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Effects of Bismuth Subsalicylate and Calcium-Ammonium Nitrate on Beef Cattle Production

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Title:
Effects of Bismuth Subsalicylate and Calcium-Ammonium Nitrate on Beef Cattle Production
Creator:
Henry, Darren D
Place of Publication:
[Gainesville, Fla.]
Florida
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University of Florida
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english
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1 online resource (163 p.)

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Animal Sciences
Committee Chair:
DILORENZO,NICOLAS
Committee Co-Chair:
LAMB,GRAHAM CLIFF
Committee Members:
JEONG,KWANG CHEOL
ADESOGAN,ADEGBOLA TOLULOPE
DUBEUX,JOSE CARLOS

Subjects

Subjects / Keywords:
ammonium -- beef -- bismuth -- calcium -- cattle -- digestibility -- fermentation -- metabolism -- methane -- nitrate -- performance -- subsalicylate
Animal Sciences -- Dissertations, Academic -- UF
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bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Animal Sciences thesis, Ph.D.

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Abstract:
To aid in the objective of becoming ever more efficient, scientists have developed strategies, such as providing nitrate as a NPN source, to produce high-quality beef and dairy products while mitigating the environmental impact of production. Another challenge in animal-protein production is elevated S concentrations in byproducts, forages, and water in certain areas of the U.S. Bismuth subsalicylate (BSS) has been observed to mitigate negative effects of S, such as H2S, using ruminal in vitro systems. The overarching objective of this dissertation was to test the hypothesis that BSS and calcium-ammonium nitrate (CAN) on would improve beef cattle performance and lessen the environmental presence. To test this hypothesis, 4 experiments were designed to evaluate their on in vitro fermentation, ruminal fermentation, enteric CH4 production and nutrient digestibility, and on performance and liver mineral concentration. In vitro ruminal fermentation was altered by both BSS and CAN. Production of H2S was reduced (P = 0.024) quadratically with the addition of up to 1.0% of the substrate DM. A linear reduction in total CH4 (P 0.05) by the addition of BSS or eCAN. As a potential NPN source, the data from this series of experiments indicates that eCAN may not promote the same ruminal fermentation and performance of cattle consuming urea. Bismuth subsalicylate appears to bind to sulfur in the ruminant; however, the negative effects on Cu concentration in the liver may impede upon further use of BSS in cattle fed low-quality forage-based, diets. ( en )
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In the series University of Florida Digital Collections.
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Includes vita.
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Includes bibliographical references.
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Description based on online resource; title from PDF title page.
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This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (Ph.D.)--University of Florida, 2017.
Local:
Adviser: DILORENZO,NICOLAS.
Local:
Co-adviser: LAMB,GRAHAM CLIFF.
Statement of Responsibility:
by Darren D Henry.

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EFFECTS OF BISMUTH SUBSALICYLATE AND CALCIUM AMMONIUM NITRATE ON BEEF CATTLE PRODUCTION By DARREN DWAYNE HENRY A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2017

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2017 Darren Dwayne Henry

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To my wife and family

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4 ACKNOWLEDGMENTS I want to give my greatest thanks to our Lord and Savior, without him in my life, I would not have had any of the success I have enjoyed in my life. My wife is the most important person in my life. It was because of her support, love, and unbelievable hard work, that I was able to complete my Doctor of philosophy degree. She has stood by my side through the often rewarding moments in a young scientist s life and held my hand when that can become seemingly u nbearable. Thank you, Beeb, I love you from here all the way around to here. Without the solid foundation provided by my parents, Sonny and Connie Henry, I would have never left Montgomery County, TX. Their love and support through my career as a professio nal student was, and always will be, more than I could ever ask for. I would also like to thank Katie Henry for her patience throughout this process as well. It would have been impossible to complete 3 experiments and plan a wedding at the same time withou t her. I need to thank my brother, little Sonny, for always being there for me. I wish to give a huge thank you to Ruth Henry (aka Mawmaw). She never gave up on me while I was away studying and she forgives me when I forget to call. I hold an undying love in my heart for my family who are no longer with us. I want to thank Joseph F. Henry, Sr. for his unconditional love and support all of my life. To Mary Gribble, I give immense thanks for her love and laughter. I thank Jerry Caveness for teaching me how to Gribble was a man I will never forget and I will miss him dearly for all of my days. I give great gratitude to Dr. Rafael C. Araujo and his employers, GRASP Ind. & Com, for providing all of the nitrate used during my PhD program. Furthermore, I wish to thank Rafael especially for his comments and valuable inputs to the design of the

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5 experiments. Mr. Randy Davis and Quality Liquid Feeds deserves my thanks for donating the molasses for these exper iments. More than that, I thank Randy for the amazing crawfish boil that he performed for Francine and myself as a wedding gift. I would like to extend my gratitude to my friend, Dr. Vitor Mercadante. Vitor was a great example to follow throughout my graduate career and the lessons I learned from him are invaluable. Pedro Fontes is also a fellow student whom I would like to give special recognition. It is easy to become content with the work you are doing, and riosity always kept me on my toes. A huge thanks goes out to Dr. Martin Ruiz Moreno for all of the insightful discussions we have had over the years. I would also like to extend my gratitude to all of my friends and colleagues who have helped me over the years: Nicky Oosthuizen, Erick Santos, David Jaramillo, Lautaro Rostoll, Mariana Garcia, Tessa Schulmeister, Carla Sanford, Luara Canal, and many others. I would like to thank the countless interns from all over the world who lent an extremely big hand in collecting the data for these experiments. In particular I would like Maria Alejandra Gutierrez Beltrn I wish to thank the members of my committee: Drs. G. Clifford Lamb Jose Dubeux, Adegbola Adesogan, and K. C. Jeong. Your patience with me and your guidance over the past years has been invaluable. I have the utmost love and respect for Dr. Nicolas DiLorenzo. As a mentor and advisor, I can think of none better than him. Thinking back on when I began my

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6 graduate career in 2012, there is absolutely no doubt in my mind that I would never have gained the knowledge necessary to be here today if it had not been for Dr. Nicolas DiLorenzo. He is one of my greatest friends and I hope that I can repay the enormous debt that I owe him someday.

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7 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ .......... 10 LIST OF FIGURES ................................ ................................ ................................ ........ 12 LIST OF ABBREVIATIONS ................................ ................................ ........................... 13 ABSTRACT ................................ ................................ ................................ ................... 15 Chapter 1 REVIEW OF THE LITERATURE ................................ ................................ ............ 17 Methane Production in Cattle ................................ ................................ .................. 17 Methanogenesis ................................ ................................ ............................... 17 Issues Related to Enteric Methane Production ................................ ................. 19 Miti gation of Enteric Methane Production ................................ ......................... 21 Tannins ................................ ................................ ................................ ...... 21 Lipids ................................ ................................ ................................ ......... 23 Ionophores ................................ ................................ ................................ 25 3 Nitrooxypropanol ................................ ................................ ..................... 28 Diet ................................ ................................ ................................ ............ 30 Potential for Nitrates in the Cattle Industry ................................ ....................... 31 Mitigation of enteric methane production with nitrate ................................ 32 Nitrate as a non protein nitrogen source ................................ .................... 33 Sulfur in Beef Cattle Production ................................ ................................ .............. 35 Issues Related to High Sulfur Diets ................................ ................................ .. 35 Mitigation of Sulfur Toxicity ................................ ................................ ............... 38 Mitigation of Hydrogen Sulfide Production with Bismuth Subsalicylate ............ 38 2 EFFECTS OF BISMUTH SUBSA LICYLATE AND CALCIUM AMMONIUM NITRATE ON IN VITRO FERMENTATION OF BAHIAGRASS HAY WITH SUPPLEMENTAL MOLASSES ................................ ................................ .............. 41 Introduction ................................ ................................ ................................ ............. 41 Materials and Methods ................................ ................................ ............................ 42 Experiment 1 ................................ ................................ ................................ .... 42 Experimental design, substrate, and treatments ................................ ........ 42 In vitro incubations ................................ ................................ ..................... 43 Total ga s production, CH 4 and H 2 S production, NH 3 N analyses ............... 44 Volatile fatty acid analysis ................................ ................................ .......... 45 In vitro OM digestibility ................................ ................................ ............... 46 Experiment 2 ................................ ................................ ................................ .... 46

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8 Experimental design, substrate, and treatments ................................ ........ 46 In vitro incubations ................................ ................................ ..................... 47 Experiment 3 ................................ ................................ ................................ .... 47 Experimental design, substrate, and treatment ................................ .......... 47 In vitro incubations ................................ ................................ ..................... 47 Statisti cal Analysis ................................ ................................ ............................ 48 Results and Discussion ................................ ................................ ........................... 49 Experiment 1 ................................ ................................ ................................ .... 49 Experiment 2 ................................ ................................ ................................ .... 53 Experiment 3 ................................ ................................ ................................ .... 58 3 EFFECTS OF BIS MUTH SUBSALICYLATE AND CALCIUM AMMONIUM NITRATE ON RUMINAL FERMENTATION OF BEEF CATTLE ............................. 70 Introduction ................................ ................................ ................................ ............. 70 Materials and Methods ................................ ................................ ............................ 71 Experimental Design, Animals, and Treatments ................................ ............... 71 Sampling Procedures ................................ ................................ ....................... 73 Ruminal gas cap sampling ................................ ................................ ......... 73 Ruminal fluid, pH, blood sampling, and rectal temperature ........................ 73 Hay and total dry matter intake ................................ ................................ .. 74 Omasal sampling ................................ ................................ ....................... 74 Bacterial pellet for purine reference ................................ ........................... 7 6 Ruminal evacuations and digesta flow mar ker dosing ............................... 76 Laboratory Analyses ................................ ................................ ......................... 78 Hydrogen sulfide concentration ................................ ................................ .. 78 Volatile fatty acid profile ................................ ................................ ............. 78 Ammonia N and blood urea N concentration ................................ ............. 79 Chemical analyses of hay, orts, ruminal, and omasal digesta fractions ..... 79 Chromium concentrations ................................ ................................ .......... 81 Purine analysis ................................ ................................ ........................... 81 Calculations ................................ ................................ ................................ ...... 82 Flow of digesta ................................ ................................ ........................... 82 Flow and efficiency of microbial N ................................ .............................. 82 Statistical Analysis ................................ ................................ ............................ 83 Results and Discussion ................................ ................................ ........................... 84 4 EFFECTS OF BISMUTH SUBSALICYLATE AND CALCIUM AMMONIUM NITRATE ON ENTERIC METHANE PRODUCTION AND NUTRIENT DIGESTIBILITY OF BEEF STEERS ................................ ................................ ..... 105 Introduction ................................ ................................ ................................ ........... 105 Mate rials and Methods ................................ ................................ .......................... 106 Experimental Design, Animals, and Treatments ................................ ............. 106 Sampling Procedures ................................ ................................ ..................... 108 Apparent total tract digestibility of nutrients and retention of S ................ 108 Methane emissions ................................ ................................ .................. 108

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9 Laboratory Analyses ................................ ................................ ....................... 109 Concentration of DM, OM, CP, NDF, ADF, and S ................................ .... 109 Concentration of iNDF ................................ ................................ ............. 110 Methane and SF 6 analyses ................................ ................................ ...... 110 Calculations ................................ ................................ ................................ .... 111 Statistical Analysis ................................ ................................ .......................... 111 Results and Discussion ................................ ................................ ......................... 112 5 EF FECTS OF BISMUTH SUBSALICYLATE AND CALCIUM AMMONIUM NITRATE ON LIVER MINERAL CONCENTRATION AND PERFORMANCE OF GROWING BEEF HEIFERS ................................ ................................ ................. 122 Introduction ................................ ................................ ................................ ........... 122 Materials and Methods ................................ ................................ .......................... 123 Experimental Design, Animals, and Treatments ................................ ............. 123 Sample and Data Collection ................................ ................................ ........... 124 Statistical Analysis ................................ ................................ .......................... 126 Results and Discussion ................................ ................................ ......................... 126 6 SUMMARY AND RECOMMENDATIONS ................................ ............................. 139 Summary ................................ ................................ ................................ .............. 139 Recommendations ................................ ................................ ................................ 143 Calcium Ammo nium Nitrate ................................ ................................ ............ 143 Bismuth Subsalicylate ................................ ................................ .................... 144 LIST OF REFERENCES ................................ ................................ ............................. 146 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 163

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10 LIST OF TABLES Table page 2 1 Analyzed 1 chemical composition of the basal substrate used in in vitro incubations (Exp. 1, 2, and 3) ................................ ................................ ............. 60 2 2 Effect of NPN and inclusion rate of CAN 1 on in vitro fermentation parameters (Exp. 1) ................................ ................................ ................................ ............... 61 2 3 Effect of NPN and inclusion of CAN 1 on in vitro ruminal VFA profile (Exp. 1) ..... 62 2 4 Effect of BSS 1 on in vitro ruminal fermentation parameters (Exp. 2) .................. 63 2 5 Effect of CAN 1 on in vitro ruminal fermentation parameters (Exp. 2) .................. 64 2 6 Effect of BSS 1 and CAN 2 on in vitro ruminal CH 4 production (Exp. 2) ................ 65 2 7 Effect of BSS 1 on in vitro ruminal VFA profile (Exp. 2) ................................ ....... 66 2 8 Effect of CAN 1 on in vitro ruminal VFA profile (Exp. 2) ................................ ....... 67 2 9 Effect of increasing inclusion rate of BSS 1 on in vitro ruminal fermentation parameters (Exp. 3) ................................ ................................ ............................ 68 2 10 Effect of increasing inclusion rate of BSS 1 on in vitro ruminal VFA profile (Exp. 3) ................................ ................................ ................................ ............... 69 3 1 Analyzed 1 chemical composition of the basal diet fed to cattle while in the dry lot and while confined in the UF NFREC Beef Unit Pavilion ............................. 93 3 2 Effect of BSS 1 and eCAN 2 on ruminal gas cap H 2 S concentration and rectal temperature ................................ ................................ ................................ ........ 94 3 3 Effect of BSS 1 and eCAN 2 on DMI and ruminal fermentation parameters .......... 95 3 4 Effect of BSS 1 and eCAN 2 on ruminal VFA concentration and A:P 3 ................... 96 3 5 Effect of BSS 1 and eCAN 2 on ruminal VFA molar proportions ............................ 97 3 6 Effect of BSS 1 and eCAN 2 on intake, flow at the omasal canal, and microbial N flow and efficiency ................................ ................................ ........................... 98 4 1 Analyzed chemical composition of bahiagrass hay and sugar cane molasses 119 4 2 Effect of BSS 1 and eCAN 2 on nutrient intake and apparent total tract digestibility, and sulfur intake and retention ................................ ...................... 120

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11 4 3 Effect of BSS 1 and eCAN 2 on enteric CH 4 production of beef steers ................ 121 5 1 Analyzed 1 chemical composition of bahiagrass hay and sugar cane molasses 135 5 2 Effect of BSS 1 and eCAN 2 on growth performance of beef heifers ................... 136 5 3 Effect of BSS 1 and eCAN 2 on carcass ultrasound measurements ................... 137 5 4 Effect of BSS 1 and eCAN 2 on liver mineral concentration ................................ 138

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12 LIST OF FIGURES Figure page 3 1 Effect of bismuth subsalicylate (BSS) and eCAN (encapsulated calcium ammonium nitrate; 65.1% N DM basis) on ruminal pH of cannulated Angus crossbred cattle fed bahiagrass hay ad libitum with 2.7 g/kg BW of molasses ... 99 3 2 Effect of bismuth subsalicylate (BSS) and eCAN (encapsulated cal cium ammonium nitrate; 65.1% N DM basis) on ruminal NH 3 N concentrations of cannulated Angus crossbred cattle fed bahiagrass hay ad libitum with 2.7 g/kg BW of molasses ................................ ................................ ........................ 100 3 3 Effect of bismuth subsalicylate (BSS) and eCAN (encapsulated calcium ammonium nitrate; 65.1% N DM basis) on blood urea nitrogen (BUN) concentrations of cannulated Angus crossbred cattle fed bahiagrass hay ad libitum with 2.7 g/kg BW of molasses ................................ ............................... 101 3 4 Effect of bismuth subsalicylate (BSS) and eCAN (encapsulated calcium ammonium nitrat e; 65.1% N DM basis) on the ruminal acetate to propionate ratio (A:P) of cannulated Angus crossbred cattle fed bahiagrass hay ad libitum with 2.7 g/kg BW of molasses ................................ ............................... 102 3 5 Effect of bismuth subsalicylate (BSS) and eCAN (encapsulated calcium ammonium nitrate; 65.1% N DM basis) on ruminal acetate molar proportions of cannulate d Angus crossbred cattle fed bahiagrass hay ad libitum with 2.7 g/kg BW of molasses ................................ ................................ ........................ 103 3 6 Effect of bismuth subsalicylate (BSS) and eCAN (encapsulated calcium ammonium nitrate; 65.1% N DM basis) on ruminal propionate molar proportions of cannulated Angus crossbred cattle fed bahiagrass hay ad libitum with 2.7 g/kg BW of molasses ................................ ............................... 104

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13 LIST OF ABBREVIATIONS [H] Metabolic hydrogen 0BSS Basal substrate without bismuth subsalicylate in Chapter 2 Experiment 3 5BSS Basal substrate with 0.05% bismuth subsalicylate in Chapter 2 Experiment 3 10BSS Basal substrate with 0.10% bismuth subsalicylate in Chapter 2 Experiment 3 33BSS Basal substrate with 0.33% bismuth subsalicylate in Chapter 2 Experiment 3 3 NOP 3 Nitrooxypropanol A:P Acetate to propionate ratio APS Adenosine phosphosulfate BSS Bismuth subsalicylate BUN Blood urea nitrogen CAN Calcium ammonium nitrate CAN1.2 Basal substrate with 0.38% urea and 1.2% CAN in Chapter 2 Experiments 1 and 2 CAN2.4 Basal substrate with 2.4% CAN in Chapter 2 Experiments 1 and 2 CO 2 eq Carbon dioxide equivalent CTRL Basal substrate with 0.75% urea and without calcium ammonium nitrate in Chapter 2 Experiment 1 eCAN Encapsulated calcium ammonium nitrate EE Ether extract GHG Greenhouse gas GSH A ntioxidant glutathione iNDF Indigestible neutral detergent fiber

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14 IVOMD In vitro organic matter digestibility MCR methyl coenzyme reductase NCTRL Basal substrate/diet without a non protein nitrogen source NIT Basal diet with encapsulated calcium ammonium nitrate (350 mg/kg of body weight) NITB Basal diet with encapsulated calcium ammonium nitrate (350 mg/kg of body weight) and bismuth subsalicylate (58.4 mg/kg of body weight) PEM Polioencephalomalacia SRB Sulfate reducing bacteria U Basal diet with urea (182 mg/kg of body weight) UB Basal diet with urea (182 mg/kg of body weight) and bismuth subsalicylate (58.4 mg/kg of body weight) UC Ulcerative colitis

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15 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy E FFECTS OF BISMUTH SUBSALICYLATE AND CALCIUM AMMONIUM NITRATE ON BEEF CATTLE PRODUCTION By Darren Dwayne Henry December 2017 Chair: Nicolas DiLorenzo Major: Animal Sciences To aid in the objective of becoming ever more efficient, scientists have developed strategies, such as providing nitrate as a NPN source, to produce high quality beef and dairy products while mitigating the environmental impact of production. Another chall enge in animal protein production is elevated S concentrations in byproducts, forages, and water in certain areas of the U.S. B ismuth subsalicylate ( BSS ) has been observed to mitigate negative effects of S, such as H 2 S using ruminal in vitro systems The overarching objective of this dissertation was to test the hypothesis that BSS and calcium ammonium nitrate ( CAN ) would improve beef cattle performance and lessen the environmental impact. Four experiments were designed to evaluate BSS and CAN effects on i n vitro and in vivo ruminal fermentation, enteric CH 4 production and nutrient digestibility, and on performance and liver mineral concentration. In vitro ruminal fermentation was alter ed by both BSS and CAN. Production of H 2 S was reduced ( P = 0.024) quadra tically with the addition of up to 1.0% of the substrate DM. A linear reduction in total CH 4 ( P < 0.001), CH 4 in mmol/g of substrate incubated ( P < 0.001) and fermented ( P < 0.001) was observed when CAN was provided up to 2.4% of the substrate DM. When pro vided to cattle in vivo, BSS had minimal effects on ruminal

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16 fermentation; however, apparent S retention was reduced ( P = 0.003) as was liver concentration of Cu ( P = 0.002). In vivo, encapsulated CAN ( eCAN ) reduced ruminal total VFA concentration ( P = 0.01 1), NH 3 N concentration ( P = 0.001), apparent total tract DM and OM digestibility ( P < 0.021), and enteric CH 4 production (g/kg of BW 0.75 ; P = 0.051) compared with urea. Performance of cattle was not affected ( P > 0.05) by the addition of BSS or eCAN. As a potential NPN source, the data from this series of experiments indicate that eCAN may not promote the same ruminal fermentation and performance of cattle consuming urea. Bismuth subsalicylate appears to bind to S in the rum en ; however, the negative ef fects on Cu concentration in the liver may impede upon further use of BSS in cattle fed low quality forage based, diets.

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17 CHAPTER 1 REVIEW OF THE LITERATURE Methane Production in Cattle Methanogenesis Methanogenesis in the rumen is a necessary process to prevent ruminal dysfunctions such as acidosis. As ruminal microorganisms degrade substrates, various redox reactions are occurring simultaneously, which alter and balance the concentration of ruminal metabolic hydrogen ( [H] ). These reactions involve many co factors, which are continuously acting as a sink or donor of [H]. For example, when glucose is converted to pyruvate in glycolysis, glyceraldehyde 3 phosphate is oxidized to 1, 3 biphosphoglycerate, NAD + is reduced to NADH and H+ is released: G 3P + Pi + NAD + 1, 3 BPG + NADH + H + There are a multitude of reactions such as this in the ruminal environment that transfer [H], which in turn prevents the pool size of H 2 from increasing to a point where acidosis becomes an issue (Janssen, 2010) The ruminal pool of H 2 is warranting more attention (Janssen, 2010; Ungerfeld, 2015; Guyader et al., 2017) related to its effects on fermentation pathways in the rumen. T he differences in ruminal fermentation between ruminants provided a high grain vs high forage diet are well r ecognized For decades, it has been known that as readily digestible carbohydrates (i.e. starch ) replace forages in ruminant diets, molar proportions of propionate increase while acetate molar proportions decrease (Balch and Rowland, 1957) In the past it has been speculated that this shift in fermentation is simply due to shifts in populations of cellulolytic and amylolytic microorganisms. Recently, it has been postulated that this shift is more likely to be driven by ruminal concentration of H 2 (Janssen, 2010) A review by Janssen (2010), investigated the

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18 notion that thermodynamics are controlling the use of these pathways. For example, when H 2 conce ntration is elevated in the rumen, non H 2 producing pathways ( 0.66 acetate + 1.33 propionate) are more thermodynamically favorable. When ruminal H 2 concentration is lesser the opposite is true; therefore, H 2 producing fermentation pathways (butyrate + 2 H 2 or 0.66 acetate + 0.66 butyrate + 2.66 H 2 ) are more thermodynamically favorable (Janssen, 2010) The concept that shifts in fermentation are largely due to H 2 concentration is supported by diur nal shifts in molar proportions of VFA in the rumen. Concentration of H 2 has been shown to peak shortly after feeding for ruminants consuming forage (Barry et al., 1977) and concentrate based diets (Robinson et al., 1981) Furthermore, when Robinson et al. (1981) analyzed H 2 concentrations in the rumen of a cow consuming a concentrate based diet, the authors observed that dissolved H 2 concentration was 15 2 concentration reduced to a steady (Robinson et al., 1981) With this shift in dissolved H 2 concentration, thermodynamics of fermentation are also going to shift, i.e. shift from H 2 producing pathways to non H 2 producing pathways (Janssen, 2010) Ciriaco et al. (2016) provided ruminally cannulated steers with bermudagrass hay and supplem ental 50:50 molasses:glycerol mixture. When evaluating the VFA profile, a reduction in acetate molar proportion after feeding the liquid feed (9% reduction 3 h post feeding) was observed, returning to pre feeding proportions 9 h post feeding (Ciriaco et al., 2016) It is unlikely that shifts in microbial populations occur this rapidly (within hours) to alter fermentation pathways. What is more probable is that when the suppl ement was

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19 provided, H 2 concentration in the rumen may have peaked caus ing acetate forming pathways to become thermodynamically unfavorable (Janssen, 2010) When investigating the impact of H 2 con centration on methanogenesis, it is pertinent to consider not only the pool of H 2 but also the flux within the pool (Janssen, 2010) The rate at which CH 4 is produced is directly related to the r ate at which H 2 passes through the pool of dissolved H 2 and the amount of CH 4 produced is determined by the amount of H 2 that passes through the pool (Janssen, 2010) Again, when H 2 concentration is elevated (as with concentrate diets), production of H 2 (i.e. the flux of H 2 through the pool of dissolved H 2 ) is reduced which in turn reduces the rate and amount of CH 4 produced per unit of substrate degraded by ruminal microorganisms (Janssen, 2010) Issues R elated to E nteric M ethane P roduction supply through meat and milk. It has been reported that of the total solar energy captured by biomass on earth, only 5% is potentially available for human consumption (Russell and Gahr, 2000) The majority of the remaining 95% can be consumed by ruminants, then fermented and enzymatically broken down by ruminal microorganisms into products s uch as VFA, which are then used as energy to produce meat and milk (Russell and Gahr, 2000) One disadvantage of the fermentation of substrates to VFA is the production of reducing equivalents, which, in turn, need to be disposed of. Disposal of reducing equivalents is perfo rmed by transferring electrons and H 2 to various acceptors in the rumen. Methane, which is formed by the reduction of CO 2 or formate, is one of the most common and important hydrogen sinks in the rumen (Russell and Gahr,

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20 2000) Methanogens, a subgroup of Archaea use hydroge n as an energy source and primarily, CO 2 as a carbon source to form CH 4 The hydrogen consuming process of methanogens reduces the concentration of hydrogen in the rumen to allow for rapid fermentation of substrates (Buddle et al., 2011) Regardless of the importance of CH 4 as a hydrog en sink in the rumen, the production of CH 4 can be detrimental to the environment by adding to the already elevated concentration of greenhouse gases ( GHG ) in the atmosphere In general, GHG are measured in equivalents of CO 2 ( CO 2 eq ) and CH 4 is considered to be 20 25 times more potent as a GHG than CO 2 (FAO, 2013) Enteric CH 4 production in ruminants varies among diets of differing levels of concentrate, digestibility, and other aspects. In general high forage diets produce 3 to 4 fold more CH 4 than high concentrate diets (Johnson and Johnson, 1995) Nearly 70% of the energy requirements for cattle are used prior to weaning, when the primary dietary component is forage (Shike, 2013) ; therefore, reducing enteric CH 4 production is a vital step in providing enough animal protein for the growing population while preserving our environment. L ife cycle assessments investigating various cattle production systems have reported that the largest influence on the carbon footprint of beef and dairy production is represented by on farm emissions, when cattle are generally grazing (Beauchemin et al., 2010; Kristensen et al., 2011) When evaluating beef production specifica lly, approximately 80% of GHG emissions are released from the cow calf phase of production (Beauchemin et al., 2011) It has been estimated that a n i ncrease greater than 6% in global enteric CH 4 production will occur between the year 2015 to

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21 2020 (2204 to 2344 MtCO 2 eq, respectively ; EPA ( 2006 ). This is a 32% increase from the year 1990 (EPA, 2006) Methane not only has an effect on the environment, but also on energy losses in cattle. It has been reported that CH 4 accounts for up to 12% of GE losses in cattle (Johnson and Johnson, 1995) The percentage of energy lost is highly related to the type of diet provided. Losses from forage based diets typically are 2 fold greater than energy losses to CH 4 from cattle cons uming high concentrate diets (Harper et al., 1999; Doreau et al., 2011) Mitigation of E nteric M ethane P roduction Many researchers have investigated the mitigation of CH 4 and potential strategies are as follows: 1) increase feed utilization efficiency b y improving the digestibility of forages and feedstuffs; 2) develop feed additives to act as H 2 sinks in the rumen; 3) reduce or inhibit methanogen populations in the rumen; 4) provide substrate to microorganisms other than methanogens to aid in the format ion of more efficient products, such as propionate; and 5) improve the efficiency of production (meat, milk, etc.) to reduce the number of livestock required (USDA, 2004) Tannins Tannins are a group of diverse poly meric flavonoids with either C C or C O C bonds and are principally found in the cell walls or vacuoles of dicotyledonous plants (McMahon et al., 2000) Tannins can improve performance of ruminants consuming diets with CP concentrations above nutritional requirements by binding to excess protein allowing for post ruminal digestion. In contrary, if CP is limiting, detrimental effects to the animal, related to reduced amino acid absorption, may be obs erved (Waghorn, 2008)

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22 Throughout the literature, there are several different sources of tannins used to alter fermentation of ruminants. These tannins vary widely in their effectiveness of CH 4 mitigation and impact on animal performance Within tannin groups, there is also large variability. For example, quebracho tannin has been fed to beef, dairy, and boer goats, as well as used in in vitro experiments with differing results (Pellikaan et al., 2011; Dickhoefer et al., 2016; Ebert et al., 2017) Pellikaan et al. (2011) compared se veral different tannins on in vitro fermentation of lucerne hay ( Medicago sativa ). The authors observed that CH 4 as a percent of total gas production and rate of CH 4 production was reduced with the addition of quebracho tannin (Pellikaan et al., 2011) Conversely, Ebert et al. (2017) provided quebracho tannin to growing beef steers (350 32 kg BW) consuming a 92% concentrate finishing diet and observed no change in enteric CH 4 production. The differences observed between the results of Ebert et al. (20 17) and Pellikaan et al. (2011) could be related to differences in in vitro and in vivo experiments or the diet provided. However, when quebracho tannin was provided to growing beef heifers (238 13 kg BW) consuming a 70% barley silage diet no change in enteric CH 4 emissions compared with control heifers was observed (Beauchemin et al., 2007) Furthermore, Beauchemin et al. (2007) reported a 15% reduction in apparent total tract digestibility of CP when quebracho tannin was provided at 2% diet DM. Other sources of tannin s have presented different results from quebracho. Puchala et al. (2005) provided Angora does with either a crabgrass ( Digitaria ischaemum )/tall fescue ( Festuca arundinacea ) mixture or sericea lespedeza ( Lespedeza cuneata ). The sericea lespedeza contained 18% condensed tannins wherea s the crabgrass/tall fescue mixture contained 0.5%. D ifferences between the

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23 forages w ith in vitro DM digestibility being 64.5 and 75.3% for sericea lespedeza and crabgrass/tall fescue, respectively were observed However, the researchers reported a 50% r eduction in CH 4 as grams per kilogram of digestible DMI along with a 40% increase in DMI for the does consuming s ericea lespedeza when compared with the crabgrass/tall fescue fed does (Puchala et al., 2005) Using 60 Holstein Friesian cows (40 cows for CH 4 measurements), Grainger et al. (2009) evaluated the effects of Acacia mearnsii tannin on enteric production of CH 4 and nitrogen excretion. Cows were grazing a mixture of diploid and tetraploid ryegrass hybrids ( Lolium boucheanum syn. L. hybridum ) and were supplemented with 4.5 kg/d of cracked triticale ( Triticosecale ) grain. Cows were dosed with either 163 or 326 g/d of tannins. Grams of CH 4 per d were significantly reduced when cows received tannins extracted from the bark of A. mearnsii ; however, when emission intensity was considered using grams of CH 4 per kg of fat + protein yield in milk, the researchers observed no differences between t reatments. This was due to the 12% reduction in milk fat + protein yield when tannin was dosed up to 326 g/d. Until researchers can find suitable tannins to reduce CH 4 production, without negatively affecting production of the animal, other routes of mitig ation should be sought after. Lipids Lipid supplementation has negative impacts on the activity of methanogens and ruminal protozoa (Johnson and Johnson, 1995) Saturated fatty acids, from sources such as coconut oil (lauric acid; C12:0), have toxic effec ts on ruminal protozoa and methanogens (Zhou et al., 2013) ; however, it appears that unsaturated fatty acids can be detrimental to methanogenic archaea as well (Prins et al., 1972) Researchers have also described a process in which electrons are accepted during biohydrogenation

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24 within the rumen, lessening the availability of H 2 for methanogens to use as an energy source (Boadi et al., 2004) Moreover, l ipid supplementation can decrease CH 4 production by limiting fermentation of OM due to a physical coating of the feed particles, inhibiting bacteria to attach and degrade the material (Johnson and Johnson, 1995) Never theless, a n issue associated with lipid supplementation is that fats should not be provided at concentrations greater than 5 to 6% of the diet DM due to negative effects on digestibility of fiber (Boadi et al., 2004) and milk fat production (Jenkins et al., 1996; Ashes et al., 1997) To evaluate differing lipid sources, a research group designed an experiment using 45 Nellore steers (419 11 kg BW; 20 steers for CH 4 emissions) consuming a 60:40 forage:concen trate ration for 118 d (Fiorentini et al., 2014) The steers were randomly assigned to 1 of 5 treatments: control (2.8% ether extract; EE ), control + palm oil (7.1% EE), control + linseed oil (7.0% EE), control + protected fat (7.0% EE), and control + whole soybean (7.0% EE). Lipid supplementation impacted DMI of the Nel l ore steers with control having greater DMI compared with palm oil (46% reduction), linseed oil (20% reduction), and whole so ybean (27% reduction) addition (Fiorentini et al., 2014) Control and protected fat fed steers had similar DMI. Protected fat did not have any effect on enteric CH 4 production as would be expected since the purpose of protecting fats is to allow passage out of the rumen without being affected by the ruminal microorganisms. All other sources of lipid provided resulted in reductions in total CH 4 production (grams per d; 56% reduction) and CH 4 produc ed as a percentage of GE intake (33% reduction). Supplementation of lipids (not including protected fat) did

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25 reduce performance of the steers with ADG being reduced by 41% (Fiorentini et al., 2014) A meta analysis was conducted to evaluate CH 4 mitigation by lipid supplementation of lactating dairy cows using data from 7 publications, resulting in 25 diets (Eugne et al., 2 008) The authors reported a 9% overall reduction in enteric CH 4 production (MJ/d) when the diets contained an average of 6.4% ether extract. This reduction in CH 4 production was attributed to a reduction (6.5%) in DMI of the cows across the 25 diets. W hen performance was evaluated, the authors observed that 4% fat corrected milk was unchanged by the addition of lipids to the diets; therefore, enteric CH 4 emission intensity, in terms of MJ per kilogram of 4% fat corrected milk, was reduced by 9% (Eugne et al., 2008) Zhou et al. (2013) used pure cultures of Methanobrevibacter rum inantium to elucidate the mode of action of saturated fatty acids on inhibition of methanogenesis. Methanobrevibacter ruminantium L of lauric (C14:0) myristic (C14:0), palmitic (C16:0), or stearic (C18:0) acid. The authors report ed greater impacts on CH 4 production with the shorter chain fatty acids. Furthermore, it was observed that cell viability (ratio of live vs dead cells) was reduced and extracellular K + concentration increased with lauric and myristic acid. The resulting increase in extracellular K + concentration indicates cell membrane permeation which can lead to cell death, thus reducing methanogenic capability of M. ruminantium (Zhou et al., 2013) Ionophores Ionophores are lipophilic substances that alter the flow of ions across the membranes of certain bacteria eventually de energizing the cell, causing necrosis (DiLorenzo and Galyean, 2010) Although several ionophores, such as lasalocid and

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26 laidlomycin (Bartley et al., 1979; Domescik and Martin, 1999; Appuhamy et al., 2013) have been investigated in ruminants with reductions in enteric CH 4 production, monensin has been investig ated more thoroughly than other ionophores and is used on a daily basis in beef and dairy production in the U.S. (FAO, 2013) ; therefore, the majority of data discussed in th is sub section will focus on monensin. Monensin has been used for decades in the beef and dairy industry (Goodrich et al., 1984; Duffield et al., 2008) It has been reported that monensin can reduce the acetate to propionate ratio ( A:P ; Bogaert et al., 1990; Guan et al., 2006; Ellis et al., 2012) and reduce protozoa populations in the rumen (Hino and Russell, 1987; Guan e t al., 2006) With these changes in ruminal fermentation, the potential for mitigating methanogenesis has been investigated. A group of researchers performed a meta analysis evaluating the anti methanogenic effects of monensin on beef and dairy production systems (Appuhamy et al., 2013) This meta analysis encompassed 13 manuscripts (6 dairy and 7 beef related) wit h 22 experiments (11 dairy and 11 beef related). The average monensin dose was 21 and 32 mg/kg of DMI, and was provided for an average of 72 and 38 d for dairy cows and beef steers, respectively. Total CH 4 production (grams per d) was reduced 15% and CH 4 as a percentage of GE lost was reduced by 9% for beef steers provided monensin (Appuhamy et al., 2013) The reduction in total CH 4 produced could be partly attributed to a reduction in DMI ( 0.41 kg reduction) of the steers ; however, when evaluated as a percentage of GE lost, DMI did not account for any reduction in CH 4 production. Enteric CH 4 production was not affected by monensin when provided to dairy cows at an average rate of 21 mg/kg of DMI; however, DMI was reduced without a

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27 negative effect on milk yield leading to the probability that emission intensity was reduced by the addition of monensin (Appuhamy et al., 2013) It has been proposed that although intake, shifts in VFA profile, and feed efficiency of beef cattle consuming monensin a re persistent over time, enteric CH 4 mitigation may not persist for the entire time that monensin is provided (Guan et al., 2006) To investigate this, rese archers provided 36 Angus steers (328 25 kg BW) either a 75:25 or 30:70 forage:concentrate ration for 16 wk. Within the 2 groups of steers, 3 treatments were randomly assigned: control (no ionophore), control + monensin (33 mg/kg diet DM), and control + monensin/lasalocid (lasalocid was provided at 36 mg/kg diet DM; steers were alternated every 2 weeks from monensin to lasalocid). The authors reported no effects of treatment on DMI for the 75:25 ration; however, DMI was reduced for steers consuming ionoph ores compared with steers in the control group consuming the 30:70 ration. This reduction in DMI was accompanied by no change in performance which led to an increase in feed efficiency. Using the SF 6 tracer technique, the researchers assessed enteric CH 4 production at w k 1, 3, 4, 5, 6, 8, 10, 12, and 16. When evaluating CH 4 as a percentage of GE intake, the authors observed marked reductions for ionophore treated cattle at wk 3 and 4 for the high concentrate diet; however, the high forage diet maintained t he reduction in CH 4 production from wk 3 to 6. By wk 8, all treatments returned to baseline values. To further investigate this short term effect of ionophores on enteric CH 4 production, the authors measured protozoa counts and observed that protozoa in th e rumen were reduced during the same wk that CH 4 was reduced. Th ese data imply that the reduction in CH 4 not accounted for by DMI, is likely due to inhibition of protozoa growth in the rumen (Guan

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28 et al., 2006) Furthermore, th ese data indicate that persistency of CH 4 mitigation may not last longer than 6 wk which could explain variability in CH 4 emissions found in the literature. 3 Nitrooxypropanol A promising molecule that has gained the regard of ruminant nutritionists is known as 3 nitrooxypropanol (C 3 H 7 NO 4 ; 3 NOP ). This small molecule impedes methanogen growth in the rumen by inhibiting the reduction of CO 2 to CH 4 (Duin et al., 2016) In the process of reducing CO 2 to CH 4 methyl coenzyme reductase ( MCR ) is a necessary catalyst. A tetrapyrrole derivative named cofactor F 430 is a portion of the nickel enzyme MCR and it is where nickel i s bound. For F 430 to be active, the nickel has to be in the Ni(I) form. Acting as an oxidant, 3 NOP, which has a similar shape as MCR, binds and inactivates MCR, halting the formation of CH 4 (Duin et al., 2016) Several research groups have reported significant reductions in enteric CH 4 production of large and small ruminants treated with 3 NOP (Haisan et al., 2014; Martnez Fernndez et al., 2014; Reynolds et al., 2014) Vyas et al. (2016) used 84 crossbred steers (319 20 kg BW) in a 238 d feeding trial. For t he first 105 d (backgrounding phase) steers were provided a barley silage based backgrounding diet followed by 28 d of adaptation to a dry rolled barley grain based diet which was provided for 105 d (finishing phase). Steers were randomly assigned to 1 of 3 treatments: control ( no addition of 3 NOP ) ; l ow 3 NOP ( 100 mg/kg of diet DM ) ; or, high 3 NOP ( 200 mg/kg of diet DM ) To determine enteric CH 4 emissions, 15 steers (5 per treatment) were evaluated using calorimetry chambers. During the backgrounding phase, there was a reduction in DMI for steers consu ming high 3 NOP when compared with control and low 3 NOP steers ; however, grams of CH 4 per

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29 kilogram of DMI was also reduced for high 3 NOP (Vyas et al., 2016) The authors attribut e d the hypophagic effects of 3 NOP to a possible increase in ruminal propionate molar proportions. Researchers have observed significant alterations in the ruminal VFA profile of ruminants treated with 3 NOP (Haisan et al., 2014; Martnez Fernndez et al., 2014; Romero Perez et a l., 2014) It is evident that 3 NOP generally increases propionate molar proportions leading to a reduction of A:P The shift in molar proportions of VFA could be due to an increase in ruminal H 2 concentration, which would transfer [H] from H 2 forming ( 2 acetate + 4 H 2 ) to non H 2 forming pathways (0.66 acetate + 1.33 propionate; Janssen, 2010 ). Throughout the finishing phase, DMI was reported to be reduced f or steers consuming high 3 NOP which lead to a tendency for a reduction of ADG for high 3 NOP as well (Vyas et al., 2016) Methane emission intensity was reduced by 81% for high3 N OP compared with control steers (Vyas et al., 2016) Other researchers focusing on 3 NOP ha ve also reported reduction of enteric CH 4 production in ruminants with improvements in pe rformance indices. Haisan et al. (2014) observed a 60% reduction in grams of CH 4 per kilogram of DMI when 2,500 mg/d (128 mg/kg diet DM assuming 19.5 kg DMI) of 3 NOP was provided to Holstein cows (580 76 KG BW) consuming barley silage and ground corn based diet. This reduction in CH 4 was accompanied by an increase in ADG of the cows without altering DMI or milk production (Haisan et al., 2014) Similarly, researchers using 48 Holstein cows (653 12 kg BW) consuming a 50:50 forage:concentrate diet observed a 29% decrease in CH 4 (g/d) with a 36% increase in ADG for cows consuming 80 mg/kg diet DM compared with control cows.

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30 Diet Another effective, but not always viable, strategy for mitigating CH 4 is by altering the composition of the diet. The composition of the diet specific ally the types of carbohydrates used, has a great impact on CH 4 production due to its effects on ruminal pH, concentration of H 2 and subsequent consequences on the ruminal milieu (Johnson and Johnson, 1995) Cellulose and hemicellulose digestibility have a greater correlation with CH 4 production when compared to soluble carbohydrates (Hook et al., 2010) Furthermore, by increasing starch concentration, decreasing cell wall components, and processing the ingredients in the diet ruminants will encounter increased passage rate which is associated with an increase in H 2 concentration, reduction of A:P, and possible reduction of the ruminal pH (Owens et al., 1998; Janssen, 2010) With these alterations in ruminal fermentation, CH 4 production is likely to decrease (Janssen, 2010) ; however, the risk of sub acute ruminal acidosis increases (Owens et al., 1998) Shifting fermentation pathways to propionate production likely is caused by greater concentrations of H 2 which in turn m akes H 2 producing pathways thermodynamically less favorable A decrease in CH 4 production will occur since the flux of H 2 through the pool will be reduced, whereas the opposite is true for substrates (high cellulose containing diets) that produce less H 2 d uring degradation and, therefore, shift to more thermodynamically favorable pathways, acetate and butyrate (Van Nevel and Demeyer, 1996; Janssen, 2010) U sing 24 growing B os taurus crossbred heifers (318 35 kg BW) in two periods (n = 48 ), a n experiment was designed to investigate the effect s of chitosan ( Henry et al., 2015) a natural bio polymer with anti microbial activity (Chung et al., 2004; Qi et al.,

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31 2004; Daetz et al., 2016) The heifers were randomly assigned to 1 of 6 treatments arranged as a 2 3 factorial with diet (64:36 and 15:85 roughage:concentrate) and chitosan inclusion rate (0.0, 0.5, and 1.0% diet DM) being the factors. Chitosan had no impact on enteric CH 4 production; however, when the main effect of diet was considered, heifers consuming the high roughage diet pr oduced nearly 5 fold more CH 4 as grams per kilogram of DM digested, when compared with heifers consuming the low roughage diet. A s a result of the dependence of grazing forages with little supplementation i t is difficult to alter ruminal fermentation by modifying the diet of cattle in the cow calf sector of the U.S.; hence, there is a need for a mitigation strategy that can be applied to the 30.3 million head of beef cows grazing forages in the U.S. Potential for N itrates in the C attle I ndustry For seve ral years, nitrate has been considered as a tool to inhibit enteric CH 4 production of ruminants (Sar et al., 2004; Leng, 2008) ; however, there has been political and social concerns related to to xicity associated with nitrite accumulation in the rumen. Nitrite can be absorbed through the rumen epithelium into the blood stream where it converts hemoglobin to methemoglobin. When methemoglobin concentration is greater than 30% of the total hemoglobin issues with transporting O 2 to tissues can be observed (Benu et al., 2016) Researchers have determined that microbial populations that reduce nitrate to nitrite have a rapid growth rate, whereas nitrite reducers have a slow growth rate leading to an accumulation of nitrite in the rumen if the microbiome is not properly adapted to nitrate inclusion (Leng, 2008) To hinder this concern, adaptation to nitrate can be achieved by providing increasing amounts over an adaptation period (Newbol d

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32 et al., 2014) Through adaptation, researchers have reported supplementing nitrate to cattle at up to 3.0% diet DM (Newbold et al., 2014) Mitigation of enteric methane production with nitrate Nitrates have been provided to sheep and cattle mitigating enteric CH 4 production between 15 and 30% (van Zijderveld et al., 2010; van Zijderveld et al., 2011; Hulshof et al., 2012) Most of the research regarding nitrate as an inhibitor of CH 4 has utilized high concentrate diets (feedlot animals) or diets with at least 50% concentrate (backgrounding and da iry animals). Very little research has focused on the effects of nitrate on enteric CH 4 production and performance of cattle consuming forage based diets (Hulshof et al., 2012; Benu et al., 2016) Methane production decrease has been proposed to be due to the reduction of nitrates to nitrites, and finally, to NH 3 (FAO, 2013) which is therm odynamically more favorable than the reduction of CO 2 to CH 4 (Ungerfeld and Kohn, 2006) Certain bacteria within the rumen are very efficient at reducing nitrates. Denitribacterium detoxificans and Wolinella succinogenes utilize nitrates within the rumen, and Wolinella succinogenes has been shown to rapidly reduce nitrate to NH 3 with little accumulation of nitrite (Morgavi et al., 2010) I t has been reported that increasing the number of nitrate reducing bacteria in the rumen decreases CH 4 production when nitrate is available for reduction (Morgavi et al., 2010) Nitrate can be reduced by 3 pathways: denitrificati on, which leads to N 2 O, assimilatory, and dissimilatory nitrate reduction to NH 3 Although N 2 O has been detected with the addition of nitrates (Latham et al., 2016) researchers have observed that only 0.02% of nitra te is converted to N 2 O in vitro (Guyader et al., 2017) Furthermore, assimilatory reductio n of nitrate to NH 3 is an energy consuming pathway that is suppressed by NH 3 whereas the dissimilatory

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33 pathway produces energy; therefore, it is more favorable for ruminal microorganisms to reduce nitrate using the dissimilatory pathway (Leng, 2008; Latham et al., 2016) Nitrate may act as a H 2 sink in the rumen, but other modes of action for its anti methanogenic activity have been investigated. The addition of nitrate has been reported to reduce populations of methanogens by up to 97% in vitro (Zhou et al., 2012) This reduction in CH 4 producing ar chaea has been shown to be due to the effects of nitrite on methanogens. Recently, while elucidating the mode of action of 3 NOP, Duin et al. (2016) reported that nitrite completely inhibits MCR. It would be assumed that nitrate, acting as a [H] sink, would reduce the concentration of H 2 in the rumen; however, there is contradicting research that reports H 2 to increase with the addition of nitrate (Guyader et al., 2015) To speculate, thi s may be related to the inhibitory action of nitrite on the reduction of CO 2 to CH 4 by ruminal methanogens, which perhaps, could increase ruminal H 2 concentration to a great enough extent that nitrate could not capture all of the [H]. Nitrate as a non prot ein nitrogen source For nitrate to not only have beneficial effects towards the carbon footprint of the cattle industry, it must also have characteristics th at will provide cattle producers with incentive to provide nitrate to their cattle. One possibility is to utilize nitrate as a NPN source to cattle consuming low nitrogen content forages or diets. To evaluate the effect of nitrate on 300 growing N el l ore bulls (392 28kg BW), Newbold et al. (2014) included incremental amounts of nitrate (0.0 to 2.4% diet DM) into a 16:84 forag e:concentrate diet. The bulls were randomly assigned to 1 of 6 treatments (0.0, 0.48, 0.96, 1.44, 1.92, or 2.40% nitrate in the diet DM) made isonitrogenous with urea, and fed for 111 d. There was no effect of nitrate on final BW, ADG, or HCW;

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34 however, DMI was linearly reduced as the inclusion of nitrate increased to 2.4% diet DM. The researchers also observed a 9% increase in G:F when comparing the 0.0% with 2.4% nitrate diets. An experiment was designed with the objective of determining the effects of nitrate, in place of urea, provided to 432 (435 kg BW) composite breed steers fed a 12:88 forage:concentrate ration for 108 d (Hegarty et al., 2016) Steers were assigned to 1 of 4 treatments that were arranged in a 2 2 factorial with NPN source (urea or nitrate) and inclusion rate of NPN (0.25 or 0.45%) being the factors Steers that were provided nitrate, regardless of inclusion rate, had lesser ADG compared to cattle supplemented urea as a NPN source. This reduction in ADG led to a reduction in HCW for nitrate fed steers compared with u rea. Furthermore, the ADG was likely affected by the 600 g reduction in DMI of steers provided nitrate compared with urea. The authors reported no effects of treatment on carcass traits or nitro compounds in the meat (Hegarty et al., 2016) It is evident that further research is needed to determine the efficacy of supplementing nitrate as a NPN in place of urea. A dditionally, very little research on the use of nitrates in a high forage or grazing system has been performed. Many areas in the U.S., such as the southeast, have forages that lack sufficient nitrogen content for not only the animal, but also production o f ruminal microorganisms. It would be valuable to U.S. producers to be able to provide a NPN to cattle grazing low protein forages while reducing the impact of their operations on the environment.

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35 Sulfur in Beef Cattle Production Issues R elated to Hi gh S ul fur D iets In the rumen, H 2 S is predominantly formed by dissimilatory sulfate reducing bacteria ( SRB ) (Drewnoski et al., 2014) Briefly, sulfate is transported into the cell via sulfate permease, where ATP sulfurylase activates the sulfate. Activated sulfate can then re activate adenosine phosphosulfate ( APS ). Reduction of APS to sulfit e occurs through catalyzation via APS reductase (Bradley et al., 2011) For the reduction from sulfite to H 2 S, there are multiple proposed models (Kobayashi et al., 1969; Rees, 1973) The Rees (1973) model proposes that sulfite is directly reduced to H 2 S via dissimilatory sulfite reductase, whereas Kobayashi et al. (1969) offered a model where intermediary products, such as trithionate, were produced and further reduced to H 2 S. It is likely tha t H 2 S toxicity occurs due to the inhalation of eructated ruminal H 2 S (Gould, 1998) Therefore, mitigation of ruminal production of H 2 S is key to reducing toxicity. Concentration of H 2 S in the gas c ap of the rumen is predominantly determined by sulfide production in the ruminal fluid (pKa of H 2 S = 7.04), pH of the rumen, frequency of eructation, and absorption of sulfide through the ruminal mucosa (Gould, 1998) It has been agreed upon that H 2 S is the culprit in the neurological disorder polioencephalomalacia ( PEM ; Gould, 1998; Cammack et al., 2010; Drewnoski et al., 2014) microscopic features not recognized for a particular etiology (Gould, 1998) Incidence of PEM when high S diets are consumed is around 1% (Nichols et al., 2012) Concentrations of H 2 S greater than 2000 mg/L have been implicated in PEM; however,

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36 studies have reported concentrations 2 to 3 fold greater with no symptoms of PEM (Drewnoski et al., 2012; Drewnoski et al., 2014) Cytotoxicity due to elevated concentrations of H 2 S may be explained in various ways (Drewnoski et al., 2014) Hydrogen sulfide can easily and hastily cross lipid bilayers, where it can inhibit cytochrome c oxidase, the last enzyme in the electron trans port chain (Truong et al., 2006) It has been reported that H 2 S also depletes th e antioxidant glutathione ( GSH ). Using hepatocytes, researchers reported that a source of H 2 S reduced the amount of GSH in the cell, eventually leading to a rapid necrosis, likely related to the involvement of reactive S and O species (Truong et al., 2006) Intracellular Fe stores are released from ferritin proteins due to increased H 2 S concentrations, respiration is inhibited, super oxide ions, and subsequently H 2 O 2 are accumulated, ATP is depleted, anaerobic metabolism increases and cellular pH decreases, eventually causing cell death (Truong et al., 2006) Adequate mineral nutrition is a component of successful cattle performance, not only due to production in dices, but also the biological roles in which minerals have a significant responsibility (NASEM, 20 16) Elevated concentrations of S can interfere with trace mineral absorption. The NASEM ( 2016) recommends concentrations of S in the diet to be at least 0.15 and no more that 0.4% of the diet DM It is likely that the concentration of S that interrupts trace mineral absorption is lower than the concentration in which issues with cattle performance become evident (Drewnoski et al., 2014) When sul fur binds to molybdate in the rumen, it can form complexes called thiomolybdates, which along with sulfide, binds Cu inhibiting its absorption and subsequent use by the animal (Suttle, 1991) In the U.S., many beef cow herds are

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37 provided with some type of forage during the winter months, such as hay or planted winter forage, and very often, a high S supplement, such as corn gluten feed It is le ss likely that these females will develop PEM on a high forage diet; however, inhibition of trace mineral absorption should be considered to ensure proper growth and health (Drewnoski et al., 2014) The effect of S concentr ation in the diet on DMI of growing cattle is more likely to be affected by S concentrations in a high concentrate rather than a high forage diet (Spears et al., 2011; Drewnoski et al., 2012) This tolerance to elevated S in forage based diets may be related to increased pH of the rumen and/or the ruminal microorganisms present in a forage fed animal (Drewnoski et al., 2014) Another consideration is the type of S provided to the animal. It has been reported that ca ttle consuming rations with more than 0.2% inorganic S have decreased DMI, ADG, and HCW (Drewnoski et al., 2014) A major provider of S to feedlot diets is When were included in feedlot rations, up to 30% of the diet DM, there was an increase in ADG; however, when surpassing 30% ADG began to decline (Klopfenstein et al., 2008) It has been suggested that due to possible negative effects on DMI and ADG, to achieve a desired H CW when providing a high S diet, cattle may need to stay on feed for longer than anticipated. Additionally, when S content was 0.46% of t he diet DM, LM area was decreased compared to cattle consuming diets with less than 0.2% S. Few investigators have cons idered the effects of H 2 S on performance indices. A negative correlation was reported for H 2 S concentrations and ADG and DMI (Uwituze et al., 2011)

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38 Mitigation of S ulfur T oxicity The availability of S in the rumen may have a significa nt impact on S toxicity. A concept known as adjusted ruminal protein S proposes that S from differing sources differs in their availability within the rumen (Sarturi et al., 2013) This concept comes from the idea that S from RUP is unavailable to SRB of the rumen. For example, of the S in wet only 71% is expected to be ru minally available, whereas 100% of the S from inorganic sources, such as ammonium sulfate, are available to SRB (Sarturi et al., 2013) Concentration of fiber in the diet can have positive effects on the risk of S toxicity. A meta analysis of feedlot data described a 19% decrease in the risk of PEM for every 1% increase of NDF ( Nichols et al., 2012 ). In accordance, investigators have observed that increasing NDF significantly decreases H 2 S production (Morine et al., 2014a) without having effects on the performance of growing cattle (Morine et al., 2014b) Ruminal pH is generally elevated in cattle consuming high forage diets compared to those consuming high concentrate diets (Counotte et al., 1979) The increase in pH may inh ibit H 2 S production to some degree due to the pKa (7.04) of H 2 S (G ould, 1998; Drewnoski et al., 2014) Some research has indicated a negative correlation between ruminal pH and H 2 S when pH of the rumen is less than 5.8; however, at a ruminal pH greater than 5.8, ruminal pH is a poor predictor of H 2 S production (Morine et al., 2014b) Mitigation of H ydrogen S ulfide P roduction with B ismuth S ubsalicylate In humans, H 2 S has been implicated as a bacterial product that could damage the gut mucosa (Mitsui et al., 2003) Production of excess H 2 S in the large intestine may be pathogenic causing ulcerative colitis ( UC ), a form of inflammatory bowel disease that is known for ulcers and open sores in the colon (Levine et al., 1998) In the past two

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39 decades, researchers have investigated the use of bismuth to aid UC. Although the mechanisms of bioactivity have not been fully elucidated for bismuth, it has been suggested, due to its thiophilic behavior, that biological molecules containing S (i.e. thiol or thiolate ) are likely candidates for bismuth binding (Phillips et al., 2007) When bismuth interacts with sulfide, it reacts to form an insoluble black salt called bismuth sulfide. This is believed to be the cause of black feces observed in patients treated with bismuth subsalicylat e ( BSS ; Suarez et al. 1998) Additionally, bismuth, in the form of BSS, has been shown to have antimicrobial effects against Helicobacter pylori a bacterium known for causing ulcers in the gastro intestinal tract of humans, and E. coli (Manhart, 1990; Suarez et al., 1998) Greater numbers of SRB can be found along with increased H 2 S production in fecal samples of humans with UC as compared to healthy patients (Levine et al., 1998; Pitcher et al., 2000) Through the use of popular over the counter medicines, such as Pepto Bis mol (262 mg of BSS per caplet), to aid in the gastrointestinal issues, the importance of bismuth in medicine is underscored (Phillips et al., 2007) In the laboratory, when BSS was fixed in a gas space with H 2 S, BSS quickly removed H 2 S by binding to the S (Suarez et al., 1998) To determine whether subsalicylate had any effect on H 2 S, res earchers designed an experiment using bismuth acetate and sodium salicylate. It was reported that bismuth acetate rapidly bound to H 2 S, whereas sodium salicylate had no effect (Suarez et al., 1998) In f ecal slurries, H 2 S was reduced up to 90% when bismuth was included (Mitsui et al., 2003) More over, results from an in vivo study indicated that humans treated with BSS had virtually no H 2 S production in feces

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40 for up to seven days of treatment. This reduction in H 2 S no longer existed after 5 days without BSS (Suarez et al., 1998) Similar to human trials, H 2 S production from feces was significantly reduced when rodents were treated with BSS (Suarez et al., 1998; Levitt et al., 2002) In ad dition, when cecum concentrations of H 2 S were considered, rats consuming rat chow with BSS had reduced production compared to non treated rats (Suarez et al., 1998) Researchers have recognized BSS as a tool to quantify H 2 S production, through the quantification of bismuth sulfide, due to the innate ability of BSS to bind to sulfur in H 2 S (Levitt et al., 2002) Few investigations of BSS on ruminal fermentation have b een made through in vitro fermentations using ruminal fluid as an inoculum (Ruiz Moreno et al., 2015) When BSS was added to a high S substrate (0.44% S, DM basis) at 1.0% of the diet DM, H 2 S concentration was decreased, as was A:P, without affecting total VFA production (Ruiz Moreno et al., 2015) No changes occurred for NH 3 N When BSS was added to a comparable substrate at 1.0% diet DM in continuous culture system, H 2 S was reduced by 98% as compared to control substrates (Ruiz Moreno et al., 2015) In the continuous culture system, BSS significantly increased degradability of NDF and ADF (Ruiz Moreno et al., 2015) In contradiction, VFA production was reduced and A:P was increased. Furthermore, NH 3 N was in creased when BSS was included (Ruiz Moreno et al., 2015) The opposing results from the two in vitro studies for VFA and NH 3 N production along with coinciding H 2 S results illustrates a need for further research into this potentially industry changing bismuth compound.

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41 CHAPTER 2 EFFECTS OF BISMUTH SUBSALICYLATE AND CALCIUM AMMONIUM NITRATE ON IN VITRO FERMENTATION OF BAHIAGRASS HAY WITH SUPPLEMENTAL MOLASSES Introduction Many researchers have focused on reducing enteric CH 4 production through dietary modification (Shibata et al., 1992; Mc Geough et al., 2010) inhibition of methanogens (Prins et al., 1972; Zeitz et al., 2013) and providing H 2 sinks within the rumen (van Zijderveld et al., 2010; Newbold et al., 2014; Lee et al., 2015a) Much of the research has been focused on enteric CH 4 production of ruminants consuming diets which contai n at least 50% concentrate. It has been determined that 80% of the greenhouse gas ( GHG ) emissions of beef production in kg of CO 2 equivalents, are associated with the cow calf sector (Beauchemin et al., 2010) where cattle are consuming forage based diets often deficient in CP Issues with high S diets are often considered in the feedlot industry in relation to polioencephalomalacia and adverse effects on performance (Gould, 1998; Felix et al., 2012; Drewnoski et al., 2014) In some areas of the world high S forages have gained attention due to issues associated with trace mineral absorption (Arthington et al., 2002) Bismuth subsalicylate ( BSS ) has been used for more than 200 years in human medicine to combat gastro intestinal pain (Phillips et al., 2007) I n the human gut, BSS binds to the S in H 2 S, a known gut irritant, forming Bi 2 S 3 (Suarez et al., 1998) F ew experiments (Ruiz Moreno et al., 2015) have focus ed on the possibility of providing BSS to reduce the negative effects of high S on ruminant production. It was hypothesized that providing calcium ammonium nitrate ( CAN ) as a NPN s ource in combination with BSS will reduce in vitro CH 4 production and H 2 S without

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42 negatively affecting fermentation. Three experiments were designed to test this hypothesis with the following objectives: Exp. 1 evaluate the effects of CAN on in vitro rum inal fermentation; Exp. 2 evaluate the effects of CAN in combination with BSS on in vitro ruminal fermentation; and Exp. 3 determine a valid inclusion rate for BSS that will reduce H 2 S production without negatively affecting fermentation. Materials and Methods All procedures involving animals were approved by the University of Florida Institutional Animal Care and Use Committee. Experiment 1 Experimental d esign, substrate, and t reatments In vitro batch culture incubations were conducted as a randomized complete block design using Pensacola bahiagrass hay ( Paspalum notatum ; 80% substrate DM) and sugar cane molasses (20% substrate DM ) as a basal substrate ( Table 2 1 ). Treatments were : 1) basal substrate ( N CTRL ) ; 2) basal substrate with 0.75% urea ( CTRL ); 3) basal substrate with 1.2% CAN and 0.38% urea ( CAN1.2 ); and 4) basal substrate with 2.4% CAN ( CA N2.4 ). Treatments CTRL, CAN1.2, and CAN2.4 were isonitrogenous. The inclusion rate of CAN was selected from several previous in vivo experiments where CAN was provided to growing sheep or cattle at these concentrations (Nolan et al., 2010; van Zijderveld et al., 2011; Newbold et al., 2014) These combinations of substrate and treatments were used to evaluate CH 4 and H 2 S production, NH 3 N concentration, VFA profile, and in vitro OM digestibility ( IVOMD ). Before inclusion into the incubations, bahiagrass hay was dried for 24 h at 55C and ground to pass a 2 mm screen in a Wiley mill (Thomas Scientific, Swedesboro, NJ). Reverse osmosis filtered water was used to dissolve CAN and urea, and the NPN

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43 solutions were added to the incubations at mL serum bottle. Bottles founding effects of dilution or lesser headspace. In vitro incubations Substrates and treatments were incubated for 48 h at 39C under constant agitation (60 rpm) in 125 mL serum bottles. Two bottles per treatment and 2 blank (without substrate or treatmen t) bottles were incubated in each of the 3 separate replicate days Two ruminally cannulated Angus crossbred steers (348 30 kg BW; average SD) were provided ad libitum access to bahiagrass hay and water, along with 2.27 kg of a 50:50 (as fed) molasses: glycerol mixture (5.5% CP; 75.0% TDN) for 2 weeks prior to the first ruminal fluid collection. To increase the amount of S the steers were receiving to approximately 0.35% DMI, 23.3 g of MgSO 4 was mixed into the liquid feed mixture daily. Steers were not a dapted to CAN to eliminate any residual effects of nitrate in the inoculum of donors A representative sample of digesta was collected and strained through 4 layers of cheese cloth, placed in a prewarmed thermos container, and transported to the laboratory within 30 min of collection. In the laboratory, ruminal fluid from the 2 steers was combined in equal proportions. buffer:ruminal fluid mixture was used as inoculum for all incubations (McDougall, 1944) Bottles contained 0.7 g DM of substrate and 50 mL of inoculum. At the end of the 48 h incubation s, final pH was recorded and fermentation was halted by adding 0.5 mL of a 20% H 2 SO 4 solution to each bottle. Two 10 mL samples were then collected from the 125 mL serum bottles and stored at 20C for subsequent VFA and NH 3 N analyses.

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44 Total gas production, CH 4 and H 2 S production, NH 3 N analyses Total gas production was measured by connecting the sealed bottle to an inverted biuret filled with water allowing the headspace of the bottle to equilibrate to atmospheric pressure. The amount of water displaced by the gas was recorded as total gas production. After the pres sure was released, 10 and 5 mL of gas was extracted from the sealed bottles for CH 4 and H 2 S analyses, respectively. To determine concentration s of CH 4 a gas chromatograph (Agilent 7820A GC, Agilent Technologies, Palo Alto, CA) using flame ionization and a capillary column (Plot Fused Silica 25 m 0.32 mm, Coating Molsieve 5A, Varian CP7536 ; Varian Inc., Lake Forest, CA ) was used. Temperatures of the injector, column, and detector were 80, 160, and 200C, respectively, and N 2 was the carrier gas flowing a t 3.3 mL/min. The split ratio for the injected CH 4 sample was 100:1. Production of H 2 S was determined by s lowly bubbling 5 mL of gas from the headspace of the serum bottles into 15 mL evacuated tubes (BD Vacutainer, Franklin Lakes, NJ) which contained 5 mL of alkaline water prepared as described by Smith et al. ( 2010) Tubes were vigorously shaken to allow for proper dispersion of the gas in the alkaline water. A 0.5 mL injection of N N dimethyl p phenylenediamine sulfate was made into the tubes foll owed by 0.5 mL of a ferric chloride solution. Tubes were vigorously shaken again and allowed to rest for 30 min for the reaction to occur. Absorbance was read in 96 well, flat bottom plates at 665 nm using a plate reader ( DU 50 0; Beckman Coulter Inc., Palo Alto, CA). The phenol hypochlorite reaction was used to determine concentration s of NH 3 as described by Broder ick and Kang ( 1980) S amples of the post incubation inoculum were centrifuged at 10,000 g for 15 min at 4C (Avanti J E, Beckman Coulter Inc.).

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45 Briefly, 1 mL of a phenol reagent was pipetted into 12 75 mm borosilicate disposable culture tubes (Fisherbrand; Thermo Fisher Sci entific Inc., Waltham, MA). A 20 aliquot of the supernatant from the centrifuged sample was then transferred to the phenol containing culture tubes. After vortexing, 0.8 mL of a hypochlorite solution was added to the mixture and vortexed again. The cult ure tubes were then covered with glass marbles and placed in a water bath at 95C for 5 min. The only modification to the original protocol was that absorbance was read in 96 well, flat bottom plates at 665 nm using a plate reader (DU 500; Beckman Coulter Inc.). Volatile fatty acid analysis Concentrations of VFA were determined in a water based solution using ethyl acetate extraction. Samples were centrifuged at 10,000 g for 15 min at 4C. Two milliliters of the supernatant was mixed with 0.4 mL (5:1 rati o) of a metaphosphoric:crotonic acid (internal standard) solution and samples were frozen overnight. Samples were then thawed and centrifuged again at 10,000 g for 15 min at 4C. Supernatant was transferred into 12 mm 75 mm borosilicate disposable cult ure tubes ( Fisherbrand; Thermo Fisher Scientific Inc., Waltham, MA ) and mixed with ethyl acetate to form a 2:1 ethyl acetate:supernatant mixture. Culture tubes were vigorously shaken and followed by a 5 min rest time to allow the separation of the ethyl ac etate. A subsample of the ethyl acetate was transferred into small vials prior to analysis. Samples were analyzed with a gas chromatograph (Agilent 7820A GC, Agilent Technologies) using a flame ionization detector and a capillary column (CP WAX 58 FFAP 25 m 0.53 mm, Varian CP7767; Varian Inc.). Column temperature was maintained at 110C, and the injector and detector temperatures were 200 and 220C, respectively.

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46 In vitro OM digestibility A modified Tilley and Terry ( 1963) procedure was used to determine IVOMD. buffer:ruminal fluid inoculum in 100 mL plastic centrifuge tubes for 48 h under constant agitation (60 rpm). Two tubes per treatment and 2 blank (without substrate or treatment) t ubes were incubated in each of the 3 separate replicate days. After the initial 48 h, 6 mL of HCl was added to the tubes along with 2 mL of a 5% pepsin solution. Tubes were then incubated for an additional 48 h. Samples were then filtered through P8 filter s ( Fisherbrand; Thermo Fisher Scientific Inc. ). Filters with wet samples were then dried at 105C in a forced air oven for 24 h to determine IVDMD. Dry samples were then placed in a muffle furnace for 6 h at 650C. The ash was then placed in a 10 5 C oven f or 24 h prior to recording weight. Experiment 2 Experimental design, substrate, and treatments In vitro batch culture incubations were conducted as a randomized complete block design with a 4 3 factorial arrangement of treatments. Bahiagrass hay (80% substrate DM) and sugar cane molasses (20% substrate DM) w as used as the basal substrate ( Table 2 1 ). Factors included 4 inclusion rates of BSS (0.0, 0.33, 0.66, and 1.0% substrate DM) and 3 inclusion rates of CAN (0.0, 1.2, and 2.4% substrate DM). All treatments were made isonitrogenous with urea. The inclusion rates of BSS were selected based on research performed by Ruiz Moreno et al. ( 2015) The authors reported that when BSS was provided to in vitro batch and continuous cultures with a high grain substrate, when added at up to 1.0% of the substrate DM, BSS reduced H 2 S

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47 witho ut affecting fermentation; however, at inclusion rates greater than 1.0%, fermentation was negatively affected (Ruiz Mo reno et al., 2015) In vitro incubations Method of incubation was described in Exp 1. Experiment 1 and 2 were performed simultaneously and the same ruminally cannulated steers were used. Bismuth subsalicylate was not soluble in water; therefore, BSS was weighed into individual incubation bottles at its respective inclusion rate All analyses performed were the same as described in Exp 1. Experiment 3 Experimental design, substrate, and treatment In vitro batch culture incubations were conducted as a randomized complete block design using bahiagrass hay ( 79.25 % substrate DM) sugar cane molasses (20% substrate DM) and urea (0.75% substrate DM) as the basal substrate ( Table 2 1 ). Treatments included: 1) basal substrate with 0.0% BSS ( 0 B ); 2) basal substrate with 0.05% BSS ( 5BSS ); 3) basal substrate with 0.10% BSS ( 10BSS ); and 4) basal substrate with 0.33% BSS ( 33BSS ). This experiment was designed to determine a m ore precise amount of BSS needed to have beneficial effects on ruminal fermentation using in vitro techniques. These combinations of substrate and treatments were used to evaluate CH 4 and H 2 S production VFA profile, and IVOMD, and these analyses were perf ormed as described in Exp 1 In vitro incubations Method of incubation has been described in Exp 1. Two ruminally cannulated Angus crossbred steers (727 11 kg BW) were provided ad libitum access to bahiagrass hay and water, along with 2.27 kg of a 50:50 (as fed) molasses:glycerol

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48 mixture (5.5% CP; 75.0% TDN) for 2 weeks prior to the first ruminal f luid collection. Steers were not supplemented with additional S The amount of BSS that needed to be added to the serum bottles was limiting (0.00035 and 0.00231 g per serum bottle for 0.05 and 0.33%, respectively); therefore, four premixes were produced u sing Cr 2 O 3 as an inert carrier and each premix was included in the respective serum bottles at 5 mg/bottle. All analyses performed were the same as described in Exp 1. Statistical A nalysis Experiment 1 was analyzed as a randomized complete block design wi th 3 replicated days (block) using the MIXED procedure of SAS ( SAS Inst. Inc., Cary, NC ) For all analyses, the average of 2 tubes within day was considered the experimental unit. The following contrasts were used to aid in data interpretation: effect of N PN (NCTRL vs. others) ; and the linear and quadratic effects of CAN. The model included the fixed effects of treatment and the random effect of day. When random factors are included into models analyzed by SAS, SE of the means are inflated; therefore, SE of the me ans presented were calculated from SE of the treatment differences Significance was declared at P Experiment 2 was ana lyzed as a randomized complete block design with a 4 3 factorial arrangement of treatments using the MIXED procedure of SAS (SAS Inst. Inc.). The experimental unit was considered the mean of two tubes within day (block; n = 3/treatment). The model included the fixed effects of treatment and the random effect of day. Contrasts were used to evaluate: effect of BSS CAN; line ar, quadratic, and cubic effect of BSS; and the linear and quadratic effect of CAN. Standard error of the means presented were calculated from SE of the treatment differences. Significance was declared at P nd Tukey Kramer adjustments were utilized

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49 Experiment 3 was analyzed as a randomized complete block design with 3 replicated days (block) using the MIXED procedure of SAS (SAS Inst. Inc.). The model included the fixed effect of BSS inclusion rate and the random effect of day. Orthogonal polynomi al contrasts adjusted for unequal spacing using the IML procedure of SAS (SAS Inst. Inc.), were used to determine the linear, quadratic, and cubic effects of BSS. Standard error of the means presented were calculated from SE of the treatment differences. Significance was declared at P Kramer adjustments were used. Results and Discussion Experiment 1 In vitro ruminal fermentation parameters can be found on Table 2 2 There was an effect ( P = 0.0437) of NPN on maximal gas production. When NPN was provided in the substrate as either urea or CAN maximal gas production was increased by 2% as compared to NCTRL; however, when CAN was provided at up to 2.4 % of the substrate DM, maximal gas production was linearly reduced. The effect of NPN was expected due to the lack of available N for microbial use in the basal substrate ( basal substrate CP = 7.4%). Other researchers have observed that when a source of ni trate was provided as a NPN source to ruminants consuming low quality forages, fermentation was often increased. For example, an experiment was conducted to evaluate the effects of CAN on CH 4 production and performance of Merino ewes consuming oaten chaff (CP = 4.1 % ) that were either defaunated or faunated (Nguyen et al., 2016) The researchers reported that when CAN was provided to the ewes fermentation was increased leading to improvements in DM digestibility. Furthermore, CH 4 production was increased with the addition of CAN, and this was speculated to be in relation to the increase in

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50 fermentation, and as a result, a reduction in CH 4 yield (g/kg of DMI; Nguyen et al., 2016 ). By providing a source of NPN, IVOMD was improved b y 10% ( P = 0.0120). In accordance with the increase in maximal gas production when NPN was provided, IVOMD was likely improved because of the increase in available N for microbial growth and fermentation; however, i t is evident that the source of NPN affec ts IVOMD. When CAN was provided at 2.4% IVOMD was reduced ( P = 0.0013) by 16% when compared to CTRL. The reduction in IVOMD with the inclusion of CAN may be partially related to a potential increase in concentration of H 2 Although, H 2 was not evaluated i n this experiment, it has been reported that inclusion of nitrate can increase ruminal concentration of H 2 (Guyader et al., 2015) While this may be counter intuitive to the concept that nitrate is a H 2 sink; however, if one considers that nitrite, an intermediary of nitrate reduction to NH 3 ha s toxic effects on methanogens (Duin et al., 2016) it can be speculated that an increase in concentration of H 2 may be observed at times throughout fermentation. When concentration of H 2 i n the rumen is elevated, the restoration of NAD + from NADH may be inhibited, which could lead to reduction in cellulolytic activity (Janssen, 2010) C oncentration s of NH 3 N was increased ( P = 0.0010) by the addition of NPN by more than 5 fold. There was a linear ( P < 0.0001) decrease of 86% when CAN was provided at up to 2.4% of the substrate DM. The reduction observed with the addition of CAN could be the result of several issues. First, the steers used in this experiment as ruminal fluid donors were not adapted to CAN Researchers have reported the need to adapt ruminants to nitrate for at least 8 d to increase the population of nitrate and nitrite

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51 reducers in the rumen (van Zijderveld et al., 2011; Newbold et al., 2014; Latham et al., 2016) By not adapting the donors to CAN, it is possible that the populations needed to redu ce nitrate to nitrite and finally NH 3 were not available in large enough percentages to convert the CAN within 48 h. Lin et al. ( 2013) reported a 40% increase in NH 3 N after 24 h of in vitro incubation when the inoculum donors w ere adapted to nitrate rather than urea for 15 d pr ior to the initiation of the in vitro experiment. All measures of CH 4 (total production m M mmol/g of substrate incubated, and mmol/g of substrate fermented) were reduced linearly ( P < 0.0001) with the addition of CAN. An 88% reduction was observed for CH 4 in mmol/g of substrate fermented when comparing CAN2.4 to CTRL Since CAN is a NPN source the large reduction in CH 4 production related to the addition of CAN caused the mean of NPN treatments to be lesser ( P likely an artifact and NPN source (i.e. urea) has not been associated with a reduction in in vitro ruminal CH 4 production. Several researchers have reported in vivo and in vitro reductions of 20 30% when nitrate was included in the diet or substrate at app roximately 2.0 to 2.4% of the DM (van Zijderveld et al., 2011; Newbold et al., 2014; Sun et al., 2017) The reduction in CH 4 was expected with the addition of a nitrate source; however, much of the research performed has focu sed on feedlot and dairy cattle consuming at least 50% concentrate. CAN on the in vitro fermentation of the poor quality forage, bahiagrass. There was no effect ( P > 0.10) of NPN or CAN on in vitro ruminal H 2 S production. Perhaps nitrate could reduce H 2 S in the same manner that it reduces CH 4 production, acting as a H 2 sink; however, in this experiment, this was not observed. It may be that

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52 the variation (SEM = 7.58 1 ) between samples was too large and masked any potential effects. The in vitro ruminal VFA profile was not affected by NPN nor CAN ( P > 0.05 ; Table 2 3 ). This concurs with other reports that have evaluated the addition of nitrate to forage based substrates in vitro (Sun et al., 2017) and in vivo with a 5 5 : 45 forage to concentrate ratio (Lee et al., 2015a) It should be noted that the exact implications of nitrate on the ruminal VFA profile is still unclear. A review by Janssen ( 2010) provided a summary of data indicating that the VFA profile and shifts are largely related to the concentration of H 2 in the rumen. Production of VFA is shi fted to acetate producing pathways, when concentration of H 2 is reduced, and to propionate producing pathways when the concentration of H 2 in the rumen i s greater. It may be speculated that because the role of nitrate in the rumen is to capture H 2 that may be utilized by methanogens to produce CH 4 an increase in acetate molar proportions may be observed. Guyader et al. ( 2016) reported a linear increase in acetate molar proportion when nitrate was provided up to 6 m M In vitro ruminal pH was not affected by NPN nor CAN ( P > 0.10 ; Table 2 2 ). Several other studies have reported no differences in either in vivo or in vitro ruminal pH when a source of nitrate is provided (Lund et al., 2014; Tomkins et al., 2016; Sun et al., 2017) ; however, other researchers have reported increased in vitro ruminal pH with an orchardgrass hay substrate (Sar et al., 2005) The similarity in pH among treatments in this experiment is possibly due to the lack of change in total concentration of VFA among treatments.

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53 Experiment 2 No interactions ( P > 0.05) between BSS and CAN were observed for maximal gas production, IVOMD, NH 3 N concentration, final inoculum pH, H 2 S production, and the VFA profile; therefore, only the main effects of these variables will be discussed. The main effects of BSS on in vitro ruminal fermentation parameters are presented on Table 2 4 Maximal gas production was linearly reduced ( P < 0.0001) by 22% as BSS was included in the substrate up to 1.0% of the DM. This indicates that fermentation was negatively affected when BSS was included in the substrate DM up to 1.0%. One study using U.S. feedlot concentrate substrates, evaluated the effects of BSS on in vitro batch and continuous culture incubation s (Ruiz Moreno et al., 2015) The authors incubated up to 4% of BSS in the substrate DM in a n in vitro batch culture experiment They observed that when provided at up to 1.0% of the substrate DM, no differences occurred in maximal gas production; however, when 2 and 4% BSS was included in the DM, a 12 and 25% reduction in gas production occurred (Ruiz Moreno et al., 2015) The difference in substrate types between the current experiment and that of Ruiz Moreno et al. (2015) may explain why the two experiments differed in effects when BSS was provided at 1.0% of the substrate DM. A 29% reduction (linear P < 0.0001) in IVOMD was observed when the inclusion rate of BSS was increased to 1.0% of the substrate DM. The linear reduction in maximal gas production when BSS was included up to 1.0% of the substrate DM followed the pattern of IVOMD. When Ruiz Moreno et al. (2015) provided a continuous culture with 1.0% of BSS in the substrate DM, apparent and true dig estibility of OM was increased. As discu ssed previously, the feedlot type substrate they used may have altered the effect of BSS on fermentation. It has been reported that BSS may have an impact on

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54 certain bacteria in the human gut and feces (Manhart, 1990; Yakoob et al., 2013) ; thus, it may be speculated that BSS, when provided at 1.0% of the substrate DM, may alter the ruminal microbiome to have a greater ability to digest OM of a high g rain substrate compared with a forage based substrate. The pH of the inoculum as reported by the authors (pH = 4.8) may have had an impact on the activity of BSS in vitro (Ruiz Moreno et al., 2015) Sox and Olson ( 1989) reported a large increase in the antimic robial effects of BSS when pH was 3 compared to 7. In vitro ruminal NH 3 N was linearly increased ( P = 0.0092) as the inclusion rate of BSS increased. C oncentration of NH 3 N is another indication that in vitro fermentation of bahiagrass hay and molasses wa s negatively affected when BSS was provided up to 1.0% of the substrate DM. The concentration of NH 3 N reported by Ruiz Moreno et al. ( 2015) was essentially unchanged as BSS increased to 4.0% of the substrate DM in the batch culture and when BSS was provided to a continuous culture at 1.0% of the substrate DM. It appears that the negative effects of BSS on in vitro fermentation may be enhanced with a forage based diet compared to a high grain diet. Final pH of the inoculum was increased linearly ( P = 0.0004) as BSS was increased to 1.0% of the substrate DM. Similar result s were reported for pH where no differences between treatments were observed until BSS was included at 2.0% of the substrate DM (Ruiz Moreno et al., 2015) The increase in pH was more substantial with an inclusion of 4.0% BSS in the substrate DM (Ruiz Moreno et al., 2015) All of the data related to BSS inclusion in a forage based substrate indicate that at 1.0% of the DM, negative effects on fermentation are likely to occur

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55 The production of H 2 S was reduced in a quadratic ( P = 0.0243) manner as BSS was increased in the substrate DM to 1.0%. When included in the substrate DM at 0.33, 0.66, and 1.0%, H 2 S was reduced by 61, 84, and 100%. This data is in agreement with several studies utilizing human and rat models (Suarez et al., 1998; Levitt et al., 2002; Mitsui et al., 2003) Ruiz Moreno et al. (2015) observed similar results to those of the current experiment. When provided at 1.0% of the subs trate DM, Ruiz Moreno et al. (2015) reported a 34% reduction in H 2 S production. The difference observed between the current experiment and that of Ruiz Moreno et al. (2015) may be due to the difference in the total amount of H 2 S produced between the 2 expe riments. In the current experiment, substrate S concentration was 0.35% of the substrate DM and the S concentration of Ruiz Moreno et al. (2015) was 0.44% of the substrate DM. The 25% increase in S concentration, and the reduced pH associated with high gra in diets likely increased H 2 S production when compared with the current experiment. If production of H 2 S was greater in the experiment reported by Ruiz Moreno et al. (2015), then it is possible that the amount of BSS was not sufficient to reduce the H 2 S at the same magnitude as the current experiment. The main effects of CAN on in vitro ruminal fermentation parameters can be found on Table 2 5 Similar to Exp. 1, CAN linearly reduced ( P = 0.0006) maximal gas production as the inclusion rate of CAN increased from 0.0 to 2.4% of the substrate DM. A 14% linear reduction ( P = 0.0009) in IVOMD was observed when CAN replaced urea as an in vitro ruminal fermentation NPN sour ce. As discussed in Exp. 1, this may be related to a shift in the concentration s of H 2 in the inoculum inhibiting cellulolytic activity (Janssen, 2010)

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56 The concentration of NH 3 N was reduced line arly ( P = 0.0001) as the inclusion rate of CAN increased to 2.4% of the substrate DM. This result mirrors what was observed in Exp. 1. To contradict the results of Exp. 1, the production of H 2 S was linearly reduced ( P = 0.0113) by 66% as CAN was included in the substrate DM up to 2.4%. Although the reason for the difference in H 2 S between the 2 experiments is unknown, the mode of action of nitrate may reduce H 2 S production as well as CH 4 Due to the ability of nitrate to act as a H 2 sink, H 2 that may have been destined for the reduction of sulfate to H 2 S, may have been captured by nitrate since the thermodynamics of nitrate reduction are 599.8 and 152.3 for nitrate and sulfate reducti on, respectively; Weimer, 1998) Furthermore, it is known that nitrate and its intermediates i n the path way to NH 4 can have toxic effects on microorganisms (Zhou et al., 2012; Latham et al., 2016) ; therefore, if sulfate reducing bacteria are negatively impacted by supplementation of nitrate, it is probabl e that H 2 S production would be reduced. There was an interaction between BSS and CAN for total CH 4 production ( P = 0.0097), CH 4 produced per gram of substrate incubated ( P = 0.0263), and CH 4 produced per gram of substrate fermented ( P = 0.0408 ; Table 2 6 ). There was an additive effect when BSS and CAN were combined in the substrate where CH 4 production was decreased in vitro It is likely that BSS was reducing fermen tation of the in vitro batch culture which led to reductions in CH 4 production. With the CH 4 being reduced due to the negative effects of BSS on fermentation, CAN continued to accept H 2 which would alternatively be used by methanogens to produce CH 4

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57 The effects of BSS on the in vitro ruminal VFA profile are presented on Table 2 7 The total concentration s of VFA was linearly reduced ( P = 0.0025) as BSS was increased to 1.0% of the substrate DM whereas the inclusion of CAN in the substrate DM did not impact total VFA concentration. The reduction in total concentration s of VFA aligns with the notion that BSS at 1.0% of the substrate DM n egatively impacts in vitro ruminal fermentation of bahiagrass hay and molasses. Whe n Ruiz Moreno et al. (2015) observed negative effects on fermentation when BSS was included at 4% of the substrate DM, a reduction in the total VFA concentration was reporte d Along with a reduction in total concentration s BSS altered the molar proportion of several key VFA A quadratic effect ( P = 0.003) was observed for acetate molar proportion where there was a reduction when BSS was included in the substrate DM up to 0. 66% ; however, when BSS was included at 1.0% of the substrate DM, the acetate molar proportion returned to control values. A similar, albeit opposite, quadratic ( P = 0.0127) pattern occurred for propionate molar proportion. The le ast molar proportion of pro pionate was observed with 0.0% BSS, and the greatest molar proportion occurred when BSS was included in the substrate DM at 0.66%. T his shift in the molar proportions of acetate and propionate produced a quadratic effect ( P = 0.0049) on the A:P, with t he V FA profile being more energetically efficient when BSS was include d at 0.66% of the substrate DM. The shifts in molar proportions of VFA may partially be due to the concentration of H 2 concentration of the inoculum. When BSS binds to H 2 S, it occurs accordi ng to the following reaction: 2 Bi 3+ + 3 H 2 2 S 3 + 6 H +

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58 T he possibility exists that the greater concentration s of H 2 may shift VFA pathways away from the acetate producing pathways, which release H 2 making these pathways less thermodynamically favora ble when concentration s of H 2 is greater, and shift them towards the energetically more efficient propionate producing pathways (Janssen, 2010) Another possibility is that BSS may be inhibitory t owards bacterial populations that produce mainly acetate causing a shift in the molar proportions. The inclusion of CAN did not affect the molar proportions of any VFA measured, nor was there any effect on the A:P ( P > 0.05 ; Table 2 8 ). This emulates the results as discussed in Exp. 1. The pH of the in vitro ruminal batch culture inoculum after 48 h was not a ffected by inclusion rate of CAN ( P > 0.05; Table 2 5 ) Experiment 3 In vitro ruminal fermentation parameters of Exp. 3 are presented i n Table 2 9 With the inclusion rates of BSS used in the current experiment no differences ( P > 0.05) were observed for IVOMD, final inoculum pH, total CH 4 production, or CH 4 produced per gram of substrate fermented. Th e data impl y that at these inclusion rates of BSS, fermentation was not affected. Furthermore, H 2 S production was linearly reduced ( P = 0.0040) by 19% as BSS was included in the substrate DM up to 0.33% Interestingly, unlike Exp. 2, maximal gas production was increa sed in a quadratic manner ( P = 0.0050) as BSS was increased in the substrate DM from 0.0 to 0.33%. Although there was no difference in IVOMD in the current experiment, Ruiz Moreno et al. (2015) reported an increase in NDF, ADF, and OM digestibility of a hi gh concentrate substrate incubated in continuous culture for 10 d. While unknown, it may be speculated that the BSS at lesser concentrations has beneficial effects on fermentation.

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59 There were no changes ( P > 0.05 ; Table 2 10 ) in the VFA profile when BSS was included in the substrate DM up to 0.33%. This adds further evidence to support the idea that at lesser concentrations, BSS does not have negative effects on in v itro ruminal fermentation of bahiagrass hay and molasses. In conclusion, the data collected from th e s e 3 experiment s indicate that CAN may be used in an effort to mitigate in vitro ruminal CH 4 production. With the inclusion rates used in these experi ments, it appears that negative effects on IVOMD concentrations of NH 3 N, and maximal gas production are disadvantage to the use of CAN to redirect metabolic H to the reduction of nitrate rather than CH 4 formation. Furthermore, it may be possible that the reduction in IVOMD concentrations of NH 3 N, and maximal gas production related to the inclusion rate of CAN is associated with the lack of adaptation to nitrate by the ruminal fluid donors. Bismuth subs alicylate when included in the substrate DM up to 1.0% appears to have drastically negative effects on in vitro fermentation of bahiagrass hay and molasses. By designing another experiment to test the effects of BSS at lower inclusion rates, the author w as able to provide data which implied that BSS could be included up to 0.33% of the substrate DM without reducing fermentation while also reducing H 2 S production by 19%. The combination of CAN and BSS may have beneficial impacts on the ruminal fermentatio n and enteric CH 4 production of ruminant animals consuming low quality, forage based diets.

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60 Table 2 1 Analyzed 1 chemical composition of the basal substrate used in in vitro incubations (Exp. 1, 2, and 3) Item Bahiagrass hay (80% substrate DM) Sugar cane molasses (20% substrate DM) DM, % 90.1 81.1 OM, % DM 92.32 87.72 CP, % DM 8.1 4.5 NDF, % DM 75.7 ADF, % DM 42.2 TDN, % DM 53.0 81.0 S, % DM 0.22 0.86 Nitrate % DM <0.03 0.04 1 Analyzed by a commercial laboratory using a wet chemistry package (Dairy One, Ithaca, NY).

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61 Treatment 2 P value 3 Item NCTRL CTRL CAN 1.2 CAN 2.4 SEM 4 NPN CAN L CAN Q M 5 mL 241.0 265.2 248.6 223.6 1.61 0.044 <0.0 01 0.077 IVOMD 6 % 38.9 46.1 43.8 38.5 0.95 0.012 0.001 0.239 NH 3 N, m M 0.20 2.40 1.18 0.33 0.160 0.001 < 0.00 1 0.381 pH 6.59 6.55 6.59 6.56 0.037 0.62 6 0.833 0.527 Total CH 4 production, m M 4.48 5.00 3.08 0.40 0.144 <0.00 1 <0.00 1 0.075 CH 4 mmol/g OM incubated 1.15 1.41 0.84 0.14 0.027 <0.0 01 <0.0 01 0.139 CH 4 mmol/g OM fermented 3.00 3.09 1.92 0.37 0.120 <0.0 01 <0. 001 0.238 H 2 S, mol /g ferm. OM 24.88 34.22 37.83 18.10 7.581 0.577 0.183 0.256 1 CAN = Calcium ammonium nitrate (5Ca(NO 3 ) 2 NH 4 NO 3 ) 75% nitrate DM basis 2 NCTRL = 80% bahiagrass hay + 20% sugar cane molasses; CTRL = NCTRL + 0.75% urea; CAN1.2 = NCTRL + 1.2% CAN + 0.38% urea; CAN2.4 = NCTRL + 2.4% CAN (DM Basis). 3 Observed significance levels for : NPN = NCTRL vs. the mean of CTRL, CAN1.2, and CAN2.4; CAN L = linear effect of CAN (excludes NCTRL) ; CAN Q = quadratic effect of CAN (excludes NCTRL) 4 n = 3 experimental units/treatment mean 5 M = Maximal gas production as mL/g of OM incubated after 48 h 6 IVOMD = In vitro OM digestibility Table 2 2 Effect of NPN and inclusion rate of CAN 1 on in vitro fermentation parameters (Exp. 1)

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62 Treatment 2 P value 3 Item NCTRL CTRL CAN 1.2 CAN 2.4 SEM 4 NPN CAN L CAN Q VFA, mol/100 mol Acetate 59.23 59.88 59.59 59.47 0.338 0.322 0.418 0.855 Propionate 31.09 30.56 30.93 30.84 0.330 0.438 0.574 0.599 Butyrate 7.82 7.71 7.69 7.89 0.115 0.653 0.326 0.449 BCVFA 5 1.06 1.08 1.04 1.07 0.036 0.974 0.798 0.428 Valerate 0.80 0.76 0.76 0.74 0.017 0.073 0.393 0.764 Total VFA 40.86 39.96 39.51 38.67 1.136 0.303 0.453 0.896 A:P 6 1.92 1.97 1.94 1.94 0.033 0.4 75 0.536 0.630 1 CAN = Calcium ammonium nitrate (5Ca(NO 3 ) 2 NH 4 NO 3 ) 75% nitrate DM basis 2 NCTRL = 80% bahiagrass hay + 20% sugar cane molasses; CTRL = NCTRL + 0.75% urea; CAN1.2 = NCTRL + 1.2% CAN + 0.38% urea; CAN2.4 = NCTRL + 2.4% CAN (DM Basis). 3 Observed significance levels for : NPN = NCTRL vs. the mean of CTRL, CAN1.2, and CAN2.4; CAN L = linear effect of CAN (excludes NCTRL); CAN Q = quadratic effect of CAN (excludes NCTRL) 4 n = 3 experimental units/treatment mean 5 BCVFA = branched chain VFA: isobutyrate + isovalerate + 2 methylbutyrate. 6 A:P = acetate to propionate ratio. Table 2 3 Effect of NPN and inclusion of CAN 1 on in vitro ruminal VFA profile (Exp. 1)

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63 BSS inclusion, % substrate DM P value 2 Item 0.0 0.33 0.66 1.0 SEM 3 BSS L BSS Q BSS C M 4 mL 245.8 225.6 203. 4 190. 1 9.32 < 0 .0 01 0.532 0.652 IVOMD 5 % 42. 8 39.6 33.1 30.4 2.0 3 <0.0 01 0.835 0.200 NH 3 N, m M 1.30 1.59 2.04 2.21 0.42 9 0.009 0.808 0.685 pH 6.56 6.63 6.70 6.78 0.05 4 <0.001 0.227 0.834 H 2 S, mol/g ferm. OM 30.05 11.66 4.68 0.00 4.910 <0. 001 0.024 0.480 1 BSS = bismuth subsalicylate 2 Observed significance levels for : BSS L = linear effect of BSS; BSS Q = quadratic effect of BSS; BSS C = cubic effect of BSS 3 n = 9 experimental units/treatment mean 4 M = Maximal gas production as mL/g of OM incubated after 48 h 5 IVOMD = In vitro OM digestibility Table 2 4 Effect of BSS 1 on in vitro ruminal fermentation parameters (Exp. 2)

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64 CAN inclusion, % substrate DM P value 2 Item 0.0 1.2 2.4 SEM 3 CAN L CAN Q M 4 mL 228.8 217.6 202.3 9.32 < 0.0 01 0.721 IVOMD 5 % 38.9 37.2 33.3 2.0 3 < 0.0 01 0.372 NH 3 N, m M 2.59 1.59 1.17 0.42 9 < 0.0 01 0.287 pH 6.63 6.68 6.70 0.05 4 0.099 0.657 H 2 S, mol/g ferm. OM 14.56 15.27 4.96 4.910 0.011 0.080 1 CAN = Calcium ammonium nitrate (5Ca(NO 3 ) 2 NH 4 NO 3 ) 75% nitrate DM basis 2 Observed significance levels for : NPN = NCTRL vs. the mean of CTRL, CAN1.2, and CAN2.4; CAN L = linear effect of CAN (excludes NCTRL); CAN Q = quadratic effect of CAN (excludes NCTRL) 3 n = 9 experimental units/treatment mean 4 M = Maximal gas production as mL/g of OM incubated after 48 h 5 IVOMD = In vitro OM digestibility Table 2 5 Effect of CAN 1 on in vitro ruminal fermentation parameters (Exp. 2)

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65 Table 2 6 Effect of BSS 1 and CAN 2 on in vitro ruminal CH 4 production (Exp. 2) BSS % substrate DM 0 0.33 0.66 1.0 CAN % substrate DM P value 3 Item 0 1.2 2.4 0 1.2 2.4 0 1.2 2.4 0 1.2 2.4 SEM 4 BSS CAN BN CH 4 m M 5 5.00 3.08 0.40 3.89 1.10 0.00 2.06 0.00 0.00 0.38 0.00 0.00 0.524 <0.01 <0.01 <0.01 CH 4 inc 6 1.41 0.84 0.14 1.07 0.35 0.00 0.58 0.00 0.00 0.09 0.00 0.00 0.160 <0.01 <0.01 0.026 CH 4 ferm 7 3.09 1.92 0.37 2.44 0.85 0.00 1.36 0.00 0.00 0.27 0.00 0.00 0.365 <0.01 <0.01 0.041 1 BSS = bismuth subsalicylate. 2 CAN = Calcium ammonium nitrate (5Ca(NO3)2 NH4NO3) 75% nitrate DM basis. 3 Observed significance levels for: BSS = main effect of BSS; CAN = main effect of CAN; BN = interaction of BSS and CAN. 4 n = 3 experimental units/treatment mean. 5 CH 4 m M = total CH 4 production after 48 h (m M ) 6 CH 4 inc = mmol of CH 4 produced per gram of OM incubated. 7 CH 4 ferm = mmol of CH 4 produced per gram of OM fermented.

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66 BSS inclusion, % substrate DM P value 2 Item 0.0 0.33 0.66 1.0 SEM 3 BSS L BSS Q BSS C VFA, mol/100 mol Acetate 59.65 58.62 58.35 59.08 0.351 0.041 0. 003 0.771 Propionate 30.78 31.80 32.45 31.90 0.501 0.005 0 .013 0.528 Butyrate 7.76 7.93 7.70 7.49 0.228 0.092 0.173 0.484 BCVFA 4 1.06 0.95 0.82 0.85 0.132 0.03 4 0 .367 0.631 Valerate 0.76 0.71 0.69 0.68 0.020 <0.0 01 0.086 0.682 Total VFA 39.38 36.47 35.49 35.23 1.520 0.003 0.146 0.7 62 A:P 5 1.95 1.86 1.81 1.86 0.041 0.007 0.005 0.558 1 BSS = bismuth subsalicylate. 2 Observed significance levels for : BSS L = linear effect of BSS; BSS Q = quadratic effect of BSS; BSS C = cubic effect of BSS 3 n = 9 experimental units/treatment mean 4 BCVFA = branched chain VFA: isobutyrate + isovalerate + 2 methylbutyrate. 5 A:P = acetate to propionate ratio. Table 2 7 Effect of BSS 1 on in vitro ruminal VFA profile (Exp. 2)

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67 CAN inclusion, % substrate DM P value 2 Item 0.0 1.2 2.4 SEM 3 CAN L CAN Q VFA, mol/100 mol Acetate 59.04 58.96 58.78 0.351 0.307 0.787 Propionate 31.60 31.62 31.97 0.501 0.302 0.595 Butyrate 7.71 7.78 7.68 0.228 0.877 0.610 BCVFA 4 0.94 0.94 0.87 0.132 0.485 0.640 Valerate 0.72 0.70 0.70 0.020 0.117 0.773 Total VFA 37.24 37.02 35.68 1.520 0.160 0.554 A:P 5 1.88 1.88 1.85 0.041 0.300 0.724 1 CAN = Calcium ammonium nitrate (5Ca(NO 3 ) 2 NH 4 NO 3 ) 75% nitrate DM basis 2 Observed significance levels for : CAN L = linear effect of CAN; CAN Q = quadratic effect of CAN 3 n = 9 experimental units/treatment mean 4 BCVFA = branched chain VFA: isobutyrate + isovalerate + 2 methylbutyrate. 5 A:P = acetate to propionate ratio. Table 2 8 Effect of CAN 1 on in vitro ruminal VFA profile (Exp. 2)

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68 BSS inclusion, % substrate DM P value 2 Item 0.0 0.05 0.10 0.33 SEM 3 Linear Quad. Cubic M 4 mL 277.9 284.7 286.8 289.0 1.11 < 0.0 01 0.005 0.256 IVOMD 5 % 53.6 53.3 53.7 55.4 0.86 0.114 0.631 0.825 pH 6.50 6.49 6.49 6.48 0.007 0.099 0.607 0.33 4 Total CH 4 production, m M 5.58 5 71 5.69 5.70 0.064 0.461 0.296 0.433 CH 4 mmol/g OM incubated 1.68 1.77 1.78 1.79 0.020 0.029 0.034 0.27 5 CH 4 mmol/g OM fermented 2.89 3.05 3.04 2.97 0.070 0.899 0.144 0.444 H 2 S, mol/g ferm. OM 25.63 25.31 25.27 20.69 0.881 0.004 0.355 0.822 1 BSS = bismuth subsalicylate 2 Observed significance levels for : Linear = linear effect of BSS; Quad. = quadratic effect of BSS; Cubic = cubic effect of BSS 3 n = 3 experimental units/treatment mean 4 M = Maximal gas production as mL/g of OM incubated after 48 h 5 IVOMD = In vitro OM digestibili ty Table 2 9 Effect of increasing inclusion rate of BSS 1 on in vitro ruminal fermentation parameters (Exp. 3)

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69 Table 2 10 Effect of increasing inclusion rate of BSS 1 on in vitro ruminal VFA profile (Exp. 3) BSS inclusion, % substrate DM P value 2 Item 0.0 0. 05 0. 10 0.33 SEM 3 Linear Quad. Cubic VFA, mol/100 mol Acetate 64.59 64.56 65.87 67.27 1.326 0.159 0.852 0.678 Propionate 23.45 23.52 21.98 20.44 1.454 0.149 0.839 0.649 Butyrate 10.06 10.05 10.08 10.01 0.071 0.627 0.806 0.794 BCVFA 4 0.90 0.90 1.08 1.26 0.178 0.162 0.837 0.670 Valerate 0.99 0.98 1.00 1.02 0.022 0.228 0.960 0.494 Total VFA 43.06 44.81 38.25 32.05 5.281 0.130 0.932 0.536 A:P 5 2.76 2.75 3.10 3.46 0.339 0.151 0.841 0.663 1 BSS = bismuth subsalicylate. 2 Observed significance levels for : Linear = linear effect of BSS; Quad. = quadratic effect of BSS; Cubic = cubic effect of BSS 3 n = 3 experimental units/treatment mean 4 BCVFA = branched chain VFA: isobutyrate + isovalerate + 2 methylbutyrate. 5 A:P = acetate to propionate ratio.

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70 CHAPTER 3 EFFECTS OF BISMUTH SUBSALICYLATE AND CALCIUM AMMONIUM NITRATE ON RUMINAL FERMENTATION OF BEEF CATTLE Introduction It is widely accepted that the addition of nitrate to ruminants can mitigate enteric CH 4 production (van Zijderveld et al., 2010; Newbold et al., 2014; Olijhoek et al., 2016) The reduction in CH 4 is due to nitrate acting as a H 2 sink in the rumen and having negative effects on methanogenic archaea (Zhou et al., 2012; Duin et al., 2016) Researchers have evaluated the impacts of nitrate supplementation on ruminal fermentation, but a majority of the research has focused on cattle consuming at least 30% concentrate. It has been speculated that nitrate could be substituted for urea as a NPN source while mitigating enteric CH 4 production (Leng, 2008) Researchers have evaluated microbial CP synthesis in vivo and in vitro with differing results (Li et al., 2012; Li et al., 2013; Guyader et al., 2016) ; however, there has not been a focus on cattle consuming low quality, forage based diets, in which case NPN would be beneficial. Bismuth subsalicylate ( BSS ) is not a novel compound in human health; however, very little research has evaluated its effects in ruminants (Ruiz Moreno et al., 2015) In humans, BSS has been reported to decrease H 2 S in the gut by binding the sulfide (Suarez et al., 1998) and to have antimicrobial effects (Manh art, 1990; Bland et al., 2004; Yakoob et al., 2013) In in vitro ruminal fermentation, BSS has been reported to reduce H 2 S and, while at lesser inclusion rates, have minimal effects on fermentation (Ruiz Moreno et al., 2015) It was hypothesized that the addition of encapsulated calcium ammonium nitrate ( eCAN ) to cattle consuming a bahiagrass hay based diet will shi ft ruminal fermentation

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71 to acetate producing pathways, and have similar microbial CP synthesis compared with urea. Providing BSS was hypothesized to reduce H 2 S concentration in the ruminal gas cap while not shifting ruminal fermentation. The objective of t his experiment was to evaluate the effects of the combination of eCAN and BSS on ruminal fermentation and microbial protein synthesis. Materials and Methods All procedures involving animals were approved by the University of Florida Institutional Animal Care and Use Committee. Experimental D esign, A nimals, and T reatments The experiment was conducted at the University of Florida North Florida Research and Education Center Beef Unit ( UF NFREC BU ) in Marianna, FL. Ten ruminally cannulated Angus crossbred steers (n = 8; 461 148 kg of BW; average BW SD) and heifers (n = 2; 337 74 kg of BW) were used in a duplicated 5 5 Latin square design with a 2 2 + 1 factorial arrangement of treatments. In each of the five 28 d periods, d 0 to 13 were for adapt ation to the diet and treatments; on d 0, 1, 2, 3, and 14 ruminal gas cap samples were collected; from d 13 to 17, the cattle were brought into the UF NFREC BU Pavilion to collect data for DMI, blood and ruminal parameters, and omasal spot samples; on d 21 and 22, ruminal evacuations were performed and the cattle were dosed with Cr EDTA and YbCL 3 for digesta flow rate analysis; and d 23 to 28 were for washout, when all the cattle only received bahiagrass hay and sugar cane molasses. At the beginning of th e first period, all cattle were randomly assigned to 1 of 5 treatments: 1) NCTRL no added NPN or BSS; 2) U urea supplemented at 182 mg/kg of BW; 3) NIT nitrate, in the form of eCAN, supplemented at 350 mg/kg of BW; 4) UB urea supplemented at 182 mg/kg of BW and BSS

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72 supplemented at 58.4 mg/kg of BW; and 5) NITB nitrate, in the form of eCAN, supplemented at 350 mg/kg of BW and BSS supplemented at 58.4 mg/kg of BW. Treatments U, NIT, UB, and NITB were isonitrogenous. Throughout the 5 periods, the cattle were kept in individual pastures ( 0.69 ha; d 0 to 13, d 17 to 21, and d 23 to 28) and pens in the UF NFREC Beef Unit Pavillion ( 13.4 m 2 ; d 13 to 17 and d 21 to 22). The cattle had ad libitum access to Pensacola bahiagrass ( Paspalum notatum ) hay and were supplemented daily with molasses at 2.7 g/kg of BW. When on pasture, cattle received full length bahiagrass hay as round bales; however, to be able to provide the same source of hay fed to cattle on dormant pastures when the cattle were in the Pavilion, bahiagrass hay was chopped using a Tub Grinder (Haybuster, Jamestown, ND) and square baled. Round bales of hay were sampled at the beginning of each period for nutrient analysis using a hand drill and core sampler and square bales were sample d by hand as the hay was fed to the cattle. The amount of molasses corresponding to each animal was weighed and offered daily using rubber containers inside pastures and pens. The sugar cane molasses was provided by Quality Liquid Feed ( Dodgeville, WI ). Ch emical composition of bahiagrass hay and sugar cane molasses while on pasture and while confined in the UF NFREC BU Pavilion are presented on Table 3 1 To reduce any negative effects of nitrate on the cattle, on d 0 cattle began an adaptation to eCAN and urea. On d 0 and 1, cattle received 20% of their total supplemental N; on d 2 and 3, cattle received 40% of their total supplemental N; on d 4 and 5, cattle receiv ed 60% of their total supplemental N; on d 6 and 7, cattle received

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73 80% of their total supplemental N; and beginning on d 8, cattle were receiving 100% of their total supplemental N as urea or eCAN. Sampling P rocedures All protocols and procedures used fo r collecting samples and data from animals were used in an identical manner throughout all 5 periods. Ruminal gas cap sampling On d 0, ruminal gas cap samples were collected at 0, 3, 6, 9, and 12 h post feeding of molasses. On d 1, 2, 3, and 14, samples we re collected at h 0, just prior to feeding molasses. To collect the sample, a 10 mL syringe fitted with a one way valve and 16 gauge needle was inserted through the cannula plug to prevent as much contamination with environmental air as possible. Once the needle was inside the rumen, the plunger of the syringe was used 5 times to thoroughly mix the gas sample surrounding the needle. Then 5 mL of gas cap were drawn into the syringe, at which point, the one way valve was closed to prevent contamination. The s ample was then injected into a 10 mL evacuated tube (BD Vacutainer, Franklin Lakes, NJ) containing 5 mL of alkaline water (pH 8.5 to 9). The samples were transported to the laboratory and immediately analyzed for H 2 S concentration. Ruminal fluid, pH, blood sampling, and rectal temperature Ruminal fluid and blood were collected, and rectal temperature and pH were recorded over a 72 h period. Briefly, on d 14 of each period samples were collected at 0800, 1400, and 2000 h; on d 15 at 0200, 1000, 1600, and 220 0 h; on d 16 at 0400, 1200, and 1800 h; and on d 17 at 0000 and 0600h. A representative sample of ruminal digesta was collected and strained through 4 layers of cheesecloth, and pH was immediately measured using a manual pH meter (Corning Pinnacle M530), C orning

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74 Inc., Corning, NY). A 10 mL sample of the ruminal fluid was taken into a 15 mL conical tube and mixed with 0.1 mL of a 20% (vol/vol) H 2 SO 4 to halt fermentation and preserve NH 3 N and VFA. Samples of ruminal fluid were stored at 20C for further ana lysis. Blood samples were collected via jugular venipuncture into 10 mL evacuated tubes which contained Na heparin (BD Vacutainer), placed on ice, and centrifuged at 1,500 g for 15 min at 4C. Plasma was then transferred to polypropylene vials (12 75 mm; Fisherbrand; Thermo Fisher Scientific Inc., Waltham, MA) and stored at 20C for further analysis. Hay and total dry matter intake On d 13 of each period, the cattle were moved from their respective pastures into the UF NFREC BU Pavilion. From d 13 t o 17, hay DMI was measured by weighing bahiagrass hay ( as is ) prior to feeding and then weighing orts after 24 h. Samples of hay and orts were collected each d for chemical analysis. The cattle consumed all of the molasses provided to them while DMI was be ing measured; therefore, there were no molasses orts to be weighed. Hay, molasses, and orts were also sampled on d 21 and 22 of each period to determine DM, OM, and indigestible NDF ( iNDF ) entering the rumen. These values were used for digesta flow calcul ations. Omasal sampling Omasal spot sampling occurred on d 14 to 17 at the same 12 time points as described in the Ruminal fluid, pH, blood sampling, and rectal temperature subsection. Omasal samples were collected with a device constructed as follows: fir st, a vacuum pump wa s connected using a rubber hose and rubber stopper to a 500 mL polypropylene side arm flask; the first side arm flask wa s connected to a second

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75 identical flask using a rubber hose, connecting the two side arms T he collection hose, a 1. 9 cm (inside diameter) clear rubber hose with fiber reinforcement, was fitted to the top of the second side arm flask using a rubber stopper; a 50 mL scintillation vial was filled with ball bearings, sealed closed, and attached to the end of the collection hose to act as a weight aiding in the placement of the hose, without hindering flow. To collect the sample, the omasal orifice was located and the weight at the end of the collection hose was inserted into the orifice. The hose was then placed further int o the omasum to attempt to collect strictly omasal samples; furthermore, the first 250 mL sample collected was discarded in case ruminal fluid had entered the collection tube. Samples were collected into the second side arm flask until the volume reached 250 mL. The 250 mL sample was then transferred into a plastic freezer storage bag and placed on ice until transported to the laboratory. Omasal samples were stored at 20C for further analysis. To separate the fractions of the omasal samples, equal volum es (150 mL) of each individual time point was combined to create one composite representative of 1 animal for 24 h post feeding in 1 period. The composites were filtered through 1 layer of cheesecloth and the residue left in the cheesecloth was considered the large particle fraction. The filtrate was centrifuged at 1,000 g for 5 min at 4C and the supernatant was considered the liquid fraction and the pellet was considered the small particle fraction. Both solid fractions were weighed wet and subsamples w ere taken for DM, OM, iNDF, N, and purines analysis. Volume and weight were recorded of the liquid fraction and a sub sample was collected for DM, OM, N, and purine analysis.

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76 Reconstruction of omasal composites, in DM, was calculated from the DM of the dif fering fractions. Bacterial pellet for purine reference On d 15 at 1000, 1600, and 2200 h, a representative sample of whole rumen content was collected to isolate particle associate bacteria and liquid associated bacteria according to Martnez et al. ( 2009) The digesta was collected and placed into plastic freezer storage bags and placed on ice until transported to the laboratory. Once in the laboratory, samples were strained through 4 layers of cheesecloth to separate the liquid and solid fractions. The li quid fraction was then centrifuged at 1000 g for 5 min. The supernatant was collected and centrifuged at 25,000 g The supernatant was discarded and the liquid associated bacterial pellet was stored at 20C for further analysis. To isolate the particl e associated bacteria from the ruminal solids, a 50 g (as is) subsample was incubated at 39C under constant agitation (60 rpm) for 15 min with 150 mL of a saline solution (0.9% NaCl) which contained 0.1% methylcellulose. After incubation, 25 mL of cold sa line methylcellulose solution (4C) was added and the solids were stored at 4C for 24 h. Once removed from the 4C environment, the solids were placed in a blender (Waring Products Division, New Hartford, CT) and homogenized for 10 s. The resulting soluti on was filtered through two layers of nylon cloth (50 particle associated bacterial pellet was stored at 20C for further analysis. Ruminal evacuations and digesta flow marker do sing On d 21, the cattle were brought into the UF NFREC BU Pavilion at 0900 h (1 h post feeding of molasses). The cattle were immediately brought through the chute for ruminal evacuation. Ruminal contents were evacuated manually into large containers so

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77 that weight and volume of the ruminal contents could be recorded. When the dimensions of the ruminal contents were recorded, the digesta was returned to the respective rumen. An aliquot representing 10% of the total digesta was separated and stored on ice until transported to the laboratory. Ruminal aliquots were stored at 20C for further analysis. To determine the ruminal nutrient pool sizes, aliquots were thawed and fractionated into 3 phases: liquid fraction, large particles, and small particles. Brie fly, the whole digesta sample (representing 10% of the total ruminal digesta) was strained through 1 layer of cheesecloth. The content remaining within the cheesecloth was considered the large particle phase. The filtrate was then centrifuged at 1000 g f or 5 min at 4C. The supernatant was considered the liquid fraction phase and the pellet was considered the small particle phase. All 3 of the phases were weighed and sub samples were taken and lyophilized for further analysis. Ruminal pool sizes (kg) of D M and OM were determined by multiplying the concentration of each component by the DM mass of each of the 3 phases. Chromium EDTA and YbCl 3 external and inert markers, were used to mark the liquid fraction and small particle phases of the digesta, respectively. Once the weight and volume of ruminal contents had been recorded and contents returned to their respective rumen, a 1 L Cr EDTA and Yb Cl 3 solution [2.77 g of Cr and 2.2 g Yb; modified from Binnerts et al. ( 1968 )] was poured into the rumen using a funnel and rubber hose (1 m). Ruminal contents were mixed for 3 min to aid in equilibration of the markers in the rumen. Whole ruminal contents were collected every 3 h post dosing for 21 h and strained through 1 layer of cheesecloth to determine a ruminal liquid and small

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78 particle dilution rates (Romero et al., 2013) Indigestible NDF was determined in the large and small particle phases (not in the liquid fraction phase; Ahvenjrvi et al., 2000 ). Laboratory A nalyses All protocols and procedures used for analyz ing samples and data from animals were used in an identical manner throughout all 5 periods. Hydrogen sulfide concentration Concentration of H 2 S in the ruminal gas cap was determined as described by Henry et al. ( 2015) Briefly, A 0.5 mL injection of N N dimethyl p phenylenediamine sulfate was made into the tubes followed by 0.5 mL of a ferric chloride solution. Tubes were shaken vigorously and allowed to rest for 30 min for the reaction to occur (Smith et al., 2010) Absorbance was read in 96 well, flat bottom plates at 665 nm using a plate reader (DU 500; Beckman Coulter Inc., Palo Alto, CA). Volatile fatty a cid profile A water based solution using ethyl acetate extraction was used to determine VFA concentrations in the ruminal fluid samples. Samples were centrifuged at 10,000 g for 15 min at 4C. Two milliliters of the supernatant was mixed with 0.4 mL (5:1 ratio) of a metaphosphoric:crotonic acid (internal standard) solution and samples were frozen overnight. Samples were then thawed and centrifuged again at 10,000 g for 15 min at 4C. Supernatant was transferred into 12 mm 75 mm borosilicate disposable culture tubes (Fisherbrand; Thermo Fisher Scientific Inc., Waltham, MA) and mixed with ethyl acetate to form a 2:1 ethyl acetate:supernatant mixture. Culture tubes were vigorously shaken and followed by a 5 min rest time to allow the separation of the eth yl acetate. A subsample of the ethyl acetate was transferred into small vials prior to analysis. Samples were analyzed with a gas chromatograph (Agilent 7820A GC, Agilent

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7 9 Technologies) using a flame ionization detector and a capillary column (CP WAX 58 FFA P 25 m 0.53 mm, Varian CP7767; Varian Inc.). Column temperature was maintained at 110C, and the injector and detector temperatures were 200 and 220C, respectively. Ammonia N and blood urea N concentration The phenol hypochlorite reaction was used to de termine NH 3 N concentration as described by Broder ick and Kang ( 1980) Ruminal fluid samples were centrifuged at 10,000 g for 15 mi n at 4C (Avanti J E, Beckman Coulter Inc.). Briefly, 1 mL of a phenol reagent was pipetted into 12 75 mm borosilicate disposable culture tubes (Fisherbrand; Thermo Fisher Scientific Inc., Waltham, MA). A 20 supernatant from the centrif uged ruminal fluid was then transferred to the phenol containing culture tubes. After vortexing, 0.8 mL of a hypochlorite solution was added to the mixture and vortexed again. The culture tubes were then covered with glass marbles and placed in a water bat h at 95C for 5 min. The only modification to the original protocol was that absorbance was read in 96 well, flat bottom plates at 665 nm using a plate reader (DU 500; Beckman Coulter Inc.). Plasma was analyzed for blood urea N ( BUN ) using a quantitative colorimetric kit (B7551 120; Pointe Scientific Inc., Canton, MI). Chemical analyses of hay, orts, ruminal, and omasal digesta fractions Hay and orts were analyzed for DM and OM on all d of collection (d 13 to 17 and d 21 and 22). On d 21 and 22, hay and or ts were analyzed for iNDF. To determine DM of hay and orts, samples were weighed prior to being placed in a 55C forced air oven for 72 h. Dry, hot weight was used to calculate DM of the sample. The sample was then ground to pass through a 2 mm screen in a Wiley mill

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80 (Thomas Scientific, Swedesboro, NJ). To determine OM, 0.5 g of ground sample (in duplicate) was weighed into ceramic crucibles and placed in a 105C forced air oven for 24 h to determine sample DM. Dried samples were then placed in a 650C muff le furnace for 6 h before returning to a 105C forced air oven. Hot, ashed samples were weighed and used to calculate OM. To determine DM of ruminal and omasal fractions, samples were weighed wet prior to lyophilization. After being lyophilized, samples w ere placed in a 55C oven for 24 h prior to being weighed. To determine OM, the same procedure as described for hay and orts was used. The concentration of iNDF in the hay, orts, ruminal, and omasal solid fractions was determined as described by Gregorini et al. (2008), Cole et al. (2011), and Krizsan and Huhtanen ( 2013) Briefly, 0.5 g of sample was weighed into Ankom F57 filter bags (Ankom Technology Corp. Macedon, NY) and then incubated into the rumen of a cannulated steer grazing a bahiagrass and bermudagrass ( Cynadon dactylon ) mixed pasture for 288 h to ensure complete digestion of potentially digestible NDF. After incubation samples were rinsed 2 times with warm tap water followed by 4 rinses with water filtered through a reverse osmosis system. The rinsed samples were then analyzed for NDF, using heat amylase and sodium sulfite as described by Van Soes t et al. ( 1991) using an Ankom 200 Fiber Analyzer (Ankom Technology Corp.). Ruminal and omasal fractions were analyzed for total N using a CHNS analyzer by the Dumas dry combustion method (Vario Micro Cube; Elementar, Hanau, Germany). Crude protein was calculated by multiplying the N concentration of the dry sample by 6.25.

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81 Chromium concentrations For concentrations of Cr, ruminal fluid samples were centrifuged at 1,000 g at 4C for 5 min. The supernatant was transferred to 15 mL conical tubes and the small particle pellet was lyophilized for further analysis. Atomic absorption spectrophotometry (359.4 nm with an air plus acetylene flame; AAnalyst 200; PerkinElmer, Walther, MA) was used to determine the concentration of Cr in the ruminal liquid fraction after dilution with deionized water. The natural logarithm of Cr concentration in the small liquid fraction was regressed against time to calculate liquid fraction flow rate (Romero et al., 2013) Purine analysis The purine content of omasal digesta and ruminal bacteria (liquid and par ticle associated) was determined as described by Zinn and Owens ( 1986) using Torula Yeast RNA as a standard. Briefly, 0.5 g of omasal sample and 0.2 g of bacterial sample were hydrolyzed by incubating the samples in 25 mL borosilicate screw cap borosilicate culture tubes (Fisherbrand; Thermo Fisher Scientific Inc.) with 2.5 mL of a 70% perchloric acid in a 95C water bath. After 15 min, the samples were vortexed and returned to the water bath for an additional 45 min. A dilute buffer (0.0285 M NH 4 H 2 PO 4 ) was added to the solution at 17.5 mL before vortexing. The tubes were then returned to the water bath for 15 min prior to filtration (Whatman #541; GE Healthcare UK Limited, Buckinghamshire, UK ). To determine the purine content, 0.5 mL of the filtrate was transferred into 50 mL round bottom centrifuge tubes followed by 0.5 mL of 0.4 M AgNO 3 and 9 mL of a 0.2 M NH 4 H 2 PO 4 buffer. The samples were then stored at 4C for 12 h before centrifugation at 10,000 g for 10 min at 4C. The supernatant was removed and discarded. Without disturbing the pellet, 10 mL of acidic reverse osmosis

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82 filtered water (made pH 2 using H 2 SO 4 ) was used to wash the pellet. The tubes were centrifuged again at 10,000 g for 10 min at 4C and the supernatant was removed and discarded. A 0.5 N HCl solution was added at 10 mL and mixed using a pasteur pipette. The tube was then incubated in a 95C water bath for 30 min. The samples were vortexed and centrifuged at 10,000 g for 10 min at 4C. The absorbance of the supernatant was then read at 260 nm (ultra violate) using a spectrophotometer (DU 530, Beckman Coulter, Palo Alto, CA). Calculations Flow of digesta Large particle digesta flow was calculated as described by Linneen et al. ( 2015) : Large particle digesta flow (%/h) = iNDF intake (g/h) [d 21 ruminal large particle iNDF (kg) + d 22 ruminal lar ge particle iNDF (kg)] 2. The flow rate of the liquid fraction was calculated by regressing the natural logarithm of the marker concentration against time. The absolute value of the slope was considered flow rate in percent of volume per h. All fraction al rates of passage were then multiplied by the mean ruminal DM mass of their respective fraction to calculate grams per hour and, further, kilograms per 24 h. Flow and efficiency of microbial N Microbial N flow to the omasum was calculated using liquid as sociated bacteria and particle associated bacteria as references and total purines as microbial markers as follows:

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83 The true digestibility of OM in the rumen was calculated as described by Gozho et al. ( 2009) : OM truly digested (% of total OM) = OM intake (kg/d) {[OM flowing to the omasum (kg/d) bacterial OM flowing to the omasum (kg/d)] OMI kg/d} 100 Microbial N efficiency was calculated as described by Gozho et al. ( 2009) : Microbial N efficiency = microbial N flowing to the omasum (g/24 h) OM truly digested (kg/24 h). Statistical Analysis Data were analyzed as a duplicated 5 5 Latin square with repeated measures for blood and rum inal fermentation parameters (H 2 S on d 0, BUN, NH 3 N, ruminal pH, and VFA) using the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC). For hay and total DMI, H 2 S on d 1, 2, and 3, digesta flow, and microbial N efficiency, the model included the fixed effe cts of treatment, square, period, and animal within square. For parameters analyzed using repeated measures, the model included the fixed effects of treatment, square, period, time, treatment time, and animal within square. The random effect of animal wi thin treatment was used to designate the denominator degrees of freedom. Animal within period was considered the subject and the covariance structure used for all parameters was compound symmetry based on the lesser Akaike information criterion value. To a id in the interpretation of data, the following contrasts were used: the effect of NPN = NCTRL vs. the mean of U, NIT, UB, and NITB; the effect of NPN source = the mean of U and UB vs. the mean of NIT and NITB; the effect of BSS = the mean of U and NIT vs. the mean of UB and NITB; and NPN source BSS = the mean of U and NITB vs. the mean of NIT and UB. Significance was declared at P 0.05

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84 Results and Discussion Nitrate has been evaluated extensively in the past in ruminants consuming diets that range fro m 50 to 90% concentrate (Olijhoek et al., 2016; Lee et al., 2017a; Lee et al., 2017b) The vast majority of this research has focused on mitigating enteric CH 4 emissions and performance of cattle (beef and dairy) and sheep (van Zijderveld et al., 2010; van Zijderveld et al., 2011; Newbold et al., 2014) There is a lack of data evaluating the effects of nitrate on forage fed animals, which produce nearly three times as much CH 4 as grain fed (Henry e t al., 2015) The current experiment utilized bahiagrass hay and molasses, a common wintering diet for beef cattle in the southeast U. S. Cattle consuming forage based diets, such as the current experiment, rarely have issues with S induced polioencephalomalacia (Gould, 1998) because the ruminal pH is not optimal for the undissociated sulfide (pK a 7.04); h owever, other negative effects of S have been reported, such as impacts on trace mineral absorption (Arthington et al., 2002; Pogge et al., 2014) Ruminal gas cap H 2 S concentration and rectal temperature data can be found on Table 3 2 There was no treatment time interaction ( P = 0.7880) for ruminal H 2 S concentration, nor was there an effect ( P > 0.05) of NPN, NPN source or BSS. A decrease in H 2 S concentration was observed for animals consuming NPN, regardless of source, on d 3 ( P = 0.0179). Neither NPN source nor BSS impacted H 2 S concentration of t he ruminal gas cap on d 1, 2, 3 or 14 ( P > 0.05). It was hypothesized that nitrate, which reduces CH 4 via two methods acting as a H 2 sink (Ungerfeld and Kohn, 2006) and toxic effects on methanogens (Zhou et al., 2012; Duin et al., 2016) could have similar effects on H 2 S production in the rumen as a H 2 sink. Bismuth subsalicylate was

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85 also expected to reduce concentration s of H 2 S in the rumen. Data from a series of in vitro experiments presented and discussed in Chapter 2 indicated that BSS wa s a strong inhibitor of H 2 S. When provided at 0.33% of a bahiagrass hay and molasses substrate (DM basis), BSS reduced production of H 2 S by 61%. Furthermore, research evaluating BSS in a high grain substrate observed a 34% reduction in production of H 2 S (Rui z Moreno et al., 2015) In the current experiment, concentration s of H 2 S w ere analyzed prior to feeding molasses on d 1, 2, 3, and 14. It is probable that the effects of treatment were no longer observable. The fermentation of molasses, where the majori ty of the dietary S is found, is rapid, and ruminal pH reached the nadir 2 h post feeding of molasses (Figure 3 1). Future studies evaluating the effects of nitrate and BSS on H 2 S production should consider evaluating ruminal concentrations 2 to 4 h post feeding. There was no treatment time interaction ( P = 0.1795) for rectal temperature. Rectal temperature was evaluated in this experiment because previous work in humans has indicated that nitrates lead to vasodilation (Butler and Feelisch, 2008) The author speculated that if providing nitrate caused vasodilation of cattle, perhaps body temperature might be lessened. That hypothesis was not supported by the data in this experiment, as the addition of eCAN in place of urea, in the diet of cannulated cattle did not affect ( P = 0.2910) rectal temperature. Interestingly, a decrease ( P = 0.0087) in rectal temper ature was observed when cattle were provided BSS. A vast majority of research focusing on bismuth compounds (i.e. BSS) has been performed in human and rat models (Suarez et al., 1998; Levitt et al., 2002) knowledge, this experiment represents the first data representing the effects of BSS in ruminants (in vivo). It may be possible that as bismuth can act as a nonsteroidal

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86 antiinflammatory drug (NSAID) in humans (Hawksworth et al., 2014) that BSS may have similar effects in cattle. Furthermore, salicylate can al ter body temperature by inhibiting both COX 1 and COX 2. Through these inhibitions, prostaglandins, which cause inflammation and increased body temperature, are reduced (Cashman, 1996) Data related to DMI, ruminal pH, NH 3 N and BUN concentration is presented on Table 3 3 There were no effects observed on bahiagrass hay or total DMI ( P > 0.05). There was a treatment time interaction for ruminal pH ( P = 0.0002; Figure 3 1 ), ruminal concentration of NH 3 N ( P < 0.0001; Figure 3 2 ), and BUN ( P < 0.0001; Figure 3 1 ). For ruminal pH, NCTRL exhibited a lesser pH compared with U, NIT, and UB 2 h postfeeding of m olasses ( P < 0.03). Cattle consuming NCTRL also had lesser ruminal pH at 6 h postfeeding when compared with U and UB ( P < 0.04). Likely NCTRL had a more acidic ruminal environment because of the lack of NPN source to be converted to NH 3 which acts as a bu ffer in the rumen. As time post feeding progressed, NCTRL had greater ruminal pH when compared with U at h 14 ( P < 0.05) and NIT at h 20 ( P < 0.04). Obvious effects of the different NPN sources on ruminal NH 3 N concentration can be observed in Figure 3 2 All treatments had similar concentration of NH 3 N at 0 h post feeding of molasses, but from h 2 to 6, U and UB had greater concentration of NH 3 N when compared with all other treatments ( P < 0.0001), and NIT and NITB were greater than NCTRL ( P < 0.02). Eight hours post feeding, NCTRL still had lesser concentration s of NH 3 N when compared with U and UB ( P < 0.05), and that difference remained until 10 h post feeding when NCTRL had lesser concentration compared with all other treatments ( P 3 N concentration is one that has been reported by other research groups. Lee et al. ( 2015b) provided ruminally

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87 cannulated be ef heifers with a barley silage based diet with either 0, 1, 2, or 3% eCAN (DM). All diets were made isonitrogenous with urea. The au thors observed a 16% reduction in NH 3 N for 3% eCAN when compared to urea and went on to report that heifers provided eCAN had lesser ruminal NH 3 N concentration at h 3 and 6 postfeeding. Furthermore, El Zaiat et al. ( 2014) reported that ruminal concentration of NH 3 N was decreased for Santa Ins lambs consuming eCAN rather than u rea in a corn based diet. In contrast, Veneman et al. ( 2015) providing dairy cattle urea or calcium ammonium nitrate, and Nolan et al. ( 2010) providing Merino w eathers oaten chaff with urea or potassium nitrate, both reported no differences between urea and nitrate treatments for concentration s of ruminal NH 3 N I n Chapter 2, reductions in NH 3 N were also observed when CAN was provided compared with urea. The red uction in NH 3 N concentration s in the current experiment may be due to the encapsulation of the nitrate which causes slow release in the rumen; however, Lee et al. ( 2015b) compared encapsulated urea and encapsulated nitrate and observed similar results. Another possibility is that nitrate may take longer to reduce to NH 3 than urea, which is immediately converted to NH 3 in the rumen, but if this was true, the sustained increase of concentration s of NH 3 N would likely have been observed after the concentration s in the urea treatments reduced to pre feeding levels; however, this was not detected. The source of NPN and BSS did not impact concentration s of BUN ( P > 0.05); however, the addition of NPN to cattle consuming bahiagrass hay and molasses increased concentrations of BUN ( P < 0.0001). This effect can easily be observed in Figure 3 3 The concentration s of BUN was lesser ( P < 0.05) for NCTRL when compared with all other treatments at 4, 6, 8, 12, 14, 16, and 20 h post feeding. Just

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88 prior to feeding molasses, NCTRL had lesser conc entrations of BUN when compared with NIT and NITB ( P < 0.05), and U had lesser concentrations of BUN when compared to NITB ( P < 0.05). Concentration s of BUN was also reduced ( P < 0.05) in NCTRL when compared with U, UB, and NITB 2 h post feeding of molasse s. Also 2 h post feeding, NIT exhibited lesser ( P < 0.05) concentration s when compared with U and UB, which carried over into 4 h post feeding of molasses. Blood urea N was greater ( P < 0.05) for U when compared with all other treatments 8 h post feeding. At 10 h post feeding of molasses, NCTRL had lesser ( P < 0.05) BUN concentration when compared with U, NIT, and UB, and U and UB had greater ( P < 0.05) concentrations when compared to NITB Eighteen hours post feeding, NIT had greater ( P < 0.05) BUN concent ration when compared to NCTRL. Finally, 22 h post feeding, NCTRL had less ( P < 0.05) BUN concentration when compared with U and NITB, and NITB had greater concentration when compared with U, NIT, and UB ( P < 0.05). No obvious differences between urea and eCAN treatments were observed in the current experiment. This was interesting due to the vast differences in ruminal NH 3 N concentration between urea and eCAN. Contrary to data from the current experiment, Lee et al. ( 2017b) reported a reduction in B UN for steers provided a silage based diet with either eCAN rather than urea as a NPN source. The authors followed these same steers to the finishing phase and observed the same patterns where steers receiving urea had greater BUN compared with those consuming eCAN (Lee et al., 2017a) There were treatment time interactions observed for acetat e ( P = 0.0227; Figure 3 5 ) and propionate ( P = 0.0059; Figure 3 6 ) molar proportions, and the acetate to propionate ratio ( A:P ; P = 0.0087; Figure 3 4 ). An important aspect of the treatment

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89 time interactions observed is the decrease in acetate and increase in propionate molar proportions for approximately 8 h post feeding. This is likely related to an increase in the ruminal concentration of H 2 that is often found shortly after feeding (Janssen, 2010) There were no observed effects of NPN ( P = 0.3585; Table 3 4 ) on ruminal concentration of total VFA; however, the molar proportion of acetate was increased at the expense of butyrate when a NPN source was provided ( P < 0.054; Table 3 5 ). Similarly, BSS did not affect total VFA concentration ( P = 0.1705). This is in agreement with data from Chapter 2 Exp. 3 when BSS was included in the substrate DM at 0.33%. Ruiz Moreno et al. ( 2015) also reported no difference in total VFA conc entration when BSS was included at up to 2% of a high grain substrate (DM basis) in an in vitro batch culture; however, in the same study, the authors observed a large decrease in VFA concentration when BSS was provided at 1% of the substrate DM in a conti nuous culture experiment. Furthermore, there was no effect ( P > 0.05) of BSS on molar proportions of VFA analyzed, which is similar to the results of Exp. 3 from Chapter 2. The additio n of eCAN to the bahiagrass hay based diet reduced ( P = 0.0106) the conc entration of total VFA. This is in agreement with Asanuma et al. ( 2015) which reported a decrease in total VFA concentration when a nitrate source was provided to goats at 9 g/d. An in vitro experiment utilizing alfalfa hay as the sole substrate had similar results with increasin g nitrate concentration decreasing total VFA concentration (Zhou et al., 2012) It should be noted that neither of these studies had isonitrogenous treatments (i.e. no urea control; Zhou et al., 2012; Asanuma et al., 2015 ). Several others have compared nitrate with urea and reported either n o differences (de Raphlis Soissan et al., 2014; Lee et al., 2015a; Veneman et al., 2015) or increases (Nolan et al.,

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90 2010; El Zaiat et al., 2014; Zhao et al., 2015) in concentration s of total VFA when a nitrate source was provided rather than urea. It is unclear why total VFA was reduced in the current expe riment for cattle receiving eCAN compared with urea. In the current experiment, NH 3 N of all treatments was lesser than what has been recommended in the literature (5.7 m M ; Satter and Slyter, 1974 ), more specifically, cattle consuming eCAN had lesser concentrations than those consuming urea; therefore, it is probable that total VFA concentr ation in the rumen was lesser for eCAN compared with urea in relation to a lack of N for microbial needed for fermentation. The addition of eCAN in place of urea, also increased ( P = 0.0049) the molar proportion of valerate when compared to urea. Effects of BSS and NPN source on digesta and microbial N flow are presented on Table 3 6 Similar to d 13 to 17, DMI and OMI were not affected ( P > 0.05) by the addition NPN, NPN source or BSS. Furthermore, flow of DM and OM to the omasum was also not impacted ( P > 0.05). In humans, NO, an intermediary of nitrate reduction to NH 4 has been reported to cause relaxation of smooth muscle tissue (Butler and Feelisch, 2008) Prior to the commencement of the current experiment, it was speculated that eCAN may h ave reduce passage rate in ruminants by causing the smooth muscle in the rumen to relax leading to less motility. The data does not support this hypothesis. Although there is very little data regarding bismuth in ruminants, scientists have exhaustedly exam ined its effects in human and mice models. In mice, bismuth ions have been reported to bind to gastrin compounds inhibiting the effects of the gastrin compound (Pannequin et al., 2004) Gastrin is a peptide hormone that is involved w ith the secretion of gastric juices and gut motility in monogastrics and also

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91 ruminants (Ozturk et al., 2013) There were numerical reductions in flow to the omasum when BSS was provided; however, no significant differences were observed in the current experiment. Organic matter truly digested in the rumen was not affected by the addition of NPN, NPN source or BSS ( P > 0.05). Microbial N flow to the omasum was not im pacted by the addition of NPN, NPN source, or BSS ( P > 0.05). This is in agreement with Li et al. ( 2013) who provide d Merino lambs with urea or calcium ammonium nitrate. The authors reported that when sheep were provided a barley based diet, no differences in microbial N flow were observed. In fact several research groups have reported that microbial N flows are similar for cattle and sheep consuming urea or nitrate as NPN sources (Nolan et al., 2010; Li et al., 2012; Olijhoek et al., 2016) It was unexpected that the addition of NPN did not increase microbi al N flow out of the rumen. Researchers have reported that when calcium ammonium nitrate was provided to sheep consuming oaten chaff (4.1% CP), microbial N flow was increased by more than 160% (Nguyen et al., 2016) It is unclear why no changes in microbial N were observed in the current experimen t. Efficiency of microbial N (g/kg of OMTDR) was unchanged ( P > 0.05) by treatment. In conclusion, BSS did not have any effects on ruminal fermentation. To the best r uminants. Further research should evaluate BSS on ruminal fermentation of cattle consuming high sulfur, concentrate based diets. The addition of eCAN had negative effects on ruminal fermentation of cattle consuming bahiagrass hay and molasses. This data in dicates that eCAN may not be a viable NPN source for ca ttle consuming poor

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92 quality hay based diets. Future research should inquire about the differences in the effect of nitrate on differing sources and conservation of forages.

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93 Table 3 1 Analyzed 1 chemical co mposition of the basal diet fed to cattle while in the dry lot and while confined in the UF NFREC Beef Unit Pavilion Item Bahiagrass hay on pasture (average SD) Bahiagrass hay while confined (average SD) Sugar cane molasses (average SD) DM, % 91.6 0.15 94.0 0.52 79.2 0.71 OM, % DM 91.4 0.11 93.9 0.51 84.2 0.35 CP, % DM 9.3 0.25 9.6 0.26 6.8 0.57 NDF, % DM 71.1 0.90 71.9 1.87 ADF, % DM 38.5 0.72 39.1 0.27 TDN, % DM 54.0 0.0 53.8 0.44 76.5 0.71 Sulfur, % DM 0.24 0.01 0.31 0.01 0.78 0.98 Nitrate % DM <0.03 <0.03 0.08 0.06 1 Analyzed by a commercial laboratory using a wet chemistry package (Dairy One, Ithaca, NY).

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94 Table 3 2 Effect of BSS 1 and eCAN 2 on ruminal gas cap H 2 S concentration and rectal temperature Treatment 3 P value 4 Item NCTRL U NIT UB NITB SEM 5 TRTT NPN NS B BN H 2 S 6 d 0.44 0.52 0.31 0.39 0.37 0.121 0.788 0.518 0.093 0.599 0.158 H 2 S 7 d 0.06 0.07 0.03 0.07 0.05 0.021 0.999 0.126 0.698 0.658 H 2 S 7 d 0.13 0.03 0.02 0.05 0.10 0.036 0.056 0.678 0.211 0.392 H 2 S 7 d 0.31 0.09 0.08 0.14 0.11 0.077 0.018 0.847 0.632 0.863 H 2 S 7 d 0.06 0.07 0.07 0.07 0.08 0.012 0.532 0.997 0.576 0.757 Temperature 8 C 38.15 38.40 38.35 38.23 38.15 0.136 0.180 0.057 0.291 0.009 0.826 1 BSS = bismuth subsalicylate. 2 eCAN = encapsulated calcium ammonium nitrate (5Ca(NO 3 ) 2 NH 4 NO 3 ) 65.1% nitrate DM basis 3 NCTRL = ad libitum bahiagrass hay + 2.7 g/kg of BW of molasses (basal diet); U = basal diet + 182 mg/kg of BW of urea; NIT = basal diet + 3 50 mg/kg of BW of NO 3 ; UB = U + 58.4 mg/kg of BW of BSS; NITB = NIT + 58.4 mg/kg of BW of BSS. 4 Observed significance levels for: TRTT = treatment by time interaction; NPN = effect of NPN, NCTRL vs the mean of U + NIT + UB + NITB; NS = effect of NPN source (excludes NCTRL); B = effect of BSS (excludes NCTRL); BN = interaction of BSS and eCAN 5 NCTRL, U, NIT, UB, and NITB had 10, 10, 10, 9, and 8 experimental units, respectively; largest SEM was provided 6 H 2 S on d 0 was analyzed in the ruminal gas cap at h 0, 3, 6, 9, and 12 post feeding molasses 7 H 2 S on d 1, 2, 3, and 14 was analyzed in the ruminal gas cap just prior to feeding molasses. 8 Rectal temperature recorded at 12 time points over 72 h to represent every 2 h post feeding for 24 h.

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95 Table 3 3 Effect of BSS 1 and eCAN 2 on DMI and ruminal fermentation and blood parameters Treatment 3 P value 4 Item 5 NCTRL U NIT UB NITB SEM 6 TRTT NPN NS B BN Hay DMI, kg/d 4.91 4.94 5.13 5.35 4.59 0.459 0.851 0.526 0.884 0.292 Total DMI, kg/d 6.09 6.12 6.31 6.52 5.98 0.475 0.767 0.708 0.932 0.429 Ruminal pH 6.66 6.62 6.63 6.66 6.62 0.056 < 0.0 01 0.502 0.690 0.667 0.505 NH 3 N, mM 0.84 3.34 2.08 3.49 2.48 0.565 <0.0 01 <0. 001 0.001 0.375 0.673 BUN, mg/dL 6.41 10.49 9.73 10.33 10.16 0.984 <0.001 <0.001 0.463 0.836 0.640 1 BSS = bismuth subsalicylate. 2 eCAN = encapsulated calcium ammonium nitrate (5Ca(NO 3 ) 2 NH 4 NO 3 ) 65.1 % nitrate DM basis 3 NCTRL = ad libitum bahiagrass hay + 2.7 g/kg of BW of molasses (basal diet); U = basal diet + 182 mg/kg of BW of urea; NIT = basal diet + 350 mg/kg of BW of NO 3 ; UB = U + 58.4 mg/kg of BW of BSS; NITB = NIT + 5 8.4 mg/kg of BW of BSS. 4 Observed significance levels for: TRTT = treatment by time interaction; NPN = effect of NPN, NCTRL vs the mean of U + NIT + UB + NITB; NS = effect of NPN source (excludes NCTRL); B = effect of BSS (excludes NCTRL); BN = interacti on of BSS and eCAN 5 Ruminal fluid and bl ood samples were collected at 12 time points over 72 h to represent every 2 h post feeding for 24 h. 6 DMI was recorded from d 13 to 17 of each period when cattle were housed in the UF NFREC Beef Unit Pavilion

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96 Treatment 4 P value 5 Item 6 NCTRL U NIT UB NITB SEM 7 TRTT NPN NS B BN VFA, m M Acetate 37.84 41.39 39.10 40.01 37.10 1.590 0.491 0.173 0.018 0.120 0.769 Propionate 9.00 9.81 9.13 9.54 8.67 0.475 0.392 0.373 0.014 0.228 0.753 Butyrate 7.90 7.39 7.04 7.67 7.21 0.500 0.613 0.087 0.190 0.473 0.862 BCVFA 8 0.25 0.24 0.19 0.25 0.21 0.031 0.324 0.185 0.009 0.267 0.878 Valerate 0.61 0.64 0.82 0.56 0.72 0.098 0.035 0.311 0.025 0.207 0.939 Total VFA, m M 55.60 59.49 56.30 58.05 53.94 2.155 0.669 0.359 0.011 0.171 0.734 A:P 4.27 4.27 4.34 4.26 4.36 0.139 0.008 0.715 0.309 0.957 0.981 1 BSS = bismuth subsalicylate. 2 eCAN = encapsulated calcium ammonium nitrate (5Ca(NO 3 ) 2 NH 4 NO 3 ) 65.1% nitrate DM basis 3 A:P = acetate to propionate ratio. 4 NCTRL = ad libitum bahiagrass hay + 2.7 g/kg of BW of molasses (basal diet); U = basal diet + 182 mg/kg of BW of urea; NIT = basal diet + 350 mg/kg of BW of NO 3 ; UB = U + 58.4 mg/kg of BW of BSS; NITB = NIT + 58.4 mg/kg of BW of BSS. 5 Observed significance levels for: TRTT = treatment by time interaction; NPN = effect of NPN, NCTRL vs the mean of U + NIT + UB + NITB; NS = effect of NPN source (excludes NCTRL); B = effect of BSS (excludes NCTRL); BN = interaction of BSS and eCAN. 6 Ruminal fluid samples collected at 12 time points over 72 h to represent every 2 h post feeding for 24 h. 7 NCTRL, U, NIT, UB, and NITB had 10, 10, 10, 9, and 8 experimental units, respectively; largest SEM was provided. 8 BCVFA = branched chain VFA: isobutyrate + isovalerate + 2 methylbutyrate Table 3 4 Effect of BSS 1 and eCAN 2 on ruminal VFA concentration and A:P 3

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97 Treatment 3 P value 4 Item 5 NCTRL U NIT UB NITB SEM 6 TRTT NPN NS B BN VFA, m ol/100 mol Acetate 67.99 69.55 69.34 68.90 68.66 0.868 0.02 3 0.054 0.670 0.220 0.97 9 Propionate 16.10 16.43 16.19 16.36 15.97 0.473 0.00 6 0.7 10 0.33 9 0.6 60 0.81 8 Butyrate 14.32 12.51 12.61 13.32 13.59 0.823 0.29 3 0.032 0.73 5 0.11 8 0.88 1 BCVFA 7 0.44 0.40 0.33 0.43 0.38 0.053 0.217 0.07 1 0.06 3 0.15 5 0.79 8 Valerate 1.12 1.10 1.50 0.97 1.35 0.18 1 0.086 0.446 0.00 5 0.29 7 0.981 1 BSS = bismuth subsalicylate. 2 eCAN = encapsulated calcium ammonium nitrate (5Ca(NO 3 ) 2 NH 4 NO 3 ) 65.1% nitrate DM basis 3 NCTRL = ad libitum bahiagrass hay + 2.7 g/kg of BW of molasses (basal diet); U = basal diet + 182 mg/kg of BW of urea; NIT = basal diet + 350 mg/kg of BW of NO 3 ; UB = U + 58.4 mg/kg of BW of BSS; NITB = NIT + 58.4 mg/kg of BW of BSS. 4 Ob served significance levels for: TRTT = treatment by time interaction; NPN = effect of NPN, NCTRL vs the mean of U + NIT + UB + NITB; NS = effect of NPN source (excludes NCTRL); B = effect of BSS (excludes NCTRL); BN = interaction of BSS and eCAN. 5 Rumina l fluid samples collected at 12 time points over 72 h to represent every 2 h post feeding for 24 h. 6 NCTRL, U, NIT, UB, and NITB had 10, 10, 10, 9, and 8 experimental units, respectively; largest SEM was provided. 7 BCVFA = branched chain VFA: isobutyrate + isovalerate + 2 methylbutyrate Table 3 5 Effect of BSS 1 and eCAN 2 on ruminal VFA molar proportions

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98 Treatment 3 P value 4 Item NCTRL U NIT UB NITB SEM 6 NPN NS B BN DM Intake, kg/d 5.99 6.19 5.92 5.47 5.17 0.495 0.554 0.553 0.134 0.969 Flow, kg/d 5.16 5.43 4.90 4.77 5.92 0.794 0.911 0.680 0.818 0.276 OM Intake, kg/d 5.58 5.77 5.52 5.09 4.80 0.473 0.556 0.551 0.134 0.969 Flow, kg/d 4.16 4.47 3.93 3.86 4.96 0.723 0.849 0.687 0.761 0.242 OMTDR 5 kg/d 1.62 1.56 1.87 1.47 1.00 0.273 0.600 0.754 0.081 0.146 Microbial N flow, g/d 14.33 15.94 14.68 16.41 21.40 2.948 0.365 0.512 0.218 0.275 Microbial efficiency, g of MN 6 / kg of OMTDR 10.63 8.84 8.77 10.86 13.26 5.549 0.973 0.827 0.549 0.818 1 BSS = bismuth subsalicylate. 2 eCAN = encapsulated calcium ammonium nitrate (5Ca(NO 3 ) 2 NH 4 NO 3 ) 65.1% nitrate DM basis 3 NCTRL = ad libitum bahiagrass hay + 2.7 g/kg of BW of molasses (basal diet); U = basal diet + 182 mg/kg of BW of urea; NIT = basal diet + 350 mg/kg of BW of NO 3 ; UB = U + 58.4 mg/kg of BW of BSS; NITB = NIT + 58.4 mg/kg of BW of BSS. 4 Observed significance levels for: TRTT = treatment by time interaction; NPN = effect of NPN, NC TRL vs the mean of U + NIT + UB + NITB; NS = effect of NPN source (excludes NCTRL); B = effect of BSS (excludes NCTRL); BN = interaction of BSS and eCAN. 5 OMTDR = OM truly digested in the rumen. 6 MN = microbial N Table 3 6 Effect of BSS 1 and eCAN 2 on intake, flow at the omasal canal, and microbial N flow and efficiency

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99 Figure 3 1 Effect of bismuth sub s alicylate (BSS) and eCAN (encapsulated calcium ammonium nitrate; 65.1% N DM basis) on ruminal pH of cannulated Angus crossbred cattle fed bahiagrass hay ad libitum with 2.7 g/kg BW of molasses. A trea tment time post feeding interaction was observed ( P = 0.0002). NCTRL = ad libitum bahiagrass hay + 2.7 g/kg of BW of molasses (basal diet); U = basal diet + 182 mg/kg of BW of urea; NIT = basal diet + 350 mg/kg of BW of NO3 ; UB = U + 58.4 mg/kg of BW of BSS; NITB = NIT + 58.4 mg/kg of BW of BSS. Error bars represent the SEM for treatment time postfeeding interaction. NCTRL, U NIT, UB, and NITB had 10, 10, 10, 9, and 8 experimental units, respectively; largest SEM was provided. = pH of NCTRL was lesser compared to U, NIT, and UB ( P < 0.03) ; U and UB had greater pH than NITB ( P < 0.04) ¤ = NCRTL and U had lesser pH compared with UB ( P < 0.05) § = NCTRL had greater pH than U ( P < 0.05) = NCTRL had greater pH compared with NIT ( P < 0.04) 6.00 6.30 6.60 6.90 0 2 4 6 8 10 12 14 16 18 20 22 pH Time postfeeding, h NCTRL U NIT UB NITB ¤ §

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100 Figure 3 2 Effect of bismuth sub s alicylate (BSS) and eCAN (encapsulated calcium ammonium nitrate; 65.1% N DM basis) on ruminal NH 3 N concentrations of cannulated Angus crossbred cattle fed bahiagrass hay ad libitum with 2.7 g/kg BW of molasses. A treatment time postfeeding interaction was observed ( P < 0.0001). NCTRL = ad li bitum bahiagrass hay + 2.7 g/kg of BW of molasses (basal diet); U = basal diet + 182 mg/kg of BW of urea; NIT = basal diet + 350 mg/kg of BW of NO3 ; UB = U + 58.4 mg/kg of BW of BSS; NITB = NIT + 58.4 mg/kg of BW of BSS. Error bars represent the SEM for t reatment time postfeeding interaction. NCTRL, U, NIT, UB, and NITB had 10, 10, 10, 9, and 8 experimental units, respectively; largest SEM was provided. = U and UB had greater concentrations of NH 3 N compared to all other treatments ( P < 0.0001); NIT an d NITB had greater concentrations of NH 3 N than NCTRL ( P < 0.02) ¤ = NCTRL had lesser concentration of NH 3 N compared with UB and NITB ( P < 0.05) § = NCTRL had lesser concentration of NH 3 N compared with all other treatments ( P 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 0 2 4 6 8 10 12 14 16 18 20 22 NH 3 N, m M Time postfeeding, h NCTRL U NIT UB NITB § ¤

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101 Figure 3 3 Effect of bismuth sub s alicylate (BSS) and eCAN (encapsulated calcium ammonium nitrate; 65.1% N DM basis) on blood urea nitrogen (BUN) concentrations of cannulated Angus crossbred cattle fed bahiagrass hay ad libitum with 2.7 g/kg BW of molasses. A treatment time postfeeding interactio n was observed ( P < 0.0001). NCTRL = ad libitum bahiagrass hay + 2.7 g/kg of BW of molasses (basal diet); U = basal diet + 182 mg/kg of BW of urea; NIT = basal diet + 350 mg/kg of BW of NO3 ; UB = U + 58.4 mg/kg of BW of BSS; NITB = NIT + 58.4 mg/kg of BW of BSS. Error bars represent the SEM for treatment time postfeeding interaction. NCTRL, U, NIT, UB, and NITB had 10, 10, 10, 9, and 8 experimental units, respectively; largest SEM was provided. ¤ = NCTRL had lesser concentrations of BUN compared with NIT and NITB ( P < 0.05); and U had lesser concentrations of BUN compared to NITB ( P < 0.05). £ = NCTRL had lesser concentrations of BUN compared with U, UB, and NITB ( P < 0.05); and NIT had lesser concentrati ons compared with U and UB ( P < 0.05). = NCTRL had lesser concentrations of BUN compared to all other treatments ( P < 0.05). § = U and UB had greater concentrations of BUN compared to NIT ( P < 0.05). = U had greater concentrations of BUN compared with NIT, UB, and NITB ( P < 0.05). = NCTRL had lesser BUN concentration compared with U, NIT, and UB ( P < 0.05) ; and U and UB had greater concentrations of BUN compared to NITB ( P < 0.05) = NIT had greater BUN concentration compared to NCTRL ( P < 0.05). ¢ = NCTRL had lesser concentration of BUN compared with U and NITB ( P ; and NITB had greater concentration of BUN compared with U, NIT, and UB ( P < 0.05) 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 0 2 4 6 8 10 12 14 16 18 20 22 BUN, mg/dL Time postfeeding, h NCTRL U NIT UB NITB * * * ¤ £ § ¢

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102 Figure 3 4 Effect of bismuth sub s alicylate (BSS) and eCAN (encapsulated calcium ammonium nitrate; 65.1% N DM basis) on the ruminal acetate to propionate ratio (A:P) of cannulated Angus crossbred cattle fed bahiagrass hay ad libitum with 2.7 g/kg BW of molasses. A treatment time postfeed ing interaction was observed ( P = 0.0087). NCTRL = ad libitum bahiagrass hay + 2.7 g/kg of BW of molasses (basal diet); U = basal diet + 182 mg/kg of BW of urea; NIT = basal diet + 350 mg/kg of BW of NO3 ; UB = U + 58.4 mg/kg of BW of BSS; NITB = NIT + 58. 4 mg/kg of BW of BSS. Error bars represent the SEM for treatment time postfeeding interaction. NCTRL, U, NIT, UB, and NITB had 10, 10, 10, 9, and 8 experimental units, respectively; largest SEM was provided. = NCTRL had a greater A:P compared with NIT ( P < 0. 05 ); NIT and NITB had greater concentrations of NH 3 N than NCTRL ( P < 0.02). ¤ = NCTRL and U had a lesser A:P compared with NITB ( P < 0.03 ). § = NCTRL U, and UB had a lesser A:P compared with NIT ( P 2.50 3.00 3.50 4.00 4.50 5.00 5.50 0 2 4 6 8 10 12 14 16 18 20 22 A:P Time postfeeding, h NCTRL U NIT UB NITB ¤ §

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103 Figure 3 5 Effect of bismuth sub s alicylate (BSS) and eCAN (encapsulated calcium ammonium nitrate; 65.1% N DM basis) on ruminal acetate molar proportions of cannulated Angus crossbred cattle fed bahiagrass hay ad libitum with 2.7 g/kg BW of molasses. A treatment time postfeeding interaction was observed ( P = 0.0 227 ). NCTRL = ad libitum bahiagrass hay + 2.7 g/kg of BW of molasses (basal diet); U = basal diet + 182 mg/kg of BW of urea; NIT = basal diet + 350 mg/kg of BW of NO3 ; UB = U + 58.4 mg/kg of BW of BSS; NITB = NIT + 58.4 mg/kg of BW of BSS. Error bars represent the SEM for treatment time postfeeding interaction. NCTRL, U, NIT, UB, and NITB had 10, 10, 10, 9, and 8 experimental units, respectively; largest SEM was provided. = NCTRL had lesser concentration than U, UB, and NITB ( P < 0.0 4 ) ¤ = NCTRL had lesser concentration compared with U, NIT, and NITB ( P < 0.03). § = NCTRL had lesser concentration than NITB ( P < 0.03 ) = NCTRL and NITB had lesser concentrations compared w ith U and NIT ( P < 0.05) CTRL, UB, and NITB had lesser concentrations than NIT ( P < 0,03); UB and NITB had lesser concentrations than U and NIT ( P < 0.05) £ = U had greater concentration compared with UB and NITB ( P < 0.05) 60.00 65.00 70.00 75.00 0 2 4 6 8 10 12 14 16 18 20 22 Acetate, mol/100mol Time postfeeding, h NCTRL U NIT UB NITB ¤ § £

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104 Figure 3 6 Effect of bismuth sub s alicylate (BSS) and eCAN (encapsulated calcium ammonium nitrate; 65.1% N DM basis) on ruminal propionate molar proportions of cannulated Angus crossbred cattle fed bahiagrass hay a d libitum with 2.7 g/kg BW of molasses. A treatment time postfeeding interaction was observed ( P = 0.0059). NCTRL = ad libitum bahiagrass hay + 2.7 g/kg of BW of molasses (basal diet); U = basal diet + 182 mg/kg of BW of urea; NIT = basal diet + 350 mg/k g of BW of NO3 ; UB = U + 58.4 mg/kg of BW of BSS; NITB = NIT + 58.4 mg/kg of BW of BSS. Error bars represent the SEM for treatment time postfeeding interaction. NCTRL, U, NIT, UB, and NITB had 10, 10, 10, 9, and 8 experimental units, respectively; large st SEM was provided. = NIT had greater concentration compared to all other treatments ( P < 0.0 3 ). ¤ = NITB had less concentration than U and UB ( P < 0.03). § = NITB had lesser concentration than U ( P = 0.02 ). = NIT had lesser concentration compared with UB ( P < 0.0 2 ) 14.00 16.00 18.00 20.00 0 2 4 6 8 10 12 14 16 18 20 22 Propionate, mol/100mol Time postfeeding, h NCTRL U NIT UB NITB ¤ §

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105 CHAPTER 4 EFFECTS OF BISMUTH SUBSALICYLATE AND CALCIUM AMMONIUM NITRATE ON ENTERIC METHANE PRODUCTION AND NUTRIENT DIGESTIBILITY OF BEEF STEERS Introduction There is a great desire to mitigate enteric CH 4 production of ruminants for environmental concerns and the potential to reduce a portion of GE that would otherwise be lost. Beauchemin et al. ( 2010) reported that approximately 80% of the total greenhouse gases ( GHG ) produced by the beef industry is related to the cow calf sector, in which, a majority of cattle are consuming a forage based diet. Nitrate has been reported to reduce enteric CH 4 production in ruminants consuming several different mixed rations (van Zijderveld et al., 2010; Newbold et al., 2014; Lee et al., 2015b) ; however, most re search disregards the time prior to weaning when 70% of the energy requirements of beef cattle are consumed (Shike, 2013) When considering the ultimate end product of any experiment, the benefit to soc iety nitrate may be a viable option for cattle producers to provide a needed NPN source while reducing the environmental impact of production. Bismuth subsalicylate ( BSS ) has been reported to aid in human digestive issues by binding to sulfide in the gastro intestinal tract, thereby reducing a known gut irritant, H 2 S, and by acting as an antimicrobial agent against Helicobacter pylori To the best of on ruminal ferment ation, in vivo. Using ruminal fermentation in vitro systems, researchers have observed that BSS can have mitigating effects on H 2 S (Ruiz Moreno et al., 2015) ; however, no peer reviewed data has evaluated the effects of BSS on in vitro CH 4 production.

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106 It was hypothesized that the addition of encapsulated calcium ammonium nitrate ( eCAN ) to a bahiagrass diet supplemented with molasses will reduce enteric methane production; furthermore, BSS may have antimicrobial effects, which may lead to a reduction in CH 4 production. The objective of this experiment was to evaluate the effects of BSS and eCAN, in combination, on appare nt total tract digestibility of nutrients, and enteric CH 4 production. Materials and Methods All procedures involving animals were approved by the Animal Care and Use Committee of the Institute of Food and Agricultural Sciences at the University of Florida Experimental D esign, A nimals, and T reatments Twenty five Bos taurus Bos indicus (3 3 5 46 kg of BW; average BW SD) steers were used in a randomized block design with a 2 2 + 1 factorial arrangement of treatments and 2 experimental periods (block) o f 20 d each with a 7 d washout period in between where cattle only received bahiagrass ( Paspalum notatum ) hay and sugar cane molasses (chemical composition presented on Table 4 1 ). On d 0 of each period, BW was recorded to determine the amounts of urea, eCAN, and BSS to provide. From d 0 to 13, steers underwent adaptation to treatment and facilities. Within this 14 d adaptation period, steers receiving NPN were adapt ed in the following manner: on d 0 and 1, cattle received 20% of their total supplemental N; on d 2 and 3, cattle received 40% of their total supplemental N; on d 4 and 5, cattle received 60% of their total supplemental N; on d 6 and 7, cattle received 80% of their total supplemental N; and beginning on d 8, cattle were receiving 100% of their total supplemental N as urea or eCAN. From d 7 to 13 steers were equipped with training CH 4 collection canisters. Starting on d 13, hay and molasses samples were coll ected at time of feeding until d 16.

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107 From d 14 to 17 at 0700 and 1500 h, fecal samples were collected. From d 14 to 18, animals were equipped with CH 4 collection canisters. Beginning with the first period, all steers were stratified by weight and breed, and randomly assigned to 1 of 5 treatments: 1) NCTRL no added NPN or BSS; 2) U urea supplemented at 182 mg/kg of BW; 3) NIT nitrate, in the form of e CAN, supplemented at 350 mg/kg of BW; 4) UB urea supplemented at 182 mg/kg of BW and BSS supplemented at 58.4 mg/kg of BW; and 5) NITB nitrate, in the form of eCAN, supplemented at 350 mg/kg of BW and BSS supplemented at 58.4 mg/kg of BW. Treatments U, N IT, UB, and NITB were isonitrogenous. The amount of and adaptation to eCAN and BSS provided was based on the metabolism experiment in Chapter 3 of this dissertation. When these amounts of eCAN and BSS were chosen, it was expected that nitrate would be prov ided at 2.0% and BSS at 0.33% of the total DMI. After evaluating the DMI during the experimental periods the steers receiving eCAN and BSS consumed 1.54% of the total DMI of nitrate and 0.25% of the total DMI of BSS. After experimental period 1, the 25 ste ers were re randomized for experimental period 2, which was identical to experimental period 1 in regards to treatments and procedures. To ensure that the additional NPN and BSS were consumed in full, urea, eCAN, and BSS were weighed and mixed into the ind had ad libitum access to hay and received 1.07 kg/d of sugar cane molasses. During the experiment, steers were housed 5 per pen in the University of Florida North Florida Research and Education Center Feed Effic iency Facility in Marianna, FL. Daily DMI of bahiagrass hay was recorded by the GrowSafe system (GrowSafe Systems Ltd.,

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108 Airdrie, Alberta, Canada). Steers were housed in pens of 108 m 2 equipped with 2 GrowSafe feed bunks and a single water trough. Sampling P rocedures All protocols and procedures used for collecting samples and data from steers were used in an identical manner throughout both experimental periods. Apparent total tract digestibility of nutrients and retention of S Apparent total tract digestib ility of nutrients was determined using indigestible NDF ( iNDF ) as an internal marker. Feed samples were collected daily from d 13 to 16 and fecal samples were collected at 0700 and 1500 h from d 14 to 17 either by rectal grab or from the ground, inside th e pen, immediately after the animal defecated. Hay samples were stored at 10C, and molasses and fecal samples were stored at 20C for further analysis. Hay and fecal samples were dried at 55C for 72 h in a forced air oven, ground in a Wiley mill (Arthur H. Thomas Co. Philadelphia, PA) to pass a 2 mm screen. Hay was pooled within pen and feces were pooled within steer to determine DM, OM, CP, NDF, ADF, iNDF, and S. Methane emissions Emissions of enteric CH 4 production were measured using the sulfur hexafl uoride ( SF 6 ) tracer technique (Johnson et al., 1994) from d 14 to 18 of each period. Brass permeation tube bodies (length = 4.4 cm, o.d. = 1.43 cm, i.d. = 0.79 cm, inside depth = 3.8 cm, and volume = 1.86 mL), nylon washers, a Teflon membrane, secured with a porous (2 it and a brass nut, were used in each steer. Permeation tubes were filled with approximately 2.3 g of SF 6 Permeation tubes were kept at 39C and weighed 12 times within 38 d. The average SF 6 release rate across steers was of 2.44 mg/d. Permeation tubes we re dosed via balling gun on d 7 of the first

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109 period. Gas collection canisters were constructed of polyvinyl chloride pipe to have a final volume of 2 L. The samples were collected by evacuating the collection canisters to 68.6 cmHg and connecting the canis ter to a halter, which was equipped with a crimped capillary tube that was positioned to sample, using a loop design, from both nostrils. The volume of the collection canisters and the crimped capillary tubes were designed to allow half of the vacuum to re main after 24 h. Three collection canisters and capillary tubes were used to determine environmental CH 4 and SF 6 concentrations. It has been proposed by Haisan et al. ( 2014) to consider any animal with at least 2 d of valid CH 4 measurements. For the current exper iment, only steers with at least 3 successful d of collection and measurement were considered in the final analysis of CH 4 variables. Laboratory A nalyses All protocols and procedures used for analyzing samples and data from steers were used in an identical manner throughout both experimental periods. Concentration of DM, OM, CP, NDF, ADF, and S All hay and fecal samples were placed in a 55C forced air oven for 72 h to obtain dry samples. Dry, hot weight was used to calculate DM of the sample. The sample wa s then ground to pass through a 2 mm screen in a Wiley mill (Thomas Scientific, Swedesboro, NJ). To determine OM, 0.5 g of ground sample (in duplicate) was weighed into ceramic crucibles and placed in a 105C forced air oven for 24 h to determine sample DM Dried samples were then placed in a 650C muffle furnace for 6 h before returning to a 105C forced air oven. Hot, ashed samples were weighed and used to calculate OM.

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110 Samples of hay and feces were weighed (0.5 g in duplicate) into F57 bags (Ankom Techn amylase and sodium sulfite. Subsequent ADF analysis was performed sequentially as described by Van Soest et al. ( 1991) in an Ankom 200 Fiber Analyzer (Ankom Technology Inc .). Hay and fecal samples were analyzed for total N and S using a C H N and S analyzer by the Dumas dry combustion method (Vario Micro Cube; Elementar, Hanau, Germany). Crude protein was calculated by multiplying the N concentration of the dry sample by 6.25. Concentration of iNDF The concentration of iNDF in hay and feces was determined as described by Gregorini et al. ( 2008 ), Cole et al. ( 2011 ), and Krizsan and Huhtanen ( 2013) Briefly, 0.5 g of sample was weighed into Ankom F57 filter bags (Ankom Technology Corp. Macedon, NY) and then incubated into the rumen of a cannulated steer grazing a bahiagrass and bermudagrass ( Cyn o don dactylon ) mixed pasture for 288 h to ensure complete digestion of potentially digestible NDF. After incubation, samples were rinsed 2 times with tap water followed by 4 rinses with water filtered through a reverse osmosis system. The rinsed samples wer e then analyzed for NDF as previously described. Methane and SF 6 analyses Methane and SF 6 concentrations in collection canisters were analyzed by gas chromatography (Agilent 7820A GC; Agilent Technologies, Palo Alto, CA). A flame ionization detector and el ectron capture detector were used for CH 4 and SF 6 analysis, respectively, with a capillary column (Plot Fused Silica 25m by 0.32mm, Coating

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111 Molsieve 5A, Varian CP7536; Varian Inc. Lake Forest, CA). Injector, column, and detector temperatures for CH 4 analys is were 80, 160, and 200C, respectively. For SF 6 Temperatures were 50, 30, and 300C for the injector, column, and detector, respectively. The carrier gas for CH 4 and SF 6 was N 2 Calculations Emission of CH 4 produced by steers was determined in relation to the SF 6 tracer gas captured in the collection canisters. The following equation was used to quantify CH 4 production: Q CH4 = Q SF6 ([CH 4 ] [CH 4 ] ) ([SF 6 ] [SF 6 ] ) in which Q CH4 is considered CH 4 emissions per animal (g/d), Q SF6 is considered SF 6 release rate (mg/d), [CH 4 ] is considered the concentration of CH 4 in the animals collection canister, [CH 4 ] is considered the concentration of CH 4 in the environmental canisters, [SF 6 ] is considered the concentration of SF 6 in the animals collection canister, and [SF 6 ] is considered the concentration of SF 6 in the environmental collection canister. Apparent total tract digestibility of DM, OM, CP, NDF, and ADF, and retention of S was calculated as follows with OM total tract digestibility as an exam ple: OM digestibility (%) = 100 100 [(iNDF concentration in the feed iNDF concentration in the feces) (OM concentration in the feces OM concentration in the feed)]. Statistical A nalysis All data were analyzed as a randomized block design with a 2 2 + 1 factorial arrangement of treatments using the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC) using steer as the experimental unit. The model included the fixed effects of

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112 treatment and the random effects of pen and period. To aid in the interpr etation of data, the following contrasts were used: the effect of NPN = NCTRL vs. the mean of U, NIT, UB, and NITB; the effect of NPN source = the mean of U and UB vs. the mean of NIT and NITB; the effect of BSS = the mean of U and NIT vs. the mean of UB a nd NITB; and NPN source BSS = the mean of U and NITB vs. the mean of NIT and UB. Significance was declared at P Results and Discussion Intake and apparent total tract nutrient digestibility data are presented on Table 4 2 There was no effect ( P > 0.05) of NPN, NPN source or BSS on intake of DM, OM, NDF, or ADF during the 4 d digestibility period. This experiment was not designed to test effects on intake; therefore, it cannot be assumed that eCAN and BSS have no impact on DMI. I t appears that the effect of nitrate on intake is conditional depending upon diet type and the quality of the diet. Data reported by Lee et al. ( 2015b) indicated that heifers consuming a diet made of 55% forage (50 and 5% of barley silage and grass hay, respectively) reduced DMI by 6% for when nitrate was used as a source of NPN rather than urea. Lee et al. ( 2017b) observed that when growing steers were placed on a backgrounding diet w ith a greater concentration of forage (65% corn silage), DMI was not affected by the inclusion of eCAN at 1.25 or 2.5% of the diet DM. The difference between these two studies may be related to the source of nitrate used: Lee et al. ( 2015b) provided heifers with an unencapsulated form of calcium ammonium nitrate, whereas Lee et al. ( 2017 b ) used an encap sulated form of the same nitrate. Encapsulation of nitrate has been observed to abate the release of nitrate in vitro, compared to unencapsulated calcium ammonium nitrate, without affecting NH 3 N or total VFA concentration (Lee et al., 2017c) Likewise, encapsulation of c alcium

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113 ammonium nitrate essentially eliminated nitrite accumulation in vitro wh ere the unencapsulated form allowed accumulation to occur. It is possible that the accumulation of nitrate and nitrite in the rumen may have negative effects on intake of rumina nts due to the inhibitory actions on fiber degrading bacteria (Zhou et al., 2012) Others have reported reductions in DMI of animals consuming 78% concentrate ( 2.57% calcium ammonium nitrate; Velazco et al. 2014) and 90% concentrate when provided 2.5% eCAN, but not 1.25% (Lee et al., 2017a) As expec ted, CP intake during the 4 d digestibility period was increased with the addition of NPN ( P = 0.0021). The addition of NPN had no effect ( P > 0.05) on apparent total tract digestibility of DM, OM, NDF, and ADF. It was hypothesized that nutrient digestibil ity would have been enhanced with the addition of a NPN source; however, this effect was not observed. T he concentration of N in the basal diet was sufficient for microbial growth and development. In the current experiment, the DMI of the NCTRL steers was composed of nearly 9.8% CP, this is a drastic difference from the 4% CP oaten chaff that was provided to Merino ewes by Nguyen et al. ( 2016) Nguyen et al. (2016) reported that the addition of NPN in the form of calcium ammonium nitrate increased DM dige stibility by 12%. The authors attributed this change in digestibility to an increase in ruminal NH 3 N. It is likely that the NH 3 N concentrations in the rumen of the NCTRL steers provided a sufficient amount of N for the microbial population. Apparent tota l tract digestibility of nutrients was not affected by BSS ( P > 0.05). In a companion in vitro study, presented and discussed in Chapter 2 Experiments 2 and 3, a linear reduction in in vitro OM digestibility ( IVOMD ) was observed when BSS was included in the substrate DM up to 1.0%. It was speculated in Chapter 2 that this

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114 reduction was one of several indications that BSS was impeding fermentation at a 1.0% inclusion rate; however, in Exp. 3, when BSS was included at 0.00, 0.05, 0.10, or 0.33% of the subst rate DM, no effect of BSS on IVOMD was observed. Another research group reported that BSS, included at 1.0% of a high concentrate substrate, did not negatively affect fermentation (Ruiz Moreno et al., 2015) In Chapter 2, it was discussed the possibility that the impact of BSS on fermentation was likely diet dependent. Furthermore, it was speculated that when provided with a high forage diet or substrate, inclusion rates of BSS must be lesser than when provided with a high concentrate diet or substrate. The data from the current experiment indicates that at approximately 0.25% of DMI of a forage based diet, BSS does not impact total tract digestibility of nutrients. Further research is needed to evaluate the effects on diets differing in quality and composition. There was a negative effect of eCAN on DM ( P = 0.0152), OM ( P = 0.0214), NDF ( P = 0.0118), and ADF ( P = 0.0125 ) total tract digestibility. In the literature, there is not a consensus on the impact of nitrate on nutrient digestibility. Researchers have reported increases in DM digestibility when nitrate was included into a 35% forage diet (Li et al., 2013) while others reported no change in nutrient digestibility of diets ranging from 50% (Olijhoek et al ., 2016) to 70% forage (Klop et al., 2017; Sun et al., 2017) and, furthermore, linear reductions in DM digestibility have been reported for diets containing 55% forage (Lee et al., 2015b) More research is needed to evaluate the effects of nitrate on nutrient digestibility of cattle consuming a range of diet compositions fed ad libitum and restrictively. The reductions in nutrient digestibility observed in the current experiment may be partially the cause of an accumulation of nitrate/nitrite in the rumen

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115 and/or an increase in the concentration of H 2 in the rumen. It is evident that in the current experiment, the reductions in DM and OM total tract digestibility were largely accounted for by the reduction in digestibility of NDF and ADF. As discussed previously, nitrate and nitrite have toxic effects on certain populations of microbial species in the rumen. Several research groups have observed reductions in the relative abundance of cellulolytic microbial populations in the rumen when nitrate was provided (Marais, 1988; Zhou et al., 2012; Asanuma et al., 2015) These inhibitory effects on fiber degrading microorganisms would be a probable explanation for the reductions in nutrient digestibility in the rumen. If it was not a direct effect of nitrate/nitrite on microbial populations, it is plausible that the increase in H 2 occasionally observed when nitrate is introduced to a ruminal environment would const rain the activity of certain cellulolytic bacteria (Janssen, 2010; Guyader et al., 2015) It is probable that the reduction in fiber digestion, leading to reduction in DM and OM, was a result of both toxic effects of nitrate/nitrite on ruminal microorganisms and an inc rease in H 2 concentration in the rumen. Total tract digestibility of CP was reduced by 8% when eCAN was provided as a NPN source compared with urea ( P = 0.0143). It is possible that this is an actual difference and eCAN does reduce the digestibility of CP in the total tract; however, an observation was made when collecting feces from the steers during the digestibility portion of this experiment: eCAN prills were found, intact, in the feces. By observing eCAN prills in the feces of these animals, it was con sidered that the amount of N analyzed in the feces would be greater, thereby, causing a reduction in the digestibility of CP in the total tract. Other researchers have reported that the percentage of intake N

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116 retention is linearly increased as eCAN is prov ided up to 3% of DMI (Lee et al., 2015b) ; however, the heifers used were provided a basal diet that had a greater inclusion of concentrate (45% concentrate) compared w ith the current experiment (approximately 14% molasses). It is possible that the encapsulation of the nitrate used in the current experiment was over protected, allowing some nitrate to pass through the total tract unmetabolized. Future research should foc us on the effects of differing degrees of protection of nitrate on ruminal fermentation, digestibility, performance, and CH 4 production. There was no effect ( P > 0.05) of NPN, NPN source or BSS on the intake of S during the 4 d digestibility portion of th is experiment; however, S retention was reduced ( P = 0.0034) by 8% with the inclusion of BSS. It has been proposed that BSS in humans reacts in the following manner: 2 Bi 3+ + 3 H 2 2 S 3 + 6 H + Bismuth sulfide is an insoluble, black salt that, theoretic ally, is not absorbed through the intestinal wall (Suarez et al., 1998) In the rumen of cattle that are consuming diets which would promote the production of H 2 S (high S, high concentrate diets; lesser ruminal pH), it may be speculated that BSS acts in a similar manner (Ruiz Mor eno et al., 2015) ; however, it is not evident what reaction is occurring when H 2 S is not present, or is present in extremely reduced concentrations, such as that observed in the metabolism experiment presented and discussed in Chapter 3. Whatever the mo de of action, the data indicates that BSS is shifting a portion of the intake S that would be apparently retained to pass though the gastro intestinal tract and be expunged in the feces of the animal.

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117 Methane production parameters are presented on Table 4 3 It has been widely accepted that nitrate reduces enteric methane emissions of ruminants (Hulshof et al., 2012; Newbold et al., 2014; Lee et al., 2015b) This reduction in CH 4 production is thought to be related to 2 factors: nitrate acting as a H 2 sink (Leng, 2008) and the inhibitory effects of nitrate, more specifically nit rite, on methanogens (Duin et al., 2016) In the current experiment, a reduction ( P = 0.0393) in CH 4 (g/d) was observed when eCAN was provided as a NPN source rather than urea. When evaluat ing grams of CH 4 produced per kilogram of OM intake and OM digested, there was no effect of NPN source ( P > 0.05). Although there was not a statistical difference in OM among the steers, which consumed eCAN rather than urea, there was a numerical reduction and since this CH 4 variable (g/kg OM intake) is calculated, the similarity among treatments is reasonable. Furthermore, when the effect of OM digestibility was considered, eCAN was unsuccessful at reducing emission intensity. When metabolic BW was taken into account, eCAN reduced ( P = 0.0513) CH 4 production (g/kg of BW 0.75 ) by 11% compared with urea. There was no effect of NPN or BSS on any of the CH 4 variables evaluated in this experiment. In conclusion, the data indicate that the addition of BSS d oes not negatively affect apparent total tract digestibility of nutrients, nor does BSS appear to mitigate enteric CH 4 emissions. Further research is needed to evaluate the effects of BSS on differing dietary compositions in regards to nutrient digestibili ty. It may be plausible in grains, to mitigate the negative effects associated with high S diets. The inclusion of eCAN in the diets of ruminants provided forage based diets may reduce daily emissions

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118 of CH 4 (g/d) ; however, the effect of nitrate on digestibility should be considered. Future research should focus on the effects of nitrate on grazing cattle and cattle consuming different forage types.

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119 Table 4 1 Analyzed chemical composition of bahiagrass hay and sugar cane molasses Item Bahiagrass hay (average SD) Sugar cane molasses 1 (average SD) DM, % 88.8 1.69 79.2 0.71 OM, % DM 94.9 0.1 8 84.2 0.35 CP, % DM 10.2 0.72 6.8 0.57 NDF, % DM 71.8 2.87 ADF, % DM 35.2 3.22 Sulfur, % DM 0.29 0.0 2 0.78 0.98 Nitrate % DM 0.08 0.06 1 Analyzed by a commercial laboratory using a wet chemistry package (Dairy One, Ithaca, NY).

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120 Treatment 3 P value 4 Item NCTRL U NIT UB NITB SEM 5 NPN NS B BN 4 d intake, kg/d DM 8.31 8.17 7.79 8.43 7.92 0.442 0.63 6 0.321 0.666 0.887 OM 7.76 7.63 7.26 7.87 7.39 0.418 0.632 0.320 0.666 0.888 CP 0.81 0.98 0.94 1.00 0.96 0.043 0.002 0.353 0.714 0.953 NDF 5.11 5.00 4.71 5.18 4.80 0.323 0.60 9 0.306 0.674 0.907 ADF 2.52 2.46 2.30 2.54 2.35 0.162 0.576 0.288 0.690 0.931 S 0.039 0.038 0.037 0.039 0.037 0.001 0.62 7 0.290 0.727 0.963 Digestibility, % DM 52.58 52.99 51.65 52.66 50.66 0.658 0.424 0.015 0.323 0.620 OM 54.09 54.87 53.59 54.28 52.69 0.601 0.727 0.021 0.222 0.795 CP 34.66 46.82 43.67 46.14 41.89 1.444 <0.00 1 0.014 0.401 0.707 NDF 52.09 52.22 50.81 52.40 50.98 0.536 0.419 0.01 2 0.744 0.990 ADF 49.36 50.16 47.25 50.25 49.30 0.737 0.881 0.013 0.155 0.193 S retention 6 % 61.94 62.41 61.31 57.49 56.01 1.642 0.158 0.437 0.003 0.908 1 BSS = bismuth subsalicylate. 2 eCAN = encapsulated calcium ammonium nitrate (5Ca(NO 3 ) 2 NH 4 NO 3 ) 65.1% nitrate DM basis 3 NCTRL = ad libitum bahiagrass hay + 2.7 g/kg of BW of molasses (basal diet); U = basal diet + 182 mg/kg of BW of urea; NIT = basal diet + 350 mg/kg of BW of NO 3 ; UB = U + 58.4 mg/kg of BW of BSS; NITB = NIT + 58.4 mg/kg of BW of BSS. 4 Ob served significance levels for: NPN = effect of NPN, NCTRL vs the mean of U + NIT + UB + NITB; NS = effect of NPN source (excludes NCTRL); B = effect of BSS (excludes NCTRL); BN = interaction of BSS and eCAN. 5 NCTRL, U, NIT, UB, and NITB had 10, 10, 10, 10 and 10 experimental units, respectively; largest SEM was provided. 6 S absorption was calculated as follows: 100 (100 (([diet iNDF] [fecal iNDF]) ([fecal S] [diet S]))) Table 4 2 Effect of BSS 1 and eCAN 2 on nutrient intake and apparent total tract digestibility, and sulfur intake and retention

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121 Treatment 3 P value 4 Item 5 NCTRL U NIT UB NITB SEM 6 NPN NS B BN CH 4 emissions, g/d 121.16 117.27 115.57 134.09 100.04 10.986 0.673 0.039 0.951 0.121 CH 4 emissions, g/kg OMI 7 15.35 15.42 16.44 15.59 13.67 1.197 0.954 0.689 0.264 0.209 CH 4 emissions, g/ kg OMD 8 28.72 28.63 30.33 29.36 25.70 2.246 0.921 0.649 0.372 0.222 CH 4 emissions, g/ kg MBW 9 1.51 1.39 1.44 1.55 1.18 0.126 0. 341 0. 051 0. 689 0. 063 1 BSS = bismuth subsalicylate. 2 eCAN = encapsulated calcium ammonium nitrate (5Ca(NO 3 ) 2 NH 4 NO 3 ) 65.1% nitrate DM basis 3 NCTRL = ad libitum bahiagrass hay + 2.7 g/kg of BW of molasses (basal diet); U = basal diet + 182 mg/kg of BW of urea; NIT = basal diet + 350 mg/kg of BW of NO 3 ; UB = U + 58.4 mg/kg of BW of BSS; NITB = NIT + 58.4 mg/kg of BW of BSS. 4 Observed significance levels for: NPN = effect of NPN, NCTRL vs the mean of U + NIT + UB + NITB; NS = effect of NPN source (excludes NCTRL); B = effect of BSS (excludes NCTRL); BN = interaction of BSS and eCAN. 5 CH 4 was determined from the average of at least three out of five 24 h periods of breath sample collection 6 NCTRL, U, NIT, UB, an d NITB had 10, 8, 7, 8, and 7 exper imental units, respectively; largest SEM was provided. 7 OMI = OM intake 8 OMD = OM digested. 9 MBW = BW 0.75 Table 4 3 Effect of BSS 1 and eCAN 2 on enteric CH 4 production of beef steers

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122 CHAPTER 5 EFFECTS OF BISMUTH SUBSALICYLATE AND CALCIUM AMMONIUM NITRATE ON LIVER MINERAL CONCENTRATION AND PERFORMANCE OF GROWING BEEF HEIFERS Introduction There is a growing desire to reduce the environmental impacts of beef production. One methodology that has been rigorousl y evaluated and reported is the addition of nitrate to the diets of cattle in place of traditional urea (van Zijderveld et al., 2011; Newbold et al., 2014; Hegarty et al., 2016) Most data indicate that nitrate can reduce enteric CH 4 production by 10 to 30% (Lee and Beauchemin, 2014; Guyader et al., 2015) Enteric CH 4 production can account for 2 to 12% of GE losses, depending upon diet type, and it has been theorized that nitrate may increase ME supply by reducing the amount of C lost as CH 4 ; however, most research has focused on the performance of cattle consuming moderate (Lee et al., 2017b) to high concentrate (Newbold et al., 2014) diets. Little is known about the effects of bismuth subsalicylate ( BSS ) on ruminant animals. For dec ades, BSS has been heralded as a mediator of H 2 S in humans, lessening pain in the gastro intestinal tract (Suarez et al., 1998; Levitt et al., 2002; Mitsui et al., 2003) In vitro ruminal fermentation has been used to evaluate the possible influence of BSS on in vivo parameters, but in vivo data is needed to truly evaluate to potential impacts on production and performance of cattle (Ruiz Moreno et al., 2015) Bismuth compound s may have a place in beef production by miti gating the negative effects of S on trace mineral absorption. By binding to S (Suarez et al., 1998) BSS may reduce thiol compounds which inhibit trace min eral absorption.

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123 The current experiment was designed to test the hypothesis that encapsulated calcium ammonium nitrate ( eCAN ) would not impact performance of growing heifers consuming a bahiagrass hay and molasses diet. The second hypothesis was that BSS w ould not alter performance of growing heifers; however, liver trace mineral content should be increased. The objective of this experiment was to evaluate the performance and liver mineral concentration of heifers provided eCAN and/or BSS. Materials and Met hods All procedures involving animals were approved by the Animal Care and Use Committee of the Institute of Food and Agricultural Sciences at the University of Florida. Experimental D esign, A nimals, and T reatments Seventy five growing Bos taurus and Bos indicus heifers (279 57 kg of initial BW; average SD) were used in a n randomized in complete block design with a 2 2 + 1 factorial arrangement of treatments at the University of Florida North Florida Research and Education Center Beef Uni t in Marian na, FL. The experiment consisted of a 28 d adaptation period followed by a 56 d data collection period in which heifers were weighed every 14 d from d 0 to d 56. On d 28 and 27, all heifers were weighed and the average weight of each heifer on those 2 d was considered initial BW. Similarly, the average BW of each heifer on d 55 and 56 was considered final BW. On d 27, heifers were stratified and blocked by weight and allotted to 25 dormant bahiagrass ( Paspalum notatum ) pastures. Pastures (1.34 ha each) w ere located in 3 different areas of the Beef Unit and were within 0.52 km of each other. The three locations were termed North Circle (n = 13 ; pastures per location ), South Circle (n = 6), and R Pens (n = 6). Pastures were stratified by location and random ly assigned to 1 of 5 treatments: 1) NCTRL no added NPN or BSS; 2) U urea supplemented at 182 mg/kg of BW; 3) NIT nitrate, in the

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124 form of eCAN, supplemented at 350 mg/kg of BW; 4) UB urea supplemented at 182 mg/kg of BW and BSS supplemented at 58.4 mg/kg of BW; and 5) NITB nitrate, in the form of eCAN, supplemented at 350 mg/kg of BW and BSS supplemented at 58.4 mg/kg of BW. Treatments U, NIT, UB, and NITB were isonitrogenous. To r educe any negative effects of nitrate on the health of the heifers, on d 27 cattle began an adaptation to eCAN and urea. From d 27 to d 14, cattle received 20% of their total supplemental N; on d 13 and 12, cattle received 40% of their total supplemen tal N; on d 11 and 10, cattle received 60% of their total supplemental N; on d 9 and 8, cattle received 80% of their total supplemental N; and beginning on d 7, cattle were receiving 100% of their total supplemental N as urea or eCAN. One pasture (NIT B) was removed from the experiment because the heifers refused to consume the molasses and treatment. Heifers had ad libitum access to bahiagrass hay and received 1.07 kg/d (DM basis) of sugar cane molasses (chemical composition is presented on Table 5 1 ) This experiment began on February 15 2017 and prior to initiation, pastures were mob grazed to remove any residual forage; therefore, bahiagrass hay was the only forage available to the heifers. Molasses was weighed and provided daily, and was used as the carrier of treatments. Sample and D ata C ollection Samples of hay and molasses were collected every 14 d and stored at 10C (hay) or 20C for further analysis. A ll feed samples were shipped to a commercial laboratory (Dairy One Forage Laboratory, Ithaca, NY) for chemical analysis, which are presented in Table 5 1.

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125 Heifers BW was recorded every 14 d starting on d 0. To calculate ADG, difference in BW was divided by the number of d between BW recordings. Liver samples were taken on d 28 and 27, and again on d 55 and 56. A random subset of heifers (2 heifers/pen) was selected to collect liver t issue. One heifer per pen was randomly selected to have liver sample t aken on d 28 and 55, and the second heifer donating liver tissue was col lected on d 27 and 56. There were no statistical differences ( P > 0.05) between cohorts; therefore, for the rest of the manuscript, liver tissue will be described as initial and fina l. The techniques described in Arthington and Corah ( 1995) were used to collect liver tissue. Briefly, A Tru Cut biopsy needle (CareFusion, 14 gauge by 15 cm; Becton, Dickinson, and Comp., Vernon Hills, IL) was inserted between the 11 th and 12 th capsule was penetrated. Three core tissue samples (25 mg each) were collected from each animal at each collection. All tissue samples were stored in Zn free microcentrifuge tubes ( Fisherbrand; Thermo Fisher Scientific Inc., Waltham, MA ) at 20C until further analysis. Liver tissue mineral concentration was determined using ICP MS (Animal Health Diagnostic Laboratory, Lansing, MI). On d 28, 27, 55, and 56, carcass measurements were taken using ultrasonography (3.5 mHz linear array transducer, Aloka 500V; Corimetrics Medical Systems Inc., Wallingford, CT ). Two thirds of the heifers were measured on d 28 and 55 (all animals not donating live r tissue plus the first cohort of liver t issue donors) and one third was measured on d 27 and 56 (the second cohort of liver tissue donors). No differences were found between d 28 and 27, nor d 55 and 56; therefore, for the remainder of the manuscript, ultrasonography measurements will be described as initial

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126 and final. Ultrasonography measurements were made between the 12 th and 13 th intercostal space. Images we re used to assess LM area and back fat thickness Statistical Analysis Data were analyzed as a randomized incomplete block design with a 2 2 + 1 factorial arrangement of treatments using pasture as the experimental unit. The model included the fixed effect of treatment and the random effects of block and location (North Circle, South Circle, and R Pens). Pens blocked (n = 5) by BW. Initial liver mineral concentration and ultrasound measurements were used as covariates for final liver mineral concentration and ultrasound measurements respectively Denominator degrees of freedom were adjusted using the Kenward Rogers adjustment. The following contrasts were used to aid in the interpretation of data: the effect of NPN = NCTRL vs. the mean of U, NIT, UB, and NITB; the effect of NPN source = the mean of U and UB vs. the mean of NIT and NITB; the effect of BSS = the mean of U and NIT vs. the mean of UB and NITB; and NPN source BSS = the mean of U and NITB vs. the mean of NIT and UB. Significance was declared at P Results and Discussion This experiment was the final of a series of experiments to examine the effects of providing eCAN as a NPN source along with BSS. Data from the previous experiments indicated that rumen fermentation was altered by the addition of eCAN inplace of urea but not BSS. Furthermore, digestibility and enteric CH 4 produc tion (g/kg of BW 0.75 ) was reduced with the addition of eCAN. In the current experiment, treatments identical to those used in Chapters 3 and 4 were provided to the heifers to consider the consequences producers may face if these compounds are fed commercia lly.

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127 Growth performance data is presented on Table 5 2 At the beginning of the current experiment, all treatments had similar BW ( P > 0.05) By the end of the experiment, BW was not affected by NPN ( P = 0.4701), NPN source ( P = 0.3836), or BSS ( P = 0.5999); however, there was an interaction ( P = 0.0484) between BSS inclusion and source of NPN. There was no effect of NPN, NPN source BSS or an interaction for ADG measured within any time points ( P > 0.05). As discussed in Chapter 2, it is possible that the CP content of the forage and molasses was sufficient to support the growth and development of a healthy and productive ruminal microbiome. The most recent NRC model (NASEM, 2016) recommends a CP content of the diet to range from 10.2 to 17.2% of the diet DM for growing animals weighing 250 to 350 kg. This range of CP concentration depends g reatly on the energy content of the ration. In the current experiment, assuming cattle were consuming 85% bahiagrass hay and 15% molasses (see Chapter 4) the CP content of the diet would have been approximately 9.8% of the DMI with nearly 57.5% TDN. For g rowing animals, the NRC gives examples and recommendations using 4 different diets ranging from 65 to 80% TDN. With less TDN in the diet of the current experiment compared with the NRC examples, it may be that the animals could not synchronize the use of a ll of the available CP due to a lack in fermentable energy (Dewhurst et al., 2000; NASEM, 2016) In a review manuscript by Dewhurst et al. (2000), the theory of rumen synchrony was discussed in depth. The authors deliberated over several data sets evaluating diets that differed in rumen available energy and N sources. The authors concluded that a balance between fermentable energy and N sources should be sought after to optimize microbial protein synthesis It could be speculated that in the current experiment, the energy available for

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128 microbial protein synthesis was not sufficient to utilize the NPN sources provided; thereby, not improving performances of animals provided NPN. When considering the proposed mode of action of the reduction of CH 4 production with the addition of nitrate, it may be speculated that performance of the ruminant being provided nitrate would increase. When nitrate is reduced in the rumen to NH 3 H 2 that would be used to reduce CO 2 to CH 4 would theoretically no longer be available; therefore, possibly a shift in the VFA profile to produce more propionate would occur, leading to increases in performance. Data from the current experiment does not support this theory nor does a majority of the literature on nitrate report suc h findings (Newbold et al., 2014; Lee et al., 2017a; Lee e t al., 2017b) It has been hypothesized that the production of propionate is not increased with the addition of nitrate because of the thermodynamics of VFA production (Ungerfeld and Kohn, 2006; Janssen, 2010; Ungerfeld, 2015) To speculate on this subject, one must assume that nitrate only acts on enteric CH 4 production by acting as a H 2 sink only and not inhibiting methanogens. When nitrate is reduced in the rumen, conceptually, on e can visualize the pool of H 2 to be lessened. In a review by Janssen (2010), evidence was presented that indicated a shift in VFA production to H 2 producing pathways (i.e. 2 acetate + 4 H 2 ); therefore, increasing the amount of C used for acetate production at the expense of butyrate and/or propionate. It is plausible that this occurred in the current experiment and C that would have been lost as CH 4 was used to produce acetate, a VFA less energetically favorable than propionate. Future research should focus on the long term effects of nitrate on ruminal f ermentation to evaluate if the shift in VFA, as seen in Chapter 3, persists over a longer forage feeding period.

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129 performance of ruminants provided BSS. A plethora of data has been reported indicating the negative effects of S on beef cattle performance (Gould, 1998; Felix et al., 2012; Drewnoski et al., 2014) It was hypothesized that BSS may be able to th wart these negative effects by binding to ruminal S and transporting it, as Bi 2 S 3 out of the gastro intestinal tract via feces. In the current experiment, the diet consisted of approximately 0.51% S (DM basis), which is marginally greater than what is con sidered safe for grazing beef c attle (NASEM, 2016) Early research has indicated that growing cattle consuming forage based diets and high S water (approximately 0.55% S in DMI) experience reductions in DMI and BW gain (Weeth and Hunter, 1971; Weeth and Capps, 1972) In the current experiment, heifers not receiving BSS did not perform less favorably compared with those which w ere provided BSS indicating that heifers were not affected by the S concentration of the diet and/or BSS did not improve performance by sequestering S in the rumen. Data from a ruminal metabolism experiment, presented and discussed in Chapter 3, indicated that BSS had minimal effects on the concentration of H 2 S in the gas cap of ruminally cannulated cattle consuming bahiagrass hay and molasses ( 0.40% S DM basis ) Previous work has indicated that the activity of BSS is impacted by pH, with more acidic envir onments being more appropriate (Sox and Olson, 1989) It is possible that the rumen environment was not as conducive for BSS activity in the current experiment compared to a ruminant fed a hig h grain diet Researchers published data from an in vitro batch culture experiment using a 90% concentrate substrate with 1% BSS (DM basis), in which fermentation was not negatively impacted by BSS, while H 2 S was reduced by 34% indicating that BSS was

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130 bind ing to S (Ruiz Moreno et al., 2015) ; however, in a separate in vitro continuous culture experiment, the same inclusi on rate of BSS reduced H 2 S by nearly 99% When evaluating the pH of each in vitro experiment, the batch culture had a final pH of approximately 6.3, whereas the mean pH of the continuous culture was approximately 5.4 (Ruiz Moreno et al., 2015) An in vitro experiment, presented and discussed in Chapter 2, was used to evaluate BSS at up to 1.0% of the DM of a bahiagrass hay and molasses substrate. At 1.0%, H 2 S was reduced by 100%; however, it was obvious from the results of other variables, that fermentation had been negatively affected. When included in the substrate at 0.33%, BSS reduced H 2 S by 61% without negatively af fecting in vitro fermentation. Currently, there is not enough data to conclude exactly what is occurring in the rumen when BSS is provided, but the data from the current experiment does not support the hypothesis that BSS will improve the performance of ca ttle consuming high S, forage based diets. Suarez et al. ( 1998) theorized that BSS lessened gastro intestinal pain in humans by binding to H 2 S, a major gut ir ritant in humans, in the following manner: 2 Bi 3+ + 3 H 2 2 S 3 + 6 H + If this hypothesis is correct, then it is possible that bismuth only binds to sulfide when it is in the undissociated form. This may explain the interaction observed between NPN sour ce and BSS. In Chapter 2 Exp. 2 the inclusion of a nitrate source reduced H 2 S in a linear manner. Although no effects of NPN source on ruminal H 2 S concentration were observed in the metabolism experiment (Chapter 3), if eCAN reduced the amount of H 2 S pr oduced in the current experiment, by acting as a H 2 sink, the pool of H 2 would likely be comparably less. Speculatively, if BSS could not bind to H 2 S, it may be more

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131 likely to act as an antimicrobial (Sox and Olson, 1989) and inhibit growth of beneficial bacterial populations in the rumen. Carcass ultrasound results are presented on Table 5 3 There was no effect of NPN, NPN source or BSS on LM area ( P > 0.05). As previously discussed, there was an expectation for NPN to improve performance, thereby, possibly increasing LM area; however, this was not observed in the current experiment. The lack of change in LM area when eCAN was provided in place of urea, is in agreement with much of the literature (Hegarty et al., 2016; Lee et al., 2017a) In general, the benefits o f nitrate on performance are observed in increases in G:F (Newbold et al., 2014; Lee et al., 2017a) As BSS was n ever r eported to have been provided to ruminants, the data from this experiment provides a good base for other researchers to build on. Surprisingly a BSS NPN source interaction ( P < 0.001) was observed for 12 th rib fat thickness on d 56 and change in fat thickness from d 28 to d 56. The data from this experiment indicates that BSS may incr ease fat deposition when urea is provided as a NPN source; however, potentially negative effects on fat thickness may occur when BSS is provided with eCAN. Although not significant, butyrate molar proportions were numerically increased, in the metabolism e xperiment presented and discussed in Chapter 3, when BSS was provided. If the heifers in the current experiment encountered shifts in ruminal fermentation to produce more butyrate, it is possible that more butyrate was utilized in energy production through the tricarboxylic acid cycle (Van Soest, 1994) Furthermore, by providing NPN, fat thickness at the 12 th rib was increased ( P = 0.042). The concentration of trace minerals in the liver from cattle in the current experiment can be found on Table 5 4 Concentration of minerals in the liver was not

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132 affected by NPN sou r ce ( P first data repres enting the effects of nitrate supplementation on liver mineral concentration. There were no interactions between BSS and source of NPN ( P > 0.05) affecting liver mineral concentration. The addition of NPN increased liver concentrations of Fe ( P = 0.0132) a nd Mn ( P = 0.0056) by 37 and 15%, respectively. The reason for this change in Fe and Mn is unknown It was hypothesized that BSS may increase the liver concentration of trace minerals when BSS was provided with a high S diet. The effects of diets with large concentrations of S on trace mineral absorption has been reported in both grazing cattle (Arthington et al., 2002) and grain fed cattle (Pogge and Hansen, 2013; Pogge et al., 2014) In the rumen, S, along with Mo, can act as an antagonist to trace mineral (i.e. Cu) absorption. The S binds with Mo to form thiomolybdates that bi nd to minerals, leaving them in absorbable (Drewnoski et al., 2014) It was speculated that BSS may reduce the amount of available S in the rumen by forming Bi 2 S 3 Without having available S to bind to trace minerals, the minerals, theoretically, would have a greater chance of absorption. The data from this experiment does not support this hypothesis. Liver mineral concentrations of Cu were reduced ( P = 0.0018) by 70% when BSS was provided to heifers consuming bahiagrass hay and molasses. Unfortunately, very little data is available evaluating the effects of bismuth on the mineral status of humans and rats. One study injected BiCl 3 subcutaneously into rats to evaluate the effects of bismuth on Cu concentration of differing rat organs (Szymanska and Zelazowski, 1979) The researchers found an increase in total body Cu content with no change in liver concentrations of C u; however, the concentration of Cu observed in the kidneys was

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133 in creased with the injection of bismuth. It may be speculated that if kidney concentrations of Cu are increased, that an increase in urine Cu may be observed; however, the authors did not eval uate such parameters (Szymanska and Zelazowski, 1979) Assuming that bismuth causes a shift in Cu transportation to the kidneys, it may be possible that the heifers receiving BSS in the cur rent experiment experienced this change in Cu accumulation. Another possibility is that formations of Bi Cu S comp o unds bound Cu, and excreted the Cu via feces. Researchers have discussed the synthesis of Cu 3 BiS 3 nanocrystals in the laboratory (Yan et al., 2013) ; however, there is no evidence that this can occur naturally in the rumen. The inclusion of BSS in the diets of heifers was also associated with a 30% increase ( P = 0.0157) in liver concentration of Fe. There has been s ome evidence that bismuth may act as an antimicrobial by inhibiting bacterial uptake of Fe (Domenico et al., 1996) More recently, other research groups have contested this theory claiming that the effec ts of bismuth on bacteria (i.e. Helicobacter pylori ) is similar to intracellular iron deprivation and causative (Bland et al., 2004) Further research needs to focus on the mode of action of bismuth on H pylori If there is a link between bismuth and the uptake of Fe by bacteria, it i s possible that more Fe is available for absorption in the duodenum; however, this is unlikely. It is more probable that the increase in liver Fe concentration is related to the depletion of Cu in the liver (Suttle, 2010) Copper is vital for the activity of ferroxidase, an enzyme which mobilizes Cu out of the liver; therefore, if Cu is limiting in the liver, Fe is likely to accumulate in the hepatic tissue (Mills et al., 1976; Hansen et al., 2010; Suttle, 2010)

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134 In conclusion, the inclusion of eCAN did not improve nor worsen performance of growing heifers unless it was provided in combination with BSS. Alone, BSS did not hinder growth of heifers consuming bahiagrass hay and molasses. The diet in the current experiment contained approximately 0.5% S (DM) which may not have been great enough to truly observe the potential benefits of BSS on performance. R egardless of performance, heifers consuming BSS exhibited reductions in liver Cu and liver Fe accumulation, which may have detrimental effects in the long term. More research is required to determine the effects of BSS on cattle consuming diets of differin g composition and S content.

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135 Table 5 1 Analyzed 1 chemical composition of bahiagrass hay and sugar cane molasses Item Bahiagrass hay Sugar cane molasses DM, % 90.0 78.7 OM, % DM 93.4 84.4 CP, % DM 10.3 7.2 NDF, % DM 73.0 ADF, % DM 45.7 TDN, % DM 54.0 77 S % DM 0.34 1.47 Nitrate % DM 0.07 0.12 1 Analyzed by a commercial laboratory using a wet chemistry package (Dairy One, Ithaca, NY).

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136 Treatment 3 P value 4 Item NCTRL U NIT UB NITB SEM 5 NPN NS B BN Initial BW 6 kg 274 275 276 279 277 3.1 0.349 0.930 0.435 0.615 Final BW 7 kg 335 335 342 352 332 6.7 0.470 0.384 0.600 0.048 ADG, kg d 28 to 0 0.8 7 0.8 0 0.9 7 1.07 0.80 0.139 0.794 0.729 0.731 0.125 d 0 to 14 0.6 7 0.69 0.83 1.01 0.80 0.141 0. 286 0. 772 0. 307 0. 218 d 0 to 28 0.70 0.75 0.75 0.8 6 0.83 0.110 0.401 0.898 0.388 0.898 d 0 to 42 0.7 8 0.76 0.74 0.9 0 0.7 6 0.092 0.891 0.398 0.347 0.508 d 0 to 56 0.65 0.6 9 0.68 0.7 4 0.55 0.061 0.865 0.125 0.545 0.139 1 BSS = bismuth subsalicylate. 2 eCAN = encapsulated calcium ammonium nitrate (5Ca(NO 3 ) 2 NH 4 NO 3 ) 65.1% nitrate DM basis 3 NCTRL = ad libitum bahiagrass hay + 2.7 g/kg of BW of molasses (basal diet); U = basal diet + 182 mg/kg of BW of urea; NIT = basal diet + 350 mg/kg of BW of NO 3 ; UB = U + 58.4 mg/kg of BW of BSS; NITB = NIT + 58.4 mg/kg of BW of BSS. 4 Observed significance levels for: NPN = effect of NPN, NCTRL vs the mean of U + NIT + UB + NITB; NS = effec t of NPN source (excludes NCTRL); B = effect of BSS (excludes NCTRL); BN = interaction of BSS and eCAN. 5 NCTRL, U, NIT, UB, and NITB had 5, 5, 5, 5, and 4 experimental units, respectively; largest SEM was provided. 6 Initial BW was the average of BW recorded on d 28 and 27 7 Final BW was the average of BW recorded on d 55 and 56. Table 5 2 Effect of BSS 1 and eCAN 2 on growth performance of beef heifers

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137 Treatment 3 P value 4 Item 6 NCTRL U NIT UB NITB SEM 5 NPN NS B BN LM area, cm 2 54.36 56.54 54.41 58.07 56.79 2.677 0.441 0.493 0.431 0.871 LM area change 7 cm 2 10.26 12.33 11.38 15.12 12.32 2.221 0.298 0.395 0.397 0.671 12 th rib fat thickness, cm 0.50 0.42 0.47 0.56 0.40 0.025 0.204 0.012 0.080 <0.001 12 th rib fat thickness change 8 cm 0.063 0.025 0.025 0.117 0.045 0.019 0.042 0.009 0.069 <0.001 1 BSS = bismuth subsalicylate. 2 eCAN = encapsulated calcium ammonium nitrate (5Ca(NO 3 ) 2 NH 4 NO 3 ) 65.1% nitrate DM basis 3 NCTRL = ad libitum bahiagrass hay + 2.7 g/kg of BW of molasses (basal diet); U = basal diet + 182 mg/kg of BW of urea; NIT = basal diet + 350 mg/kg of BW of NO 3 ; UB = U + 58.4 mg/kg of BW of BSS; NITB = NIT + 58.4 mg/kg of BW of BSS. 4 Observed significance levels for: NPN = effect of NPN, N CTRL vs the mean of U + NIT + UB + NITB; NS = effect of NPN source (excludes NCTRL); B = effect of BSS (excludes NCTRL); BN = interaction of BSS and eCAN. 5 NCTRL, U, NIT, UB, an d NITB had 5 5 5 5 and 4 experimental units, respectively; largest SEM was provided. 6 LM area and 12 th rib fat thickness was measured on d 56 using d 28 as a covariate 7 Difference in LM area between d 28 and 56 8 Difference in 12 th rib fat thickness between d 28 and 56 Table 5 3 Effect of BSS 1 and eCAN 2 on carcass ultrasound measurements

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138 Treatment 3 P value 4 Item NCTRL U NIT UB NITB SEM 5 NPN NS B BN Final liver mineral 6 mg/kg DM Cu 54.98 55.69 53.93 11.35 21.66 9.771 0.088 0.666 0.002 0.533 Fe 189.33 213.63 235.73 268.00 318.15 24.274 0.013 0.116 0.016 0.527 Zn 123.56 128.83 134.93 137.20 131.60 4.983 0.087 0.958 0.604 0.251 Mn 11.14 12.56 12.34 13.00 13.45 0.487 0.006 0.815 0.134 0.475 Se 0.33 0.38 0.37 0.31 0.33 0.035 0.613 0.921 0.125 0.637 1 BSS = bismuth subsalicylate. 2 eCAN = encapsulated calcium ammonium nitrate (5Ca(NO 3 ) 2 NH 4 NO 3 ) 65.1% nitrate DM basis 3 NCTRL = ad libitum bahiagrass hay + 2.7 g/kg of BW of molasses (basal diet); U = basal diet + 182 mg/kg of BW of urea; NIT = basal diet + 350 mg/kg of BW of NO 3 ; UB = U + 58.4 mg/kg of BW of BSS; NITB = NIT + 58.4 mg/kg of BW of BSS. 4 Observed significance levels for: NPN = effect of NPN, NCTRL vs the mean of U + NIT + UB + NITB; NS = effect of NPN source (excludes NCTRL); B = effect of BSS (excludes NCTRL); BN = interaction of BSS and eCAN. 5 NCTRL, U, NIT, UB, an d NITB had 5 5 5 5 and 4 experimental units, respectively; largest SEM was provided. 6 Liver mineral analyzed on d 56 using d 28 as a covariate. Table 5 4 Effect of BSS 1 and eCAN 2 on liver mineral concentration

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139 CHAPTER 6 SUMMARY AND RECOMMENDATIONS Summary This series of experiments was designed to evaluate the effects of calcium ammonium nitrate ( CAN ) and bismuth s ubsalicylate ( BSS ) a novel feed additive for ruminants, first work conducted in which ruminants received BSS in vivo. Much of the published research on BSS focuses on monogastr ics, more specifically human and rat models, and gut health. One research group from the University of Minnesota included BSS in the substrate used for in vitro batch and continuous culture fermentations hypothesizing that BSS would inhibit production of H 2 S. In fact, H 2 S was reduced by 34 and 99% when provided at 1% of the substrate DM with in vitro batch and continuous cultures, respectively. Furthermore, BSS did not appear to inhibit fermentation using the batch culture system when provided up to 1% of the substrate DM. Providing a NPN source to ruminants is not a novel idea within the confines of protein and fiber production (Owen et al., 1942) Urea has been provided to cattle for years as an inexpensive source of N that ruminal microorganisms can convert to true protein. In recent years, nitrate has been investigated as a potential strategy to reduce the productio n of enteric CH 4 from ruminants by acting as a H 2 sink ( Leng, 2008; van Zijderveld et al., 2010; Hulshof et al., 2012) It has been proposed that nitrate could also act as a NPN source for ruminal microorganisms through the same pathway as the proposed mode of action for the mitigation of CH 4 capturing H 2 a nd reducing nitrate to NH 3 (Leng, 2008; Newbold et al., 2014) A majority of the research published evaluating the CH 4 mitigating effects and potential role as a NPN source has focused on cattle

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140 consuming at least 5 0% concentrate diets (Nolan et al., 2010; Newbold et al., 2014; Lee et al., 2017a) ; therefore, the current series of experiments was designed to evaluate nitrate supplementation to cattle consuming forage based diets. In the current series of experiments, 3 objectives were developed to test the hypothesis that CAN and BSS would improve beef cattle production while lessening environmental impacts. The objectives were: 1) to evaluate the effect of increasing doses of BSS in combination with CA N on in vitro ruminal fermentation; 2) Determine the effects of BSS and encapsulated CAN ( eCAN ) on in vivo ruminal fermentation, metabolism nutrient digestibility, and enteric CH 4 production of beef cattle consuming a bahiagrass hay diet with supplemental molasses; and 3) Asses the effects of BSS in combination with eCAN on performance of growing cattle consuming a bahiagrass hay diet with supplemental molasses. The first objective involved a series of 3 in vitro ruminal fermentation experiments. The first in vitro experiment evaluated CAN on fermentation, the second in vitro experiment considered the effects of CAN and BSS on fermentation parameters, and the third experiment was designed to determine a dose of BSS that would allow for reduction in H 2 S prod uction without having negative effects on other fermentation parameters. These experiments provided data that implied that CAN has the ability to effectively reduce in vitro CH 4 production; however, negative effects on in vitro OM digestibility ( IVOMD ), co ncentrations of NH 3 N, and gas production were also observed. There were few interactions between CAN and BSS when evaluated in vitro, with the exception of CH 4 production. When BSS was provided along with CAN, in place of urea,

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141 in vitro CH 4 production (mm ol/g of fermented OM) was decreased in an additive manner. The production of H 2 S in vitro was reduced by 61% when BSS was provided at 0.33% of the substrate DM. When included a t 1.0% of the substrate DM, BSS had detrimental effects on fermenta tion with reductions of 11, 29, and 23% for production of total VFA, IVOMD, and maximal gas production, respectively. In the third in vitro experiment, when BSS was provided alone at lesser concentrations, it was observed that at up 0.33% of the substrate DM BSS reduced production of H 2 S without impeding fermentation in vitro. The second objective was composed of 2 in vivo experiments: 1) an experiment using 10 ruminally cannulated cattle to evaluate the effects of eCAN and BSS on ruminal metabolism and microb ial N flow out of the rumen; and 2) an experiment using 25 crossbred steers to investigate the impacts of eCAN and BSS on enteric CH 4 production and total tract digestibility of nutrients. The metabolism experiment provided data that implied that eCAN alte red ruminal fermentation by decreasing the production of total VFA. There were no differences observed between cattle that received urea or eCAN as NPN sources for microbial N flow; furthermore, no differences were observed between cattle that received a N PN source and those that did not for microbial N flow. There was no observed impact of BSS on ruminal metabolism ; however, rectal temperature was significantly reduced in cannulated steers and heifers that were receiving BSS. The explanation of this resul t is still unknown, but it allows for the opportunity of future research into the effects of BSS on inflammation.

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142 The experiment designed to evaluate the effects of eCAN and BSS on total tract digestibility of nutrients and enteric CH 4 production provided results that support the data gained in the metabolism experiment. There was no impact of BSS on enteric CH 4 production nor nutrient digestibility; however, apparent retention of S was decreased with the addition of BSS to a bahiagrass hay diet supplemente d with molasses. Enteric CH 4 production was decreased (g/d and g/kg of BW 0.75 ) with the addition of eCAN to the diet of steers. Total tract digestibility of nutrients (DM, OM, NDF, ADF, and CP) was also reduced with the addition of eCAN. Considering the re duction in digestibility, when CH 4 production was expressed as grams per kilogram of OM digested, there were no observable differences between cattle receiving urea and eCAN. Finally, the third objective included a performance experiment which was composed of 75 growing heifers housed in 25 dormant bahiagrass pastures. Data from this experiment implied that eCAN and BSS, alone, did not impact performance of growing cattle consuming forage based diets; however, when provided in combination, a NPN source B SS interaction was observed where final BW of heifers consuming eCAN and BSS appeared to be negatively affected. Furthermore, those heifers consuming both BSS and eCAN had lesser back fat thickness. It was hypothesized that BSS would increase concentration s of trace minerals in the liver of heifers; however, data gained from this experiment indicated that concentrations of Cu in the liver were nearly depleted with the addition of BSS in the diet of growing heifers. This was accompanied by an increase in con centrations of liver Fe.

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143 Recommendations The data collected from the experiments described here within create a solid foundation for future research involving ruminants consuming forage based diets. Not only did the data regarding the use of BSS and eCAN answer several pressing questions, but it also opened the door for researchers to pursue different avenues of research with nitrate and bismuth subsalicylate Calcium A mmonium N itrate It appears from the data gained in this series of experiment s and that from other researchers, that eCAN may inhibit ruminal degradation of fiber due to inhibitory effects on cellulolytic bacteria (Asanuma et al., 2015; Latham et al., 2016) This likely explains why nitrate has been successfully used in feedlot and dairy diets to reduce enteric CH 4 production while either maintaining performance (Olijhoek et al., 2016) or improving feed efficiency (Newbold et al., 2014) After evaluating the data, it is not recommended to use eCAN as a NPN for cattle consuming low quality, hay based diets. It is p ossible that the high concentration of fiber in the hay is not being degraded as rapidly or extensively as it would be when urea is used as a NPN source. With concentrate based diets, the limited amount of fiber may not be digested as well, but as it is a minor fraction of the diet, it may not affect animal performance. Future research should focus on the use of eCAN on differing types of forage and conservation methods (fresh vs. haylage or hay). Another potential for eCAN in ruminant diets is to mitigate negative effects of high S, feedlot diets, more specifically H 2 S. Although the experiments described within this dissertation did not fully elucidate the impact of nitrate on production of H 2 S, there is s free energy for change is

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144 143.4 kcal while the reduction of thermodynamically favorable reaction; Weimer, 1998 ). With the reduction of nitrate being more thermodynamically favorable than t he reduction of sulfate, which is more thermodynamically favorable than methanogenesis ( 36.4 vs. 33.2 kcal), it is probable that nitrate could be used as a dual purpose additive with multiple outcomes: NPN source and hydrogen sink with negative effects o n methanogenesis and production of H 2 S. Bismuth S ubsalicylate It was hypothesized, prior to the commencement of this study, that BSS would have benefits on 2 fronts of beef cattle production: 1) reduce production of H 2 S; and 2) mitigate negative effects of S on trace mineral concentration in the liver. This series of experiments did not fully define the potential of BSS to act as a limiter of production of H 2 S, mainly due to the forage content of the diet provided to the cattle; however, in the third experi ment (digestibility and enteric CH 4 production), data indicated that more S was being excreted from steers receiving BSS compared with those not receiving BSS. It would be beneficial to the field of ruminant nutrition to determine the impact of BSS on catt le receiving a high S, high concentrate diet. Data from the performance experiment implied that BSS did not improve trace mineral accumulation in the liver of growing heifers, in fact, liver Cu was nearly depleted after 70 d of receiving BSS. This indicates that BSS should not be used by beef producers to improve trace mineral absorption when cattle are consuming high S diets; however, there is a potential for BSS in another ruminant species: sheep. It has been recognized for decades that sheep are susceptible to disease and death caused by over

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145 accumulation of Cu in the liver (Goodrich and Tillman, 1966) With the data observed in the performance experiment of this study, it could be speculated that BSS, when fed at concentrations that do not affect ruminal fermentation, could provide feed producers another avenue to combat Cu toxicity in sheep.

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146 LIST OF REFERENCES Ahvenjrvi, S., a Vanhatalo, P. Huhtanen, and T. Varvikko. 2000. Determination of reti culo rumen and whole stomach digestion in lactating cows by omasal canal or duodenal sampling. Br. J. Nutr. 83:67 77. doi:10.1017/S0007114500000106. Appuhamy, J. A. D. R. N., A. B. Strathe, S. Jayasundara, C. Wagner Riddle, J. Dijkstra, J. France, and E. K ebreab. 2013. Anti methanogenic effects of monensin in dairy and beef cattle: A meta analysis. J. Dairy Sci. 96:5161 5173. doi:10.3168/jds.2012 5923. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23769353 Arthington, J. D., and L. R. Corah. 1995. Live r biopsy procedures for determining the trace mineral status in beef cows. Part II. Arthington, J. D., J. E. Rechcigl, G. P. Yost, L. R. Mcdowell, and M. D. Fanning. 2002. Effect of ammonium sulfate fertilization on bahiagrass quality and copper metabolism in grazing beef cattle. J. Anim. Sci. 80:2507 2512. Asanuma, N., S. Yokoyama, and T. Hino. 2015. Effects of nitrate addition to a diet on fermentation and microbial populations in the rumen of goats, with special reference to Selenomonas ruminantium havin g the ability to reduce nitrate and nitrite. Anim. Sci. J. 86:378 384. doi:10.1111/asj.12307. Ashes, J. R., S. K. Gulati, and T. W. Scott. 1997. Potential to alter the content and composition of milk fat through nutrition. J. Dairy Sci. 80:2204 2212. doi:1 0.3168/jds.S0022 0302(97)76169 1. Available from: http://dx.doi.org/10.3168/jds.S0022 0302(97)76169 1 Balch, D. A., and S. J. Rowland. 1957. Volatile fatty acids and lactic acid in the rumen of dairy cows receiving a variety of diets. Br. Journalof Nutr. 1 1:288 298. Barry, T. N., A. Thompson, and D. Armstrong. 1977. Rumen fermentation studies on two contrasting diets. 1. Some characteristics of the in vivo fermentation, with special reference to the composition of the gas phase, oxidation/reduction state an d volatile fatty acid proportions. J. Agric. Sci. 89:183 195. Bartley, E. E., E. L. Herod, R. M. Bechtle, D. A. Sapienza, and B. E. Brent. 1979. Effect of monensin or lasalocid, with and without niacin or amicloral, on rumen fermentation and feed efficienc y. J. Anim. Sci. 49:1066 1075. Beauchemin, K. A., H. Henry Janzen, S. M. Little, T. A. McAllister, and S. M. McGinn. 2010. Life cycle assessment of greenhouse gas emissions from beef production in western Canada: A case study. Agric. Syst. 103:371 379. doi :10.1016/j.agsy.2010.03.008. Available from: http://linkinghub.elsevier.com/retrieve/pii/S0308521X10000387 Beauchemin, K. A., H. H. Janzen, S. M. Little, T. A. McAllister, and S. M. McGinn. 2011.

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163 BIOGRAPHICAL SKETCH D arren grew up in southeast Texas in a small town called Porter. From a young age he was heavily involved with agriculture calf operations or through 4 H and FFA. After graduating from New Caney High School, Darren began his Bachelor of Science degree in College Station, TX at Texas A&M University where he received degrees in Animal Science and Agricultural Leadership and Education. Darren lived and studied for 6 months in Pirassun u n ga, SP, Brazil. It was while working at a research feedlot in Brazil that Darren discovered his passion for research. Following graduation in December 2011, Darren travelled to Marianna, FL, the home of the North Florida Research and Education Center, whe re he lived and worked throughout his M aster of Science and Doctor of P hilosophy degrees. Darren worked under the advisement and mentorship of Dr. Nicolas DiLorenzo throughout both advanced degrees while at the University of Florida. Darren is happily marr ied to his beautiful wife, Francine Henry. After completing his Doctor of Philosophy in December 2017, Darren joined the faculty at Texas Tech University as an Assistant Professor of Sustainable Livestock Grazing Management