Compositions and methods for improving hen welfare with beta-mannanase and probiotics

Abstract

The present disclosure relates to a composition comprising capsicum, a functional mineral compound, and yeast cell walls. Furthermore, a method of feeding an animal, such as a cattle, with the composition is provided to prove a beneficial effect on the animal. The compositions and methods described herein can benefit overall animal health and future productivity, especially in young animals.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 400,098, filed on August 23, 2022, under 35 U.S.C. §119(e), the entire disclosure of which is incorporated herein by reference. Technical Field

[0003] The present disclosure relates to supplements containing beta-mannanase, the supplements containing beta-mannanase and / or probiotics. In addition, methods of administering the supplements to poultry, such as chickens, are provided to demonstrate beneficial effects on poultry. The compositions and methods described herein can benefit overall animal health and future productivity, especially in laying hens. Background Art

[0004] Improving poultry animal health is an important consideration for the agriculture and food production industries. In modern commercial operations, poultry feeds can be supplemented to improve animal health and welfare.

[0005] For example, being able to maintain healthy birds, being able to improve their eating, walking and consuming behaviors, is generally beneficial to overall bird health and future productivity. In addition, in commercial poultry operations, improving egg production productivity and the resulting egg quality is an important consideration. Therefore, new compositions and methods are needed to help manage birds to promote healthy behaviors and improve egg production characteristics.

[0006] Probiotics can reduce heat stress and abnormal behavior in broiler chickens and improve health status. Such responses come from the regulatory ability of probiotics under the microbiota-gut-brain axis. Probiotics can also alleviate the stress response of the hypothalamus-pituitary-adrenal axis and reduce the levels of corticotropin-releasing hormone, adrenocorticotropic hormone and corticosterone in plasma or brain.

[0007] Enzyme supplementation is another strategy that can improve intestinal health by reducing the impact of anti-nutritional components. The use of β-mannanase can help non-ruminant animals process non-starch polysaccharides that can reduce nutrient digestibility. This component is found in plant cell walls and is present in many ingredients used in large quantities in animal feed, such as soy. The main hemicellulose found in plant cell walls is β-mannan, which can also be found on the surface of microorganisms. Therefore, when a diet containing β-mannan is ingested, the animal's innate immune system is activated, and the response is the proliferation of monocytes, macrophages, dendritic cells and increased cytokine production. Such factors produce unnecessary energy expenditure and an increase in inflammatory responses. By hydrolyzing β-mannan, this enzyme can improve the digestibility of mannan, increase beneficial bacteria, improve immunity, digestion and absorption of nutrients, and in addition limit the proliferation of potential pathogens in the intestine. Summary of the invention

[0008] The present disclosure provides supplements comprising β-mannanase and / or probiotics and related methods. The compositions and methods of the present disclosure surprisingly provide a synergistic effect of improving avian behavior and egg quality. The present disclosure describes a composition comprising β-mannanase (e.g., Hemicell HT TM ) and probiotics (e.g., Protexin TM ) supplements that can be added to feed to improve animal health and animal welfare.

[0009]

[0013] Additional features of the disclosure will become apparent to those skilled in the art after considering the illustrative embodiments illustrating the best mode of carrying out the disclosure as presently understood. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The detailed description makes particular reference to the accompanying drawings, in which:

[0011] Figure 1 An example of an egg with a small amount of feces present is shown.

[0012] Figure 2 An example of an egg with a large amount of feces is shown.

[0013] Figure 3 Shown are ultramicroscopy images collected in a single eggshell during the first stage.

[0014] Figure 4 Shown are ultramicroscopy images collected in a single eggshell during the second stage.

[0015] Figure 5 Shown are ultramicroscopy images collected in a single eggshell during the third stage. DETAILED DESCRIPTION

[0016] In an illustrative aspect, a method is provided comprising the step of administering one or more supplements to a non-human animal. In one embodiment, wherein the animal is a poultry. In one embodiment, the poultry is selected from the group consisting of: a chicken, a turkey, and a duck. In one embodiment, the poultry is a chicken. In one embodiment, the chicken is a hen. In one embodiment, the hen is an egg-laying hen. In one embodiment, the poultry is present in a cage system.

[0017] In one embodiment, the supplement comprises Hemicell TM In one embodiment, the supplement comprises Hemicell HT TM In one embodiment, the supplement comprises β-mannanase, also referred to herein as "β-mannanase."

[0018] In one embodiment, the β-mannanase is provided by fermentation of Paenibacillus lentus. In one embodiment, the β-mannanase is applied at a dosage of 100 g / ton to 500 g / ton. In one embodiment, the β-mannanase is applied at a dosage of 100 g / ton. In one embodiment, the β-mannanase is applied at a dosage of 200 g / ton. In one embodiment, the β-mannanase is applied at a dosage of 300 g / ton. In one embodiment, the β-mannanase is applied at a dosage of 400 g / ton. In one embodiment, the β-mannanase is applied at a dosage of 500 g / ton.

[0019] In one embodiment, the supplement comprises Protexin TM. In one embodiment, the supplement comprises a probiotic supplement. In one embodiment, the probiotic supplement comprises Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium bifidum, Enterococcus faecium and Streptococcus thermophiles. In one embodiment, the supplement comprises a probiotic supplement, and the probiotic supplement comprises Enterococcus faecium. In one embodiment, the Enterococcus faecium is the National Collection of Industrial, Food and Marine Bacteria (NCIMB) culture number 11181.

[0020] In one embodiment, the probiotic supplement is administered at a dosage of 10 g / ton to 100 g / ton. In one embodiment, the probiotic supplement is administered at a dosage of 10 g / ton. In one embodiment, the probiotic supplement is administered at a dosage of 20 g / ton. In one embodiment, the probiotic supplement is administered at a dosage of 30 g / ton. In one embodiment, the probiotic supplement is administered at a dosage of 40 g / ton. In one embodiment, the probiotic supplement is administered at a dosage of 50 g / ton. In one embodiment, the probiotic supplement is administered at a dosage of 60 g / ton. In one embodiment, the probiotic supplement is administered at a dosage of 70 g / ton. In one embodiment, the probiotic supplement is administered at a dosage of 80 g / ton. In one embodiment, the probiotic supplement is administered at a dosage of 90 g / ton. In one embodiment, the probiotic supplement is administered at a dosage of 100 g / ton.

[0021] In one embodiment, the supplement comprises i) β-mannanase and ii) probiotics. In one embodiment, the β-mannanase and the probiotics provide a synergistic effect in improving the health of the animal.

[0022] In one embodiment, the supplement is present in the animal's feed. In one embodiment, the feed is a free-choice feed.

[0023] In one embodiment, said administering provides an improvement in the health of said animal. In one embodiment, said improvement in the health of said animal is an improvement in animal welfare. In one embodiment, said improvement in the health of said animal comprises an improvement in feeding behavior. In one embodiment, said improvement in the health of said animal comprises an improvement in walking behavior. In one embodiment, said improvement in the health of said animal comprises an improvement in standing behavior. In one embodiment, said improvement in the health of said animal comprises an improvement in egg-laying behavior.

[0024] In one embodiment, the improvement in the health of the animal comprises an improvement in liquid consumption behavior. In one embodiment, the improvement in the health of the animal comprises an improvement in feather exploration behavior. In one embodiment, the improvement in the health of the animal comprises an improvement in head scratching behavior. In one embodiment, the improvement in the health of the animal comprises an improvement in wing flapping behavior. In one embodiment, the improvement in the health of the animal comprises an improvement in leg stretching behavior. In one embodiment, the improvement in the health of the animal comprises an improvement in stretching behavior. In one embodiment, the improvement in the health of the animal comprises an improvement in aggressive pecking behavior. In one embodiment, the improvement in the health of the animal comprises an improvement in non-aggressive pecking behavior.

[0025] In one embodiment, said improvement in the health of said animal comprises a reduction in lesions on said animal. In one embodiment, said lesions are present on the neck. In one embodiment, said lesions are present on the tail. In one embodiment, said lesions are present on the cloaca. In one embodiment, said lesions are present on the coronal gland.

[0026] In one embodiment, the improvement in the health of the animal comprises improvement in the intestinal health of the animal. In one embodiment, the improvement in the health of the animal comprises prevention of intestinal dysbiosis in the animal.

[0027] In one embodiment, the improvement in the health of the animal comprises a reduction in pro-inflammatory effects in the animal. In one embodiment, the improvement in the health of the animal comprises an increase in the release of manno-oligosaccharides (MOS) in the animal.

[0028] In one embodiment, the administration provides an improvement in egg quality of an egg among a plurality of eggs produced by the animal. In one embodiment, the improvement in egg quality is indicated by a change in a quality selected from the group consisting of weight, specific gravity, albumen height, albumen weight, yolk height, yolk length, yolk index, yolk weight, Haugh unit, shell weight, shell cracking strength, albumen pH, yolk pH, yolk color score, yolk brightness, yolk redness, yolk yellowness, chroma, and any combination thereof.

[0029] In one embodiment, the improvement in egg quality is indicated by a change in weight. In one embodiment, the improvement in egg quality is indicated by a change in specific gravity. In one embodiment, the improvement in egg quality is indicated by a change in albumen height. In one embodiment, the improvement in egg quality is indicated by a change in albumen weight. In one embodiment, the improvement in egg quality is indicated by a change in yolk height. In one embodiment, the improvement in egg quality is indicated by a change in yolk length. In one embodiment, the improvement in egg quality is indicated by a change in yolk index. In one embodiment, the improvement in egg quality is indicated by a change in yolk weight. In one embodiment, the improvement in egg quality is indicated by a change in Haugh units. In one embodiment, the improvement in egg quality is indicated by a change in shell weight. In one embodiment, the improvement in egg quality is indicated by a change in shell cracking strength. In one embodiment, the improvement in egg quality is indicated by a change in albumen pH. In one embodiment, the improvement in egg quality is indicated by a change in yolk pH. In one embodiment, the improvement in egg quality is indicated by a change in yolk color score. In one embodiment, the improvement in egg quality is indicated by a change in the brightness of the yolk. In one embodiment, the improvement in egg quality is indicated by a change in the redness of the yolk. In one embodiment, the improvement in egg quality is indicated by a change in the yellowness of the yolk. In one embodiment, the improvement in egg quality is indicated by a change in chromaticity. In one embodiment, the change is an increase in a measured value. In one embodiment, the change is a decrease in a measured value.

[0030] In one embodiment, the administration provides an improvement in reactive oxygen species (ROS) in the animal. In one embodiment, the improvement in ROS is in the intestine of the animal. In one embodiment, the improvement in ROS is in the fallopian tube of the animal.

[0031] In one embodiment, the improvement of the ROS is indicated by changes in thiobarbituric acid reactive substances (TBARS), glutathione S-transferase (GST), protein thiol (TSH) and any combination thereof. In one embodiment, the improvement of the ROS is indicated by changes in thiobarbituric acid reactive substances (TBARS). In one embodiment, the improvement of the ROS is indicated by changes in glutathione S-transferase (GST). In one embodiment, the improvement of the ROS is indicated by changes in protein thiol (TSH). In one embodiment, the change is an increase in a measured value. In one embodiment, the change is a reduction in a measured value.

[0032] In one embodiment, the administration provides an increase in egg production rate of the animal. In one embodiment, the administration provides an increase in fresh egg weight of eggs laid by the animal. In one embodiment, the administration provides a decrease in the coefficient of variation of egg weight of eggs laid by the animal. In one embodiment, the administration provides an increase in egg component quality of eggs laid by the animal. In one embodiment, the egg component quality is egg quality. In one embodiment, the egg component quality is yolk quality. In one embodiment, the egg component quality is protein quality. In one embodiment, the egg component quality is eggshell quality.

[0033] In one embodiment, the administration provides an increase in the incidence of clean eggs produced by the animal. In one embodiment, the increase in the incidence of clean eggs is a decrease in the presence of feces on the eggs.

[0034] In one embodiment, the administration provides a change in eggshell thickness in eggs laid by the animal. In one embodiment, the change in eggshell thickness is an increase in eggshell thickness. In one embodiment, the change in eggshell thickness is a decrease in eggshell thickness.

[0035] In one embodiment, the change in eggshell thickness is observed during a first period of about 40 weeks. In one embodiment, the change in eggshell thickness is observed during a second period of about 44 weeks. In one embodiment, the change in eggshell thickness is observed during a third period of about 48 weeks.

[0036] The following numbered examples are contemplated and are non-limiting:

[0037] 1. A method comprising the step of administering one or more supplements to a non-human animal.

[0038] 2. The method of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the animal is poultry.

[0039] 3. The method of clause 2, any other suitable clause, or any combination of suitable clauses, wherein the poultry is selected from the group consisting of chicken, turkey, and duck.

[0040] 4. The method of clause 2, any other suitable clause, or any combination of suitable clauses, wherein the poultry is a chicken.

[0041] 5. The method of clause 4, any other suitable clause, or any combination of suitable clauses, wherein the chicken is a hen.

[0042] 6. The method of clause 5, any other suitable clause, or any combination of suitable clauses, wherein the hen is an egg-laying hen.

[0043] 7. The method of clause 2, any other suitable clause, or any combination of suitable clauses, wherein the birds are present in a cage system.

[0044] 8. The method of any one of clauses 1 to 7, any other suitable clause, or any combination of suitable clauses, wherein the supplement comprises Hemicell HT TM .

[0045] 9. The method of any one of clauses 1 to 8, any other suitable clause, or any combination of suitable clauses, wherein the supplement comprises β-mannanase.

[0046] 10. The method of clause 9, any other suitable clause, or any combination of suitable clauses, wherein the β-mannanase is provided by fermentation of Paenibacillus lentus.

[0047] 11. The method of clause 9, any other suitable clause or any combination of suitable clauses, wherein the β-mannanase is applied at a dosage of 100 g / ton to 500 g / ton.

[0048] 12. The method of clause 9, any other suitable clause or any combination of suitable clauses, wherein the β-mannanase is applied at a dosage of 100 g / ton.

[0049] 13. The method of clause 9, any other suitable clause or any combination of suitable clauses, wherein the β-mannanase is applied at a dosage of 200 g / ton.

[0050] 14. The method of clause 9, any other suitable clause or any combination of suitable clauses, wherein the β-mannanase is applied at a dosage of 300 g / ton.

[0051] 15. The method of clause 9, any other suitable clause or any combination of suitable clauses, wherein the β-mannanase is applied at a dosage of 400 g / ton.

[0052] 16. The method of clause 9, any other suitable clause or any combination of suitable clauses, wherein the β-mannanase is applied at a dosage of 500 g / ton.

[0053] 17. The method according to any one of clauses 1 to 16, any other suitable clause, or any combination of suitable clauses, wherein the supplement comprises Protexin TM .

[0054] 18. The method of any one of clauses 1 to 17, any other suitable clause, or any combination of suitable clauses, wherein the supplement comprises a probiotic supplement.

[0055] 19. The method of clause 18, any other suitable clause, or any combination of suitable clauses, wherein the probiotic supplement comprises Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium bifidum, Enterococcus faecium, and Streptococcus thermophilus, or

[0056] wherein the supplement comprises a probiotic supplement comprising Enterococcus faecium, optionally wherein the Enterococcus faecium is National Collection of Industrial, Food and Marine Bacteria (NCIMB) culture number 11181, United Kingdom.

[0057] 20. The method of clause 18, any other suitable clause or any combination of suitable clauses, wherein the probiotic supplement is administered at a dosage of 10 g / ton to 100 g / ton.

[0058] 21. The method of clause 18, any other suitable clause, or any combination of suitable clauses, wherein the probiotic supplement is administered at a dose of 10 g / ton.

[0059] 22. The method of clause 18, any other suitable clause, or any combination of suitable clauses, wherein the probiotic supplement is administered at a dose of 20 g / ton.

[0060] 23. The method of clause 18, any other suitable clause, or any combination of suitable clauses, wherein the probiotic supplement is administered at a dose of 30 g / ton.

[0061] 24. The method of clause 18, any other suitable clause, or any combination of suitable clauses, wherein the probiotic supplement is administered at a dose of 40 g / ton.

[0062] 25. The method of clause 18, any other suitable clause, or any combination of suitable clauses, wherein the probiotic supplement is administered at a dose of 50 g / ton.

[0063] 26. The method of clause 18, any other suitable clause, or any combination of suitable clauses, wherein the probiotic supplement is administered at a dose of 60 g / ton.

[0064] 27. The method of clause 18, any other suitable clause, or any combination of suitable clauses, wherein the probiotic supplement is administered at a dose of 70 g / ton.

[0065] 28. The method of clause 18, any other suitable clause, or any combination of suitable clauses, wherein the probiotic supplement is administered at a dose of 80 g / ton.

[0066] 29. The method of clause 18, any other suitable clause, or any combination of suitable clauses, wherein the probiotic supplement is administered at a dose of 90 g / ton.

[0067] 30. The method of clause 18, any other suitable clause, or any combination of suitable clauses, wherein the probiotic supplement is administered at a dose of 100 g / ton.

[0068] 31. The method of any one of clauses 1 to 30, any other suitable clause, or any combination of suitable clauses, wherein the supplement comprises i) β-mannanase and ii) a probiotic.

[0069] 32. The method of clause 31, any other suitable clause, or any combination of suitable clauses, wherein the β-mannanase and the probiotic provide a synergistic effect in improving the health of the animal.

[0070] 33. The method of any one of clauses 1 to 32, any other suitable clause, or any combination of suitable clauses, wherein the supplement is present in the feed of the animal.

[0071] 34. The method of clause 33, any other suitable clause or any combination of suitable clauses, wherein the feed is a free choice feed.

[0072] 35. The method of any one of clauses 1 to 34, any other suitable clause, or any combination of suitable clauses, wherein the administering provides an improvement in the health of the animal.

[0073] 36. A method as described in clause 35, any other suitable clause or any combination of suitable clauses, wherein said improvement in the health of said animal is an improvement in animal welfare.

[0074] 37. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein said improvement in the health of said animal comprises an improvement in feeding behavior.

[0075] 38. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein said improvement in the health of said animal comprises an improvement in walking behavior.

[0076] 39. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein said improvement in the health of said animal comprises an improvement in standing behavior.

[0077] 40. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein said improvement in the health of said animal comprises an improvement in egg-laying behavior.

[0078] 41. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein said improvement in the health of said animal comprises an improvement in fluid consumption behavior.

[0079] 42. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein said improvement in the health of said animal comprises an improvement in feather probing behavior.

[0080] 43. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein said improvement in the health of said animal comprises an improvement in head scratching behavior.

[0081] 44. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein said improvement in the health of said animal comprises an improvement in wing flapping behavior.

[0082] 45. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein said improvement in the health of said animal comprises an improvement in leg extension behavior.

[0083] 46. ​​The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein said improvement in the health of said animal comprises an improvement in stretching behavior.

[0084] 47. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein said improvement in the health of said animal comprises improvement in aggressive pecking behavior.

[0085] 48. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein said improvement in the health of said animal comprises an improvement in non-aggressive pecking behavior.

[0086] 49. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein said improvement in the health of said animal comprises a reduction in lesions on said animal.

[0087] 50. The method of clause 49, any other suitable clause or any combination of suitable clauses, wherein the lesion is present on the neck.

[0088] 51. The method of clause 49, any other suitable clause or any combination of suitable clauses, wherein the lesion is present on the tail.

[0089] 52. The method of clause 49, any other suitable clause or any combination of suitable clauses, wherein the lesion is present on the cloaca.

[0090] 53. A method according to clause 49, any other suitable clause or any combination of suitable clauses, wherein the lesion is present on the corona.

[0091] 54. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein the improvement in the health of the animal comprises an improvement in the intestinal health of the animal.

[0092] 55. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein said improving health of said animal comprises preventing intestinal dysbiosis in said animal.

[0093] 56. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein said improvement in the health of said animal comprises a reduction in pro-inflammatory effects in said animal.

[0094] 57. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein said improvement in the health of said animal comprises increased release of manno-oligosaccharides (MOS) in said animal.

[0095] 58. The method of any one of clauses 1 to 57, any other suitable clause, or any combination of suitable clauses, wherein said administering provides an improvement in egg quality of an egg among a plurality of eggs laid by said animal.

[0096] 59. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a change in a quality selected from the group consisting of weight, specific gravity, albumen height, albumen weight, yolk height, yolk length, yolk index, yolk weight, Haugh units, shell weight, shell cracking strength, albumen pH, yolk pH, yolk color score, yolk brightness, yolk redness, yolk yellowness, chroma, and any combination thereof.

[0097] 60. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a change in weight.

[0098] 61. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a change in specific gravity.

[0099] 62. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a change in albumen height.

[0100] 63. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a change in egg white weight.

[0101] 64. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a change in yolk height.

[0102] 65. The method of clause 58, any other suitable clause or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a change in yolk length.

[0103] 66. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a change in Yolk Index.

[0104] 67. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a change in yolk weight.

[0105] 68. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a change in Haugh Units.

[0106] 69. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a change in shell weight.

[0107] 70. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a change in cracking strength.

[0108] 71. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a change in albumen pH.

[0109] 72. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a change in egg yolk pH.

[0110] 73. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a change in yolk color score.

[0111] 74. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a change in yolk brightness.

[0112] 75. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a change in the redness of the egg yolk.

[0113] 76. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a change in the yellowness of the egg yolk.

[0114] 77. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a change in colour.

[0115] 78. A method as described in any one of clauses 59 to 77, any other suitable clause, or any combination of suitable clauses, wherein the change is an increase in a measured value.

[0116] 79. A method as described in any one of clauses 59 to 77, any other suitable clause, or any combination of suitable clauses, wherein the change is a decrease in a measured value.

[0117] 80. The method of any one of clauses 1 to 79, any other suitable clause, or any combination of suitable clauses, wherein said administering provides an improvement in reactive oxygen species (ROS) in said animal.

[0118] 81. The method of clause 80, any other suitable clause, or any combination of suitable clauses, wherein the amelioration of ROS is in the intestine of the animal.

[0119] 82. The method of clause 80, any other suitable clause, or any combination of suitable clauses, wherein the amelioration of ROS is within the oviduct of the animal.

[0120] 83. A method according to any one of clauses 80 to 82, any other suitable clause, or any combination of suitable clauses, wherein the improvement in ROS is indicated by changes in thiobarbituric acid reactive substances (TBARS), glutathione S-transferase (GST), protein thiols (TSH), and any combination thereof.

[0121] 84. The method of any one of clauses 80 to 82, any other suitable clause, or any combination of suitable clauses, wherein the improvement in ROS is indicated by a change in thiobarbituric acid reactive substances (TBARS).

[0122] 85. The method of any one of clauses 80 to 82, any other suitable clause, or any combination of suitable clauses, wherein the improvement in ROS is indicated by a change in glutathione S-transferase (GST).

[0123] 86. The method of any one of clauses 80 to 82, any other suitable clause, or any combination of suitable clauses, wherein the improvement in ROS is indicated by changes in protein thiols (TSH).

[0124] 87. A method as described in any one of clauses 83 to 86, any other suitable clause, or any combination of suitable clauses, wherein the change is an increase in a measured value.

[0125] 88. A method as described in any one of clauses 83 to 86, any other suitable clause, or any combination of suitable clauses, wherein the change is a decrease in a measured value.

[0126] 89. The method of any one of clauses 1 to 88, any other suitable clause, or any combination of suitable clauses, wherein said administering provides an increase in egg production rate of said animal.

[0127] 90. The method of any one of clauses 1 to 89, any other suitable clause, or any combination of suitable clauses, wherein said administering provides an increase in fresh egg weight of eggs produced by said animal.

[0128] 91. The method of any one of clauses 1 to 90, any other suitable clause or any combination of suitable clauses, wherein said administering provides a reduction in the coefficient of variation of egg weight of eggs produced by said animal.

[0129] 92. The method of any one of clauses 1 to 91 , any other suitable clause, or any combination of suitable clauses, wherein said administering provides an increase in the quality of egg components of eggs produced by said animal.

[0130] 93. The method of clause 92, any other suitable clause, or any combination of suitable clauses, wherein the egg component mass is egg mass.

[0131] 94. A method as described in clause 92 or clause 93, any other suitable clause or any combination of suitable clauses, wherein the egg component mass is egg yolk mass.

[0132] 95. The method of any one of clauses 92 to 94, any other suitable clause or any combination of suitable clauses, wherein the egg component quality is protein quality.

[0133] 96. The method of any one of clauses 92 to 95, any other suitable clause, or any combination of suitable clauses, wherein the egg component mass is egg shell mass.

[0134] 97. The method of any one of clauses 1 to 96, any other suitable clause, or any combination of suitable clauses, wherein said administering provides an increase in the incidence of clean eggs produced by said animal.

[0135] 98. The method of clause 97, any other suitable clause, or any combination of suitable clauses, wherein the increase in the incidence of clean eggs is a decrease in the presence of feces on the eggs.

[0136] 99. The method of any one of clauses 1 to 98, any other suitable clause, or any combination of suitable clauses, wherein said administering provides a change in eggshell thickness in eggs laid by said animal.

[0137] 100. A method as described in clause 99, any other suitable clause, or any combination of suitable clauses, wherein the change in eggshell thickness is an increase in eggshell thickness.

[0138] 101. A method as described in clause 99, any other suitable clause, or any combination of suitable clauses, wherein the change in eggshell thickness is a decrease in eggshell thickness.

[0139] 102. The method of any one of clauses 1 to 101, any other suitable clause, or any combination of suitable clauses, wherein the change in eggshell thickness is observed during a first period of about 40 weeks.

[0140] 103. The method of any one of clauses 1 to 101 , any other suitable clause, or any combination of suitable clauses, wherein the change in eggshell thickness is observed during a second phase of about 44 weeks.

[0141] 104. The method of any one of clauses 1 to 101 , any other suitable clause, or any combination of suitable clauses, wherein the change in eggshell thickness is observed during a third stage of about 48 weeks.

[0142] Examples

[0143] Example 1

[0144] Behavioral assessment

[0145] For the current example, poultry was evaluated at a commercial farm in Salvador do Sul, Rio Grande do Sul, southern Brazil. The experimental unit was randomly selected from hens raised in a commercial farm with approximately 28,000 lightweight egg-laying hens (Hyline W 36 pedigree, 36 weeks of age). Replicates were assigned to four treatments in a completely randomized design, including i) a control treatment (basic diet without any other additives), ii) β-mannanase (control diet supplemented with 300 g / ton β-mannanase), iii) probiotics (control diet supplemented with 50 g / ton polycepa probiotic additives), and iv) β-mannanase plus probiotic treatment (control diet supplemented with 300 g / ton β-mannanase and 50 g / ton polycepa probiotic additives).

[0146] The β-mannanase (Hemicell HT TM , Elanco Animal Health) is an exogenous enzyme produced by fermentation of Paenibacillus lentiviridae bacteria. TM concentrate, Elanca Animal Health) including Lactobacillus acidophilus (2.06 × 10 8 UFC / g), Lactobacillus bulgaricus (2.06×10 8 UFC / g), Lactobacillus plantarum (1.26×10 8 UFC / g), Lactobacillus rhamnosus (2.06×10 8 UFC / g), Bifidobacterium (2.0×10 8 UFC / g), Enterococcus faecium (6.46×10 8 UFC / g) and Streptococcus thermophilus (4.10×10 8 UFC / g).

[0147] The basal diet consisted of a corn-soybean meal based feed formulated according to nutritional requirements, and an inert material (kaolin) was included in the basal feed in place of β-mannanase and / or probiotic additives. Feed and water were provided ad libitum throughout the experiment using nipple drinkers and trough feeders.

[0148] The birds were housed in conventional sheds arranged in an east-west direction with concrete floors and masonry walls complemented by steel mesh on the ceiling. The sheds were equipped with side curtains, which were managed according to weather conditions to provide thermal comfort. The average minimum and maximum values ​​of temperature and relative humidity of the air recorded were 18°C ​​and 36°C and 35.8% and 94.7%, respectively. The lighting regime consisted of 16 hours of light and 8 hours of darkness per day.

[0149] During the entire experimental period, birds were housed in galvanized wire cages (100-cm length × 40-cm width × 45-cm height, yielding a 500-cm 2 / floor space of hens). Birds were assigned to each cage. Birds were supplemented for 84 days and evaluated in the last week of the trial.

[0150] Behavioral assessment was performed by image capture in conjunction with local feather range and crown abnormality assessment. For behavioral assessment, six birds (one per cage) were randomly selected for each treatment for observation. The captured images were performed over 7 consecutive days, divided into two time periods corresponding to the hottest and coldest times of the day in the 15-minute morning (e.g., peak of avian egg production) and 30-minute afternoon time periods. Images were recorded and analyzed by visual counting and frequency methods.

[0151] Lesion scoring was performed by visual scoring, attributed to three body areas (neck, tail and anus) of 25 birds from each treatment group randomly selected. Possible lesions and different severity were analyzed, with a scale from 0 to 5 (the best score was 0, indicating that the feathers were intact and without lesions, and the worst score was 5, indicating that the feathers were intact but with areas of skin lesions). Comb abnormalities were observed in the same birds using a scale from 0 to 3, with the best score being 0 (no evidence of comb abnormalities) and the worst score being 3 (3 or more comb areas with evidence of abnormalities).

[0152] All behavioral tests were performed during the last week of the experiment, which allowed the birds to be exposed to the treatments over a longer period. The same animal was used for only one of the tests, thus preventing one test from interfering with the results of another.

[0153] The frequencies of the main behaviors observed in laying hens fed β-mannanase and / or probiotics are shown in Table 1.

[0154] Table 1.

[0155]

[0156]

[0157] 1 Means followed by different capital letters are statistically different by 5%, while lowercase letters are used to indicate differences of 10%. Treatment lines within each observation time A、B、C、D The comparisons were made between the 4 treatments and the averages obtained when the three observation times were polled together were also compared (indicated as 'Average Treatment'). The averages obtained when the fourth treatment was polled together at each observation time are also presented (indicated as 'Average Time') and are listed in Table 1. XYZ for comparison.

[0158] 2 Probability of treatment effects (Treatment), observation time (Time), and interaction (TxT).

[0159] The time (minutes / bird) spent in each major behavior by laying hens fed β-mannanase and / or probiotics is shown in Table 2.

[0160] Table 2.

[0161]

[0162]

[0163] 1 Means followed by different capital letters are statistically different by 5%, while lowercase letters are used to indicate differences of 10%. Treatment lines within each observation time A、B、C、D The comparisons were made between the 4 treatments and the averages obtained when the three observation times were polled together were also compared (indicated as 'Average Treatment'). The averages obtained when the fourth treatment was polled together at each observation time are also presented (indicated as 'Average Time') and are listed in Table 1. XYZ for comparison.

[0164] 2 Probability of treatment effects (Treatment), observation time (Time), and interaction (TxT).

[0165] The time (minutes / bird) spent on other activities by laying hens fed β-mannanase and / or probiotics is shown in Table 3.

[0166] Table 3.

[0167]

[0168]

[0169] 1 Means followed by different capital letters are statistically different by 5%, while lowercase letters are used to indicate differences of 10%. Treatment lines within each observation time A、B、C、D The comparisons were made between the 4 treatments and the averages obtained when the three observation times were polled together were also compared (indicated as 'Average Treatment'). The averages obtained when the fourth treatment was polled together at each observation time are also presented (indicated as 'Average Time') and are listed in Table 1. XYZ for comparison.

[0170] 2 Probability of treatment effects (Treatment), observation time (Time), and interaction (TxT).

[0171] The frequencies (%) of birds with lesions observed in the groups of laying hens fed β-mannanase and / or probiotics, irrespective of the score, are shown in Table 4.

[0172] Table 4.

[0173]

[0174] The lesion scores observed in the groups of laying hens fed β-mannanase and / or probiotics are shown in Table 5. Scores from 0 (no lesions) to 5 (severe lesions) were attributed to the birds individually, except for the crests, which had scores ranging from 0 (no lesions) to 2 (severe lesions). Means (LS / mean) followed by different capital letters were statistically different by 5%.

[0175] Table 5.

[0176]

[0177] Example 2

[0178] Egg quality assessment

[0179] For this example, egg quality of birds in different treatment groups was assessed.Egg quality was assessed during the storage period.

[0180] Experimental units were randomly selected from hens raised on a commercial farm (approximately 28,000 lightweight laying hens, 36 weeks old, Hyline W36 lineage) and assigned to treatments, namely i) control (basal diet, no supplementation); ii) supplemented with 300 g / ton β-mannanase iii) supplemented with 50 g / ton probiotic additives iv) a diet supplemented with 50 g / ton probiotic additive and 300 g / ton β-mannanase (Hemicell+Protexin).

[0181] The basal diet was formulated according to the nutrient requirements of the gene (Hy-Line, 2020). Corn and soybean meal were the main ingredients in the diet formulation. Feed and water were provided ad libitum using nipple drinkers and trough feeders throughout the experimental period.

[0182] Three 28-day production cycles were performed, and on the last day of each cycle 480 eggs (120 per treatment group) were randomly collected to assess egg quality. Eggs with cracks were excluded from the experiment.

[0183] A portion of the eggs were immediately analyzed for quality to characterize fresh eggs (storage day 0). The remaining eggs were stored at room temperature and randomly separated at each storage interval (7 days, 14 days, 21 days, 28 days, 35 days, and 42 days) for quality evaluation.

[0184] During the storage period, fifteen eggs were randomly selected from each treatment and weighed individually at weekly intervals. The weight loss (%) of eggs during storage was calculated using the following equation:

[0185]

[0186] Three measurements were taken at different points on the albumen at 10 mm from the yolk using a digital caliper (TMX PD-150, China), and the albumen height was estimated by the average of the three measurements. Thus, the Haugh unit (HU) was obtained by the equation proposed by Haugh (1937), where h = albumen thickness (mm); W = mass of the entire egg (g):

[0187]

[0188] The yolk width and height (mm) of 15 eggs from each treatment were measured at weekly intervals using a digital caliper (TMX PD-150, China). Afterwards, the yolk index was calculated as follows:

[0189]

[0190] The yolk color of 15 eggs in each treatment was determined at weekly intervals using the Roche color fan (DSM, Sao Paulo, Brazil), with scores ranging from 1 (light yellow) to 15 (reddish orange). In addition, a spectrophotometer device (DeltaVista model 450G, Delta Color, Sao Leopoldo, Brazil) was also used for this evaluation, which determined the colorimetric coordinates of luminosity (L*), red intensity (a*) and yellow intensity (b*). The following equation was considered to estimate the chromaticity:

[0191] C=(a* 2 +b* 2 ) 1 / 2

[0192] After separation of yolk and white, the thick and runny whites were homogenized for 20 seconds and then the pH was determined using a digital pH meter (Kasvi model k39-2014B, Paraná, Brazil) previously calibrated with buffer solutions of pH 4, pH 7 and pH 10. The pH of the yolk was determined using the same pH meter.

[0193] The specific gravity value is based on Archimedes' principle using the following equation:

[0194]

[0195] The total solids content in egg white and egg yolk was determined separately. Five grams of egg white and egg yolk were weighed separately in a pre-dried ceramic crucible. The egg white and egg yolk samples were kept in an oven at 60°C for 12 hours and weighed. After weighing, the samples were kept at 105°C for 12 hours and weighed again. Seven eggs per treatment were evaluated to determine the total solids.

[0196] Shell percentage was obtained after shell separation, washing, drying and weighing. Fifteen eggs per treatment were evaluated for these variables on days 0, 21 and 42.

[0197] Thiobarbituric acid-reactive substances (TBARS) were used to assess lipid oxidation in three egg yolk pools per treatment collected during three storage periods (0, 21 and 42 days). Due to the results obtained previously in the egg quality analysis, only the treatments using Hemicell and Protexin alone were tested.

[0198] A summary of the fresh egg quality analysis of laying hens fed β-mannanase and / or probiotics is shown in Table 6. The values ​​represent the average of the three evaluation periods, considering fresh eggs only.

[0199] Table 6.

[0200]

[0201] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0202] Table 7 shows the weight loss (g) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the first trial.

[0203] Table 7.

[0204]

[0205]

[0206] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0207] Table 8 shows the weight loss (g) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the second trial.

[0208] Table 8.

[0209]

[0210]

[0211] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0212] Table 9 shows the weight loss (g) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the third trial.

[0213] Table 9.

[0214]

[0215]

[0216] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0217] Table 10 shows the specific gravity (g / ml) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the first trial.

[0218] Table 10.

[0219]

[0220]

[0221] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0222] Table 11 shows the specific gravity (g / ml) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the second trial.

[0223] Table 11.

[0224]

[0225]

[0226] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0227] Table 12 shows the specific gravity (g / ml) of eggs from laying hens fed with β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the third trial.

[0228] Table 12.

[0229]

[0230]

[0231] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0232] Table 13 shows the albumen height (mm) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the first trial.

[0233] Table 13.

[0234]

[0235]

[0236] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0237] Table 14 shows the albumen height (mm) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the second trial.

[0238] Table 14.

[0239]

[0240]

[0241] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0242] Table 15 shows the albumen height (mm) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the third trial.

[0243] Table 15.

[0244]

[0245]

[0246] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0247] Table 16 shows the yolk length (mm) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the first trial.

[0248] Table 16.

[0249]

[0250]

[0251] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0252] Table 17 shows the yolk length (mm) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the second trial.

[0253] Table 17.

[0254]

[0255]

[0256] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0257] Table 18 shows the yolk length (mm) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the third trial.

[0258] Table 18.

[0259]

[0260]

[0261] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0262] Table 19 shows the yolk height (mm) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the first trial.

[0263] Table 19.

[0264]

[0265]

[0266] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0267] Table 20 shows the yolk height (mm) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the second trial.

[0268] Table 20.

[0269]

[0270]

[0271] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0272] Table 21 shows the yolk height (mm) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the third trial.

[0273] Table 21.

[0274]

[0275]

[0276] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0277] Table 22 shows the yolk index of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the first trial.

[0278] Table 22.

[0279]

[0280]

[0281] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0282] Table 23 shows the yolk index of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the second trial.

[0283] Table 23.

[0284]

[0285]

[0286] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0287] Table 24 shows the yolk index of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the third trial.

[0288] Table 24.

[0289]

[0290]

[0291] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0292] Table 25 shows the yolk weight (g) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the first trial.

[0293] Table 25.

[0294]

[0295]

[0296] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0297] Table 26 shows the yolk weight (g) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the second trial.

[0298] Table 26.

[0299]

[0300]

[0301] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0302] Table 27 shows the yolk weight (g) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the third trial.

[0303] Table 27.

[0304]

[0305]

[0306] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0307] Table 28 shows the Haugh units of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the first trial.

[0308] Table 28.

[0309]

[0310]

[0311] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0312] Table 29 shows the Haugh units of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the second trial.

[0313] Table 29.

[0314]

[0315]

[0316] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0317] Table 30 shows the Haugh units of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the third trial.

[0318] Table 30.

[0319]

[0320]

[0321] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0322] Table 31 shows the egg white weight (g) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the first trial.

[0323] Table 31.

[0324]

[0325]

[0326] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0327] Table 32 shows the egg white weight (g) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the second trial.

[0328] Table 32.

[0329]

[0330]

[0331] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0332] Table 33 shows the egg white weight (g) of the laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the third trial.

[0333] Table 33.

[0334]

[0335]

[0336] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0337] Table 34 shows the shell weight (g) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the first trial.

[0338] Table 34.

[0339]

[0340]

[0341] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0342] Table 35 shows the shell weight (g) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the second trial.

[0343] Table 35.

[0344]

[0345]

[0346] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0347] Table 36 shows the shell weight (g) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the third trial.

[0348] Table 36.

[0349]

[0350]

[0351] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0352] Table 37 shows the cracking strength (N) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the first trial.

[0353] Table 37.

[0354]

[0355] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0356] Table 38 shows the cracking strength (N) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the second trial.

[0357] Table 38.

[0358]

[0359] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0360] Table 39 shows the cracking strength (N) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the third trial.

[0361] Table 39.

[0362]

[0363]

[0364] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0365] Table 40 shows the albumen pH of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the first trial.

[0366] Table 40.

[0367]

[0368]

[0369] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0370] Table 41 shows the albumen pH of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the second trial.

[0371] Table 41.

[0372]

[0373]

[0374]

[0375] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0376] Table 42 shows the albumen pH of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the third trial.

[0377] Table 42.

[0378]

[0379]

[0380] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0381] Table 43 shows the yolk pH of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the first trial.

[0382] Table 43.

[0383]

[0384]

[0385] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0386] Table 44 shows the yolk pH of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the second trial.

[0387] Table 44.

[0388]

[0389]

[0390]

[0391] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0392] Table 45 shows the yolk pH of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the third trial.

[0393] Table 45.

[0394]

[0395]

[0396] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0397] Table 46 shows the yolk color scores (color palette) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the first trial.

[0398] Table 46.

[0399]

[0400]

[0401] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0402] Table 47 shows the yolk color scores (color palette) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the second trial.

[0403] Table 47.

[0404]

[0405]

[0406] 1Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0407] Table 48 shows the yolk color scores (color palette) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the third trial.

[0408] Table 48.

[0409]

[0410]

[0411] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0412] Table 49 shows the yolk brightness (L* color) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the first trial.

[0413] Table 49.

[0414]

[0415]

[0416] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0417] Table 50 shows the yolk brightness (L* color) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the second trial.

[0418] Table 50.

[0419]

[0420]

[0421] 1 Standard error. 2The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0422] Table 51 shows the yolk brightness (L* color) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the third trial.

[0423] Table 51.

[0424]

[0425]

[0426] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0427] Table 52 shows the yolk redness (a*color) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the first trial.

[0428] Table 52.

[0429]

[0430]

[0431] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0432] Table 53 shows the yolk redness (a* color) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the second trial.

[0433] Table 53.

[0434]

[0435]

[0436] 1 Standard error. 2The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0437] Table 54 shows the yolk redness (a* color) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the third trial.

[0438] Table 54.

[0439]

[0440]

[0441] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0442] Table 55 shows the yolk yellowness (b* color) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the first trial.

[0443] Table 55.

[0444]

[0445]

[0446] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0447] Table 56 shows the yolk yellowness (b* color) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the second trial.

[0448] Table 56.

[0449]

[0450]

[0451] 1 Standard error. 2The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0452] Table 57 shows the yolk yellowness (b* color) of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to different storage times in the third trial.

[0453] Table 57.

[0454]

[0455]

[0456] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0457] Example 3

[0458] Oxidant and Antioxidant Assessment

[0459] For this example, the evaluation of oxidant and antioxidant levels in birds was assessed.Conditions in the intestines and oviducts of laying hens from one of the four treatment groups described in Example 2 were assessed.

[0460] To determine the level of reactive oxygen species (ROS), the method described by Halliwell and Gutteridge (2007) was used. The level of lipid peroxidation in heterogeneous muscle was determined by measuring the level of antibodies reactive to thiobarbituric acid (TBARS) by the absorbance of the red product at 532 nm according to the method described by Ohkawa et al. (1978) and expressed as nmol MDA / mg protein.

[0461] GST activity was measured spectrophotometrically at 340 nm according to the method of Habig et al. (1974). The mixture contained the supernatant of the muscle homogenate as tested, 0.1 M potassium phosphate buffer (pH 7.4) as substrate, 100 mM GSH and 100 mM CDNB. Enzyme activity was expressed as μmol / CDNB / mg protein. Protein thiols (PSH) were determined by the method described by Sedlak and Lindsay (1968), a technique using DTNB (5,5-dithiobis-2-nitrobenzoic acid; Sigma). Protein thiols were measured by the precipitate formed by the precipitated protein, the material was resuspended, and the PSH content was determined using homogenization buffer. Absorbance (405 nm) was read using a fluorescence spectrophotometer (Biotek, Synergy HT).

[0462] Table 58 shows the oxidant and antioxidant status in the intestine and oviduct of laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin). The variables evaluated included TBARS (thiobarbituric acid reactive substances; mmol MDA / mg protein); GST (glutathione S-transferase; micromoles CDNB / minute / mg protein); and TSH (protein thiols; μmol TSH / mg protein).

[0463] Table 58.

[0464]

[0465] 1 Standard error. 2 Probability of a treatment effect (model does not include an effect of storage time). Means followed by different capital letters are statistically different at 5%, while lower case letters are used to indicate a difference of 10%.

[0466] Example 4

[0467] Performance Evaluation

[0468] For this example, various performances were performed. Experimental units were randomly selected from hens raised on a commercial farm (approximately 28,000 lightweight laying hens, 36 weeks old, Hyline W 36 lineage) and assigned to treatments, i.e., i) control (basal diet, no supplementation); ii) supplemented with 300 g / ton β-mannanase iii) supplemented with 50 g / ton probiotic additives iv) a diet supplemented with 50 g / ton probiotic additive and 300 g / ton β-mannanase (Hemicell+Protexin).

[0469] The basal diet was formulated according to the nutrient requirements of the gene (Hy-Line, 2020). Corn and soybean meal were the main ingredients in the diet formulation. Feed and water were provided ad libitum using nipple drinkers and trough feeders throughout the experimental period.

[0470] The birds were housed in conventional sheds arranged in an east-west direction with concrete floors and masonry walls complemented by steel mesh on the ceiling. The sheds were equipped with side curtains, which were managed according to weather conditions to provide thermal comfort. The average maximum and minimum values ​​of temperature and relative humidity of the air recorded were 36°C and 18.1°C and 94.7% and 35.8%, respectively. Only natural light was used for illumination, with a lighting schedule that allowed 16 hours and 30 minutes of light per day.

[0471] The example started when the birds were 36 weeks old and lasted for 144 days. Egg production in subgroups of 120 cages (4 birds per cage), corresponding to 30 replicates per treatment, was recorded separately at weeks 4, 8 and 12. All eggs produced in these subgroups were weighed individually and analyzed for the presence of feces in the shell, which was classified by visual analysis by the same observer as: absent (clean eggs), slightly present (e.g., small spots present; see Figure 1 ) or in large numbers (e.g., the presence of large spots; see Figure 2 ).

[0472] The egg laying rate and egg quality were calculated taking into account all eggs (including unsaleable eggs) of each replicate (cage). The coefficient of variation was calculated for each cage taking into account the individual weights of all eggs produced each week. A similar procedure was used to evaluate egg quality.

[0473] At the end of the project, blood samples were collected from eight birds per treatment (selected randomly). Paulo, Brazil)) and semi-automatic analyzers to process and analyze the samples (Bio- , biochemical analyzer, Bioplus, São Paulo, Brazil) total protein, albumin, uric acid, total cholesterol, triglyceride, glucose, alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase.

[0474] Table 59 shows the performance of laying hens fed a diet supplemented with β-mannanase (Hemicell) and / or probiotics (Protexin).

[0475] Table 59.

[0476]

[0477] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%. 3 No treatment was provided from Week 48 to Week 62. Therefore, the last assessment was performed after 14 weeks in the absence of supplementation.

[0478] Table 60 shows the quality of egg components produced by hens fed a diet supplemented with β-mannanase (Hemicell) and / or probiotics (Protexin).

[0479] Table 60.

[0480]

[0481] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0482] Table 61 shows the incidence of clean eggs or low / high presence of feces (%) in eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin).

[0483] Table 61.

[0484]

[0485] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%. 3 No treatment was provided from Week 48 to Week 62. Therefore, the last assessment was performed after 14 weeks in the absence of supplementation.

[0486] Table 62 shows serum biochemistry and intestinal morphology of laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin).

[0487] Table 62.

[0488]

[0489]

[0490] 1 Standard error.2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0491] Example 5

[0492] Egg microstructural assessment

[0493] For this example, egg microstructure was evaluated.Eggshells obtained from eggs of laying hens from one of the four treatment groups described in Example 2 were evaluated.

[0494] Scanning electron microscopy analysis was performed to evaluate the microstructure of the eggshell. For this evaluation, 20 shells from the last trial were used, of which approximately 5 cm 2 The fragments were taken out and stored in falcon tubes. After this period, the samples were mounted on short posts, coated with 35nm of gold-palladium for 3 minutes (sputtering coater SCD 050 from Balzers, Germany), and analyzed by scanning electron microscope (JEOL 6060, Japan) at a magnification of 100 to 2000×. In the images, the thickness of the mastoid layer, palisade layer, membranous layer, and vertical crystal layer were measured in three positions arbitrarily selected in each eggshell image. The size of the columnar or mastoid body and the distance between the mastoid nodes can also be obtained, but due to the large variability between the measurements, five measurements were carried out in each image.

[0495] Table 63 summarizes the eggshell traits obtained by ultrastructure of eggs from laying hens fed β-mannanase (Hemicell) and / or probiotics (Protexin) according to storage time.The evaluation was conducted in the first phase (40 weeks), the second phase (44 weeks) and the third phase (48 weeks).

[0496] Table 63.

[0497]

[0498] 1 Standard error. 2 The probability of a treatment effect. Means followed by different capital letters are statistically different at 5%, while lowercase letters are used to indicate a difference of 10%.

[0499] Figure 3 Shown are ultramicroscopy images collected in a single eggshell at the first stage. Figure 4 Shown are ultramicroscopy images collected in a single eggshell at the second stage. Figure 5 Shown are ultramicroscopy images collected in a single eggshell at the third stage.

Claims

1. A method comprising the step of administering one or more supplements to a non-human animal, wherein the supplements comprise i) β-mannanase and ii) a probiotic.

2. The method of claim 1, wherein the animal is a bird.

3. The method of claim 2, wherein the poultry is selected from the group consisting of chicken, turkey and duck.

4. The method of claim 2, wherein the poultry is chicken.

5. The method of claim 3, wherein the chicken is a hen.

6. The method of claim 4, wherein the hen is an egg-laying hen.

7. The method of claim 1, wherein the β-mannanase is administered at a dosage of 300 g / ton, and wherein the probiotic is administered at a dosage of 50 g / ton.

8. The method of claim 1, wherein the β-mannanase and the probiotic provide a synergistic effect in improving the health of the animal.

9. The method of claim 1, wherein the β-mannanase is applied at a dosage of 100 g / ton to 500 g / ton.

10. The method of claim 1, wherein the β-mannanase is applied at a dosage of 300 g / ton.

11. The method of any one of claims 1 to 5, wherein the supplement comprises a probiotic supplement comprising Enterococcus faecium.

12. The method according to claim 11, wherein the Enterococcus faecium is the National Collection of Industrial, Food and Marine Bacteria (NCIMB) culture number 11181.

13. The method of any one of claims 1 to 5, wherein the probiotic supplement comprises Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium bifidum, Enterococcus faecium, and Streptococcus thermophiles.

14. The method of claim 12, wherein the probiotic supplement is administered at a dosage of 50 g / ton.

15. The method of any one of claims 1 to 8, wherein the supplement is present in the animal's feed.

16. The method of claim 8, wherein the feed is a free-choice feed.

17. The method of any one of claims 1 to 8, wherein the administration provides an improvement in the health of the animal.

18. The method of claim 17, wherein the improvement in the health of the animal is an improvement in animal welfare.

19. The method of claim 17, wherein the improvement in the health of the animal comprises a reduction in lesions on the animal.

20. The method of claim 17, wherein the improvement in the health of the animal comprises an improvement in the intestinal health of the animal.

21. The method of claim 17, wherein the improvement in the health of the animal comprises preventing intestinal dysbiosis in the animal.

22. The method of claim 17, wherein the improvement in the health of the animal comprises a reduction in pro-inflammatory effects in the animal.

23. The method of claim 17, wherein the improvement in the health of the animal comprises an increased release of manno-oligosaccharides (MOS) in the animal.

24. The method of any one of claims 1 to 8, wherein the administering provides an improvement in egg quality of an egg among a plurality of eggs laid by the animal.

25. The method of claim 24, wherein the improvement in egg quality is indicated by a change in a quality selected from the group consisting of weight, specific gravity, albumen height, albumen weight, yolk height, yolk length, yolk index, yolk weight, Haugh unit, shell weight, shell cracking strength, albumen pH, yolk pH, yolk color score, yolk brightness, yolk redness, yolk yellowness, chroma, and any combination thereof.

26. The method of any one of claims 1 to 8, wherein the administering provides an improvement in reactive oxygen species (ROS) in the animal.

27. The method of claim 26, wherein the improvement in ROS is indicated by changes in thiobarbituric acid reactive substances (TBARS), glutathione S-transferase (GST), protein thiols (TSH), and any combination thereof.

28. The method of any one of claims 1 to 8, wherein the administration provides an increase in egg production rate of the animal.

29. The method of any one of claims 1 to 8, wherein the administration provides an increase in fresh egg weight of eggs produced by the animal.

30. The method of any one of claims 1 to 8, wherein the administration provides a reduction in the coefficient of variation of egg weight of eggs produced by the animal.

31. The method of any one of claims 1 to 8, wherein the administration provides an increase in the quality of egg components of eggs produced by the animal.

32. The method of any one of claims 1 to 8, wherein the administering provides an increase in the incidence of clean eggs produced by the animal.

33. The method of any one of claims 1 to 8, wherein the administering provides a change in eggshell thickness in eggs laid by the animal.

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