FEED SUPPLEMENT AND ITS USE

Abstract

The present invention relates to feed supplements comprising vitamins and canthaxanthin and the use of such supplements for improving performance and health in poultry, in particular in poultry pullets. It has been found surprisingly that feed additive compositions as hereinafter defined improves immune status, bone and skeletal development, flock uniformity, growth and feed conversion of pullets.

The feed additive composition according to the present invention comprises canthaxanthin, at least one vitamin selected from the group consisting of vitamin C and vitamin E, a trace mineral, in particular selenium, and optionally a mixture of at least two compounds selected from the group consisting of thymol, eugenol, vanillin and -terpinene.

Claims

1. A method for improving feed intake, weight gain, feed conversion ratio and/or bone development in pullets and/or for boosting the immune system of pullets comprising administering to a pullet a feed composition comprising canthaxanthin in combination with at least one vitamin selected from the group consisting of vitamin C and vitamin E, selenium and optionally a mixture of at least two compounds selected from the group consisting of thymol, eugenol, vanillin and -terpinene.

2. The method according to claim 1, wherein the feed composition comprises canthaxanthin in combination with vitamin C, vitamin E, selenium and a mixture of essential oils comprising thymol and eugenol.

3. A feed supplement composition for pullets, comprising canthaxanthin, at least one vitamin selected from the group consisting of vitamin C and vitamin E, a trace mineral, and optionally a mixture of essential oils comprising at least two compounds selected from the group consisting of thymol, eugenol, vanillin and -terpinene. A feed supplement composition according to claim 3, comprising canthaxanthin, vitamin C, vitamin E, selenium and a mixture of essential oils comprising as main ingredients thymol and eugenol

4. The feed supplement composition according to claim 3, comprising canthaxanthin, vitamin C, vitamin E, selenium and a mixture of essential oils comprising thymol and eugenol.

5. A premix or regular animal feed which comprises a feed supplement composition according to claim 3.

6. An animal feed comprising: 2 to 100 ppm canthaxanthin, preferably 2 to 10 ppm; 20 to 200 ppm vitamin E, preferably 30 to 150 ppm; 20 to 200 ppm vitamin C, preferably 50 to 150 ppm; 0.01 to 0.5 ppm selenium, preferably 0.05 to 0.2 ppm; and 20 to 250 ppm of a mixture of essential oils, preferably 30 to 150 ppm.

7. An animal feed according to claim 6 comprising: 2, 4 or 6 ppm canthaxanthin; 35, 70 or 105 ppm vitamin E; 50, 100 or 150 ppm vitamin C; 0.05, 0.1 or 0.15 ppm selenium; and 34, 67 or 101 ppm of a mixture of essential oils.

8. An animal feed according to claim 6 comprising: 2% to 10% thymol, preferably 5%; 10% to 20% eugenol, preferably 17%; 0.5% to 5% -terpinene, preferably 1%; 5% to 10% vanillin, preferably 7%; and 55% to 82.5% fillers, carriers and emulsifying surfactants.

Description

EXAMPLE

1. EXPERIMENTAL DESIGN

Global Design

[0073] The experiment concerns 4 groups of 260 pullets. Each group is divided into 10 repeats of 26 birds. Animals are placed in cages during rearing period.

[0074] Bodyweight measurements are planned at day-old and at 3, 8, 12 and 17 weeks of age. Feed intake is also calculated at the same ages. Thus, we can determine feed conversion ratio for each period.

[0075] Immune status is evaluated at 6, 12, 15 and 17 weeks of age.

[0076] Bone and skeletal development is estimated through tibia strength measurements coordinated at 3 different ages: 4, 8 and 17 weeks of age.

Experimental Groups

[0077] 4 treatments are compared: [0078] A batch: control with no supplement, [0079] B batch: with feed supplement composition (FSC) in the feed (dosage of 2 kg/t), [0080] C batch: with feed supplement composition (FSC) (dosage of 4 kg/t), [0081] D batch: with feed supplement composition (FSC) (dosage of 6 kg/t).

TABLE-US-00001 B C final final D dosage dosage final dosage in feed in feed in feed FSC comprises: ppm ppm ppm Vitamin E: ROVIMIX E50 35 70 105 Vitamin C: ROVIMIX C 50 100 150 Canthaxanthin: CAROPHYLLRed 2 4 6 Selenium: SELSAF2000 0.05 0.1 0.15 Essential oils: CRINA100.696 34 68 102

2. MANAGEMENT OF THE BIRDS DURING THE TEST

Vaccine Program

[0082] Vaccine program is attached to protocol (annex 1).

Feeding

[0083] Feeding program is divided into 3 periods: [0084] From day-old to 3 weeks of age: Starter feed (PLD), [0085] From 4 to 10 weeks of age: Grower feed (PLC), [0086] From 11 to 17 weeks of age: Pullet feed (PLE).

[0087] Full description of different feeds used during rearing period is attached to protocol (annex 2).

[0088] Detail of feed composition is presented in table below:

TABLE-US-00002 Starter Grower Pullet 0-3 weeks 4 to 10 weeks 11 to 18 weeks Kg feed/bird 0.5 2.5 4 Composition % Corn 41.6 35.8 35 Wheat 25 32.5 34 wheat bran 1.2 7.5 7.5 Soya meal 48 27.5 20 19 Carbonate 0 0.65 1 Bical Phosph 1.65 1.3 1.25 NaCl 0.2 0.25 0.25 sodium bicarbonate 0.2 0.1 0.1 methionine DL 0.15 0.1 0.1 Lysine Hcl 78 0.1 0 Choline premix 20% 0.2 0.2 0.2 Trace elements premix 1 1 1 Vitamin premix 0.6 0.6 0.6 FSC 0-2-4-6 kg/t 0-2-4-6 kg/t 0-2-4-6 kg/t % protein 18.5 16 16 Fat 3 2.5 2.5 Fiber 3.5 3.5 3.5 methio 0.45 0.37 0.36 lysine 1.04 0.79 0.77 Ash 6 6 6 Na 0.15 0.14 0.14 Ca 1 1.1 1.3 Phosphorus 0.6 0.6 0.6 ME Kcal 2845 2753 2749 mg/kg except vit A et D in International units Vitamin A 7800 7800 7800 Vitamine D3 3000 3000 3000 Vitamin E 15 15 15 Vitamin B 1 1.26 1.26 1.26 Vitamin B2 4.8 4.8 4.8 Vitamin B6 2.4 2.4 2.4 Vitamine B12 0.009 0.009 0.009 Vitamin K3 2.4 2.4 2.4 Niacin 37.2 37.2 37.2 Panthothenic acid 8.7 8.7 8.7 Folic acid 0.72 0.72 0.72 Biotin 0.12 0.12 0.12 Vitamin C 12 12 12 Choline 400 400 400 cobalt 0.4 0.4 0.4 Copper 15 15 15 Iodium 1.5 1.5 1.5 Iron 65 65 65 Manganese 90 90 90 Selenium 0.2 0.2 0.2

[0089] The FSC is produced with wheat middlings as carrier.

3. EXPERIMENTAL PROCEDURE AND TRAITS MEASURED

Calendar of the Test

[0090] Data collection takes place between 1 and 123 day-old. The main events are listed below:

TABLE-US-00003 Age (weeks) Events During all the Mortality recording rearing period Day 1 Individual wingbanding Distribution of birds among repeats Individual bodyweight measurement Beginning of supplementation Week 3 Feed intake measurement Individual bodyweight measurement Week 4 Blood sampling (for oxidative test) Tibia sampling Week 6 Blood sampling (for ND IHA analyses) Week 8 Feed intake measurement Individual bodyweight measurement Tibia sampling Week 9 Blood sampling (for oxidative test) Week 12 Feed intake measurement Individual bodyweight measurement Blood sampling (for ND IHA) Week 14 Blood sampling (for oxidative test) Week 15 Blood sampling (for ND IHA analyses) Week 16 Blood sampling (for oxidative test) Week 17 Feed intake measurement Individual bodyweight measurement Blood sampling (for ND IHA analyses and IBD ELISA analyses) Tibia sampling

Bone and Skeletal Development

[0091] Bone and skeletal development is characterized evaluating tibia strength.

[0092] Synergie 200 MTS compression machine allows to measure stiffness, maximum force and fracture force of the tibia. 20 left tibia per treatment are analysed at 3 different ages: 4, 8 and 17 weeks old.

4. CRITERIA TO ESTIMATE EFFECT OF THE EXPERIMENTAL PRODUCT

[0093] Bodyweight: Bodyweights (average and uniformity) are compared at 4 different ages: 3, 8, 12 and 17 weeks of age. [0094] Feed consumption: Feed intakes are compared at 4 different ages: 3, 8, 12 and 17 weeks of age. [0095] Feed Conversion Ratio is ratio between feed intake and bodyweight. Treatments can also be compared for this trait at 4 different ages: 3, 8, 12 and 17 weeks of age. [0096] Immune status: Immune status is compared at 4 different ages: 6, 12, 15 and 17 weeks of age. [0097] Bone and skeletal development: Tibia strengths (stiffness and fracture force) are compared at 3 different ages: 4, 8 and 17 weeks of age.

5. STATISTICAL ANALYSIS

Experimental Unit/Statistical Unit

[0098] Experimental unit is the repeat of 26 pullets.

[0099] Statistical unit is the bird or the cage.

Comparability of Initial Groups

[0100] Day-old chicks are randomly distributed among different repeats.

[0101] Furthermore, day-old bodyweight measurements inform us about comparability of initial groups for average and uniformity of bodyweights.

Statistical Tests

[0102] Alpha risk will be 5%.

[0103] SAS software will be used to run statistical tests. [0104] Quantitative traits analysis

[0105] For quantitative traits as bodyweight, mathematical model is:

X.sub.ijk=+.sub.i+E.sub.ij [0106] X.sub.ij=measured trait [0107] =average [0108] .sub.i=fixed effect of i.sup.th treatment [0109] E.sub.ij=residual

[0110] Constraints of above mathematical model are: [0111] data from each population have to be normally distributed and to get same variance, [0112] recordings are supposed to be independent.

[0113] Normality of the traits is tested with residuals of Kolmogorov-Smirnov test.

[0114] Evenness of variances is tested with Bartlett test.

[0115] Fixed effects are tested using variance analysis. Then, Fisher's LSD (Least Significant Difference test) is used to compare the treatment groups means. [0116] Qualitative traits analysis

[0117] .sup.2 test is used to compare proportions (mortality trait for example)

6. RESULTS

[0118] Feeding animals with a feed supplement composition according to the invention shows significant effect on [0119] average bodyweight: experimental treatments>control treatment, [0120] feed conversion ratio: control treatments>experimental treatments, [0121] tibia strength: experimental treatments>control treatment.

[0122] Increasing dosage does not improve the effects. Therefore the dosage B looks to be satisfactory in improving bodyweight, feed conversion and tibia strength.

[0123] Supplements have also a positive impact on immune status for Newcastle disease offering a better and more homogeneous protection for the birds of C and D treatments.

MORTALITY AND BODYWEIGHT MEASUREMENTS

[0124] Bodyweight according to treatment at 1, 22, 56, 85 and 123 days of age

TABLE-US-00004 Age (days) Bodyweight (g) Mortality (weeks) Treatment (CV %) rate (%) Pullets # Statistical analysis 1 d A 34.1 2.5 (7.3) a 260 Kolmogorov-Smirnov Test NS.sup.1 B 34.1 2.9 (8.4) a 260 Bartlett Test NS C 34.2 2.6 (7.6) a 260 Variance analysis: NS D 33.9 2.5 (7.3) a 260 Treatment comparison 22 d A 167.6 19.3 (11.5) b 0.8% 258 Kolmogorov-Smirnov Test HS (3 wk) B 162.8 21.6 (13.3) a 1.9% 254 Bartlett Test NS C 167.8 21.1 (12.6) b 0.8% 258 Variance analysis: HS D 169.1 21.4 (12.6) b 0.8% 258 Treatment comparison B < A, C, D 56 d A 622.2 67.4 (10.8) a 1.5% .sup.235.sup.2 Kolmogorov-Smirnov Test HS (8 wk) B 634.6 67.2 (10.6) ab 2.3% 232 Bartlett Test NS C 640.7 72.0 (11.2) b 1.2% 237 Variance analysis: HS D 644.9 66.9 (10.4) b 1.2% 237 Treatment comparison A < C, D 85 d A 1056.6 93.4 (8.8) a 1.5% .sup.216.sup.3 Kolmogorov-Smirnov Test NS (12 wk) B 1078.5 94.1 (8.7) b 2.3% 214 Bartlett Test NS C 1080.7 94.7 (8.8) b 1.2% 217 Variance analysis: HS D 1083.8 86.7 (8.0) b 1.2% 217 Treatment comparison A < B, C, D 123 d A 1479.5 110.7 (7.5) a 1.5% 216 Kolmogorov-Smirnov Test NS (17 wk) B 1524.8 114.8 (7.5) b 2.3% 214 Bartlett Test NS C 1520.2 116.8 (7.7) b 1.2% 217 Variance analysis: VHS D 1515.0 107.6 (7.1) b 1.2% 217 Treatment comparison A < D, C, B .sup.1NS = Non significant, S = Significant difference (P < 0.05), HS = Highly Significant difference (P < 0.01), VHS = Very Highly Significant difference (P < 0.001) .sup.220 animals per treatment are killed at 27 d for taking tibia sample .sup.320 animals per treatment are killed at 56 d for taking tibia sample

[0125] Generally speaking, mortality rate is quite low for every treatment. It varies between 1.2% and 2.3% according to group. There are no statistical significant difference between treatments at 17 weeks-old (.sup.2: p=0.677).

[0126] At Day 1, average bodyweights of the different treatments are comparable (around 34 g). At day 22, B group shows lower bodyweight than the 3 other groups. Then, at the 3 other ages (56, 85 and 123 day-old), average bodyweights of the 3 experimental batches (B, C and D) are higher than the control group (A). Difference is around 40 g at 17 weeks-old ( half a week of growth).

[0127] Flock uniformity is comparable for the 4 groups. Thus, Bartlett test that compares evenness of variances is not significant. At 123 day-old, bodyweight CV vary between 7.1 and 7.7% according to the group.

AILT FEED CONSUMPTION

[0128] Daily Feed Consumption according to treatment at 22, 56, 85 and 123 days of age

TABLE-US-00005 Feed Consumption Age (days) Treatment (g/bird/day) (CV %) Cages # Statistical analysis 22 d A 17.6 1.0 (5.8) a 10 Kolmogorov-Smirnov Test NS.sup.1 (3 wk) B 17.4 0.8 (4.7) a 10 Bartlett Test NS C 17.2 0.5 (3.0) a 10 Variance analysis: NS D 17.6 1.1 (6.4) a 10 Treatment comparison 56 d A 36.4 0.6 (1.6) a 10 Kolmogorov-Smirnov Test S (8 wk) B 35.8 1.0 (2.7) a 10 Bartlett Test NS C 36.2 1.0 (2.7) a 10 Variance analysis: NS D 36.6 1.1 (3.1) a 10 Treatment comparison 85 d A 45.3 0.5 (1.1) a 10 Kolmogorov-Smirnov Test NS (12 wk) B 46.0 1.0 (2.1) a 10 Bartlett Test NS C 45.8 0.9 (2.0) a 10 Variance analysis: NS (p = 0.098) D 46.3 1.0 (2.1) a 10 Treatment comparison 123 d A 53.8 0.7 (1.3) a 10 Kolmogorov-Smirnov Test NS (17 wk) B 54.5 1.0 (1.8) a 10 Bartlett Test NS C 53.8 1.1 (2.0) a 10 Variance analysis: NS D 54.1 1.0 (1.9) a 10 Treatment comparison .sup.1NS = Non significant, S = Significant difference (P < 0.05), HS = Highly Significant difference (P < 0.01), VHS = Very Highly Significant difference (P < 0.001)

[0129] There is no statistical significant difference between the different groups for Daily Feed

[0130] Consumption (DFC) trait. Average DFC is 54.0 g/bird/day at 123 day-old for the whole sample.

FEED CONVERSION RATIO

[0131] Feed Conversion Ratio accordina to treatment at 22. 56. 85 and 123 days of age

TABLE-US-00006 Age (days) Treatment FCR (CV %) Cages # Statistical analysis 22 d A 2.21 0.14 (6.5) a 10 Kolmogorov-Smirnov Test NS.sup.1 (3 wk) B 2.25 0.11 (4.8) a 10 Bartlett Test NS C 2.15 0.09 (4.1) a 10 Variance analysis: NS D 2.19 0.20 (9.0) a 10 Treatment Effect 56 d A 3.36 0.15 (4.5) a 10 Kolmogorov-Smirnov Test NS (8 wk) B 3.24 0.09 (2.7) a 10 Bartlett Test NS C 3.24 0.12 (3.7) a 10 Variance analysis: NS (p = 0.06) D 3.24 0.10 (3.2) a 10 Treatment Effect 85 d A 3.74 0.08 (2.2) a 10 Kolmogorov-Smirnov Test NS (p = 0.06) (12 wk) B 3.72 0.07 (1.8) a 10 Bartlett Test NS C 3.69 0.13 (3.4) a 10 Variance analysis: NS D 3.72 0.11 (3.0) a 10 Treatment Effect 123 d A 4.54 0.06 (1.4) b 10 Kolmogorov-Smirnov Test NS (17 wk) B 4.46 0.08 (1.8) a 10 Bartlett Test NS C 4.39 0.08 (1.9) a 10 Variance analysis: HS D 4.45 0.10 (2.2) a 10 Treatment Effect C, D, B < A .sup.1NS = Non significant, S = Significant difference (P < 0.05), HS = Highly Significant difference (P < 0.01), VHS = Very Highly Significant difference (P < 0.001)

[0132] Bodyweights of dead birds (2 origins: mortality or tibia sampling at 27 or 56 days of age) are included to calculate Feed Conversion Ratio (FCR).

[0133] At 123 days-old, the 3 experimental groups (B, C and D) show better FCR than control group (A). Differences between treatments are not significant at younger ages (22, 56 and 85 days of age).

IMMUNE STATUS

Newcastle Disease

[0134] Newcastle Disease antibodies titres according to treatment at 40, 81, 105 and 123 days of age

TABLE-US-00007 Age (days) Log(titre) (CV (weeks) Treatment %) Pullets # Statistical analysis 40 d A 0.6 0 (0) 20 Kolmogorov-Smirnov Test (6 wk) B 0.6 0 (0) 20 Bartlett Test C 0.6 0 (0) 20 D 0.6 0 (0) 20 81 d A 1.6 0.4 (25.0) 19 Kolmogorov-Smirnov Test HS (12 wk) B 1.6 0.5 (31.4) 19 Bartlett Test NS C 1.8 0.6 (34.8) 20 D 1.6 0.5 (31.4) 20 105 d A 2.6 0.7 (28.2) 19 Kolmogorov-Smirnov Test HS (15 wk) B 2.7 0.7 (24.6) 19 Bartlett Test NS C 2.4 0.6 (26.6) 20 D 2.4 0.9 (36.0) 20 123 d A 3.9 0.4 (11.1) 18 Kolmogorov-Smirnov Test HS (17 wk) B 3.8 0.4 (10.0) 18 Bartlett Test NS (p = 0.098) C 3.9 0.2 (6.0) 20 D 3.9 0.3 (8.5) 20 .sup.1NS = Non significant, S = Significant difference (P < 0.05), HS = Highly Significant difference (P < 0.01), VHS = Very Highly Significant difference (P < 0.001)

[0135] Newcastle Disease antibodies titres classes according to treatment at 123 days of age

TABLE-US-00008 Age (days) Class (weeks) Treatment 0 1 Pullets # Chi2 123 d A 5 13 18 NS.sup.1 (17 wk) B 5 13 18 (p = 0.069) C 0 20 20 D 3 17 20 .sup.1NS = Non significant, S = Significant difference (P < 0.05), HS = Highly Significant difference (P < 0.01), VHS = Very Highly Significant difference (P < 0.001)

[0136] At 40 days-old (before 1.sup.st ND vaccination), no antibodies are detected.

[0137] Statistics are run on logarithm of titres. Normality of the trait is not accepted at any age then results of variance analysis are not presented.

[0138] Bartlett test is close to be significant at 123 day-old. Thus, titres of A and B treatments seems to show higher CV at this age.

[0139] To compare treatments at 123 day-old, we choose to create 2 titres categories:

[0140] Class 0: titre value<4096,

[0141] Class 1: titre value4096.

[0142] We use .sup.2 test to compare proportions of birds in each class. Differences between groups are, here, close to be significant (p=0.069). With a larger sample size, we would probably get significant differences (p<0.05). Thus, A and B treatments would show a higher proportion of birds with lower antibodies titres values.

Infectious Bursal Disease

[0143] Infectious Bursal Disease antibodies titres accordina to treatment at 123 days of age

TABLE-US-00009 Age (days) (weeks) Treatment IBD Titre (CV %) Pullets # Statistical analysis 123 d A 4564 616 (13.5) 16 Kolmogorov-Smirnov Test NS (p > 0.15) (17 wk) B 4686 704 (15.0) 18 Bartlett Test NS (p = 0.93) C 4557 609 (13.4) 20 Variance analysis: NS (p = 0.90) D 4540 655 (14.4) 20 .sup.1NS = Non significant, S = Significant difference (P < 0.05), HS = Highly Significant difference (P < 0.01), VHS = Very Highly Significant difference (P < 0.001)

[0144] Immune status is not statistically different between treatments for IBD.

BONE AND SKELETAL DEVELOPMENT

[0145] Fracture Force, Maximum Force, and Stiffness of tibia according to treatment at 27, 56 and 123 days of age

TABLE-US-00010 Age (days) Fracture Force (N) Maximum Force (N) Stiffness (N/mm) (weeks) Treatment (CV %) (CV %) (CV %) Pullets # 27 d A 24.4 13.4 (55.0) 50.7 10.0 (19.7) 50.2 10.1 (20.1) 20 (4 wk) B 19.3 6.9 (35.9) 49.4 7.4 (15.0) 49.3 9.1 (18.4) 20 C 21.0 14.1 (67.3) 47.4 9.2 (19.5) 45.2 10.4 (23.0) 20 D 25.5 15.0 (58.9) 52.3 10.4 (19.9) 49.6 12.4 (25.1) 20 56 d A 83.5 44.0 (52.7) 132.5 24.9 (18.8) 97.0 21.0 (21.7) 19 (8 wk) B 95.7 49.2 (51.4) 145.6 31.8 (21.8) 103.1 18.5 (18.0) 20 C 93.3 44.8 (48.0) 141.4 26.5 (18.7) 104.3 20.2 (19.4) 20 D 77.8 44.9 (57.7) 134.1 35.6 (26.5) 95.8 25.6 (26.7) 20 123 d A 80.2 41.3 (51.4) 173.8 26.2 (15.1) 136.1 17.1 (12.5) 20 (17 wk) B 93.5 47.2 (50.5) 191.4 18.1 (9.5) 148.4 17.6 (11.9) 20 C 91.3 42.8 (46.8) 180.2 23.7 (13.1) 143.3 17.0 (11.9) 20 D 90.5 58.7 (64.9) 188.7 25.1 (13.3) 147.3 18.2 (12.4) 20 .sup.1NS = Non significant, S = Significant difference (P < 0.05), HS = Highly Significant difference (P < 0.01), VHS = Very Highly Significant difference (P < 0.001)

[0146] Statistical analysis for tibia strength measurements at 27 days of age

TABLE-US-00011 Fracture Maximum Force Force (N) (N) Stiffness (N/mm) Kolmogorov-Smirnov HS.sup.1 NS (p = 0.083) NS (p = 0.095) Test Bartlett Test S NS NS Variance analysis NS NS NS Treatment comparison .sup.1NS = Non significant, S = Significant difference (P < 0.05), HS = Highly Significant difference (P < 0.01), VHS = Very Highly Significant difference (P < 0.001)

[0147] Statistical analysis for tibia strength measurements at 56 days of age

TABLE-US-00012 Fracture Maximum Force Force (N) (N) Stiffness (N/mm) Kolmogorov-Smirnov S.sup.1 NS NS Test Bartlett Test NS NS NS Variance analysis NS NS NS Treatment comparison .sup.1NS = Non significant, S = Significant difference (P < 0.05), HS = Highly Significant difference (P < 0.01), VHS = Very Highly Significant difference (P < 0.001)

[0148] Statistical analysis for tibia strength measurements at 123 days of age

TABLE-US-00013 Maximum Fracture Force Stiffness Force (N) (N) (N/mm) Kolmogorov-Smirnov NS.sup.1 NS NS Test Bartlett Test NS NS NS Variance analysis NS (p = 0.873) NS (p = 0.080) NS (p = 0.117) Treatment comparison .sup.1NS = Non significant, S = Significant difference (P < 0.05), HS = Highly Significant difference (P < 0.01), VHS = Very Highly Significant difference (P < 0.001)

[0149] Bodyweight and tibia weight of the pullets sacrificed for tibia strength evaluation at 27, 56 and 123 days of age

TABLE-US-00014 Age (days) Bodyweight (g) Tibia weight (g) Pullets (weeks) Treatment (CV %) (CV %) # 27 d A 190 21 (10.9) 20 (4 wk) B 194 21 (10.6) 20 C 176 17 (9.6) 20 D 186 32 (17.0) 20 56 d A 613 74 (12.1) 5.2 0.7 (12.5) 19 (8 wk) B 623 58 (9.4) 5.3 0.6 (12.0) 20 C 655 60 (9.2) 5.3 0.6 (12.2) 20 D 620 76 (12.3) 5.1 0.8 (15.2) 20 123 d A 1476 113 (7.6) 10.9 0.9 (7.8) 20 (17 wk) B 1558 86 (5.5) 11.5 0.7 (6.4) 20 C 1490 109 (7.3) 11.2 0.7 (6.0) 20 D 1517 121 (8.0) 11.3 0.5 (4.6) 20 .sup.1NS = Non significant, S = Significant difference (P < 0.05), HS = Highly Significant difference (P < 0.01), VHS = Very Highly Significant difference (P < 0.001)

[0150] There is no significant difference between treatments for tibia fracture force at any age.

[0151] Tibia maximum force and stiffness differences are close to be significant (with, respectively, p=0.080 and p=0.117) at 123 days-old when they are not at younger ages (27 and 56 days-old). Thus, control group is characterized by weaker tibia compared to experimental batches. With a larger sample size, we would probably get significant differences (p<0.05). Higher values for the 3 experimental treatments are partially explained by higher bodyweights and tibia weights for these groups. Thus, introducing these traits in the mathematical model, we significantly affect p value:

[0152] Bodyweight as covariate in the model: [0153] Maximum force: p value for treatment effect=0.271, [0154] Stiffness: p value for treatment effect=0.368,

[0155] Tibia weight as covariate in the model: [0156] Maximum force: p value for treatment effect=0.265, [0157] Stiffness: p value for treatment effect=0.481.

TABLE-US-00015 Annex 1 WEEK DAY VACCINES DETAILS Supplier ADMINISTRATION COMMENTS 1 1 MAREK (Rispens) Rispens: This vaccine contains the MERIAL (Sanofi INJECTION INACTIVATED Rispens CVI 988 strain of chicken herpes group) VACCINE virus. This product is used as an aid in the prevention of very virulent Marek's disease in chickens. 1 1 NOBILIS BI MA5 A live vaccine for the immunization of INTERVET SPRAY LIVE VACCINE chickens against Infectious Bronchitis Hatchery serotype Massachusetts (strain Ma5) 1 1 VAXXITEK HVT + IBD This vaccine contains a live strain of MERIAL INJECTION INACTIVATED serotype 3 vectored virus that has been VACCINE shown to aid in the prevention of Bursal disease and Marek's disease. This vaccine is recommended for in ovo vaccination of 18 to 19 day old embryonated chicken eggs. 2 3 21 POULVAC IB QX 2000 Infectious bronchitis virus variant D388 PFIZER SPRAY LIVE VACCINE (QX) 4 5 35 NEMOVAC 1000 D a freeze-dried lie vaccine against Swollen MERIAL SPRAY LIVE VACCINE Head Syndrome prepared from a modified live virus (PL21 strain) multiplied in VERO cells. It is the first and only chicken- derived live Avian Pneumovirus vaccine. As an aid in the control of respiratory disease in chickens associated with Avian Pneumovirus 6 42 BIORAL H 120 2000 D Modified live vaccine against avian MERIAL SPRAY LIVE VACCINE infections bronchitis H 120 strain 6 43 AVINEW 1000 DOSES NewCastle Disease MERIAL SPRAY LIVE VACCINE 7 8 54 POULVAC LARYNGO LARYNGO TRACHEITIS INFECTIOUS PFIZER WATER LIVE VACCINE 9 10 70 NEMOVAC 1000 D SWOLLEN HEAD SYNDROME MERIAL SPRAY LIVE VACCINE 11 77 NOBILIS BI 4 91 live vaccine for the immunization of INTERVET SPRAY LIVE VACCINE chickens against infectious bronchitis serotype 4-91 and related serotypes. 12 13 91 NOBILIS LA SOTA Live vaccine for the immunization of INTERVET SPRAY LIVE VACCINE chickens against Newcastle Disease 14 98 POULVAC Avian encephalomyelitis PFIZER WATER LIVE VACCINE ENCEPHALO 15 106 NOBILIS RT PONTE EGG DROP SYNDROM, IB, RT, INTERVET INJECTION INACTIVATED NEWCASTLE VACCINE