RU2798969C2 - Method of animal feeding - Google Patents

Abstract

FIELD: animal husbandry.

SUBSTANCE: invention can be used in feeding animals of the pig family. The method of feeding an animal consists in introducing a composition containing vitamin E and a source of polyphenols into the composition of its feed ration.

EFFECT: increased productivity of the pig animal family.

8 cl, 11 dwg, 48 tbl, 10 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This international patent application titled "Method of Feeding an Animal" claims priority and includes by reference in its entirety the following patent applications: U.S. Provisional Application No. 62/115412 entitled "Method of Feeding an Animal" filed February 12, 2015 by M. Hilbert and others and assigned to CAN Technologies, Inc. (Registry Number N00255USP3), U.S. Provisional Patent Application No. 62/031977 entitled "Method of Feeding an Animal of the Poultry Class", filed Aug. 1, 2014 by M. Hilbert et al. and assigned to CAN Technologies, Inc. (registry number N00255USP2); and U.S. Provisional Application No. 62/005084 entitled "Method of Feeding an Animal of the Poultry Class" filed May 30, 2014 by M. Hilbert et al. and assigned to CAN Technologies, Inc. (registry number N00255USP1).

FIELD OF TECHNOLOGY

The present invention generally relates to feeding an animal. Aspects of the present invention particularly relate to feeding a monogastric animal a diet containing vitamin E with a source of polyphenols.

BACKGROUND OF THE INVENTION

To obtain eggs in integrated broiler breeding farms, the eggs are hatched in their own incubator and the offspring are raised to slaughter. Income is largely dependent on the amount of poultry meat produced, minus the costs associated with feeding broiler breeders in the hatchery, feeding the offspring, and other costs (eg poultry).

The normal growth and development of the embryo depends on the supply of all the necessary nutrients inside the egg. It is known that the supply of nutrients in the egg arises through the nutrition of the parent flock and the metabolism of the breeding birds. If it is possible to make changes to the diet of the parent stock that will affect the development of the embryo and the growth of the offspring, then this can be much more cost effective than changing the diet of the offspring to increase productivity. For example, European Patent Application No. EP2358214 titled "Use of canthaxanthin and/or 25-OH-D3 to improve the hatchability of birds", assigned to DSM IP Assets B.V., relates to the use of canthaxanthin to improve the hatchability of birds.

One breeding broiler can produce about a hundred broiler chickens per year. Thus, a small improvement in progeny performance due to a change in the nutrition of a breeding bird can have a significant impact on the yield obtained from the offspring.

Similarly, the success and profitability of livestock farms for cattle, pigs, sheep and many other animals depends to a large extent on the ability to produce a large number of viable offspring. Therefore, it is required to increase the birth rate, ensuring the development of a large percentage of fertilized eggs into viable offspring.

SUMMARY OF THE INVENTION

Disclosed is a method of feeding an animal with a composition that contains vitamin E and a source of polyphenols. Animals may include monogastric animals such as those of the avian class or the pig family. In some embodiments, the implementation of feeding vitamin E and a source of polyphenols increases the productivity of animals, which is determined by their reproductive capacity and resulting offspring. In other embodiments, the animal shows an increase in the antioxidant status of the animal, as well as its offspring. In some embodiments, vitamin E can be replaced with a source of polyphenols.

In one aspect of the invention, a method is disclosed for increasing the productivity of an animal of the avian class, the method comprising:

feeding an animal of the bird class as part of its feed ration a composition containing:

vitamin E; And

source of polyphenols;

collecting fertilized eggs laid by an animal of the avian class after the composition has been fed to the animal of the avian class;

incubation of fertilized eggs until hatching to ensure offspring; And

rearing offspring;

at the same time, productivity is increased compared to an animal of the bird class, which was fed a feed ration that did not contain the composition.

In another aspect of the invention, a method is disclosed for increasing the productivity of an animal of the bird class, the method comprising:

feeding an animal of the bird class as part of its feed ration a composition containing:

vitamin E; And

source of polyphenols;

wherein vitamin E is present in the diet at an inclusion level of about 10 ppm (parts per million) to about 200 ppm (parts per million), and the ratio of vitamin E inclusion to polyphenol source inclusion in the composition is about 1:1 vitamin E to a source of polyphenols,

collection of fertilized eggs laid by an animal of the avian class, about 8 weeks after the start of feeding the composition to an animal of the avian class;

incubation of fertilized eggs until hatching to ensure offspring; And

rearing offspring;

at the same time, productivity is increased compared to an animal of the bird class, which was fed a feed ration that did not contain the composition.

In still other aspects of the invention, a method for improving the productivity of a porcine animal is disclosed, comprising:

feeding an animal of the pig family as part of its feed ration with a composition containing:

vitamin E; And

a source of polyphenols, while productivity is increased compared to an animal fed a feed ration that does not contain the composition.

Other aspects of the invention include a method for increasing the productivity of an animal of the pig family, including:

feeding an animal of the pig family as part of its feed ration with a composition containing:

vitamin E; And

source of polyphenols;

wherein vitamin E is present in the diet at an inclusion level of about 1 ppm to about 300 ppm, and the ratio of vitamin E inclusion to polyphenol source inclusion in the composition is about 1:1 vitamin E to polyphenol source.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph of body weight gain compared to the performance standards of Cobb 500 FF broilers in Example 1.

Figure 2 is a plot of egg production compared to the Cobb 500 FF broiler production standards in Example 1.

Figure 3 is a graph of egg weights compared to Cobb 500 FF broiler performance standards in Example 1.

Figure 4 is a graph of hatchability versus overall results based on the treatment of breeding broilers in Example 5.

Figure 5 is a graph of the percentage of empty eggs, early, medium, late and total embryonic mortality based on the treatment of breeding broilers in example 5.

Figure 6 is a graph showing the effect of treatment of breeding broilers on feed to growth ratios for the offspring as a whole and feed to growth adjusted for final body weight in Example 6.

Figure 7 is a graph showing the amount of food provided during the pre-experimental period (49-56 weeks) and the experimental period (56-64 weeks) in Example 7.

Figure 8 is a graph showing the average percentage of egg production of breeding birds fed various diets during the experimental period (56-64 weeks) in Example 7.

Figure 9 is a graph showing the response of IgG titers to LPS with respect to treatment and age of breeding broilers in Example 9.

Figure 10 is a graph showing the response of HuSA-specific titers versus age of the chicks in Example 9.

Figure 11 is a graph showing the response of LPS titers to the treatment of breeding broilers in Example 9.

DETAILED DESCRIPTION OF THE INVENTION

Specific details of several embodiments of the invention are described below with reference to an animal. Other specific details of several embodiments of the invention are described below with reference to a method for increasing productivity (e.g., offspring productivity or reproductive capacity in an animal (e.g., an animal of the avian class or the pig family). For example, in one embodiment, the disclosed method is a method comprising feeding to an animal in its diet composition containing vitamin E and a source of polyphenols.In some embodiments, the method of feeding an animal its diet includes vitamin E present in the diet at an inclusion level of from about 10 ppm to about 100 ppm, and the ratio of the vitamin inclusion level E to the level of inclusion of the source of polyphenols in the composition, which is about 1:1 vitamin E to the source of polyphenols, collection of fertile eggs laid by an animal of the bird class, about five weeks after the start of feeding the composition to an animal of the bird class, incubation of fertilized eggs until hatching to ensure offspring and rearing offspring, while the productivity of the offspring is increased in comparison with the offspring obtained from an animal class of birds, which were fed a feed ration that did not contain the composition.

The term "animals" as used in this invention includes monogastric and ruminant animals. Used in this invention, the term "monogastric" means any organism that has a simple single-chambered stomach. Such monogastric animals include, but are not limited to, pigs, horses, goats, sheep, animals bird class, commercial marine animals (aquaculture).

The monogastric avian animals used in the present invention include poultry. The term "poultry" as used in this invention means poultry, including chickens, turkeys, geese, ducks, ostriches, quails and pheasants raised for meat or eggs. Chickens include "laying hens" raised for laying eggs and "broilers" for meat production. Chickens also include "breeding birds", that is, birds that have reached the age of sexual maturity and can lay eggs. In some embodiments, the bird class animal is selected from the group consisting of chicken, turkey, duck, and goose. In some embodiments, the bird class animal is a chicken.

Chickens suitable for use in embodiments of the present invention may be one of many possible breeds of chickens including, for example, Ross, Cobb, Isa Brown, Hubbard, Shaver, Arbor Airex, Indian River, Peterson, and DeKalb White. In some embodiments, the chicken may be a Ross chicken.

Monogastric pigs include, for example, fattening pigs and breeding pigs, including piglets, sows, gilts, boars and boars. As used herein, the term "ruminant" means any mammal that has a multi-chambered stomach and is associated with digestion of food by regurgitation and re-chewing of a bolus or cud. Such ruminant mammals include, but are not limited to, cattle, such as buffalo, bison, and all livestock, including calves, bulls, heifers, cows, and bulls.

Although the description is given primarily with reference to animals of the avian class and the pig family, it is not limited as such, and it should be understood that the invention is applicable to other monogastric and ruminant animals.

Antioxidants, when included in the diet of animals, are known to protect cells from damage caused by free radicals and peroxides. There are various types of antioxidants suitable for feeding an animal (for example, the bird class and the pig family), including natural dietary antioxidants. Natural dietary antioxidants include, for example, vitamins (eg E, C), minerals (eg Se, Zn), carotenoids and polyphenols.

Polyphenols are known to promote vitamin E regeneration in addition to their free radical scavenging function. The polyphenols used in embodiments of the present invention are commercially available and can be obtained from a variety of sources including, for example, fruits, vegetables, legumes, nuts, seeds, tea extracts, herbs, spices, and tree barks. Fruits from which polyphenols can be extracted include, for example, apples (red or green), apricots, currants (black or red), blackberries, blueberries, cherries (sweet or sour), aronia, cranberries, dates, elderberries, gooseberries, grapes (red or purple) kiwi, lemons, lingonberries, limes, mangoes, marion berries, nectarines, olives, oranges (e.g. Navel, tangelo, tangerines, kinglet), peaches, pears, plums, pomegranates, quinces, raspberries, rhubarb, strawberries and tomatoes. Vegetables from which polyphenols can be extracted include, for example, artichokes, broccoli, celery, corn, eggplant, fennel, garlic, leafy greens (such as collard greens and rape), kohlrabi, leeks, lovage, onions (red and yellow ), parsnips, peppers, spinach, red cabbage, rutabagas, green onions, shallots, sweet potatoes, and watercress. Legumes, nuts, seeds from which polyphenols can be extracted include, for example, seeds of any of the fruits listed above (e.g., grape seeds), almonds, cashews, chickpeas, beans (e.g., black beans, red beans, pinto beans, cow peas), English peas, broad beans, flax seeds, green peas, hazelnuts, lentils, pecans, peanuts, pistachios, pumpkin seeds, common beans, sunflower seeds and walnuts. Teas from which polyphenols can be extracted include, for example, green tea, black tea, oolong tea, Earl Gray tea, Ceylon tea and Darjeeling tea. Herbs and spices from which polyphenols can be extracted include, for example, basil, chives, capers (red and green), cinnamon, curry, fennel, horseradish, oregano, parsley, rosemary, sage, tarragon, and thyme. In some embodiments, the source of polyphenols includes at least one of the following: onion extract, grape seed extract, and rosemary extract. Wood bark from which polyphenols can be extracted includes, for example, arjuna bark, aspen bark, birch bark, conifer bark (eg, cedar, cypress, fir, larch, pine, spruce, yew), eucalyptus bark, and maple bark.

In some embodiments, the polyphenol source may include onion extract, grape seed extract, and rosemary extract.

A source of polyphenols suitable for use in embodiments of the present invention is commercially available under the trade name PROVIOX 50 available from Cargill, Incorporated, Wayzata, MN, USA. PROVIOX 50 is formulated with a blend of polyphenols from grape seed and skin extracts, onion extracts and rosemary extracts. PROVIOX 50 is a standardized product for its total polyphenol content. PROVIOX 50 contains a source of compounds with antioxidant properties and can replace or supplement vitamin E 50, which can save costs and provide animals with antioxidant status and performance. The replacement ratio depends on the level of vitamin E in the diet. In some embodiments, vitamin E levels above National Research Council (NRC) requirements (see Tables A and B for swine and poultry) may be substituted or replaced with a vitamin E equivalent, such as a polyphenol. In some embodiments, the polyphenol is PROVIOX 50.

One aspect of the present invention relates to feeding an animal, as part of its diet, a composition containing vitamin E and a source of polyphenols, such as PROVIOX 50.

In some embodiments for feeding an animal of the avian class, vitamin E is present in the diet at an inclusion level of about 10 ppm to about 200 ppm, about 20 ppm to about 100 ppm, about 30 ppm to about 90 ppm, or about 35 ppm to about 85 ppm.

In some embodiments for feeding a porcine animal, vitamin E is present in the diet at an inclusion level of about 1 ppm to about 300 ppm, about 20 ppm to about 250 ppm, about 30 ppm to about 200 ppm, or about 35 ppm to about 150 ppm.

In some embodiments, the ratio of vitamin E incorporation to polyphenol source incorporation in the composition is about 1:1 vitamin E to polyphenol source. Vitamin E in the diet of animals is usually at least at (equal to) the minimum requirement of the National Research Council (NRC) (see tables A and B below for pigs and poultry).

In some embodiments, vitamin E levels above the minimum NRC levels may be substituted with a vitamin E equivalent, such as a polyphenol. In some embodiments, the polyphenol is PROVIOX 50. While the composition is referred to in terms of a 1:1 ratio of vitamin E to vitamin E equivalent, it should be understood that the application contemplates various other ratios of vitamin E to vitamin E equivalent, such as a polyphenol. In some embodiments, the ratio of vitamin E to polyphenol source may be in the range of about 1:2 to about 1:5, such as 1:2, 1:3, 1:4, or 1:5.

Table A

Pigs (Vit. E) Minimum NRC Requirement (ME) Piglets 3-5 kg 20 IU/kg Piglets 5-10 kg 20 IU/kg Piglets 10-20 kg 14 IU/kg Pigs 20-50 kg 14 IU/kg Pigs 50-80 kg 14 IU/kg Pigs 80-12 kg 14 IU/kg Sows in gestation 55 IU/kg Sows during lactation 55 IU/kg

Table B

Bird (Vit.E) Minimum NRC Requirement (ME) Broiler 0-7 days 19 Broiler 0-18 days 18 Broiler 19-35 days 15 Broiler 36-42 days 13 Laying hens 0-6 weeks 15 Laying hens 7-16 weeks 12 Layer >17 weeks 10

The level of inclusion of a source of polyphenols in a complete feed varies from 1 ppm to 300 ppm (from 10 g to 300 g of a source of polyphenols per ton of complete feed). For example, from 10 ppm to 100 ppm, also, for example, from 20 ppm to 95 ppm, also, for example, from 30 ppm to 90 ppm, also, for example, from 35 ppm to 85 ppm.

In some embodiments, an animal of the bird class is fed a composition containing vitamin E and a source of polyphenols to improve the performance of the bird. Poultry productivity includes breeding bird productivity, offspring productivity and antioxidant status.

In some embodiments, an animal of the bird class is fed a composition that contains vitamin E and a source of polyphenols for at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks. weeks prior to collection of fertilized eggs for incubation. In one embodiment, fertilized eggs are harvested about 6-10 weeks after the start of feeding the composition to an animal of the avian class. In other embodiments, fertilized eggs are harvested about 8 weeks after the start of feeding the composition.

Methods for preparing poultry feed are known in the art and are described, for example, in Poultry Nutrition: The Classic Guide to Poultry Nutrition for Chickens, Turkeys, Ducks, Geese, Gamebirds, and Pigeons, G.F. Heauser, Norton Creek Press, 2003 and Commercial Poultry Nutrition, 3rd Edition, Leeson et al., University Books, 2005.

Feed rations for poultry animals may contain a variety of ingredients such as, for example, corn, wheat, soy flour, fats and oils (e.g. soybean oil), limestone, monocalcium phosphate, sodium bicarbonate, sodium chloride, L-lysine HCl, DL- methionine, L-threonine and starter premix or growth premix including, for example, vitamin A (retinyl acetate), vitamin D3 (cholecalciferol), vitamin E (DL-α-tocopherol), vitamin K3 (menadione), vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B6 (pyridoxine HCI), vitamin B12 (cyanocobalamin), nicotinic acid, D-pantothenic acid, choline chloride, folic acid, biotin, KI, FeSO ₄ ⋅H ₂ O; CuSO ₄ × 5H ₂ O, MnO, ZnSO ₄ × H ₂ O and Na ₂ SeO ₃ .

In some embodiments, an animal of the avian class may be fed a composition containing vitamin E and a source of polyphenols starting at hatching starting at 1 week of age from hatching starting at 2 weeks of age starting at 3 weeks of age starting at 4 weeks of age starting at 5 weeks old, 6 weeks old, 7 weeks old, 8 weeks old, 9 weeks old, 10 weeks old, 11 weeks old, 12 weeks old, 13 weeks old weeks starting at 14 weeks old starting at 15 weeks old starting at 16 weeks old starting at 17 weeks old starting at 18 weeks old starting at 19 weeks old starting at 20 weeks old starting at 21 weeks old from 22 weeks of age, from 23 weeks of age, from 24 weeks of age, from 25 weeks of age, from 26 weeks of age, from 27 weeks of age, from 28 weeks of age, from 29 weeks of age, or from 30 weeks old from hatching. In some embodiments, an animal of the avian class may be fed a composition containing vitamin E and a source of polyphenols throughout its life cycle.

Progeny from fertilized eggs laid by an avian animal fed a composition containing vitamin E and a source of polyphenols may show increased productivity compared to progeny from an avian animal fed a diet without the composition, which can be determined according to methods known in the art. average specialist in the respective fields.

In some embodiments, increased progeny productivity is at least one of: increased progeny hatch weight, increased final progeny body weight, increased average daily progeny gain, increased progeny feed conversion rate, increased progeny feed intake, and antioxidant status. Antioxidant status can be determined by any of the known methods, including determination of superoxide dismutase or glutathione peroxidase activities and vitamin A and vitamin E levels.

In some embodiments, increased offspring productivity in a bird is at least 1% higher, at least 1.5% higher, at least 2% higher, at least 2.5% higher at least 3% higher, at least 3.5% higher, at least 4% higher, at least 4.5% higher, at least 5% higher, at least 5.5% higher, at least 6% higher, at least 6.5% higher, at least 7% higher, at least 7.5% higher at least 8% higher, at least 8.5% higher, at least 9% higher, at least 9.5% higher, or at least 10% higher compared with the productivity of offspring from an animal class of birds, which were fed a feed ration that did not contain the composition. In other embodiments, the offspring of a bird fed a disclosed composition containing a source of polyphenols is the same as an animal of the avian class fed vitamin E.

In some embodiments, the performance of a breeding bird is increased compared to a breeding bird fed a diet without vitamin E. In some embodiments, feeding a composition containing vitamin E and a source of polyphenols may result in a similar or higher performance of the breeding bird compared to the breeding bird. , which was fed only vitamin E. The productivity of breeding birds includes, for example, the hatchability of fertilized eggs and damage (breakage) of the eggshell. In some embodiments, hatchability has improved. In other embodiments, eggshell damage has been reduced. In other embodiments, the implementation of the antioxidant status was increased.

A similar method may apply to feeding an animal of the pig family, as described later in the examples. In some embodiments, a porcine animal is fed a composition containing vitamin E and a source of polyphenols to improve porcine performance. Pig productivity includes reproductive capacity, litter productivity and antioxidant status.

Reproductive capacity includes, for example, farrowing percentage, actual litter size, mating, farrowing and weaning, and other indicators as defined in Table 44. Litter productivity includes, for example, the number of pigs born live, weak, or born mummified, litter weight ( for example, at weaning or after lactation) and other indicators, which are defined in table 45. Antioxidant status includes the determination of the activity of superoxide dismutase, glutathione peroxidase and the determination of the content of vitamins A and E.

Feeding a composition containing vitamin E and a source of polyphenols may result in increased performance in pigs relative to pigs fed a diet without vitamin E. In some embodiments, feeding a composition containing vitamin E and a source of polyphenols may result in similar or higher performance in pigs. compared to pigs fed only vitamin E.

Feeding of the composition can be carried out daily during the initial period of gestation up to lactation and farrowing. In some embodiments, the implementation of feeding can begin from the first day of mating and even in the period up to 120 days before mating (premating period). The food fed during the pre-mating period and during mating is well known in the art. The concentration of the composition containing vitamin E and polyphenol may be essentially the same during the pre-partum period as that fed during the gestation period.

The base diet can be any typical pig diet known in the art, including diets specifically designed for pregnant or lactating pigs. For example, the diet will include a variety of components, described in table 42 of the application. Detailed guidance on formulating diets for feeding pigs can be found in Nutrient Requirements of Swine, Nutrient Requirements of Domestic Animals, Number 3, 9th rev. ed. (National Academy of Science, Washington, D.C.: 1988).

The feeding schedule and feeding rates used can be any of the standard feeding schedules and rates used in the art. Typically, pregnant pigs are fed about 4 to about 6 pounds of ration per day and about 4 to about 5 pounds per day. Lactating pigs are typically fed about 9 to about 15 pounds of ration per day and about 13 to about 14 pounds per day. Typically, feeding is carried out 1 to 2 times a day for pregnant pigs and 1 to 2 and up to a maximum of 4 times a day for lactating pigs.

In some embodiments for feeding a porcine animal, vitamin E is present in the diet at an inclusion level of about 1 ppm to about 300 ppm, about 20 ppm to about 250 ppm, about 30 ppm to about 200 ppm, or about 35 ppm to about 150 ppm.

EXAMPLES

Aspects of specific methods in accordance with aspects of the invention are illustrated in the following examples. Examples 1-9 are for feeding an animal bird class, and example 10 is intended for feeding an animal of the pig family.

EXAMPLE 1. PARTIAL REPLACEMENT OF VITAMIN E WITH PROVIOX 50 IN BROILER DIETS

Two different broiler diets (Table 1) were tested in randomized block design. Breeders received a standard control diet or a diet in which 50% vitamin E was replaced with PROVIOX 50. The two diets were randomized into six cages, receiving three replications per treatment with 28, 29 or 30 females and three or four males per replication.

Table 1

Diets for breeding broilers

Turn-on level (ppm) Vitamin E Dietary Equivalence (ppm) Chicken diet Male diet No. Treatment Vit.E 50 PROVIOX 50 Vit.E 50 PROVIOX 50 Hen Cockerel 1 Control 160 - 120 - 80 60 2 PROVIOX 50 80 80 60 60 80 60

The experimental period lasted four weeks; the breeding birds received the starter diet of laying hens during the entire period according to the experimental design. Food and water were limited. The amount of food depended on the increase in body weight. The feeding schedule for Example 1 is shown in Table 2. Water was provided for two hours a day, crops were checked regularly to ensure adequate water supply.

table 2

Feeding Schedule

Amount of feed, g/bird/day Hen Cockerel aged 22-27 weeks 154 100.7 aged 27-28 weeks 156 105 aged 28-29 weeks 158 110.7 aged 29-30 weeks 160 115

A total of 24 males and 173 female Cobb 500 FF breeding chicks at 20 weeks of age were provided. Males and females were randomly assigned to six floor cages (four males per cage, 29 hens per cage 1-3, 30 hens per cage 4, and 28 hens per cage 5 and 6). During the pre-experimental period, all breeding birds received the control diet.

Throughout the experiment, the breeding birds were placed in floor cages for broiler breeding (2.60×2.40 m) on bedding (flax). Each cage was equipped with two height-adjustable bell drinkers. The hens were fed by two hopper feeders on a raised mesh floor. The cocks were fed through a trough feeder on the other side of the cage.

The length of daylight was set at 14 hours per day until peak egg laying was reached (70%, day 15). After reaching the peak of oviposition, the daylight hours were set to 15 hours per day. Temperature and ventilation were controlled by computer. The temperature was set at 20°C during the entire experimental period.

The diets were produced by Research Diet Services, Wijk bij Duurstede, The Netherlands. All diets were produced separately using one batch of basic ration for female diets and one batch of basic ration for male diets. The rations were produced in the form of compound feed.

Prior to the preparation of the diet composition, batches of soybean meal and wholemeal corn and wheat were reserved and analyzed by wet chemistry for crude protein, calcium and phosphorus. In addition, soybean meal was analyzed for potassium content. Near infrared spectrometry ("NIR") using a Bruker MPA spectrometer according to ISO 12099, manufactured by Bruker Optik GmbH, Ettlingen, Germany, was used to determine the content of crude ash, crude fat, crude fiber, moisture, and to cross-check crude protein. The formulating of the diets was based on the analyzed nutrient content of the reserved components.

The basic diet (corn - soybean meal - wheat and wheat bran) was based on Provimi B.V. nutritional recommendations. for breeding broilers. The premix for breeding broilers was used without the addition of vitamin E and a special antioxidant product. Received different diets for cockerels and hens, while using the same experimental design. This was done to be able to feed males according to their nutrient requirements (lower requirements for crude protein, energy and calcium, and various premixes) without deviating from the experimental design. The composition of the experimental diets is shown in Table 3. The results of the analysis of the experimental diets are presented in Table 4.

Table 3

Component and nutritional composition of experimental diets

Feed code LT1202-1 LT1202-2 LT1202-3 LT1202-4 Treatment 1 2 1 2 Floor Hen Hen Cockerel Cockerel Diet Form compound feed compound feed compound feed compound feed Component composition (%) Corn (A5043) 44.5 44.5 30.0 30.0 Wheat (A4976) 20.0 20.0 26.7 26.7 Soybean meal (A4971) 16.5 16.5 13.2 13.2 wheat bran 7.5 7.5 25.0 25.0 coarse limestone 5.0 5.0 1.1 1.1 Fine limestone 2.5 2.5 0.6 0.6 Monocalcium phosphate 1.4 1.4 1.0 1.0 Soybean oil 1.0 1.0 1.0 1.0 Premix for hens ¹ (1%) 1.0 1.0 - - Male premix ² (1%) - - 1.0 1.0 Salt 0.12 0.12 0.26 0.26 Na bicarbonate 0.26 0.26 0.07 0.017 DL-Methionine 0.148 0.148 0.045 0.045 L-lysine HCL 0.109 0.109 - - Vitamin E 50% 0.016 0.016 0.010 0.005 PROVIOX 50 - - - 0.005 Nutrient composition (%) - based on the Provimi Nutrient Code System 1120 Crude protein 14.9 14.9 15.4 15.4 1130 Crude fat 3.43 3.43 3.53 3.53 2250 Crude fiber 2.65 2.65 3.84 3.84 1150 Ash 11.3 11.3 5.9 5.9 1110 Dry matter 88.4 88.4 87.4 87.4 1511 Calcium 3.15 3.15 0.90 0.90 1513 Phosphorus P 0.66 0.66 0.72 0.72 3312 R dost. 0.42 0.42 0.35 0.35 3311 P usv. laying hens 0.39 0.39 0.35 0.35 1521 Sodium 0.16 0.16 0.15 0.15 1522 Potassium 0.67 0.67 0.82 0.82 1523 Chloride 0.14 0.14 0.20 0.20 1530 dB 200 200 218 218 3110 OE for birds 2713 2713 2686 2686 3112 OE for birds (FS-R) 2743 2743 2710 2710 3120 OE for layers 2750 2750 2723 2723 1210 lys 0.759 0.759 0.673 0.673 1211 met 0.383 0.383 0.288 0.288 1213 SA 0.657 0.657 0.583 0.583 1214 thr 0.534 0.534 0.539 0.539 1215 TRP 0.170 0.170 0.187 0.187 1216 isle 0.609 0.609 0.602 0.602 1219 arg 0.903 0.903 0.948 0.948 1218 val 0.690 0.690 0.713 0.713 3200 AFD LYSp 0.630 0.630 0.540 0.540 03201 AFD METp 0.346 0.346 0.247 0.247 3203 AFD SAAp 0.560 0.560 0.480 0.480 3204 AFD THRp 0.422 0.422 0.419 0.419 3205 AFD TRPp 0.145 0.145 0.157 0.157 3206 AFD ILEp 0.516 0.516 0.503 0.503 3209 AFD ARGp 0.791 0.791 0.827 0.827 3208 AFD VALp 0.564 0.564 0.577 0.577 3240 TFD LYSp 0.671 0.671 0.572 0.572 3241 TFD METp 0.358 0.358 0.258 0.258 3243 TFD SAAP 0.590 0.590 0.503 0.503 3244 TFD THRp 0.456 0.456 0.450 0.450 3245 TFD TRPP 0.148 0.148 0.160 0.160 3246 TFD ILEp 0.546 0.546 0.531 0.531 3249 TFD ARGp 0.825 0.825 0.860 0.860 3248 TFD VALp 0.612 0.612 0.617 0.617 1327 C18:2 1.61 1.61 1.61 1.61 Vitamin E equivalent (IU) 80.0 80.0 50.0 50.0

¹ Given per kg of diet: riboflavin, 9.0 mg; niacinamide, 40 mg; D-pantothenic acid, 12 mg; choline chloride, 600 mg; DL-α-tocopherol, - IU; menadione, 2.5 mg; retinyl acetate, 12500 IU; cholecalciferol, 2500 IU; biotin, 150 mcg; folic acid, 1.5 mg; thiamine, 2.0 mg; pyridoxine HCl, 4.5 mg; cyanocobalamin, 30 mcg; FeSO ₄ ×H ₂ O, 147 mg; MnO ₂ , 135 mg; CuSO ₄ × 5H ₂ O, 40 mg; ZnSO ₄ ×H ₂ O, 275 mg; Se (org.), 0.88 mg; KI, 2.6 mg; antioxidant (Oxytrap PXN), - mg.

² Given per kg of diet: riboflavin, 7.5 mg; niacinamide, 30 mg; D-pantothenic acid, 9 mg; choline chloride, 500 mg; DL-α-tocopherol, - IU; menadione, 3.5 mg; retinyl acetate, 10,000 IU; cholecalciferol, 2000 IU; biotin, 100 mcg; folic acid, 1.0 mg; thiamine, 2.0 mg; pyridoxine HCI, 3.0 mg; cyanocobalamin, 25 mcg; FeSO ₄ ×H ₂ O, 147 mg; MnO ₂ , 106 mg; CuSO ₄ × 5H ₂ O, 40 mg; ZnSO ₄ ×H ₂ O, 206 mg; Se (org.), 0.88 mg; KI, 1.9 mg; antioxidant (Oxytrap PXN), - mg.

Table 4

Analyzed nutritional composition of experimental diets

chickens Petushki Diet code LT1202-1 LT1202-2 LT1202-3 LT1202-4 Calculated amount of nutrients, % Crude protein 14.9 14.9 15.4 15.4 crude fat 3.4 3.4 3.5 3.5 crude fiber 2.7 2.7 3.8 3.8 Dry matter 88.4 88.4 87.4 87.4 Ca 3.15 3.15 0.90 0.90 P 0.66 0.66 0.72 0.72 Nutrients Analyzed Crude protein 15.3 16.1 16.6 15.8 Crude protein (NIKS) 15.8 16.0 16.3 15.7 Crude fat (NIKS) 3.2 3.2 3.9 3.7 Crude fiber (NIKS) 2.8 2.6 3.9 3.9 Moisture (NICS) 11.4 11.1 11.1 11.5 Dry matter 88.6 88.9 88.9 88.5 Ca 3.07 3.08 1.13* 0.87 P 0.65 0.66 0.70 0.64* % expected amount Crude protein 103 104 105 103 crude fat 93 93 110 105 crude fiber 106 98 101 101 Dry matter 100 101 102 101 Ca 98 98 126 97 P 99 100 98 89 *Parallel analysis

The individual weight of the birds was recorded weekly from the start of the experiment. The amount of food provided was recorded daily. Egg production and egg quality data were recorded daily. % egg production was calculated as the number of eggs laid per cage divided by the number of bird days of the experimental period. Gross egg production per cage per week was calculated as % egg production (including category II eggs) multiplied by average egg weight per cage. The average egg weight was determined per cell once a week. The eggs of the second category were divided into dirty eggs, broken eggs, eggs without shell, two-yolk eggs, floor eggs and the rest of the eggs of the second category. Mortality was monitored every day; dead animals were not replaced during the experimental period.

To compare different treatments, all scores were subjected to a mixed model procedure using SAS version 9.2, 2008 (SAS Institute Inc., Cary, NC, USA) according to the following statistical model:

Y _ij =μ+α _i +ε _ij

Where:

Y _ij =specific trait per experimental unit (cell);

μ=general mean value for a particular trait;

α _i =fixed treatment effect (i=1 or 2);

ε _ij =error vector.

Differences between means were considered statistically significant based on a P<0.05 probability (Student's t-test for difference in means) unless another probability value was specified.

The nutritional composition of the diets was reasonably as expected (Table 4) with the exception of calcium levels in the control male diet and phosphorus levels in the PROVIOX 50 male diet (based on a parallel analysis). However, since the same ration batch was used to produce both rations, and the same reserved and analyzed components were used as for the hen diets, it is assumed that these data are due to analysis or sampling errors, since the rations were fed as compound feed.

The observed health status of the birds was good throughout the experiment. Mortality, including culling, was 1.5% (Table 5), in line with actual levels (1.6% between 26 and 30 weeks of age). The technical performance of the birds met the standards of breeding birds of the Cobb 500 FF breed (figure 1-3 and table 6).

Table 5

Mortality and culling calculated as percentage of animals at start of trial (total 195)

Period Mortality culling Total 26-30 days 0.5 1 1.5

Table 6

Technical results of breeding birds fed a control diet or a PROVIOX 50 diet in which half of the vitamin E level was replaced with PROVIOX 50 (26 to 30 weeks of age).

Control PROVIOX SEM p-value Percentage of egg production,% 83.6 82.4 1.654 0.631 Eggs of the first category, % 77.9 77.0 1.655 0.709 Eggs of the second category Broken eggs, % 1.6 1.2 0.408 0.550 Dirty eggs, % 0.7 0.8 0.110 0.615 Eggs without shell, % 0.1 0.0 0.060 0.375 Two-yolk eggs, % 1.3 1.9 0.409 0.355 Floor eggs, % 1.4 0.9 0.291 0.283 The rest, % 0.5 0.5 0.142 0.976 Egg weight, g 56.4 55.5 0.581 0.328 Egg mass, g/layer/day 47.1 45.7 1.039 0.382 MT 26 weeks chickens, kg 3.50 3.49 0.026 0.787 MT 30 weeks chickens, kg 3.68 3.69 0.035 0.756 MT 26 weeks cocks, kg 3.91 3.90 0.107 0.939 MT 30 weeks cocks, kg 4.19 4.38 0.111 0.273

As shown in Figures 1-3 and Table 6, no statistically significant differences were found between the two treatment groups in terms of egg production of broiler breeders. This may be related to the number of iterations per treatment. Some changes were expected as the breeding bird was in the early stages of egg production.

No statistically significant differences were found between the two treatments, indicating that breeding bird performance was not affected by replacing 50% vitamin E levels with PROVIOX 50.

EXAMPLE 2. PRODUCTION OF CHICKS FROM EGGS OF EXAMPLE 1

For nine days, eggs were collected from the broiler breeding flock of Example 1. Eggs were stored based on the breeding bird cage and day of storage. The eggs were divided into twelve trays and nine blocks, with each block representing a storage day. At E17, all eggs were checked by candling, and at E18, eggs were transferred from the hatcher chambers to the hatcher baskets.

A total of 540 eggs (all eggs laid within nine days) from the 28-29 week old breeding broilers of Example 1 were collected and placed in an incubator at 18°C (64.4°F) and 75 RH. %. At the beginning of the incubation period, the eggs were weighed and divided into blocks, each block representing a day of storage. Each block consists of twelve experimental units, with each experimental unit representing a group of ten eggs. Included only intact eggs; cracked, dirty or deformed eggs were excluded from the experiment.

The eggs were placed in a NatureForm NMC 2340 incubator with automatic temperature and relative humidity control. E0 to E18 eggs were placed in setter trolleys, which consist of two shelves each holding thirteen trays. Each tray has three spacers for thirty eggs. During this period, the initial temperature in the incubator was set to 37.5° C. and the relative humidity to 54%. The temperature was set based on the temperature profile, starting at 37.5°C at E0 and gradually decreasing from E12 to E18 from 37.5°C to 36.7°C.

After an incubation period (E0-E18), the eggs were transferred to the hatcher baskets using the block structure in the incubator. The eggs were placed in the hatcher section for repetition, and then the tray in order in the block. Each hatcher trolley has twelve baskets holding 180 eggs each. During this period, the temperature in the incubator was set to 36.7° C. and the relative humidity to 70%.

Egg weights were recorded for each experimental unit at E0. At the hatching stage (E21), the number of dead chicks, dead embryos and pipped eggs (external and internal) was recorded. The remaining unhatched eggs were dissected to determine age and cause of death. The number of hatched chicks was recorded for each repetition, as well as the average weight of the chick. Gender was determined by a chick cloacal examination conducted by a professional chick sexing operator. Hatching was calculated as a percentage of total and inseminated eggs (eggs that contained live embryos after E17). A total of 102 cockerel chicks per treatment were randomly selected for Example 3 based on broiler breeding cages. Only chicks with an average body weight, completely dry, showing active movement and with a closed umbilical ring were selected. Two male chicks from each experimental unit (a total of 108 per treatment) were selected for necropsy. Selected chicks were individually weighed and killed by cervical dislocation to determine residual yolk sac weight, yolk-free body weight ("YFBM"), chick length, and leg length for both legs. Relative asymmetry was calculated using the following formula:

RA=(|L-R|/[(L+R)/2])×100

Where:

L=left foot length

R=right leg length.

To compare different treatments, all scores were subjected to a mixed model procedure using SAS version 9.2, 2008 (SAS Institute Inc., Cary, NC, USA) according to the following statistical model:

Y _ijkl =μ+α _i +b _j +c _k +ac _ik +ε _ijkl

Where:

Y _ijkl =specific feature;

μ=general mean value for a particular trait;

α _i =fixed effect of treatment of breeding broilers (i=1 or 2);

b _j =random effect block/day of storage (j=1-9);

c _k =random effect of the entire block diagram (k=1, 2 or 3);

ac _ik = random interaction effect of the entire block diagram and processing;

ε _ijkl =error vector.

The interaction factor of the entire block diagram with treatment was included in the model to adjust the number of experimental units used to treat breeding broilers. Taking into account the design of the experiment, the effect of the day of storage was fully combined with the effect of the block (location in the incubator). Fertility, total hatchability and hatchability of fertilized eggs were considered as binomial data. Differences between means were considered statistically significant based on a P<0.05 probability (Student's t-test for difference in means) unless another probability value was specified.

The average egg weight at the beginning of the experiment was 57.9 grams per egg. Overall fecundity (94%) was in line with the Cobb breeding standard, and hatchability (91%) was above average in accordance with the breeding standard (95% and 85% for 28-29 week old breeding birds, respectively). Higher than normal hatching results can result from small incubators used compared to those used in practice, leading to an optimized incubation process and therefore less embryonic mortality.

Broiler treatments did not statistically significantly affect fertility and hatchability (table 7). However, quantitatively, the treatment of eggs laid by breeding broilers, carried out by feeding PROVIOX 50, allowed a 4.6% increase in hatchability in general, which is mainly associated with an increase in fecundity by 3.6%.

Table 7

Influence of broiler breeding treatment on hatchability, fertility, chick weight and relative chick weight

Vit.E 50% PROVIOX SEM p-value n 3 3 - - General hatchability, % 88.6 92.7 - 0.2546 Hatching of fertilized eggs, % 95.6 96.6 - 0.5820 Fertility,% 92.6 95.9 - 0.1727 Weight of chickens, g 42.4 41.2 0.545 0.3039 Relative weight of chickens ¹ ,% 72.5 71.9 0.309 0.2351 ¹ expressed as a percentage of the average egg weight

Broiler breeder treatments did not statistically significantly affect any of the following: hen performance and quality scores that were evaluated (Table 8), indicating that 50% replacement of the vitamin E level in the broiler breeder diet with PROVIOX 50 did not affect the performance of hatched chicks.

Table 8

Influence of breeding broiler treatment on chick weight, yolk sac weight, relative yolk sac weight, yolk-free body weight, chick length, and relative leg asymmetry ("RA") of opened chicks

Vit.E 50% PROVIOX SEM p-value n 3 3 - - Chicken weight, g 41.3 40.5 0.6239 0.558 Yolk weight, g 4.92 4.36 0.1778 0.248 Relative weight of chicken ¹ , % 70.7 70.7 0.4332 0.973 Relative weight of chicken ² , % 11.8 10.7 0.2955 0.180 YFBM ³ , g 36.2 36.0 0.4554 0.808 Chick length, cm 17.9 17.8 0.0772 0.760 Relative asymmetry, % 1.59 1.52 0.1894 0.828

¹ expressed as a percentage of the average egg weight

² expressed as a percentage of chick weight

³ body weight without yolk.

The results show that 50% replacement of vitamin E in the diet of broiler breeders with PROVIOX 50 has no statistically significant effect on the performance of hatched chicks (eg fertility, hatchability and hatching characteristics).

EXAMPLE 3. GROWING CHICKS FROM EXAMPLE 2

In this example, hatched chicks were raised to slaughter age and performance was evaluated. The aim of the current experiment was to evaluate the effect on offspring performance of a broiler breeder diet with vitamin E compared with a 50% replacement of vitamin E with PROVIOX 50.

Two different treatments were tested on broiler breeders as shown in Example 1. In this example, the hatched chicks from Example 2 were raised from the eggs obtained in Example 1, with the breeder receiving a control diet or a diet in which vitamin E levels were 50% replaced PROVIOX 50.

The experimental period was divided into the starting phase (0-14 days) and the growth phase (14-35 days). Food and water were provided ad libitum.

A total of 204 Cobb 500 fast fledgling day old male chicks from 28-29 week old breeding broilers (Example 1) were selected from the total number of hatched male chicks from Example 2. Chicks were selected at random from various experimental units in the incubator, ensuring that the broiler breeder unit is not damaged by placing chicks from one broiler breeder unit into two rearing cages. Seventeen chickens were kept in a cage. To distribute seventeen chicks per cage, trays of day-old chicks from the defined experimental unit of Example 2 were placed in a line; Chicks were randomly selected from each pallet and placed in a specific cage. This procedure was repeated for all cells of this example.

After placing the chicks in the cages, the individual weights of all chicks in the cage were recorded to determine the starting weight of the chicks. There was a marked difference in initial weight between the various treatments, respectively 40.9 g (±1.6) and 39.9 g (±0.9) for treatments 1 (vitamin E only) and 2 (50% replacement of vitamin E with PROVIOX 50).

Throughout the experiment, chickens were kept in individual broiler cages (100×110 cm) on bedding (wood chips). Each cage was equipped with two height-adjustable nipple drinkers. During the first fourteen days, the feeder was inside the cage, and from the fourteenth day, food was fed through a feeder in the front of the cage.

Daylength was set to 23 hours a day for the first three days, 20 hours a day from day four to day seven, and 18 hours a day for the remainder of the experiment. Temperature, humidity and ventilation were controlled by computer. The temperature was gradually reduced by 2.5°C per week, from 35°C on the day of admission to a final temperature of 20.5°C at the end of the experiment (day 35). The relative humidity in the experimental house was set at 50 percent. Birds were spray vaccinated against Newcastle disease (Poulvac NDW vaccine manufactured by Intervet, Boxmeer, The Netherlands) at 14 days of age.

Prior to ration preparation, batches of wheat, corn, and soybean meal were reserved and analyzed. The diets were produced by Research Diet Services, Wijk bij Duurstede, The Netherlands. Starter and grower rations were produced using the same base ration batch (for each period). Starter rations were granulated into 2.5 mm granules and growth rations into 3 mm granules with the addition of steam (about 80°C). The composition of the diets was based on the analyzed nutrient content of the components. Rations were formulated to meet the nutrient requirements of broilers (CVB, 2006). Starter and grower rations contained 2750 and 2850 kcal CFU⋅kg ^-1 and 10.20 and 9.70 g×kg ^-1 apparent fecally digestible (AFD) lysine, respectively. The composition of the experimental diets is shown in table 9, the results of the analysis are presented in table 10.

Table 9

Component and nutritional composition of the starter and growth diet

Diet code BG1301-1 BG1301-11 Phase Home Rostovaya Diet Form Granule - 2.5 mm Granule - 3 mm Component composition (%) Corn 42.79 44.77 Wheat 20.00 20.00 Soybean meal >48% 30.28 27.97 Fats/Oils, Soybean Oil 2.049 3.010 Limestone 1.694 1.349 Monocalcium phosphate 1.355 1.149 Bicarbonate of soda 0.246 0.181 Salt 0.217 0.213 L-lysine HCL 0.143 0.153 DL-Methionine 0.211 0.195 L-Threonine 0.018 0.018 Starter premix for broilers ¹ 1,000 - Growth premix for broilers ² - 1,000 Nutritional composition (%)-PNC 1120 Crude protein 20.6 19.7 1130 OIL (EE) 4.3 5.3 1140 crude fiber 2.6 2.6 1150 Ash 6.2 5.5 1110 SW 88.0 88.0 1511 Calcium 0.97 0.80 1513 Phosphorus, total 0.67 0.62 1521 Na 0.16 0.14 1522 K 0.82 0.78 1523 Cl 0.20 0.20 1530 deb 223 205 3310 P usv. broilers 0.40 0.35 3312 R dost. for broilers 0.43 0.38 3110 CFU for poultry ³ 2924 3022 3112 CFU for poultry (FS-R) 2934 3031 3130 CFU for broilers 2750 2850 1210 LYS 1.183 1.127 1211 MET 0.514 0.487 1213 SAA 0.850 0.810 1214 THR 0.777 0.740 1215 TRP 0.244 0.230 1216 ILE 0.885 0.839 1218 VAL 0.961 0.916 1219 ARG 1.334 1.261 3200 AFD LYSp ⁴ 1.020 0.970 3201 AFD METp 0.477 0.451 3203 AFD SAAP 0.745 0.708 3204 AFD THRp 0.643 0.611 3205 AFD TRPP 0.212 0.200 3206 AFD ILEp 0.769 0.727 3208 AFD VALp 0.816 0.776 3209 AFD ARGp 1.180 1.115 3240 TFD LYSp ⁵ 1.058 1.008 3241 TFD METp 0.483 0.457 3243 TFD SAAP 0.768 0.732 3244 TFD THRp 0.671 0.639 3245 TFD TRPP 0.214 0.202 3246 TFD ILEp 0.795 0.754 3248 TFD VALp 0.859 0.819 3249 TFD ARGp 1.221 1.154 1327 C18:2 2.1 2.6

¹ Given per kg of diet: vitamin A (retinyl acetate), 12,000 IU; vitamin D3 (cholecalciferol), 5000 IU; vitamin E (DL-α-tocopherol), - mg; vitamin K3 (menadione), 2.3 mg; vitamin B1 (thiamine), 1.0 mg; vitamin B2 (riboflavin), 4.5 mg; vitamin B6 (pyridoxine HCl), 2.7 mg; vitamin B12 (cyanocobalamin), 20 mcg; niacin, 40 mg; D-pantothenic acid, 9 mg; choline chloride, 500 mg; folic acid, 0.5 mg; biotin, 100 mcg; FeSO ₄ ×H ₂ O, 150 mg; CuSO ₄ × 5H ₂ O, 40 mg; MNO, 100 mg; ZnSO ₄ ×H ₂ O, 145 mg; KI, 2.0 mg; Na ₂ SeO ₃ , 0.56 mg; antioxidant (Oxytrap PXN), - mg.

² Given per kg of diet: vitamin A (retinyl acetate), 10,000 IU; vitamin D3 (cholecalciferol), 2000 IU; vitamin E (DL-α-tocopherol), - mg; vitamin K3 (menadione), 2.3 mg; vitamin B1 (thiamine), 0.8 mg; vitamin B2 (riboflavin), 4.5 mg; vitamin B6 (pyridoxine HCl), 1.9 mg; vitamin B12 (cyanocobalamin), 20 mcg; niacin, 30 mg; D-pantothenic acid, 8 mg; choline chloride, 400 mg; folic acid, 0.5 mg; biotin, 50 mcg; FeSO ₄ ×H ₂ O, 150 mg; CuSO ₄ × 5H ₂ O, 40 mg; MNO, 100 mg; ZnSO ₄ ×H ₂ O, 145 mg; KI, 1.9 mg; Na ₂ SeO ₃ , 0.50 mg; antioxidant (Oxytrap PXN), - mg.

³ Based on CVB (2006) calculations

⁴ AFD=apparent faecal digestibility, CVB (2006) calculations.

⁵ TFD=true faecal digestibility.

Table 10

Analyzed nutritional composition of experimental diets

Home Rostovaya Diet code BG1301-1 BG1301-11 Calculated amount of nutrients, % Crude protein 20.6 19.7 crude fat 4.3 5.3 crude fiber 2.6 2.6 Dry matter 88.0 88.0 Ca 0.97 0.80 P 0.67 0.60 Nutrients Analyzed Crude protein 20.8 20.0 Crude protein (NIKS) 21.3 19.7 Crude fat (NIKS) 4.6 5.4 Crude fiber (NIKS) 2.5 2.6 Moisture (NICS) 12.3 12.4 Dry matter 87.7 87.6 Ca 0.97 0.77 P 0.68 0.61 % expected amount Crude protein 102 101 crude fat 107 102 crude fiber 96 102 Dry matter 100 100 Ca 100 96 P 101 99

The individual weight of the birds was recorded at the beginning of the experiment (day 0) and at the age of 3, 14, 28 and 35 days. In addition, the feed intake for each cage was recorded on the same day that the birds were weighed. Based on the calculated weight gain and feed intake, the feed to gain ratio (F:G) was calculated as kg feed intake/kg weight gain. The total feed intake per cage was adjusted for mortality, culling and deviations. The European Bird Index was calculated using the following formula:

European Bird Index = (final body weight (g)×(100%-mortality%))/((10×day period)×total RCC)

The European Poultry Index, excluding mortality, was calculated using the following formula:

European Bird Index = (final body weight (g) × 100) / ((10 × period in days) × total RCC)

On the 35th day of the experiment, five birds in a cage were randomly selected (excluding obvious deviations). The birds were slaughtered the next day. On the morning of day 36, these selected birds were killed with CO ₂ /O ₂ and blood samples were collected, after which they were bled.

Statistical analysis was performed according to Provimi standards for testing hypotheses and divergence of means using ANOVA analysis of variance (version 1.3). To compare different treatments, all scores were subjected to a mixed model procedure using SAS (Version 9.3, 2008, SAS Institute Inc., Cary, NC) according to the following statistical model:

Y _ijkl =μ+α _i +b _j +c _k +ac _ik +d _l +ε _ijkl

Where:

Y _ijkl =specific trait measured for each experimental unit;

μ=general mean value for a particular trait;

α _i =fixed effect of treatment of breeding broilers (i=1 or 2);

b _j =random storage effect (j=short or long);

c _k =random effect of the entire block diagram (k=1, 2 or 3);

ac _ik = random processing interaction with the entire block diagram;

d _l = random room effect (l=A or B)

ε _ijkl =error vector.

The entire block diagram is a block of two experimental broiler breeding house units. The interaction between the entire block diagram and the treatment was included in the model to adjust for the number of actual replicates per broiler breeding unit. Mortality was considered as a binomial value. Effects were considered statistically significant based on P<0.05 probability unless another probability value was specified.

The nutritional composition of the rations was in line with the expected values (maximum deviation of 5% above or below the calculated value, table 10). The observed health status of the birds was good throughout the experiment, although mortality, including culling, of 6.9% was high compared to actual levels (3 to 4%) as well as previous broiler farming studies (average 2012: 4.4%). However, no statistically significant differences in mortality were found between the two broiler treatments.

Table 11 shows the effects of breeding broiler diets (80 ppm vitamin E or 40 ppm vitamin E + 40 ppm PROVIOX 50) on mortality, European Poultry Index, average daily weight gain, average daily feed intake and feed:gain for Cobb 500 FF broilers.

Table 11

Influence of the diet of breeding broilers

Vitamin E Vitamin E+PROVIOX Total SEM p-value n ¹ 3 3 - - Mortality, % 4.6 9.2 - 0.444 EPI, excluding mortality 369 344 15.3 0.438 EPI 388 382 6.0 0.606 MT 0 days, g 40.9 39.9 0.55 0.275 MT 3 days, g 69.4 67.3 1.29 0.087 MT 14 days, g 451 436 10.0 0.449 MT 35 days, g 2219 2181 22.5 0.114 CV 3 days, % 7 8 1.0 0.445 CV 14 days, % 8.2 8.6 0.82 0.815 CV 35 days, % 7.1 8.0 0.62 0.490 ADG 0-3 days, g 9.5 9.1 0.49 0.224 ADG 3-14 days, g 34.7 33.5 0.86 0.511 ADG 14-28 days, g 79.6 78.9 1.68 0.508 ADG 28-35 days, g 93.6 91.5 3.14 0.680 ADG 0-14 days, g 29.3 28.3 0.73 0.490 ADG 0-28 days, g 54.4 53.6 0.97 0.496 ADG 0-35 days, g 62.2 61.2 0.64 0.123 ADG 14-35 days, g 84.2 83.1 0.93 0.370 ADFI 0-3 days, g 10.1 10.4 0.39 0.374 ADFI 3-14 days, g 44.5 43.2 1.14 0.421 ADFI 14-28 days, g 127.0 124.6 2.71 0.537 ADFI 28-35 days, g 180.3 178.3 1.71 0.146 ADFI 0-14 days, g 37.1 36.2 0.97 0.474 ADFI 0-28 days, g 82.1 80.4 1.66 0.505 ADFI 0-35 days, g 101.7 100.0 1.63 0.443 ADFI 14-35 days, g 144.8 142.5 2.28 0.451 F:G 0-3 days, g 1.088 1.151 0.024 0.151 F:G 3-14 days, g 1.283 1.290 0.018 0.836 F:G 14-28 days, g 1.598 1,580 0.024 0.647 F:G 28-35 days, g 1.929 1.958 0.071 0.809 F:G 0-14 days, g 1.268 1.279 0.015 0.679 F:G 0-28 days, g 1.509 1,500 0.020 0.791 F:G 0-35 days, g 1.635 1.634 0.020 0.984 F:G 14-35 days, g 1.720 1.715 0.025 0.924 F:G adjusted to MT ² 1.627 1.634 0.020 0.883

EPI=European Poultry Index (final body weight (g)×(100%-mortality%))/((10×period in days)×total RCC): EPI excluding mortality=European Poultry Index excluding mortality (final body weight ( d)×100%)/((10×period in days)×total BCC): ADG=average daily weight gain: ADFI=average daily feed intake: F:G=feed to gain ratio (kg feed intake:kg gain):

¹ Cages of breeding birds with 27-30 layers and 3-4 males

² F:G adjusted for 2.181 grams of body weight at 35 days of age. Correction by -0.02 for every 100 g overweight.

As shown in Table 11, progeny performance, average daily weight gain (“ADG”), average daily feed intake (“ADFI”), and feed-to-growth ratio (F:G) for offspring are not statistically significantly affected by breeding broiler diets.

The results of the experiment on breeding broilers showed that the productivity of breeding birds is not statistically significantly affected by a 50% replacement of vitamin E levels with PROVIOX 50.

EXAMPLE 4. PARTIAL REPLACEMENT OF VITAMIN E WITH PROVIOX 50 IN BROILER DIETS

All breeding birds received the same standard control diet during the pre-experimental period (19-30 weeks of age). During the experimental period, the breeding birds received a control diet, a diet with a higher vitamin E content, or a diet in which the level of vitamin E was 50% replaced with PROVIOX 50 (table 12).

Table 12

Experimental Design of Treatments for Broiler Breeders

Turn-on level (ppm) Treatment Vit.E 50 PROVIOX 50 Vitamin E Dietary Equivalence (ppm) Vit.E dose 1 160 - 80 Vit.E dose 2 320 - 160 PROVIOX dose 1 80 80 80 PROVIOX dose 2 160 160 160

There were a total of 24 floor cages with 30 breeding hens and 3 males (after distribution). The experimental period lasted 9 weeks; the breeding birds received the laying phase diet during the entire experimental period. Food and water were limited. The amount of food depended on the increase in body weight. Water was provided for two hours a day, crops were checked regularly to ensure that sufficient water was provided.

Before formulating the diet composition, batches of soybean meal, wheat bran, wholemeal corn and wheat were backed up and wet chemistry analyzed for crude protein, dry matter ("DM"), calcium and phosphorus. In addition, soybean meal was analyzed for potassium content, and wheat bran was analyzed by wet chemistry for crude fiber, potassium, and starch. NIRS was used to determine the content of crude ash, crude fat, crude fiber and moisture and to recheck crude protein. The formulating of the diets was based on the analyzed nutrient content of the reserved components. One batch of food was used during the entire experimental period.

The base diet (corn - soybean meal - wheat and wheat bran) was based on Provimi nutrient recommendations for broiler breeders. The diet compositions were optimized according to the Provimi PNC nutrient coding system. The broiler breeder premix was used without vitamin E and without the addition of other antioxidants. Received different diets for cockerels and hens, while using the same experimental design and using one premix. This was done to be able to feed the males according to their nutritional requirements (lower requirements for crude protein, energy and calcium). The composition of the experimental diets and the results of the analysis are presented in table 13 and table 14, respectively. The diets were produced by Research Diet Services, Wijk bij Duurstede, The Netherlands. Both diets were produced separately using one batch of basic hen diets and one batch of basic male diets. The rations were produced in the form of compound feed.

Table 13

Component and nutritional composition of experimental diets

Feed code LT1302-1 LT1302-2 LT1302-3 LT1302-4 LT1302-5 LT1302-6 LT1302-7 LT1302-8 LT1302-9 LT1302-10 LT1302-11 Phase before oviposition start of oviposition start of oviposition laying phase laying phase laying phase laying phase laying phase laying phase laying phase laying phase Treatment - - - 1 1 2 2 3 3 4 4 Floor Hens and roosters chickens Petushki chickens Petushki chickens Petushki chickens Petushki chickens Petushki Diet Form compound feed compound feed compound feed compound feed compound feed compound feed compound feed compound feed compound feed compound feed compound feed Component composition (%) Corn (A5294) 37.6 43.2 39.0 44.1 39.0 44.1 39.0 44.1 39.0 44.1 39.0 Wheat (A5295) 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 Soybean meal (A5282) 10.6 15.8 10.8 14.5 10.8 14.5 10.8 14.5 10.8 14.5 10.8 Wheat bran (A5296) 25.0 10.0 25.0 10.0 25.0 10.0 25.0 10.0 25.0 10.0 25.0 Coarse limestone 2.2 5.0 1.1 5.2 1.1 5.2 1.1 5.2 1.1 5.2 1.1 Fine limestone 1.1 2.5 0.6 2.6 0.6 2.6 0.6 2.6 0.6 2.6 0.6 Monocalcium phosphate 1.0 1.1 1.0 1.1 1.0 1.1 1.0 1.1 1.0 1.1 1.0 Soybean oil 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 Premix ¹ (1%) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 Salt 0.17 0.25 0.19 0.21 0.19 0.21 0.19 0.21 0.19 0.21 0.19 Na bicarbonate 0.15 0.01 0.13 0.063 0.13 0.063 0.13 0.063 0.13 0.063 0.13 DL-Methionine 0.075 0.118 0.068 0.120 0.068 0.120 0.068 0.120 0.068 0.120 0.068 L-lysine HCL 0.089 0.049 0.053 0.063 0.053 0.063 0.053 0.063 0.053 0.063 0.053 L-threonine 0.004 - - - - - - - - - - Vitamin E 50% 0.010 0.012 0.012 0.016 0.016 0.032 0.032 0.008 0.008 0.016 0.016 PROVIOX 50 - - - - - - - 0.008 0.008 0.016 0.016 Nutrient composition (%) - based on the Provimi Nutrient Code System 1120 Crude protein 14.5 15.0 14.6 14.5 14.6 14.5 14.6 14.5 14.6 14.5 14.6 1130 Crude fat 3.80 3.53 3.85 3.53 3.85 3.53 3.85 3.53 3.85 3.53 3.85 1140 Crude fiber 3.75 2.86 3.79 2.83 3.79 2.83 3.79 2.83 3.79 2.83 3.79 1150 Ash 7.2 11.2 5.7 11.5 5.7 11.5 5.7 11.5 5.7 11.5 5.7 1110 Dry matter 88.1 88.5 87.9 88.6 87.9 88.6 87.9 88.6 87.9 88.6 87.9 1511 Calcium 1.50 3.10 0.90 3.25 0.90 3.25 0.90 3.25 0.90 3.25 0.90 1513 Phosphorus P 0.70 0.62 0.70 0.61 0.70 0.61 0.70 0.61 0.70 0.61 0.70 3312 R dost. 0.35 0.36 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 3311 P usv. laying hens 0.35 0.34 0.35 0.33 0.35 0.33 0.35 0.33 0.35 0.33 0.35 1521 Sodium 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 1522 Potassium 0.73 0.68 0.73 0.66 0.73 0.66 0.73 0.66 0.73 0.66 0.73 1523 Chloride 0.16 0.20 0.17 0.18 0.17 0.18 0.17 0.18 0.17 0.18 0.17 1530 dB 200 178 200 177 200 177 200 177 200 177 200 3110 OE for birds 2710 2712 2759 2712 2759 2712 2759 2712 2759 2712 2759 3112 OE for birds (FS-R) 2713 2733 2763 2732 2763 2732 2763 2732 2763 2732 2763 3120 OE for layers 2750 2750 2800 2750 2800 2750 2800 2750 2800 2750 2800 1210 lys 0.689 0.729 0.670 0.705 0.670 0.705 0.670 0.705 0.670 0.705 0.670 1211 met 0.304 0.354 0.301 0.349 0.301 0.349 0.301 0.349 0.301 0.349 0.301 1213 SA 0.577 0.623 0.577 0.611 0.577 0.611 0.577 0.611 0.577 0.611 0.577 1214 thr 0.506 0.535 0.509 0.515 0.509 0.515 0.509 0.515 0.509 0.515 0.509 1215 TRP 0.170 0.173 0.172 0.166 0.172 0.166 0.172 0.166 0.172 0.166 0.172 1216 isle 0.538 0.590 0.546 0.566 0.546 0.566 0.546 0.566 0.546 0.566 0.546 1218 val 0.664 0.693 0.674 0.669 0.674 0.669 0.674 0.669 0.674 0.669 0.674 1219 arg 0.887 0.933 0.898 0.893 0.898 0.893 0.898 0.893 0.898 0.893 0.898 3200 AFD LYSp 0.560 0.600 0.540 0.580 0.540 0.580 0.540 0.580 0.540 0.580 0.540 03201 AFD METp 0.267 0.320 0.263 .0316 0.263 0.316 0.263 0.316 0.263 0.316 0.263 3203 AFD SAAp 0.480 0.530 0.480 0.520 0.480 0.520 0.480 0.520 0.480 0.520 0.480 3204 AFD THRp 0.390 0.424 0.393 0.406 0.393 0.406 0.393 0.406 0.393 0.406 0.393 3205 AFD TRPp 0.141 0.147 0.143 0.140 0.143 0.140 0.143 0.140 0.143 0.140 0.143 3206 AFD ILEp 0.448 0.501 0.456 0.480 0.456 0.480 0.456 0.480 0.456 0.480 0.456 3208 AFD VALp 0.538 0.572 0.547 0.551 0.547 0.551 0.547 0.551 0.547 0.551 0.547 3209 AFD ARGp 0.771 0.817 0.782 0.781 0.782 0.781 0.782 0.781 0.782 0.781 0.782 3240 TFD LYSp 0.592 0.636 0.572 0.616 0.572 0.616 0.572 0.616 0.572 0.616 0.572 3241 TFD METp 0.275 0.328 0.272 0.324 0.272 0.324 0.272 0.324 0.272 0.324 0.272 3243 TFD SAAP 0.502 0.556 0.501 0.546 0.501 0.546 0.501 0.546 0.501 0.546 0.501 3244 TFD THRp 0.421 0.455 0.423 0.438 0.423 0.438 0.423 0.438 0.423 0.438 0.423 3245 TFD TRPP 0.145 0.150 0.146 0.144 0.146 0.144 0.146 0.144 0.146 0.144 0.146 3246 TFD ILEp 0.474 0.528 0.481 0.507 0.481 0.507 0.481 0.507 0.481 0.507 0.481 3248 TFD VALp 0.572 0.613 0.581 0.591 0.581 0.591 0.581 0.591 0.581 0.591 0.581 3249 TFD ARGp 0.803 0.852 0.814 0.815 0.814 0.815 0.814 0.815 0.814 0.815 0.814 1327 C18:2 1.75 1.65 1.77 1.66 1.77 1.66 1.77 1.66 1.77 1.66 1.77 Vitamin E equivalent (IU) 50 60 60 80 80 160 160 80 80 160 160

¹ Given per kg of pre-lay diet: riboflavin, 7.5 mg; niacinamide, 30 mg; D-pantothenic acid, 9 mg; choline chloride, 500 mg; DL-α-tocopherol, - IU; menadione, 3.5 mg; retinyl acetate, 10,000 IU; cholecalciferol, 2000 IU; biotin, 100 mcg; folic acid, 1.0 mg; thiamine, 2.0 mg; pyridoxine HCl, 3.0 mg; cyanocobalamin, 25 mcg; FeSO ₄ ×H ₂ O, 147 mg; MnO ₂ , 106 mg; CuSO ₄ × 5H ₂ O, 40 mg; ZnSO ₄ ×H ₂ O, 206 mg; Na ₂ SeO ₃ , 0.88 mg; KI, 1.9 mg; antioxidant (Oxytrap PXN)), - mg.

Given per kg of ration at the start of lay: riboflavin, 9.0 mg; niacinamide, 40 mg; D-pantothenic acid, 12 mg; choline chloride, 600 mg; DL-α-tocopherol, - IU; menadione, 2.5 mg; retinyl acetate, 12500 IU; cholecalciferol, 2500 IU; biotin, 150 mcg; folic acid, 1.5 mg; thiamine, 2.0 mg; pyridoxine HCl, 4.5 mg; cyanocobalamin, 30 mcg; FeSO ₄ ×H ₂ O, 147 mg; MnO ₂ , 130 mg; CuSO ₄ × 5H ₂ O, 40 mg; ZnSO ₄ ×H ₂ O, 206 mg; Na ₂ SeO ₃ , 0.88 mg; KI, 2.6 mg; antioxidant (Oxytrap PXN), - mg.

Given per kg of laying phase ration: riboflavin, 6.0 mg; niacinamide, 30 mg; D-pantothenic acid, 9 mg; choline chloride, 350 mg; DL-α-tocopherol, - IU; menadione, 2.5 mg; retinyl acetate, 12500 IU; cholecalciferol, 2500 IU; biotin, 100 mcg; folic acid, 1.0 mg; thiamine, 1.0 mg; pyridoxine HCl, 2.5 mg; cyanocobalamin, 25 mcg; FeSO ₄ ×H ₂ O, 147 mg; MnO ₂ , 114 mg; CuSO ₄ × 5H ₂ O, 40 mg; ZnSO ₄ ×H ₂ O, 206 mg; Na ₂ SeO ₃ , 0.88 mg; KI, 2.6 mg; antioxidant (Oxytrap PXN), - mg.

Table 14

Analyzed nutritional composition of experimental diets ¹

Feed code LT1302-1 LT1302-2 LT1302-3 LT130-4 LT1302-4 LT1302-5 LT1302-6 LT1302-6 LT1302-7 LT1302-8 LT1302-8 LT1302-9 LT1302-10 LT1302-10 LT1302-11 Party 1 Party 2 Party 1 Party 2 Party 1 Party 2 Party 1 Party 2 Calculated amount of nutrients, % Crude protein 14.5 15.0 14.6 14.5 14.5 14.6 14.5 14.5 14.6 14.5 14.5 14.6 14.5 14.5 14.6 crude fat 3.8 3.5 3.9 3.5 3.5 3.9 3.5 3.5 3.9 3.5 3.5 3.9 3.5 3.5 3.9 crude fiber 3.7 2.9 3.8 2.8 28 3.8 2.8 2.8 3.8 2.8 2.8 3.8 2.8 2.8 3.8 Dry matter 88.1 88.5 87.9 88.6 88.6 87.9 88.6 88.6 87.9 88.6 88.6 87.9 88.6 88.6 87.9 Ca 1.50 3.10 0.90 3.25 3.25 0.90 3.25 3.25 0.90 3.25 3.25 0.90 3.25 3.25 0.90 P 0.70 0.62 0.70 0.61 0.61 0.70 0.61 0.61 0.70 0.61 0.61 0.70 0.61 0.61 0.70 Nutrients Analyzed Crude protein 14.6 14.9 14.6 15.0 14.6 15.4 15.2 14.8 15.0 14.7 14.5 14.8 15.4 14.8 14.8 Crude protein (NIKS) 14.7 14.8 14.5 15.4 15.0 15.4 15.4 15.3 15.0 15.2 15.2 15.3 15.6 15.5 15.0 Crude fat (NIKS) 3.5 2.9 3.6 2.9 2.8 3.6 2.8 2.9 3.6 2.9 2.9 3.6 3.0 2.9 3.7 Crude fiber (NICS) 3.7 2.9 4.0 2.9 3.0 4.0 2.8 3.0 4.0 3.0 3.0 3.8 2.9 2.9 4.0 Moisture (NICS) 11.7 11.4 11.6 11.5 11.4 11.8 11.7 11.4 11.9 11.5 11.6 11.8 11.4 11.5 11.9 Dry matter 88.3 88.6 88.4 88.5 88.6 88.2 88.3 88.6 88.1 88.5 88.4 88.2 88.6 88.5 88.1 Ca 1.6 2.8 0.8 3.4 3.6 1.1 3.2 3.6 1.1 3.6 3.0 1.1 3.5 3.2 1.0 P 0.7 0.6 0.6 0.6 0.6 0.7 0.6 0.6 0.7 0.7 0.6 0.7 0.7 0.6 0.7 % expected amount Crude protein 101 99 99 105 102 105 106 104 103 103 102 103 107 104 102 crude fat 92 82 94 82 79 94 79 82 94 82 82 94 85 82 96 crude fiber 99 101 106 102 106 106 99 106 106 106 106 100 102 102 106 Dry matter 100 100 101 100 100 100 100 100 100 100 100 100 100 100 100 Ca 105 90 92 106 111 123 99 111 120 111 93 117 109 99 108 P 104 99 91 102 104 105 101 104 105 114 99 104 111 98 105

¹ The number of hen diets for the laying period was too large to be produced in one batch, so two batches were produced with the same ingredients.

A total of 730 hens and 92 males of 19-week-old Ross 308 breeding birds were provided. On admission (at the start of the pre-experimental period), hens and males were randomly assigned to floor cages (3-4 males and 30-31 females per cage). Breeding birds were redistributed at the beginning of the experimental period (30 hens and 3 males per cage, the rest of the healthy males were placed in a free cage, the rest of the hens were culled).

Throughout the experiment, breeding birds were kept in separate floor cages for breeding broilers (2.60×2.40 m) on bedding (flax). Each cage was equipped with one height-adjustable bell drinker. The hens were fed by two hopper feeders on a raised mesh floor; cockerel food was fed through a trough feeder to the other side of the cage. The breeding bird was stimulated with light during the preliminary experimental period in accordance with the breed standards. During the experimental period, the length of daylight hours was set to 15 hours per day. Temperature and ventilation were controlled by computer. The temperature was set at 20°C during the entire period. Individual bird weights were recorded weekly from the start of the pre-experimental period. At 31, 33, and 36 weeks of age, 50% of the hens were weighed, and at 35, 37, and 38 weeks of age, only the first four cages were weighed to determine the desired weight gain. The amount of food provided was recorded daily. Egg production and egg quality data were recorded daily. % egg production was calculated as the number of eggs laid per cage divided by the number of bird days of the experimental period. Gross egg production per cage per week was calculated as % egg production (including category II eggs) multiplied by average egg weight per cage. The average egg weight was determined per cell once a week. The eggs of the second category were divided into dirty eggs, broken eggs, eggs without shell, two-yolk eggs, floor eggs and the rest of the eggs of the second category. Mortality was monitored every day; dead animals were not replaced during the experimental period.

Eggs were sent to Ghent University, Belgium for ORAC and vitamin E analysis in the yolk. The method used to determine the content of polyphenols was the method of Folin-Ciocalteu, which is described in the European Pharmacopoeia, using UV-visible spectrophotometry. For ORAC analysis, the method described by Ou et al. "Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe", Journal of Agricultural and Food Chemistry 49: 4619-4626, (2001) and Huang and al. "High-throughput assay of oxygen radical absorbance capacity (ORAC) using a multichannel liquid handling system coupled with a microplate fluorescence reader in 96-well format", Journal of Agricultural and Food Chemistry 50: 4437-4444, (2002).

Statistical analysis was performed according to Provimi standards for testing hypotheses and divergence of means using ANOVA analysis of variance (version 1.3). To compare different treatments, all scores were subjected to a mixed model procedure using SAS (Version 9.3, 2008, SAS Institute Inc., Cary, NC, USA) according to the following statistical model:

Y _ij =μ+α _i + b _j + ε _ij

Where:

Y _ij =specific feature for the experimental unit (cells);

μ=general mean value for a particular trait;

α _i =fixed treatment effect (i=1, 2, 3 or 4);

b _j =random block effect (j=1, 2,..., 6);

ε _ij =error vector.

Contrasts were used to compare processing effects. Descriptions of the contrasts are provided below, and the characteristics of the contrasts to answer each question are shown in Table 15.

1) Was there a linear effect of vitamin E?

2) Was there an effect of replacing PROVIOX 50?

3) Was the effect of changing to PROVIOX 50 dose dependent?

4) Was there an additive effect of adding PROVIOX 50 on the background of vitamin E?

Table 15

Description of contrasts used for statistical analysis

Vit.E Vit.E PROVIOX PROVIOX dose 1 dose 2 dose 1 dose 2 1) -1 1 0 0 2) -1 -1 1 1 3) -1 1 1 -1 4) -1 0 0 1

The nutritional composition of the diets significantly corresponded to the expected values (table 14). The observed health status of the birds was good throughout the experiment. Mortality, including culling, was 6.1%, which is high compared to breeding standards (total mortality and culling during lay 8%). This is mainly due to the relatively high culling rate due to leg problems around 30 weeks of age, which is more common in Ross breeding birds. The technical parameters of the birds corresponded to the standards of the breeding bird of the Ross 308 breed (data not shown). During the entire experimental period (at 30-39 weeks of age) the percentage of broken eggs was significantly below 47% with partial replacement (50%) of vitamin E with PROVIOX 50 (Table 16). This suggests that there has been an improvement in eggshell strength, which is important as the number of hatching eggs will increase, resulting in higher profits from broiler breeders for the farmer.

The technical results of the breeding birds fed the experimental diets (aged 30 to 39 weeks) are shown in Table 16.

Table 16

Technical results of breeding birds fed experimental diets (aged 30 to 39 weeks)

Vitamin E Equivalence (ppm) Vit. E dose 1
80 Vit. E dose 2
160 PROVIOX 50%
80 PROVIOX 50%
160 Total SEM General
p-value Linear effect vit. E Substitution effect on PROVIOX Change to PROVIOX was dose dependent The additive effect of adding PROVIOX to the background n 6 6 6 6 - - - - - - Egg production in percent 30-34 weeks,% 87.1 87.0 83.7 84.8 1.1842 0.143 0.931 0.029 0.604 0.175 Egg production in percent 34-39 weeks,% 81.5 83.0 77.0 80.5 1.6050 0.087 0.511 0.041 0.532 0.657 Egg production in percent 30-39 weeks,% 84.3 85.0 80.4 82.7 1.1941 0.054 0.689 0.015 0.494 0.319 Eggs of the first category 30-34 weeks, % 80.2 78.6 76.5 76.5 1.6123 0.299 0.476 0.081 0.601 0.115 Eggs of the first category 34-39 weeks, % 73.8 74.4 69.5 71.1 2.0875 0.254 0.824 0.060 0.807 0.317 Eggs of the first category 30-34 weeks, % 77.0 76.5 73.0 73.8 1.6706 0.198 0.818 0.040 0.671 0.149 Eggs of the second category Broken eggs 30-34 weeks, % 1.2 1.2 0.4 0.7 0.4041 0.359 0.877 0.101 0.592 0.330 Broken eggs 34-39 weeks, % 1.2 1.5 0.4 1.1 0.3242 0.026 0.429 0.024 0.303 0.843 Broken eggs 30-39 weeks, % 1.2 1.3 0.4 0.9 0.3186 0.071 0.795 0.025 0.377 0.411 Dirty eggs 30-34 weeks, % 1.4 1.8 1.6 1.6 0.2805 0.836 0.372 0.942 0.502 0.634 Dirty eggs 34-39 weeks, % 1.9 1.7 1.6 1.4 0.2631 0.711 0.600 0.343 0.918 0.267 Dirty eggs 30-39 weeks, % 1.7 1.8 1.6 1.5 0.2360 0.944 0.821 0.618 0.740 0.717 Eggs without shell 30-34 weeks, % 0.2 0.3 0.2 0.1 0.0901 0.751 0.524 0.731 0.315 0.745 Eggs without shell 34-39 weeks, % 0.0 0.2 0.1 0.1 0.0537 0.116 0.021 0.992 0.167 0.138 Eggs without shell 30-39 weeks, % 0.1 0.2 0.2 0.1 0.0579 0.429 0.124 0.779 0.162 0.705 Two-yolk eggs 30-34 weeks, % 1.3 1.1 1.3 1.3 0.2363 0.923 0.590 0.720 0.804 0.911 Two-yolk eggs 34-39 weeks, % 0.4 0.5 0.5 0.4 0.1216 0.888 0.759 0.959 0.473 0.867 Two-yolk eggs 30-39 weeks, % 0.9 0.8 0.9 0.8 0.1698 0.960 0.788 0.790 0.939 0.893 Floor eggs 30-34 weeks, % 2.3 3.3 3.0 3.5 0.9608 0.508 0.255 0.426 0.685 0.162 Floor eggs 34-39 weeks, % 3.4 4.1 4.3 5.4 1.3350 0.572 0.608 0.291 0.862 0.175 Floor eggs 30-39 weeks, % 2.8 3.7 3.7 4.5 1.1179 0.526 0.430 0.310 0.967 0.148 Other 30-34 weeks, % 0.5 0.8 0.5 1.0 0.2043 0.334 0.342 0.675 0.672 0.133 Other 34-39 weeks, % 0.8 0.6 0.6 0.9 0.1630 0.485 0.543 0.616 0.176 0.469 Other 30-39 weeks, % 0.6 0.7 0.6 0.9 0.1499 0.262 0.756 0.546 0.257 0.133 Egg weight 30-34 weeks, g 60.1 60.3 60.2 59.6 0.3530 0.520 0.695 0.431 0.264 0.336 Egg weight 34-39 weeks, g 62.4 62.9 63.1 62.4 0.3324 0.392 0.318 0.752 0.103 0.975 Egg weight 30-39 weeks, g 61.2 61.6 61.7 61.0 0.3284 0.497 0.472 0.803 0.156 0.626 Egg mass 30-34 weeks, g/nes/day 52.4 52.5 50.4 50.5 0.7345 0.088 0.931 0.013 0.994 0.076 Egg mass 34-39 weeks, g/nes/day 50.9 52.2 48.7 50.2 1.0783 0.169 0.379 0.069 0.919 0.688 Egg mass 30-39 weeks, g/nes/day 51.6 52.4 49.5 50.4 0.7964 0.089 0.519 0.020 0.948 0.272 Body weight 30 weeks, kg 3.51 3.48 3.51 3.51 0.0230 0.513 0.192 0.476 0.351 0.869 Body weight 39 weeks, kg 3.88 3.83 3.85 3.81 0.0239 0.307 0.176 0.348 0.765 0.078 CV body weight 30 weeks, % 7.9 7.5 7.7 7.3 0.4641 0.888 0.657 0.723 0.951 0.464 CV body weight 39 weeks, % 8.6 8.2 8.7 7.8 0.5199 0.673 0.663 0.841 0.624 0.361 Average daily gain 30-34 weeks, g 5.36 5.65 5.35 4.94 0.4223 0.654 0.610 0.377 0.384 0.461 Average daily gain 34-39 weeks, g 5.96 5.40 5.25 4.66 0.3624 0.081 0.240 0.041 0.966 0.012 Average daily gain 30-39 weeks, g 5.79 5.60 5.38 4.86 0.2857 0.022 0.500 0.010 0.403 0.004

Table 17 presents the results of the analysis of egg yolk composition for ORAC and vitamin E level. As shown in Table 17, the evaluation of egg yolk for ORAC and vitamin E content did not find a statistically significant difference between the two treatment groups for ORAC, vitamin E content, however, found a linear increase due to increased levels of vitamin E in the diet. The proportionality index was found to be about 50% of the dietary vitamin E transfer to the egg, which is consistent with previous studies (Hossain et al., 1998).

Table 17

Egg Yolk Composition Analysis Results for ORAC and Vitamin E Levels

Vitamin E Equivalence (ppm) Vit. E dose 1
80 Vit. E dose 2
160 PROVIOX 50%
80 PROVIOX 50%
160 Total SEM General
p-value Linear effect vit. E Replacement effect on PROVIOX Change to PROVIOX was dose dependent The additive effect of adding PROVIOX to the background n 6 6 6 6 - - - - - - ORAC, -mol TU/g 33.8 33.5 34.2 35.1 1.35 0.7682 0.8417 0.3789 0.6124 0.4341 Vitamin E, µg α-tocopherol/g 139.7 231.6 92.0 165.5 15.0 0.0004 0.0018 0.0047 0.5989 0.3052

The results of this example did not show a beneficial effect on any of the evaluated parameters when PROVIOX 50 was used in breeding poultry diets.

EXAMPLE 5. PRODUCTIVITY OF CHICKS FROM EGGS OF EXAMPLE 4

Within six days, eggs were collected from the broiler breeder flock of Example 4 above. The eggs were stored based on the breeder cage and day of storage for an additional three days before the start of the incubation period. All eggs were incubated until the chicks hatched. All eggs were divided into 24 trays and 6 blocks, with each block representing a storage day. The breeding bird received one of the four experimental diets as shown in Table 12.

A total of 2160 eggs from 37-38 week old broiler breeders (Ross 308 breed) of Example 4 were collected and stored in the broiler breeder egg storage unit. At the beginning of the incubation period, the eggs were weighed and divided into blocks, each block representing a day of storage. Each block consisted of 24 experimental units, with each experimental unit representing a group of 15 eggs. Only intact eggs were included, and eggs that were cracked, dirty, or that had deformed shells were excluded from the test.

The eggs were placed in a NatureForm NMC 2340 incubator with automatic temperature and relative humidity control. From E0 to E18, eggs were placed in setter trolleys, which consist of two shelves each containing 13 trays. Each tray has three spacers for 30 eggs. During this period, the initial temperature in the incubator was set to 37.5° C. and the relative humidity to 54%. The temperature was initially 37.5°C at E0 and gradually decreased from E12 to E18 from 37.5°C to 36.7°C.

After an incubation period (E0-E18), the eggs were transferred to the hatcher baskets using the block and tray structure in the incubator. The eggs were placed in the hatcher section (15 eggs per experimental unit) and then the tray in order in the block (from 1AO to 24CI). Each hatcher trolley has twelve baskets holding 180 eggs each. Eggs from trays 1-12 were placed in setter 1, eggs from tray 13-24 were placed in setter 2. During this period, the temperature in the incubator was set to 36.7° C. and the relative humidity to 70%.

The weight of the egg groups was recorded at E0. At E7 and E18, all eggs were checked by candling, and empty eggs or eggs containing dead embryos (false neoplasms E0-E7, frozen E8-E18) were removed. At E18, the eggs were moved to hatcher baskets after being checked by candling. At the hatching stage (E21), the number of dead chicks, dead chicks (E19-E21) and hatched eggs were recorded. For each repetition, the number of hatched chicks was recorded, as well as the average weight of the chick. Hatching was calculated as a percentage of the total number of eggs laid. Sex was established by feather sexing, female chickens were killed by CO ₂ and disposed of in accordance with local regulations. Five male chicks per experimental unit were selected for the rearing period and allocated to broiler rearing cages. Only chicks with an average body weight, completely dry, showing active movement and with a closed umbilical ring were selected. Following this selection, one cockerel chick from each experimental unit was selected for necropsy. Selected chicks were individually weighed and killed by cervical dislocation to determine residual yolk sac weight, yolk-free body weight ("YFBM"), liver weight, chick length, and leg length for both legs. The liver was immediately placed on dry ice and, after opening, stored at -20° C. for further analysis ("SOD and GSSG").

Relative asymmetry was calculated using the following formula:

RA=(|L-R|/[(L+R)/2])×100

Where:

L=left foot length

R=right leg length.

In order to assess whether there were statistically significant differences between different broiler treatments, all variables were also subjected to mixed model analysis using SAS (Version 9.3, SAS Institute Inc., Cary, NC, 2008) according to the following statistical model:

Y _ijk =μ+α _i +b _j +α(c) _ik +ε _ijk

Where:

Y_ijk=specific feature per experimental unit (rep. 15 eggs);

μ=general mean value for a particular trait;

α _i =fixed treatment effect (i=1-4);

b _j =random effect block/day of storage (j=1-6);

α(c) _ik =random hierarchical effect per cage for keeping breeding birds within treatment (k=1-24);

ε _ijk =error vector.

The hierarchical effect per cage for keeping breeding birds within a treatment was included in the model to adjust for the number of experimental units used per treatment of broiler breeders. Taking into account the design of the experiment, the effect of the day of storage was fully combined with the effect of the block (location in the incubator). Contrasts were used to compare processing effects. Descriptions of the contrasts are provided below and characteristics of the contrasts to answer each question are shown in Table 18.

1) Was there a linear effect of vitamin E?

2) Was there a quadratic effect of vitamin E?

3) Was there an effect of replacing PROVIOX 50?

4) Was the effect of changing to PROVIOX 50 dose dependent?

5) Was there an additive effect of adding PROVIOX 50 on the background of vitamin E?

Table 18

Description of contrasts used for statistical analysis

Vit.E Vit.E PROVIOX PROVIOX dose 1 dose 2 dose 1 dose 2 1) -1 1 0 0 2) -1 -1 1 1 3) -1 1 1 -1 4) -1 0 0 1

The average egg weight at the beginning of the experiment was 63.1 grams per egg. Average hatchability (90.4% of total) was above average compared to the standards of 38 week old Ross 308 breeding birds (87.2% of total). These results indicate that the average fecundity and hatchability and incubation process are good in the current experiment.

Candling results showed a relatively high early embryonic mortality and a very low number of empty eggs (respectively 5.5 and 0.5% of the total number of eggs laid, Table 19), indicating that the sperm production of the males was good. No statistically significant differences in hatchability, empty eggs, percentage of early, middle or late embryonic mortality were observed (figures 4 and 5, table 19).

Table 19

Effect of Broiler Breeder Treatment on Embryonic Mortality and Hatching Rate of Ross 308 Embryos

Vitamin E Equivalence (ppm) Vit. E dose 1
80 Vit. E dose 2
160 PROVIOX 50%
80 PROVIOX 50%
160 General
p-value Linear effect vit. E Quadratic effect vit. E Substitution effect on PROVIOX Change to PROVIOX was dose dependent The additive effect of adding PROVIOX to the background n 24 24 24 24 - - - - - - General hatchability, % 90.6 90.2 89.9 90.9 0.974 0.994 0.659 0.988 0.669 0.898 Hatching of fertilized eggs, % 91.8 90.2 90.4 90.9 0.898 0.803 0.548 0.831 0.522 0.692 Empty eggs, % 1.2 0.0 0.6 0.0 0.274 0.373 0.954 0.906 0.841 0.204 Early embryonic mortality, % 4.0 6.6 6.7 4.8 0.363 0.680 0.099 0.676 0.092 0.583 Average embryonic mortality, % 1.6 0.5 0.6 2.1 0.125 0.432 0.030 0.569 0.023 0.600 Late embryonic mortality, % 2.7 1.8 1.3 1.6 0.423 0.666 0.364 0.210 0.418 0.226 Total embryonic mortality, % 7.7 8.6 8.5 8.5 0.978 0.907 0.790 0.841 0.775 0.740

The hatched chicks did not show any statistically significant differences in mean and relative chick weights (Table 20). In addition, no treatment effect was found for several chick quality measures (YFBM, chick length and relative skewness). The OXISELECT Superoxide Dismutase (“SOD”) Assay Kit and the OXISELECT Total Glutathione Assay (“GSSG”) Assay Kit (GOS/GSSG) commercially available from Cell Biolabs, Inc., San Diego, CA, USA were used for liver analysis chickens. No statistically significant effect was found in the analysis of SOD and HSSH of the liver. However, when comparing quantitative differences, it was found that partial replacement of vitamin E with PROVIOX 50 at a low level (80 ppm vitamin E) increased the antioxidant status of the embryos.

Table 20

Influence of treatment of breeding broilers on embryonic development and quality indicators of chickens when hatching embryos of the Ross 308 breed

Vitamin E Equivalence (ppm) Vit. E dose 1
80 Vit. E dose 2
160 PROVIOX 50%
80 PROVIOX 50%
160 Total SEM General
p-value Linear effect vit. E Quadratic effect vit. E Substitution effect on PROVIOX Change to PROVIOX was dose dependent The additive effect of adding PROVIOX to the background n 24 24 24 24 - - - - - - - Chicken weight EQ, g 62.9 63.0 63.3 63.1 0.3212 0.701 0.472 0.473 0.306 0.593 0.553 Average chicken weight, g 43.8 43.7 44.1 43.6 0.2831 0.428 0.268 0.299 0.641 0.447 0.503 Relative weight of chicken ¹ , % 69.7 69.4 69.7 69.1 0.4038 0.255 0.377 0.430 0.655 0.571 0.103 Chicken weight after opening, g 42.8 43.6 43.8 43.0 0.5057 0.409 0.952 0.102 0.657 0.108 0.763 Yolk weight, g 4.2 4.2 4.2 4.0 0.3299 0.846 0.889 0.553 0.655 0.538 0.530 Relative weight of yolk ² ,% 9.7 9.4 9.5 9.2 0.6998 0.846 0.888 0.975 0.599 0.950 0.385 Liver weight, g 1.2 1.2 1.2 1.2 0.0361 0.708 0.816 0.544 0.677 0.591 0.337 Relative weight of the liver ² , % 2.8 2.7 2.7 2.9 0.0942 0.470 0.554 0.188 0.533 0.158 0.539 YFBM ³ , g 39.6 40.6 40.5 40.1 0.4181 0.127 0.424 0.059 0.517 0.045 0.222 Chick length, cm 18.3 18.4 18.4 18.4 0.1534 0.802 0.895 0.846 0.431 0.872 0.344 Relative asymmetry, % 2.4 2.3 2.7 2.7 0.3456 0.760 0.384 0.848 0.300 0.990 0.557 SOD, units 0.776 0.777 0.777 0.776 0.0010 0.663 0.981 0.227 0.945 0.241 0.836 GSSG, -M 4.27 4.48 4.69 4.56 1.010 0.688 0.667 0.432 0.331 0.505 0.424

No statistically significant effect was found on any of the evaluated indicators. In addition, no statistically significant effect was found on the performance of hatched chicks. No statistically significant effect was found on the results of the analysis of liver enzymes for SOD and HSSH. However, when vitamin E levels are low, substitution has been shown to be effective in increasing the antioxidant capacity of the embryos. It can be concluded that replacing up to 50% of vitamin E in the diet with PROVIOX 50 can be used to reduce the overall cost of feeding for these diets without compromising hatchability and fertility or quality of hatched chicks, at least in the experimental period at 30-38 weeks of age.

EXAMPLE 6. GROWING CHICKS FROM EXAMPLE 5

In this example, hatched chicks were raised to slaughter age and performance was evaluated. The aim of the current experiment was to evaluate the effect on offspring performance of a broiler breeder diet with vitamin E compared with a 50% replacement of vitamin E with PROVIOX 50.

Four types of processing of breeding broilers were evaluated (table 12). The hatched chicks of Example 5 were raised from eggs from the broiler breeder experiment of Example 4, with the breeder receiving a control diet, a high vitamin E diet, or a diet with vitamin E levels (at both levels) at 50% replaced PROVIOX 50. In the current experiment, the same basic diet was fed to all broilers. The experimental period was divided into a starting phase (0-14 days) and a growth phase (14-35 days). At the age of 14 days, all birds were transferred to their growing diet.

Prior to formulating the rations, components (corn and soybean meal) were reserved and analyzed for crude protein (“CP”) (ISO 16634) and calcium (ISO 27085:2009, duplicate) at the Provimi B.V. laboratory, Rotterdam, The Netherlands. In addition, maize was analyzed for phosphorus (ISO 27085:2009; duplicates) and soybean meal for potassium (ISO 27085:2009). Near infrared reflectance spectroscopy analysis (ISO 12099) was used to determine the dry matter content (“DM”), crude fat, crude fiber and crude ash.

Starter and grower diets were produced by Research Diet Services (RDS, Wijk bij Duurstede, The Netherlands). Starter rations were granulated into 2.5 mm granules and growth rations into 3 mm granules with the addition of steam (about 80°C). Diets were formulated based on the components analyzed and to meet the nutrient requirements of the broilers (Cargill, Incorporated CPN EMEA guidelines). Starter and grower rations contained 2750 and 2850 kcal broiler CFU g×kg ^-1 and 10.20 and 9.70 g×kg ^-1 apparent faecal digestible (AFD) lysine, respectively. The compositions of the experimental starter and growth diets are shown in Table 21. The results of the analysis are presented in Table 22.

Table 21

Component and nutritional composition of starter and growth rations

feeding phase Home Rostovaya Feed code BG1305-1 BG1305-11 Component composition (%) Corn 45.58 47.20 Wheat 20.00 20.00 Soybean meal >48% 28.87 26.71 Limestone 1.56 1.22 Monocalcium phosphate 1.35 1.14 Premix for broilers 1% ¹ 1.00 1.00 Fat, animal origin 0.41 1.48 Fats/Oils, Soybean Oil 0.41 0.49 Bicarbonate of soda 0.253 0.186 Salt 0.212 0.209 DL-Methionine 0.199 0.184 L-lysine HCL 0.152 0.160 L-Threonine 0.016 0.017 Nutritional composition (%) 1120ac Crude protein 20.7 19.8 1130ac crude fat 3.3 4.4 1140 crude fiber 2.3 2.3 1150ac Ash 6.2 5.5 1110ac SW 88.7 88.8 1511ac Ca 0.97 0.80 1513ac P 0.67 0.61 1521ac Na 0.16 0.14 1522ac K 0.86 0.82 1523ac Cl 0.20 0.20 1530 deb 233 214 3310 P usv. broilers 0.393 0.349 3312 R dost. broilers 0.43 0.38 3110 CFU for poultry 2931 3032 3112 CFU for poultry (FS-R) 2969 3069 3130 CFU for broilers (CVB) ² 2750 2850 1210 LYS 1.182 1.125 1211 MET 0.509 0.482 1213 SAA 0.851 0.810 1214 THR 0.776 0.740 1215 TRP 0.244 0.231 1216 ILE 0.862 0.818 1218 VAL 0.960 0.915 1219 ARG 1.357 1.283 3240 TFD LYSp ³ 1.057 1.007 3241 TFD METp 0.477 0.451 3243 TFD SAAP 0.767 0.730 3244 TFD THRp 0.671 0.639 3245 TFD TRPP 0.215 0.203 3246 TFD ILEp 0.775 0.735 3248 TFD VALp 0.859 0.818 3249 TFD ARGp 1.242 1.174 3200 AFD LYSp ⁴ 1.020 0.970 3201 AFD METp 0.471 0.445 3203 AFD SAAP 0.745 0.708 3204 AFD THRp 0.643 0.611 3205 AFD TRPP 0.213 0.201 3206 AFD ILEp 0.749 0.710 3208 AFD VALp 0.816 0.776 3209 AFD ARGp 1.201 1.134 1327 C18:2 1.374 1.528

¹ Given per kg of starting diet: vitamin A (retinyl acetate), 12,000 IU; vitamin D3 (cholecalciferol), 5000 IU; vitamin E (DL-α-tocopherol), 30 mg; vitamin K3 (menadione), 2.3 mg; vitamin B1 (thiamine), 1.0 mg; vitamin B2 (riboflavin), 4.5 mg; vitamin B6 (pyridoxine HCl), 2.7 mg; vitamin B12 (cyanocobalamin), 20 mcg; niacin, 40 mg; D-pantothenic acid, 9 mg; choline chloride, 500 mg; folic acid, 0.5 mg; biotin, 100 mcg; FeSO ₄ ×H ₂ O, 150 mg; CuSO ₄ × 5H ₂ O, 40 mg; MNO, 100 mg; ZnSO ₄ ×H ₂ O, 145 mg; KI, 2.0 mg; Na ₂ SeO ₃ , 0.56 mg; antioxidant (Oxytrap PXN), 125 mg. Given per kg of growth diet: vitamin A (retinyl acetate), 10,000 IU; vitamin D3 (cholecalciferol), 2000 IU; vitamin E (DL-α-tocopherol), 20 mg; vitamin K3 (menadione), 2.3 mg; vitamin B1 (thiamine), 0.8 mg; vitamin B2 (riboflavin), 4.5 mg; vitamin B6 (pyridoxine HCl), 1.9 mg; vitamin B12 (cyanocobalamin), 20 mcg; niacin, 30 mg; D-pantothenic acid, 8 mg; choline chloride, 400 mg; folic acid, 0.5 mg; biotin, 50 mcg; FeSO ₄ ×H ₂ O, 150 mg; CuSO ₄ × 5H ₂ O, 40 mg; MNO, 100 mg; ZnSO ₄ ×H ₂ O, 145 mg; KI, 1.9 mg; Na ₂ SeO ₃ , 0.50 mg; antioxidant (Oxytrap PXN), 125 mg.

Table 22

Calculated and analyzed nutritional composition of starter and growth rations

Rations BG1305-1 BG1305-11 Calculated amount of nutrients, % joint venture 20.7 19.8 crude fat 3.28 4.44 crude fiber 2.31 2.27 SW 88.7 88.8 Ca 0.97 0.80 P 0.67 0.61 Analyzed amount of nutrients, % joint venture 21.5 20.2 SP ¹ 20.6 19.1 Raw fat ¹ 3.40 4.3 Crude fiber ¹ 2.50 2.6 SW ¹ 89.3 88.4 Ca 0.92 0.76 P 0.68 0.60 % expected amount joint venture 103.7 102.1 crude fat 103.8 96.8 crude fiber 108.2 114.3 SW 100.7 99.6 Ca 95.0 95.2 P 102.3 98.5 ¹ NIRS analysis

The experiment was carried out in a broiler house unit consisting of two rooms with 36 cages each. A total of 720 day old Ross 308 cockerel chicks from broiler breeders at 38 weeks of age (Example 4) were randomly selected from the total number of hatched cockerel chicks from Example 5. Chicks were selected at random from various experimental units in the selection. incubator to ensure that the broiler breeder unit is not damaged by placing the chicks from one broiler breeder unit in one cage for rearing. Each cage contained 30 chickens with an initial individual BW of 43.3±0.81 and 43.3±0.68 g for unit A and unit B, respectively.

The cages (90×225 cm) had a raised floor consisting of plastic nets covered with a 2 cm layer of wood shavings. Each cage was equipped with two sets of two adjustable nipple drinkers and a feeder, which were placed inside the cage for the first 13 days. Starting on day 14, food was fed through a trough feeder at the front of the cage. Both food and water were provided ad libitum throughout the study. Continuous artificial lighting was maintained for 23 hours per day during the first three days of the experiment, 20 hours per day from days four to seven, and 18 hours per day for the remainder of the experiment. Temperature, relative humidity and ventilation were controlled by computer, with the first thirteen days the temperature was gradually reduced by 0.5°C per day, from the fourteenth day to day 35 the temperature was gradually reduced by about 0.4°C from 34.0°C. C on the day of entry (day-old chicks) to a final temperature of 20.4°C at the end of the experiment (day 35).

Chickens were vaccinated against Newcastle disease by spraying (Poulvac NDW vaccine, Intervet, Boxmeer, The Netherlands) at 15 days of age.

The individual weight of the birds was recorded at the beginning of the experiment (day 0) and at the age of 14, 21, 28 and 35 days. In addition, the feed intake for each cage was recorded on the same day that the birds were weighed. Based on the calculated weight gain and feed intake, the feed to gain ratio (F:G) was calculated as kg feed intake/kg weight gain. The total feed intake per cage was adjusted for mortality, culling and deviations. The European Bird Index was calculated using the following formula:

European Bird Index = (final body weight (g)×(100%-mortality%))/((10×day period)×total RCC)

The European Poultry Index, excluding mortality, was calculated using the following formula:

European Bird Index = (final body weight (g) × 100) / ((10 × period in days) × total RCC)

All parameters were subjected to a mixed model procedure by SAS (version 9.3, SAS Institute Inc., Cary, NC, USA) according to the following statistical model:

Y _ij =μ+α _i + b _j + ε _ij

Where:

Y _ij =specific feature measured for each experimental unit;

μ=general mean value for a particular trait;

α _i =fixed effect of treatment of breeding broilers (i=1-4);

b _j =random block effect (j=1-6);

ε _ij = vector of residual errors.

Contrasts were used to compare processing effects. Descriptions of the contrasts are provided below, and the characteristics of the contrasts to answer each question are shown in Table 23.

1) Was there a linear effect of vitamin E?

2) Was there an effect of replacing PROVIOX 50?

3) Was the effect of changing to PROVIOX 50 dose dependent?

4) Was there an additive effect of adding PROVIOX 50 on the background of vitamin E?

Table 23

Description of contrasts used for statistical analysis

Vit.E Vit.E PROVIOX PROVIOX dose 1 dose 2 dose 1 dose 2 1) -1 1 0 0 2) -1 -1 1 1 3) -1 1 1 -1 4) -1 0 0 1

The nutritional composition of the rations corresponded to the expected values (Table 22). The observed health status of the birds was good throughout the experiment. Mortality, including culling, was 1.4%, which is low compared to actual levels (3 to 4%) as well as previous studies on broiler farming (2012 average: 4.5%).

Unexpectedly, the final offspring body weight, average daily gain and feed intake were significantly higher when partial replacement of vitamin E with PPROVIOX 50 in the diet of broiler breeders (respectively 4.2%, 4.8% and 3.8%), this does not depend on the level of inclusion, as shown in table 24.

Table 24

Effect of Broiler Breeder Treatment on Offspring Mortality, BW, European Poultry Index (EPI), Average Daily Growth (ADG), Average Daily Feed Intake (ADFI) and Feed to Growth Ratio (F:G)

Vit. E dose 1 Vit. E dose 2 PROVIOX dose 1 PROVIOX dose 2 Total SEM p-value contrasts linear effect vit. E replacement effect on PROVIOX Dose dependent substitution Additive PROVIOX effect on the background n ¹ 6 6 6 6 - - - - - - Mortality, % 2.9 0.9 2.4 3.4 - 0.469 nd nd nd nd MT 0 day, g 43.5 42.9 43.5 43.2 0.30 0.518 nd nd nd nd MT 35 day, g ² 2206 2133 2276 2245 46.90 0.094 nd ** nd nd CV 35 day, % 11.5 11.8 12.0 10.6 1.06 0.712 nd nd nd nd EPI ³ 403 392 414 419 8.47 0.009 nd *** nd * EPI, excluding mortality ³ 419 396 427 434 11.15 0.011 ** *** * nd ADG 0-14 day, g 27.3 26.7 28.5 28.2 0.81 0.241 nd * nd nd ADG 14-21 days, g ⁴ 59.5 53.3 60.4 63.1 4.04 0.097 nd * nd nd ADG 21-28 days, g ⁴ 83.0 84.3 88.6 88.2 2.71 0.167 nd ** nd * ADG 28-35 day, g ³ 113.2 107.5 113.0 111.5 3.44 0.493 nd nd nd nd ADG 0-35 day, g ³ 62.3 59.7 63.8 64.1 1.50 0.017 * *** nd nd ADG 14-35 day, g ³ 84.7 81.7 87.3 86.2 1.94 0.146 nd * nd nd ADFI 0-14 day, g 33.5 33.0 35.3 35.0 0.90 0.121 nd ** nd nd ADFI 14-21 days, g 89.9 83.9 91.6 93.8 4.14 0.069 nd ** nd nd ADFI 21-28 days, g 131.9 133.0 138.1 135.6 3.46 0.300 nd * nd nd ADFI 28-35 day, ⁵ g 183.0 176.1 185.9 180.6 3.60 0.179 nd nd nd nd ADFI 0-35 day, ⁵ g 94.3 91.8 97.2 96.0 1.86 0.029 nd ** nd nd ADFI 14-35 day, ⁵ g 134.9 131.0 138.5 136.7 2.64 0.043 nd ** nd nd F:G 0-14 day 1.228 1.236 1.237 1.242 0.008 0.696 nd nd nd nd F:G 14-21 days 1.524 1.606 1.526 1.491 0.047 0.177 nd nd nd nd F:G 21-28 day ⁴ 1.589 1,580 1.561 1.547 0.023 0.458 nd nd nd nd F:G 28-35 day ³ 1.614 1.641 1.647 1.641 0.029 0.827 nd nd nd nd F:G 0-35 day ³ 1.517 1.537 1.524 1.508 0.009 0.022 ** * *** nd F:G 14-35 day ³ 1.579 1.604 1.587 1.564 0.012 0.017 ** ** *** nd F:G adjusted for MT ^5.6 1.507 1.546 1.504 1.485 0.016 0.007 ** *** ** nd

Mortality=percentage of total mortality and culling relative to the number of birds on day 0; EPI=((final body weight (g)×(100%-mortality%))/((10×period in days)×total BCC): EPI without mortality=((final body weight (g)×100%) /((10xperiod in days)xtotal RCC) F:G=feed intake r: gain r).

¹ Separate cage with 30 birds.

² n=5 for vit. E dose 1 and Proviox dose 2. ³ n=5 for vit. E dose 1 and n=4 for Proviox dose 2. ⁴ n=5 for Proviox dose 2. ⁵ n=5 for vit. E doses 1.

⁷ F:G adjusted for BW=F:G adjusted for 2.176 grams of body weight at 35 days of age. Correction by -0.02 for every 100 g overweight.

nd=P>0.10; *=P<0.10; **=P<0.01.

As shown in Table 24, the increase in feed to growth ratio was statistically significantly more significant when replacing the high inclusion level, with the replacement resulting in a 1.9% more efficient feed use. This effect was even more pronounced when adjusted for final body weight (3.9% more efficient offspring; FIG. 6). Overall, the feed-to-growth ratio was statistically significantly (1.4%) worse for the high vitamin E treatment compared to the low vitamin E treatment. In addition, this effect was more pronounced when adjusted for final body weight (2 .6%).

It is not clear why a higher level of vitamin E in the diet of breeding broilers results in less efficient offspring. Published studies of broiler breeder diets based on offspring production generally report vitamin E levels between 0 and 100 ppm, the current study has a high inclusion level of 160 ppm. In practice, even higher levels are sometimes used, but the focus is on the number of chicks hatched per breeder and, to a lesser extent, or even absent, on offspring productivity. A possible explanation for lower offspring performance could be that vitamin E was overdosed in the breeding bird's diet, thereby programming the offspring for less efficient vitamin E uptake, or less effective antioxidant defense. By providing PROVIOX 50 in the diet of broiler breeders, other antioxidant sources are available to the offspring (water soluble sources). This can increase the antioxidant status of the offspring, hence allowing them to grow more and more efficiently.

The results of the current study show that increasing vitamin E in the diet of broiler breeders from 80 to 160 ppm results in less efficient offspring. Surprisingly, it was found that a partial 50% replacement of vitamin E with PROVIOX 50 led to statistically significantly higher overall growth and feed intake (regardless of dose), as well as more efficient offspring at high inclusion levels.

EXAMPLE 7. PARTIAL REPLACEMENT OF VITAMIN E IN DIETS OF BREEDING BROILERS

All breeding birds received the same diet during the preliminary experimental period (49-56 weeks of age). During the experimental period, the breeding birds received a control diet or a diet in which 50% of vitamin E was replaced with PROVIOX 50 (table 25).

Table 25

Experimental Design of Treatments for Broiler Breeders

Treatment Vitamin E 50 (ppm) PROVIOX 50 (ppm)* Control 160 - PROVIOX 80 80

*Vitamin E equivalence.

There were a total of twelve floor cages with approximately thirty hens and three male breeding birds per cage used. The experimental period lasted eight weeks; the breeding birds received the laying phase diet during the entire experimental period. Food and water were limited. The amount of food depended on the average weight gain. The feeding levels during the pre-experimental period and the experimental period are presented in Fig.7. Water was provided for two hours a day, crops were checked regularly to ensure that sufficient water was provided.

Before formulating the diet composition, batches of soybean meal, wheat bran, wholemeal corn and wheat were backed up and wet chemistry analyzed for crude protein, dry matter ("DM"), calcium and phosphorus. In addition, soybean meal was analyzed for potassium content, and wheat bran was analyzed by wet chemistry for crude fiber, potassium, and starch. NIR reflectance spectroscopy analysis was used to determine the content of crude ash, crude fat, crude fiber and moisture and to recheck crude protein. The formulating of the diets was based on the analyzed nutrient content of the reserved components. One batch of food was used during the entire experimental period.

The base diet (corn - soybean meal - wheat and wheat bran) was based on Provimi nutrient recommendations for broiler breeders. The diet compositions were optimized according to the Provimi PNC nutrient coding system. The broiler breeder premix was used without vitamin E and without the addition of other antioxidants. Received different diets for cockerels and hens, while using the same experimental design and composition of the premix. This was done to be able to feed the males according to their nutritional requirements (lower requirements for crude protein, energy and calcium). The composition of the experimental diets and the results of the analysis are presented in table 26 and table 27, respectively. The diets were produced by Research Diet Services (RDS, Wijk bij Duurstede, The Netherlands). Diets were produced separately using one batch of basic ration for female diets and one batch of basic ration for male diets. The rations were produced in the form of compound feed.

Table 26

Component and nutritional composition of experimental diets

Feed code LT1304-prev-1 LT1304-prev-2 LT1304-1 LT1304-2 LT1304-3 LT1304-4 Treatment - - 1 2 1 2 Floor Hen Cockerel Hen Hen Cockerel Cockerel Diet Form compound feed compound feed compound feed compound feed compound feed compound feed Component composition (%) Corn 44.8 38.2 44.98 44.98 40.08 40.08 Wheat 20.0 20.0 20.0 20.0 20.0 20.0 Soybean meal 13.3 6.6 14.7 14.7 6.6 6.6 wheat bran 10.0 30.0 9.1 9.1 28.0 28.0 Coarse limestone 5.6 - 5.2 5.2 0.0 Fine limestone 2.8 1.7 2.6 2.6 1.7 1.7 Monocalcium phosphate 1.1 1.0 1.1 1.1 1.3 1.3 Soybean oil 1.0 1.0 1.0 1.0 1.0 1.0 Premix ¹ (1%) 1.0 1.0 1.0 1.0 1.0 1.0 Salt 0.21 0.17 0.21 0.21 0.22 0.22 Na bicarbonate 0.06 0.16 0.062 0.062 0.092 0.092 L-lysine HCL 0.072 0.024 0.060 0.060 0.009 0.009 DL-Methionine 0.118 0.039 0.123 0.123 0.047 0.047 Vitamin E 50% - - 0.016 0.008 0.016 0.008 PROVIOX 50 - - - 0.008 - 0.008 Nutrient composition (%) - based on the Provimi Nutrient Code System 1120 Crude protein 14.5 13.5 14.5 14.5 13.0 13.0 1130 Crude fat 3.43 3.87 3.59 3.59 3.88 3.88 2250 Crude fiber 2.58 3.91 2.80 2.80 4.06 4.06 1150 Ash 11.9 5.6 11.3 11.3 5.7 5.7 1110 Dry matter 88.4 87.7 88.1 88.1 87.2 87.2 1511 Calcium 3.43 0.90 3.20 3.20 0.90 0.90 1513 Phosphorus P 0.59 0.72 0.59 0.59 0.75 0.75 3312 R dost. 0.35 0.35 0.35 0.35 0.40 0.40 3311 P usv. 0.33 0.35 0.33 0.33 0.39 0.39 1521 Sodium 0.14 0.14 0.14 0.14 0.14 0.14 1522 Potassium 0.63 0.68 0.64 0.64 0.67 0.67 1523 Chloride 0.18 0.16 0.18 0.18 0.18 0.18 1530 dB 171 190 174 174 180 180 3110 OE for poultry 2712 2730 2710 2710 2707 2707 3112 OE for poultry (FS-R) 2733 2735 2741 2741 2737 2737 3120 OE for laying hens 2750 2772 2750 2750 2750 2750 1210 lys .0705 0.563 0.704 0.704 0.530 0.530 1211 met 0.348 0.259 0.352 0.352 0.260 0.260 1213 SA 0.611 0.524 0.613 0.613 0.515 0.515 1214 thr 0.513 0.462 0.516 0.516 0.446 0.446 1215 TRP 0.165 0.158 0.165 0.165 0.151 0.151 1216 isle 0.564 0.488 0.568 0.568 0.470 0.470 1218 val 0.667 0.620 0.669 0.669 0.598 0.598 1219 arg 0.886 0.809 0.892 0.892 0.776 0.776 3200 AFD LYSp 0.580 0.440 0.580 0.580 0.410 0.410 03201 AFD METp 0.315 0.222 0.319 0.319 0.224 0.224 3203 AFD SAAp 0.520 0.429 0.523 0.523 0.423 0.423 3204 AFD THRp 0.404 0.349 0.408 0.408 0.338 0.338 3205 AFD TRPp 0.139 0.130 0.140 0.140 0.124 0.124 3206 AFD ILEp 0.478 0.402 0.482 0.482 0.388 0.388 3208 AFD VALp 0.549 0.497 0.552 0.552 0.479 0.479 3209 AFD ARGp 0.774 0.701 0.781 0.781 0.673 0.673 3240 TFD LYSp 0.616 0.470 0.616 0.616 0.440 0.440 3241 TFD METp 0.324 0.230 0.327 0.327 0.232 0.232 3243 TFD SAAP 0.547 0.449 0.549 0.549 0.444 0.444 3244 TFD THRp 0.436 0.380 0.439 0.439 0.367 0.367 3245 TFD TRPP 0.143 0.133 0.143 0.143 0.127 0.127 3246 TFD ILEp 0.505 0.427 0.509 0.509 0.412 0.412 3248 TFD VALp 0.590 0.529 0.592 0.592 0.511 0.511 3249 TFD ARGp 0.808 0.730 0.815 0.815 0.700 0.700 1327 C18:2 1.62 1.78 1.70 1.70 1.80 1.80 Vitamin E equivalent (IU) 80.00 80.00 80.00 80.00 80.00 80.00

¹ Given per kg of laying phase diet: riboflavin, 6.0 mg; niacinamide, 30 mg; D-pantothenic acid, 9 mg; choline chloride, 350 mg; DL-α-tocopherol, - IU; menadione, 2.5 mg; retinyl acetate, 12500 IU; cholecalciferol, 2500 IU; biotin, 100 mcg; folic acid, 1.0 mg; thiamine, 1.0 mg; pyridoxine HCl, 2.5 mg; cyanocobalamin, 25 mcg; FeSO ₄ ×H ₂ O, 147 mg; MnO ₂ , 114 mg; CuSO ₄ , 40 mg; ZnSO ₄ ×H ₂ O, 206 mg; Na ₂ SeO ₃ , 0.88 mg; KI, 2.6 mg; antioxidant (Oxytrap PXN), - mg.

Table 27

Analyzed nutritional composition of experimental diets

Feed code LT1304-prev-1 LT1304-prev-2 LT1304-1 LT1304-2 LT1304-3 LT1304-4 Calculated amount of nutrients, % Crude protein 14.5 13.5 14.5 14.5 13.0 13.0 crude fat 3.4 3.9 3.6 3.6 3.9 3.9 crude fiber 2.6 3.9 2.8 2.8 4.1 4.1 Dry matter 88.4 87.7 88.1 88.1 87.2 87.2 Ca 3.43 0.90 3.20 3.20 0.90 0.90 P 0.59 0.72 0.59 0.59 0.75 0.75 Nutrients Analyzed Crude protein 14.7 13.7 14.9 14.9 13.4 13.2 Crude protein (NIKS) 14.7 13.7 14.8 15.0 13.0 13.2 Crude fat (NIKS) 3.2 4.1 3.5 3.4 3.8 3.8 Crude fiber (NIKS) 2.8 4.5 3.1 2.9 4.2 4.4 Moisture (NICS) 11.8 12.1 11.9 12.0 12.4 12.4 Dry matter 88.2 87.9 88.1 88.0 87.6 87.6 Ca 3.62 1.02 3.40 3.42 0.94 0.92 P 0.65 0.79 0.65 0.62 0.80 0.82 % expected amount Crude protein 101 101 102 103 102 102 crude fat 93 106 97 95 98 98 crude fiber 109 115 111 104 103 108 Dry matter 100 100 100 100 100 100 Ca 106 113 106 107 104 102 P 110 110 109 104 106 109

A total of 730 hens and 92 males of Ross 308 breeding birds were purchased through an industrial breeding organization (Pluvita B.V., Apeldoorn, The Netherlands). On admission (at the start of the pre-experimental period), hens and males were randomly assigned to floor cages (3-4 males and 30-31 females per cage). Breeding birds were redistributed at the beginning of the laying period (30 hens and 3 males per cage). The rest of the healthy cockerels were placed in a free cage, and the rest of the hens were culled. Animals were not reallocated at the beginning of the following (preliminary) experimental periods.

Throughout the experiment, breeding birds were kept in floor cages for broiler breeding (2.60×2.40 m) on litter (flax). Each cage was equipped with one automatic drinker, adjustable in height. The hens were fed by two hopper feeders on a raised mesh floor; cockerel food was fed through a trough feeder to the other side of the cage.

The breeding bird was stimulated with light during the first preliminary experimental period in accordance with the breed standards. During the experimental period, the length of daylight hours was set to 15 hours per day. Temperature and ventilation were controlled by computer. The temperature was set at 20°C during the entire period. The room temperature was continuously recorded throughout the experiment using a data logger located inside the poultry house. Individual bird weights were recorded at 49, 55, 60 and 64 weeks of age. At 51, 53, 58, and 62 weeks of age, 50% of the hens and all males were weighed, and in the remaining weeks, only the first four cages were weighed to determine the required weight gain. The amount of food provided was recorded daily. Egg production and egg quality data were recorded daily. % egg production was calculated as the number of eggs laid per cage divided by the number of bird days of the experimental period. Gross egg production per cage per week was calculated as % egg production (including category II eggs) multiplied by average egg weight per cage. The average egg weight was determined per cell once a week. The eggs of the second category were divided into dirty eggs, broken eggs, eggs without shell, two-yolk eggs, floor eggs and other eggs of the second category. Mortality was monitored every day; dead animals were not replaced during the experimental period.

Statistics

Statistical analysis was performed according to Provimi standards for testing hypotheses and divergence of means using ANOVA analysis of variance (version 1.3). To compare different treatments, all scores were subjected to a mixed model procedure using SAS (Version 9.3, SAS Institute Inc., Cary, NC, 2008) according to the following statistical model:

Y _ij =μ+α _i + b _j + ε _ij

Where:

Y _ij =specific feature for the experimental unit (cells);

μ=general mean value for a particular trait;

α _i =fixed treatment effect (i=1 or 2);

b _j =random block effect (j=1, 2,..., 6);

ε _ij =error vector.

Contrasts were used to compare processing effects. Descriptions of the contrasts are given below, and the characterization of the contrasts to answer the question is given in Table 28.

1) Was there an effect of PROVIOX 50?

Table 28

Description of the contrast used for statistical analysis

Control PROVIOX 1) -1 1

The nutritional composition of the diets was as expected, as shown in Table 27. The observed health status of the birds was good throughout the experiment. Mortality, including culling, was 6.7%, which is average compared to the standards of breeding birds (total mortality and culling during the 40 weeks of the laying period 8%). Technical performance was in line with Ross 308 breeding standards. Variation within treatments (LSD), expressed as a percentage of average bird weight gain, was 13.0% for the entire experimental period and 1.6% for egg percentage and egg weight, respectively.

Table 29

Technical results of breeding birds fed experimental diets

Control PROVIOX Total SEM General
p-value PROVIOX effect n 6 6 - - - Percentage of egg production 56-64 weeks,% 50.4 54.8 2.2122 0.458 0.193 Percentage of egg production of the 1st category 56-64 weeks,% 45.1 49.0 2.4724 0.600 0.241 Eggs of the second category Broken eggs 56-64 weeks, % 1.5 0.8 0.4942 0.662 0.277 Dirty eggs 56-64 weeks, % 2.1 1.8 0.2878 0.184 0.551 Eggs without shell 56-64 weeks, % 0.0 0.1 0.0908 0.371 0.936 Two-yolk eggs 56-64 weeks, % 0.2 0.1 0.0720 0.650 0.469 Floor eggs 56-64 weeks, % 0.4 1.7 1.0741 0.708 0.382 Other eggs 56-64 weeks, % 1.0 1.3 0.1897 0.750 0.312 Egg weight 56-64 weeks, g 71.3 71.9 0.3499 0.385 0.294 Egg mass 56-64 weeks g/nes./day 35.9 39.4 1.5316 0.401 0.134 Body weight 56 weeks, kg 4.29 4.25 0.0403 0.175 0.433 Body weight 64 weeks, kg 4.45 4.37 0.0442 0.066 0.237 CV body weight 56 weeks, % 9.4 11.3 0.6126 0.115 0.057 CV body weight 64 weeks, % 10.1 11.3 0.6111 0.372 0.211

However, as shown in FIG. 8, when PROVIOX 50 was used in breeding diets, the percentage of egg production quantitatively increased by 8.8% compared to the control diet. For the entire experimental period, no statistically significant effect on egg weight was found, which is consistent with previous studies.

As shown in Table 30, relative eggshell weight increased statistically significantly by 3.8% with partial replacement (50%) of vitamin E with PROVIOX 50. This also applies to the quantitative increase in eggshell strength (7.9%). In quantitative terms, there were also 50% fewer broken eggs during the experimental period with partial replacement of vitamin E with PROVIOX 50.

Table 30

The results of the analysis of indicators of the quality of eggs of breeding birds, which were fed experimental diets

Control PROVIOX Total SEM General
p-value PROVIOX effect n 6 6 - - - Breaking strength, N 33.7 36.3 1.2540 0.492 0.183 Dense protein height, cm 6.66 6.39 0.2863 0.002 0.298 Egg weight, g 70.8 72.2 0.6862 0.118 0.152 Egg white weight, g 36.7 37.9 0.8763 0.794 0.348 Egg yolk weight, g 23.7 23.7 0.3320 0.778 0.956 Eggshell weight, g 6.27 6.64 0.0832 0.039 0.008 Relative weight of egg white, % 51.4 52.6 1.0871 0.790 0.409 Relative weight of egg yolk, % 33.7 33.3 0.3373 0.285 0.541 Relative weight of eggshell, % 8.89 9.23 0.0883 0.082 0.031 color fan 7.72 7.85 0.1849 <,0001 0.580 Minolta L 62 weeks 52.6 52.0 0.3665 <,0001 0.194 Minolta a 62 weeks 1.24 1.19 0.2461 <,0001 0.820 Minolta b 62 weeks 38.4 37.9 0.4640 <,0001 0.209 Minolta L 64 weeks 51.9 52.4 0.2602 <,0001 0.194 Minolta a 64 weeks 0.92 0.90 0.2513 <,0001 0.944 Minolta b 64 weeks 37.2 37.3 0.4207 <,0001 0.865

The percentage of egg production quantitatively increased by 8.8% with partial replacement (50%) of vitamin E with PROVIOX 50. No statistically significant effect on the total average egg weight was found. The relative weight of the eggshell increased by 3.8% with a partial replacement (50%) of vitamin E with PROVIOX 50; this also applies to the quantitative increase in egg shell strength (7.9%).

EXAMPLE 8. PRODUCTIVITY OF CHICKS FROM EGGS OF EXAMPLE 7

For eleven days, eggs were collected from the broiler breeding stock of Example 7. They were stored based on the breeding bird cage and day of storage. All eggs were incubated until the chicks hatched. The eggs were divided into 12 trays and 6 blocks, with each block representing a storage day. The breeding bird received one of the two experimental diets as shown in Table 31.

Table 31

Experimental Design of Treatments for Broiler Breeders

No. Processing of breeding birds Vitamin E 50 (ppm) PROVIOX 50 (ppm)* 1 Control 160 - 2 PROVIOX 80 80 * Vitamin E Equivalence 50

A total of 1080 eggs from the 63 week old broiler breeders of Example 7 were collected and stored in the egg storage unit of the broiler breeder house at the Velddriel Innovation Center. At the beginning of the incubation period, the eggs were weighed and divided into blocks, each block being a combination of a storage day. Each block consisted of 12 experimental units, with each experimental unit representing a group of 15 eggs. Included only intact eggs; cracked, dirty or deformed eggs were excluded from the experiment.

The eggs were placed in a NatureForm NMC 2340 incubator with automatic temperature and relative humidity control. From E0 to E18, eggs were placed in setter trolleys, which consist of two shelves each containing 13 trays. Each tray has three spacers for 30 eggs. During this period, the initial temperature in the incubator was set to 37.5° C. and the relative humidity to 54%. The temperature was set based on the temperature profile, starting at 37.5°C at E0 and gradually decreasing from E12 to E18 from 37.5°C to 36.7°C.

After an incubation period (E0-E18), the eggs were transferred to the hatcher baskets using the block and tray structure in the incubator. The temperature in the incubator was set to 36.7° C. and the relative humidity to 70%. The temperature in the incubator was continuously recorded throughout the experiment using data loggers located at various locations within the incubator.

Egg group weights were recorded at E0, E7 and E18. At E7 and E18, all eggs were checked by candling, and empty eggs or eggs containing dead embryos (false neoplasms E0-E7, frozen E8-E18) were removed. At E18, the eggs were moved to hatcher baskets after being checked by candling. At the hatching stage (E21), the number of dead chicks, suffocated embryos (E19-E21) and pecked eggs was recorded. The number of hatched chicks was recorded for each repetition, as well as the average weight of the chick. Hatching was calculated as a percentage of the total number of eggs laid. Sex was established by sexing by feathers, chickens were used for autopsy. All cockerel chicks were selected for the rearing period and allocated to broiler cages. Only completely dry chickens showing active movement and with a closed umbilical ring were selected. One female chick from each experimental unit was selected for necropsy. The rest of the chickens were killed by CO ₂ and disposed of in accordance with local regulations. Chicks used for autopsy were individually weighed and killed by cervical dislocation to determine residual yolk sac weight, yolk-free body weight ("YFBM"), chick length, and leg length for both legs. Relative asymmetry was calculated using the following formula:

RA=(|L-R|/[(L+R)/2])×100

Where:

L=left foot length

R=right leg length.

To compare different treatments, rates of weight gain (normally distributed) and hatchability and embryonic mortality (binomially distributed) were subjected to mixed model analysis using SAS version 9.3, 2008 (SAS Institute Inc., Cary, NC) according to the following statistical model:

Y _ijk =μ+α _i +b _j +α(c) _ik +ε _ijk

Where:

Y_ijk=specific feature per experimental unit (rep. 15 eggs);

μ=general mean value for a particular trait;

α _i =fixed effect of breeding poultry treatment (i=1-2);

b _j =random effect block/day of storage (j=1-6);

α(c) _ik =random hierarchical effect per cage for keeping breeding birds within treatment (k=1-12);

ε _ijk =error vector.

The hierarchical effect per cage for keeping breeding birds within a treatment was included in the model to adjust for the number of experimental units used to treat breeding broilers. Taking into account the design of the experiment, the effect of the day of storage was fully combined with the effect of the block (location in the incubator). Contrasts were used to compare processing effects. A description of the contrast is given below, and the contrast characteristic for answering the question is given in Table 32.

1) Was there an effect of PROVIOX 50?

Table 32

Description of the contrast used for statistical analysis

Control PROVIOX 1) -1 1

The average egg weight at the start of the trial was 71.2 grams per egg, slightly above the 63 week old Ross breed standard of 70.3 grams per egg. Average hatchability (72.0% of total) was slightly below average compared to 63 week old Ross breeding standards (73% of total). Variation within treatments (LSD), expressed as a percentage of average bird weight gain, was high for chick and yolk-free body weights (6.7% and 6.4%, respectively) compared to previous trials (3.5%, respectively). and 3.7%) using nine replicates per treatment).

As shown in Table 33, no statistically significant effect on overall hatchability and hatchability of fertilized eggs was found.

Late embryonic mortality (E18-E21) was statistically significantly lower with PROVIOX 50 treatment compared to the control diet. However, in the early stages of incubation (E0-E7), mortality was slightly higher when treated with PROVIOX 50 compared to the control diet; thus, no statistically significant effect on hatchability was found.

Table 33

Effect of Broiler Breeder and Hatchery Treatment on Embryonic Mortality and Hatching*

Control PROVIOX General
p-value PROVIOX effect n 6 6 - - General hatchability, % 78.4
(69.4-85.3) 72.2
(62.0-80.5) 0.490 0.297 Hatching of fertilized eggs, % 88.7
(83.4-92.5) 89.2
(83.9-93.0) 0.400 0.867 Empty eggs, % 10.8
(6.2-18.2) 18.7
(11.4-29.0) 0.401 0.126 Early embryonic mortality, % 2.7
(1.4-5.0) 3.6 (2.1-6.4) 0.303 0.436 Average embryonic mortality, % 2.2 (1.1-4.6) 2.3 (1.1-4.8) 0.438 0.943 Late embryonic mortality, % 2.6 (1.5-4.5) 0.8 (0.3-2.1) 0.077 0.042 Total embryonic mortality, % 7.6 (5-11.4) 6.7 (4.3-10.4) 0.218 0.674

As shown in Table 34, mean chick weight at hatch was statistically significantly higher when treated with PROVIOX 50 compared to the control diet. After adjusting for the initial weight of the eggs, it was still statistically significantly higher compared to the control treatment. The relative weight of the residual yolk sac at hatch was statistically significantly lower with PROVIOX 50 treatment compared to the control treatment, suggesting an increase in yolk utilization.

Table 34

Influence of treatment of breeding broilers on embryonic development and chick quality indicators when hatching embryos of the Ross 308 breed

Control PROVIOX General SEM General
p-value PROVIOX effect n 6 6 - - - Egg weight E0, g 70.8 71.4 0.4998 0.783 0.322 E7 egg weight, g 68.0 68.6 0.5049 0.733 0.380 E18 egg weight, g 64.3 65.0 0.5232 0.531 0.383 Moisture loss E0-E7, % 3.9 3.9 0.2394 0.374 0.865 Moisture loss E0-E18, % 9.1 9.1 0.2842 0.111 0.854 Average chicken weight, g 47.7 50.1 0.7240 0.079 0.018 Relative weight of chicken ¹ , % 67.3 70.2 0.9620 0.049 0.012 Chicken weight after opening, g 48.9 49.0 0.5955 0.168 0.883 Yolk weight, g 6.1 5.4 0.3023 0.239 0.067 Relative weight of yolk ² ,% 12.5 10.9 0.5553 0.084 0.032 YFBM ³ , g 42.5 43.2 0.4874 0.010 0.281 Chick length, cm 19.4 19.4 0.0994 0.540 0.404 Relative asymmetry, % 1.4 1.3 0.1301 0.089 0.444 Metatarsus coloration Minolta L (lightness) 61.7 61.6 0.3775 <,0001 0.684 Minolta a (redness) 8.3 8.2 0.3580 <,0001 0.765 Minolta b (yellowness) 13.2 14.2 0.4386 <,0001 0.116 ¹ Expressed as a percentage of egg weight at E0
² Expressed as a percentage of chick weight
³ Body weight without yolk

No statistically significant effect on overall hatchability or hatchability of fertilized eggs was found. Enriching the diet of breeding birds with PROVIOX 50 statistically significantly resulted in heavier offspring and increased yolk utilization.

EXAMPLE 9. GROWING CHICKS FROM EXAMPLE 8

Two feed treatments for breeding broilers were evaluated (Table 35).

Table 35

Experimental Design of Treatments for Broiler Breeders

Treatment Vitamin E 50 (ppm) PROVIOX 50 (ppm)* Control 160 - PROVIOX 80 80

*Vitamin E equivalence.

The hatched chicks of Example 8 were raised from eggs from the broiler breeder experiment of Example 7, with the breeder fed a control diet or a diet in which 50% vitamin E was replaced with PROVIOX 50. In this example, the same basic diet was fed to all broilers. The experimental period was divided into a starting phase (0-14 days) and a growth phase (14-35 days). At the age of 14 days, all birds were transferred to their growing diet.

Prior to formulating the rations, components (corn and soybean meal) were reserved and analyzed for crude protein (“CP”) (ISO 16634) and calcium (ISO 27085:2009) at the Provimi B.V. laboratory, Rotterdam, The Netherlands. Corn was analyzed for phosphorus (ISO 27085:2009) and soybean meal for potassium (ISO 27085:2009). Near infrared reflectance spectroscopy analysis (ISO 12099) was used to determine the content of dry matter, crude fat, crude fiber and crude ash.

Starter and grower diets were optimized within and based on the new Cargill Nutrient System (“CNS”) and practiced poultry feeds (TD 2014 1). The starter and grower diet compositions were derived from the practice diets P_BRHIGH51 and P_BRHIGH52, respectively. According to the CNS matrix, the increase in CFU is explained by the use of the NSP enzyme (Hostazyme X, 100 g/Mt; +40 kcal/kg). The enzyme phytase (Phzyme 10000 TPT) was included in both diets at PHY IDX 9. The compositions of the experimental starter and grower diets are shown in Table 36. The results of the analysis are shown in Table 37. The starter and grower diets were produced by Research Diet Services (RDS, Wijk bij Duurstede , Netherlands). Starter rations were granulated into 2.5 mm granules and growth rations into 3 mm granules with the addition of steam (about 80°C).

Table 36

Component and nutritional composition of diets

feeding phase Home Rostovaya Feed code BG1402-1 BG1402-10 Diet Form Granule - 2.5 mm Granule - 3.0 mm Component composition (g/kg) Wheat (A5734) 20,000 20,000 Corn (A5733) 41.394 44.212 Soybean Meal HP (A5714) 33.665 30.915 Fat, animal origin 0.498 0.636 Fats/Oils, Soybean Oil 0.498 0.636 Bicarbonate of soda 0.000 0.010 Salt 0.379 0.347 Monocalcium phosphate 0.737 0.612 Limestone 1.378 1.191 L-Threonine 0.037 0.044 L-lysine HCL 0.166 0.164 DL-Methionine 0.230 0.215 PHY9 Natup D10000P 0.008 0008 ENZ C HOstx D 100 P - corn 0.010 0.010 Premix for broilers 1% ¹ 1,000 1,000 General content 100.00 100.00 Nutritional composition (%) 1110ac Dry matter 87.5 87.4 1111ac Moisture 12.5 12.6 1120ac Crude protein 21.8 20.7 1130ac crude fat 3.6 3.9 1140 crude fiber 2.6 2.6 1140ac <Fiber 2.5 2.5 1511ac Calcium 0.78 0.68 1513ac Phosphorus R 0.55 0.51 1521ac Sodium 0.16 0.15 1522ac Potassium 0.97 0.92 1523ac Chloride 0.30 0.28 1530 deb 233 221 3317 PO RETP 0.39 0.36 3335 <PLT DEB 230 218 6300 PHY-IDX 9.0 9.0 3110 OE for poultry 2858 2912 3130 OE for broilers 2669 2727 3132ac PME 0 21 2800 2850 3134ac PME 22 42 2880 293 3136ac PME42+ 2950 3006 1209ac <LYSINE 1,300 1.225 1211ac <METHIONINE 0.571 0.543 1213ac <MET+CYS 0.971 0.928 1214ac <THREONINE 0.823 0.789 1215ac <TRIPTOFAN 0.278 0.261 1216ac <ISOLEUCINE 0.918 0.867 1218ac <VALIN 1.037 0.985 1219ac <ARGININE 1.430 1.347 3200ac PO TFD LYS 1,200 1.130 3201ac PO TFD MET 0.545 0.518 3203ac PO TFD MC 0.890 0.850 3204ac PO TFD THR 0.740 0.710 3205ac PO TFD TRP 0.255 0.240 3206ac PO TFD ILE 0.844 0.798 3208ac PO TFD VAL 0.936 0.890 3209ac PO TFD ARG 1.345 1.267 x3201aa PO TFD MET/LYS 0.454 0.458 x3203a PO TFD M+C/LYS 0.742 0.752 x3204a PO TFD THR/LYS 0.617 0.628 x3205a PO TFD TRP/LYS 0.212 0.212 x3206a PO TFD ILE/LYS 0.703 0.706 x3208a PO TFD VAL/LYS 0.780 0.788 x3209a PO TFD ARG/LYS 1.120 1.121 1210 Lysine 1.274 1.199 1211 Methionine 0.557 0.529 1213 Meth.+Cyst. 0.919 0.877 1214 Threonine 0.842 0.807 1215 tryptophan 0.256 0.241 1216 Isoleucine 0.921 0.869 1218 Valine 1.021 0.970 1219 Arginine 1.454 1.368 1230 Glycine+serine 1.979 1.877 3200 Usv. lysine (bird) 1.105 1.037 3201 Usv. methionine (poultry) 0.517 0.491 3203 Usv. Meth.+Cyst. (by bird) 0.808 0.769 3204 Usv. threonine (bird) 0.703 0.673 3205 Usv. tryptophan (bird) 0.224 0.210 3206 Usv. isoleucine (poultry) 0.802 0.756 3208 Usv. valine (bird) 0.870 0.824 3209 Usv. arginine (poultry) 1.287 1.211 3219 Usv. glycine + serine (bird) 1.657 1,570 1327 C18:2 (linoleic acid) 1.5 1.6 1327ac < Linoleic acid 1.8 1.9

¹ Given per kg of starting diet: vitamin A (retinyl acetate), 12,000 IU; vitamin D3 (cholecalciferol), 5000 IU; vitamin E (DL-α-tocopherol), 30 mg; vitamin K3 (menadione), 2.3 mg; vitamin B1 (thiamine), 1.0 mg; vitamin B2 (riboflavin), 4.5 mg; vitamin B6 (pyridoxine HCl), 2.7 mg; vitamin B12 (cyanocobalamin), 20 mcg; niacin, 40 mg; D-pantothenic acid, 9 mg; choline chloride, 500 mg; folic acid, 0.5 mg; biotin, 100 mcg; FeSO ₄ ×H ₂ O, 150 mg; CuSO ₄ × 5H ₂ O, 40 mg; MNO, 100 mg; ZnSO ₄ ×H ₂ O, 145 mg; KI, 2.0 mg; Na ₂ SeO ₃ , 0.56 mg; antioxidant (Oxytrap PXN), 125 mg.

Given per kg of growth diet: vitamin A (retinyl acetate), 10,000 IU; vitamin D3 (cholecalciferol), 2000 IU; vitamin E (DL-α-tocopherol), 20 mg; vitamin K3 (menadione), 2.3 mg; vitamin B1 (thiamine), 0.8 mg; vitamin B2 (riboflavin), 4.5 mg; vitamin B6 (pyridoxine HCl), 1.9 mg; vitamin B12 (cyanocobalamin), 20 mcg; niacin, 30 mg; D-pantothenic acid, 8 mg; choline chloride, 400 mg; folic acid, 0.5 mg; biotin, 50 mcg; FeSO ₄ ×H ₂ O, 150 mg; CuSO ₄ × 5H ₂ O, 40 mg; MNO, 100 mg; ZnSO ₄ ×H ₂ O, 145 mg; KI, 1.9 mg; Na ₂ SeO ₃ , 0.50 mg; antioxidant (Oxytrap PXN), 125 mg.

² Based on CVB (2006) calculations; ³ AFD=apparent faecal digestibility; ⁴ TFD=true faecal digestibility.

Table 37

Calculated and analyzed nutritional composition of starter and growth rations

Diet code BG1402-1 BG1402-10 Calculated amount of nutrients, % Crude protein 21.8 20.7 crude fat 3.6 3.9 crude fiber 2.5 2.5 Dry matter 87.5 87.4 Ca 0.78 0.68 P 0.55 0.51 Nutrients Analyzed Crude protein 22 21.3 Crude protein (NIKS) 22.1 20.8 Crude fat (NIKS) 3.5 3.8 Crude fiber (AOCS Ba 6a-05) 2.2 2.1 Crude fiber (NIKS) 2.6 2.4 Moisture (NICS) 11.6 12.2 Dry matter 88.4 87.8 Ca 0.86 0.67 P 0.61 0.55 % expected amount Crude protein 101 103 crude fat 97 97 crude fiber 102 96 Dry matter 101 100 Ca 110 98 P 111 108

A total of 252 day old Ross 308 cockerel chicks from the 63 week old breeding broilers of Example 7 were selected from the total hatched cockerel chicks of Example 8. damage to a Broiler Breeder Pilot Unit by placing chicks from one Broiler Breeder Pilot Unit in one cage for rearing. Each cage contained 30 chickens with an initial individual body weight of 49.1 g ± 1.0 g and 48.5 g ± 1.0 g for units A and B, respectively.

The cages (90 x 225 cm) had a raised stainless steel floor covered with a 2 cm layer of wood shavings. Each cage was equipped with two sets of two adjustable nipple drinkers and a feeder, which were placed inside the cage for the first 13 days. Starting on day 14, food was fed through a trough feeder at the front of the cage. Both food and water were provided ad libitum throughout the study. Continuous artificial lighting was maintained for 23 hours/day during the first three days of the experiment, 20 hours/day from the fourth day to the seventh day, and 18 hours/day during the remainder of the experiment. Temperature, relative humidity and ventilation were controlled using a computer, while in the first 13 days the temperature was gradually reduced by 0.5°C per day, from day 14 to day 35 the temperature was gradually reduced by about 0.4°C from 34.0°C. C on the day of arrival (day-old chicks) to a temperature of 19.6°C on day 35 and maintained constant at 19.6°C until the end of the experiment (day 35). The room temperature was continuously recorded using data loggers and the relative humidity was set at 50% throughout the experiment. Chicks were spray vaccinated against Newcastle disease using a Poulvac NDW vaccine (Poulvac NDW Vaccine, Intervet, Boxmeer, The Netherlands) at 14 days of age.

The room temperature was continuously recorded throughout the experiment using data loggers placed inside the poultry house, one per room. Individual body weights were recorded at the beginning of the experiment (0 day) and at the age of 7, 14, 21, 28 and 35 days. In addition, feed intake for each cage was recorded on the same day that the chicks were weighed. Based on the calculated weight gain and feed intake, the feed to gain ratio (F:G) was calculated as kg feed intake/kg weight gain. The total feed intake per cage was adjusted for mortality, culling and deviations. The European Poultry Index (De Herdt et al., 1999) was calculated with (1) and without (2) mortality using the following formulas:

European Bird Index = (final body weight (g)×(100%-mortality%))/((10×day period)×total RCC)

1. The European Poultry Index, excluding mortality, was calculated using the following formula:

2. European Bird Index=(final body weight (g)×100)/((10×day period)×total RCC)

To assess the development of the immune status in chickens, all chickens were individually inoculated with HuSA according to the method described by Parmentier et al. and 34 days. Blood plasma was stored for further analysis for IgT, IgA, IgY, and IgM (Wageningen University Laboratory, Wageningen, The Netherlands). On a weekly basis, starting at 21 days of age, several experts rated the quality of the litter from 1 to 5. On the 34th day of the experiment, five birds per cage were randomly selected (excluding obvious deviations). Faeces were collected during the night and taken in the morning (day 35).

After collection, the faeces were thoroughly mixed and a 10 ml plastic tube (heparin blood tube) was filled for free water analysis and an NIR container was filled for NIR analysis (Provimi laboratory, Rotterdam, The Netherlands). The free water content was analyzed by centrifuging the filled tubes at 3000 rpm for 12 minutes. Water (g) on the surface of the solid phase was expressed as a percentage of the weight of faeces (g) before centrifugation. At the age of 35 days, the birds were kept on a starvation diet during the night (about 8 hours) and slaughtered the next day. On the morning of day 36, these selected birds were sequentially weighed, killed with CO ₂ /O ₂ , bled and feathers removed. Carcass weights were determined (whole birds without feathers, blood, organs, intestines, head and legs below the knee joint) and carcass slaughter yield was calculated as a percentage of the weight of the bird deprived of feed (weight of the whole bird before slaughter). In addition, the weight of the breast (pectoralis major, pectoralis minor, sternum and collarbone) and abdominal adipose tissue was determined and their yield calculated as a percentage of the carcass weight. Also determined the weight of the spleen and liver, and also relatively expressed as a percentage of individual body weight.

All normally distributed performance measures were subjected to the PROC MIXED description procedure in SAS version 9.3 (SAS Institute Inc., Cary, NC) according to the following statistical model:

Y _ij =μ+α _i + b _j + ε _ij

Where:

Y _ij =specific feature measured for each experimental unit;

μ=general mean value for a particular trait;

α _i =fixed effect of treatment of breeding broilers (i=1-2);

b _j =random block effect (j=1-6);

ε _ij = vector of residual errors.

Mortality was analyzed as binomial, litter scores as ordinal data using the PROC GLIMMIX description procedure in SAS version 9.3 (SAS Institute Inc., Cary, NC) using the same model. To account for the effect of the day of repeated litter performance for this analysis, the day was taken into account in the statistical model as a covariate. To adjust for subjective influence in litter evaluation, the subject variable was treated as a random effect in the statistical model. The results of the blood test were analyzed using repeated measurements, with a single bird as the subject. Contrasts were used to compare processing effects. A description of the contrast is given below, and the contrast characteristic for answering the question is given in Table 38.

1) Was there an effect of PROVIOX 50?

Table 38

Description of the contrast used for statistical analysis

Control PROVIOX 1) -1 1

The nutritional composition of the rations corresponded to the expected values (Table 37). The observed health status of the birds was good throughout the experiment. Although mortality, including culling, was 5.7%, slightly higher than actual levels (3 to 4%) as well as previous broiler farming studies (mean 3.9%). The mean technical weight gain (mean body weight at 34 days: 2516 g) was higher compared to other experiments conducted in the Velddriel Innovation Center broiler houses (mean BW: 2383 g). Within-block variation (LSD), expressed as a percentage of average bird weight gain, was the average for weight gain and RCC of 4.2% and 1.7%, respectively (max. 21 chicks per cage and 6 replicates - data not shown) compared to previous data (mean LSD MT and CCC: 4.0% and 1.8%).

The growth rates of offspring are not statistically significantly affected by any of the diets of breeding broilers. A previous study evaluating the effect of PROVIOX 50 in breeder diets on offspring growth performance found a 2.4% increase in ADG at inclusion level, which was also used in the current trial. Now the quantitative increase was only 1.3% (see table 39). In a previous study, the substitution effect was more pronounced with high levels of vitamin E (160 ppm, increased ADG and F:G). Current and previous results indicate that the combination of antioxidants in the diet of broiler breeders is effective for offspring growth performance, and the effects appear to be most pronounced for the offspring of younger broiler breeders and at a baseline vitamin E level of 80 ppm.

Table 39

Effect of breeding broiler treatment on offspring mortality, body weight, European poultry index (EPI), average daily gain (ADG), average daily feed intake (ADFI) and feed to growth ratio (F:G) of Ross 308 broiler males

Control PROVIOX p-value Influence of PROVIOX n ¹ 6 6 - - - Mortality, % 4.0 5.0 - 0.063 nd MT 0 days, g 49.0 48.5 0.43 0.790 nd MT 34 days, g 2491 2523 35.03 0.580 nd CV 0 days, % 7 7 0.62 0.491 nd CV 34 days, % 7.1 7.4 1.01 0.911 nd EPI 476 472 10.29 0.183 nd EPI, excluding mortality 495 501 8.48 0.456 nd ADG 0-14 days, g 37.8 37.5 0.65 0.545 nd ADG 14-34 days, g 95.6 97.5 1.40 0.379 nd ADG 0-34 days, g 71.8 72.8 1.03 0.575 nd ADFI 0-14 days, g 44.2 43.6 0.82 0.416 nd ADFI 14-34 days, g 149.7 152.9 2.01 0.570 nd ADFI 0-34 days, g 106.3 107.9 1.44 0.782 nd F:G 0-14 days, g 1.169 1.162 0.008 0.346 nd F:G 14-34 days, g 1.565 1.569 0.011 0.696 nd F:G 0-34 days, g 1.479 1.482 0.009 0.617 nd F:G adjusted to MT ² 1.479 1.476 0.013 0.422 nd

Mortality=percentage of total mortality and culling relative to the number of birds on day 0; EPI=((final body weight (g)×(100%-mortality%))/((10×period in days)×total BCC): EPI without mortality=((final body weight (g)×100%) /((10xperiod in days)xtotal RCC) F:G=feed intake r: gain r).

¹ Individual cage with 21 birds.

² F:G adjusted for BW=F:G adjusted for 2.491 grams of body weight at 34 days of age.

Correction by -0.02 for every 100 g of excess weight.

nd=P>0.10; *=P<0.05; ***=P<0.01.

When evaluating the autopsy rates and the results of the analysis of feces (table 40), no statistically significant differences were found.

Table 40

Influence of breeding broiler treatment on necropsy rates, litter rates and fecal composition of males of the Ross 308 broiler breed

Control PROVIOX p-value PROVIOX effect n ¹ 6 6 - - - MT for 36 days. 2581 2560 40 0.656 nd Carcass, g 1766 1728 25 0.414 nd Carcass, % MT 68.3 67.5 0.44 0.640 nd Breast 36 days, g 725 710 11.3 0.192 nd Breast, % carcass 41.1 41.1 0.28 0.180 nd Breast as % carcass adjusted for BW ² 40.9 40.9 0.28 0.269 nd Abdominal adipose tissue, g 18 20 1.14 0.524 nd Abdominal adipose tissue, % carcass 1.0 1.2 0.07 0.529 nd Liver weight, g 62.0 62.2 1.314 0.062 nd Liver weight, % carcass 2.40 2.44 0.048 0.001 nd Spleen weight, g 2.7 2.6 0.261 0.775 nd Spleen weight, % carcass 0.10 0.10 0.010 0.695 nd Likelihood of an overall lower litter score ^{of 3} 33.4 35.3 - 0.784 nd The content of free water in faeces,% 28.4 29.7 2.71 0.847 nd Feces analyzed using NIRS, g/kg N 12.1 12.6 0.62 0.365 nd NDF 42.2 43.9 5.03 0.546 nd Ca 3.82 3.70 0.195 0.428 nd P 0.19 0.18 0.017 0.716 nd Moisture 794 793 4.88 0.732 nd

¹ On average 5 birds per cage, n is the number of cages.

² Breast meat % adjusted for 2.491 grams of body weight at 42 days of age. Correction by 0.2% for every 100 g of excess weight.

³ Average per treatment (including cell level)

Anti-HuSA IgA titers have been reported to be very low and therefore unreliable, and therefore these results are not discussed. No statistically significant interaction was found between treatment of breeding birds and day for HuSA specific plasma titers (see Table 41).

Table 41

Interaction effects between breeding broiler treatment and screening day for HuSA and LPS titers in plasma

Treatment Control PROVIOX Day 14 21 28 34 14 21 28 34 SEM p-value n ¹ 6 6 6 6 6 6 6 6 - - IgA to HuSA -1.6 0.1 1.4 1.5 -1.6 0.5 0.9 1.8 0.230 0.079 IgG to HuSA 4.2 9.1 8.9 8.5 3.6 9.4 8.9 8.8 0.399 0.400 IgM to HuSA 0.8 3.7 4.0 2.9 0.7 4.4 3.7 3.2 0.194 0.149 IgT to HuSA 1.7 6.0 5.8 5.1 1.3 6.3 5.9 5.2 0.344 0.634 IgA to LPS -2.0 -1.5 -0.3 0.6 -2.1 -1.1 -0.3 0.7 0.173 0.115 IgG to LPS 0.9 0.5 0.8 1.9 1.3 0.7 1.3 1.8 0.115 0.014 IgM to LPS -0.5 1.0 2.5 3.8 0.0 1.3 2.8 3.6 0.128 0.138 IgT to LPS 1.0 1.1 1.8 2.7 1.3 1.6 2.2 2.6 0.111 0.367 ¹ Average 6 birds per cage

For the IgG titer to LPS, a statistically significant interaction was found, which is explained by the relatively higher gain at the age of 34 days for the offspring of the breeding birds receiving the control diet (Fig.9).

HuSA specific titers were statistically significantly day dependent, indicating that values were lower before HuSA inoculation and increased after inoculation, then decreased again as the chicks got older (see FIG. 10). This is in line with what was expected. LPS titers increased as the chicks got older, this is also in line with what was expected.

As shown in Figure 11, LPS titers (IgG, IgM and IgT) were statistically significantly higher in offspring of breeding birds receiving the partially substituted vitamin E with PROVIOX 50. These results indicate that immune activity was higher in offspring of broilers, receiving PROVIOX 50.

Based on the results of the present and previous rearing studies, it can be assumed that broiler growth rate is improved when fed a combination of antioxidants in the diet of breeding broilers. The effects appear to be most pronounced for the offspring of younger breeding broilers and with a baseline vitamin E level of 80 ppm. An analysis of the immune parameters of blood plasma indicates an increase in non-specific titers, with a partial (50%) replacement of vitamin E with PROVIOX 50 in the diet of breeding broilers.

EXAMPLE 10. METHOD FOR FEEDING A PIG FAMILY ANIMAL

The purpose of this example is to prove the efficacy of PROVIOX 50 in pigs. Product efficacy was assessed by comparing the PROVIOX 50 supplemented group based on sow response criteria, litter response criteria, antioxidant status and vitamin E status with a negative control group and groups with different levels of vitamin E.

The test was carried out on a farm in Poland. In general, the conditions of detention and care at the selected enterprise were considered typical for the geographical region. The farm production facilities for mating, gestation and farrowing were completely enclosed buildings with ventilated and heated rooms to maintain the minimum temperature of the comfort zone for sows.

The ambient temperature in the sow gestation room and the farrowing room was maintained at a relatively constant level of 18-20°C, and the relative humidity at 60%. In general, all production facilities comply with the requirements for comparative group tests and the requirements of the current regulations regarding the protection of animals.

The test was carried out on sows (primiparous and multiparous) of the same type of crossing (PIC). The trial included 52 sows in a continuous farrowing system. Each group consisted of 13 animals. The test ran from day 1 of gestation to re-mating in the next cycle.

There were two diet formulations: LP 0-90 days, LK 90 days. gestation-weaning. The feed fed throughout the study was produced at the farm's feed mill. The feed produced was a complete feed in accordance with the standard feeding procedures of the participating farm. The consistency of the food was compound feed. All diets were analyzed for nutrient and vitamin E content (AOAC, 2000). Feed is shown in table 42.

Table 42

Specification LP LK Barley % 40.0 10.0 Triticale % 25.5 20.5 Wheat % 5.0 10.0 Corn % 15.0 32.5 Soybean meal % 9.0 19.0 Vegetable oil (EVO) % - 2.5 Lonocel (lignocellulose product) % 3.0 1.5 Premix LP % 2.5 - Premix LK % - 4.0 nutritional value Clean energy kcal/kg 2155 2320 Crude protein % 14.0 17.0 Lysine % 0.70 1.05 Methionine + cystine % 0.51 0.62 Threonine % 0.51 0.69 tryptophan % 0.16 0.20 Calcium % 0.70 0.95 Digestible phosphorus % 0.36 0.47 Sodium % 0.14 0.25

Test product name: PROVIOX 50.

Each group was fed a complete diet for gestating sows (0-90 days) and for lactating sows (90 days gestation-weaning) with various additions of vitamin E and test product (Table 43A) and lactation diet (see Table 43B).

Table 43A

Diets during pregnancy (0-90 days of gestation) -LP diets

Addition of vitamin E and PROVIOX 50 Group 1 Vitamin E - 0 mg/kg Group 2 Vitamin E - 50 mg/kg Group 3 Vitamin E - 100 mg/kg Group 4 Vitamin E 50 mg/kg + PROVIOX 50 mg/kg

Table 43B

Diets during lactation (90 days of gestation-28 days of lactation) -LK diets

Addition of vitamin E and PROVIOX 50 Group 1 Vitamin E - 0 mg/kg Group 2 Vitamin E - 75 mg/kg Group 3 Vitamin E - 150 mg/kg Group 4 Vitamin E 75 mg/kg+PROVIOX 50 75 mg/kg

The following performance criteria were monitored during the trial: (i) sow response criteria (actual parity, farrowing percentage, sows culled - number and causes, weaning-weaning interval, farrowing percentage next cycle); (ii) litter response criteria (number of piglets, total, live, stillborn, mummified; piglet and litter weight, survival). During the test, the following biochemical criteria (blood) for sows (mating, farrowing and weaning), piglets on day 21 were monitored: (i) glutathione peroxidase (GP) activity; (ii) superoxide dismutase (SOD) activity; (iii) total antioxidant status (TOS); (iv) vitamin E content; (v) vitamin A content.

To determine the oxidative status, blood samples were taken from sows and piglets in test tubes with lithium anticoagulant - whole blood was obtained for analysis. At the same time, blood samples were taken for a blood clot. They were centrifuged (1500 rpm, 15' temp. 4°C) and the resulting serum was pipetted into separate tubes.

In studies of the oxidative status of sows and piglets, reagent kits from Sigma-Aldrich were used corresponding to each of the indicators. The determination was carried out in accordance with the description of the rules and procedures established by the manufacturer.

TOS (Total Antioxidant Status) was determined using Antioxidant Assay Kit (Cat. No. CS0790), SOD (Superoxide Dismutase) activity was determined using SOD 19160 Assay Kit, GP Activity (Glutathione Peroxidase Activity Kit) Cat. No. CGP1, vitamin A and E content according to McMurray and Blanchflower 1979 (Journal of Chromatography 178, 525-531.).

SLA

Serum samples (10 µl) were incubated in duplicate at room temperature for 5 minutes with a working solution of ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (150 Pl) and a solution with myoglobin (20 Pl) At the same time, a calibration curve was prepared based on serial dilutions of Trolox equivalent in the range of 0.015 to 0.42 mM (mmol).

The resulting ferryl radical from myoglobin and hydrogen peroxide causes the oxidation of ABTS and the formation of the ABTS* radical cation. The resulting chromogen has a blue-green color. In the presence of antioxidants, the radical cation is suppressed to a degree dependent on the activity of the antioxidant, and the intensity of the color decreases proportionally. Color was measured using a spectrophotometer at a wavelength of 405 nm.

Serum samples (20 µl) were incubated in duplicate at 37°C for 20 minutes with a working solution of WST (tetrazolium sodium salt, 2-(4-lodophenyl)-3-(4-nitrophenyl)-5-(2.4 -disulfophenyl)-2H-tetrazolium monosodium salt) (200 Pl) and enzyme working solution (20 Pl). Simultaneously, blank samples (two reagents, and a blank for each sample) and a calibration curve based on serial dilutions of SOD in the range of 200 to 0.001 U/mL were obtained. The optical density of a blank sample, standard and test samples was determined on a spectrophotometer at a wavelength of 450 nm. WST forms a water-soluble formazan dye upon reduction of the superoxide anion. The rate of O ₂ recovery is linearly related to xanthine oxidase (KO) activity. SOD activity is measured as the degree of blocking the formation of formazan dye.

Whole blood samples (blood sampling from animals in tubes with lithium anticoagulant) were added in duplicate (10 Pl) to 930 ml of buffer (pH 8), mixed (Vortex 10'), and then 10 Pl of a 70% aqueous solution of tert-hydroperoxide was added. butyl, stirred (inversion) and measured the optical density directly on the spectrophotometer at a wavelength of 340 nm. Measurements were repeated after 1 minute. Simultaneously, blanks (reagent) and a calibration curve were obtained based on serial dilutions of HP in the range of 20 to 50 units/ml. The decrease in the optical density of NADPH (within 1 minute), measured at a wavelength of 340 nm as a result of the oxidation of NADPH to NADP+, is an indicator of HP activity.

Serum samples (1 ml) were deproteinized with anhydrous ethanol (1 ml), extracted with 5 ml of n-hexane (Vortex 5') and then centrifuged (3000 rpm, 10', temp. 4°C). The resulting 4 ml of supernatant was evaporated to dryness under nitrogen atmosphere and then dissolved in 1 ml of 96% ethanol and analyzed by high performance liquid chromatography (HPLC).

The content of retinol and tocopherol was determined by reverse phase high performance liquid chromatography (HPLC) using a SHIMADZU chromatograph. A Nucleosil C18 column was used, the mobile phase was methanol-water (95:5, v/v). The measurement was carried out using a UV detector at a wavelength of 326 nm to determine the content of retinol and a fluorescent detector (excitation wavelength - 295 nm, emission wavelength - 330 nm) to determine the content of tocopherol. The concentration of retinol and tocopherol was calculated from the peaks of external standards (Sigma-Aldrich (±)-α-tocopherol, retinol vitamin A-alcohol).

Experimental data were statistically processed using one-way analysis of variance and Duncan's test. The results obtained were determined using the arithmetic mean (x), standard error of the mean (SEM) and significance level (P). All calculations were carried out using STATISTICA 10 software.

The results of the analysis of the reproductive capacity of sows are shown in table 44 and table 45.

Table 44

Sow productivity

Groups SEM R 1 2 3 4 Sows, n 13 13 13 13 - primiparous 1 1 1 1 - multiparous 12 12 12 12 Average sow parity 3.23 3.85 3.46 3.50 0.183 0.690 Non-pregnant sows after 1 mating, n 5 3 2 2 - - Non-pregnant sows after 2 mating, n 3 2 2 2 - - Farrowing percentage (%) 76.92 84.62 84.62 84.62 5.143 0.999 Culled sows, n 3 2 2 2 - incapable of fertilization 3 2 2 2 Subsequent productivity Weaning-estrus interval, days 5.40 5.09 5.64 5.00 0.102 0.093 Interval weaning-re-mating, days 14.86 9.89 7.70 5.10 1.373 0.100 Farrowing percentage (%) 70.00 80.00 90.91 90.91 5,820 0.544

Table 45

Litter productivity

Groups SEM R 1 2 3 4 Duration of lactation, days 28.00 28.00 28.00 28.00 Litter Response Criteria
1st day
Number of piglets in a litter live births 11.50 12.82 13.18 13.00 0.308 0.224 Weak 0.80 0.36 0.40 0.45 0.104 0.450 stillborn 1.70 1.36 1.09 1.27 0.173 0.677 mummified 0.20 0.18 0.20 0 0.055 0.500 Litter weight, kg ^15.59b 18.50 ^{a 19.07a 19.34a} 0.522 0.037 Piglet weight, kg 1.36 1.44 1.45 1.49 0.023 0.223 weaning Number of piglets in a litter 9.80 11.73 11.82 11.55 0.173 0.055 Litter weight, kg ^{67.85b 83.96b 84.33b 85.98b} 2.329 0.014 Piglet weight, kg 6.94 7.16 7.31 7.48 0.152 0.657 Mortality, % 13.25 8.37 10.64 11.06 0.852 0.261

a, b - P<0.05.

As shown in tables 44 and 45, the mean parities of treated and control sows were 3.85, 3.46, 3.50 and 3.23, respectively. Sows fed diets with vitamin E or vitamin E supplemented with PROVIOX 50 had better fertility and mating efficiency compared to the sows of the negative control group (number of non-pregnant sows after the first and second mating, percentage of farrowing). However, the observed differences are not statistically significant. In this experiment, the addition of a polyphenol product (PROVIOX 50) did not have a statistically significant effect on the number of sows culled, but showed a trend towards a shorter weaning-re-mating interval. The addition of various levels of vitamin E and PROVIOX 50 to the diet of sows increased the percentage of farrowing in the next cycle, but the differences between groups are not statistically significant. Based on the results, it can be concluded that the sows fed diets supplemented with PROVIOX 50 had similar fertility and mating performance compared to the group of sows fed diets with vitamin E.

There were no differences (P<0.05) in the total number of piglets born alive, weak, or born mummified (Table 45). Depending on the treatment, the average number of piglets born alive from the sows studied varied between 11.50 and 13.18 and the average number of weak piglets varied from 0.36 to 0.8. The experimental groups had a higher number of live births per litter (12.82, 13.18, 13.00) than the control group (11.50). The observed differences are not statistically significant (P<0.05). The average number of mummified piglets varied from 0 to 0.2. The number of weaned piglets per litter tended to increase (P=0.06) in sows fed diets with vitamin E supplemented with PROVIOX 50 (11.73, 11.82, 11.55 vs 9.80). The experimental groups statistically significantly (P<0.05) had a higher litter weight (18.50, 19.07, 19.34 kg) than the control group (15.59 kg). The birth weight of piglets in the experimental group was 1.44 (group 2), 1.45 (group 3) and 1.49 (group 4), respectively, while in the control animals (group 1) averaged 1.46 kg. The observed differences are not statistically significant (P<0.05). The vitamin E and PROVIOX 50 supplemented groups statistically significantly (P<0.05) had higher litter weights at weaning (85.05, 84.33, 85.98 kg) than the control group (67.85 kg). After the lactation period, the final body weight of piglets from treatments 2, 3 and 4 was 7.16, 7.31 and 7.48 kg, respectively, while animals from the control group (1) weighed an average of 6.94 kg. Differences in weight between groups at weaning are not statistically significant. The use of vitamin E and PROVIOX 50 in the diets of sows reduces the mortality of experimental piglets, but the differences between the groups are not statistically significant. The three main causes of death were weakness, crushing by the sow, and dehydration.

Based on the results, it can be concluded that sows fed diets supplemented with PROVIOX 50 had similar fertility, mating efficiency and litter performance compared to the group of sows fed diets supplemented with vitamin E.

At each measurement period during gestation and lactation, serum α-tocopherol (vitamin E) concentrations were higher (P<0.001) when vitamin E was added to the diet of sows. As the level of vitamin E supplementation increased, serum α-tocopherol concentrations increased (Table 46). Although an increase in serum vitamin E was demonstrated in both periods (farrowing and weaning), the results showed a greater increase at farrowing than later. In group IV (vitamin E 50/75 mg/kg + PROVIOX 50), serum α-tocopherol concentrations at farrowing and weaning were similar to group III (vit. E 100/150 mg/kg). There was a decrease in serum α-tocopherol from mating to farrowing, but only in groups 1 and 2. With the basal diet, serum vitamin A (retinol) concentrations decreased from mating to farrowing. When vitamin E supplementation was provided, a linear increase (P<0.001) in serum vitamin A concentration at farrowing was observed. Compared to pigs fed 50/75 mg vitamin E/kg diet (group 2), pigs fed diets 3 (100/150 mg vit. E/kg) and 4 (vit. E+PROVIOX 50) had higher serum vitamin A concentrations. Fortification of the diet with vitamin E and PROVIOX 50 had a positive effect (P<0.05) on serum vitamin A concentrations in sows. Vitamin E had no effect on serum vitamin A concentrations at weaning.

Conducted various analyzes to evaluate the antioxidant effect of vitamin E and PROVIOX 50 in the diet, as shown in table 46.

Table 46

The content of vitamins E and A (pg / ml) in the serum of sows

Specification Groups SEM R 1 2 3 4 Vitamin E mating 1.04 1.09 1.11 1.06 0.012 0.106 Farrow 0.42 ^{C 0.74b 1.16a 1.14a} 0.050 <0.001 weaning ^{0.75C 1.02b} 1.13 ^{A 1.06ab} 0.026 <0.001 Vitamin A mating 0.14 0.15 0.16 0.16 0.003 0.100 Farrow ^{0.07C 0.11B} 0.15 ^Ab 0.17 ^Aa 0.007 <0.001 weaning 0.16 0.15 0.15 0.16 0.002 0.091

a, b - P<0.05 A, B - P<0.01.

The total antioxidant status (TOS) of serum was used as an indicator of the antioxidant status of the animal. The addition of different levels of vitamin E and PROVIOX 50 changes OSA. For OSA, differences were observed between control (0 vit. E) and experimental groups. Serum OSA increased more in sows fed vitamin E with PROVIOX 50 than in sows fed vitamin E alone. mating. Serum SOD activity values were influenced by dietary vitamin E levels at farrowing and weaning (P<0.001). The highest SOD activity values were observed in sows fed vitamin E and PROVIOX 50, especially at weaning (P<0.001). The value of GP (glutathione peroxidase) activity in the blood of sows from the control group was statistically significantly lower (P<0.001) than in sows receiving vitamin E. The highest values of GP activity were observed in sows fed with vitamin E and PROVIOX 50 in both periods (farrowing and weaning) (P<0.001).

On the concentration of α-tocopherol in the serum of piglets aged 21 days. influenced both the source of antioxidants (vitamin E and PROVIOX 50) and the level of vitamin E fed to reproductive females (see tables 47 and 48).

Table 47

General antioxidant status (OSA mmol/l serum), activity of superoxide dismutase (SOD u/ml serum) and glutathione peroxidase (GP u/ml blood) in the serum and blood of sows

Specification Groups SEM R 1 2 3 4 SLA mating 1.27 1.26 1.29 1.29 0.007 0.304 Farrow ^{1.14Bb 1.18a 1.19a 1.20a} 0.007 0.007 weaning 1.16 ^{Bb 1.18B} 1.20 ^{Ba 1.29a} 0.010 <0.001 SOD mating 70.99 71.43 73.28 71.53 0.676 0.610 Farrow 61.13 ^{C 77.14b 79.60AB 82.82a} 1.458 <0.001 weaning ^{61.88d 79.50C 90.93b 140.27a} 4.715 <0.001 GP mating 37.24 37.73 38.85 38.92 0.271 0.059 Farrow ^27.00D 32.77 ^{C 37.64B} 40.11 ^A 0.835 <0.001 weaning ^27.23D 34.78 ^{C 36.48B 40.27A} 0.766 <0.001

Vitamin E supplementation in sow diets resulted in an increase (P<0.001) in serum α-tocopherol concentration. When lactating sows were fed 150mg/kg vitamin E, their piglets had higher (P<0.001) serum α-tocopherol concentrations than piglets from sows fed 75mg/kg vitamin E. Piglets from sows fed PROVIOX 50 had higher (P<0.001) serum α-tocopherol concentrations at day 21 than piglets of lactating sows fed 75 mg/kg vit. E. Serum retinol (vitamin A) concentrations increased (P<0.001) as vitamin E levels in the sow diet increased. The highest serum vitamin A concentrations were observed in piglets from sows fed vitamin E and PROVIOX 50 (P<0.001).

The addition of different levels of vitamin E and PROVIOX 50 alters OSA. For OSA, there were differences between control and experimental groups (P<0.001). Mean serum OSA concentrations were higher in the vitamin E supplemented groups than in the non-supplemented groups. Serum SOD activity values were highest in piglets from sows supplemented with vitamin E and PROVIOX 50 compared to piglets from sows supplemented with vitamin E alone. lower (P<0.001) than piglets from sows fed vitamin E. When lactating sows were fed 75mg/kg vitamin E and PROVIOX 50, their piglets had a higher (P<0.001) blood GP activity than piglets from sows fed 75 or 150 mg vitamin E/kg diet.

Based on the results, it can be concluded that sows fed diets supplemented with PROVIOX 50 had similar or higher vitamin E status and higher antioxidant status compared to sows fed diets supplemented with vitamin E only. It was concluded that PROVIOX 50 can replace 50% vitamin E with the same (productivity) or better (antioxidant status) effect.

Unless the context clearly requires otherwise, throughout the description and claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is, in the sense of "including but not limited to". Words using singular or plural also include plural or singular, respectively. When the word "or" is used in a claim with respect to a list of two or more items, the word encompasses all of the following interpretations of the word: any of the items in the list, all items in the list, and any combination of items in the list.

The foregoing detailed description of the embodiments of the invention is not intended to be exhaustive or to limit the invention to the precise form disclosed above. Although specific embodiments of the invention and examples thereof are described above for illustrative purposes, various equivalent modifications are possible within the scope of the invention, which should be clear to experts in the relevant field. For example, while the steps are presented in a specific order, in alternative embodiments, the steps may be performed in a different order. The various embodiments described herein may also be combined to provide additional embodiments.

In general, the terms used in the following claims should not be construed as limiting the invention to the particular embodiments disclosed in the specification, unless the above detailed description clearly defines them. Although certain aspects of the invention are set forth below in certain claims forms, the inventors contemplate various aspects of the invention in any number of claims forms. Accordingly, the inventors reserve the right to add additional claims after filing the application in order to extend such additional forms of the claims to other aspects of the invention.

Claims (11)

1. A method for increasing the productivity of an animal family of a pig, including: feeding an animal of the pig family as part of its feed ration with a composition containing: vitamin E; And a source of polyphenols, while productivity is increased compared to an animal fed a feed ration that does not contain the composition, and vitamin E is present in the feed ration at an inclusion level of from about 1 to 300 ppm. 2. The method of claim 1, wherein the performance of the porcine animal includes reproductive capacity, litter performance, and antioxidant status. 3. The method according to claim 1 or 2, wherein the porcine animal is a fattening pig, a breeding pig, a piglet, a sow, a gilt, a boar, and a boar. 4. A method according to one of the preceding claims, wherein the animal is fed during gestation, lactation, mating or before mating. 5. The method according to one of the preceding claims, in which the source of polyphenols includes at least one of the following: onion extract, grape seed extract and rosemary extract. 6. The method according to one of the preceding claims, wherein the ratio of the level of inclusion of vitamin E to the level of inclusion of the source of polyphenols in the composition is 1:1 of vitamin E to the source of polyphenols. 7. The method according to one of the preceding claims, wherein the ratio of vitamin E incorporation to polyphenol source incorporation in the composition may be in the range of 1:2 to 1:5 vitamin E to polyphenol source. 8. The method according to one of the preceding claims, wherein the antioxidant status includes superoxide dismutase, glutathione peroxidase, and vitamin A and vitamin E.

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