosis in hens fed ad libitum.

FIG. 15 shows the effect of dietary supplementation of 25-OH-D3+antioxidants/anti-inflammatories on plasma IL-6 (top graphs) and IL-1β concentration (lower graphs) of broiler hens with restricted or ad libitum feed intake. Results were expressed with mean±SEM (n=6). Means with different letters over the bars are significantly different (P<0.05)

Conclusions and annotations from FIG. 15:

• • 25-OH-D3+antioxidants/anti-inflammatories ameliorated chronic systemic inflammation in hens fed ad libitum.

FIG. 16 shows the effect of dietary supplementation of 25-OH-D3+antioxidants/anti-inflammatories on cardiac cell apoptosis of broiler hens with restricted or ad libitum feed intake. (at age of 35 weeks). Results were expressed with mean±SEM (n=3). Means with different letters over the bars are significantly different (P<0.05)

Conclusions and annotations from FIG. 16:

• 1. 25-OH-D3+antioxidants/anti-inflammatories ameliorated cardiac cell apoptosis in hens with restricted or ad libitum feed intake.

Conclusions:

Supplemental 25-OH D3 and antioxidants/anti-inflammatories ameliorated deleterious effects associated with overfeeding of broiler breeder females by:

• • lowering mortality rate and improving ovary function and therefore reproductive performance of overfed broiler breeder hens.
  • improving endocrine (insulin) signaling
  • reducing lipotoxic development and systemic inflammation
  • activating cardio-protective mechanisms against fuel-overload induced cardiac pathogenesis.

Claims

1. A food suitable for a companion animal comprising the combination comprising 25-Hydroxy vitamin D (25-OH D), Vitamin C, Vitamin E and a carotenoid selected from the group consisting of: lycopene, astaxanthin, cryptoxanthin, beta-carotene, lutein, zeaxanthin and canthaxanthin.

2. A food according to claim 1 comprising: 25-OH D, beta carotene, vitamin C and Vitamin E and

optionally further comprises at least one further bio-active ingredient selected from the group consisting of: Vitamin D, Vitamin B2, Vitamin B6, Niacin, Zinc, Copper, Manganese, and Selenium.

3. A combination according to claim 1 wherein the amount of Vitamin E or Vitamin C to beta carotene may range from 40:1 to 1:1; preferably from 20:1 to 1:1; and more preferably from 10:1 to 1:1

4. A combination according to claim 1 wherein the 25-hydroxy vitamin D is 25-hydroxy vitamin D3 (25-OH D3).

5. A feed comprising the combination or according to claim 1 wherein:

the amount of 25-OH D3 is from 15-200 μm/kg

the amount of Vitamin E is from 40-400 mg/kg

the amount of ascorbic acid is from 40-400 mg/kg; and

the amount of beta carotene is from 1-15 mg/kg

6. A feed according to claim 1 wherein:

the amount of 25-OH D3 is from 80-150 μm/kg;

the amount of Vitamin E is from 30-300 mg/kg;

the amount of ascorbic acid is from 80-300 mg/kg; and

the amount of beta-carotene is from 1-15 mg/kg.

7. A feed according to claim 1 comprising:

25-OH D3: 35-150 m/kg;

Vitamin E: 80-300 mg/kg;

Beta carotene: 3-12 mg/kg; and

Ascorbic acid: 100-300 mg/kg.

8. A feed according to claim 1 comprising:

25-0H D3: 35 μg/kg

Vitamin E: 80 mg/kg

Beta-Carotene: 3 mg/kg; and

Ascorbic acid: 100 mg/kg.

9. A feed according to claim 1 comprising:

25-0H D3: 69 μg/kg

Vitamin E: 150 mg/kg

Beta Carotene: 6 mg/kg; and

Ascorbic acid: 150 mg/kg.

10. A feed according to claim 1 comprising:

25-0H D3: 150 m/kg

Vitamin E: 300 mg/kg

Beta Carotene: 12 mg/kg, and

Ascorbic acid: 300 mg/kg.

11. A method of ameliorating the amount of weight gained by a companion animal which is fed ad libitum comprising: administering the combination of 25-OH D, a carotenoid, Vitamin C and Vitamin E to the animal.

12. A method according to claim 11 further comprising administering at least one further bio-active ingredient selected from the group consisting of:

Vitamin D, Vitamin B2, Vitamin B6, Niacin, Zinc, Copper, Manganese, Selenium and combinations thereof.