COMPOSITIONS

MANNANASE AND PROBIOTICS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 USC § 119(e) of U.S. Provisional Application Serial No. 63/400,098, filed on August 23, 2022, the entire disclosure of which is incorporated herein by reference.

HELD OF THE INVENTION

[0002] The present disclosure relates to supplements comprising beta-mannanase comprising beta-mannanase and/or a probiotic. Furthermore, methods of administering the supplements to avians such as chickens are provided to demonstrate beneficial effects on the avians. The compositions and methods described herein can benefit overall animal wellbeing and future productivity, especially in egg-laying chickens.

BACKGROUND

[0003] Improvement in animal health in avians is an important consideration for the agriculture and food production industries. In modern commercial operations, supplementation of avian feed can be undertaken in order to improve animal health and welfare.

[0004] For instance, the ability to maintain healthy avians that are able to improve their eating, walking, and consumption behavior is typically beneficial to overall avian wellbeing and future productivity. Furthermore, in commercial avian operations, improving egg laying productivity and the resulting quality of eggs is an important consideration. Thus, there exists a need for new compositions and methods to assist with management of avians in order to promote healthy behaviors and improve egg laying characteristics.

[0005] Probiotics can reduce heat stress and abnormal behavior as well as improve health in broiler chickens. Such responses occur from the regulatory power of probiotics under the micro biota-gut-brain axis. Probiotics also alleviate the stress response along the hypothalamic-pituitary-adrenal axis, reducing plasma or brain levels of corticotropin-releasing hormone, adrenocorticotropic hormone, and corticosterone.

[0006] Enzyme supplementation is another strategy that can benefit the gut health status by reducing the impacts of anti-nutritional components. The use of -mannanase can help nonruminant animals dealing with the non-starch polysaccharides, which can reduce nutrient digestibility. Such components are found in plant cell walls and are present in many ingredients largely used in animal feeding, such as soybeans. Among the main hemicelluloses found in plant cell walls are P-mannans, which can also be found on the surface of microorganisms. Thus, the animal's innate immune system is activated when foods that contain P-mannans are ingested, which responds with the proliferation of monocytes, macrophages, dendritic cells, and increased production of cytokines. Such factors generate an unnecessary energy expenditure and an increase in inflammatory responses. By hydrolyzing the P-mannans, this enzyme can improve the digestibility of mannans, increasing the population of beneficial bacteria, improving immunity, digestion and absorption of nutrients, in addition to limiting the proliferation of potential pathogens in the intestine.

SUMMARY

[0007] The present disclosure provides supplements comprising beta-mannanase comprising beta-mannanase and/or a probiotic as well as associated methods. The compositions and methods of the present disclosure surprisingly provide a synergistic effect for improve avian behaviors and egg laying qualities. The present disclosure describes supplements comprising beta-mannanase (e.g., Hemicell HT™) and a probiotic (e.g., Protexin™) that can be added to feed to improve animal health and animal welfare.

[0008] Additional features of the present disclosure will become apparent to those skilled in the art upon consideration of illustrative embodiments exemplifying the best mode of carrying out the disclosure as presently perceived.

BRIEF DESCRIPTIONS OF THE DRAWINGS

[0009] The detailed description particularly refers to the accompanying figures in which: [0010] Figure 1 shows examples of eggs with minor presence of feces.

[0011] Figure 2 shows examples of eggs with major presence of feces.

[0012] Figure 3 shows ultramicroscopy images collected in a single eggshell during the first phase.

[0013] Figure 4 shows ultramicroscopy images collected in a single eggshell during the second phase.

[0014] Figure 5 shows ultramicroscopy images collected in a single eggshell during the third phase.

DETAILED DESCRIPTION

[0015] In an illustrative aspect, a method comprising a step of administering one or more supplements to a non-human animal is provided. In an embodiment, the animal is an avian. In an embodiment, the avian is selected from the group consisting of a chicken, a turkey, and a duck. In an embodiment, the avian is a chicken. In an embodiment, the chicken is a hen. In an embodiment, the hen is a laying hen. In an embodiment, the avian is present in a cage system.

[0016] In an embodiment, the supplement comprises Hemicell™. In an embodiment, the supplement comprises Hemicell HT™. In an embodiment, the supplement comprises beta- mannanase, also referred to herein as “0- mannanase”.

[0017] In an embodiment, the beta-mannanase is provided by fermentation of Paenibacillus lentus. In an embodiment, the beta-mannanase is administered at a dose between 100 g/ton and 500 g/ton. In an embodiment, the beta-mannanase is administered at a dose of 100 g/ton. In an embodiment, the beta-mannanase is administered at a dose of 200 g/ton. In an embodiment, the beta-mannanase is administered at a dose of 300 g/ton. In an embodiment, the beta-mannanase is administered at a dose of 400 g/ton. In an embodiment, the beta-mannanase is administered at a dose of 500 g/ton.

[0018] In an embodiment, the supplement comprises Protexin™. In an embodiment, the supplement comprises a probiotic supplement. In an embodiment, the probiotic supplement comprises Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium bifidum, Enterococcus faecium, and Streptococcus thermophiles. In an embodiment, the supplement comprises a probiotic supplement comprising Enterococcus faecium. In an embodiment, the Enterococcus faecium is National Collection of Industrial, Food and Marine Bacteria (NCIMB) culture number 11181

[0019] In an embodiment, the probiotic supplement is administered at a dose between 10 g/ton and 100 g/ton. In an embodiment, the probiotic supplement is administered at a dose of 10 g/ton. In an embodiment, the probiotic supplement is administered at a dose of 20 g/ton. In an embodiment, the probiotic supplement is administered at a dose of 30 g/ton. In an embodiment, the probiotic supplement is administered at a dose of 40 g/ton. In an embodiment, the probiotic supplement is administered at a dose of 50 g/ton. In an embodiment, the probiotic supplement is administered at a dose of 60 g/ton. In an embodiment, the probiotic supplement is administered at a dose of 70 g/ton. In an embodiment, the probiotic supplement is administered at a dose of 80 g/ton. In an embodiment, the probiotic supplement is administered at a dose of 90 g/ton. In an embodiment, the probiotic supplement is administered at a dose of 100 g/ton.

[0020] In an embodiment, the supplements comprise i) beta-mannanase and ii) a probiotic. In an embodiment, the beta-mannanase and the probiotic provide a synergistic effect in improvement in health of the animal.

[0021] In an embodiment, the supplement is present in a feed for the animal. In an embodiment, the feed is a free choice feed. [0022] In an embodiment, the administration provides an improvement in health of the animal. In an embodiment, the improvement in health of the animal is an improvement in animal welfare. In an embodiment, the improvement in health of the animal comprises an improvement in eating behavior. In an embodiment, the improvement in health of the animal comprises an improvement in walking behavior. In an embodiment, the improvement in health of the animal comprises an improvement in standing behavior. In an embodiment, the improvement in health of the animal comprises an improvement in sitting behavior.

[0023] In an embodiment, the improvement in health of the animal comprises an improvement in liquid consumption behavior. In an embodiment, the improvement in health of the animal comprises an improvement in feather exploring behavior. In an embodiment, the improvement in health of the animal comprises an improvement in head scratching behavior. In an embodiment, the improvement in health of the animal comprises an improvement in wing flapping behavior. In an embodiment, the improvement in health of the animal comprises an improvement in leg stretching behavior. In an embodiment, the improvement in health of the animal comprises an improvement in stretching behavior. In an embodiment, the improvement in health of the animal comprises an improvement in aggressive pecking behavior. In an embodiment, the improvement in health of the animal comprises an improvement in non- aggressive pecking behavior.

[0024] In an embodiment, the improvement in health of the animal comprises a decrease in lesions on the animal. In an embodiment, the lesions are present on the neck. In an embodiment, the lesions are present on the tail. In an embodiment, the lesions are present on the cloaca. In an embodiment, the lesions are present on the comb.

[0025] In an embodiment, the improvement in health of the animal comprises an improvement in intestinal health in the animal. In an embodiment, the improvement in health of the animal comprises prevention of intestinal dysbiosis in the animal.

[0026] In an embodiment, the improvement in health of the animal comprises a reduction in pro-inflammatory effect in the animal. In an embodiment, the improvement in health of the animal comprises increased release of mannan-oiligosaccharides (MOS) in the animal.

[0027] In an embodiment, the administration provides an improvement in egg quality of one of more eggs laid by the animal. In an embodiment, the improvement in egg quality is indicated by a modification of 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 breaking strength, albumen pH, yolk pH, yolk color score, yolk lightness, yolk redness, yolk yellowness, chroma, and any combination thereof. [0028] In an embodiment, the improvement in egg quality is indicated by a modification of weight. In an embodiment, the improvement in egg quality is indicated by a modification of specific gravity. In an embodiment, the improvement in egg quality is indicated by a modification of albumen height. In an embodiment, the improvement in egg quality is indicated by a modification of albumen weight. In an embodiment, the improvement in egg quality is indicated by a modification of yolk height. In an embodiment, the improvement in egg quality is indicated by a modification of yolk length. In an embodiment, the improvement in egg quality is indicated by a modification of yolk index. In an embodiment, the improvement in egg quality is indicated by a modification of yolk weight. In an embodiment, the improvement in egg quality is indicated by a modification of Haugh unit. In an embodiment, the improvement in egg quality is indicated by a modification of shell weight. In an embodiment, the improvement in egg quality is indicated by a modification of shell breaking strength. In an embodiment, the improvement in egg quality is indicated by a modification of albumen pH. In an embodiment, the improvement in egg quality is indicated by a modification of yolk pH. In an embodiment, the improvement in egg quality is indicated by a modification of yolk color score. In an embodiment, the improvement in egg quality is indicated by a modification of yolk lightness. In an embodiment, the improvement in egg quality is indicated by a modification of yolk redness. In an embodiment, the improvement in egg quality is indicated by a modification of yolk yellowness. In an embodiment, the improvement in egg quality is indicated by a modification of chroma. In an embodiment, the modification is an increase in measurement. In an embodiment, the modification is a decrease in measurement. [0029] In an embodiment, the administration provides an improvement in reactive oxygen species (ROS) in the animal. In an embodiment, the improvement in ROS is in the intestine of the animal. In an embodiment, the improvement in ROS is in the oviduct of the animal.

[0030] In an embodiment, the improvement in ROS is indicated by a modification of thiobarbituric acid reactive substances (TBARS), glutathione S-transferase (GST), protein thios (TSH), and any combination thereof. In an embodiment, the improvement in ROS is indicated by a modification of thiobarbituric acid reactive substances (TBARS). In an embodiment, the improvement in ROS is indicated by a modification of glutathione S-transferase (GST). In an embodiment, the improvement in ROS is indicated by a modification of protein thios (TSH). In an embodiment, the modification is an increase in measurement. In an embodiment, the modification is a decrease in measurement.

[0031] In an embodiment, the administration provides an increase in egg laying rate of the animal. In an embodiment, the administration provides an increase in fresh egg weight for eggs laid by the animal. In an embodiment, the administration provides a reduction in coefficient of variability of egg weight for eggs laid by the animal. In an embodiment, the administration provides an increase in an egg component mass for eggs laid by the animal. In an embodiment, the egg component mass is egg mass. In an embodiment, the egg component mass is yolk mass. In an embodiment, the egg component mass is albumen mass. In an embodiment, the egg component mass is egg shell mass.

[0032] In an embodiment, the administration provides an increase in occurrence of clean eggs laid by the animal. In an embodiment, the increase in occurrence of clean eggs is a reduction in the presence of feces on the eggs.

[0033] In an embodiment, the administration provides a modification in eggshell thickness in eggs laid by the animal. In an embodiment, the modification in eggshell thickness is an increase in eggshell thickness. In an embodiment, the modification in eggshell thickness is a decrease in eggshell thickness.

[0034] In an embodiment, the modification in eggshell thickness is observed during a first phase of about 40 weeks. In an embodiment, the modification in eggshell thickness is observed during a second phase of about 44 weeks. In an embodiment, the modification in eggshell thickness is observed during a third phase of about 48 weeks.

[0035] The following numbered embodiments are contemplated and are non-limiting:

1. A method comprising a step of administering one or more supplements to a nonhuman animal.

2. The method of clause 1 , any other suitable clause, or any combination of suitable clauses, wherein the animal is an avian.

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

4. The method of clause 2, any other suitable clause, or any combination of suitable clauses, wherein the avian is a chicken.

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

6. The method of clause 5, any other suitable clause, or any combination of suitable clauses, wherein the hen is a laying hen.

7. The method of clause 2, any other suitable clause, or any combination of suitable clauses, wherein the avian is present in a cage system.

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™. 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 beta-mannanase.

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

11. The method of clause 9, any other suitable clause, or any combination of suitable clauses, wherein the beta-mannanase is administered at a dose between 100 g/ton and 500 g/ton.

12. The method of clause 9, any other suitable clause, or any combination of suitable clauses, wherein the beta-mannanase is administered at a dose of 100 g/ton.

13. The method of clause 9, any other suitable clause, or any combination of suitable clauses, wherein the beta-mannanase is administered at a dose of 200 g/ton.

14. The method of clause 9, any other suitable clause, or any combination of suitable clauses, wherein the beta-mannanase is administered at a dose of 300 g/ton.

15. The method of clause 9, any other suitable clause, or any combination of suitable clauses, wherein the beta-mannanase is administered at a dose of 400 g/ton.

16. The method of clause 9, any other suitable clause, or any combination of suitable clauses, wherein the beta-mannanase is administered at a dose of 500 g/ton.

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

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.

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 thermophiles, or 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.

20. The method of clause 18, any other suitable clause, or any combination of suitable clauses, wherein the probiotic supplement is administered at a dose between 10 g/ton and 100 g/ton.

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.

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. 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.

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.

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.

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.

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.

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.

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.

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.

31. The method of any one of clauses 1 to 30, any other suitable clause, or any combination of suitable clauses, wherein the supplements comprise i) beta-mannanase and ii) a probiotic.

32. The method of clause 31 , any other suitable clause, or any combination of suitable clauses, wherein the beta-mannanase and the probiotic provide a synergistic effect in improvement in health of the animal.

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 a feed for the animal.

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

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

36. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein the improvement in health of the animal is an improvement in animal welfare.

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

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

40. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein the improvement in health of the animal comprises an improvement in sitting behavior.

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

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

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

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

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

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

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

48. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein the improvement in health of the animal comprises an improvement in non- aggressive pecking behavior. 49. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein the improvement in health of the animal comprises a decrease in lesions on the animal.

50. The method of clause 49, any other suitable clause, or any combination of suitable clauses, wherein the lesions are present on the neck.

51. The method of clause 49, any other suitable clause, or any combination of suitable clauses, wherein the lesions are present on the tail.

52. The method of clause 49, any other suitable clause, or any combination of suitable clauses, wherein the lesions are present on the cloaca.

53. The method of clause 49, any other suitable clause, or any combination of suitable clauses, wherein the lesions are present on the comb.

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

55. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein the improvement in health of the animal comprises prevention of intestinal dysbiosis in the animal.

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

57. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein the improvement in health of the animal comprises increased release of mannan- oiligosaccharides (MOS) in the animal.

58. The method of any one of clauses 1 to 57, any other suitable clause, or any combination of suitable clauses, wherein the administration provides an improvement in egg quality of one of more eggs laid by the animal.

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 modification of 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 breaking strength, albumen pH, yolk pH, yolk color score, yolk lightness, yolk redness, yolk yellowness, chroma, and any combination thereof.

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 modification of weight. 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 modification of specific gravity.

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 modification of albumen height.

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 modification of albumen weight.

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 modification of yolk height.

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 modification of yolk length.

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 modification of yolk index.

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 modification of yolk weight.

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 modification of Haugh unit.

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 modification of shell weight.

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 modification of shell breaking strength.

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 modification of albumen pH.

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 modification of yolk pH.

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 modification of yolk color score.

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 modification of yolk lightness.

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 modification of yolk redness. 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 modification of yolk yellowness.

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 modification of chroma.

78. The method of any one of clauses 59 to 77, any other suitable clause, or any combination of suitable clauses, wherein the modification is an increase in measurement.

79. The method of any one of clauses 59 to 77, any other suitable clause, or any combination of suitable clauses, wherein the modification is a decrease in measurement.

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

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

82. The method of clause 80, any other suitable clause, or any combination of suitable clauses, wherein the improvement in ROS is in the oviduct of the animal.

83. 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 modification of thiobarbituric acid reactive substances (TBARS), glutathione S-transferase (GST), protein thios (TSH), and any combination thereof.

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 modification of thiobarbituric acid reactive substances (TBARS).

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 modification of glutathione S-transferase (GST).

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 a modification of protein thios (TSH).

87. The method of any one of clauses 83 to 86, any other suitable clause, or any combination of suitable clauses, wherein the modification is an increase in measurement.

88. The method of any one of clauses 83 to 86, any other suitable clause, or any combination of suitable clauses, wherein the modification is a decrease in measurement. 89. The method of any one of clauses 1 to 88, any other suitable clause, or any combination of suitable clauses, wherein the administration provides an increase in egg laying rate of the animal.

90. The method of any one of clauses 1 to 89, any other suitable clause, or any combination of suitable clauses, wherein the administration provides an increase in fresh egg weight for eggs laid by the animal.

91. The method of any one of clauses 1 to 90, any other suitable clause, or any combination of suitable clauses, wherein the administration provides a reduction in coefficient of variability of egg weight for eggs laid by the animal.

92. The method of any one of clauses 1 to 91, any other suitable clause, or any combination of suitable clauses, wherein the administration provides an increase in an egg component mass for eggs laid by the animal.

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

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

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 mass is albumen mass.

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.

97. The method of any one of clauses 1 to 96, any other suitable clause, or any combination of suitable clauses, wherein the administration provides an increase in occurrence of clean eggs laid by the animal.

98. The method of clause 97, any other suitable clause, or any combination of suitable clauses, wherein the increase in occurrence of clean eggs is a reduction in the presence of feces on the eggs.

99. The method of any one of clauses 1 to 98, any other suitable clause, or any combination of suitable clauses, wherein the administration provides a modification in eggshell thickness in eggs laid by the animal.

100. The method of clause 99, any other suitable clause, or any combination of suitable clauses, wherein the modification in eggshell thickness is an increase in eggshell thickness.

101. The method of clause 99, any other suitable clause, or any combination of suitable clauses, wherein the modification in eggshell thickness is a decrease in eggshell thickness. 102. The method of any one of clauses 1 to 101, any other suitable clause, or any combination of suitable clauses, wherein the modification in eggshell thickness is observed during a first phase of about 40 weeks.

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

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

EXAMPLES

Example 1 Behavioral Assessments

[0036] For the instant example, birds were evaluated at a commercial farm in Salvador do Sul, state of Rio Grande do Sul, in southern Brazil. The experimental units were randomly selected among hens housed in a commercial farm with about 28 thousand lightweight laying hens (Hyline W 36 lineage, 36 weeks old). The replicates were assigned in a completely randomized design to the four treatments, that included i) control treatment (a basal diet without supplementation with any other additive), ii) ; P-mannanase (control diet supplemented with 300 g/ton of -mannanase), hi) probiotic (control diet supplemented with 50 g/ton of a multi- cepa probiotic additive), and iv) P-mannanase plus probiotic treatment (control diet supplemented with 300 g/ton of P-mannanase and 50 g/ton of a multi-cepa probiotic additive). [0037] The P-mannanase (Hemicell HT™, Elanco Animal Health) for the instant example was an exogenous enzyme from the fermentation of the Paenibacillus lentus bacteria. The probiotic additive (Protexin™ Concentrate, Elanco Animal Health) for the instant example includes Lactobacillus acidophilus (2.06 x 10 ⁸ UFC/g), Lactobacillus bulgaricus (2.06 x 10 ⁸ UFC/g), Lactobacillus plantarum (1.26 x 10 ⁸ UFC/g), Lactobacillus rhamnosus (2.06 x 10 ⁸ UFC/g), Bifidobacterium bifdum (2.0 x 10 ⁸ UFC/g), Enterococcus faecium (6.46 x 10 ⁸ UFC/g), and Streptococcus thermophilus (4.10 x 10 ⁸ UFC/g).

[0038] The basal diet comprised a com-soybean meal-based feed formulated according to nutritional requirements and inert material (kaolin) was included in basal feed to replace - mannanase and/or probiotic additives. Feed and water were both provided ad libitum throughout the experimental period using nipple drinkers and gutter feeders. [0039] The birds were housed in conventional sheds, arranged in an east-west direction, with concrete floors and masonry walls complemented with wire mesh to the ceiling. The shed was equipped with side curtains, which were managed according to weather conditions to provide thermal comfort. The average minimum and maximum temperature and air relative humidity values recorded were 18 and 36 °C, and 35.8 and 94.7%, respectively. The lighting regime was composed of 16 hours of light and 8 hours of dark per day.

[0040] The birds remained in galvanized- wire cages (100-cm long x 40-cm wide x 45- cm high, resulting in a floor area of 500 cm ² /hen) throughout the experimental period. Birds were allocated in each cage. Birds were supplemented for 84 days and the assessments were performed in the last week of the trial.

[0041] Behavioral assessments were performed via image capture combined with local feather scoping and comb abnormalities assessments. For the behavior assessment, six birds per treatment (one per cage) were randomly selected for observation. The captured images were carried out for 7 consecutive days, in a period of 15 minutes in the morning (e.g., the highest peak of laying of the birds) and 30 minutes in the afternoon divided into two periods corresponding to the hottest and cooler times of the day. Images were recorded and analyzed by visual counting and frequency methods.

[0042] The lesion scoring was performed through visual scoring attributed to three body regions (neck, tail, and vent) from 25 birds per treatment group that were randomly selected. Possible injuries and different degrees of severity were analyzed on a scale from 0 to 5 (best score being 0, indicating complete plumage and no damage, and the worst score being 5, indicating completely feathered areas with 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).

[0043] All behavioral tests were carried out in the last week of the experiment, allowing the birds to remain exposed to the treatments for a longer period. The same animal was used only in one of the tests, thus preventing one test from interfering with the result of the other.

[0044] The frequency of the main behaviors observed in laying hens fed P-mannanase and/or probiotics are shown in Table 1.

Table 1.

Treatments ¹ P-value ²

. T..rai .t.s C „ont .ro -l He _Z m _II i,cell Protexin H .. Avg . . . • .. ,

_ (H) _ (P) _ + P time _ Treat Time T x T Eating times were polled together (indicated at ‘Avg treat’. The averages obtained when the fourth treatments were polled together in each observation time are also presented (indicated as ‘Avg time’) and compared within the column ^XYZ .

² Probability of treatment effect (treat), time of observation (time), and interaction (T x T). [0045] The times expended (minutes/bird) in each of the main behaviors by laying hens fed P-mannanase and/or probiotics are shown in Table 2.

Table 2. Walking

^eans followed b different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%. Comparisons were performed among treatments - line A.,B,C,D each observation time and also for averages obtained when the three observation times were polled together (indicated at ‘Avg treat’. The averages obtained when the fourth treatments were polled together in each observation time are also presented (indicated as ‘Avg time’) and compared within the column ^XYZ .

² Probability of treatment effect (treat), time of observation (time), and interaction (T x T).

[0046] The times expended (minutes/bird) in other behaviors by laying hens fed p- mannanase and/or probiotics are shown in Table 3.

Table 3. Avg treat 0.33 0.09 0.01 0.09

^eans followed b different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%. Comparisons were performed among treatments - line A ^B ,C,D _w jp]j _{n eac} h observation time and also for averages obtained when the three observation times were polled together (indicated at ‘Avg treat’. The averages obtained when the fourt treatments were polled together in each observation time are also presented (indicated as ‘Avg time’) and compared within the column ^XYZ .

² Probability of treatment effect (treat), time of observation (time), and interaction (T x T).

[0047] The frequency (%) of birds with lesions (disregarding the score) observed in groups of laying hens fed P-mannanase and/or probiotics are shown in Table 4.

Table 4.

[0048] The lesion score observed in groups of laying hens fed 3-mannanase and/or probiotics are shown in Table 5. Scores from 0 (no lesion) to 5 (severe lesion) were attributed to the birds individually, except for crest in which the scores ranged from 0 (no lesion) to 2 (severe lesion). Means (LS/means) followed by different uppercase letters differ statistically at

5%.

Table 5.

Example 2 Egg Quality Assessments

[0049] For the instant example, the egg quality of birds in various treatment groups was evaluated. The egg quality was evaluated during storage periods.

[0050] The experimental units were randomly selected among the hens housed in the commercial farm (about 28,000 light-weight laying hens, 36 weeks old, Hyline W 36 lineage) and assigned to the treatments, that were i) control (basal diet, without supplementation); ii) diet supplemented with 300 g/ton of the P-mannanase Hemicell®; iii) diet supplemented with 50 g/ton of the probiotic additive Protexin®; iv) diet supplemented with 50 g/ton of probiotic additive and 300 g/ton of P-mannanase (Hemicell + Protexin).

[0051] The basal feed was formulated according to the nutritional requirements for the genetic (Hy-Line, 2020). Corn and soybean meal were the main ingredients in the feed formulas. Feed and water were both provided ad libitum throughout the experimental period using nipple drinkers and gutter feeders.

[0052] Three 28-day production cycles were carried out and 480 eggs (120 from each treatment group) were randomly collected on the last day of each cycle for evaluation of egg quality. Cracked eggs were excluded from the experiment.

[0053] Part of the eggs were immediately subjected to quality analysis to represent the characteristics of fresh eggs (zero days of storage). The other eggs were stored at room temperature and randomly separated for quality assessment at each storage interval (7, 14, 21, 28, 35 and 42 days).

[0054] Fifteen eggs from each treatment were randomly chosen and weighed individually at weekly intervals during the storage period. The weight loss (%) of eggs during storage was calculated using the following equation: ^

[0055] The albumen height was estimated by the average of three measurements taken at different points on the albumen at a distance of 10 mm from the yolk using a digital caliper (TMX PD - 150, China). Thus, the Haugh Unit (HU) was obtained through the equation proposed by Haugh (1937), where h= thickness of albumen (mm); W= mass of the entire egg (g):

[0056] Yolk width and height (mm) were measured with a digital caliper (TMX PD -

150, China) on 15 eggs from each treatment at weekly intervals. After, the yolk index was calculated as:

[0057] Yolk color was determined in fifteen eggs per treatment at weekly intervals using the Roche colorimetric fan (DSM, Sao Paulo, Brazil), with a score ranging from 1 (light yellow) to 15 (reddish orange). Complementarily, a spectrophotometer equipment (Delta Vista model 450G, Delta Color, Sao Leopoldo, Brazil) was also used for this evaluation, which determined colorimetric coordinates of luminosity (L*), red intensity (a*) and yellow intensity (b*). Chroma was estimated considering the following equation:

[0058] After yolk and albumen separation, the dense and the fluid albumen were homogenized for 20 seconds and then the pH was determined using a digital pH meter (Kasvi model k39-2014B, Parana, 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.

[0059] The value of specific gravity was based on Archimedes’ principle, using the equation: [0060] Total solid content was determined separately in albumen and yolk. Five grams of albumen and yolk were weighed separately in previously dried porcelain crucibles. The albumen and 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 from each treatment were evaluated to determine the total solids.

[0061] The shell percentage was obtained after shell separation, washing, drying and weighing. Fifteen eggs from each treatment were evaluated on day 0, 21 and 42 for these variables.

[0062] The substances that react to thiobarbituric acid (TBARS) were used to access the lipid oxidation in a pool of three yolks per treatment collected in three storage periods (0, 21, and 42 days). Only treatments with Hemicell and Protexin isolated were tested due to the previous results obtained in egg quality analysis.

[0063] Table 6 displays a summary of fresh egg quality analyses from laying hens fed P-mannanase and/or probiotics. The values represent averages from the three evaluated periods, considering only fresh eggs.

Table 6.

Treatments

, Hemicell Protexin ,, _ ,, , ,

Storage time Control . H + P SE H) (r) ¹ P-value

( ²

Weight 62.74 63.11 62.51 62.71 0.290 0.904

Specific gravity (g/ml) 1.006B 1.007A 1.006B 1.006B 0.001 <0.001

Albumen height (mm) 8.04 8.06 8.18 8.17 0.104 0.129

Albumen weight (g) 36.82 37.39 36.30 36.59 0.239 0.424

Yolk height (mm) 17.98B 18.15A 18.27A 18.18A 0.063 0.037

Yolk length (mm) 40.67B 41.62A 41.25AB 41.82A 0.118 0.002

Yolk index 0.443 0.435 0.443 0.435 0.017 0.184

Yolk weight (g) 15.33B 15.70AB 15.45B 16.08A 0.096 0.004

Haugh unit 89.40 90.10 89.88 89.55 0.558 0.132

Shell weight (g) 5.81B 6.15A 5.96AB 6.11A 0.041 0.09

Shell breaking strength (N) 2.47 2.39 2.17 2.25 0.106 0.563

Albumen pH 8.41 8.40 8.38 8.44 0.028 0.178

Yolk pH 6.04B 5.96A 5.99A 6.00A 0.013 0.002

Yolk color score (palette) 5.60B 5.98A 5.77 AB 5.87AB 0.052 0.032

Yolk lightness (L* color) 50.86B 50.66B 51.33AB 52.16A 0.161 0.002

Yolk redness (a* color) 7.12B 7.66A 7.67A 7.66A 0.100 <0.001

Yolk yellowness (b* color) 57.41 58.88 58.93 58.75 0.354 0.122

Chroma 57.85B 59.67A 59.67A 59.25A 0.357 0.003

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at f0%. [0064] Table 7 shows weight loss (g) of eggs from laying hens fed beta-mannanase

(Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.

Table 7.

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[0065] Table 8 shows weight loss (g) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.

Table 8 (H) (P) value ²

^Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[0066] Table 9 shows weight loss (g) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.

Table 9. Only 3 treatments 0.062 0.300

Only Hemicell 0.085 0.152

Only Protexin 0.055 0.670

Day 21 1.523 1.562 1.436 1.719

All treatments AB AB A B 0.051 0.022

Only 3 treatments 0.062 0.424

Only Hemicell 0.058 0.708

Only Protexin 0.061 0.402

Day 28 2.137 2.300 2.018 2.243

All treatments 0.053 0.169

Only 3 treatments 0.065 0.170

Only Hemicell 0.074 0.241

Only Protexin 0.080 0.476

Day 35 2.439 2.121 2.539 2.592

All treatments 0.052 0.182

Only 3 treatments 0.058 0.218

Only Hemicell 0.077 0.291

Only Protexin 0.057 0.358

Day 42 3.675 3.263 3.132 3.382

All treatments B B A AB 0.049 0.007

Only 3 treatments B A A 0.057 0.003

Only Hemicell B A 0.069 0.020

Only Protexin B A 0.076 <0.001

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[0067] Table 10 shows specific gravity (g/ml) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.

Table 10.

Treatments

.. . Hemicell Protexin „ „ P-

Storage time Control _{(H) (p)} H + P SE ¹

Day l 1.066 1.059 1.064 1.057

All treatments A B B B 0.007 <0.001

Only 3 treatments A B A 0.007 <0.001

Only Hemicell A B 0.009 <0.001

Only Protexin 0.008 0372

Day 7 1.051 1.057 1.041 1.054

All treatments 0.259 0.997

Only 3 treatments 0.345 0.983

Only Hemicell 0.520 0.959

Only Protexin 0.503 0.922

Day 14 1.054 1.048 1.050 1.049

All treatments 0.016 0.653

Only 3 treatments 0.016 0.348 Only Hemicell 0.025 0.242

Only Protexin 0.025 0.381

Day 21 1.062 1.050 1.051 1.060

All treatments 0.028 0.293

Only 3 treatments 0.036 0.345

Only Hemicell 0.008 0.226

Only Protexin 0.008 0.353

Day 28 1.042 1.042 1.039 1.041

All treatments 0.018 0.954

Only 3 treatments 0.021 0.845

Only Hemicell 0.022 0.976

Only Protexin 0.026 0.522

Day 35 1.042 1.042 1.055 1.057

All treatments 0.018 0.954

Only 3 treatments 0.021 0.845

Only Hemicell 0.022 0.976

Only Protexin 0.026 0.622

Day 42 1.047 1.056 1.055 1.057

All treatments B A A A 0.006 <0.001

Only 3 treatments B A A 0.007 <0.001

Only Hemicell B A 0.009 <0.001

Only Protexin B A 0.009 <0.001

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[0068] Table 11 shows specific gravity (g/ml) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.

Table 11.

Treatments

.. . i Hemicell Protexin ,, .. P-

Storage time Control H + P SE ¹ _yalue2

Day l 1.075 1.083 1.073 1.074

All treatments B A B B 0.008 <0.001

Only 3 treatments B A B 0.010 <0.001

Only Hemicell B A 0.011 <0.001

Only Protexin 0.010 0.246

Day ? 1.069 1.066 1.071 1.066

All treatments AB AB A B 0.007 0.026

Only 3 treatments AB B A 0.008 0.031

Only Hemicell a b 0.008 0.065

Only Protexin 0.010 0.415

Day 14 1.059 1.059 1.053 1.062

All treatments A A B A 0.0065 <0.001

Only 3 treatments A A B 0.0071 <0.001

Only Hemicell 0.0067 0.526

Day 28 1.059 1.063 1.061 1.071

All treatments B B B A 0.007 <0.001

Only 3 treatments B A AB 0.006 0.040

Only Hemicell B A 0.007 0.011

Only Protexin 0.007 0.251

Day 35 1.036 1.039 1.038 1.038

All treatments B A AB AB 0.004 0.022

Only 3 treatments B A A 0 0.004 0.016

Only Hemicell B A 0.006 0.020

Only Protexin B A 0.005 0.011

Day 42 1.048 1.049 1.050 1.049

All treatments 0.004 0.301

Only 3 treatments 0.005 0.143

Only Hemicell 0.006 0.657

Only Protexin b a 0.006 0.054

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[0069] Table 12 shows shows specific gravity (g/ml) of eggs from laying hens fedbeta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.

Table 12.

Treatments

, Hemicell Protexin _ _T _ P-

Storag se time Control H + P SB (H) (P) ¹ , value ₇ 2

Day l 1.059 1.065 1.063 1.160

A B A A B 0.004 <0.001

Only 3 treatments B A A 0.005 <0.001

Only Hemicell B A 0.007 <0.001

Only Protexin B A 0.006 0.001

Day 7 1.064 1.061 1.068 1.064

All treatments BC C A B 0.004 <0.001

Only 3 treatments B C A 0.005 <0.001

Only Hemicell A B 0.004 0.001

Only Protexin B A 0.005 <0.001

Day 14 1.052 1.053 1.053 1.056

All treatments B AB AB A 0.004 0.036

Only 3 treatments 0.005 0.607

Only Hemicell 0.006 0.347

Only Protexin 0.007 0.742

Day 21 1.038 1.046 1.042 1.047

All treatments C A B A 0.005 <0.001

Only 3 treatments C A B 0.005 <0.001

Only Hemicell B A 0.008 <0.001

Only Protexin B A 0.005 <0.001

Day 28 1.031 1.033 1.031 1.032

All treatments 0.003 0.223

Only 3 treatments 0.004 0.104

Only Hemicell b a 0.005 0.079

Only Protexin 0.004 0.780

Day 35 1.038 1038 1.040 1.039

All treatments 0.004 0.954

Only 3 treatments 0.004 0.319

Only Hemicell 0.004 0.862

Only Protexin 0.005 0.240

Day 42 1.032 1.037 1.035 1.037

All treatments B A A A 0.005 <0.001

Only 3 treatments B A A 0.005 <0.001

Only Hemicell B A 0.007 <0.001

Only Protexin B A 0.005 0.003

¹ Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[0070] Table 13 shows albumen height (mm) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.

Table 13.

Treatments e. .. , Hemicell Protexin ,, „ P-

Storag ⁶ e time Control (H) (P) H + P SE ¹ va .lue ₂ ²

Day l 9.34 9.69 9.73 8.55

All treatments AB A A B 0.14 0.009

Only 3 treatments 0.15 0.524

Only Hemicell 0.18 0.347

Only Protexin 0.17 0.250

Day 7 6.03 5.90 6.33 5.18

All treatments A A A B 0.08 <0.001

Only 3 treatments 0.08 0.102

Only Hemicell 0.08 0.461

Only Protexin 0.10 0.161

Day 14 4.74 4.30 4.79 4.39

All treatments AB B A AB 0.07 0.035

Only 3 treatments AB B A 0.09 0.048

Only Hemicell B A 0.11 0.044

Only Protexin 0.11 0.827

Day 21 3.97 3.67 3.86 3.56 All treatments A AB AB B 0.05 0.041

Only 3 treatments 0.06 0.186

Only Hemicell a b 0.08 0.096

Only Protexin 0.06 0.433

Day 28 3.24 3.16 3.31 3.09

All treatments 0.04 0.397

Only 3 treatments 0.05 0.511

Only Hemicell 0.06 0.538

Only Protexin 0.07 0.666

Day 35 2.98 2.88 2.77 2.77

All treatments 0.04 0.343

Only 3 treatments 0.05 0.339

Only Hemicell 0.08 0.534

Only Protexin 0.06 0.144

Day 42 2.70 2.76 2.58 2.71

All treatments 0.04 0.545

Only 3 treatments 0.04 0.285

Only Hemicell 0.05 0.644

Only Protexin 0.05 0.298

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[0071] Table 14 shows albumen height (mm) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.

Table 14.

Treatments e. .. , Hemicell Protexin ,, „ P-

St r tim C ntr l H + P SE ¹

Day l 6.83 6.65 7.05 6.52

All treatments 0.11 0.384

Only 3 treatments 0.14 0.519

Only Hemicell 0.13 0.504

Only Protexin 0.20 0.595

Day 7 5.33 4.91 5.26 5.08

All treatments A B AB B 0.06 0.064

Only 3 treatments A B AB 0.06 0.006

Only Hemicell A B 0.08 0.007

Only Protexin 0.08 0.710

Day 14 3.92 3.58 3.73 3.34

All treatments A AB AB B 0.06 0.006

Only 3 treatments 0.07 0.163

Only Hemicell a b 0.08 0.058

Only Protexin 0.09 0.336

Day 21 3.23 2.89 3.06 2.75

All treatments A AB AB B 0.05 0.007 Only 3 treatments A B AB 0.05 0.061

Only Hemicell A B 0.07 0.016

Only Protexin 0.06 0.219

Day 28 2.74 2.58 2.72 2.75

All treatments 0.04 0.470

Only 3 treatments 0.05 0.406

Only Hemicell 0.06 0.242

Only Protexin 0.06 0.893

Day 35 2.75 2.57 2.71 3.06

All treatments AB B B A 0.05 0.004

Only 3 treatments 0.04 0.325

Only Hemicell 0.06 0.173

Only Protexin 0.06 0.762

Day 42 2.62 2.67 2.74 2.51

All treatments AB AB A B 0.03 0.069

Only 3 treatments 0.03 0.404

Only Hemicell 0.0o4 0.531

Only Protexin 0.04 0.160

¹ Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[0072] Table 15 shows albumen height (mm) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.

Table 15.

Treatments

.. „ . , Hemicell Protexin .. P-

Storag

^& e time Control , _T „ H + P SE ) (P) ¹ . (H value ₂ 2

Day l 7.97 7.83 7.56 7.64

All treatments 0.07 0.209

Only 3 treatments 0.09 0.201

Only Hemicell 0.12 0.576

Only Protexin 0.11 0.119

Day 7 5.49 5.28 5.50 5.25

All treatments 0.06 0.303

Only 3 treatments 0.07 0.394

Only Hemicell 0.09 0.249

Only Protexin 0.10 0.975

Day 14 4.74 4.30 4.79 4.39

All treatments A AB A B 0.05 0.011

Only 3 treatments a b a 0.07 0.085

Only Hemicell a b 0.08 0.051

Only Protexin 0.08 0.925

Day 21 3.67 3.13 3.51 3.31

All treatments A B AB AB 0.06 0.033

Only 3 treatments A B AB 0.07 0.012 Only Hemicell A B 0.01 0.016

Only Protexin 0.06 0.214

Day 28 2.96 2.82 2.93 2.73

All treatments a a a b 0.03 0.098

Only 3 treatments 0.04 0.424

Only Hemicell 0.05 0.245

Only Protexin 0.05 0.791

Day 35 2.66 2.48 2.52 2.77

All treatments AB B B A 0.03 0.018

Only 3 treatments 0.04 0.303

Only Hemicell a b 0.04 0.057

Only Protexin 0.06 0.295

Day 42 2.88 2.47 2.48 2.36

All treatments A AB AB B 0.04 0.050

Only 3 treatments 0.06 0.176

Only Hemicell 0.06 0.106

Only Protexin 0.08 0.130

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[0073] Table 16 shows yolk length (mm) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.

Table 16.

Treatments

, Hemicell Protexin ....| P-

Storage time Control H + P SE ¹ _value 2

Day l 40.63 41.25 41.32 41.50

All treatments 0.25 0.565

Only 3 treatments 0.24 0.467

Only Hemicell 0.30 0.319

Only Protexin 0.29 0.261

Day 7 43.49 44.38 43.50 44.38

All treatments 0.23 0.319

Only 3 treatments 0.27 0.323

Only Hemicell 0.39 0.262

Only Protexin 0.27 0.986

Day 14 45.02 45.58 44.48 44.52

All treatments 0.23 0.285

Only 3 treatments 0.24 0.188

Only Hemicell 0.31 0.391

Only Protexin 0.27 0.328

Day 21 49.84 46.85 45.71 47.91

All treatments A B B AB 0.42 0.00

Only 3 treatments A B B 0.52 0.001

Only Hemicell A B 0.73 0.005

Only Protexin A B 0.73 0.005

Day 28 47.91 48.00 46.71 46.00

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[0074] Table 17 shows yolk length (mm) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.

Table 17. Only Protexin A B 0.50 0.003

Day 35 47.90 48.50 48.47 47.96

All treatments 0.28 0.816

Only 3 treatments 0.34 0.733

Only Hemicell 0.35 0.416

Only Protexin 0.45 0.536

Day 42 51.59 51.91 51.02 49.76

All treatments 0.39 0.238

Only 3 treatments 0.48 0.739

Only Hemicell 0.56 0.782

Only Protexin 0.66 0.680

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[0075] Table 18 shows yolk length (mm) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.

Table 18.

Treatments o. , Hemicell Protexin ,, „ P-

Storage time Control H + P SE ¹ _yalue2

Day l 39.94 41.10 41.40 41.96

All treatments B AB AB A 0.19 0.002

Only 3 treatments b a a 0.22 0.051

Only Hemicell B A 0.26 0.024

Only Protexin B A 0.27 0.032

Day 7 42.03 42.63 42.76 44.01

All treatments B AB AB A 0.23 0.017

Only 3 treatments 0.23 0.400

Only Hemicell 0.28 0.301

Only Protexin 0.28 0.208

Day 14 46.63 45.40 45.93 46.21

All treatments 0.24 0.355

Only 3 treatments 0.31 0.248

Only Hemicell a b 0.37 0.099

Only Protexin 0.39 0.378

Day 21 46.08 46.10 46.18 47.66

All treatments 0.25 0.107

Only 3 treatments 0.27 0.989

Only Hemicell 0.37 0.977

Only Protexin 0.29 0.872

Day 28 46.08 45.47 47.16 48.99

All treatments B B AB A 0.31 <0.001

Only 3 treatments 0.33 0.108

Only Hemicell 0.30 0.328

Only Protexin 0.41 0.217

Day 35 48.06 47.96 48.39 47.44

All treatments 0.35 0.809 Only 3 treatments 0.42 0.913

Only Hemicell 0.54 0.928

Only Protexin 0.60 0.790

Day 42 50.00 49.04 48.80 49.71

All treatments 0.35 0.611

Only 3 treatments 0.43 0.502

Only Hemicell 0.58 0.418

Only Protexin _ 0.47 _ 0.206

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[0076] Table 19 shows yolk height (mm) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.

Table 19.

Treatments

.. , Hemicell Protexin ,, „ P-

Storag

⁶ e time Control H + P SE (H) (P) ¹ . value ₂ 2

Day l 18.66 18.44 19.13 18.34

All treatments AB AB A B 0.10 0.035

Only 3 treatments b b a 0.12 0.060

Only Hemicell 0.12 0.367

Only Protexin 0.16 0.147

Day 7 15.77 15.22 15.36 14.98

All treatments A AB AB B 0.10 0.57

Only 3 treatments 0.11 0.154

Only Hemicell 0.13 0.132

Only Protexin 0.15 0.184

Day 14 13.15 13.09 13.10 13.05

All treatments 0.10 0.987

Only 3 treatments 0.12 0.974

Only Hemicell 0.14 0.820

Only Protexin 0.18 0.873

Day 21 11.82 12.08 11.94 11.96

All treatments 0.07 0.717

Only 3 treatments 0.08 0.520

Only Hemicell 0.11 0.295

Only Protexin 0.11 0.633

Day 28 10.51 10.48 11.21 11.40

All treatments B B A A 0.09 <0.001

Only 3 treatments B B A 0.10 0.003

Only Hemicell 0.11 0.875

Only Protexin B A 0.12 0.002

Day 35 10.07 10.03 11.01 9.81

All treatments B B A B 0.11 <0.001

Only 3 treatments B B A 0.13 0.001

Only Hemicell 0.14 0.894

Only Protexin B A 0.14 <0.001 Day 42 9.96 9.51 10.07 10.26

All treatments b b a ab 0.11 0.088

Only 3 treatments 0.13 0.193

Only Hemicell 0.14 0.119

Only Protexin 0.17 0.776

^Standard error. ² Prohability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[0077] Table 20 shows yolk height (mm) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.

Table 20.

Treatments

Day l 17.88 17.79 17.94 18.11

All treatments 0.08 0.273

Only 3 treatments 0.09 0.551

Only Hemicell 0.11 0.633

Only Protexin 0.10 0.215

Day 7 15.50 15.77 16.27 16.01

All treatments B AB A AB 0.09 0.018

Only 3 treatments B AB A 0.11 0.020

Only Hemicell 0.13 0.327

Only Protexin B A 0.15 0.013

Day 14 13.47 13.42 13.78 13.10

All treatments 0.10 0.122

Only 3 treatments 0.11 0.400

Only Hemicell 0.13 0.880

Only Protexin 0.15 0.306

Day 21 12.33 11.83 12.23 12.17

All treatments 0.08 0.180

Only 3 treatments a b a 0.09 0.067

Only Hemicell 0.13 0.127

Only Protexin 0.09 0.510

Day 28 10.86 10.46 10.83 10.88

All treatments 0.09 0.431

Only 3 treatments 0.09 0.205

Only Hemicell 0.11 0.103

Only Protexin 0.11 0.900

Day 35 10.88 11.00 11.39 10.96

All treatments 0.09 0.266

Only 3 treatments 0.12 0.206

Only Hemicell 0.15 0.700

Only Protexin A B 0.12 0.040

Day 42 9.64 9.59 10.49 10.07

All treatments B B A AB 0.10 0.002

Only 3 treatments B B A 0.12 0.001 Only Hemicell 0.14 0.860

Only Protexin _ B _ A _ 0.16 _ 0.007

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[0078] Table 21 shows yolk height (mm) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.

Table 21.

Treatments o. .. „ . , Hemicell Protexin ,, „ P-

Stora e time Control H + P SE ¹

Day l 17.59 17.92 17.75 18.11

All treatments 0.10 0.333

Only 3 treatments 0.12 0.542

Only Hemicell 0.15 0.304

Only Protexin 0.16 0.636

Day 7 15.97 15.70 15.81 16.27

All treatments 0.09 0. 194

Only 3 treatments 0.10 0.592

Only Hemicell 0.13 0.324

Only Protexin 0.13 0.564

Day 14 14.05 13.61 13.65 13.81

All treatments 0.08 0.247

Only 3 treatments 0.09 0.131

Only Hemicell 0.12 0.102

Only Protexin 0.12 0.103

Day 21 13.07 12.96 12.67 12.87

All treatments 0.10 0.569

Only 3 treatments 0.12 0.392

Only Hemicell 0.12 0.756

Only Protexin 0.13 0.131

Day 28 12.39 12.28 12.38 11.95

All treatments 0.08 0.221

Only 3 treatments 0.08 0.863

Only Hemicell 0.08 0.525

Only Protexin 0.12 0.961

Day 35 11.48 11.28 11.23 11.36

All treatments 0.08 0.802

Only 3 treatments 0.09 0.557

Only Hemicell 0.10 0.373

Only Protexin 0.11 0.315

Day 42 10.74 10.41 10.79 10.39

All treatments 0.10 0.385

Only 3 treatments 0.12 0.432

Only Hemicell 0.17 0.351

Only Protexin 0.14 0.872 Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[0079] Table 22 shows yolk index of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.

Table 22. Standard error. Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%. [0080] Table 23 shows yolk index of eggs from laying hens fed beta-mannanase

(Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.

Table 23.

Treatments

. - Hemicell Protexin _rT „ P-

Storage time Control ( _/T H _T ). ( _ZT P _V ) _l H + P SE ¹ va ,lue ₂ ²

Day l 0.429 0.418 0.433 0.435

All treatments ab a b b 0.002 0.083

Only 3 treatments 0.003 0.142

Only Hemicell 0.003 0.113

Only Protexin 0.003 0.600

Day 7 0.347 0.369 0.383 0.369

All treatments B A A A 0.002 <0.001

Only 3 treatments B A A 0.003 <0.001

Only Hemicell B A 0.003 0.002

Only Protexin B A 0.004 <0.001

Day 14 0.293 0.294 0.312 0.289

All treatments B B A B 0.002 0.002

Only 3 treatments B B A 0.003 0.012

Only Hemicell 0.002 0.947

Only Protexin B A 0.004 0.020

Day 21 0.260819 0.248324 0.270605 0.258164

All treatments AB B B AB 0.002 0.032

Only 3 treatments AB B B 0.003 0.013

Only Hemicell 0.003 0.101

Only Protexin 0.003 0.190

Day 28 0.214 0.218 0.228 0.225

All treatments 0.002 0.286

Only 3 treatments 0.003 0.156

Only Hemicell 0.003 0.602

Only Protexin 0.003 0.074

Day 35 0.228 0.232 0.236 0.229

All treatments 0.002 0.730

Only 3 treatments 0.003 0.631

Only Hemicell 0.004 0.622

Only Protexin 0.003 0.240

Day 42 0.186 0.185 0.205 0.202

All treatments B B A A 0.003 0.023

Only 3 treatments AB B A 0.003 0.033

Only Hemicell 0.003 0.907

Only Protexin b a 0.005 0.064

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%. [0081] Table 24 shows yolk index of eggs from laying hens fed beta-mannanase

(Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.

Table 24.

Treatments

, Hemicell Protexin .. , P-

Storag ⁶ e time Control (H) (P) H + P SE ¹ va .lue ₂ ²

Day l 0.441 0.436 0.432 0.432

All treatments 0.002 0.623

Only 3 treatments 0.003 0.571

Only Hemicell 0.004 0.618

Only Protexin 0.004 0.340

Day 7 0.380 0.369 0.366 0.370

All treatments 0.003 0.407

Only 3 treatments 0.003 0.266

Only Hemicell 0.003 0.148

Only Protexin 0.004 0.155

Day 14 0.314 0.299 0.301 0.299

All treatments 0.002 0.156

Only 3 treatments 0.003 0.146

Only Hemicell 0.004 0.108

Only Protexin 0.004 0.147

Day 21 0.284 0.271 0.275 0.270

All treatments 0.003 0.486

Only 3 treatments 0.004 0.435

Only Hemicell 0.003 0.291

Only Protexin 0.003 0.197

Day 28 0.271 0.271 0.263 0.243

All treatments A A AB B 0.003 0.003

Only 3 treatments 0.003 0.589

Only Hemicell 0.002 0.947

Only Protexin 0.004 0.399

Day 35 0.239 0.235 0.229 0.240

All treatments 0.002 0.434

Only 3 treatments 0.003 0.416

Only Hemicell 0.003 0.598

Only Protexin 0.004 0.27

Day 42 0.220 0.214 0.222 0.210

All treatments 0.003 0.483

Only 3 treatments 0.003 0.682

Only Hemicell 0.005 0.575

Only Protexin 0.003 0.848

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[0082] Table 25 shows yolk weight (g) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial. Table 25. y

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[0083] Table 26 shows yolk weight (g) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.

Table 26.

' Slandard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[0084] Table 27 shows yolk weight (g) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.

Table 27.

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[0085] Table 28 shows Haugh unit of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.

Table 28. Only Hemicell b a 1.06 0.080

Only Protexin 0.82 0.921

Day 7 76.76 75.55 78.39 70.26

All treatments A A A B 0.74 <0.001

Only 3 treatments 0.73 0.292

Only Hemicell 0.84 0.484

Only Protexin 0.38 0.369

Day 14 85.55 60.84 67.17 61.36

All treatments AB B A 0.89 0.126

Only 3 treatments 1.08 0.044

Only Hemicell 1.44 0.120

Only Protexin 1.16 0.496

Day 21 58.18 53.70 53.91 51.21

All treatments A AB AB B 0.94 0.075

Only 3 treatments 1.09 0.176

Only Hemicell 1.48 0.132

Only Protexin 1.16 0.163

Day 28 48.49 44.55 48.51 48.09

All treatments 1.02 0.458

Only 3 treatments 1.17 0.294

Only Hemicell 1.52 0.200

Only Protexin 1.49 0.994

Day 35 44.22 42.70 42.03 42.41

All treatments 1.05 0.309

Only 3 treatments 1.24 0.185

Only Hemicell 1.56 0.152

Only Protexin 1.63 0.112

Day 42 39.62 41.97 39.88 42.16

All treatments 0.93 0.582

Only 3 treatments 109 0.857

Only Hemicell 1.56 0.466

Only Protexin 1.22 0.117

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[0086] Table 29 shows Haugh unit of eggs from laying hens fed beta-mannanase

(Hemicell) and/or probiotics (Protexin), according to various storage Limes in a second trial.

Table 29.

Treatments

.. z . . Hemicell Protexin .. ,, P-

Storage time Control H + P SE ¹

Day l 84.42 82.53 85.31 79.85

All treatments A AB A B 0.58 0.003

Only 3 treatments 0.62 0.181

Only Hemicell 0.76 0.221

Only Protexin 0.76 0.566

Day 7 70.58 69.01 69.00 66.96 All treatments 0.69 0.343

Only 3 treatments 0.81 0.195

Only Hemicell 0.90 0.140

Only Protexin 1.00 0.443

Day 14 58.24 53.89 57.42 48.30

All treatments A AB A B 1.01 0.001

Only 3 treatments 1.07 0.213

Only Hemicell 1.34 0.106

Only Protexin 1.23 0.746

Day 21 47.09 43.03 46.60 41.53

All treatments A A A B 0.92 0.094

Only 3 treatments 1.12 0.287

Only Hemicell 1.41 0.154

Only Protexin 1.40 0.863

Day 28 41.03 39.52 41.68 38.71

All treatments 0.77 0.543

Only 3 treatments 0.87 0.587

Only Hemicell 1.18 0.533

Only Protexin 1.04 0.759

Day 35 43.40 41.17 39.60 46.44

All treatments ABA B B A 0.88 0.030

Only 3 treatments 0.96 0.292

Only Hemicell 1.12 0.331

Only Protexin 1.35 0.163

Day 42 38.10 41.18 39.96 38.45

All treatments 0.85 0.593

Only 3 treatments 1.09 0.557

Only Hemicell 1.39 0.280

Only Protexin 1.31 0.497

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[0087] Table 30 shows Haugh unit of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.

Table 30.

Treatments

. Hemicell Protexin P-

Storag

⁶ e time Control H + P SE ¹ . (H) (P) value ₂ 2

Day l 86.93 87.22 87.38 87.14

All treatments 0.42 0.990

Only 3 treatments 0.55 0.58

Only Hemicell 0.76 0.858

Only Protexin 0.66 0.770

Day 7 71.65 68.71 70.85 67.55

All treatments 0.88 0.134

Only 3 treatments 0.78 0.297

Only Hemicell 0.98 0.139 Only Protexin 0 96 0 577

¹ Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[0088] Table 31 shows albumen weight (g) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.

Table 31. Only Protexin 0.55 0.200

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[0089] Table 32 shows albumen weight (g) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.

Table 32.

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[0090] Table 33 shows albumen weight (g) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.

Table 33. Only Hemicell 0.62 0.153

Only Protexin 0.61 0.966

Day 21 38.56 38.43 36.77 37.07

All treatments 0.43 0.342

Only 3 treatments 0.52 0.294

Only Hemicell 0.66 0.926

Only Protexin 0.58 0.132

Day 28 36.07 37.92 37.18 35.89

All treatments 0.46 0.369

Only 3 treatments 0.56 0.411

Only Hemicell 065 0.162

Only Protexin 0.71 0.445

Day 35 34.49 35.36 34.86 34.55

All treatments 0.43 0.900

Only 3 treatments 0.49 0.92

Only Hemicell 0.68 0.536

Only Protexin 0.61 0.775

Day 42 33.99 33.36 33.12 33.80

All treatments 0.43 0.891

Only 3 treatments 0.49 0.761

Only Hemicell 0.57 0.589

Only Protexin 0.65 0.518

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[0091] Table 34 shows shell weight (g) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.

Table 34.

Treatments

... .. , Hemicell Protexin ,, „ P-

Storage time Control H + P SE ¹ _yalue2

Day l 5.78 6.07 6.05 6.03

All treatments 0.07 0.482

Only 3 treatments 0.09 0.371

Only Hemicell 0.12 0.262

Only Protexin 0.07 0.071

Day 7 6.17 6.10 6.15 6.19

All treatments 0.04 0.919

Only 3 treatments 0.05 0.873

Only Hemicell 0.07 0.635

Only Protexin 0.06 0.901

Day 14 6.01 6.02 6.18 6.07

All treatments 0.05 0.636

Only 3 treatments 0.06 0.489

Only Hemicell 0.06 0.931

Only Protexin 0.08 0.300

Day 21 5.81 6.08 5.93 6.11

y

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[0092] Table 35 shows shell weight (g) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.

Table 35.

Day 35 5.95 5.90 5.75 6.13

All treatments 0.05 0.112

Only 3 treatments 0.05 0.278

Only Hemicell 0.06 0.737

Only Protexin 0.06 0.130

Day 42 6.22 6.26 6.01 6.39

All treatments 0.06 0.245

Only 3 treatments 0.07 0.394

Only Hemicell 0.09 0.859

Only Protexin 0.10 0.307

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[0093] Table 36 shows shell weight (g) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.

Table 36.

Treatments

.. „ . , Hemicell Protexin .. _ P-

Storag ^s e time Control (H) (P) H + P SE ¹ val .ue ² ,

Day l 5.40 6.18 5.90 6.13

All treatments B A AB A 0.07 <0.001

Only 3 treatments B A A 0.09 0.002

Only Hemicell B A 0.12 0.001

Only Protexin B A 0.10 0.016

Day 7 5.32 6.26 5.84 4.97

All treatments C A B AB 0.06 <0.001

Only 3 treatments C A B 0.08 <0.001

Only Hemicell B A 0.11 <0.001

Only Protexin B A 0.09 0.003

Day 14 5.42 6.17 6.03 6.09

All treatments B A A A 0.07 <0.001

Only 3 treatments B A A 0.09 0.001

Only Hemicell B A 0.11 0.001

Only Protexin B A 0.10 0.002

Day 21 5.70 6.03 5.83 6.21

All treatments B AB B A 0.05 0.003

Only 3 treatments B A B 0.05 0.089

Only Hemicell b a 0.08 0.051

Only Protexin 0.06 0.320

Day 28 5.76 6.18 6.09 6.17

All treatments 0.06 0.101

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[0094] Table 37 shows shell breaking strength (N) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.

Table 37. Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[0095] Table 38 shows shell breaking strength (N) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial. Table 38. y

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[0096] Table 39 shows shell breaking strength (N) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.

Table 39. ' Slandard error. Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[0097] Table 40 shows albumen pH of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.

Table 40.

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%. [0098] Table 41 shows albumen pH of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.

Table 41.

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%. [0099] Table 42 shows albumen pH of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.

Table 42.

Treatments

, Hemicell Protexin , P-

Storag ⁶ e time Control (H) (P) H + P SE ¹ va .lue ₂ ²

Day l 8.13 8.05 8.09 8.10

All treatments 0.01 0.178

Only 3 treatments 0.01 0.163

Only Hemicell 0.02 0.101

Only Protexin 0.02 0.481

Day 7 8.30 8.35 8.35 8.37

All treatments 0.01 0.127

Only 3 treatments 0.01 0.239

Only Hemicell 0.01 0.171

Only Protexin 0.01 0.155

Day 14 8.46 8.48 8.49 8.50

All treatments 0.01 0.175

Only 3 treatments 0.01 0.552

Only Hemicell 0.01 0.545

Only Protexin 0.01 0.361

Day 21 8.57 8.59 8.60 8.58

All treatments 0.01 0.417

Only 3 treatments 0.01 0.274

Only Hemicell 0.01 0.437

Only Protexin 0.01 0,125

Day 28

All treatments 0.01

Only 3 treatments 0.01

Only Hemicell 0.01

Only Protexin 0.01

Day 35 8.75 8.77 8.78 8.76

All treatments 0.01 0.490

Only 3 treatments 0.01 0.336

Only Hemicell 0.01 0.403

Only Protexin 0.01 0.192

Day 42 9.08 9.09 9.09 9.09

All treatments 0.01 0.946

Only 3 treatments 0.01 0.879

Only Hemicell 0.01 0.669

Only Protexin 0.01 0.704

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[00100] Table 43 shows yolk pH of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial. Table 43. Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[00101] Table 44 shows yolk pH of eggs from laying hens fed beta-mannanase

(Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.

Table 44.

y

' Slandard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[00102] Table 45 shows yolk pH of eggs from laying hens fed beta-mannanase

(Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.

Table 45.

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[00103] Table 46 shows yolk color score (palette) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.

Table 46. All treatments 0.10 0.100

Only 3 treatments 0.13 0.254

Only Hemicell b a 0.13 0.075

Only Protexin 0.17 0.999

Day 7 4.87 4.87 4.80 5.21

All treatments 0.07 0.221

Only 3 treatments 0.08 0.935

Only Hemicell 0.10 0.999

Only Protexin 0.10 0.764

Day 14 5.42 5.13 6.00 5.27

All treatments B B A B 0.08 <0.001

Only 3 treatments B B A 0.10 0.001

Only Hemicell 0.08 0.102

Only Protexin B A 0.13 0.031

Day 21 6.36 6.07 6.20 6.07

All treatments 0.08 0.574

Only 3 treatments 0.08 0.430

Only Hemicell 0.10 0.166

Only Protexin 0.12 0.525

Day 28 5.53 6.64 6.60 6.62

All treatments B A A A 0.07 0.001

Only 3 treatments B A A 0.08 <0.001

Only Hemicell B A 0.11 <0.001

Only Protexin B A 0.10 0.001

Day 35 6.38 6.67 6.64 6.29

All treatments 0.08 0.289

Only 3 treatments 0.09 0.446

Only Hemicell 0.10 0.202

Only Protexin 0.13 0.348

Day 42 6.18 6.91 6.82 6.38

All treatments B A A AB 0.08 <0.001

Only 3 treatments B A A 0.09 0.001

Only Hemicell B A 0.12 0.002

Only Protexin B A 0.10 0.001

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[00104] Table 47 shows yolk color score (palette) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.

Table 47.

Treatments

... , Hemicell Protexin .. „ P-

S to rag

⁶ e time Control , ( _T _ H + P SE H) (P) ¹ , value ₇ 2

Day l 5.33 6.00 5.93 5.60

All treatments B A A AB 0.07 0.004

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[00105] Table 48 shows yolk color score (palette) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.

Table 48.

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[00106] Table 49 shows yolk lightness (L* color) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.

Table 49.

Only Protexin 0.45 0.849

Day 7 58.18 58.72 57.51 57.20

All treatments a a ab b 0.21 0.051

Only 3 treatments 0.24 0.125

Only Hemicell 0.27 0.336

Only Protexin 0.32 0.300

Day 14 57.60 58.70 58.27 61.36

All treatments 0.61 0.133

Only 3 treatments 0.61 0.772

Only Hemicell 0.89 0.548

Only Protexin 0.39 0.408

Day 21 57.13 58.45 57.48 58.16

All treatments B B AB AB 0.18 0.036

Only 3 treatments B A AB 0.21 0.032

Only Hemicell B A 0.23 0.003

Only Protexin 0.26 0.512

Day 28 55.27 58.72 57.86 58.54

All treatments B A A A 0.313 <0.001

Only 3 treatments B A A 0.377 <0.001

Only Hemicell B A 0.505 <0.001

Only Protexin B A 0.491 0.006

Day 35 55.38 58.60 57.53 56.25

All treatments C A AB BC 0.31 0.001

Only 3 treatments B A A 0.35 <0.001

Only Hemicell B A 0.49 <0.001

Only Protexin B A 0.43 0.010

Day 42 57.77 59.14 58.96 58.53

All treatments 0.22 0.121

Only 3 treatments b a ab 0.26 0.071

Only Hemicell B A 0.32 0.029

Only Protexin b a 0.35 0.092

¹ Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[00107] Table 50 shows yolk lightness (L* color) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.

Table 50.

Treatments

.. z , . Hemicell Protexin .. ,, P-

Storage time Control H + P SE ¹

Day l 50.99 51.65 52.06 53.73

All treatments B B AB A 0.27 0.001

Only 3 treatments 0.27 0.300

Only Hemicell 0.32 0.311

Only Protexin a 0.30 0.079

Day 7 55.77 56.35 56.45 56.07 All treatments 0.23 0.741

Only 3 treatments 0.26 0.544

Only Hemicell 0.26 0.276

Only Protexin 0.35 0.343

Day 14 57.87 57.87 58.54 59.04

All treatments 0.19 0.101

Only 3 treatments 0.22 0.377

Only Hemicell 0.29 0.995

Only Protexin 0.30 0.284

Day 21 56.62 58.01 58.93 58.32

All treatments B AB A AB 0.25 0.008

Only 3 treatments B AB A 0.32 0.008

Only Hemicell b a 0.41 0.099

Only Protexin B A 0.43 0.005

Day 28 58.25 58.11 58.31 59.82

All treatments B B B A 0.18 0.003

Only 3 treatments 0.16 0.885

Only Hemicell 0.21 0.757

Only Protexin 0.18 0.870

Day 35 58.73 58.82 58.89 58.97

All treatments 0.17 0.970

Only 3 treatments 0.21 0.954

Only Hemicell 0.28 0.878

Only Protexin 0.24 0.750

Day 42 59.07 58.84 59.10 59.88

All treatments 0.22 0.385

Only 3 treatments 0.24 0.899

Only Hemicell 0.37 0.762

Only Protexin 0.26 0.948

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[00108] Table 51 shows yolk lightness (L* color) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.

Table 51.

Treatments

, Hemiccll Protexin _¥¥ „ P-

Storage time Control H + P SE ¹

Day l 50.26 50.01 50.45 50.88

All treatments 0.23 0.777

Only 3 treatments 0.25 0.776

Only Hemicell 0.29 0.677

Only Protexin 0.32 0.768

Day 7 53.37 56.34 54.60 55.78

All treatments B A AB 0.32 0.003

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[00109] Table 52 shows yolk redness (a* color) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.

Table 52. Only Hemicell b a 0.19 0.081

Only Protexin 0.18 0.572

Day 14 7.21 7.62 7.28 6.69

All treatments 0.12 0.067

Only 3 treatments 0.14 0.494

Only Hemicell 0.17 0.265

Only Protexin 0.10 0.860

Day 21 7.02 7.41 6.71 6.36

All treatments 0.15 0.112

Only 3 treatments 0.18 0.298

Only Hemicell 0.17 0.266

Only Protexin 0.12 0.534

Day 28 6.57 7.46 7.15 6.20

All treatments AB A A B 0.1 0.003

Only 3 treatments B A AB 0.14 0.034

Only Hemicell B A 0.17 0.007

Only Protexin 0.16 0.201

Day 35 5.80 6.79 7.28 6.27

All treatments B AB A AB 0.15 0.005

Only 3 treatments B AB A 0.18 0.005

Only Hemicell B A 0.24 0.040

Only Protexin B A 0.17 0.003

Day 42 7.01 6.95 7.45 7.24

All treatments 0.17 0.757

Only 3 treatments 0.13 0.245

Only Hemicell 0.16 0.862

Only Protexin 0.14 0.203

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[00110] Table 53 shows yolk redness (a* color) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.

Table 53.

Treatments

.. , Hemicell Protexin ,, „ P-

Storage time Control H + P SE ¹

Day l 6.98 7.20 7.97 6.02

All treatments AB A A B 0.16 <0.001

Only 3 treatments B B A 0.13 0.003

Only Hemicell 0.14 0.460

Only Protexin B A 0.10 0.002

Day 7 6.17 6.98 7.16 6.87

All treatments B A A AB 0.11 0.017

Only 3 treatments B A A 0.14 0.008

Only Hemicell B A 0.18 0.025

Only Protexin B A 0.12 0.006

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[00111] Table 54 shows yolk redness (a* color) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.

Table 54. All treatments 0.14 0.370

Only 3 treatments 0.17 0.900

Only Hemicell 0.20 0.920

Only Protexin 0.17 0.751

Day 21 6.09 6.04 6.38 5.09

All treatments AB AB A B 0.18 0.058

Only 3 treatments 0.21 0.777

Only Hemicell 0.28 0.937

Only Protexin 0.10 0.512

Day 28 5.49 6.05 6.11 5.93

All treatments 0.14 0.420

Only 3 treatments 0.18 0.332

Only Hemicell 0.22 0.233

Only Protexin 0.12 0.235

Day 35 5.58 6.33 6.22 5.29

All treatments AB AB A B 0.14 0.024

Only 3 treatments b a a 0.14 0.088

Only Hemicell b a 0.20 0.069

Only Protexin 0.10 0.125

Day 42 5.39 6.38 5.89 5.48

All treatments B A AB B 0.12 0.011

Only 3 treatments B A AB 0.12 0.002

Only Hemicell B A 0.16 0.001

Only Protexin b a 0.11 0.097

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[00112] Table 55 shows yolk yellowness (b* color) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.

Table 55.

Treatments

.. , , . , Hemicell Protexin .. P-

Storag

⁶ e time Control , _T „ H + P SE (H) (P) ¹ , , value ²

Day l 6.05 6.16 5.96 6.12

All treatments 0.33 0.167

Only 3 treatments AB A B 0.34 0.060

Only Hemicell 0.43 0.224

Only Protexin 0.33 0.178

Day 7 5.98 6.12 6.00 5.98

All treatments 0.35 0.445

Only 3 treatments 0.40 0.302

Only Hemicell 0.45 0.118

Only Protexin 0.53 0.880

Day 14 6.02 6.40 5.98 5.98

All treatments B A B B 0.53 0.013 Only 3 treatments B A B 0.67 0.018

Only Hemicell B A 0.89 0.030

Only Protexin 0.64 0.799

Day 21 5.96 6.23 5.96 5.85

All treatments AB A AB B 0.43 0.011

Only 3 treatments B A B 0.48 0.025

Only Hemicell B A 0.50 0.005

Only Protexin 0.55 0.997

Day 28 5.63 6.16 5.98 5.87

All treatments C A AB BC 0.45 <0.001

Only 3 treatments B A A 0.55 <0.001

Only Hemicell B A 0.78 <0.001

Only Protexin B A 0.85 0.006

Day 35 5.53 5.83 5.61 5.36

All treatments ab a ab b 0.66 0.072

Only 3 treatments 0.73 0.204

Only Hemicell b a 0.90 0.089

Only Protexin 0.97 0.688

Day 42 5.55 5.67 5.77 5.69

All treatments 0.60 0.666

Only 3 treatments 0.72 0.487

Only Hemicell 0.88 0.507

Only Protexin 0.92 0.252

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[00113] Table 56 shows yolk yellowness (b* color) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.

Table 56.

Treatments o. , Hemicell Protexin ,, „ , _1T ,. P-

Storage time Control H + P SE ¹

Day l 5.70 5.68 6.07 5.66

All treatments b b a b 0.62 0.055

Only 3 treatments B B A 0.70 0.034

Only Hemicell 0.86 0.895

Only Protexin B A 0.90 0.036

Day 7 5.25 5.45 6.02 5.70

All treatments B B A AB 0.68 0.012

Only 3 treatments B B A 0.82 0.006

Only Hemicell 1.03 0.752

Only Protexin B A 1.07 0.015

Day 14 5.62 5.80 5.80 5.53

All treatments 0.55 0.210

Only 3 treatments 0.63 0.422

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[00114] Table 57 shows yolk yellowness (b* color) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.

Table 57.

Day 28 5.21 5.82 5.68 5.54

All treatments B A A AB 0.64 0.003

Only 3 treatments B A A 0.80 0.003

Only Hemicell B A 1.16 0.006

Only Protexin B A 1.08 0.026

Day 35 5.55 5.80 5.74 5.50

All treatments 0.51 0.102

Only 3 treatments 0.54 0.156

Only Hemicell b a 0.74 0.091

Only Protexin 0.74 0.219

Day 42 5.41 5.78 5.65 5.36

All treatments B A AB B 0.51 0.007

Only 3 treatments B A AB 0.53 0.011

Only Hemicell B A 0.73 0.009

Only Protexin B A 0.59 0.036

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

Example 3

Oxidant and Antioxidant Assessments

[00115] For the instant example, assessment of oxidant and antioxidant levels birds was evaluated. The status in intestines and oviducts from laying hens in one of the four treatment groups described in Example 2 was evaluated.

[00116] To determine the levels of reactive oxygen species (ROS), the methodology described by Halliwell and Gutteridge (2007) was followed. The levels of non-homogeneous muscle lipid peroxidation were determined as levels of antibodies reactive to thiobarbituric acid (TBARS), measured 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 of protein.

[00117] GST activity was measured using spectrophotometry at 340 nm, according to Habig et al. (1974). The mixture contained muscle homogenate supernatant as a test, 0.1 M potassium phosphate buffer (pH 7.4), 100 mM GSH and 100 mM CDNB, used as substrate. Enzyme activity was expressed as pmol/CDNB/mg protein. Protein thiols (PSH) were determined by the method described by Sedlak and Lindsay (1968), a technique that uses DTNB (5,5-dithiobis- 2- nitrobenzoic acid; Sigma). Protein thiols were measured by the sediment formed by the precipitated protein that material was resuspended, using a homogenization buffer to determine the PSH content. The readings absorbances (405 nm) were performed using a spectrofluorometer (Biotek, Synergy HT).

[00118] Table 58 shows oxidant and antioxidant status in intestines and oviducts from laying hens fed beta-mannanase (Hemicell), and/or probiotics (Protexin). The variables evaluated included TBARS (thiobarbituric acid reactive substances; mmol MDA/mg proteins); GST (glutathione S-transferase; pmolCDNB/min/mg protein); and TSH (protein thios; pmol TSH/mg protein).

Table 58.

Treatments

Variables Control P H H + P SE ¹ P- value ²

Intestine

TBARS 1.84A 1.79A 1.39AB 1.06B 0.12 0.018

GST 975. IB 912.6B 914.9B 1251A 10.1 0.006

TSH 1.49G 2.81A 1.78B 1.70B 0.04 <0.001

Oviduct

TBARS 0.70 0.907 0.77 0.81 0.08 0.440

GST 861.2a 794.9a 741.7b 832.9a 15.3 0.061

TSH 1.25 1.30 1.05 1.22 0.14 0.420

Standard error. ² Probability of treatment effect (model did not included the effect of storage time). Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

Example 4

Performance Assessments

[00119] For the instant example, various performance were undertaken. The experimental units were randomly selected among the hens housed in the commercial farm (about 28,000 light-weight laying hens, 36 weeks old, Hyline W 36 lineage) and assigned to the treatments, that were i) control (basal diet, without supplementation); ii) diet supplemented with 300 g/ton of the P-mannanase Hemicell®; iii) diet supplemented with 50 g/ton of the probiotic additive Protexin®; iv) diet supplemented with 50 g/ton of probiotic additive and 300 g/ton of -mannanase (Hemicell+Protexin).

[00120] The basal feed was formulated according to the nutritional requirements for the genetic (Hy-Line, 2020). Corn and soybean meal were the main ingredients in the feed formulas. Feed and water were both provided ad libitum throughout the experimental period using nipple drinkers and gutter feeders.

[00121] The birds were housed in conventional sheds, arranged in an east-west direction, with concrete floors and masonry walls complemented with wire mesh to the ceiling. The shed was equipped with side curtains, which were managed according to weather conditions to provide thermal comfort. Average maximum and minimum temperature and air relative humidity values recorded were 36 and 18.1 °C, and 94.7 and 35.8%, respectively. Only natural light illumination was used, allowing a lighting program of 16 hours and 30 minutes of light per day.

[00122] The instant example started when birds were 36 weeks old and lasted 144 days. Egg production was individually recorded at weeks 4, 8, and 12 in a subgroup of 120 cages with 4 birds each, corresponding to 30 replicates per treatment. All eggs produced in these subgroups were individually weighed and analyzed for the presence of feces in the shells, which was classified by the same observer through visual analysis as: absent (clean eggs), minor presence (e.g., presence of small spots; see Figure 1), or major presence (e.g., presence of large spots; see Figure 2).

[00123] Laying rate and egg mass were calculated considering all eggs (including non- marketable eggs) for each replicate (cage). The coefficient of variability was calculated for each cage considering the individual weight of all the eggs produced in each week. A similar procedure was adopted for evaluation of egg masses.

[00124] Blood samples were collected at the end of the project from 8 birds per treatment (random selection). Samples were processed and analyzed (Bio-Plus®, Biochemical Analyzer, Bioplus, Sao Paulo, Brazil) for total protein, albumin, uric acid, total cholesterol, triglycerides, glucose, alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase using commercial kits (Wiener Lab Group, Sao Paulo, Brazil) and semiautomatic analyzer.

[00125] Table 59 shows performance of laying hens fed diets supplemented with beta- mannanase (Hemicell) and/or probiotics (Protexin).

Table 59.

Treatments

.. „ . , Hemicell Protexin .. „ P-

Responses Control H + P SE ¹ ₂

Laying rate (%)

36 - 40 wk 85.31 C 93.71 B 83.25 C 97.66 A 0.11 <0.001

41 - 44 wk 78.34 B 96.69 A 96.36 A 90.55 A 0.11 <0.001

45 - 48 wk 91.05 B 92.59 AB 92.60 AB 95.90 A 0.09 <0.001

36 - 48 wk 84.90 C 94.33 A 90.74 B 94.70 A 0.59 <0.001

Weight of fresh eggs (g)

36 - 40 wk 61.95 B 62.36 B 61.38 B 63.18 A 0.14 <0.001

41 - 44 wk 61.28 C 63.09 A 61.98 B 62.79 A 0.15 <0.001

45 - 48 wk 64.13 B 65.21 A 63.76 B 65.52 A 0.16 <0.001

36 - 48 wk 62.47 B 63.55 A 62.37 B 63.83 A 0.09 <0.001

62 wk (no treat.) ³ 62.53 B 64.40 A 64.10 A 64.28 A 0.13 0.013 Coefficient of variability in egg weight (%)

36 - 40 wk 5.944b 5.897 b 5.193 a 5.292 ab 0.014 0.072

41 - 44 wk 7.152 B 5.625 A 5.750 A 5.734 A 0.018 0.007

45 - 48 wk 7.088 B 5.397 A 5.608 A 5.405 A 0.019 0.004

36 - 48 wk 6.728 B 5.640 Q 5.517 A 5.477 A 0.010 <0.001

62 wk (no treat.) ³ 7.940 8.285 8.272 7.891 0.124 0.564

'Slandard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%. treatments were not provided from wk 48 to 62. Thus, the last evaluation was performed after 14 wk without supplementation.

[00126] Table 60 shows egg component masses produced by hens fed diets supplemented with beta-mannanase (Hemicell) and/or probiotics (Protexin).

Table 60.

Treatments

Hemicell Protexin _¥¥ P-

Responses Control H + P SE ^{1 2}

Egg mass (g/hen/day)

36 - 40 wk 5B2.85 B 58.44 A 51.10 B 61.70 A 0.67 <0.001

41 - 44 wk 48.01 C 61.00 A 59.72 AB 56.86 B 0.39 <0.001

45 - 48 wk 58.30 B 60.38 A 59.04 AB 62.83 A 0.63 <0.001

36 - 48 wk 53.08 C 59.94 A 56.62 B 60.46 A 0.39 <0.001

Yolk mass (g/hen/day)

36 - 40 wk 15.89 B 17.36 A 14.57 B 18.46 A 0.19 <0.001

41 - 44 wk 13.45 B 16.69 A 16.71 A 16.24 A 0.11 <0.001

45 - 48 wk 14.81 C 15.58 B 15.67 B 16.70 A 0.18 <0.001

36 - 48 wk 14.77 V 16.56 A 15.66 B 17.14 A 0.11 <0.001

Albumen mass (g/hen/day

36 - 40 wk 31.81 B 35.22 A 31.35 B 36.98 A 0.42 <0.001

41 - 44 wk 29.54 B 38.05 A 36.95 A 34.72 A 0.23 <0.001

45 - 48 wk 38.06 B 38.54 A 37.30 B 39.69 A 0.49 <0.001

36 - 48 wk 33.09 C 37.26 A 35.18 B 37.12 A 0.24 <0.001

Egg shell mass (g/hen/day)

36 - 40 wk 5.14 B 5.78 A 5.21 B 6.26 A 0.07 <0.001

41 - 44 _W k 5.02 B 6.26 A 6.06 A 5.90 A 0.04 <0.001

45 - 48 wk 5.42 C 6.26 A 6.06 B 6.44 A 0.06 <0.001

36 - 48 wk 5.22 C 6.13 A 5.78 B 6.20 A 0.04 <0.001

¹ Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[00127] Table 61 shows occurrence of clean eggs or minor/major presence of feces (%) in eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin). Table 61. ' Si andard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%. treatments were not provided from wk 48 to 62. Thus, the last evaluation was performed after 14 wk without supplementation.

[00128] Table 62 shows serum biochemistry and intestinal morphometry of laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin).

Table 62. Villi area, pm ² 367039 288838 343272 450579 1798 0.670 Crypt depth, pm 224.9 186.4 227.6 202.1 4.96 0.397 Villi height : Crypt depth 6.839 B 7.245 AB 6.304 B 8.295 A 0.171 0.007

Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

Example 5

Egg Microstructure Assessments

[00129] For the instant example, microstructure of the eggs was evaluated. The eggshells obtained from eggs of laying hens in one of the four treatment groups described in Example 2 were evaluated.

[00130] Scanning electron microscopy analyzes were performed to assess the microstructure of the eggshells. For the evaluations, 20 shells from de last trial were used, in which fragments of approximately 5 cm ² were removed and stored in falcon tubes. After this period, the samples were mounted on a stubs, coated with gold-palladium of 35 nm for 3 minutes (Sputter Coater - SCD 050 Balzers, Germany) and analyzed through a scanning electron microscope (JEOL 6060, Japan) at magnifications from 100 to 2000x. In the images, the thickness of mammillary layer, palisade layer, membrane layer, and vertical crystal layer were measured in three locations aleatorily chosen in each eggshell image. The size of column or mammillary bodies and the distance between mammillary knobs were also accessed, but five measurements were made in each image due to the larger variability among measurements.

[00131] Table 63 summarizes eggshell traits accessed through ultramicroscopy in eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin) depending on storage times. Evaluations were obtained at the first phase (40 weeks), second phase (44 weeks), and third phase (48 weeks).

Table 63.

Treatments

First Phase - 40 wk

Mammillary layer, pm 73.29 71.81 59.94 69.26 24.17 0.155

Palisade layer, pm 238.6B 260.2AB 262.7A 254.0AB 22.50 0.035

Mammill. -rPalisade, pm 317.8 332.0 315.4 315.7 28.10 0.319

Membrane layer, pm 57.39 29.84 68.88 68.20 20.01 0.294

Crystal layer, pm 28.39B 26.89B 26.80B 30.92A 19.32 0.429

Mammillary bodies, pm 68.18B 65.82B 62.42B 71.34A 6.928 0.009

Mam. Knobs distance, pm 83.89 82.34 68.13 78.30 21.41 0.148

Second Phase - 44 wk

Mammillary layer, pm 50.86B 66.81A 65.56A 59.88A 12.03 0.003

Palisade layer, pm 259.7B 284.3A 266.7 A 270.7 A 9.953 0.013

¹ Standard error. ² Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.

[00132] Figure 3 shows ultramicroscopy images collected in a single eggshell at the first phase. Figure 4 shows ultramicroscopy images collected in a single eggshell at the second phase. Figure 5 shows ultramicroscopy images collected in a single eggshell at the third phase.