Effects of Feeding a Natural Biopolymer (Chitosan) on Methane Emissions and Performance in Beef Cattle

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Effects of Feeding a Natural Biopolymer (Chitosan) on Methane Emissions and Performance in Beef Cattle
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1 online resource (69 p.)
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english
Creator:
Henry, Darren D
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University of Florida
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Gainesville, Fla.
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Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Animal Sciences
Committee Chair:
DILORENZO,NICOLAS
Committee Co-Chair:
LAMB,GRAHAM CLIFF
Committee Members:
HERSOM,MATTHEW J

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Subjects / Keywords:
chitosan -- fermentation -- methane -- ruminant
Animal Sciences -- Dissertations, Academic -- UF
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Animal Sciences thesis, M.S.
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theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
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Abstract:
Recent in vitro experiments indicate that the natural biopolymer chitosan could modify ruminal fermentation and decrease methane production. Our objective was to evaluate the natural biopolymer chitosan as a feed additive to mitigate in vivo methane emissions and increase performance in beef cattle. Twenty four crossbred heifers (BW = 252 ± 24 kg) were used in a randomized block design replicated in two periods. Heifers were randomly assigned to 12 pens (2 heifers/pen) and pens were randomly assigned to one of six treatments in a 2 × 3 factorial arrangement. Factors included diet high concentrate (HC) or low concentrate (LC) and either 0.0, 0.5 or 1.0% of chitosan dietary inclusion on a DM basis. Heifers were housed in the University of Florida-Feed Efficiency Facility in Marianna, FL. Diets were offered ad libitum and intake was recorded by a GrowSafe system (GrowSafe Systems, Airdrie, Alberta, Canada). After at least a 14-d adaptation period, methane emissions were measured using the SF6 tracer technique for 5 consecutive d in each sampling period. Performance data were collected for the heifers. Data were analyzed using the MIXED procedure of SAS (SAS Institute, Cary, NC). No effects (P > 0.10) of chitosan or chitosan × diet interaction were found for DMI or methane emissions. A diet effect (P BW0.75, and as g/kg of DMI. Heifers consuming a LC diet produced 130 g of CH4/d vs. 45 g of CH4/d in the HC diet. When adjusted for intake, heifers produced 18.2 and 7.1 g of CH4/kg of DMI in LC and HC, respectively. The addition of up to 1 % of chitosan did not affect (P > 0.10) methane emissions or growth performance in heifers consuming either diet. Heifers consuming a LC diet had decreased (P P 2O3 and TiO2 were utilized as external markers, no differences (P > 0.10) were found in nutrient digestibility when chitosan was included. However, using indigestible NDF as an internal marker, DM and OM digestibility were improved (P P > 0.10) when chitosan was included in HC diets. Chitosan, included up to 1% with a HC substrate, had no effect (P > 0.10) on in vitro fermentation parameters, except for pH which was linearly increased (P 4 production was not affected (P > 0.10) by the inclusion of chitosan when added to a HC substrate; however, when incubated with a LC substrate, CH4 production was increased (P P P = 0.08) caproate linearly. In vitro incubations of the LC substrate with chitosan had no effect (P > 0.10) on VFA production.
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In the series University of Florida Digital Collections.
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Includes vita.
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Statement of Responsibility:
by Darren D Henry.
Thesis:
Thesis (M.S.)--University of Florida, 2013.
Local:
Adviser: DILORENZO,NICOLAS.
Local:
Co-adviser: LAMB,GRAHAM CLIFF.

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1 EFFECTS OF FEEDING A NATURAL BIOPOLYMER (CHITOSAN) ON METHANE EMISSIONS AND PERFORMANCE IN BEEF CATTLE By DARREN DWAYNE HENRY A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT O F THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 201 3

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2 2013 Darren Dwayne Henry

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3 To my family

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4 ACKNOWLEDGMENTS First and foremost, I must thank our Lord Jesus Christ. It is be cause of His love and blessings that I am here today. I hold the utmost admiration for Dr. Nicolas DiLorenzo. He has not only been a great advisor and mentor, but he has also become a dear friend of mine. It was Nicolas who believed in me and my poten tial as a graduate student, and for that, I am ever grateful. I would also like to thank Dr. G. Cliff Lamb for his valued mentorship and friendship. Since the first time we met, I have realized his door will always be open to his students, no matter the need. I have had the privilege to listen a number of discussions by Dr. Matt J. Hersom and it will be difficult to find a scientist who can communicate with producers as eloquently. I consider myself extremely fortunate to have a committee with such a di versity of expertise. I am forever thankful for the copious amounts of knowledge I have gained from these three men. Although my time in Florida has been brief, the relationships I have made here continue to grow. I have had the honor to work closely wi th some outstanding lab mates, but more importantly friends. Ms. Francine Messias Ciriaco Silva and Mr. Vitor Rodrigues Gomes Mercadante have been there from the beginning, and I I would also like to show my appreciation to Ms. Tessa Schulmeister, Mrs. Tina Gwin and Mrs. Gina Arnett and everyone at the NFREC who has helped me throughout these last two years. And to all of my friends that I have made along the way, I am thankful. Without a doubt, I have one of the most l oving and supportive families a man could ask for. I received nothing but praise and encouragement for every decision I made about my only want to go to s chool for four years, but maybe six or even ten. This was not an issue I had because I knew I had always had my parents behind me. I thank my grandparents for instilling

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5 in me a love for agriculture and the manners and respect every young man needs. I c an see my me my entire life, but he has taught me the value of work, and I know I will never find someone who works harder at some thing he wants than him. There are a thousand other family and friends who should be named, but I would never have enough room to truly express my undying gratitude.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ........... 4 LIST OF TABLES ................................ ................................ ................................ ...................... 8 LIST OF ABBREVIATIONS ................................ ................................ ................................ ...... 9 ABSTRACT ................................ ................................ ................................ ............................. 11 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ ............. 13 2 LITERATURE REVIEW ................................ ................................ ................................ ... 16 Enteric Methane Production ................................ ................................ ............................... 16 Methanogenesis ................................ ................................ ................................ ........... 16 Methanogens ................................ ................................ ................................ ............... 18 Potential Mitigation Efforts ................................ ................................ ................................ 19 Tannins ................................ ................................ ................................ ....................... 20 Fats ................................ ................................ ................................ ............................. 21 Ionophores ................................ ................................ ................................ .................. 22 Nitrates ................................ ................................ ................................ ....................... 24 Chitosan ................................ ................................ ................................ ............................. 27 Mode of Action ................................ ................................ ................................ .................. 31 3 EFFECTS OF FEE DING A NATURAL BIOPOLYMER (CHITOSAN) ON METHANE EMISSIONS AND PERFORMANCE IN BEEF CATTLE ............................. 32 Materials and Methods ................................ ................................ ................................ ....... 32 Experiment 1 ................................ ................................ ................................ ............... 32 Experimental design, animals, and treatments ................................ ....................... 32 Apparent digestibility ................................ ................................ ........................... 33 Methane emissions ................................ ................................ ............................... 33 Laboratory analyses ................................ ................................ .............................. 34 Experiment 2 ................................ ................................ ................................ ............... 36 Treatments and substrates ................................ ................................ ..................... 36 In vitro incubations ................................ ................................ ............................... 37 Methane, H 2 S production, and ammonia nitrogen analysis ................................ .... 37 VFA analyses ................................ ................................ ................................ ....... 38 In vitro true dry matter digestibility ................................ ................................ ...... 38 Calculations and Statistical Analysis ................................ ................................ ........... 39 Results and D iscussion ................................ ................................ ................................ ....... 40 Experiment 1 ................................ ................................ ................................ ............... 40 Experiment 2 ................................ ................................ ................................ ............... 43

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7 Conclusion ................................ ................................ ................................ ......................... 47 LIST OF REFERENCES ................................ ................................ ................................ .......... 61 BIOGR APHICAL SKETCH ................................ ................................ ................................ ..... 69

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8 LIST OF TABLES Table page 3 1 Ingredients and chemical composition of the basal diets ................................ ................ 48 3 2 Composition and analyzed nutrient content (DM basis) of substrates used for in vitro incubations ................................ ................................ ................................ .................... 49 3 3 Effect of diet and inclusion level of chitosan on DMI and CH 4 emissions in beef heifers ................................ ................................ ................................ ........................... 50 3 4 Effect of diet and inclusion level of chitosan on nutrient digestibility when Cr 2 O 3 was utilized as an external marker ................................ ................................ ................. 51 3 5 Effect of diet and inclusion level of chitosan on nutrient digestibility when TiO 2 was utilized as an external marker ................................ ................................ ........................ 52 3 6 Effect of diet and inclusion level of chitosan on nutrient digestibility when iNDF was utilized as an internal marker ................................ ................................ ......................... 52 3 7 Effect of monensin and inclusion level of chitosan on in vitro fermentation parameters with a HC subs trate 1 ................................ ................................ .................... 54 3 8 Effect of monensin and inclusion level of chitosan on in vitro CH 4 production with a high concentrate substrate 1 ................................ ................................ ............................ 55 3 9 Effect of monensin and inclusion level of chitosan on in vitro fermentation parameters with a LC substrate 1 ................................ ................................ ..................... 56 3 10 Effect of monensin and inclusion level of chitosan on in vitro CH 4 pro duction with a LC substrate 1 ................................ ................................ ................................ ................. 57 3 11 Effects of monensin and inclusion level of chitosan on in vitro proportions of VFA (mol/100 mol), total VFA concentration (m M ), and the acetate:propionate ratio with a HC substrate 1 ................................ ................................ ................................ .............. 58 3 12 Effect of monensin and inclusion level of chitosan on in vitro VFA concentrations (m M ) and the acetate:propionate ratio with a LC substrate 1 ................................ ............ 59 3 13 Effect of monensin and inclusion level of chitosan on in vitro VFA proportions (mol/100 mol) with a LC substrate 1 ................................ ................................ ............... 60

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9 LIST OF ABBREVIATIONS ADF Acid detergent fiber ADG Average daily gain BW Body weight CH 3 S CoM Methyl coenzyme M CP Crude protein DM Dry matter DMI Dry matter intake DPD N, N dimethyl p phenylenediamine sulfate EPA Environmental Protection Agency FA Fatty acid FCM Fat corrected milk FEF Fee d Efficiency Facility GE Gross energy G:F Gain:feed ratio GHG Greenhouse gas GIT Gastro intestinal tract GRAS Generally recognized as safe HC High concentrate diet H 4 MPT tetrahydromethanopterin HS CoM Coenzyme M HS HTP 7 mercaptoheptanoyltheron ine iNDF Indigestible neutral detergent fiber IVOMD In vitro organic matter digestibility IVTDMD In vitro true dry matter digestibility Kf Rate of gas production (%/h) LC Low concentrate diet

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10 LD50 Lethal dose 50 M Maximal gas production MBW Metabol ic body weight (BW 0.75 ) NADH Nicotinamide adenine dinucleotide NDF Neutral detergent fiber OM Organic matter rpm Revolutions per minute SD Standard deviation VFA Volatile fatty acid

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11 Abstract of Thesis Presented to the Graduate School of the Unive rsity of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EFFECTS OF FEEDING A NATURAL BIOPOLYMER (CHITOSAN) ON METHANE EMISSIONS AND PERFORMANCE IN BEEF CATTLE By Darren Dwayne Henry December 201 3 Chair: Nicolas DiLorenzo Co chair: Graham Cliff Lamb Major: Animal Sciences We evaluated chitosan as a feed additive to mitigate in vivo CH 4 emissions and increase performance in beef cattle. Twenty four crossbred heifers (BW=25224 kg) were used in a randomized block design replicated in two periods. The design included a 23 factorial which included diet [high concentrate (HC) or low concentrate (LC)] and 0.0, 0.5 or 1.0% of chitosan inclusion (DM basis). Diets were offered ad libitum and individual intake was recorded. An in kinetics was performed. A diet effect ( P < 0.01) was determined for methane emissions expressed as g/d, g/kg of BW 0.75 and g/kg of DMI. Heifers consuming LC produced 130 g of CH 4 /d vs. 45 g of CH 4 /d with HC. A digestibility marker diet interaction occurred ( P < 0.05) for DM, OM, CP, NDF, and ADF digestibility. When using Cr 2 O 3 and TiO 2 as external markers, no differences existed (P > 0 .10) in nutrient digestibility when chitosan was included. However, using indigestible NDF as an internal marker DM and OM digestibility were improved ( P < 0.05) when chitosan was included in LC. In the HC diet, pH was increased ( P < 0.05) linearly when chitosan was included. In vitro CH 4 production was not affected ( P > 0.10) by chitosan in

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12 HC substrate; however, when incubated with LC substrate, CH 4 production increased quadratically ( P < 0.01) as chitosan inclusion increased.

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13 CHAPTER 1 INTRODUC TION Methane (CH 4 ), in the eyes of a paleoclimatologist, may be viewed as a nuisance in our environment adding to the copious amount of greenhouse gases ( GHG ) being e mitted into the oleum engineer, CH 4 may be a budding energy source with the potential to power and warm the homes of those who seek alternative fuel sources. An animal scientist, more specifically a ruminant nutritionist, understands CH 4 to be a hydrogen (H 2 ) sink, used t o capture H 2 to prevent fermentation from coming to a halt, due to a pH lower than what the microbial ecosystem of the rumen can function in, or a basis of energy loss in the rumen. Today, scientists must work together to alleviate these issues which are burdened by CH 4 (climate change, alternative energy sources, and the growing need of meat and milk production accompanied by the growing population) and determine the most beneficial level of production from livestock. In the animal kingdom, the primary p roducers of CH 4 are ruminants. The rumen, a large fore stomach which occupies over 70% of the total gastro intestinal tract ( GIT ), acts as a continuous fermentation vat. The rumen can hold volumes of 100 to 150 L in beef cattle (up to 210 L in dairy catt le (Russell and Gahr, 2000) ) and about 15 L in small ruminants, such as sheep and goats (Hofmann, 1993) In this fermentation vat, microbial biomass and VFA are produced when feed digestion is initiated by rumen microbes, leaving behind C H 4 as an end product (Leng, 2008) Through fermentation of feedstuffs, cattle produce ( annually ) about seven to nine times more CH 4 than sheep and goats, due to rumen size (Shibata et al., 1992) The United Stat es Environmental Protection Agency ( EPA ; direct contribution to global non CO 2 emissions. The EPA claimed to have calculated enteric CH 4 emissions at 2,079 Mt CO 2 eq/yr for 2010 and they also predicted emissions to reach 2,34 4

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14 Mt CO 2 eq/yr by the 2020 (CH 4 has a global warming potential of 25 times that of CO 2 ). This contribution from livestock will account for about 7.3 to 7.5% of total global GHG emissions between the years 2010 and 2020 (EPA, 2006) Researchers reported CH 4 emitted from livestock to only have a global impact on GHG of about four perce nt (Forster et al., 2007) Others have suggested livestock production to have an 8 to 11% contribution to the total GHG production Another report claimed livestock in the United States account ed for 3.1% of the total GHG emissions in the U.S. in 2009 and 28% of the total CH 4 emissions the second greatest in the country (EPA, 2011) Compar ed with Brazil (a country with a grass based production system) with livestock producing 12% of the total GHG emissions in 2005 (Cerri et al., 2009) Leng (2008) concur ed with the global EPA data claiming ruminants produce about 28% of the CH 4 emissions globally. Pasture based production systems, such as Brazil, Argentina, and New Zealand, have a considerably greater percentage of livestock produced GHG emissions than countries such as the U.S., with a strong grain based cattle feeding system Methane gas is not the only GHG produced from ruminants NO 2 (global warming potential of 310) is also produced in the rumen. Researchers determined that there is about 103 fold less NO 2 in the rumen headspace than CH 4 (Hristov et al., 2010, 2011) The cow calf phase of beef cattle production is when a majority of the GHG emitted from beef cattle occurs (about 80% of total GHG emissions of beef cattle ; Beauchemin et al., 2011) This is likely due to the predominantly forage based diets of the cow calf industry. This is supported by a number of life cycle assessments (Roy et al., 2009; Beauchemin et al., 2010; Thoma et al., 2013) in which on farm emissions accounted for the largest proportion of GHG from the dairy and beef industries.

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15 Therefore, t he objective of this study wa s t o determine the effects of increasing levels of chitosan on nutrient digestibility, enteric methane production, and in vitro batch culture fermentation parameters.

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16 CHAPTER 2 LITERATURE REVIEW Enteric Methane Production Methanogenesis In the ruminant, C H 4 is mostly produced in the reticulo rumen. The process of reducing CO 2 to CH 4 is necessary to prevent ruminal dysfunctions. When H 2 is not utilized by methanogens for producing CH 4 Nicotinamide adenine dinucleotide ( NADH ) can re oxidize with the he lp of dehydronases from bacteria in the rumen to form products such as lactate or ethanol (Moss et al., 2000) When this occurs, usually in animals fed great amounts of highly fermentable carbohydrate (i.e. starches f rom corn), the ruminant can enter a physiological state called acidosis. With acidosis come a number of other metabolic and deleterious conditions. Therefore, without another H 2 sink, CH 4 is of great importance to the ruminant. Moss and co worker (2000 ) describes the elimination of H 2 through the formation of CH 4 with the following equation: CO 2 + 4H 2 4 + 2H 2 O Rouviere and Wolf (1988) described the reduction of CO 2 to CH 4 in a more detailed manner. Formyl methanofuran formation is the first established step of CO 2 reduction. This first product is formed when electrons from oxidized substrates, such as formate and methanol, combine with the CO 2 and methanofuran. The formyl group is transferred to tetrahydromethanopterin ( H 4 MPT ) once methanofuran is cleaved. This transfer occurs due to an appropriately named transferase called formyl methanofura n:tetrahydromethanopterin formyltransferase. Tetrahydromethanopterin is a pterin exclusively produced by methanogens (Donnelly and Wolfe, 1986) The formyl group is transformed into a methenyl group by 5, 10 methenyltetrahydromethanopterin cyclohydrolase (Dimarco et al., 1986) A H 2 is donated by a

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17 reduced coenzyme F 420 for the reduction of the double bond of the methenyl group forming a methylene group. The enzyme methylene tetrahydromethanopterin:coenzyme F 420 oxido reductase is used to catalyze the reduction reaction (Hartzell et al., 1985) Again, electrons from oxidized substrates are donated to form a methyl group from the methylene group. At this point the methylreductase sys tem is initiated. Coenzyme M ( HS CoM ) cleaves the H 4 MPT from the methyl group leaving methyl coenzyme M ( CH 3 S CoM ) (Rouviere and Wolfe, 1988) A phosphate called 7 mercaptoheptanoyltheronine ( HS HTP ) reduces CH 3 S CoM producing a heterodisulfide of HS HTP and HS CoM (CoM S S HTP) and CH 4 (Noll and Wolfe, 1987; Ellermann et al., 1988) The process in which CH 4 is produced occurs simultaneously with the utilization of feed energy; therefore, it is noted by changing the efficienc y of the energy utilization, CH 4 emissions can be influenced (Shibata and Terada, 2010) Animal type, feed quality and quantity, and environmental conditions affect feed energy utilization (Shibata and Terada, 2010) Johnson and Johnson ( 1995) found in ruminants, as the DMI increases, a mean decrease in the percentage of gross energy lost as CH 4 of 1.6% per level of intake. Similar t o this, when a limited amount of a highly available carbohydrate is fed, greater fractions of gross energy are lost as CH 4 However, with increased intakes of highly digestible feedstuffs, lesser fractional CH 4 losses occur (Johnson and Johnson, 1995) Production of VFA plays a key role in the production of CH 4 in the rumen. Volatile fatty acids, such as acetate and b utyrate, promote the production of CH 4, while the formation of propionate impedes the yields of CH 4 Acetate and butyrate encourage methanogenesis by releasing CO 2 during their formation. Propionate, the most energy efficient VFA used for gluconeogenesis does not produce CO 2 when formed through the pyruvate/lactate pathway.

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18 The pyruvate/lactate pathway is the most common conduit of propionate formation in grain fed animals. An in vitro study with monensin found CH 4 was not correlated to acetate; howev er, there was a negative association between CH 4 and propionate along with a slight association between CH 4 and the acetate:propionate ratio. The ratio of (acetate + butyrate)/propionate had an improved relationship with CH 4 production. Moss et al. ( 2000) provides evidence that propioneogenesis and methanogenesis compete. If the acetic to propionic ratio was 0.5, the percentage of energy loss by CH 4 would be 0.0%. Alternativ ely, if all the carbohydrates in a theoretically be a 33% loss of energy due to the reduction of carbon dioxide to CH 4 Methanogens In the rumen, microorganisms are needed to carry out anaerobic fermentation in which feedstuffs are digested to produce microbial protein. M icroorganisms which produce CH 4 ( specialized methanogenic prokaryotes ) are strictly anaerobic and belong to the archaea kingdom (Jones et al., 1987) Archaea differ from other bacteria by lacking peptidoglycan polymer s in their cell walls (Moss et al., 2000). The composition of intracellular lipids of archaea are also differe nt from other bacteria, ether linkages replace triacylglycerol between glycerol and polyisoprenoid chains (Moss et al., 2000) It has been discovered through ribosomal RNA nucleotide sequencing that archaea and other b acteria diverged early during evolution. Also, in the phylum of methanogens, the different genera and species have many different shapes and physical characteristics: cocci, rods spirilla, thermophylic, and mesophylic species, motile and non motile cells (Moss et al., 2000) Methanogens have been found attached to the external pellicle of protozoa (Stumm et al., 1982; Krumholz et al., 1983) Since protozoa produce H 2 and methanogens can potentially utilize H 2 (Iannotti et al., 1973) Researchers conclude, methanogens outcompete other microbes

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19 for the use of H 2 (Ellis et al., 2008) There are some bacteria, which utilize starch, that have the ability to compete with methanogens for H 2 ; these starch digesting bacteria use the H 2 for propioneogenesis (Russell, 1998) Lactic bacteria, that unlike cellulolytic bacteria and methanogens can tolerate low pH, can also utilize H 2 making them competitor s of methanogens, even in unfavorable conditions (Moss et al., 2000) Methanogens use H 2 formate, acetate, methanol, mono di and tri methylamine as potential substrates, but only H 2 CO 2 and small quantities of formate are the substrates used as precursors to CH 4 in the rumen (Miller, 1995) For the reduction of carbon dioxide to CH 4 to be completed by methanogens, the potential aforementioned substrate s are oxidized, donating electrons (Rouviere and Wolfe, 1988) The reduction of CO 2 by a pr eponderance of methanogens occurs through the stages of formyl, methenyl, methylene, and methyl to CH 4 (Rouviere and Wolfe, 1988) Potential Mitigation Efforts For almost two decades, researchers have been attempting to mitigate CH 4 production in ruminant animals. Many strategies and theories have been devised, but only a few currently show pot ential as a viable source of CH 4 mitigation. The following include only some of the more feasible approaches. Joblin (1999) described three possible schemes to reduce enteric CH 4 production: 1) remove methanogens from the rumen; 2) reduce H 2 production; and 3) provide an alternative H 2 sink in the rumen. Additional potential strategies are: 1) increasing efficiency by improving the digestibility of forages and feedstuffs; 2) developing feed additives to act as H 2 sinks; 3) reduce or inhibit methanogen po pulations in the rumen; 4) provide nourishment to microbes other than methanogens to aid in the formation of more efficient substrates, such as propionate, instead of CH 4 ; and 5) improve the efficiency of production (meat, milk, etc.) to reduce the number of livestock required (USDA, 2004)

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20 Tannins T annins ( proanthocyanidins ) are a group of diverse polymeric flavonoids with either C C or C O C bonds (McMahon et al., 2000) Found in cell walls or in vacuoles of stems, bark, leaves, flowers or seeds, tannins are principally found in dicotyledonous plants (McMahon et al., 2000) Condensed tannins ( similar to lignin ) are synthesized from precu rsors of the acetate pathway (McMahon et al., 2 000) Tannins have been shown to decrease CH 4 up to 20% (Zhou et al., 2011; Staerfl et al., 2012) T he production of CH 4 in g per kg of DMI was reduced by 29% in sheep grazing ryegrass ( Lolium perenne L. ) supplemented with a tannin containing lotus ( Lotus pedunculatus ; Woodward et al., 2001) The se researchers also witnessed a 23% decrease in g of CH 4 per kg of DMI in dairy cows fed lotus silage rather than ryegrass silage. Dairy cows in New Zealand grazing sulla ( Hedysarum coronarium ) vs. ryegrass had a 21% decrease in L of CH 4 produced per kg of DMI and a decrease of 27% for L of CH 4 produced per kg of milk solids (Waghorn et al., 2002) In addition sheep fed a 60:40 forage:concentrate diet had a n 11% decrease in L of CH 4 produced per d when supplemented with tea saponins. This was accompanied by a 13% decrease in the acetate to propionate ratio (Zhou et al., 2011) Zhou et al. (2011) attributed the reduction in methanogenesis to tannins inhibiting protozoa, increasing molar proportions of propionate and by decreasing the acetate to propionate rati o. In growing bulls fed a corn based diet, CH 4 emissions in L per d were reduced by 30%, L per kg of DMI was reduced by 28% and L per kg of BW gain was reduced by 22% when tannins where fed along with the diet (Staerfl et al., 2012) Comparing a tannin rich forage, sericea lespedeza ( Lespedeza cuneata ) with a mixture of crabgrass ( Digitaria ischaemum ) and Kentucky 31 tall fescue ( Festuca arundinacea ), goats produced 30% less g of CH 4 per d and 57% less g per kg of DMI when fed sericea lespedeza (Puchala et al., 2005)

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21 There are, however, some shortcomings that come along with tannins in ruminant diets. Tannins were reported nutriti absorption (Waghorn, 2008) McMahon et al. (2000) describe d attributed to a low palatability of some diets containing greater levels of tannins. Tannins fed to dairy cows reduced CH 4 production per kg of DMI by 22%, but experienced a 10% decrease in milk production (Grainger et al., 2009) Consumption of tannins may be a potential strategy for mitigating CH 4 production in rumin ants, but further research must be performed to determine if the overshadowing reduction in digestion and production of animals outweighs the reduction in CH 4 Fats Dietary lipids have been evaluated to determine effects on production qualities such as mi lk yield, milk fats, and fat corrected milk ( FCM ). R ecent attention has been placed on the inclusion of fats to mitigate CH 4 production, especially in dairy cows. Lipids may reduce enteric CH 4 production by a number of methods, including reduction of DMI ; however, g/kg DMI may or may not be affected (Zeitz et al., 2013) In performing a meta analysis it was determined that a constant decrease in DMI of dairy cows across many diets supplemented with dietary lipids (tallow, various calcium salts of fatty acids [ FA ] oilseeds, prilled fat) with a maintained or increased milk production (Rabiee et al., 2012) One may extrapolate further on these results and assume, with consistent or increase milk production and lower feed inta ke, an increase in feed efficiency and, subsequently, a reduction in CH 4 production (FAO, 2013) Another meta analysis consider ed fat supplementation on CH 4 production of dairy cows reported similar results to that of Rabiee et al. (2012 ; Eugene et al., 2008) Eugene et al. (2008) analyzed seven publications (25 diets) and report ed a 9% decrease in enteric CH 4 production in dairy cows when supplemented with lipids. Along with the decrease in CH 4 a 6.4% decrease in

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22 DMI was also noted. When CH 4 production was calculated per unit of FCM, there was a 9% decrease in CH 4 energy produ ction (0.82 vs. 0.75 MJ CH 4 energy/kg 4% FCM). Although it is possible for unsaturated fats to act as a H 2 sink during biohydrogenation, it is unlikely this will make a significant impact on methanogenesis because it has been proposed that only one to two percent of H 2 in the rumen is used in this fashion (Czerkawski and Clapperton, 1984) In a study comparing animal fat (tallow) and sunflower oil (both supplemented at 3.4% of diet DM), there was a 12% reduction in CH 4 with both lipid sources. This reduction of CH 4 was accompanied by no significant change in DM or NDF digestibility, feed intake, ADG or quantity of saturated FA (Beauchemin et al., 2007) Another study fed differing quantities of crushed sunflower seed (9 and 10% of diet DM [6.7 to 7.3% cr ude fat]) to dairy cows to evaluate CH 4 and milk production and rumen fermentation (Beauchemin et al., 2009) When evaluating CH 4 per unit of FCM, there was approximately a 15% decrease compared to a 45:55 forage:concentrate control diet. There may be a potential to abate CH 4 production in cattle using dietary lipids, but it must be evaluated further to determine the detrimental effects to pro duction and intake. Ionophores Ionophores, such as monensin, have been used heavily in the beef feeding industry since (Schelling, 1984) In more recent years, in the United States, monensin has also been introduced into the dairy industry (Russell and Houlihan, 2003) A number of modes of action which try to explain how ionophores like monensin alter the rumen ecosystem causing an increase in feed efficiency and a decrease in CH 4 production. The basic mode of action of an ionop is to modify the movement of ions across the membranes of microbes in the rumen (Schelling, 1984) With this transfer of ions, most notably sodium, monensin terminates certain micro organisms most notably gram positive Modification of VFA

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23 production has been shown in the rumen of monensin fed animals (Schelling, 1984) Studies have shown a decrease in the acetate:propionate (Richardson et al., 1976) This decrease can be attributed to a decrease in acetate production and an increase in propionate (Richardson et al., 1976) As researchers have found, propioneogenesis competes with methanogenesis (Moss et al., 2000) ; therefore, it can be concluded that increasing propionate, to compete with CH 4 production, and decreasing acetate, which is a promoter of CH 4 can mitigate CH 4 to some extent. Monensin is known to decrease f e ed intake of rumin ants (Schelling, 1984) This decrease in feed intake could possibly be considered a system mode of action or a response (Schelling, 1984) Either way, it has been well documented (Johnson and Johnson, 1995; Shibata and Terada, 2010) that CH 4 production is positively correlated to DMI and by decreasing feed intake, without causing detrimental effects to production, CH 4 production could be altered Hydrogen production has been shown to be affected by monensin. It was reported that moenesin selects against some H 2 producing microbes (Chen and Wolin, 1977) The se authors also reported that monensin affect the physiology of bacteria which for m succinate and Selenomonas ruminantium a microbe which decarboxylates succinate to form propionate. By decreasing the H 2 production, methanogenesis could be reduced as well (USDA, 2004) Through an analysis of several in vitro and in vivo studies Schellin (1984) reported a change in CH 4 producti o n which ranged from 4% to 31% when monensin was fed, with an average decrease of 18%. Monensin improve d feed efficiency in fed cattle by 7.5% (Goodrich et al., 1984) growing cattle on pasture by 15% (Potter et al., 1986) and dai ry cows by 2.5% (Duffield et al., 2008) This improvement in feed efficie ncy may lead to a decrease in CH 4 emissions (FAO,

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24 2013) Monensin also consi stently decrease d the acetate:propionate in cattle consuming high grain diets which could cause a decrease in CH 4 production (FAO, 2013) A more recent meta analysis of anti methanogenic properties of monensin in beef and dairy cattle showed when monensin (administered at 32 mg/kg diet DM) was fed to beef steers, CH 4 was decrease d by 19 4 g steer 1 d 1 (Appuhamy et al., 2013) Although there were significant changes in methanogenesis for dairy cows, when beef steers and dairy cows were analyzed together, there was a 13 g animal 1 d 1 decrease. Furtherm ore, when considering the conversion rate of GE to CH 4 beef steers receiving monensin had a decrease of 0.54 0.14%. As one may expect, there was a consistent decrease of DMI and no change in production factors such as milk yield (Appuhamy et al., 2013) Nitrates In the past quarter century, nitrates have been chastised and cherished by ruminant nutritionists. Some scientists fear the potential toxicities which may accompany nitrate feeding where others explore the use nitrates for anti methanogenic properties. FAO (2013) reports a need to further explore nitrates as potential H 2 sinks, especially alongside low protein diets where microbes of the rumen may benefit from non protein nitrogen (NPN). It is possible that nitrates could replace urea in diets as a nitrogen source for the production of microbial protein (Leng, 2008) The following equation demonstrates how nitrates may be reduced to ammonia; therefore, acting as a H 2 sink and NPN source (Leng, 2008) : NO 3 + 4H 2 +2H + 4 + + 3H 2 O In theory, one mol of nitrate would produce one mol of ammonia and, consequently, decrease CH 4 by one mol (16 g or 22.4 L ; Leng, 2008) There is the potential for nitrite poisoning, methaemoglobinaemia, which is associated with greater quantities of dietary nitr ates. Nitrite toxicity occurs when the rate at which nitrite is

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25 absorbed is greater than the capacity of the red blood cells to bind and oxidize nitrite to nitrate. At this point, methaemoglobin concentration s increase in the blood cells. With great er c oncentrations of methaemoglobin, the ability of the red blood cells to carry oxygen i s decreased and hypoxia of organs can occur and eventually result in death (Leng, 2008) Nitrite toxicity is found to be linked to diets high in protein with supplemental nitrate to increase the CP content of the diet. For this reason, Leng (2008) claim ed there may be an interaction between nitrate s and protein to cause the toxicity. T oxic doses for ruminants range from 198 to 998 mg/kg of BW (Hibberd et al., 1994) A lethal dose 50 ( LD50 ) is defined abruptly injected, is lethal to 50% of the experiment T he rumen can eventually adapt to great er concentrations of nitrate/nitrites. A report focusing on the bacteria in the rumen found an increase in nitrate reducing microbes when nitrates were added to the diet (Allison and Reddy, 1984) Several studies have shown nitrates to be fed gradually to allow t he rumen to adapt with no detrimental health problems (Nolan et al., 2010; van Zijderveld et al., 2010, 2011a b) In addition when sheep were dosed with nitrate s at 2.5 g kg 1 BW 1 d 1 an acclimation of the rumen occurred (Alaboudi and Jones, 1985) ; however, when the nitrate (fed as KN O 3 ) was removed from the diet, the nitrate reducing ability of the sheep d ecreased to original levels. This could be a potential issue of using nitrates in countries or regions where feed sources and diets are variable (FAO, 2013) The utilization of nitrate s as an anti methanogenic supplement would be most successful with diet s low in protein (Leng, 2008) Nitrite accrual may increas e when diets are high in CP associated with increased ammonia nitrogen and hydrogen sulfide concentrations (Leng, 2008) A majority of documented nitrite toxicity cases occurred when CP was great (18 to 38%) and when ammonia concentrations in the rumen were great er (3 recommended level for microbial

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26 growth ; Preston and Leng, 1985) Leng (2008) summarized data from Faulkner and Hutjens (1989) describing pos sible amounts of nitrates in certain feedstuffs. Some forages may have nitrate levels upwards of 2.6 and 2.9% for corn silage and green chopped sudan, respectively (Faulkner and Hutjens, 1989) Leng (2008) reported when corn silage has 2% of the diet DM as nitrates, with 25% inclusion of corn silage, a dairy cow consuming 25 kg DM d 1 may intake 125 g/d of nitrates from corn silage alone. Furthermore, the level of nitrates in forages can be impacted depending on the amount of nitrogen fertilizer applied (Leng, 2008) An in vitro study with perennial ryegrass, showed for each percent age increase in CP (from 13 to 23%), the concentration of nitrate s in forage increased linearly by 0.035 g kg 1 DM (Lovett et al., 2004) This indicates ruminants are exposed to nitrates in nature and when considering using nitrates as a strategy for CH 4 mitigation, basal levels of nitrates in the feed must be accounted for (Leng, 2008) Several studies have shown the potential mitigation effects of nitrates. Bos indicus cattle fed a 60:40 forage:concentrate diet was either supplemented with 0 or 22 g kg 1 DM of nitrate (Hulshof et al., 2012) These researchers reported a 32% decrease in g/d of CH 4 (125 g d 1 for control vs. 85 g/d for nitrate ), a 27% decrease in g of CH 4 per kg DMI (18.2 g kg 1 DMI for control vs. 13.3 g/kg DMI for nitrate) and a decrease in percent of GE intake lost as CH 4 from 5.9% to 4.2%. Another study with sheep on a high forage diet investigated supplementing 4% or 0% of diet DM with KNO 3 (Nolan et al., 2010) This study showed an average of 23% decrease in L of CH 4 per kg DMI. In addition researchers investigated sheep on a corn s ilage being supplemented with 2.6% diet DM of nitrate (van Zijderveld et al., 2010) Methane production in L per d was reduced by 32%, as well was CH 4 produced in L per kg of BW 0.75 Similarly, L of CH 4 produced per kg of DMI was decreased by 3 1%. The same researchers performed a similar

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27 experiment with dairy cows consuming a 66:34 forage:concentrate diet and supplemented with zero or 21 g per kg DM of nitrate. Production of CH 4 in g per d, g per kg DMI and as a percent of GE intake lost was d ecreased by 16% (van Zijderveld et al., 2011b) There was no change in milk production. None of these studies showed deleterious effects of feeding nitrates Chitosan Chitosan (N acetyl d glucosamine polymer) is a natural biopolymer formed from the deacetylation of chitin. Chitin, the second most abundant organic compound on earth, can be found in the cell walls of lower plants and the exoskeletons of some arthropods and crustaceans (e.g., crab and shrimp). Chitosan has been studied for various applications in food preservation and medicine due to its antimicrobial actions (Cuero, 1999; Shahidi et al., 1999) In the U.S., GRAS the way for chitosan to be used as an alternative to antibiotics. Chi tosan should not be considered a single compound, but rather a series of compounds with differing levels of deac e tylation and other physic chemical characteristics (Goiri et al., 2009a) Chitosan, as an in vivo CH 4 inhibitor, is a novel product for ruminants; however, there have been studies with monogastrics (i.e., poultry and swine) to alter protein fe rmentation of intestinal microbiota (Han 2011) An experiment was conducted to determine the effects of including chitosans of differing deacetylation degrees and viscosities on in vitro batch culture fermentation (Goiri et al., 2009a) This study utilized four different deacetylation degrees (75, 85, a combination of 75 to 90, and > 90%) and viscosities which ranged from 20 to 2000 mPa. Res earchers utilized rumen fluid from four ruminally fistulated sheep consuming a high forage diet (70:30 forage:concentrate) as buffer solution as inoculum. Corn s ilage, 0.5 g per 50 mL of culture fluid, was used as the

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28 substrate for the in vitro incubation. Each of the six chitosans (CHI1, CHI2, CHI3, CHI4, CHI5, and CHI6) was administered at 750 mg L 1 of culture fluid. The bottles were incubated for 24 h. Over all, chitosan significantly decreased total gas production. Chitosans which were 70 to 90% deacetylated (CHI5) and > 95% deacetylated (CHI6) decreased total VFA production. The reduction of total VFA by CHI6 may have been caused by a decline in the molar proportion of acetate compared to control s (57.1 vs. 51.3 mmol). There was an 18% increase in the molar proportion of propionate with CHI6 as well and the decrease in acetate and increase in propionate concentrations lead to a significantly lower acetate :propionate. All types of chitosan decreased the true digestibility compared to control samples. Treatments CHI3 (deacetylation degree of 85%), CHI5, and CHI6 decreased production of CH 4 compared with control (control vs. CHI3, CHI5, and CHI6; 0.8 mmol v s. 0.61, 0.65, and 0.56). There was no change in ammonia nitrogen concentration. From the results, it appears that CHI5 and CHI6 seem to have the most potential effects on CH 4 production mitigation. It has been suggested that with increased percentage o f deacetylation, there is a increased level of interaction between the glucosamine radicals of chitosan and the bacterial cell wall components (Chung et al., 2004) Another study from the same group (Goiri et al., 2009b) utilized the rumen simulation technique (Rusitec) to develop a culture medium for an in vitro experiment. To inoculate the R usitec, the researchers collected rumen fluid and solids from four ruminally fistulated sheep consuming a 70:30 forage:concentrate diet. The substrate for the R usitec was a 50:50 diet and the inoculum was 400 mL of rumen fluid and 300 mL of buffer solution. Two rates of chitosan (deacetylation degree of > 95% ) were administered to the Rusitec system daily after an 8 d adaptation period. The increased dose of chitosan (CHI H) was 1380 mg 700 mL 1 d 1 and the low dos e (CHI L) was 690 mg 700 mL 1 d 1 from d 8 to d 16 of the Rusitec study. Samples were

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29 taken over a nine d collection period and averaged. Both CHI H and CHI L decreased DM, OM and CP disappearance, while NDF disappearance was only affected by CHI H. Treatments CHI H and CHI L decreased total gas production and mmol per d of CH 4 by about 27 and 34%, respectively. Production of CH 4 reported in mmol/g of DM disappeared, was decrease d by 23% by CHI H. Neither CHI H nor CHI L had any effect on acetate production. However, both treatments increased propionate production by a mean of 56%. This was fol lowed by a significant decrease in the acetate:propionate. There was a 46% decrease in ammonia nitrogen associated with CHI H. After the 9 d collection period, fluid from the Rusitec was used as inoculum for a 96 h in vitro experiment, with starch (corn) cellulose, and the 50:50 diet used with the R usitec. Asymptotic gas production of with starch, cellulose and the 50:50 diet was decreased, as compared with control, when CHI L was administered Treatment CHI H was associated with a decrease in asymptot ic gas production for starch and the 50:50 diet. Both treatments were associated with a decrease in gas production rate and lag time for the cellulose substrate. A third study from the same group (Goiri et al., 2009c) investigated a number of variables such as, deacetylation degree (75, 85, and > 95%; CHI1, CHI2, and CHI3, respectively), dose response of each type of chitosan (0, 325, 750, and 1500 mg/L of culture fluid), and three diets differing in forage quantity (forage:concentrate; 80:20, 50:50, and 20:80). For the high forage diet, all deac etylation degrees decreased in vitro organic matter digestibility ( IVOMD ) compared to control with a linear effect of dose. There was no change in total VFA production among the treatments nor was there a change in acetate concentration. Propionate conce ntrations were increased the greatest by CHI3 with a linear effect of dose. Suitably, CHI3 at 1500 mg/L of incubation fluid had the largest decrease in the acetate:propionate and CH 4

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30 production among the treatments. For the 50:50 diet, all deacetylation degrees decreased IVOMD and increased propionate with a linear effect of dose. T here was no change in acetate production among the treatments. The acetate:propionate was decreased by all deacetylation degrees with a linear effect of dose. The only treat ment which lowered CH 4 production was CHI2 at 325 mg/L of incubation fluid. The low forage diet had similar results. All deacetylation degrees decreased IVOMD with a linear effect of dose. There was a decrease in total VFA production associated with CHI 2 at 325 mg/L of incubation fluid. Acetate concentration was lowered by both CHI2 and CHI3 with a linear effect of dose. Similar to the other diets, deacetylation degrees increased propionate concentrations with a linear effect of dose. T he acetate:prop ionate and CH 4 production were reduced by all deacetylation degrees with a linear effect of dose. C hange in ammonia nitrogen was not deteremined for any of the treatments among the diets. These results supported the hypothesis of Goiri et al. (2009a) tha t CHI3 had the greatest impact among the analyses. The same group of investigators (Goiri et al., 2010) considered the effects of chitosan (> 95% deacetylated) on ruminal and cecal fermentation parameters, such a s VFA in rumen fluid and feces. Four ruminally fistulated sheep (mean BW 62 kg) were fed a 50:50 forage:concentrate diet along with either zero or 136 mg/kg BW of chitosa n. Fermentation parameters were measured then rumen fluid from these four sheep was used as inoculum for an in vitro study using starch, cellulose and the 50:50 diet as substrates. Chitosan caused a decrease in ammonia in the rumen fluid of the sheep. P ropionate was increased but, no change was found for acetate. However, the acetate:propionate was decreased with the addition of chitosan. In the feces of the sheep, chitosan caused decreases in acetate concentrations and the acetate:propionate. There w as an increase in molar proportion of propionate due to chitosan in

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31 the feces. The in vitro study showed the following results: for the starch substrate, the addition of chitosan increased the propionate concentration, and decreased the acetate:propionate and CH 4 production. Total VFAs were decreased when chitosan was added to the cellulose substrate. The propionate proportion was increased and CH 4 was decreased with the addition of chitosan along with the acetate:propionate. For the 50:50 diet, no chan ges were noted for the acetate or propionate concentrations, yet there was a decrease in CH 4 production. Mode of Action There are a number of theories which suggest the mode of action for chitosan to decrease CH 4 production in ruminants. One theory sugg ests positively charged chitosan may interact with negatively charged microorganisms and cause a leakage of proteinaceous and other intracellular constituents from the cytosol (Rabea et al., 2003) Another notion of the mode of action of chitosan is through agglutination. It has been demonstrated (Chung et al., 2004) that chitosan has a positive charge and at lower concentrations (< 0.2 mg/mL of incubation fluid), may bind to negatively charged bacterial surfaces to cause agglutination.

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32 CHAPTER 3 EFFECTS OF FEEDING A NATURAL BIOPOLYMER (CHITOSAN) ON METHANE EMISSIONS AND PERFORMANCE IN BEEF CATTLE Materials and Methods Experiment 1 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 Florid a. Experimental design, animals, and t reatments Twenty four crossbred heifers (318 35 kg of initial BW; mean SD) were used in a randomized block design with a 2 3 factorial arrangement of treatments and two experimental periods. Factors were diet type (high and low concentrate) and chitosan inclusion level (0.0, 0.5 and 1.0% of dietary DM). Heifers were stratified by weight and randomly assigned to treatments i n the first period. After experimental period one, the 24 heifers were subjected to a 14 d washout period during which they received bahiagrass ( Paspalum notatum ) hay, and were then re randomized for the second experimental period. During the experiment, heifers were housed two per pen in the Feed Efficiency Facility ( FEF ) at the North Florida Research and Education Center in Marianna. To provide the desired concentrations in the experimental diets, chitosan (90% deacetylation degree; PharmaNutrients, In c. Lake Forest, IL ) was mixed with soybean meal to provide a chitosan premix that was included at 2% of the dietary DM. The high concentrate ( HC ) basal diet (85% concentrate) was comprised of (all in DM basis): 40.0% corn gluten feed, 39.3% soybean hulls pellets, 15.0% peanut hulls, 4.0% liquid supplement, and 1.7% mineral and vitamin supplement. The low concentrate ( LC ) basal diet (36% concentrate) was comprised of (all in DM basis): 15.0% corn gluten feed, 15.0% soybean hulls pellets, 64.0% peanut hulls 3.8% liquid supplement, and 2.2% meal supplement. Analyzed nutrient content of diets is

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33 described in Table 3 1. Both diets were formulated to meet or exceed the CP, minerals, and vitamins requirements of the heifers (NRC, 1996) Experimental diets were offered ad libitum usin g a tractor pulled mixer/delivery unit (Roto Mix 84 8, Dodge City, KS ). Daily ad libitum DMI was recorded by the GrowSafe system ( GrowSafe Systems Ltd., Airdrie, Alberta, Canada ) Heifers were housed in pens of 108 m 2 each, equipped with 2 GrowSafe feed b unks each, and water troughs. Heifers were weighed at the beginning of each period and at the end of each period to determine mean BW, G:F, and ADG for each period. Apparent digestibility Apparent total tract digestibility of nutrients was determined u sing two external markers (chromic oxide and titanium dioxide) and indigestible NDF ( iNDF ) as an internal marker. From d 7 to d 16 of each period, 10 g of Cr 2 O 3 and 10 g of TiO 2 were mixed with 0.25 kg of brown sugar to ensure total consumption, and top dr essed to allow for adaptation and constant excretion rate. From d 17 to d 20 of each period, 5 g of Cr 2 O 3 and 5 g of TiO 2 were dosed twice daily at 0700 h and 1500 h via a gelatin capsule using a balling gun. Fecal samples of each heifer were collected by rectal grab twice daily (0700 h and 1500 h) from d 18 to d 21 of each period. Immediately after collection, fecal samples were stored frozen at to d 20 for determination of nutrient content and iNDF concentra tion. for at least 48 h in a forced air oven. Feed and fecal samples were ground in a Wiley mill (Arthur H. Thomas Co Philadelphia, PA) to pass a 2 mm screen. Feed samples were composited in pen. Fecal samples were composited in the individual heifer. Methane emissions Methane emissions were measured using the sulfur hexafluoride (SF 6 ) tracer technique (Johnson et al., 1994) Permeation tubes consisted of a brass body ( length = 3.14 cm; outside

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34 diameter = 1.1 cm; inside diameter = 0.52 cm; inside depth = 2.6 cm; final volume = 0.5 ml ) with a Teflon membra steel frit and a SwageLok nut. Permeation tubes were dosed via balling gun on d 0 of the first period. Permeation tubes were filled with approximately 600 mg of SF 6 and an average permeation rate of 1.745 mg of SF 6 per d was measured after a 3 mo calibration at 39 C. Gas samples were collected into pre evacuated polyvinyl chloride canisters (2 L volume), through a capillary tube attached to a 15 fixed near the nostrils of the heifer. Canisters were fitte d with an on/off valve and a female quick connect and were replaced daily at 0700 h. The volume of the canister and length of the capillary tubes were designed to allow for half of the vacuum to remain after 24 h of collection. Two canisters and capillar ies, identical to the units on the heifers, were utilized to capture ambient CH 4 and SF 6 concentrations in the FEF. All heifers were adapted to the canisters for a week prior to collections. Laboratory a nalyses For concentrations of Cr approximately 0.5 g of ground feces were dried in a forced air The method of Williams et al. (1962) was used to digest Cr 2 O 3 in the samples. C oncentration of chromium was then d etermined by atomic absorption spectrophotometry ( 358 nm with an air plus acetylene flame; AAanlyst 200; Perkin Elmer Walther, MA). For concentrations of TiO 2 approximately 0.5 g of ground feces were dried in a forced OM. Titanium dioxide samples were analyzed using a modification of the method developed by Titgemeyer et al. (2001) Briefly, TiO 2 in the samples was digested by bringing 10 mL of 7.4 M

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35 sulfuric aci d to a gentle boil for approximately 30 min (or until translucent) using a hot plate under a fume hood. After the samples had cooled, the contents of each beaker were rinsed into tared 120 mL sample cups. Ten mililiters of 30% H 2 O 2 was added and the weig ht of each cup was brought to 100 g using distilled water. Samples were then mixed and filtered (Fischerbrand P8 Grade, Fisher Scientific, Pittsburgh, PA ) and analyzed for concentration of TiO 2 measuring absorbance at 405 nm wavelength in a Beckman DU 530 Spectrophotometer (Beckman Coulter, Palo Alto, CA). To analyze concentrations of iNDF in feed and feces, the method described by Cole et al. (2011) with the modification proposed by Krizsan and Huhtanen et al. (2013) was used. Briefly, samples were wei ghed (0.5 g) into F57 filter bags, and then incubated at 39 C using a 4:1 ratio II incubator (Ankom Technology, Macedon, NY) for 288 h to ensure complete digestion of potentially digestible NDF ( Krizsan and Huhta nen 2013). After incubation, samples were rinsed and analyzed for NDF concentration in an Ankom 200 Fiber Analyzer (Ankom Technology) using sodium sulfate and heat stable amylase. C oncentration s of crude protein in feed and feces was determined by rapid combustion using a macro elemental N analyzer (Vario Max CN, Elementar Americas Inc., Mt. Laurel, NJ) following official method 992.15 (AOAC, 1995) Methane and SF 6 concentrations in canisters were analyzed by gas chromatography (Agilent 7820A GC, Agilent Techno logies, Palo Alto, CA) using a flame ionization detector and a capillary column ( Plot Fused Silica 25 m 0.32 mm, Coating Molsieve 5A, Varian CP7536 ).

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36 Experiment 2 Treatments and s ubstrates In vitro batch culture incubations were conducted using a high and low concentrate substrate ( HC and LC respectively). The LC substrate consisted of 64.0% peanut hulls, 15.0% soybean hulls, 15.0% corn gluten feed, 5.1% soybean meal, and 0.9% urea (all in DM basis). The HC substrate consisted of 84.0% corn, 5.2% so ybean meal, 10.0% bermudagrass ( Cynodon dactylon ) hay and 0.8% urea (all in DM basis). Analyzed nutrient content of diets is described in Table 3 2. Chitosan (same source as in Exp. 1) was included into both HC and LC substrates at 0.0, 0.5, and 1.0% DM. M onensin was included in one of the treatments as a positive control and was added dissolved in ethanol at a dose of 4 mg/L of incubation volume and with a final concentration of 0.2% ethanol (v/v) in the incubation fluid These combinations of substrates and treatments were used to evaluate in vitro true dry matter digestibility (IVTDMD), gas production kinetics, H 2 S and CH 4 production, NH 3 N, and VFA concentrations. Before inclusion into the substrates, all ingredients were air dried for at least 48 h an d ground to pass a 2 mm screen (except chitosan that was in a powdered form, and urea which was ground using mortar and pestle), in a Wiley mill (Arthur H. Thomas Co.). Substrates were sent to a commercial laboratory (Dairy One Forage Laboratory, Ithaca, NY) for nutrient composition analyses.

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37 In vitro incubations Substrates were incubated for 24 h at 39C under constant agitation (60 rpm) in 250 mL bottles, and gas production kinetics was recorded using the Ankom Gas Monitoring System (Ankom Technolo gies ). Bottles containing 1.4 g of substrate and 100 mL of inoculum were incubated for 24 h and volatile fatty acid concentrations were measured at the end of the incubation period along with NH 3 N concentrations, final pH, H 2 S, and methane production. A t the end of the incubation the fermentation was stopped by adding 1 mL of a 20% H 2 SO 4 solution to each bottle. After that, a 10 mL sample was taken and frozen for subsequent VFA and NH 3 N analyses. Ruminal fluid was collected from 2 ruminally cannulated Angus crossbred steers ( 601 16. 0 kg of BW). The steers were grazing a bahiagrass ( Paspalum notatum ) pasture for at least two weeks before the collection or rumen fluid for inoculum for the LC substrates. For the HC substrate incubations, ruminal fluid donor steers grazed a bahiagrass pasture with ad libitum access to 85% concentrate diet, consisting mainly of corn gluten feed and soybean hulls, for at least two weeks before the incubations. Ruminal fluid was strained from a representative sample of dig esta through four layers of cheese cloth, placed in a pre warmed thermos container, and fluid mixture was utilized as inoculum for the LC substrate incubations, wh ile a 2:1 buffer:ruminal fluid mixture was used for the HC substrate incubations. Methane, H 2 S p roduction, and ammonia n itrogen a nalysis Total gas produced in the 250 mL bottles was collected in a 1 L Tedlar gas collection bag attached to the Ankom Gas Mo nitoring modules. The gas in the Tedlar bag was analyzed for CH 4 and H 2 S concentration. To determine H 2 S production, 5 mL of gas from the Tedlar bag were bubbled slowly into 15 mL evacuated tubes (BD Vacutainer, Franklin Lakes, NJ) containing 5 mL of alk aline water

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38 prepared as described by Smith et al. (2010). The tubes were then shaken vigorously to ensure proper dispersion of the gas in the alkaline water. An injection, 0.5 mL, of N, N dimethyl p phenylenediamine sulfate ( DPD ) was made into the tubes followed by 0.5 mL of ferric chloride. Tubes were again shaken vigorously and allowed to rest for 30 min for the reaction to occur (Smith et al., 2010) Absorbance was read at 665 nm using a spectrophotometer (DU 530, Beckman Coulter Inc.). Concentration s of NH 3 N in the incubation fluid were measured after centrifuging at 10,000 x g for 15 min at 4C (Avanti J E, Beckman Coulter Inc., Palo Alto, CA) following the phenol hypochlorite technique described by Broderick and Kang (1980) with the following modi fication: absorbance was read at 620 nm in flat bottom 96 well plates using a plate reader (DU 500, Beckman Coulter Inc.). VFA a nalyses Volatile fatty acids of the samples were determined in a water based solution using ethyl acetate extraction. Samples w ere centrifuged for 10 min at 10,000 g Five mililiters of the ruminal fluid supernatant was mixed with one mL of a meta phosphoric acid:crotonic acid (internal standard) solution and samples were frozen overnight, thawed and centrifuged for 10 min at 1 0,000 g Supernatant was transferred into vials and mixed with ethyl acetate in a 2:1 ratio of ethyl acetate to supernatant. After shaking vials vigorously, the ethyl acetate fraction rose to the top and a sub sample was transferred to a vial. Samples were analyzed by gas chromatography (Agilent 7820A GC, Agilent Technologies, Palo Alto, CA) using a flame ionization detector and a capillary column ( CP WAX 58 FFAP 25 m 0.53 mm Varian CP7 767 ). I n vitro true dry matter digestibility In vitro true dry mat ter digestibility ( IVTDMD ) was measured using 200 mL plastic scintillation vials with rubber stoppers fitted with a 16 gauge needle for gas release. Tared vials

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39 containing 0.7 g of substrate and 50 mL of a 3:1 or a 2:1 l fluid for LC and HC substrates, respectively, were incubated for 24 h at 39C under constant agitation (60 rpm). Two vials per treatment and 2 blank vials (without substrate) were incubated in each of 3 separate replicate days. After incubation vials we re immediately frozen at 20C for a minimum of 16 h and freeze dried for 24 h (FreeZone 6 Liter Freeze Dryer System, Labconco Corp., Kansas City, MO). Following freeze drying, vials were placed overnight in a 100C oven and weighed to calculate remaining dry matter. To calculate IVTDMD for each vial, the dry residue weight (corrected for the contribution of solids in the blank) was subtracted from the incubated dry matter and the result (i. e., digested dry matter) was divided by the dry weight of substr ate incubated. Calculations and S tatistical A nalysis Apparent total tract dig estion of DM, OM, CP, NDF, and ADF were calculated as follows: 100 100 [( marker concentration in feed/ marker concentration in feces) (nutrient concentration in feces /nutrien t concentration in feed)]. For Exp. 1, d ata were analyzed using MIXED procedure of SAS (SAS Inst. Inc., Cary, NC) with heifer as the experimental unit and including the fixed effects of diet and chitosan inclusion level, and the random effect of period O rthogonal polynomial contrasts were conducted to determine linear and quadratic effects of chitosan inclusion level For Exp. 2, g as production kinetics parameters were quantified using a modified Gompertz model (Schofield et al., 1994) and were fitted to time course data obtained from the Ankom wireless module system by the NLIN procedure of SAS. The parameters of this model were lag time, asymptotic maximal gas production ( M ), and fractional rate of gas production ( Kf %/h ) cal culated by dividing the absolute rate of gas production (mL h 1 ) by M. The IVDMD gas production kinetics, NH 3 N, final pH and VFA data were analyzed using the

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40 MIXED procedure of SAS, with day (block) as a random effect. Orthogonal pol ynomial contrasts wer e used to determine linear and quadratic effects of chitosan inclusion level In both Exp. 1 and Exp. 2, significance was declared at P 0.05 and tendencies were discussed when 0.05 < P < 0.10 considered tendencies. Results and discussion Experiment 1 Dry matter intake was not affected by chitosan (8.55, 8.5, and 8.75 kg/d for 0, 0.5, and 1% chitosan, respectively; P = 0.90) or diet (8.5 and 8.7 kg/d for LC and HC, respectively; P = 0.66). It is reasonable to believe that the LC diet may create a great er gut fill effect and the HC diet may reach a chemostatic control of intake, in which both situations, intake is controlled. Similar results were reported when three diets with differing amounts of concentrate (51, 37, and 87%) were administered to finis hing beef cattle and with no differences in DMI (Doreau et al., 2011) Thus, it is probable th at in the current experiment, peanut hulls may not have caused a gut fill effect, due to particle size and fiber fragility. It was also found by Goiri et al. (2010) that chitosan had no effect on the DMI of sheep fed 136 mg/kg BW (Table 3 3). No effect ( P > 0.10) of chitosan was found on the digestibility of DM, OM, CP, NDF, or ADF when using Cr 2 O 3 as an external marker (Table 3 4). This is supported by Goiri et al. (2010) as the researchers fed sheep (62 kg BW) 136 mg kg 1 BW of chitosan (approximately 1% of diet DM) along with a 50:50 diet consisting mainly of alfalfa hay and concentrate made of corn, wheat and soybean meal. Digestibility of OM and CP for sheep fed chitosan was not different from controls. Conversely, NDF digestibility was decreased when chitosan was included. One reason for the difference in NDF digestibilities could be the difference s in forage or roughage types. In th is study, peanut hulls were the main roughage source with about 75% NDF and 63% crude fiber. Alfalfa hay used by Goir i et al. (2010) would have had about 50%

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41 NDF and 30% crude fiber. One could speculate the increased fiber and NDF content diluted the effects of chitosan. Researchers descibed an increase in cellulolytic bacteria, such as Fibrobacter succinogenes Rumino coccus fl avefaciens and Ruminococcus albus after grass hay was consumed by sheep compared to alfalfa hay (Saro et al., 2012) There may not have been sufficient adequately inhibit the possible increase in fiber digesting bacteria. Digestibility of DM, OM, and CP was increased when HC was fed as compared to LC (56.5 vs. 42.8% [ P < 0.001 ], 58.1 vs. 43.5% [ P < 0.001 ], and 66.1 vs. 60.1% [ P = 0.006 ] for DM, OM, and CP, respectively). Diet had no effect on NDF or ADF digestibility. No effect of chitosan was found on the digestibility of DM, OM, CP, NDF, or ADF when using TiO 2 as an external marker (Table 3 5). The nutrient digestibilities associated with TiO 2 are similar to those determi ned using Cr 2 O 3 No differences were reported for digestibility of three different winter forages when TiO 2 and Cr 2 O 3 were used as external markers (Njombwa, 2012) Heifers fed HI diet had greater ( P < 0.01) digestibility values for DM and OM. The same diet also tended ( P = 0.09) to have increased CP digestibility. When iNDF was utilized as an internal marker, interactions between diet and chitosan were found for digestibility of DM ( P = 0.05) and OM ( P = 0.04) (Table 3 6). It could be speculated th at, even though not significant, the numerical increase (21.3% to 25.7% for 0% chitosan and1% chitosan, respectively) in digestibility of NDF when chitosan was present contributed to the increases in DM and OM digestibility. The LC diet consisted of about two thirds NDF, it is likely that the small increase in fiber digestibility had a larger impact on OM and DM digestibility due to the large fraction of fiber in the diet. W hen nano particles of chitosan were introduced to gram positive bacteria bacterial death occurs, most likely due to changes in cell permeability (Qi et al., 2004) No differences ( P > 0.10) were found

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42 in the current study for digestibility of CP, NDF, or ADF when iNDF was used as an indigestible marker. Grams of CH 4 produced per heifer per d were influenced by diet ( P < 0.01) with heifers fed HC producing approximately three times less CH 4 than those heifers fed LC (Table 3 3). Doreau et al. (2011) reported similar results between a corn based diet and a corn silage based diet fed to bulls (417 25 kg BW). Bulls fed the high corn silage (63.5:36.5, forage:concentrate) produced approximately 2.5 times more methane per day than those cattle fed the diet consisting mainly of ground corn grain (13.5:86.5, forage:conc entrate). This decrease in CH 4 production could be explained by a decrease in the ruminal acetate:propionate ratio. It is well documented that the ruminal VFA profile shifts towards increased propionate molar proportions as the percentage of concentrate increases (Loncke et al., 2009) Also, when a ruminant consumes high concentrate diets, the pathway for propionate pr oduction may be shifted towards lactate, which accepts hydrogen and does not produce CO 2 Chitosan had no effect on daily methane production of heifers ( P = 0.71 ; Table 3 3) in contrast to the studies of Goiri et al. ( 2009a ,c) and Goiri et al. ( 2010) which performed in vitro methane measurements. The lack of agreement between in vivo and in vitro studies is common in the literature and may be in part due to the nature of the inoculum (i.e., absence of feed particle associated microorganisms in most in vitr o incubations). When evaluating CH 4 production in g kg 1 of DMI, no effect of chitosan was observed ( P = 0.78 ; Table 3 3). Diet had an effect ( P < 0.01) on CH 4 production in g kg 1 of DMI, where heifers consuming HC produced 7.1 g kg 1 of DMI while those on the LC diet produced 18.2 g kg 1 of DMI. Increasing intake of highly digestible feedstuffs can decrease the fractional loss of energy to methane; therefore, because the HC diet had a greater digestibility, the production of CH 4 per kg of DMI c ould be low er than those heifers consuming

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43 a LC diet with a lower digestibility. Similarly, CH 4 emission expressed as g/kg of DM digested was not affected by chitosan inclusion ( P = 0.99 ; Table 3 3). An increase ( P < 0.01) in grams of CH 4 per kg of DM digested was ob served when heifers were fed LC compared to HC (47.7 vs. 9.2 g kg 1 of DM digested, respectively). Heifers consuming HC produced 2.84 times less CH 4 expressed in g kg 1 of MBW than heifers being fed LC ( P < 0.01; 0.55 vs. 1.56 g kg 1 of MBW, respectively) (Table 3 3). The addition of chitosan to either diet had no effect on grams of CH 4 per kg of MBW. A summary of research findings supports claims that a high forage diet fed to cattle produces about 2.5 to 3 times more CH 4 than those cattle fed a high conc entrate diet (Harper et al., 1999) Chitosan had no effect on either ADG or G:F ( P = 0.77 and P = 0.90, respectively) (data not shown). As expected, heifers fed HC had increased ADG and G:F ( P < 0.01 and P < 0.01, respectively). This study was not meant as a performance experiment; therefore, ADG and G:F were only used to deter mine if the diets performed as designed. Experiment 2 For the HC substrate, no treatment effects ( P 2 S production, rate of gas production, lag time or NH 3 N (Table 3 7). Smith et al. (2010) performed in vitro batch culture incubations to determine effects of monensin and additional sulfur on fermentation parameters. When monensin was added at 4 mg L 1 of incubation fluid, which is the same amount as the present study, along with a 90% concentrate substrate, no changes occurred for IVTDMD nor H 2 S production (Smith et al., 2010) For the current study, t here was a treatment effect ( P < 0.01) f or pH. As the level of chitosan increased, pH of the incubation fluid after 24 h increased linearly ( P < 0.01 ). Addition of monensin at 4 mg L 1 of incubation fluid decreased asymptotic maximal gas produced ( P = 0.05). Inclusion of monensin in in vitr o incubations has been shown to decrease total gas production (Schelling, 1984; Goiri et al., 2009c; Smith et al., 2010)

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44 When the LC substrate was incubated with differing rates chitosan and monensin, unlike the HC no treatment effects were found for pH (Table 3 9 ). Significant decreases ( P < 0.05) occurred for IVTDMD, H 2 S production, maximal gas production, rate of gas production, lag time, and NH 3 N when monensin was included (Table 3 9). A study using Rusitec fermenters found that when monensin was added to a low concentrate substrate, incubated in ruminal fluid from sheep, a decrease in digestibility, gas production, and NH 3 N was observed. Therefore, monensin appears to have anti microbial effects and targets gram positive bacteria which produce gases such a s CH 4 No differences ( P > 0.10) were described for any of the CH 4 measurements taken when chitosan or monensin was included with a HC substrate (Table 3 8). An in vitro batch culture incubation study reported that monensin, at the level used in the curren t study, did not have an effect on CH 4 production (measured stoichiometrically based on VFA concentrations) for high concentrate substrates (Smith et al., 2010). However, when monensin was included at 1.5 times this concentration, a linear decrease in CH 4 produced was observed (Smith et al., 2010). Studies performed using chitosan in vitro (Goiri et al., 2009b; Goiri et al., 2009c; and Goiri et al., 2010) found decreases in CH 4 production when chitosan was included with a high concentrate substrate. The q uantity of chitosan used in the present study was determined by the amount used in the in vivo study which was approximately 2.3 times less chitosan than used by Goiri et al. (2009b). Differences in the effects observed between our study and that by Goir i et al. (2009b) may be related to the differences in chitosan inclusion levels, or inoculum source as it will be discussed subsequently. Methane production in the current in vitro study using LC as a substrate, was increased quadratically ( P < 0.01) when chitosan was included, with 0.5% inclusion producing the most

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45 CH 4 (27% more than control) and 1.0% inclusion producing 17% more than control (Table 3 10). Studies performed using chitosan in vitro (Goiri et al., 2009a; Goiri et al., 2009c; and Goiri et al. 2010) had a decrease in CH 4 production when chitosan was included. The reason for the dissimilarity could be differences in diets where the in vitro studies used alfalfa hay as the primary forage with wheat and corn grain for a concentrate. Researchers reported that when alfalfa was used as an in vitro substrate rather than wheat straw, molar proportion of propionate was increased while acetate remained the same (Doane et al., 1997) One can speculate peanut hulls may have a similar fiber and energy concentration of wheat straw; therefore, producing less propionate, which directly competes with methanogenesis. Monensin decreased methane production in vitro when LC was used as a substrate which has been demonstrated in many in vivo and in vitro studies (Bogaert et al., 1990; Castro Montoya et al., 2012; Appuhamy et al., 2013) due to the targeting of gram positive bacteria such as methanogens. When a HC substrate was incubated, molar proportions of isobutyrate, butyrate, isovalerate + 2 meth ylbutyrate, and acetate were all decreased ( P < 0.05) when monensin was added while chitosan had no effect (Table 3 11). Monensin also increased ( P < 0.01) molar proportions of propionate and valerate. This increase in propionate and decrease in acetate mo lar proportions explains the decrease (28%; P < 0.01) in acetate:propionate (Table 3 11). There was no treatment effect on total concentration s of VFA Goiri et al. (2009a,b,c) described increases in propionate, along with decreases in the acetate:propiona te ratio and BCVFA such as isobutyrate and isovalerate. A possible reason these researchers reported differences while none were discovered for the current experiment is the source of inoculum. In the Goiri et al. (2009a,b,c) studies, ruminal fluid from sheep was used while ruminal fluid from steers was used for the current study. R uminal microorganisms from the sheep could differ enough from the

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46 steers to cause a difference in the VFA profile. A meta analysis of rumen fluid samples reported differences between large ruminants and small ruminants for parameters such as acetate, propionate total VFA concentrations, pH, NH 3 N, and urea (Jentsch and Wittenburg, 1993) Researchers also determined that when monensin was included at 0 to 40 mg kg 1 of DM in a high grain diet, the VFA profile of cattle shifted towards greater molar proportions of propionate and a decrease in acetate and butyrate (Ellis et al., 2012) They also described that total VFA did not change when monensin was added to a high grain diet. In addition, when monensin was included at 5 ppm with ground corn as a substrate, monensin had no effect on molar proportions of isobutyerate or isovalerate (Domescik and Martin, 1999) However, the acetate:propionate ratio was decreased with the addition of monensin. There was a tendency ( P = 0.08) for a linear effect of chitosan on caproa te when HC was used as a substrate (Table 3 11), while no caproate was reported in LC substrates (Tables 3 12 and 13). Allison et al. (1964) described that when sheep were fed wheat grain, caproate increased. It has been shown that when increased levels of c aproate are present usually greater numbers of Peptostreptococcus elsdenii are present in the rumen (Elsden et al., 1956; Gutierrez et al., 1959). Potentially, feeding chitosan may have selected for P. elsdenii when combined with a high concentrate subst rate, in vitro. A treatment effect ( P = 0.02) occurred for total VFA concentration when monensin was added in LC substrate in vitro incubations (Table 3 12). Other studies reported no change in total VFA concentration when monensin was added to low concentr ate substrates in vitro and pasture diets in vivo (Richardson et al., 1976; Goiri et al., 2009c) Chitosan had no effect ( P > 0.10) on concentrations of any of the VFA measured. Monensin decreased ( P < 0.05) concentrations of acetate, isobutyrate, butyrate, isovaleric + 2 methylbutyrate, and valerate.

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47 There was no change ( P > 0.10) in prop ionate concentration when chitosan or monensin was added; therefore the acetate:propionate ratio decreased ( P = 0.02) when monensin was added. Monensin tended to decrease ( P = 0.06) the molar proportion of acetate while increasing ( P = 0.02) the molar pro portion of propionate (Table 3 13). Monensin also decreased ( P < 0.05) proportions of isobutyric, butyric, and isovaleric + 2 methylbutyric acid. A linear increase ( P < 0.01) in H 2 S occurred as chitosan levels increased in a LC substrate. An in vitro study (Kung et al., 2000) reported an increase in sulfide (gas and liquid forms) when monensin was added to high sulfur diets. The authors claimed monensin depleted methanogens; therefore, sulfide producing microbes had less competition. One could speculate chitosan, although not significantly evident in terms of methane production, may have decreased the methanogenic archaea population, which allowed sulfide producing bacteria to thrive more than when no chitosan was included. Conclusion Inclusion of chit osan at rates of 0.5% and 1.0% of diet DM had no effect on enteric methane emissions in beef heifers. When fed a high concentrate diet, heifers produced about 2.5 times less methane than when fed low concentrate diets. Chitosan also had no effect on in v itro methane production when incubated with a high concentrate substrate. However, when incubated with a low concentrate substrate, chitosan increased in vitro methane production. When iNDF was used as an internal marker, a diet chitosan interaction wa s witnessed for DM and OM digestibility. Further investigation should be made to determine effect of different particle sizes (micro and nano particles) of chitosan fed to ruminants in vivo on CH 4 production

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48 Table 3 1 Ing redients and chemical composition of the basal diets %, DM basis Item 36% concentrate 85% concentrate Ingredient composition Corn gluten feed, pelleted 15.0 40.0 Soybean hulls, pelleted 15.0 39.3 Peanut hulls 64.0 15.0 Liquid supplement 1 3.8 4.0 Meal supplement 2 2.2 1.7 Chemical composition (DM basis) DM, % 91.3 91.0 Ash, % 9.8 9.8 NEg, Mcal/kg of diet DM 0.77 0.97 C P, % 14.9 19.2 NDF, % 59.7 47.3 ADF, % 46.2 27.7 Ether extract % 1.7 2.9 Ca % 1.30 1.49 P % 0.33 0.62 1 Westway Converter SR Southeast 32 (Westway Feed Products Inc., New Orleans, LA). Chemical composition of the supplement (as fed basis): 62% DM; 32.0% CP; 0.1% fat; contained trace minerals (cobalt, copper, iron, manganese, zinc, iodine, and selenium). 2 Supplied vitamins (A, D) and minerals (Beef Four Plus, W.B. Fleming Co. Tifton, GA).

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49 Table 3 2 Composition and analyzed nutrient content (DM basis) of substrates used for in vitro incubations %, DM basis Item LC HC Ingredient composition Corn grain, ground 84.0 Corn gluten feed, pelleted 15.0 Soybean hulls, pelleted 15.0 Peanut hulls 64.0 Soybean, meal 44% CP 5.1 5.2 Urea 0.9 0.8 Bermudagrass, hay 10.0 Chemical composition (DM basis) 1 DM, % 89.7 87. 9 NEg, Mcal/kg of diet DM 0.75 1.34 C P, % 16.8 15.3 NDF, % 67.2 14.7 ADF, % 56.2 6.6 Ether extract % 2.5 4.3 Ca % 0.18 0.06 P % 0.22 0.31 1 Dairy One Forage Testing Laboratory, Ithaca, NY.

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50 Table 3 3 Effect o f diet and inclusion level of chitosan on DMI and CH 4 emissions in beef heifers H C diet 1 L C diet 2 Chitosan inclusion, % of diet DM Chitosan inclusion, % of diet DM P value 3 Item 0 0.5 1.0 0 0.5 1.0 SEM 4 DIET CHIT CHIT DIET DMI 5 kg/d 8.7 8.1 9.4 8.5 8.9 8.1 0.64 0.66 0.90 0.20 CH 4 emissions, g/d 50.6 41.7 41.3 121.3 114.8 155.2 54.83 < 0.01 0.71 0.61 CH 4 emissio ns, g/kg DMI 8.4 6.5 6.5 18.0 15.3 21.4 8.60 < 0.01 0.65 0.63 CH 4 emissions, g/kg DM digested 6 9.0 7.4 7.3 43.9 33.4 41.4 12.63 < 0.01 0.71 0.80 CH 4 emissions, g/kg MBW 7 0.62 0.52 0.52 1.50 1.35 1.84 0.355 < 0.01 0.79 0.75 1 Comprised of 40% corn glut en feed pellets, 39% soybean hulls pellets, 15% peanut hulls, and 6% vitamins and minerals supplement; all in DM basis. 2 Comprised of 64% peanut hulls,15% corn gluten feed pellets, 15% soybean hulls pellets, and 6% vitamins and minerals supplemen t; all in DM basis. 3 Observed significance levels for the main effects of: DIET = diet (n = 24 heifers/mean), CHIT = chitosan inclusion level (n = 16 heifers/treatment), CHIT x DIET = interaction between chitosan inclusion level and diet. 4 Standard error of the mean n = 8 heifers/treatment. 5 DMI = Dry matter intake average during the methane collection period. 6 Apparent total tract digestibility measured using iNDF as indigestible marker. 7 MBW = Metabolic body weight.

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51 Table 3 4 Effect of diet and inclusion level of chitosan on nutrient digestibility when Cr 2 O 3 wa s utilized as an external marker HC diet 1 LC di et 2 Chitosan inclusion, % of diet DM Chitosan inclusion, % of diet DM P value 3 Item 0 0.5 1.0 0 0.5 1.0 SEM 4 DIET CHIT CHIT DIET DMI 5 kg/d 8.7 8.1 9.4 8.5 8.9 8.1 0.64 0.66 0.9 1 0.20 DM digestibility, % 58.9 54.4 56.1 38.4 46.3 43.8 4.08 < 0.01 0.91 0.32 OM digestibility, % 59.9 57.1 57.2 39.2 47.3 44.1 4.5 0 < 0.0 1 0.84 0.47 CP digestibility, % 67.8 64.6 66.6 59.4 60.9 60.1 2.71 < 0.0 1 0.9 5 0.69 NDF digestibility, % 41.6 32.6 36.5 26.7 35.3 28.4 5.27 0.1 3 0.94 0.25 ADF digestibility, % 35.7 25.0 29.1 24.0 33.5 25.2 6.22 0.64 0.90 0.2 8 1 Comprised of 40% corn gluten feed pellets, 39% soybean hulls pellets, 15% peanut hulls, and 6% vitamins and minerals supplement; all in DM basis. 2 Comprised of 64% peanut hulls,15% corn gluten feed pe llets, 15% soybean hulls pellets, and 6% vitamins and minerals supplement; all in DM basis. 3 Observed significance levels for the main effects of: DIET = diet (n = 24 heifers/mean), CHIT = chitosan inclusion level (n = 16 heifers/treatment), CHIT x DIET = interaction between chitosan inclusion level and diet. 4 Standard error of the mean, n = 8 heifers/treatment. 5 DMI = Dry matter intake average during the methane collection period.

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52 Table 3 5 Effect of diet and inclusion level of chitosan on nutrient digestibili ty when TiO 2 was utilized as an external marker Table 3 6 Effect of diet and inclusion level of chitosan on nutrient digestibility when iNDF was utilized as an internal marker HC diet 1 LC diet 2 Chitosan inclusion, % of diet DM Chitosan inclus ion, % of diet DM P value 3 Item 0 0.5 1.0 0 0.5 1.0 SEM 4 DIET CHIT CHIT DIET DMI 5 kg/d 8.7 8.1 9.4 8.5 8.9 8.1 0.64 0.66 0.90 0.20 DM digestibility, % 52.6 50.8 50.2 37.4 44.0 39.9 3.65 < 0.01 0.76 0.52 OM digestibility, % 53. 7 53.7 51.4 38.2 45.1 40.3 4.03 < 0.01 0.61 0.69 CP digestibility, % 62.8 61.8 61.9 58.2 58.6 56.4 3.1 0.09 0.89 0.93 NDF digestibility, % 32.5 27.3 28.1 25.9 32.9 24.2 5.48 0.71 0.75 0.51 ADF digestibility, % 25.5 19.2 19.8 23.1 31.0 20.7 7.4 7 0.50 0.70 0.49 1 Comprised of 40% corn gluten feed pellets, 39% soybean hulls pellets, 15% peanut hulls, and 6% vitamins and minerals supplement; all in DM basis. 2 Comprised of 64% peanut hulls,15% corn gluten feed pellets, 15% soybean hulls pellets, and 6% vitamins and minerals supplement; all in DM basis. 3 Observed significance levels for the main effects of: DIET = diet (n = 24 heifers/mean), CHIT = chitosan inclusion level (n = 16 heifers/treatment), CHIT x DIET = interaction between chitosan inclu sion level and diet. 4 Standard error of the mean, n = 8 heifers/treatment. 5 DMI = Dry matter intake average during the methane collection period.

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53 H C diet 1 L C diet 2 Chitosan inclusion, % of diet DM Chitosan inclusion, % of diet DM P value 3 Item 0 0.5 1.0 0 0.5 1.0 SEM 4 DIET CHIT CHIT DIET DMI 5 kg/d 8.7 8.1 9.4 8.5 8.9 8.1 0.64 0.66 0.90 0.20 DM digestibility, % 62.1 c 62.8 c 60.9 c 33.2 a 35.7 ab 40.3 b 3.95 < 0.01 0.25 0. 05 OM digestibility, % 63.0 c 65.2 c 61.9 c 34.5 a 37.7 ab 41.1 b 2.61 < 0.01 0.16 0.04 CP digestibility, % 70.1 71.2 69.8 55.3 51.7 55.6 5.62 < 0.01 0.87 0.60 NDF digestibility, % 46.3 45.2 43.7 21.3 23.8 25.7 2.45 < 0.01 0.84 0.11 ADF digestibility, % 4 1.0 39.2 37.6 18.4 21.6 22.3 3.19 < 0.01 0.93 0.16 a,b ,c Within a row, means with different superscripts differ, P < 0.05. 1 Comprised of 40% corn gluten feed pellets, 39% soybean hulls pellets, 15% peanut hulls, and 6% vitamins and minerals supplement; al l in DM basis. 2 Comprised of 64% peanut hulls,15% corn gluten feed pellets, 15% soybean hulls pellets, and 6% vitamins and minerals supplement; all in DM basis. 3 Observed significance levels for the main effects of: DIET = diet (n = 24 heifers/mean), CHIT = chitosan inclusion level (n = 16 heifers/treatment), CHIT x DIET = interaction between chitosan inclusion level and diet. 4 Standard error of the mean, n = 8 heifers/treatment. 5 DMI = Dry matter intake average during the methane collection period.

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54 Table 3 7 Effect of monensin and inclusion level of chitosan on in vitro fermentation parameters with a HC substrate 1 Treatments 2 P value 4 Item 0.0 0.5 1.0 MON SEM 3 TRT Linear Quad. IVDMD, % 79.20 79.12 78.74 78.46 3.484 0.42 0.36 0.72 H 2 S, mol/24 h/g fermented DM 8.89 10.43 10.28 9.56 1.186 0.34 0.16 0.30 Gas production kinetics 5 M, mL/g of DM incubated 266 267 258 219 12.7 0.05 0.61 0.74 Kf, %/h 0.072 b 0.072 ab 0.068 a 0.069 ab 0.0013 0.11 0.04 0.39 L ag h 1.18 1.20 1.09 1.29 0.405 0.97 0.83 0.86 NH 3 N, mM 4.12 4.03 4.02 4.28 0.285 0.42 0.54 0.80 pH 5.657 a 5.660 a 5.733 b 5.773 b 0.0229 < 0.01 < 0.01 0.08 a,b Within a row, means with different supe rscripts differ, P < 0.05. 1 Comprised of 84 % cracked corn 10 % Bermuda grass hay 5.2% soybean meal (44% CP), and 0.8 % urea ; all in DM basis. 2 0.0 = Chitosan inclusion level of 0.0% of substrate DM; 0.5 = Chitosan inclusion level of 0.5% of substrate DM; 1 .0 = Chitosan i nclusion level of 1.0% of substrate DM; MON = 4 mg of monensin per L of incubation fluid. 3 SE of treatment means, n = 2 reps/treatment on 3 separate days. 4 Obse rved significance levels for treatment effects and for linear and quadratic effec ts of increasing chitosan dose. 5 Parameters estimated by fitting to a modified Gompertz function, where M = Maximal gas production; K = fractional gas production; and L ag = duration of the lag phase.

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55 Table 3 8 Effect of mone nsin and inclusion level of chitosan on in vitro CH 4 production with a HC substrate 1 Treatments 2 P value 4 Item 0.0 0.5 1.0 MON SEM 3 TRT Linear Quad. IVDMD, % 79.20 79.12 78.74 78.46 3.484 0.42 0.36 0.72 CH 4 mg/100 mL incubation fluid 4.19 5.18 4.38 4.05 0.483 0.32 0.76 0.13 CH 4 mmol/g of incubated DM 2.19 2.62 2.22 1.88 0.197 0.15 0.93 0.12 CH 4 mM/g of fermented DM 2.76 3.35 2.82 2.40 0.291 0.17 0.88 0.12 1 Comprised of 84 % cracked corn 10 % Bermuda grass hay 5.2% soybean meal (44% CP), and 0.8 % urea ; all in DM basis. 2 0.0 = Chitosan inclusion level of 0.0% of substrate DM; 0.5 = Chitosan inclusion level of 0.5% of substrate DM; 1.0 = Chitosan inclusion level of 1.0% of substrate DM; MON = 4 mg of monensin per L of incubat ion fluid. 3 SE of treatment means, n = 2 reps/treatment on 3 separate days. 4 Obse rved significance levels for treatment effects and for linear and quadratic effects of increasing chitosan dose.

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56 Table 3 9 Effect of monensin and inclusion level of chitosan on in vitro fermentation parameters with a LC substrate 1 Treatments 2 P value 4 Item 0.0 0.5 1.0 MON SEM 3 TRT Linear Quad. IVDMD, % 48.56 48.67 48.26 46.39 2.071 0.04 0.65 0.65 H 2 S, mol/24 h/g fermented DM 1.80 a 2.72 b 2.82 b 1.37 a 0.354 < 0.01 < 0.01 0.11 Gas production kinetics 5 : M, mL/g of DM incubated 135 138 138 103 4.3 < 0.01 0.65 0.79 Kf, %/h 0.047 0.048 0.048 0.038 0.0038 0.04 0.72 0.74 L ag h 0.210 0.487 0.678 0.030 0.3193 < 0.05 0.06 0.81 NH 3 N, mM 3.19 3.21 3.19 2.40 0.191 0.03 0.93 0.90 pH 6.678 6.635 6.627 6.653 0.0251 0.25 0.08 0.43 a,b Within a row, means with different superscripts differ, P < 0 .05. 1 Comprised of 64 % peanut hulls 15 % corn gluten feed 15% soybean hulls, 5.1% soybean meal (44% CP) and 0.9 % urea ; all in DM basis. 2 0.0 = Chitosan inclusion level of 0.0% of substrate DM; 0.5 = Chitosan inclusion level of 0.5% of substrate DM; 1.0 = Chitosan inclusion level of 1.0% of substrate DM; MON = 4 mg of monensin per L of incubation fluid. 3 SE of treatment means, n = 2 reps/treatment on 3 separate days. 4 Obse rved significance levels for treatment effects and for linear and quadratic effects o f increasing chitosan dose. 5 Parameters estimated by fitting to a modified Gompertz function, where M = Maximal gas production; K = fractional gas production; and L ag = duration of the lag phase.

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57 Table 3 10 Effect of monens in and inclusi on level of chitosan on in vitr o CH 4 production with a LC substrate 1 Treatments 2 P value 4 Item 0.0 0.5 1.0 MON SEM 3 TRT Linear Quad. IVDMD, % 79.20 79.12 78.74 78.46 3.484 0.42 0.36 0.72 CH 4 mg/100 mL incubation fluid 3.49 b 4.40 d 4.09 c 2.75 a 0.14 6 < 0.01 < 0.01 < 0.01 CH 4 mmol/g of incubated DM 0.91 b 1.16 d 1.07 c 0.62 a 0.037 < 0.01 < 0.01 < 0.01 CH 4 mM/g of fermented DM 1.16 b 1.48 d 1.37 c 0.81 a 0.092 < 0.01 < 0.01 < 0.01 a,b ,c,d Within a row, means with diffe rent superscripts differ, P < 0.05. 1 Comprised of 64 % peanut hulls 15 % corn gluten feed 15% soybean hulls, 5.1% soybean meal (44% CP) and 0.9 % urea ; all in DM basis. 2 0.0 = Chitosan inclusion level of 0.0% of substrate DM; 0.5 = Chitosan inclusion level of 0.5% of substrate DM; 1.0 = Chitosan inclusion level of 1.0% of substrate DM; MON = 4 mg of monensin per L of incubation fluid. 3 SE of treatment means, n = 2 reps/treatment on 3 separate days. 4 Obse rved significance levels for treatment effects and for linear and quadratic effects of increasing chitosan dose.

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58 Table 3 11 Effects of monensin and inclusion level of chitosan on in vitro proportions of VFA (mol/100 mol), total VFA concentration (m M ), and the acetate:propionate ratio with a HC substrate 1 Treatments 2 P value 4 Item 0.0 0.5 1.0 MON SEM 3 TRT Linear Quad. Acetate 63.2 63.6 63.5 58.8 0.44 < 0.01 0.43 0.58 Propionate 21.6 21.4 21.4 27.7 0.82 < 0.01 0.56 0.80 Isobutyric 0.8 0.8 0.8 0.7 0.03 0.01 0.47 0.55 Butyric 11.0 10.8 10.7 9.0 0.50 < 0.01 0.30 0.62 Isovaleric + 2 MB 5 1.4 1.4 1.4 1.3 0.08 < 0.01 0.82 0.49 Valeric 1.6 1.6 1.7 2.1 0.08 < 0.01 0.75 0.70 Caproic 0.40 a 0.45 ab 0.47 ab 0.53 b 0.029 0.04 0.08 0.54 Total VFA 140.8 134.7 131.4 142.0 5.29 0.49 0.26 0.84 A:P 2.95 2.98 2.98 2.13 0.126 < 0.01 0.68 0.83 a,b Within a row, means with different superscripts differ, P < 0.05. 1 Comprised of 84 % crac ked corn 10 % Bermuda grass hay 5.2% soybean meal (44% CP), and 0.8 % urea ; all in DM basis. 2 0.0 = Chitosan inclusion level of 0.0% of substrate DM; 0.5 = Chitosan inclusion level of 0.5% of substrate DM; 1.0 = Chitosan inclusion level of 1.0% of substrat e DM; MON = 4 mg of monensin per L of incubation fluid. 3 SE of treatment means, n = 2 reps/treatment on 3 separate days. 4 Obse rved significance levels for treatment effects and for linear and quadratic effects of increasing chitosan dose. 5 MB = Methylbutyr ic

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59 Table 3 12 Effect of monensin and inclusion level of chitosan on in vitro VFA concentrations (m M ) and the acetate:propionate ratio with a LC substrate 1 Treatments 2 P value 4 Item 0.0 0.5 1.0 MON SEM 3 TRT Linear Quad. Acetate 43.4 42.2 42.8 37.0 2.59 < 0.01 0.62 0.41 Propionate 9.9 9.1 9.0 10.5 0.78 0.29 0.30 0.62 Isobutyric 0.5 0.6 0.5 0.4 0.02 < 0.01 0.84 0.59 Butyric 4.7 4.5 4.4 3.8 0.17 0.02 0.25 0.85 Isovaleric + 2 MB 5 0.8 0.9 0.8 0.6 0.04 < 0.01 0.59 0.60 Valeric 0.6 0.6 0.6 0.5 0.02 < 0.10 0.13 0.69 Caproic 0.0 0.0 0.0 0.0 0.0 Total VFA 60.0 57.8 58.1 52.8 3.29 0.02 0.29 0.38 A:P 4.41 4.66 4.77 3.55 0.212 0.02 0.26 0.79 1 Comprised of 64 % peanut hulls 15 % corn gluten feed 15% soybean hulls, 5.1% soybean meal (44% CP) and 0.9 % urea ; all in DM basis. 2 0.0 = Chitosan inclusion level of 0.0% of substrate DM; 0.5 = Chitosan inclusion level of 0.5% of substrate DM; 1.0 = Chitosan inclusion level of 1.0% of substrate DM; MON = 4 mg of monensin per L of incubation fluid. 3 SE of treatment means, n = 2 reps/treatment on 3 separate days. 4 Obse rved significance levels for treatment effects and for linear and quadratic effects of increasing chitos an dose. 5 MB = Methylbutyric

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60 Table 3 13 Effect of monensin and inclusion level of chitosan on in vitro VFA proportions (mol/100 mol) with a LC substrate 1 Treatments 2 P value 4 Item 0.0 0.5 1.0 MON SEM 3 TRT Linear Quad. Acetate 72.2 73.0 73.5 69.8 0.81 0.06 0.27 0.90 Propionate 16.7 15.7 15.5 20.1 0.80 0.02 0.33 0.71 Isobutyric 0.9 1.0 0.9 0.8 0.05 < 0.01 0.37 0.10 Butyric 7.8 7.9 7.7 7.2 0.34 0.02 0.44 0.38 Isovaleric + 2 MB 5 1.4 1.5 1. 4 1.2 0.09 < 0.01 0.57 0.06 Valeric 1.0 1.0 1.0 1.0 0.04 0.49 0.15 0.88 Caproic 0.0 0.0 0.0 0.0 0.0 1 Comprised of 64 % peanut hulls 15 % corn gluten feed 15% soybean hulls, 5.1% soybean meal (44% CP) and 0.9 % urea ; all in DM basis. 2 0.0 = Chitosan inclusion level of 0.0% of substrate DM; 0.5 = Chitosan inclusion level of 0.5% of substrate DM; 1.0 = Chitosan inclusion level of 1.0% of substrate DM; MON = 4 mg of monensin per L of incubation fluid. 3 SE of treatment means, n = 2 reps/treatment on 3 separate days. 4 Obse rved significance levels for treatment effects and for linear and quadratic effects of increasing chitosan dose. 5 MB = Methylbutyric

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69 BIOGRAPHICAL SKETCH Darren Dwayne Henry was born in the year 1988 in the southeast Texas town of Porter. From a young age Darren was involved in product calf o perations. In 2006, Darren began his freshman year at Texas A&M University where he received degrees in Animal Science and Agricultural Leadership and Development. After graduati on, Darren mov ed to Marianna, FL to pursue a m methane to improve the eff iciency of beef cattle. (Titgemeyer et al., 2001; Cole et al., 2011; Krizsan and Huhtanen, 2013)