CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S.

Provisional Application Serial No. 63/208,145, filed on June 8, 2021, the entire disclosure of which is incorporated herein by reference.

TECHNICAL FIELD

[0002] The present disclosure relates to compositions comprising i) an animal feed composition and ii) iodoform. The disclosure includes compositions, methods, and formulations for decreasing methane production in the rumen of a ruminant animal.

BACKGROUND AND SUMMARY OF THE INVENTION

[0003] Iodoform is an organoiodine compound with the formula CHE:

[0004] Iodoform has previously been used as a disinfectant composition, for example in a hospital setting. Iodoform has now been identified as a compound that reduces enteric methane production from ruminant animals. In particular, it is believed iodoform provides inhibition of the final enzymatic step in methanogenesis in the rumen of a ruminant animal, known as methyl -coenzyme M reductase that is present in the methanogenic archae microorganisms in the rumen.

[0005] Methane as it relates to digestion of feed the rumen of ruminant animals is a greenhouse gas of interest. Reduction of methane (i.e., C¾) in this capacity is of high interest due to the increasing effects of climate change in the world.

[0006] Therefore, there exists a need for new compositions and formulations that are capable of reducing methane production by ruminant animals. Moreover, new and effective methods of reducing methane will desirably not substantially affect rumen fermentation in a ruminant animal and/or will desirably increase propionate production in the rumen of a ruminant animal. Accordingly, the present disclosure provides compositions comprising i) an animal feed composition and ii) iodoform and methods of using the compositions which exhibit desirable properties and provide related advantages for improvement in reducing methane production by ruminant animals.

[0007] The compositions and methods of the present disclosure provide several advantages over known compositions and methods in the art. In particular, inhibition of the final enzymatic step in methanogenesis in the rumen of a ruminant animal prevents methane formation and diverts energy into alternative hydrogen sinks such as propionate, which are useful to the animal. As a result, a reduction in methane by the animal is observed, as well as improved capture of energy (feed efficiency). Further, the compositions and methods of the present disclosure will not provide substantially negative effects on rumen fermentation in a ruminant animal. This is highly desirable because complete inhibition of methane formation in the rumen is associated with negative impacts on feed intake, rumen fermentation, and animal production. For instance, various compositions and methods of the present disclosure will not substantially affect digestibility of diet in a ruminant animal. In addition, various compositions and methods of the present disclosure will not substantially affect volatile fatty acid production in a ruminant animal in a negative manner (e.g., the compositions and methods will not negatively affect volatile fatty acid production).

[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 DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 shows doses of iodoform resulted in significant reduction in methane estimated stoichiometrically by VFA profiles or by direct measurement.

[0010] Figure 2 shows addition of iodoform reduced VFA by 14.5% and 11%, respectively, for naive and YS adapted inoculum.

[0011] Figure 3 shows iodoform reduced CH4/g DM truly digested by 30% and 27%, respectively, for naive and YS adapted inoculum. [0012] Figure 4 shows changes in acetate (mM) over time in various formulations.

[0013] Figure 5 shows changes in propionate (mM) over time in various formulations.

[0014] Figure 6 shows changes in total VFA (mM) over time in various formulations.

[0015] Figure 7 shows changes in acetate percentage of total VFA over time in various formulations.

[0016] Figure 8 shows changes in propionate percentage of total VFA over time in various formulations.

[0017] Figure 9 shows minor or no differences in VFA concentration or proportions in a 6 hour post-feeding analysis.

[0018] Figure 10 shows minor or no differences in VFA concentration or proportions in a 6 hour post-feeding analysis.

[0019] Figure 11 shows a reduction in methane in treated animals.

DETAILED DESCRIPTION

[0020] The following numbered embodiments are contemplated and are non-limiting:

1. A composition comprising i) an animal feed composition and ii) iodoform.

2. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the animal feed composition is a ruminant feed composition.

3. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the animal feed composition does not comprise seaweed.

4. The composition of clause 3, any other suitable clause, or any combination of suitable clauses, wherein the seaweed is red seaweed.

5. The composition of clause 3, any other suitable clause, or any combination of suitable clauses, wherein the seaweed is a Porphyra sp.

6. The composition of clause 3, any other suitable clause, or any combination of suitable clauses, wherein the seaweed is an Asparagopsis sp.

7. The composition of clause 3, any other suitable clause, or any combination of suitable clauses, wherein the seaweed is Asparagopsis armata.

8. The composition of clause 3, any other suitable clause, or any combination of suitable clauses, wherein the seaweed is present at 10% of the animal feed composition. 9. The composition of clause 3, any other suitable clause, or any combination of suitable clauses, wherein the animal feed composition does not comprise seaweed at less than 10% of the animal feed composition.

10. The composition of clause 3, any other suitable clause, or any combination of suitable clauses, wherein the animal feed composition does not comprise seaweed at more than 10% of the animal feed composition.

11. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at an amount of about 0.35 g/hd/d.

12. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 1 mg/hd/d and 1000 mg/hd/d.

13. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 1 mg/hd/d and 500 mg/hd/d.

14. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 100 mg/hd/d and 500 mg/hd/d.

15. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 500 mg/hd/d and 1000 mg/hd/d.

16. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 1 mg/hd/d and 300 mg/hd/d.

17. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 300 mg/hd/d and 700 mg/hd/d.

18. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 700 mg/hd/d and 1000 mg/hd/d. 19. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 1 mg/hd/d and 100 mg/hd/d.

20. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 100 mg/hd/d and 200 mg/hd/d.

21. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 200 mg/hd/d and 300 mg/hd/d.

22. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 300 mg/hd/d and 400 mg/hd/d.

23. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 400 mg/hd/d and 500 mg/hd/d.

24. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 500 mg/hd/d and 600 mg/hd/d.

25. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 600 mg/hd/d and 700 mg/hd/d.

26. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 700 mg/hd/d and 800 mg/hd/d.

27. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 800 mg/hd/d and 900 mg/hd/d.

28. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 900 mg/hd/d and 1000 mg/hd/d. 29. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the composition is configured to decrease methane production in a rumen of a ruminant animal.

30. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the composition is configured to not provide substantially negative effects on rumen fermentation in a ruminant animal.

31. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the composition is configured to not substantially affect digestibility of diet in a ruminant animal.

32. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the composition is configured to not substantially affect volatile fatty acid production in a ruminant animal.

33. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the composition is configured to increase propionate production in a rumen of a ruminant animal.

34. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the composition is configured to be fed to a ruminant animal.

35. The composition of clause 34, any other suitable clause, or any combination of suitable clauses, wherein the ruminant animal is selected from the group consisting of cattle, goats, sheep, giraffes, American Bison, European bison, yaks, water buffalo, deer, camels, alpacas, llamas, wildebeest, antelope, pronghorn, and nilgai.

36. The composition of clause 34, any other suitable clause, or any combination of suitable clauses, wherein the ruminant animal is raised for meat production.

37. The composition of clause 34, any other suitable clause, or any combination of suitable clauses, wherein the ruminant animal is raised for dairy production.

38. The composition of clause 34, any other suitable clause, or any combination of suitable clauses, wherein the ruminant animal is selected from the group consisting of cattle, buffalo, sheep, and goats.

39. The composition of clause 34, any other suitable clause, or any combination of suitable clauses, wherein the ruminant animal is a bovine. 40. The composition of clause 34, any other suitable clause, or any combination of suitable clauses, wherein the ruminant animal is a buffalo.

41. The composition of clause 34, any other suitable clause, or any combination of suitable clauses, wherein the ruminant animal is a sheep.

42. The composition of clause 34, any other suitable clause, or any combination of suitable clauses, wherein the ruminant animal is a goat.

43. The composition of clause 34, any other suitable clause, or any combination of suitable clauses, wherein the ruminant animal is a beef cattle.

44. The composition of clause 34, any other suitable clause, or any combination of suitable clauses, wherein the ruminant animal is a dairy cattle.

45. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the composition comprises one or more mineral supplements.

46. The composition of clause 45, any other suitable clause, or any combination of suitable clauses, wherein the mineral supplements are free-choice mineral supplements.

47. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the composition is configured as a slow release formulation.

48. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the composition is configured as a block formulation.

49. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the composition is configured as a bolus formulation.

50. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the composition comprises a hydrogen sink compound.

51. The composition of clause 50, any other suitable clause, or any combination of suitable clauses, wherein the hydrogen sink compound is selected from the group consisting of a nitrate salt, fumaric acid, fumerate, an unsaturated fatty acid, and any combination thereof.

52. The composition of clause 50, any other suitable clause, or any combination of suitable clauses, wherein the hydrogen sink compound is a nitrate salt.

53. The composition of clause 52, any other suitable clause, or any combination of suitable clauses, wherein the nitrate salt is sodium nitrate.

54. The composition of clause 52, any other suitable clause, or any combination of suitable clauses, wherein the nitrate salt is potassium nitrate. 55. The composition of clause 50, any other suitable clause, or any combination of suitable clauses, wherein the hydrogen sink compound is fumaric acid.

56. The composition of clause 50, any other suitable clause, or any combination of suitable clauses, wherein the hydrogen sink compound is fumerate.

57. The composition of clause 50, any other suitable clause, or any combination of suitable clauses, wherein the hydrogen sink compound is an unsaturated fatty acid.

58. The composition of clause 50, any other suitable clause, or any combination of suitable clauses, wherein the hydrogen sink compound and the iodoform provide a synergistic effect in a rumen of a ruminant animal.

59. The composition of clause 50, any other suitable clause, or any combination of suitable clauses, wherein the hydrogen sink compound and the iodoform provide a synergistic methane inhibition in a ruminant animal.

60. A method of reducing methane production in a rumen of a ruminant animal, said method comprising the step of administering a composition comprising i) an animal feed composition and ii) iodoform to the ruminant animal, wherein the composition reduces methane production in the rumen of the ruminant animal.

61. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the method does not provide substantially negative effects on rumen fermentation in the ruminant animal.

62. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the method does not substantially affect digestibility of diet in the ruminant animal.

63. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the method does not substantially affect volatile fatty acid production in the ruminant animal.

64. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the method increases propionate production in the rumen of the ruminant animal.

65. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the reduction of methane production is a reduction between 10%-95% compared to a ruminant animal not administered the composition. 66. The method of clause 65, any other suitable clause, or any combination of suitable clauses, wherein the reduction of methane production is a reduction of about 10% compared to a ruminant animal not administered the composition.

67. The method of clause 65, any other suitable clause, or any combination of suitable clauses, wherein the reduction of methane production is a reduction of about 20% compared to a ruminant animal not administered the composition.

68. The method of clause 65, any other suitable clause, or any combination of suitable clauses, wherein the reduction of methane production is a reduction of about 30% compared to a ruminant animal not administered the composition.

69. The method of clause 65, any other suitable clause, or any combination of suitable clauses, wherein the reduction of methane production is a reduction of about 40% compared to a ruminant animal not administered the composition.

70. The method of clause 65, any other suitable clause, or any combination of suitable clauses, wherein the reduction of methane production is a reduction of about 50% compared to a ruminant animal not administered the composition.

71. The method of clause 65, any other suitable clause, or any combination of suitable clauses, wherein the reduction of methane production is a reduction of about 60% compared to a ruminant animal not administered the composition.

72. The method of clause 65, any other suitable clause, or any combination of suitable clauses, wherein the reduction of methane production is a reduction of about 70% compared to a ruminant animal not administered the composition.

73. The method of clause 65, any other suitable clause, or any combination of suitable clauses, wherein the reduction of methane production is a reduction of about 80% compared to a ruminant animal not administered the composition.

74. The method of clause 65, any other suitable clause, or any combination of suitable clauses, wherein the reduction of methane production is a reduction of about 90% compared to a ruminant animal not administered the composition.

75. The method of clause 65, any other suitable clause, or any combination of suitable clauses, wherein the reduction of methane production is a reduction of about 95% compared to a ruminant animal not administered the composition. 76. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the animal feed composition is a ruminant feed composition.

77. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the animal feed composition does not comprise seaweed.

78. The method of clause 77, any other suitable clause, or any combination of suitable clauses, wherein the seaweed is red seaweed.

79. The method of clause 77, any other suitable clause, or any combination of suitable clauses, wherein the seaweed is a Porphyra sp.

80. The method of clause 77, any other suitable clause, or any combination of suitable clauses, wherein the seaweed is an Asparagopsis sp.

81. The method of clause 77, any other suitable clause, or any combination of suitable clauses, wherein the seaweed is Asparagopsis armata.

82. The method of clause 77, any other suitable clause, or any combination of suitable clauses, wherein the seaweed is present at 10% of the animal feed composition.

83. The method of clause 77, any other suitable clause, or any combination of suitable clauses, wherein the animal feed composition does not comprise seaweed at less than 10% of the animal feed composition.

84. The method of clause 77, any other suitable clause, or any combination of suitable clauses, wherein the animal feed composition does not comprise seaweed at more than 10% of the animal feed composition.

85. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at an amount of about 0.35 g/hd/d.

86. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 1 mg/hd/d and 1000 mg/hd/d.

87. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 1 mg/hd/d and 500 mg/hd/d.

88. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 100 mg/hd/d and 500 mg/hd/d. 89. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 500 mg/hd/d and 1000 mg/hd/d.

90. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 1 mg/hd/d and 300 mg/hd/d.

91. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 300 mg/hd/d and 700 mg/hd/d.

92. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 700 mg/hd/d and 1000 mg/hd/d.

93. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 1 mg/hd/d and 100 mg/hd/d.

94. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 100 mg/hd/d and 200 mg/hd/d.

95. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 200 mg/hd/d and 300 mg/hd/d.

96. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 300 mg/hd/d and 400 mg/hd/d.

97. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 400 mg/hd/d and 500 mg/hd/d.

98. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 500 mg/hd/d and 600 mg/hd/d. 99. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 600 mg/hd/d and 700 mg/hd/d.

100. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 700 mg/hd/d and 800 mg/hd/d.

101. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 800 mg/hd/d and 900 mg/hd/d.

102. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is present in the composition at a dose rate between 900 mg/hd/d and 1000 mg/hd/d.

103. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is administered to the ruminant animal at a concentration between 0.01 mg/kg and 2 mg/kg of body weight of the ruminant animal.

104. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is administered to the ruminant animal at a concentration between 0.01 mg/kg and 1 mg/kg of body weight of the ruminant animal.

105. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is administered to the ruminant animal at a concentration between 0.1 mg/kg and 1 mg/kg of body weight of the ruminant animal.

106. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is administered to the ruminant animal at a concentration between 1 mg/kg and 2 mg/kg of body weight of the ruminant animal.

107. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is administered to the ruminant animal at a concentration between 0.01 mg/kg and 0.3 mg/kg of body weight of the ruminant animal.

108. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is administered to the ruminant animal at a concentration between 0.3 mg/kg and 0.6 mg/kg of body weight of the ruminant animal. 109. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is administered to the ruminant animal at a concentration between 0.6 mg/kg and 0.9 mg/kg of body weight of the ruminant animal.

110. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is administered to the ruminant animal at a concentration between 0.9 mg/kg and 1.2 mg/kg of body weight of the ruminant animal.

111. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is administered to the ruminant animal at a concentration between 1.2 mg/kg and 1.5 mg/kg of body weight of the ruminant animal.

112. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is administered to the ruminant animal at a concentration between 1.5 mg/kg and 1.8 mg/kg of body weight of the ruminant animal.

113. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is administered to the ruminant animal at a concentration between 1.8 mg/kg and 2 mg/kg of body weight of the ruminant animal.

114. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is administered to the ruminant animal at a concentration between 0.01 mg/kg and 0.1 mg/kg of body weight of the ruminant animal.

115. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is administered to the ruminant animal at a concentration between 0.1 mg/kg and 0.2 mg/kg of body weight of the ruminant animal.

116. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is administered to the ruminant animal at a concentration between 0.2 mg/kg and 0.3 mg/kg of body weight of the ruminant animal.

117. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is administered to the ruminant animal at a concentration between 0.3 mg/kg and 0.4 mg/kg of body weight of the ruminant animal.

118. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is administered to the ruminant animal at a concentration between 0.4 mg/kg and 0.5 mg/kg of body weight of the ruminant animal. 119. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is administered to the ruminant animal at a concentration between 0.5 mg/kg and 0.6 mg/kg of body weight of the ruminant animal.

120. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is administered to the ruminant animal at a concentration between 0.6 mg/kg and 0.7 mg/kg of body weight of the ruminant animal.

121. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is administered to the ruminant animal at a concentration between 0.7 mg/kg and 0.8 mg/kg of body weight of the ruminant animal.

122. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is administered to the ruminant animal at a concentration between 0.8 mg/kg and 0.9 mg/kg of body weight of the ruminant animal.

123. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is administered to the ruminant animal at a concentration between 0.9 mg/kg and 1.0 mg/kg of body weight of the ruminant animal.

124. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is administered to the ruminant animal at a concentration between 1.0 mg/kg and 1.1 mg/kg of body weight of the ruminant animal.

125. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is administered to the ruminant animal at a concentration between 1.0 mg/kg and 1.1 mg/kg of body weight of the ruminant animal.

126. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is administered to the ruminant animal at a concentration between

1.1 mg/kg and 1.2 mg/kg of body weight of the ruminant animal.

127. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is administered to the ruminant animal at a concentration between

1.2 mg/kg and 1.3 mg/kg of body weight of the ruminant animal.

128. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is administered to the ruminant animal at a concentration between

1.3 mg/kg and 1.4 mg/kg of body weight of the ruminant animal. 129. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is administered to the ruminant animal at a concentration between

1.4 mg/kg and 1.5 mg/kg of body weight of the ruminant animal.

130. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is administered to the ruminant animal at a concentration between

1.5 mg/kg and 1.6 mg/kg of body weight of the ruminant animal.

131. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is administered to the ruminant animal at a concentration between

1.6 mg/kg and 1.7 mg/kg of body weight of the ruminant animal.

132. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is administered to the ruminant animal at a concentration between

1.7 mg/kg and 1.8 mg/kg of body weight of the ruminant animal.

133. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is administered to the ruminant animal at a concentration between

1.8 mg/kg and 1.9 mg/kg of body weight of the ruminant animal.

134. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the iodoform is administered to the ruminant animal at a concentration between

1.9 mg/kg and 2.0 mg/kg of body weight of the ruminant animal.

135. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the ruminant animal is selected from the group consisting of cattle, goats, sheep, giraffes, American Bison, European bison, yaks, water buffalo, deer, camels, alpacas, llamas, wildebeest, antelope, pronghorn, and nilgai.

136. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the ruminant animal is raised for meat production.

137. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the ruminant animal is raised for dairy production.

138. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the ruminant animal is selected from the group consisting of cattle, buffalo, sheep, and goats.

139. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the ruminant animal is a bovine. 140. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the ruminant animal is a buffalo.

141. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the ruminant animal is a sheep.

142. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the ruminant animal is a goat.

143. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the ruminant animal is a beef cattle.

144. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the ruminant animal is a dairy cattle.

145. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the composition comprises one or more mineral supplements.

146. The method of clause 145, any other suitable clause, or any combination of suitable clauses, wherein the mineral supplements are free-choice mineral supplements.

147. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the composition is configured as a slow release formulation.

148. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the composition is configured as a block formulation.

149. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the composition is configured as a bolus formulation.

150. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the composition comprises a hydrogen sink compound.

151. The method of clause 150, any other suitable clause, or any combination of suitable clauses, wherein the hydrogen sink compound is selected from the group consisting of a nitrate salt, fumaric acid, fumerate, an unsaturated fatty acid, and any combination thereof.

152. The method of clause 150, any other suitable clause, or any combination of suitable clauses, wherein the hydrogen sink compound is a nitrate salt.

153. The method of clause 152, any other suitable clause, or any combination of suitable clauses, wherein the nitrate salt is sodium nitrate.

154. The method of clause 152, any other suitable clause, or any combination of suitable clauses, wherein the nitrate salt is potassium nitrate. 155. The method of clause 150, any other suitable clause, or any combination of suitable clauses, wherein the hydrogen sink compound is fumaric acid.

156. The method of clause 150, any other suitable clause, or any combination of suitable clauses, wherein the hydrogen sink compound is fumerate.

157. The method of clause 150, any other suitable clause, or any combination of suitable clauses, wherein the hydrogen sink compound is an unsaturated fatty acid.

158. The method of clause 150, any other suitable clause, or any combination of suitable clauses, wherein the hydrogen sink compound and the iodoform provide a synergistic effect in the rumen of the ruminant animal.

159. The method of clause 150, any other suitable clause, or any combination of suitable clauses, wherein the hydrogen sink compound and the iodoform provide a synergistic methane inhibition in the ruminant animal.

160. The method of clause 60, any other suitable clause, or any combination of suitable clauses, wherein the method comprises administration of a hydrogen sink compound to the ruminant animal.

161. The method of clause 160, any other suitable clause, or any combination of suitable clauses, wherein the hydrogen sink compound is selected from the group consisting of a nitrate salt, fumaric acid, fumerate, an unsaturated fatty acid, and any combination thereof.

162. The method of clause 160, any other suitable clause, or any combination of suitable clauses, wherein the hydrogen sink compound is a nitrate salt.

163. The method of clause 162, any other suitable clause, or any combination of suitable clauses, wherein the nitrate salt is sodium nitrate.

164. The method of clause 162, any other suitable clause, or any combination of suitable clauses, wherein the nitrate salt is potassium nitrate.

165. The method of clause 160, any other suitable clause, or any combination of suitable clauses, wherein the hydrogen sink compound is fumaric acid.

166. The method of clause 160, any other suitable clause, or any combination of suitable clauses, wherein the hydrogen sink compound is fumerate.

167. The method of clause 160, any other suitable clause, or any combination of suitable clauses, wherein the hydrogen sink compound is an unsaturated fatty acid. 168. The method of clause 160, any other suitable clause, or any combination of suitable clauses, wherein the hydrogen sink compound and the iodoform provide a synergistic effect in the rumen of the ruminant animal.

169. The method of clause 160, any other suitable clause, or any combination of suitable clauses, wherein the hydrogen sink compound and the iodoform provide a synergistic methane inhibition in the ruminant animal.

[0021] In an illustrative aspect, a composition comprising i) an animal feed composition and ii) iodoform is provided.

[0022] In an embodiment, the animal feed composition is a ruminant feed composition.

In an embodiment, the animal feed composition does not comprise seaweed. In an embodiment, the seaweed is red seaweed. In an embodiment, the seaweed is a Porphyra sp.

In an embodiment, the seaweed is an Asparagopsis sp. In an embodiment, the seaweed is Asparagopsis armata. In an embodiment, the seaweed is present at 10% of the animal feed composition. In an embodiment, the animal feed composition does not comprise seaweed at less than 10% of the animal feed composition. In an embodiment, the animal feed composition does not comprise seaweed at more than 10% of the animal feed composition. [0023] In an embodiment, the iodoform is present in the composition at an amount of about 0.35 g/hd/d. In an embodiment, the iodoform is present in the composition at a dose rate between 1 mg/hd/d and 1000 mg/hd/d. In an embodiment, the iodoform is present in the composition at a dose rate between 1 mg/hd/d and 500 mg/hd/d. In an embodiment, the iodoform is present in the composition at a dose rate between 100 mg/hd/d and 500 mg/hd/d.

In an embodiment, the iodoform is present in the composition at a dose rate between 500 mg/hd/d and 1000 mg/hd/d. In an embodiment, the iodoform is present in the composition at a dose rate between 1 mg/hd/d and 300 mg/hd/d. In an embodiment, the iodoform is present in the composition at a dose rate between 300 mg/hd/d and 700 mg/hd/d. In an embodiment, the iodoform is present in the composition at a dose rate between 700 mg/hd/d and 1000 mg/hd/d. In an embodiment, the iodoform is present in the composition at a dose rate between 1 mg/hd/d and 100 mg/hd/d. In an embodiment, the iodoform is present in the composition at a dose rate between 100 mg/hd/d and 200 mg/hd/d. In an embodiment, the iodoform is present in the composition at a dose rate between 200 mg/hd/d and 300 mg/hd/d. In an embodiment, the iodoform is present in the composition at a dose rate between 300 mg/hd/d and 400 mg/hd/d. In an embodiment, the iodoform is present in the composition at a dose rate between 400 mg/hd/d and 500 mg/hd/d. In an embodiment, the iodoform is present in the composition at a dose rate between 500 mg/hd/d and 600 mg/hd/d. In an embodiment, the iodoform is present in the composition at a dose rate between 600 mg/hd/d and 700 mg/hd/d. In an embodiment, the iodoform is present in the composition at a dose rate between 700 mg/hd/d and 800 mg/hd/d. In an embodiment, the iodoform is present in the composition at a dose rate between 800 mg/hd/d and 900 mg/hd/d. In an embodiment, the iodoform is present in the composition at a dose rate between 900 mg/hd/d and 1000 mg/hd/d.

[0024] In an embodiment, the composition is configured to decrease methane production in a rumen of a ruminant animal. In an embodiment, the composition is configured to not provide substantially negative effects on rumen fermentation in a ruminant animal. In an embodiment, the composition is configured to not substantially affect digestibility of diet in a ruminant animal. In an embodiment, the composition is configured to not substantially affect volatile fatty acid production in a ruminant animal. In an embodiment, the composition is configured to increase propionate production in a rumen of a ruminant animal. In an embodiment, the composition is configured to be fed to a ruminant animal.

[0025] In an embodiment, the ruminant animal is selected from the group consisting of cattle, goats, sheep, giraffes, American Bison, European bison, yaks, water buffalo, deer, camels, alpacas, llamas, wildebeest, antelope, pronghorn, and nilgai. In an embodiment, the ruminant animal is raised for meat production. In an embodiment, the ruminant animal is raised for dairy production.

[0026] In an embodiment, the ruminant animal is selected from the group consisting of cattle, buffalo, sheep, and goats. In an embodiment, the ruminant animal is a bovine. In an embodiment, the ruminant animal is a buffalo. In an embodiment, the ruminant animal is a sheep. In an embodiment, the ruminant animal is a goat. In an embodiment, the ruminant animal is a beef cattle. In an embodiment, the ruminant animal is a dairy cattle.

[0027] In an embodiment, the composition comprises one or more mineral supplements. In an embodiment, the mineral supplements are free-choice mineral supplements. In an embodiment, the composition is configured as a slow release formulation. In an embodiment, the composition is configured as a block formulation. In an embodiment, the composition is configured as a bolus formulation.

[0028] In an embodiment, the composition comprises a hydrogen sink compound. In an embodiment, the hydrogen sink compound is selected from the group consisting of a nitrate salt, fumaric acid, fumerate, an unsaturated fatty acid, and any combination thereof. In an embodiment, the hydrogen sink compound is a nitrate salt. In an embodiment, the nitrate salt is sodium nitrate. In an embodiment, the nitrate salt is potassium nitrate. In an embodiment, the hydrogen sink compound is fumaric acid. In an embodiment, the hydrogen sink compound is fumerate. In an embodiment, the hydrogen sink compound is an unsaturated fatty acid. [0029] In an embodiment, the hydrogen sink compound and the iodoform provide a synergistic effect in a rumen of a ruminant animal. In an embodiment, the hydrogen sink compound and the iodoform provide a synergistic methane inhibition in a ruminant animal. [0030] In an illustrative aspect, a method of reducing methane production in a rumen of a ruminant animal is provided. The method comprises the step of administering a composition comprising i) an animal feed composition and ii) iodoform to the ruminant animal, wherein the composition reduces methane production in the rumen of the ruminant animal. Various embodiments of the method are described herein, for instance in the list of non-limiting numbered embodiments. The previously described embodiments of the composition are applicable to the method of reducing methane production in a rumen of a ruminant animal described herein.

[0031] In an embodiment, the method does not provide substantially negative effects on rumen fermentation in the ruminant animal. In an embodiment, the method does not substantially affect digestibility of diet in the ruminant animal. In an embodiment, the method does not substantially affect volatile fatty acid production in the ruminant animal. In an embodiment, the method increases propionate production in the rumen of the ruminant animal. [0032] In an embodiment, the reduction of methane production is a reduction between

10%-95% compared to a ruminant animal not administered the composition. In an embodiment, the reduction of methane production is a reduction of about 10% compared to a ruminant animal not administered the composition. In an embodiment, the reduction of methane production is a reduction of about 20% compared to a ruminant animal not administered the composition. In an embodiment, the reduction of methane production is a reduction of about 30% compared to a ruminant animal not administered the composition. In an embodiment, the reduction of methane production is a reduction of about 40% compared to a ruminant animal not administered the composition. In an embodiment, the reduction of methane production is a reduction of about 50% compared to a ruminant animal not administered the composition. In an embodiment, the reduction of methane production is a reduction of about 60% compared to a ruminant animal not administered the composition. In an embodiment, the reduction of methane production is a reduction of about 70% compared to a ruminant animal not administered the composition. In an embodiment, the reduction of methane production is a reduction of about 80% compared to a ruminant animal not administered the composition. In an embodiment, the reduction of methane production is a reduction of about 90% compared to a ruminant animal not administered the composition. In an embodiment, the reduction of methane production is a reduction of about 95% compared to a ruminant animal not administered the composition.

[0033] In an embodiment, the animal feed composition is a ruminant feed composition.

In an embodiment, the animal feed composition does not comprise seaweed. In an embodiment, the seaweed is red seaweed. In an embodiment, the seaweed is a Porphyra sp.

In an embodiment, the seaweed is an Asparagopsis sp. In an embodiment, the seaweed is Asparagopsis armata. In an embodiment, the seaweed is present at 10% of the animal feed composition. In an embodiment, the animal feed composition does not comprise seaweed at less than 10% of the animal feed composition. In an embodiment, the animal feed composition does not comprise seaweed at more than 10% of the animal feed composition. [0034] In an embodiment, the iodoform is present in the composition at an amount of about 0.35 g/hd/d. In an embodiment, the iodoform is present in the composition at a dose rate between 1 mg/hd/d and 1000 mg/hd/d. In an embodiment, the iodoform is present in the composition at a dose rate between 1 mg/hd/d and 500 mg/hd/d. In an embodiment, the iodoform is present in the composition at a dose rate between 100 mg/hd/d and 500 mg/hd/d.

In an embodiment, the iodoform is present in the composition at a dose rate between 500 mg/hd/d and 1000 mg/hd/d. In an embodiment, the iodoform is present in the composition at a dose rate between 1 mg/hd/d and 300 mg/hd/d. In an embodiment, the iodoform is present in the composition at a dose rate between 300 mg/hd/d and 700 mg/hd/d. In an embodiment, the iodoform is present in the composition at a dose rate between 700 mg/hd/d and 1000 mg/hd/d. In an embodiment, the iodoform is present in the composition at a dose rate between 1 mg/hd/d and 100 mg/hd/d. In an embodiment, the iodoform is present in the composition at a dose rate between 100 mg/hd/d and 200 mg/hd/d. In an embodiment, the iodoform is present in the composition at a dose rate between 200 mg/hd/d and 300 mg/hd/d. In an embodiment, the iodoform is present in the composition at a dose rate between 300 mg/hd/d and 400 mg/hd/d. In an embodiment, the iodoform is present in the composition at a dose rate between 400 mg/hd/d and 500 mg/hd/d. In an embodiment, the iodoform is present in the composition at a dose rate between 500 mg/hd/d and 600 mg/hd/d. In an embodiment, the iodoform is present in the composition at a dose rate between 600 mg/hd/d and 700 mg/hd/d. In an embodiment, the iodoform is present in the composition at a dose rate between 700 mg/hd/d and 800 mg/hd/d. In an embodiment, the iodoform is present in the composition at a dose rate between 800 mg/hd/d and 900 mg/hd/d. In an embodiment, the iodoform is present in the composition at a dose rate between 900 mg/hd/d and 1000 mg/hd/d.

[0035] In an embodiment, the iodoform is administered to the ruminant animal at a concentration between 0.01 mg/kg and 2 mg/kg of body weight of the ruminant animal. In an embodiment, the iodoform is administered to the ruminant animal at a concentration between 0.01 mg/kg and 1 mg/kg of body weight of the ruminant animal. In an embodiment, the iodoform is administered to the ruminant animal at a concentration between 0.1 mg/kg and 1 mg/kg of body weight of the ruminant animal. In an embodiment, the iodoform is administered to the ruminant animal at a concentration between 1 mg/kg and 2 mg/kg of body weight of the ruminant animal. In an embodiment, the iodoform is administered to the ruminant animal at a concentration between 0.01 mg/kg and 0.3 mg/kg of body weight of the ruminant animal. In an embodiment, the iodoform is administered to the ruminant animal at a concentration between 0.3 mg/kg and 0.6 mg/kg of body weight of the ruminant animal. In an embodiment, the iodoform is administered to the ruminant animal at a concentration between 0.6 mg/kg and 0.9 mg/kg of body weight of the ruminant animal. In an embodiment, the iodoform is administered to the ruminant animal at a concentration between 0.9 mg/kg and 1.2 mg/kg of body weight of the ruminant animal. In an embodiment, the iodoform is administered to the ruminant animal at a concentration between 1.2 mg/kg and 1.5 mg/kg of body weight of the ruminant animal. In an embodiment, the iodoform is administered to the ruminant animal at a concentration between 1.5 mg/kg and 1.8 mg/kg of body weight of the ruminant animal. In an embodiment, the iodoform is administered to the ruminant animal at a concentration between 1.8 mg/kg and 2 mg/kg of body weight of the ruminant animal.

[0036] In an embodiment, the iodoform is administered to the ruminant animal at a concentration between 0.01 mg/kg and 0.1 mg/kg of body weight of the ruminant animal. In an embodiment, the iodoform is administered to the ruminant animal at a concentration between 0.1 mg/kg and 0.2 mg/kg of body weight of the ruminant animal. In an embodiment, the iodoform is administered to the ruminant animal at a concentration between 0.2 mg/kg and 0.3 mg/kg of body weight of the ruminant animal. In an embodiment, the iodoform is administered to the ruminant animal at a concentration between 0.3 mg/kg and 0.4 mg/kg of body weight of the ruminant animal. In an embodiment, the iodoform is administered to the ruminant animal at a concentration between 0.4 mg/kg and 0.5 mg/kg of body weight of the ruminant animal. In an embodiment, the iodoform is administered to the ruminant animal at a concentration between 0.5 mg/kg and 0.6 mg/kg of body weight of the ruminant animal. In an embodiment, the iodoform is administered to the ruminant animal at a concentration between 0.6 mg/kg and 0.7 mg/kg of body weight of the ruminant animal. In an embodiment, the iodoform is administered to the ruminant animal at a concentration between 0.7 mg/kg and 0.8 mg/kg of body weight of the ruminant animal. In an embodiment, the iodoform is administered to the ruminant animal at a concentration between 0.8 mg/kg and 0.9 mg/kg of body weight of the ruminant animal. In an embodiment, the iodoform is administered to the ruminant animal at a concentration between 0.9 mg/kg and 1.0 mg/kg of body weight of the ruminant animal. In an embodiment, the iodoform is administered to the ruminant animal at a concentration between 1.0 mg/kg and 1.1 mg/kg of body weight of the ruminant animal. In an embodiment, the iodoform is administered to the ruminant animal at a concentration between 1.0 mg/kg and 1.1 mg/kg of body weight of the ruminant animal. In an embodiment, the iodoform is administered to the ruminant animal at a concentration between 1.1 mg/kg and 1.2 mg/kg of body weight of the ruminant animal. In an embodiment, the iodoform is administered to the ruminant animal at a concentration between 1.2 mg/kg and 1.3 mg/kg of body weight of the ruminant animal. In an embodiment, the iodoform is administered to the ruminant animal at a concentration between 1.3 mg/kg and 1.4 mg/kg of body weight of the ruminant animal. In an embodiment, the iodoform is administered to the ruminant animal at a concentration between 1.4 mg/kg and 1.5 mg/kg of body weight of the ruminant animal. In an embodiment, the iodoform is administered to the ruminant animal at a concentration between 1.5 mg/kg and 1.6 mg/kg of body weight of the ruminant animal. In an embodiment, the iodoform is administered to the ruminant animal at a concentration between 1.6 mg/kg and 1.7 mg/kg of body weight of the ruminant animal. In an embodiment, the iodoform is administered to the ruminant animal at a concentration between 1.7 mg/kg and 1.8 mg/kg of body weight of the ruminant animal. In an embodiment, the iodoform is administered to the ruminant animal at a concentration between 1.8 mg/kg and 1.9 mg/kg of body weight of the ruminant animal. In an embodiment, the iodoform is administered to the ruminant animal at a concentration between 1.9 mg/kg and 2.0 mg/kg of body weight of the ruminant animal.

[0037] In an embodiment, the ruminant animal is selected from the group consisting of cattle, goats, sheep, giraffes, American Bison, European bison, yaks, water buffalo, deer, camels, alpacas, llamas, wildebeest, antelope, pronghorn, and nilgai. In an embodiment, the ruminant animal is raised for meat production. In an embodiment, the ruminant animal is raised for dairy production.

[0038] In an embodiment, the ruminant animal is selected from the group consisting of cattle, buffalo, sheep, and goats. In an embodiment, the ruminant animal is a bovine. In an embodiment, the ruminant animal is a buffalo. In an embodiment, the ruminant animal is a sheep. In an embodiment, the ruminant animal is a goat. In an embodiment, the ruminant animal is a beef cattle. In an embodiment, the ruminant animal is a dairy cattle.

[0039] In an embodiment, the composition comprises one or more mineral supplements. In an embodiment, the mineral supplements are free-choice mineral supplements. In an embodiment, the composition is configured as a slow release formulation. In an embodiment, the composition is configured as a block formulation. In an embodiment, the composition is configured as a bolus formulation.

[0040] In an embodiment, the composition comprises a hydrogen sink compound. In an embodiment, the hydrogen sink compound is selected from the group consisting of a nitrate salt, fumaric acid, fumerate, an unsaturated fatty acid, and any combination thereof. In an embodiment, the hydrogen sink compound is a nitrate salt. In an embodiment, the nitrate salt is sodium nitrate. In an embodiment, the nitrate salt is potassium nitrate. In an embodiment, the hydrogen sink compound is fumaric acid. In an embodiment, the hydrogen sink compound is fumerate. In an embodiment, the hydrogen sink compound is an unsaturated fatty acid. [0041] In an embodiment, the hydrogen sink compound and the iodoform provide a synergistic effect in a rumen of a ruminant animal. In an embodiment, the hydrogen sink compound and the iodoform provide a synergistic methane inhibition in a ruminant animal.

EXAMPLES

Example 1

In Vitro Fermentation Model

[0042] The instant example was performed in Alltech’s in vitro fermentation model

(IFM). Results of the instant example are shown in Figure 1.

[0043] Briefly, a 2% iodoform solution in ethanol was evaluated via IFM with basal total mixed ration (TMR) control at various dose concentrations. In particular, the iodoform solution was evaluated at 0, 1.8, 3.6, 5.4 and 18 ppm of iodoform per sample. Each sample was evaluated in quadruplicate with three blanks and control TMR. Gas samples also were also collected for GHG on the GC.

[0044] As shown in Figure 1, all doses of iodoform resulted in significant reduction in methane estimated stoichiometrically by VFA profiles or by direct measurement. As shown in Figure 1:

• The concentration of total volatile fatty acid (“Tot VFA”) in millimolars was evaluated for each concentration of iodoform solution (i.e., 0, 1.8, 3.6, 5.4 and 18 ppm of iodoform).

• Percentages of true dry matter digestibility (“TDMD”) were evaluated for each concentration of iodoform solution (i.e., 0, 1.8, 3.6, 5.4 and 18 ppm of iodoform).

• Percentages of propionate of the total volatile fatty acid were evaluated for each concentration of iodoform solution (i.e., 0, 1.8, 3.6, 5.4 and 18 ppm of iodoform).

• The amount of methane produced per gram of dry matter (“CH4/g DM gas”) in milliliters was evaluated for each concentration of iodoform solution (i.e., 0, 1.8, 3.6, 5.4 and 18 ppm of iodoform). The amount of methane gas was measured directly in the headspace gas. • Percentages of methane reduction due to intervention (“CH4 reduction”) were evaluated for each concentration of iodoform solution (i.e., 0, 1.8, 3.6, 5.4 and 18 ppm of iodoform)

[0045] Higher iodoform doses resulted in significant reductions in digestibility and

VFA production. Further, a 100% inhibition of methane was achieved at the 18 ppm iodoform concentration, with only a 7% reduction in diet digestibility.

Example 2

Iodoform Dosing Evaluation

[0046] The instant example evaluates various dose/concentrations of iodoform in the rumen of a ruminant animal. Evaluation of methane formation in the rumen of a ruminant animal following administration of a composition comprising iodoform at low levels can be performed. Further, since complete methane inhibition is associated with negative impacts on feed intake, rumen fermentation, and animal production, other endpoints can be evaluated. [0047] In the instant example, ruminally fistulated steers (3 years old, -1000 lb BW) can be evaluated. Steers can be fed grass hay, corn, soybean meal, minerals/vitamins twice a day at approximately 2% BW.

[0048] Several different compositions comprising iodoform can be fed to the animals.

The various compositions can comprise between 0-1000 mg/d iodoform. The various compositions can achieve rumen fluid concentrations between 0-10 ppm, feed concentration between 0-100 ppm, and up to 2 mg/kg BW.

[0049] In vitro testing can be used to determine the lowest dose to achieve desired outcome (e.g., >30% reduction in methane). In vitro rumen fermentation testing indicates that an iodoform dose rate equivalent to 360 mg/d (36ppm in feed, 0.72 mg/kg BW) can achieve the target reduction in methane. Further adjustments to the dose rate can be contemplated when feeding in vivo.

[0050] Initially, idoform-containing compositions can be fed to animals at 0 and 360 mg/d iodoform (e.g., two steers assigned to each treatment group). If the steers tolerate the compositions but a 30% C¾ reduction is not achieved, the dose of iodoform can be increased incrementally up to 1000 mg/d until the goal can be reached. Conversely, if at least 30% CH ₄ reduction is achieved with the initial dose of iodoform, the dose of iodoform can be reduced incrementally to identify the lowest possible dose that can achieve 30% CH4 reduction. The target dose of iodoform can be contemplated at 360mg/d and iodoform doses up to lOOOmg/d are well below the lowest toxic iodoform doses known in the art.

[0051] Crossover or Latin square experimental design can be utilized to compare control groups (0 mg/d iodoform) with iodoform treatment groups (up to 1000 mg/d). Each steer can receive each treatment of iodoform-containing compositions with washout periods of at least 2 weeks between adaptation periods. Adaptation can occur when the rumen VFA profile stabilizes (e.g., 14 days).

[0052] Following adaption (e.g., between 0-14 days), samples can be collected from the animals on days 15-21. Each treatment period of iodoform-containing compositions can be performed for 21 days. Treatment of iodoform-containing compositions can be split equally in each of two meals per day. Iodoform can be top-dressed on the concentrate to insure complete consumption.

[0053] Rumen fluid can be collected daily from the animals starting at 2 days prior to beginning treatment of iodoform-containing compositions and can last until adaption is achieved in the animals (e.g., between 7-14 days). Prior to starting treatments, rumen contents can be completely removed, weighed, subsampled, and returned to the rumen in order to determine rumen pool size. Rumen contents can be removed manually. Rumen fluid can be analyzed for VFA to estimate CO2 and CFL gas production. In addition, a luer adapter can be placed in the cannula cap of each animal to facilitate the sampling of headspace gas in the rumen without having to open it to the outside environment.

Example 3

Iodoform and Hydrogen Sink Compound Evaluation [0054] The instant example evaluates the administration of iodoform and hydrogen sink compounds to a ruminant animal. Evaluation of rumen metabolites and enteric methane potential in the rumen of a ruminant animal following administration of compositions comprising iodoform and/or hydrogen sink compounds can be performed. Further, the synergistic interaction of iodoform and hydrogen sink compounds such as nitrate salts, fumaric acid/fumerate, and unsaturated fatty acids can be evaluated. [0055] In the instant example, ruminally fistulated steers (3 years old, -1000 lb BW) can be evaluated. Steers can be fed grass hay, corn, soybean meal, minerals/vitamins twice a day at approximately 2% BW.

[0056] Several different compositions comprising iodoform can be fed to the animals.

The various compositions can comprise between 0-1000 mg/d iodoform. The various compositions can achieve rumen fluid concentrations between 0-10 ppm, feed concentration between 0-100 ppm, and up to 2 mg/kg BW. The dosing information of iodoform described in Example 2 can be utilized for the administration of iodoform-containing compositions in the instant example.

[0057] In the instant example, iodoform-containing compositions can be fed to the animals in conjunction with hydrogen sink compounds (e.g., nitrate salts, fumaric acid/fumerate, and unsaturated fatty acids). The administration of one or more hydrogen sink compounds, either separate from an iodoform-containing composition or combined with an iodoform-containing composition, can be evaluated. For instance, a slow release formulation (e.g., including both iodoform and one or more hydrogen sink compounds) can be evaluated. [0058] Evaluations can include the efficacy of iodoform-containing compositions and hydrogen sink compositions in two different ways. First, hydrogen sink compounds can be evaluated with respect to the depression in rumen fermentation that is observed using higher doses of iodoform. In this way, it can be evaluated if higher inhibition of methanogenesis in the rumen can be achieved without significantly inhibiting fermentation processes.

[0059] Second, hydrogen sink compounds can be evaluated regarding a synergistic effect with iodoform-containing compositions. For instance, the reduction in total methane emission can be evaluated for co-administration of iodoform/hydrogen sink compounds compared to iodoform and hydrogen sink compounds administered alone.

[0060] In addition, or alternatively, the co-administration of iodoform/hydrogen sink compounds can result in lowering of the dose of the iodoform, the hydrogen sink compound, or both to achieve a desired effect in the ruminant animal. For instance, the dose of the iodoform, the hydrogen sink compound, or both could be lowered to provide a sufficient inhibition of methanogenesis in the rumen while synergistically providing a reduction in negative effects that would be expected to be observed. [0061] Crossover or Latin square experimental design can be utilized to compare control groups (0 mg/d iodoform) with iodoform treatment groups (up to 1000 mg/d) and effects when administered with hydrogen sink compounds. Each steer can receive each treatment of iodoform-containing compositions with washout periods of at least 2 weeks between adaptation periods. Adaptation can occur when the rumen VFA profile stabilizes (e.g., 14 days).

[0062] Following adaption (e.g., between 0-14 days), samples can be collected from the animals on days 15-21. Each treatment period of iodoform-containing compositions and/or hydrogen sink compounds can be performed for 21 days. Treatment of iodoform- containing compositions can be split equally in each of 2 meals per day. Iodoform can be top- dressed on the concentrate to insure complete consumption. If voluntary consumption does not occur, treatments can be dosed directly into the rumen through the cannula.

[0063] Rumen fluid can be collected daily from the animals starting at 2 days prior to beginning treatment of iodoform-containing compositions and can last until adaption is achieved in the animals (e.g., between 7-14 days). Prior to starting treatments, rumen contents can be completely removed, weighed, subsampled, and returned to the rumen in order to determine rumen pool size. Rumen contents can be removed manually. Rumen fluid can be analyzed for VFA to estimate CO2 and CFL gas production. In addition, a luer adapter can be placed in the cannula cap of each animal to facilitate the sampling of headspace gas in the rumen without having to open it to the outside environment.

Example 4

Evaluation of the interaction between Iodoform and Yea-sacc conditioned rumen fluid on rumen fermentation and methane production from beef and dairy rations evaluated in vitro

[0064] One objective of the instant example was to evaluate effects of iodoform under different dietary conditions such as high concentrate beef vs. dairy rations. Another objective was to evaluate potential interactions between iodoform treatment and use of yeast cultures by testing iodoform in conditioned rumen fluid compared to non- conditioned rumen fluid (Yea- Sacc Value Test). [0065] Evaluations included the analyzing the effects of a 0.7% solution of iodoform added at 50 uL/100 mL of fermentation volume using various diets. For example, diets could differ in NDF:NFC and also could be formulated with and without yeast. Briefly, five culture replicates of each total mixed ration (TMR) were made. The final dose of iodoform was 3.5 ppm (based on a 50 L rumen liquid volume). Standard in vitro batch culture conditions were used to evaluate rumen fermentation and methane production (Alltech IFM system).

[0066] The experimental design of the instant example was completely randomized with a 2x2x2 factorial treatment structure:

^■ Factor 1 : Ration - Two rations were compared to evaluate ration specific responses to iodoform

• Dairy TMR (DAIRY); composition shown in Table 1.

• Feedlot beef TMR (BEEF); composition shown in Table 1.

^■ Factor 2: Rumen fluid source. Rumen fluid was harvested from animals that were either naive (NAIVE) or exposed to Yea-Sacc (YS)

^■ Factor 3: Presence or absence of Iodoform

• CON: No iodoform

• IODO: Iodoform added to cultures at 3.5ppm Table 1. Composition of tested TMR.

[0067] As shown in Table 2 and Figure 2, addition of iodoform reduced VFA by

14.5% and 11%, respectively, for naive and YS adapted inoculum. Furthermore, as shown in Table 2 and Figure 3, iodoform reduced CFk/g DM truly digested by 30% and 27%, respectively, for naive and YS adapted inoculum.

Table 2. Effects of adding Iodoform at 3.5 ppm to different TMR with different starch content

Table 2 (continued) Effects of adding Iodoform at 3.5 ppm to different TMR with different starch content

[0068] Moreover, Yea-Sacc allowed 85% of methane decrease obtained by iodoform in naive animals but only 50% of the drop in VFA seen in naive animals. Therefore, Yea-Sacc may be an option for reducing possibly undesirable effects of methane inhibition on rumen fermentation and on production. Iodoform was demonstrated to be highly effective in both beef and dairy rations.

Example 5

Iodoform Dosing Evaluations

[0069] Various evaluations were performed using an in vitro gas production technique to simulate rumen fermentation. A brief description of the experimental method utilized for the instant example are as follows.

[0070] A substrate (0.5g) was ground through a 2 mm screen and incubated with

100 mL of buffered rumen fluid for 48 hours in quadruplicate. Incubation flasks were maintained under continuous flushing of CO2. At the end of the incubation period, residues were dried at 104 °C for 4 hours to estimate apparent dry matter disappearance. [0071] To estimate the amount of substrate that was truly degraded, residues were treated with neutral detergent solution. The difference between substrate apparently degraded (ADMD) and substrate truly degraded (TDMD) was considered microbial biomass (MBM). Total VFA production and headspace gas composition was determined at the end of 48 hours of incubation.

[0072] Terms used according to the instant example include: ^■ Slow pool (SP; mL/g DM): gas potentially produced by the fermentation of the neutral detergent insoluble carbohydrates which consist of fiber (NDF). This pool may contain some of the slowly fermented starch.

^■ 2-Pool total (mL/ g DM): total gas produced from all ruminally fermented material which is the sum of the fast and slow pools

^■ Fast rate (%/h): degradation rate constant of the fast pool. Indicative of degradation of starches, sugars, and other quickly degrading carbohydrate

^■ Slow rate (%/h): degradation rate constant of the slow pool. Indicative of degradation of fiber. Quantitative value of forage quality.

^■ Starch kd (%/h): degradation rate constant of the starch in the feed.

^■ Fast pool (% total): proportion of the gas produced from the fast pool relative to total gas.

^■ Slow pool (% total): proportion of the gas produced from the slow pool relative to total gas.

^■ Time to max o Fast rate (h): time required for the fast pool to reach maximal degradation rate, not necessarily maximum gas production. o Slow rate (h): time required for the slow pool to reach maximal degradation rate. o The difference between time to max SP and FP is a good indicator of the synchrony between the fast and slow pool and overall energy availability to rumen microbes.

^■ ADMD (%): apparent dry matter digestibility, the percent of incubated feed DM that is left after 48h incubation, the residue contains microbial biomass.

^■ TDMD (%): true dry matter digestibility measured after solubilisation of the microbial biomass in the undigested residue, TDMD> ADMD.

^■ Microbial biomass (mg/ g DM): microbial mass synthesized during the 48 h incubation, estimated as the difference between ADMD and TDMD and corrections to account for microbial growth associated with the liquid phase. ^■ PF : ratio between dry matter truly digested (mg/g DM) and total gas production (mL/ g DM). Microbial biomass production is positively correlated with PF. Measurement of the efficiency of rumen fermentation to convert feed energy and protein in to MBM and VFA

^■ Total VFA (m M): net production of total volatile fatty acids (VFA produced from the culture corrected for a blank that is included in each incubation run).

^■ VFA, % of total: relative molar proportions of the different VFA (acetic, propionic, isobutyric, butyric, isovaleric, and valeric acid). The proportion of each organic acid depends on the substrate available.

^■ C02 and CH4: measured directly via headspace gas analysis (C02 gas and CH4 gas) and calculated by VTA profile (C02 VTA and CH4 VTA).

Study 1 A: Initial dose response in vitro study

[0073] An iodoform solution was prepared in ethanol and then added to rumen fluid inoculum at 3.5, 35, and 350 ppm. As shown in Table 3, a small decrease was observed in total gas and TDMD for the 3.5ppm Iodoform group compared to control. Further, a similar total VFA but higher acetate and lower prionate was shown for the 3.5ppm Iodoform group while there was a large decrease in total gas, TDMD, and total VTA for the 35 and 350ppm Iodoform groups. There was a 15-16% reduction in CH4 (measured either by VTA or by gas) for the 3.5ppm Iodoform group.

Table 3.

Study IB: Refining in vitro dose

[0074] An iodoform solution was prepared in ethanol and then added to rumen fluid inoculum at 0-35 ppm. As shown in Table 4, total gas production appeared to decrease at any concentration of iodoform and decreased as iodoform concentration increased. The TDMD and total VFA did not change as Iodoform was added at 0 vs. 3.5 ppm, but propionate and CH4 (VFA and gas measured) decreased by 5-10%.

Table 4.

Table 4 (continued)

Study 1C: Fine tuning of in vitro dose rate

[0075] An iodoform solution was prepared in ethanol and then added to rumen fluid inoculum at 0-18 ppm. As shown in Table 5, small decreases in total gas, digestibility (TDMD), and total VFA for were observed for the Iodoform groups at 1.8, 3.6, and 5.4 ppm compared to control. Larger decreases in these parameters were observed for the Iodoform group at 18ppm compared to control.

[0076] Acetate decreased and propionate and butyrate increase as iodoform concentration increased, which is reflected in VFA calculated CH4. In addition, CH4 calculated from VFA and gas analysis generally were observed to trend in the same direction as iodoform increased, but absolute concentration and percentage change from control were observed to be different.

Table 5.

Study ID: Stability testing of iodoform solution in ethanol [0077] This study tested iodoform solutions in ethanol prepared 5 months previously. Iodoform solutions were added to rumen fluid inoculum at 0-18 ppm. As shown in Table 6, results observed previously (e.g. increased propionate, decreased acetate, decreased CH4 VFA and gas) were not able to be reproduced. The table shows that little change in total gas production, total VFA, acetate, propionate or CH4 (VFA or gas) were observed. These data indicate the iodoform solution in ethanol may not be stable for extended periods of time.

Table 6.

Study IE: Testing iodoform efficacy of iodoform in powder form [0078] In this study, iodoform was mixed with cornstarch and added to rumen inoculum at 0-18 ppm. As shown in Table 7, the effects of iodoform concentration were similar to previous runs with iodoform solutions. A small decrease in total gas and TDMD were observed at lower concentrations and larger reduction at highest concentrations. Further, a similar total VFA was observed at all concentrations as well as an increase in propionic acid as iodoform increased. Further, data showed a trend in decreased CH4 predicted by VFA profile, which is a similar trend to CH4 measured in headspace gas. Finally, the percentage decrease in CH4 compared to control was similar for both VFA and gas CH4 measurements.

Table 7 Averages.

[0079] Table 8 shows that standard deviations were different among treatment groups for the same parameter. However, treatments including iodoform powder were not necessarily greater than control treatment in all instances and use of iodoform powder does not decrease precision amongst treatment groups.

Table 8. Standard deviation amongst treatment groups to test accuracy of using iodoform powder

Study 2 A: Evaluation of interactions with hydrogen sink compounds: Iodoform x Fumaric acid

[0080] In this study, various concentrations of iodoform (0, 1.8, 3.6, 5.4 ppm) were combined with various concentrations of fumaric acid (0, 10, 20, 20 mM). As shown in Tables 9 and 10, no interactions between iodoform and fumaric acid were detected.

Table 9.

Table 10.

Study 2B: Evaluation of interactions with hydrogen sink compounds: Iodoform x Nitrate [0081] In this study, iodoform (0-3.6 pmm) and/or nitrate (0-20 mM) were added to fermentation bottles. As shown in Table 11, no interactions between iodoform and nitrate were detected and both compounds were observed to reduce CH4.

Table 11.

Study 3 : Adapting rumen fluid donor to iodoform - evaluation of rumen adaptation to iodoform intervention

[0082] In this study, steers were fed rations including iodoform (3 lppm and a target of 3.7ppm in the rumen) for at least 2 weeks prior to in vitro analysis. Thereafter, a comparison of rumen fluid from control (no iodoform) vs. iodoform-fed steers was performed. For the instant study, 0 or 3.6ppm iodoform was added to in vitro fermentation.

[0083] As shown in Table 12, using inoculum from steers adapted to iodoform did not impact the change in CH4 compared to controls. CH4 was reduced when a similar concentration of iodoform was added to the inoculation bottle, regardless of the inoculation source. This suggests that the effect of iodoform may be short term and it must be present to affect CH4 formation.

Table 12.

Study 4: Effect of feeding iodoform on ruminal VFA profile in vivo study)

[0084] In this study, steers (n = 4) were fed 0 or 35ppm iodoform and rumen samples were collected at a time point of 2 hours post-feeding, 3 times weekly for 2 months. Rumen fluid samples were analyzed for VFA.

[0085] Figures 4-8 demonstrate some fluctuation in total VFA and individual concentrations over time, but little change in VFA profile as the percentage of total VFA as observed in vitro. This suggests that iodoform does not modify ruminal VFA in a long term fashion, but rather causes transient short term changes in rumen VFA proportions.

Study 5: Short term effects of feeding iodoform on ruminal VFA (in vivo) [0086] In this study, steers (n = 4) were fed 0 or 35ppm iodoform for at least 21 days. Rumen samples were collected at time points of 0, 2, 4, and 6 hours post-feeding. Rumen fluid samples were analyzed for VFA.

[0087] Figures 9-10 demonstrate either minor differences or no differences in

VFA concentration or proportions in a 6 hour post-feeding analysis.

Study 6: Direct measurement of methane via Greenfeed apparatus

[0088] In this study, heifers (n = 4) were fed 0 or 35ppm iodoform for at least 21 days. Animals were sampled over a period of days to represent a time course curve of methane emissions. The Greenfeed system (a system designed to measure gas fluxes of

Methane (CFF), Carbon Dioxide (CO2), and optionally, Oxygen (O2), and Hydrogen (¾) from individual animals; C-Lock, Rapid City, South Dakota) was used to quantify emissions.

[0089] Figure 11 shows a reduction in methane in treated animals. The methane emissions of iodoform-treated animals remained flat after eating, while the methane emissions of control animals increased.