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Evaluation of Brassica Carinata Meal on Animal Performance and Metabolism in Beef Cattle

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Title:
Evaluation of Brassica Carinata Meal on Animal Performance and Metabolism in Beef Cattle
Creator:
Schulmeister, Tessa M
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[Gainesville, Fla.]
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University of Florida
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english
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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:
DUBEUX,JOSE CARLOS

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Subjects / Keywords:
beef -- carinata -- heifers -- metabolism
Animal Sciences -- Dissertations, Academic -- UF
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bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
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Electronic Thesis or Dissertation
Animal Sciences thesis, M.S.

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Abstract:
Brassica carinata, a novel oilseed crop, yields high-quality biofuel, resulting in a high-protein byproduct. This meal has not been tested as a cattle protein supplement, therefore our objective was to evaluate the effects of supplementation of B. carinata meal. Eight Angus crossbred steers were utilized in a duplicated 4 by 4 Latin square design, over 4 periods of 28 d each, to assess ruminal fermentation parameters, nutrient digestibility, and blood profile. Within period, steers were assigned to one of four treatments: 1.39 kg d-1 of Brassica carinata meal pellets (BCM), 1.62 kg d-1 of cottonseed meal (CSM), 2.15 kg d-1 of dry distillers grains plus solubles (DDGS), or 1.17 kg d-1 of soybean meal (SBM). No effect of treatment (P greater than 0.05) was observed for ruminal pH, NH3-N, total VFA, plasma glucose, DMI, or apparent total tract digestibility of nutrients. Steers receiving SBM had the greatest (P less than 0.01) concentration of BUN. A ruminal in situ degradability study was performed utilizing steers from the previous experiment. Supplement remaining after 16 h of incubation was subjected to serial solutions simulating post-ruminal digestion. Rate of degradation of DM and CP was greatest (P less than 0.01) in SBM; total tract digestibility was greater in SBM. Nearly equivalent amounts of RDP and RUP were observed in CSM and DDGS, which differed (P less than 0.01) from SBM and BCM. Compared with DDGS, SBM had a greater IDP (P less than 0.01), with CSM having the greatest IADP (P less than 0.01). Animal performance, attainment of puberty, and blood profile was evaluated in 64 Angus crossbred heifers. Stratified and blocked by initial BW, heifers were randomly allocated into 18 pens over 2 consecutive years. Pens were randomly assigned, within block, to one of two treatments: 0 (CTL) or 0.3% of BW d-1 (as fed) of BCM pellets. Bermudagrass hay was provided ad libitum. Compared with CTL, BCM increased (P less than 0.01) ADG; treatment did not affect interval to attainment of puberty (P = 0.68). Concentrations of ceruloplasmin were greater (P less than 0.01) in CTL heifers, with an effect of day (P less than 0.01) observed for T3, T4, and ceruloplasmin. ( en )
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In the series University of Florida Digital Collections.
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Includes vita.
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Description based on online resource; title from PDF title page.
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This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (M.S.)--University of Florida, 2017.
Local:
Adviser: DILORENZO,NICOLAS.
Local:
Co-adviser: LAMB,GRAHAM CLIFF.
Statement of Responsibility:
by Tessa M Schulmeister.

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1 EVALUATION OF BRASSICA CARINATA MEAL ON ANIMAL PERFORMANCE AND METABOLISM IN BEEF CATTLE By TES SA MARIE SCHULMEISTER A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UN IVERSITY OF FLORIDA 2017

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2 2017 T essa Marie Schulmeister

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3 To my children

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4 ACKNOWLEDGMENTS I would be remiss in not acknowledging my Savior, first and foremost. It is because of His love, grace, and mercy that I am here today. It is with sincere gratitude that I acknowledge Dr. Nicolas DiLorenzo. Through the years he has expressed patience, kindness, and guidance, all of which have contributed to a deeper appreciation for him, not only as an employer and advisor, but as a friend. I would like to thank Drs. Cliff Lamb and Jose Dubeux for their support. It was because of Dr. Lamb that I was introduced to the NFREC family and found not only a place of employment and education, but a home. When I arrived at the station, I knew nothing about animal science, but through many conversations with Dr. Martin Ruiz-Moreno, and his contagious passion, I developed an excited interest which led to this point. He will always be one of my favorite flowers in the garden of life, and I am eternally grateful for the support and encouragement he provided, especially when I doubted myself. There are far too many interns and students who have journeyed with me over the years to thank individually, but I hold them in high regards and am very grateful for the opportunity to have work ed with each of them. I would like to specifically thank Darren Henry, Francine Messias Ciriaco Henry, Mariana Garcia Ascolani, Gleise Medeiros, Lautaro Rostoll, and Carla Sanford for the challenges they provided, as well as the support. Through many conversations, trials, and error they have enriched my understanding of animal science and challenged me at every opportunity, for which I am continually gratef ul. I would like to express my appreciation to Mrs. Tina Gwin and Mrs. Gina Arnett for their love, support, and humor throughout my time at NFREC. Without our morning chats, I would never have made it this far. My sincere

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5 appreciation is expressed to all o f the staff at NFREC, especially Ray Morgan, for without them, none of this would have been possible. Finally, I would like to express my love and appreciation for my family. Without the support and discipline from my momma, Tammy McGlamory, I would not be the woman I am today. All that I am, I owe to her, my hero. Infinite appreciation and affection are expressed to my children. They have not only provided me with silver hair and worry wrinkles, but countless hours of laughter, encouragement, and patience. It is to Andy and Gus that I owe all of my success, however, they will always be my greatest investment.

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6 TABLE OF CONTENTS P age ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES ...........................................................................................................................8 LIST OF FI GURES .........................................................................................................................9 LIST OF ABBREVIATIONS ........................................................................................................11 ABSTRACT ...................................................................................................................................16 CHAPTER 1 INTRODUCTION ..................................................................................................................18 2 LITERATURE REVIEW .......................................................................................................20 Brassica carinata ....................................................................................................................20 Favorable Long chai n Fatty Acid Profile ........................................................................20 Potential Effects of Utilizing Renewable Resources .......................................................21 Previous Research on Utilization of Carinata as a Su pplement for Livestock ................23 Protein Supplementation in Ruminants ..................................................................................24 Fractionation of Dietary Protein ......................................................................................24 Cattle Production in the Southeast U.S. ..........................................................................26 Protein requirements of beef cattle in cow -calf operations of the southeastern U.S. .......................................................................................................................28 Nutrition and attainment of puberty in beef heifers .................................................30 Glucosinolates .........................................................................................................................30 Evolution and Degradation ..............................................................................................31 Glucosinolates in Carinata: Sinigrin and Progoitrin ........................................................31 Thyroid Metabolism ...............................................................................................................33 Thyroid Hormones ...........................................................................................................33 Disorders of the Thyroid .................................................................................................35 Immune Response ...................................................................................................................36 Acute Phase Response .....................................................................................................37 Acute Phase Proteins .......................................................................................................37 3 EVALUATION OF BRASSICA CARINATA MEAL ON RUMINAL METABOLISM AND NUTRIENT DIGESTIBILITY OF BEEF CATTLE ....................................................43 Introduction .............................................................................................................................43 Materials and Methods ....................................................................................................44 Experimental design and sample collection .............................................................44 Laboratory analyses ..................................................................................................45 Calculations and statistical analysis .........................................................................47

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7 Results and Discussion ....................................................................................................48 Conclusion .......................................................................................................................53 4 CHARACTERIZATION OF THE DIETARY PROTEIN IN BRASSICA CARINATA .....67 Introduction .............................................................................................................................67 Materials and Methods ....................................................................................................68 Experimental design and sample collection .............................................................68 Laboratory analyses ..................................................................................................69 Calculations and statistical analysis .........................................................................70 Results and Discussion ....................................................................................................72 Conclusion .......................................................................................................................76 5 EVALUATION OF BRASSICA CARINATA MEAL AS A PROTEIN SUPPLEMENT FOR GROWING BEEF HEIFERS ........................................................................................83 Introduction .............................................................................................................................83 Materials and Methods ....................................................................................................84 Experimental design and sample collection .............................................................84 Laboratory analyses ..................................................................................................84 Statistical analysis ....................................................................................................86 Results and Discussion ....................................................................................................86 Conclusion .......................................................................................................................91 6 SUMMARY ............................................................................................................................99 LITERATURE CITED ................................................................................................................103 BIOGRAPHICAL SKETCH .......................................................................................................117

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8 LIST OF TABLES Table P age 2 -1 Summary of nutrient requirements of beef cattle1. ............................................................41 3-1 Analyzed1 chemical and nutrient composition (DM basis) of hay and protein supplements fed to ruminallycannulated Angus crossbred steers. ...................................54 3-2 Effects of protein supplementation on nutrient intake and apparent total tract digestibility1 of ruminally -cannulated Angus crossbred steers fed bahiagrass hay ad libitum with iNDF utilized as an internal marker. .............................................................55 3-3 Effects of protein supplementation on ruminal fermentation parameters and blood profile of ruminally-cannulated Angus crossbred stee rs fed bahiagrass hay ad libitum. ...............................................................................................................................56 3-4 Effects of protein supplementation on proportions of VFA (mol 100 mol-1), total VFA concentrations (m M ), and acetate to propionate ratio (A: P) in rumin ally cannulated Angus crossbred steers fed bahiagrass hay ad libitum. ...................................59 4-1 Analyzed1 chemical and nutrient composition (DM basis) of hay and protein supplements fed to ruminallycannulated An gus crossbred steers. ...................................77 4-2 Characterization of protein supplements fed to ruminally-cannulated Angus crossbred steers1 fed bahiagrass hay ad libitum. ................................................................78 4-3 In situ digestion kinetics on DM, OM, and CP of protein supplements fed to ruminally -cannulated Angus crossbred steers1 fed bahiagrass hay ad libitum. .................79 4-4 Amino acid composition of Brassica carinata meal pellets in original meal, ruminally incubated residue, and post-ruminal residue. ....................................................80 4-5 Total tract digestibility of amino acids from Brassica carinata meal pellets and contribution of RUP to intestinally absorbable amino acids. ............................................81 4-6 Total tract digestibility of essential amino acids of Brassica carinata meal pellets and contribution of RU P to intestinally absorbable essential amino acids. .......................82 5-1 Analyzed1 chemical and nutrient composition (DM basis) of diet fed to growing Angus crossbred heifers. ....................................................................................................92 5-2 Effects of protein supplementation on average daily gain, initial and final BW, and attainment of puberty in Angus crossbred heifers fed bermudagrass hay ad libitum. .......93 5-3 Effects of protein supplementation on thyroid hormone1 metabolism and acute phase protein response in Angus crossbred heifers fed bermudagrass hay ad libitum. ...............96

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9 LI ST OF FIGURE S Figure P age 2 -1 Breakdown products of a general glucosinolate structure under the action of myrosinase. ........................................................................................................................42 3-1 Effects of protein supplementation post-feeding in ruminal pH and concentrations of ruminal ammonia nitrogen ( P < 0.0001) in ruminally-cannulated Angus crossbred steers fed bahiagrass hay ad libitum. .................................................................................57 3-2 Effects of protein s upplementation post-feeding in concentrations of plasma glucose ( P < 0.0001) in ruminally-cannulated Angus crossbred steers fed bahiagrass hay ad libitum. ...............................................................................................................................58 3-3 Effects of protein supplementation post-feeding on molar proportions of acetate (mol 100 mol-1) in ruminally -cannulated Angus crossbred steers fed bahiagrass hay ad libitum. Treatment time interaction observed ( P < 0.0001). BCM: Brassica carinata meal pellets (1.39 kg d-1); CSM: cotto nseed meal (1.62 kg d-1); DDGS: dry distillers grain plus solubles (2.15 kg d-1); SBM: soybean meal (1.17 kg d-1). .................60 3-4 Effects of protein supplementation post-feeding on molar proportions of propionate (mol 100 mol-1) in ruminally -cannulated Angus crossbred steers fed bahiagrass hay ad libitum. Treatment time interaction observed ( P < 0.0001). BCM: Brassica carinata meal pellets (1.39 kg d-1); CSM: cottonseed meal (1.62 kg d-1); DDGS: dry distillers grain plus solubles (2.15 kg d-1); SBM: soybean meal (1.17 kg d-1). .................61 3-5 Effects of protein supplementation post-feeding on molar proportions of butyrate (mol 100 mol-1) in ruminall y-cannulated Angus crossbred steers fed bahiagrass hay ad libitum. Treatment time interaction observed ( P < 0.0001). BCM: Brassica carinata meal pellets (1.39 kg d-1); CSM: cottonseed meal (1.62 kg d-1); DDGS: dry distillers grain plus solubles (2.15 kg d-1); SBM: soybean meal (1.17 kg d-1). .................62 3-6 Effects of protein supplementation post-feeding on molar proportions of BCVFA ( P = 0.004) in ruminally-cannulated Angus crossbred steers fed bahi agrass hay ad libitum. ...............................................................................................................................63 3-7 Effects of protein supplementation post-feeding on molar proportions of valerate (mol 100 mol-1) in ruminally -cannulated Angus crossbred steers fed bahiagrass hay ad libitum. Treatment time interaction observed ( P < 0.0001). BCM: Brassica carinata meal pellets (1.39 kg d-1); CSM: cottonseed meal (1.62 kg d-1); DDGS: dry distillers grain plus solubles (2.15 kg d-1); SBM: soybean meal (1.17 kg d-1). .................64 3-8 Effects of protein supplementation post-feeding on concentrations of total VFA ( P = 0.025) in ruminally-cannulated Angus crossbred steers fed bahiagrass hay ad libitum. ...65

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10 3-9 Effects of protein supplementation post-feeding on acetate to propionate ratio in ruminally -cannulated Angus crossbred steers fed bahiagrass hay ad libitum. Treatment time interaction observed ( P < 0.0001). BCM: Brassica carinata meal pellets (1.39 kg d-1); CSM: cottonseed meal (1.62 kg d-1); DDGS: dry distillers grain plus solubles (2.15 kg d-1); SBM: soybean meal (1.17 kg d-1). .........................................66 5-1 Effects of block ( P < 0.0001) on initial and final BW of Angus crossbred heifers fed bermudagrass hay ad libitum over two consecutive years. No effect of treatment ( P = 0.96) or treatment block interaction ( P = 0.80) was observed for initial BW. No effect of treatment ( P = 0.088) or treatment block interaction ( P = 0.97) was observed for final BW. .......................................................................................................94 5-2 Effects of block ( P < 0.0001) on days to attainment of puberty in Angus crossbred heifers fed bermudagrass h ay ad libitum over two consecutive years. No effect of treatment ( P = 0.68) or treatment block interaction ( P = 0.49) was observed. ..............95 5-3 Effects of protein supplementation on thyroid hormones concentrations in plasma in Angus crossbred heifers fed bermudagrass hay ad libitum over two consecutive years. No effect of treatment was observed for T3 ( P = 0.31) or T4 ( P = 0.94), nor was an effect of treatment day observed for T3 ( P = 0.98) or T4 ( P =0.78). Day effect was observed ( P < 0.0001). .....................................................................................97 5-4 Effects of protein supplementation on day ( P < 0.0001) on acute phase protein ceruloplasmin in Angus crossbred heifers fed bermudagrass hay ad libitum over two consecutive years. An effect of treatment was observed ( P < 0.0001), however no treatment day interaction ( P = 0.56) was observed. .......................................................98

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11 LIST OF ABBREVIATIONS A A Amino acid AAD Amino acid digestib ility AAFC Agriculture and Agri Food Canada ADF Acid detergent fiber ADFI Acid detergent fiber intake ADG Average daily gain A:P Acetate to propionate ratio APP Acute phase protein APR Acute phase response BCAA Branched chain amino acid BCM Brassi ca carinata meal BCVFA Branched chain volatile fatty acid BMR Basal metabolic rate BUN Blood urea nitrogen BW Body weight CNS Central nervous system CP Crude protein CPI Crude protein intake Cp Ceruloplasmin CRP C reactive protein CSM Cottonseed meal CTL Control Cu Copper D Potentially degradable fraction

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12 DDGS Dry d istillers grains plus solubles DIT Diiodotyrosine DM Dry matter DMI Dry matter intake D o D Department of defense DPD N, N dimethyl p phenylenediamine sulfate EFSA European Food Safety Authority EIA U.S. Energy Information Administration EPA Environmental Protection Agency Exp Experiment FA Fatty acid FAO Food and Agriculture Organization of the United Nations Fe Iron FEF Feed Efficiency Facility GE Gross energy G:F Gai n to feed ratio GH Growth hormone GHG Greenhouse gas emissions Hb Hemoglobin Hp Haptoglobin HPLC High performance liquid chromatography H 2 SO 4 Sulfuric acid IAAA Intestinally absorbable amino acids IADP Intestinally absorbable dietary protein IDP I ntestinally digestible protein

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13 IGFs Insulin like growth factors IL Interleukin ILUC Indirect land use change iNDF Indigestible neutral detergent fiber IPS Inter Press Service IVOMD In vitro organic matter digestibility IVTDMD In vitro true dry matt er digestibility K + Potassium K d Rate of degradation (% h 1 ) K p Rate of passage (% h 1 ) L Lag time LCA Life cycle assessment MBW Metabolic body weight (BW 0.75 ) MCP Microbial crude protein MIT Monoiodotyrosine MP Metabolizable protein N Nitrogen Na + Sodium NAABB National Alliance for Advanced Biofuels and Bio products NADH Nicotinamide adenine dinucleotide NDF Neutral detergent fiber NDFI Neutral detergent fiber intake NFREC North Florida Research and Education Center NH 3 N Ruminal ammonia nitrogen NIMSS National Information Management and Support System

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14 NRC National Research Council Canada OM Organic matter OMI Organic matter intake PAMPs Pathogen associated molecular patterns REAP Rural Energy for America Program RDP Rumen degradabl e protein rpm Revolutions per minute r T 3 Reverse triiodothyronine RUP Rumen undegradable protein S Sulfur SAA Serum amyloid A SF Soluble fraction SBM Soybean meal SD Standard deviation SEM Standard error of means T 3 Triiodothyronine T 4 Thyroxin e TDN Total digestible nutrients TDP Total dietary protein TTDP T otal tract digestibility of protein Tg Thyroglobulin TNF

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15 VFA Volat ile fatty acid VLCFA Very long chain fatty acid

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16 Abstract of Thesis Presented to the G raduate S chool of t he U niversity of Florida in P artial Fulfillment of the Requirements f or t he Degree of Master of S cience EVALUATION OF B RASSICA CARINATA MEAL ON ANIMAL P ERFORMANCE AND METABOLISM IN B EEF C ATTLE By TESSA MARIE SCHULMEISTER De cember2017 Chair: Nicolas D iLorenzo Major: A nimal S ciences Brassica carinata, a nove l oilseed cr op, yields highquality bi of uel, resulting in a highprotein byproduct. T his meal ha s not be en t ested as a cattle p rotein supplement t herefore our objective was t o evaluate t he effects of s upplementation of B. carinata m eal Eight Angus crossbred st eers w ere ut ilized in a dupl icated 4 4 Latin square de sign, over 4 periods of 28 d each, to a ssess r uminal fermentation pa rameters, n utrient digestibility, a nd blood profile. Within period, steers w ere a ssigne d to one of f our t reatments: 1.39 kg d -1 of Brassica carinata meal pe llets ( BCM), 1.62 k g d -1 of c ottonseed meal ( CSM), 2.15 kg d -1 of dr y distillers grains plus solubles ( DDGS), or 1.17 k g d -1 of s oybea n m eal ( SBM). No effect of treatment ( P > 0.05 ) w as observed for r uminal pH N H 3 N, total V FA pl asma glucose, DMI, or apparent total tract di gestibility of nut rients. S teers r eceiving S BM h ad t he greatest ( P < 0.01) concentration of B UN. A ruminal in s itu de gradability s tudy was p erformed utilizing steers from the pr evious e xpe riment Supplement r emaining after 16 h of incubation was s ubjecte d to serial s olutions simulating pos truminal digestion. R ate of d egradation of D M an d C P w as greatest ( P in SBM; total tract digestibility was greater i n S BM Nearly e quivalent a mounts of R DP a nd RUP

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17 were observed in CSM and DDGS which differed ( P from SBM and BCM Compared with DDGS, SBM had a greater IDP ( P < 0.01), with CSM having the greatest IADP ( P < 0.01) A nimal performance, attainment of puberty, and blood profile was evaluated in 64 Angus crossbred heifers S tratified and blocked by initial BW heifers were randomly allocated into 18 pens over 2 consecutive years P ens were randomly assigned within block, to one of two treatments: 0 (CTL) or 0.3% of BW d1 (as fed) of BCM pellets B ermudagrass hay was provi ded ad libitum. Compared with CTL, BCM increased ( P ADG; treatment did not affect interval to attainment of puberty ( P = 0.68) C oncentrations of c eruloplasmin w ere greater ( P 0.01) in CTL heifers with a n effect of day ( P < 0.01) observed for T3, T4, and ceruloplasmin.

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18 CHAPTER 1 INTRODUCTION Greenhouse gas emissions in the US are principally anthropogenic, resulting from burning fossil fuels for heat, electricity, and transportation (EPA, 2015). Replacing petroleum based products with renewable resources offers probable solutions in reducing emissions (EIA, 2017), and technological advances have enabled an increasingly efficient conversion of renewable resources into fuels and chemicals (IPS, 2014). Alternatives have been employed to decrease dependency on fossil fuels. The current ethanol industry was revitalized in the 1970s in response to increasing fuels costs (EIA, 2017), and began utilizing corn as a feedstock for the production of this biofuel. The ethanol industry is insuff icient in meeting demands, partially due to EPA (2011) regulation allowing only 10 to 15% ethanol mixtures in gasoline, decreased fuel efficiency compared with gasoline alone (Knoll et al., 2009), and competition in land allocated for increased crop produc tion with land utilized to produce food for humans and livestock (FAO, 2008). Algal oil has been successfully converted to biofuel, however acquisition of materials, pricing, and efficiency of conversion have presented challenges (NAABB, 2014). Similarly, lignocellulose has been successfully converted to biofuel and used to generate electricity in local industries where biomass is abundant. Despite success, the conversion of lignocellulose to biofuel for use on a large scale is expensive, less efficient, an d environmentally taxing (Carroll and Somerville, 2009). Recently incurring greater interest are non food oilseeds, which may provide a sustainable alternative without competing directly with human food, or livestock production systems. Non food oilseeds undergo efficient treatments for oil extraction, accounting for 10% of the total costs of the final fuel (NIMSS, 2016). Due to a favorable fatty acid profile, Brassica carinata has been successfully used as a 100% dropin biofuel (NRC, 2013). After oil ext raction,

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19 a residual meal with approximately 40% CP results. Additional benefits include cold and drought tolerance, heat and disease resistance, and potential use as a pesticide and rotational crop (AAFC, 2015), which offer great promise in the Southeast U S. Brassica carinata has not been extensively tested as a protein supplement for beef cattle, therefore, research is necessary to evaluate the effects of supplementation on metabolism, feed intake, nutrient digestibility, ruminal fermentation, and performa nce in beef cattle.

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20 CHAPTER 2 LITERATURE REVIEW B rassica c arinata Concern over stability and negative effects of fossil fuels has increased awareness and motivated an effort to replace petroleum derived sources with renewable and sustainable resources (EIA, 2017). Brassica carinata is a nonfood oilseed crop, belonging to the mustard family, Brassicaceae and originates from the highlands of Ethiopia. Ethiopian mustard, or more commonly carinata, results from interspecific hybridization between B. nigr a L. and B. oleracea L. (Prakash and Hinata, 1980). As an amphidiploid (BBCC, 2n = 34), carinata possesses a complete diploid set of chromosomes from each parent, thereby acquiring valuable traits inherent to each parent. Carinata has many potential benefi ts including: a favorable very longchain fatty acid (VLCFA) profile for conversion to biofuel, a low indirect land use change (ILUC) rating, a favorable Life Cycle Assessment (LCA), drought and cold tolerance, heat and disease resistance, usage as a rotat ional crop, and a high concentration of crude protein within the meal residue after oil extraction (AAFC, 2015). F avorable Longchain Fatty Acid Profile The VLCFA profile of carinata consists of erucic and nervonic acid, favorable alternatives for not onl y biofuel production, but also bioplastics, lacquers, and paints ( Carlsson, 2009; Impallomeni et al., 2010; Newson et al., 2013). Genetically modifying the fatty acid (FA) composition of native B. carinata seed oil, approximately 40% of seed DM (Warwick e t al., 2006), significantly increased concentration of erucic acid or proportions of nervonic acid resulting in more than 40% of erucic or nervonic acid in second generation lines (Marillia et al., 2013). Erucic acid (22:1; C22) is a precursor to nervonic acid (24:1; C24), both resultant from elongation of oleic acid (18:1; Taylor et al., 2010). The increase in C22 and C24 resulted in a

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21 decrease of C18 FA proportions (Marillia et al., 2013), which is uncommon for an oilseed, considering soybean and canola m eal typically have FA profiles of C20 or less. This alteration of the FA profile is beneficial as an increase in VLCFA improves performance properties such as cold temperature flow characteristics, oxidative stability, and NOx emissions in biodiesel (Durre tt et al., 2008); decreases GHG emissions by more than 80%, reduces fuel consumption, and decreases black carbon, or incomplete combustion of biofuels, up to 50% in jet biofuel (ARA, 2017). Potential E ffects of U tilizing R enewable R esources Land use chang e, or diversion of land from its existing uses (Searchinger et al., 2008), is a concern when considering potential growth of renewable resource industries utilizing feedstock for biofuels. Potential GHG mitigation through utilization of feedstock is promis ing, however unintended effects of ILUC are not taken into account when calculating emissions, destruction of forests and grasslands, or competition between human and livestock for feed (Milazzo et al., 2013). A Life Cycle Assessment (LCA) is an intensive study used to evaluate environmental impacts, energetic balance, and economic performance of a biomass crop regarding cultivation, collection, transportation, and conversion of biomass to energy in the form of fuel or electricity ( Gasol et al., 2007; Butn ar et al., 2010) A LCA was conducted for carinata in southern Europe (Gasol et al., 2007) Spain (Butnar et al., 2010) and Italy (Cardone et al., 2003) as a lignocellulosic biomass crop for energy use and biofuel Butnar et al. (2010) analyzed the impact s of carinata and a native crop on six categories: global warming, acidification, human toxicity, ozone layer depletion, abiotic depletion, and photochemical oxidation. B iomass crops used for generating power were found to be more environmentally harmful t han current electricity producing systems however Butnar et al. (2010) observed that negative impacts

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22 decreased with an increase in biomass productivity. The use of carinata was deemed as a viable source for production of energy in southern Europe, and Ga sol et al. (2009) concluded that varying management practices to improve crop production and performance may further increase energy and environmental benefits. Fertilizers used for carinata presented the greatest impact, however utilizing an alternative, such as livestock waste, was suggested as a potential improvement (Gasol et al., 2009). Agronomic performance and energetic balance resulted in a favorable analysis in Italy concerning use of carinata as a biofuel (Cardone et al., 2003). Carinata required less fertilizer, weed control, and tillage compared with Brassica napus and due to the tolerance of carinata to harsher environments outperformed B. napus in production in coastal regions Further, biofuel from carinata exhibited positive characteristics similar to commercial biodiesel, with the potential of decreasing costs associated with production of biofuel (Cardone et al., 2003). Utilizing renewable resources for national energy security, climate change mitigation, and sustainability are motivation s behind majority of studies evaluating feedstocks as potential biofuels (Seepaul et al., 2016). Carinata eliminates competition for land allocated for food crop production through its ability to be utilized in unfavorable environments as it is drought and cold tolerant, heat and disease resistant (AAFC, 2015), and potentially able to be used in rotation with other crops, or for use in fallow land, when food production crops would not normally be present (Marois et al., 2015). Additionally, use of carinata in the southeastern U.S. promotes the agenda of Rural Energy for America Program (REAP) established by United States Department of Agriculture (USDA, 2013), through domestically grown renewable and sustainable resources. Residual meal waste may also be ben eficial in the southeastern U.S., due to high concentration of CP as a potential protein supplement for livestock.

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23 Previous R esearch on U tilization of C arinata as a S upplement for L ivestock Although carinata is not a new crop, the residual meal after oil extraction has not been extensively investigated as a protein supplement for livestock. Tadelle et al. (2003) evaluated the performance of Hubbard broiler chicks fed carinata as a rap eseed cake at various inclusion rates (0, 7, 14, 21, 28, and 35%) of the basal diet. Additionally, no differences were observed in intake of feed or water, feed to gain ratio, or in dressing percentage. Interestingly, percentage of mortality was increased for inclusion of 28% compared with 7%, but was similar for all other tre atments; however, an increase in size of thyroid follicles was observed for broiler chicks receiving 35% inclusion. Wheater (1987) observed a negative correlation regarding thyroid activity, i.e., an increase in thyroid follicle size results in a decrease in thyroid hormone secretion. Tadelle et al. (2003) concluded that the effects observed were related to the concentration of rapeseed cake inclusion; however, comparing effects of the control with dietary inclusion amounts of 7 through 21% indicated no dif ferences were observed for these treatments. Further, the economical aspect of feeding broiler chicks seemed to be the focus and it was concluded that the inclusion of 28% required the least quantity of feed per unit gain and promoted greatest dressing per centage. Considering mortality was highest for 28% inclusion, with no significance observed in dressing percentage, intake, or gain, the conclusion is questionable. Inclusion of 10% carinata meal as a protein supplement in a diet of barley silage and grain compared with canola meal at the same rate, was evaluated for yearling steers, in which no differences in live weight, ADG, DMI, or subcutaneous rib or rump fat were observed (McKinnon et al., 2012). A tendency was observed for decreased gain:feed ( P = 0 .09) in carinata compared with canola (0.09 vs 0.10, respectively), however it was concluded that inclusion of

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24 10% carinata meal of diet DM resulted in similar effects on animal performance, compared with canola meal, at the same rate of inclusion. Ruminal degradation, intestinal, and total tract digestion characteristics, and metabolizable protein supply of carinata meal was evaluated, in comparison with canola meal, using dry Holstein cows. An in situ study was performed to estimate the nutritive value of carinata meal and ruminal degradation kinetics, observing an increase in the rate of degradation of OM and CP for carinata meal when compared with canola meal (Xin and Yu, 2014). Proportions of degradable and undegradable fractions were similar for OM and CP, with soluble fractions similar for OM, but increased for CP in carinata, compared with canola. Undegradable OM tended to be decreased for carinata, compared with canola, however effective degradability of OM, CP, and NDF tended to be increased for car inata, compared with canola. Carinata and canola meals differed only in intestinally digestible rumen bypass OM, with canola being greater; therefore carinata meal performed similarly in intestinal digestibility of nutrients, except for rumen bypass OM com pared with canola meal with an increased rate of ruminal degradation for carinata compared with canola meal. P rotein Supplementation in Ruminants F ractionation of Dietary Protein Dietary CP results from the assumption that the average N content of p roteins is 16%, and is partitioned in two fractions based on derivation of N: true protein (TP) or nonprotein nitrogen (NPN). However, when calculating dietary CP content differences between TP and NPN are not delineated. Upon consumption, dietary CP is further separated by ruminal activity, resulting in potentially degradable and undegraded fractions (Orskov and McDonald, 1979). Specifically in ruminants, CP can be divided into two fractions: r uminally degradable protein (RDP) or ruminally undegradable protein (RUP). The RDP fraction includes NPN and

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25 TP and is often referred to as fraction A is composed of water soluble proteins and NPN, or nitrogenous containing entities, such as nucleic acids, urea, NH3, and nitrates, which are rapidly degradable (Dry den, 2008). A potentially degradable fraction B is comprised of TP, i.e., proteins, peptides, and amino acids, a portion of which may escape ruminal degradation. Ruminally undegradable CP is often referred to as fraction C. Thus, fraction A and degraded pr otein in fraction B compose RDP, whereas the undegraded portion of fraction B, and all of fraction C constitute RUP. Microbial degradation of fraction B includes hydrolysis of proteins to oligopeptides by proteolytic enzymes, with subsequent degradation to smaller peptides and AA for cellular uptake to occur (NRC, 2016). Short peptides and AA are preferentially utilized by ruminal microorganisms, particularly fibrolytic bacteria (NRC, 2016), however NH3 resulting from soluble NPN or deamination of AA, can b e assimilated into microbial protein synthesis (Hungate, 1966). Excessive NH3 is absorbed across the ruminal epithelium into the portal vein system, and subsequently converted to urea in the liver, to avoid toxicity. Urea, which is relatively non toxic, is either excreted in urine, returned to the rumen via saliva, or diffused through the ruminal epithelium wall where it will be converted into NH3 for utilization by ruminal microorganisms, or further recycled. Thus, efficiency of nitrogen recycling is dependent upon dietary intake of nitrogen, i.e., when nitrogen intake is decreased, efficiency of nitrogen recycling increases (NRC, 2016). Ruminally undegraded protein escapes degradation by microorganisms with subsequent digestion and absorption occurring wi thin the small intestine, and further utilized by the animal for tissue growth or lactation. Importantly, protozoa are capable of deamination, however their involvement with regards to protein metabolism include engulfing and degradation of insoluble

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26 parti culate proteins, bacterial, and fungal cells. Thus, contribution of ciliate protozoa, autolysis and activity of bacteriophages results in nearly 50% of ruminal microbial crude protein (MCP; NRC, 2016). The RDP, which is degraded and ultimately transformed into MCP, in combination with RUP and endogenous protein, comprises metabolizable protein (MP), which more succinctly defines the availability of true protein for use by the ruminant (Dryden, 2008). Ruminal microbes are able to utilize NPN sources for gro wth, however MCP yield is poor (Hume, 1970); therefore, provision of TP is essential for effective ruminal degradation and fermentation of feedstuffs. Further, maximizing ruminal MCP synthesis yields high quality protein, specifically amino acids, availabl e to the small intestine, however additional RUP is necessary for optimizing yield in highproducing animals (Stern et al., 2006). It has been suggested that approximately 80 to 90% of dietary protein is inefficiently utilized and subsequently excreted as waste (NRC, 2016). Thus, despite the importance of protein, it is secondary to energy requirements, as efficiency of MCP synthesis is a function of energy utilized by ruminal microbes for maintenance and growth (NRC, 2016). Therefore, feeding cattle involves careful synchronization of energy and protein provided, complementing the needs of the animal, environment in which they live, and resources available. It is therefore important to understand dietary requirements of cattle, specifically protein, which vary with stage of production, animal size, and expected performance, as well as to recognize the needs of meeting the requirements of both the ruminant host and its microorganisms. C attle Production in the Southeast U.S. Cattle production in the Southeast U.S. is typically comprised of cow calf operations (McBride and Matthews, 2011), with management of a cow calf herd based upon breeding cattle to suit conditions within a certain region. Often this is accomplished through crossbred cattle (Greiner, 2009), thereby increasing hybrid vigor through utilization of the F 1 hybrid, and

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27 employing breeds which are most economical and complementary to management and available resources. I n Florida alone, there are approximately 1,700,000 cattle, and nearly half of t hat total represent beef cows and replacement heifers (USDA, 2017). Geographical location defines available resources, i.e., beef cattle production in southern Florida differs from practices in northern Florida. Due to heat stress, production in southern Florida typically results in calving season beginning in late fall (Vendramini and Arthington, 2008) with earlier weaning of calves. Cattle production in the Florida panhandle differs slightly with heifers calving, in the majority of the operations in whic h a breeding season is defined, anywhere from October to midDecember, and mature cows from November to January. Cow calf production systems throughout the state, and Southeast U.S. in general, may deviate slightly from these two examples, however, these w ill be used for discussion of resources available at each stage of production. Warm season perennial grasses are the basis for beef production in the southeastern U.S., with bahiagrass ( Paspalum notatum ) being the most common forage resources throughout t he majority of Florida. In an attempt to improve the quality of the forage base over that of bahiagrass, bermudagrasses ( Cynodon dactylon) are often planted, with Tifton 85 being preferred to Coastal bermudagrass due to increased digestibility and conseque ntly, animal performance (Vendramini et al., 2008). During warmer months, Tifton 85 may be an ideal option for grazing cattle, however long periods of regrowth decrease the nutritive value of Tifton 85. Regarding forages to be used in the cool season, annu al ryegrass is commonly utilized in central and north Florida, due to productivity and increased nutritive value, with the addition of small grains mixed in to provide sufficient forage until ryegrass is most productive (Dubeux et al., 2016). As previously mentioned, calving season occurs earlier in southern Florida and is often

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28 accompanied with earlier weaning which is suggested as an effective method of preparing first calf heifers for rebreeding (Arthington and Kalmbacher 2003). In northern Florida, however, calves are not weaned until approximately 6 to 10 months of age. Though climates differ, resulting in diverse growth patterns of forages and available resources, both regions face a common challenge: feeding beef cattle at various stages of producti on, size, and levels of nutrient requirements, with calf production, lactating cows, and developing heifers relying on supplementation and grazing (Banta et al., 2016). In order to determine the protein supplementation needed for these animals, it is neces sary to review their dietary requirements. Protein r equirements of b eef c attle in c ow calf operations of the s outheastern U.S. The publication Nutrient Requirements of Beef Cattle (NRC, 2016), provides indepth, general information resulting from an abunda nce of research for provision of nutrients to beef cattle. With consideration to the prevalent production system in the Southeast U.S., energy and protein requirements are discussed for early weaned calves, growing steer calves, lactating cows, and heifers A summary of nutrient requirements for growing and lactating cattle is presented in Table 21 (NRC, 2016). Vendramini et al. (2006) reported a decrease in DMI in early weaned calves grazing wheat, and inefficient N utilization when consuming highprotei n, annual cool season pastures. Performance was improved, however, with addition of concentrate supplemented in synchronization with crude protein. Early weaned calves should therefore be provided with a diet containing at least 1 5% CP and concentrate when grazing pastures to facilitate improved ruminal degradation and N utilization, and subsequent feed intake. Growing steer calves, depending upon expected performance, should be supplemented with protein coinciding with recommended TDN values. Calves expect ed to weigh 550 kg at finishing vary in requirements depending on length of time for gain, i.e., calves with an expected

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29 ADG of 1.17 kg d1 require less DMI, but increased TDN, CP, and MP compared with calves with an ADG of 1.04 kg d1 (Table 21; NRC, 2016). Yearling stocker calves are typically grown on highroughage rations (Field, 2016), however the quality grade of beef is improved with marbling, which increases linearly with growth, genetic predisposition, and time on feed (Kern et al., 2014). Therefore, to attain expected growth and marbling, supplementation of protein may be necessary, depending on the basal diet provided. Lactating cows in southern Florida present a challenge in terms of nutrition, as intake requirements increase approaching the peak milk yield (~ 60 DIM) and subsequent rebreeding, which corresponds with the cool season and limited pasture growth. During this critical period w arm season pastures are typically poor in quantity and quality, and therefore insufficient in meeting nutritional demands (Bohnert et al., 2011), without additional supplementation. Cool season annuals in northern and southern Florida are typically sufficient to meet nutrient requirements due to increased concentration of protein. However, weather variability (m ainly their dependence on precipitation) may lead to failure during the establishment of cool season annual forages, having to incur additional supplementation. Protein supplementation for lactating cows should include sources higher in RUP to optimize mil k production (Stern et al., 2006). Conversely, dry cows and midgestation, older cows in adequate to good condition can be sustained on lower quality feed (Field, 2016). The success of a cow calf operation is dependent upon management of cattle, specifica lly replacement heifers, as they are an important resource and their requirements differ from calves and mature cows. Nutrition of yearling heifers is similar to that of yearling steers, however diets are modified slightly to prevent excessive adipose tiss ue accretion, as this may affect development of the mammary gland and subsequent lactation (Field, 2016). Heifers are expected

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30 to attain a certain weaning weight, become pregnant in the first estrous cycle, carry a calf to full term without complications, remain disease free, and achieve a successful lactation while maintaining or increasing BW, despite not having reached maturity (Bellows et al., 2002). Nutrition and attainment of puberty in beef heifers The onset of puberty, primarily a function of age a nd weight, is the first ovulation that is accompanied by visual signs of estrus and normal luteal function (Perry and Cushman, 2013). Attainment of puberty in heifers is expected at approximately 15 months of age, depending on breed ( Day and Nogueira, 2013). Funston and Deutscher (2004) suggest breeding at a target weight of 50 to 57 % of mature BW, as body conditions are primed for initiation of the estrous cycle. In order to meet that goal, heifers should gain approximately 0.22 to 0.68 kg d1, depending on weaning weight. In a cow calf operation, the length of breeding season is reduced to optimize production of a uniform calf crop. Heifers are expected to calve at 24 months, and therefore require quality feed and increased intake to ensure reproductive p erformance, as the first calving tends to set precedence for subsequent parturitions and calving events (Patterson et al., 1992). Further, heifers calving earlier in the season tend to produce heavier calves and have more time to prepare for rebreeding (Ar thington and Kalmbacher, 2003). Additionally, an increase in ADG has been observed when calving occurs 2 to 3 months prior to availability of green forage production, as opposed to calves born earlier or later (Field, 2016). Calving 2 to 3 months prior to green forage production correlates with a peak in milk yield of dams, thereby supplying calves with adequate nutrition, and displacing production pressure from the cow. Glucosinolates Carinata has been classified as a botanical impurity, with further clas sification in the Annex to Directive 2002/32/EC of the European Parliament and of the Council of 7 May 2002 as an undesirable substance in animal feed due to glucosinolates present (EFSA, 2008).

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31 Glucosinolates are a common occurrence in oilseed crops, espe cially those of Brassicaceae, and are found throughout the plant, with higher concentrations often found in seeds. Glucosinolates are a general term for any alkyl aldoximeO D thioglucopyranoside group, which vary in structure and configuration of their side chain (EFSA, 2008). E volution and Degradation Glucosinolates, possibly evolved to prevent herbivory and are inactive until, upon mastication of plant leaves, myrosinase is released, unfolding the toxicity of the compound (van Doo rn et al., 1998; Collett et al., 2014). Additionally, bacterial myrosinases are present in the gut of humans and animals (Fahey et al., 2001). Glucosinolates are hydrophilic and rather stable, therefore remaining in the seed after solvent extraction, until myrosinase acts to convert glucosinolates into various products through hydrolysis (Tripathi and Mishra, 2007). More than 140 glucosinolates have been identified, however this review will focus on those relevant to carinata, primarily sinigrin, representi ng more than 95% of total glucosinolate content, and in lower concentration, progoitrin (Bellostas et al., 2007; Marillia et al., 2013). Glucosinolates in C arinata: S inigrin and P rogoitrin The hydrolysis of sinigrin by myrosinase produces an unstable thiohydroximate O sulfate and glucose, in which the glucose is subsequently removed resulting in spontaneous decomposition of thiohydroximate O sulfate to allyl isothiocyanate and free sulfate (Yuan et al., 2016) and 3butenenitrile, also known as allyl cyanide (Duncan and Milne, 1992). The content of sinigrin in carinata seed meal was evaluated by Yuan et al. (2016) and found to contain 46.72 0.92 g mg1 compared with a crude oil concentration of 0.075 0.05 g mg1, through a reversed phase HPLC method. P rogoitrin is hydrolyzed by myrosinase to ( S) goitrin, with activity of myrosinase being pH dependent, thus increasing concentration of ( S) goitrin under biologically relevant pH conditions (6.5 to 8; Xie et al., 2011), and goitrin is a sulfur containing

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32 ox azolidine (Ishikawa et al., 2014). Additionally, progoitrin can be hydrolyzed to nitriles (Forss and Barry, 1983; Collett et al., 2014). Sinigrin and progoitrin breakdown products : Sinigrin and progoitrin are related to the characteristically bitter taste found in mustard plants (van Doorn et al., 1998), with palatability being an issue when crops containing high concentrations (~ 90 to 140 mol g1; Lardy and Kerley, 1994) of glucosinolates are consumed by humans and animals. However, the effects on healt h of the breakdown products of sinigrin and progoitrin are of greater concern. Thiocyanate (or the isothiocyanate ion) and goitrin alter thyroid metabolism, including potential enlargement of the thyroid gland (Spiegel et al., 1993), through selectively bi nding iodine, which can be remedied with iodine supplementation; however, goitrin inhibits the synthesis of thyroid hormones, which cannot be corrected with supplementation (Zukalova and Vasak, 2002). Isothiocyanate and nitriles have been confirmed to be i nvolved in growth retardation (Schone et al., 1997), inhibition of copper (Cu) and selenium absorption and metabolism, and fertility impairment (Taljaard, 1993). Further, damage to endothelium and epithelium, cell membranes, liver damage, transient impairment of locomotion, and disorientation (Schmid and Schmid, 1992) have been observed as effects of isothiocyanate and nitriles. D ecreased intake and low amino acid absorption (Barry, 2013) in addition to irritation and edema of gastro intestinal mucosa (Mas on and Lucas, 1983) have also been reported. Isothiocyanate has been implicated in the tainting of animal products consumed by humans, referring to unpleasant taste or odor as the consumption by the animal increases (Fenwick et al., 1983). Furthermore thiocyanate is absorbed into the blood and secreted into the milk, which may affect lactation performance in cows and growth of calves (Tayo et al., 2012).

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33 T hyroid Metabolism The thyroid gland consist of follicular cells, arranged into fluidfilled spheres, forming a follicle, which encloses an inner lumen filled with colloid. Thyroglobulin (Tg), a large, complex glycoprotein produced by the endoplasmic reticulum/Golgi complex of the thyroid follicular cells incorporates the amino acid tyrosine, and is expor ted via exocytosis into the colloid (Sherwood et al., 2013). The thyroid, stimulated by thyroid stimulating hormone from the adenohypophysis ( Chiamolera and Wondisford, 2009), captures iodide from blood, trapping it within the thyroid, and transfers it int o the colloid via a Na+/K+ pump located at the basolateral membrane (Dukes et al., 1993). Before reaching the colloid, iodide is oxidized by thyroperoxidase, thus indicating its active state (Sherwood et al., 2013). Once active iodide is within the colloid it is coupled to a tyrosine yielding monoiodotyrosine, or two iodide coupling to a tyrosine yield diiodotyrosine. The coupling of iodide to tyrosine is attached to Tg through peptide bonding, and remains stored until cleaved off and secreted (Sherwood et al., 2013). T hyroid Hormones Iodination of tyrosine resulting in monoiodotyrosine (MIT) and diiodotyrosine (DIT) is the precursor to formation of thyroid hormones. Triiodothyronine (T3) results from the coupling of one MIT and DIT, with thyroxine (T4) res ulting from coupling of two DITs, and these products being biologically active and stored until needed (Boelaert and Franklyn, 2005). Once stimulated for secretion, a portion of thyroglobulinhormone complex is internalized by follicular cells and phagocyt osis results in a membrane bound droplet of colloid. This droplet will coalesce with lysosomes, whose enzymes will split off biologically active T3 and T4, as well as any inactive iodotyrosines for recycling (Sherwood et al., 2013). Released T3 and T4 will enter blood where they will quickly bind with plasma proteins, except for a small concentration which will remain in the unbound form, and are biologically effective. Most of the secreted hormone is T4,

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34 though it is subsequently converted to T3 in periphe ral tissues by a deiodinase enzyme, as T3 is the major biologically active form (van der Spek et al., 2017) at the cellular level due to its affinity of binding to receptors, thereby giving peripheral cells the ability to activate their own hormone stimula tion (Zhang and Lazar, 2000). Selecting for inactive hormone, reverse triiodothyronine (r T3), allows for conservation of energy during limited food availability. Effects of thyroid hormones : Thyroid hormones have no specific target, rather they affect every tissue in the body through crossing the plasma membrane and binding to nuclear receptors bound to thyroidresponse element of DNA (Gerebens et al., 2015). Binding alters transcription of specific mRNAs, thus synthesizing specific new proteins for cell ular response (Yen et al., 2006). Thyroid hormone synthesis is c onsidered slow compared to other hormones, thus elevated concentrations of T3 and T4 are not detectable for several hours, with maximal response taking days to detect (Sherwood et al., 2013). Thyroid hormones increase basal metabolic rate (BMR) by regulating mitochondrial function and certain mitochondrial proteins (WrutniakCabello et al., 2001), and decrease BMR through regulating the rate of oxygen consumption and energy expenditure under r esting conditions. Effects on metabolism are complex, as T3 and T4 can influence synthesis and degradation of carbohydrate, fat, protein, yet varying concentrations of hormones may have opposite effects (Sherwood et al., 2013). Fluctuations in thyroid horm ones may vary by day and season, depending on animal species (Yoshimura, 2013). Thyroid hormones increase target cell responsiveness to catecholamines, thus a sympathomimetic effect is observed (Silva, 2009). In the cardiovascular system, thyroid hormones increase cardiac output through increased responsiveness to circulating catecholamines (Jabbar et al., 2016). Essential for normal growth and development (Mullur et al.,

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35 2014), thyroid hormones act permissively in concert with other hormones in stimulating growth process. Required for growth hormone (GH) secretion, T3 and T4 also promote effects of GH and IGFs onsynthesis of new structural proteins and on skeletal growth (Nilsson et al., 2005), with stunted growth observed in thyroiddeficient animals. Th yroid hormones play a crucial role in development of the nervous system, especially the central nervous system (CNS) (Zoeller and Rovet, 2005), with thyroid hormones being essential for normal CNS activity in adult animals (Beydoun et al., 2015), as well a s in conduction velocity of peripheral nerves which varies directly with availability of thyroid hormones (Zhang et al., 2015). Thyroid hormones also act on the development of steroidogenesis, testicular development, and spermatogenesis, as indicated by the presence of thyroid hormone receptors (Wagner et al., 2008). D isorders of the Thyroid Disorders of the thyroid gland are mainly grouped in two categories which reflect excess of thyroid hormone secretion, hyperthyroidism, or the deficient secretion of th yroid hormones, hypothyroidism. Hyperthyroidism results in an elevated BMR resulting in increased heat production, and subsequent excessive perspiration or panting and poor tolerance of heat. An increased BMR can also result in increased appetite and inta ke, yet decreased weight gain, leading to loss of skeletal muscle protein and weakness, and negative effects on the cardiovascular system (Klein and Ojamaa, 2001). Hyperthyroidism affects the cardiovascular system, resulting in abnormalities from direct effects of thyroid hormones, as well as their interactions with catecholamines, potentially leading to heart failure (Klein and Danzi, 2007). Effects on the nervous system are observed in hyperthyroidism, manifested by excessive alertness, potentially result ing in irritability and anxiety.

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36 Hypothyroidism, or low thyroid activity, is also characterized by BMR activity although markedly decreased (Reinehr, 2010) compared with hyperthyroidism. Hypothyroidism results in poor tolerance to cold, loss of hair or fu r, fatigue, tendency for excessive weight gain, slow and weak pulse, slow reflexes, and decreased mental awareness and memory (Roberts and Ladenson, 2004). Hypothyroidism may be a result of thyroid failure, a deficiency of thyroid releasing or secreting hormone, an inadequate supply of dietary iodine, or certain chemicals or compounds which may affect uptake or trapping of iodide (Zoeller, 2010). I mmune Response Under immunological stress, the first line of defense is the innate, antigen nonspecific response, which can be elicited immediately or within several hours (Gruys et al., 2005). Innate immunity includes chemical and physical barriers preventing entry of pathogenic substances, such as skin, tears, urine, and stomach acid, in addition to benefi cial microorganisms which will compete with foreign invaders for resources. Should pathogens penetrate these barriers, cellular defenses are activated with a release of phagocytic cells, natural killer cells, and cells that release inflammatory mediators ( Sherwood et al., 2013). Phagocytic cells are activated at the site of infection and able to recognize pathogen associated molecular patterns (PAMPs) through pattern recognition receptors. Binding of PAMPs to these receptors initiates a killing mechanism in macrophages and neutrophils (Carroll and Forsberg, 2007). Natural killer cells do not attack pathogens, but attack and kill cells which have been contaminated by pathogens through chemically perforating the cell membrane leading to an influx of fluids and rupturing of the cell. Natural killer cells also release cytokines, or interleukins (IL), thereby initiating further immunological response (Carroll and Forsberg, 2007).

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37 A cute P hase R esponse An acute phase response (APR) is a component of the innate immunity defense system, in which the response is activated due to stressors from infection, inflammation, disease, trauma, stress, and bacterial components, and is detected for several days after the stimulus (Petersen et al., 2004). Indicators of an APR incl ude fever, decreased food and water intake, catabolism of muscle proteins, decreased sexual and social behavior, alterations in plasma iron (Fe), zinc, Cu, calcium, and vitamin A, increases in circulating leukocytes, and increased sensitivity to pain (Gruy s et al., 2005, 2006; Carroll and Forsberg, 2007; Moriel and Arthington, 2013). Induced by pro inflammatory cytokines (IL 1, IL 6, and TNF ), secretions are predominantly from monocytes which have been activated as a response to bacterial toxins or local tissue damage, and subsequently released into the blood stream (Sherwood et al., 2013). Cytokines communicate between sites of infection or inflammation and hepatocytes within the liver synthesizing acute phase proteins. Proinflammatory cytokines act in tandem through multiple, overlapping pathways exhibiting effects on both cells surrounding the affected area, as well as systemic effects through transportation in the blood stream. A transient increase occurs in serum concentrations of proinflammatory cy tokines a few hours after initial stimulus, however concentration decreases within several hours (Carroll and Forsberg, 2007). A cute Phase Proteins Proteinase inhibitors, enzymes, coagulation proteins, and metal binding and transport proteins (APP) are al l produced in hepatocytes of the liver at a relatively steady rate under normal conditions, however IL 1, IL 6, and TNF mediate hepatocyte synthesis and secretion of APP during an APR (Carroll and Forsberg, 2007). In response to stimuli, protein production is either increased (positive acute phase protein) or decreased (negative acute phase protein), with hepatic mRNA upr egulation of APP produced in response to stressors negatively correlated with

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38 normal production proteins (Gruys et al., 2005), in addition to production of APP being species specific (Petersen et al., 2004). Due to the increase in concentrations of positive APPs during an APR, APPs such as haptoglobin (Hp), serum amyloidA (SAA), C reactive protein (CRP), and ceruloplasmin (Cp) have been used as potential indicators of bovine acute and chronic inflammation, however this review will only focus on Hp and Cp. It is important to note that while APPs are useful in assessing an APR, a single APP is not sufficient to evaluate healthy vs. nonhealthy conditions, and should therefore be combined with other indicators to obtain a broader perspective (Gruys et al., 2006). Haptoglobin : Haptoglobin is a positive APP, indicating an increase in synthesis and secretion is observed during an APR (Lomborg et al., 2008), therefore, Hp has been used as an indicator of health in cattle (Gruys et al., 2006), as diagnostic biomarkers and prognostic aids in veterinary medicine, and evaluation of inflammatory response (Nazifi et al., 2009). Haptoglobin is an 2globulin synthesized by the liver during APR, with IL 6 inducing synthesis of Hp within hepatocytes (Yoshioka et al., 2002). Haptoglobin strongly binds hemoglobin (Hb; Gruys et al., 2005) acting as a scavenger of free Hb in blood and assisting in an anti oxidant role of Fe stabilization, thereby reducing oxidative effects on albumin, lipids, and kidneys (Ceciliani et al., 2012). Additionally, Hp has anti inflammatory capabilities and binds to CD11b/CD18 integrines on cell membranes of leukocytes (Gruys et al., 2005), in addition to binding of Hp Hb complex to CD163 of monocytes, thus increasing upregulation of anti inflamma tory mediators (Ceciliani et al., 2012). In cattle, circulating concentrations of Hp are negligible during nonpathogenic conditions, however an increase of more than a 100fold may be observed during an APR (Cooke and Arthington, 2013). Eckersall and Bel l (2010) observed Hp serum concentrations in

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39 cattle without display of pathological conditions to be < 20 mg L1 with potential to increase 1000fold within 2 d of infection, however Tourlomoussis et al. (2004) observed mean concentrations of Hp in plasma of beef cattle without pathological conditions to be 0.11 0.08 mg mL1, and those displaying pathological conditions had increased concentrations of Hp in plasma, 0.27 0.40 mg mL1. In addition to Hp being used effectively in the diagnosis and prognos is of mastitis (Akerstedt et al., 2008), respiratory disease (Yoshioka et al., 2002), and endometritis (Eckersall and Bell, 2010), elevated concentrations of Hp have also been reported in cows at parturition (Trevisi et al., 2012), during transport stress (Lomborg et al., 2008), and exposure to stressful management practices (Cooke and Arthington, 2013). Haptoglobin is therefore an indicator of stress, in addition to APR, through potential activation of the hypothalamus pituitary adrenal axis resulting in increased production of glucocorticoids and subsequent induction of hepatic APP synthesis (Lomborg et al., 2008). Ceruloplasmin : Ceruloplasmin is a positive APP, indicating an increase in synthesis and secretion is observed during an APR (Nazifi et al., 2009), although less commonly used as a diagnostic marker of APR compared with Hp. A metalloenzyme with oxidase activity, Cp is associated with Fe and Cu metabolism (Blakely and Hamilton, 1985), and similar to Hp, Cp scavenges for free Hb in blood, thereby r educing Fe availability f or bacterial growth (Weinberg, 1984). Additionally, Cp has been used to evaluate Cu deficiency in cattle as Cp transports approximately 90 to 95% of serum Cu, an essential nutrient (Carroll and Forsberg, 2007). Arthington et al. ( 1996) found that inducing Cu deficiency with molybdenum supplementation, resulted in decreased concentrations of Cp, compared with heifers without Cu deficiency. Dietary sulfur and molybdenum are known to induce Cudeficiency in cattle, through

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40 decreasing absorption of Cu (Carroll and Forsberg, 2007). When steers were provided increasing levels of metabolizable protein (MP) following vaccination (Moriel and Arthington, 2013), decreased concentrations of ceruloplasmin were observed for the greatest level of supplementation (115% MP), compared with 85 and 100% MP, from d 10 to the end of the study (d 29). This decrease in concentration of ceruloplasmin, approximately 10 to 16 mg dL1 in 115% MP, compared with approximately 16 to 24 mg dL1 in 85 and 100% MP tr eatments, appears to be related to the increased content of molybdenum and sulfur, however this is speculative. Trevisi et al. (2012) observed an increase in concentrations of IL 6 which subsequently led to an increase in concentrations of ceruloplasmin in response to dystocic calving, placental retention, ketosis, fever, diarrhea, mastitis, delayed uterine involution, swelling of joints and lameness both pre/post calving. This differed from concentrations of Hp, which were slightly elevated before calving and markedly increased post calving, indicating that Cp may be a more effective tool in evaluating some responses compared with Hp. Further, increase in positive APP, Cp, was at the expense of negative APPs, albumin, lipoproteins and bilirubin, indicating the use of Cp to asses liver function during an APR (Trevisi et al., 2012). Concentrations of Cp in dairy cows exhibiting low liver function ranged from 3 to 3.5 mol L1, compared with dairy cows exhibiting high liver function, 2.5 to 3 mol L1, with an increase in concentrations occurring around calving.

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41 Table 21. Summary of nutrient requirements of beef cattle1. Description SBW (kg) ADG ( kg d 1 ) DMI ( kg d 1 ) TDN (% DM) CP (% DM) RDP (% CP) MP ( g d 1 ) Growing and finis hing cattle (550 kg at finishing) 250 1.04 5.93 70 14.2 48.1 607 1.17 5.72 75 15.7 46.3 652 1.25 5.42 80 17.2 45.0 680 Peak Milk ( kg d 1 ) Lactating cow, 90 d post calving 550 8 12.2 60 10.0 55.5 376 12.8 65 11.7 50.8 13.5 70 13.5 4 7.5 1 Nutrient Requirements of Beef Cattle, 2016.

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42 Figure 21. Breakdown products of a general glucosinolate structure under the action of myrosinase.

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43 CHAPTER 3 EVALUATION OF BRASSICA CARINATA MEAL ON RUMINAL METABOLIS M AND NUTRIENT DIGESTIBILITY OF BEEF CATTLE I ntroduction Brassica carinata is a nonfood oilseed crop with a favorable very long chain fatty acid composition for conversion to biofuel (Marillia et al., 2013). Oil extracted from the seed has been utilized a s a 100% dropin jet biofuel, promoting the use of B. carinata as a renewable and potentially sustainable resource (AAFC, 2015). In the southeastern U.S., B. carinata would be an ideal candidate for use in crop rotation and cover crop due to its heat and d rought tolerance, and cold and disease resistance (AAFC, 2015; Seepaul et al., 2016). A high protein meal (~40% CP) is obtained as a byproduct of oil extraction; however, this meal has not been extensively tested as a potential protein supplement for cattl e. As the southeastern U.S. is typically comprised of cow calf operations cattle often graze pastures of limited nutritive value which are not adequate to support high levels of production, especially during critical periods, necessitating supplementation of protein ( Hersom et al., 2011; McBride and Matthews, 2011). Common protein supplements in this region result from byproducts of various industries and in conjunction with the poor quality hay available in winter, provide an opportunity to meet the nutri tional requirements of growing cattle (Schulmeister et al., 2015) Brassica carinata meal has been evaluated as a high quality source of crude protein for ruminants utilizing an in situ procedure (Xin and Yu, 2014) however research in feeding B. carinata to cattle is limited Thus, the objective of this study was to evaluate the effects of supplementation with B. carinata meal in comparison with common protein supplements on ruminal fermentation parameters, metabolism, and blood profile in Angus crossbred steers consuming bahiagrass hay.

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44 M aterials and Methods All procedures involving animals were approved by the Animal Care and Use Committee of the Institute of Food and Agricultural Sciences at the University of Florida, study # 201308011. E xperimental d e sign and s ample c ollection The experiment was conducted at the University of Florida, Feed Efficiency Facility (FEF) in Marianna, FL, beginning in October, 2014. Eight ruminally cannulated Angus crossbred steers (473 119 kg of initial BW) were used in a duplicated 4 4 L atin square design conducted over four consecutive 28d periods. Steers were randomly allocated to 8 pens, and within each period steers were randomly assigned to one of four treatments: 1.39 kg d1 B. carinata meal pellets (BCM), 1.62 kg d1 cottonseed meal (CSM), 2.15 kg d1 dry distillers grain plus solubles (DDGS), or 1.17 kg d1 soybean meal (SBM), supplemented daily. Treatments were calculated to be isonitrogenous based on total N provided by supplementation of 1.39 kg d1 of BCM. On d 0, steers were shrunk weighed (after 16 h of feed and water withdrawal) and housed individually in pens at the FEF with ad libitum access to water and bahiagrass hay ( Paspalum notatum ). Each pen at the FEF was equipped with 2 GrowSafe feed bunks (GrowSa fe System Ltd., Airdrie, Alberta, Canada) to record individual hay intake by weight change measured to the nearest gram. Steers were acclimated to the facility, hay, and supplements from d 0 to 14, and d 14 through 18 consisted of a digestibility period in which hay and fecal samples were collected twice daily for four d each. Day 19 involved a 24 h collection of ruminal fluid, blood, and ruminal pH, every 3 h. Day 28 was the final and initial day of a period in which BW was measured, and the adaptation per iod began for the next supplement. Ruminal fluid and blood samples were collected before feeding (0 h) and every 3 h post feeding for 24 h. Ruminal fluid was strained from a representative sample of digesta through 4 layers of cheesecloth and pH was immed iately measured using a manual pH meter (Corning

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45 Pinnacle M530, Corning Inc., Corning, NY). A 10 mL sample was taken and 0.1 mL of a 20% (vol/vol) H2SO4 solution was added to stop fermentation. Ruminal fluid samples were stored at 20C for further analysi s. Blood samples were collected from jugular venipuncture in 10mL evacuated tubes containing sodium heparin, placed on ice following collection, and centrifuged for 15 min at 4,000 g at 4 C. After centrifugation, plasma was transferred into polypropyle ne vials (12 mm 75 mm; Fisherbrand; Thermo Fisher Scientific Inc., Waltham, MA) and stored at 20 C for further analysis. Beginning on d 14 and d 15, feed and fecal samples were collected, respectively, for 4 consecutive days to determine apparent total tract digestibility of DM, OM, CP, NDF, and ADF. Feed samples were collected twice daily immediatel y after delivery of protein supplement and stored a t 20C. Fecal samples were collected twice daily at 0800 h and 1600 h from the ground, inside the pen, immediately after the animal defecated. After collection, fecal samples were stored at 20C. At the end of each period, hay and fecal samples were thawed and dried at 55 C for 48 h in a forced air oven, ground in a Wiley mill (Arthur H. Thomas Co., Philadelphia, PA) to pass a 2 mm screen, and pooled within steer for further determination of nutrient cont ent and digestibility marker concentration. Indigestible NDF (iNDF) was used as an internal indigestible marker ( Cole et al., 2011; Krizsan and Huhtanen, 2013) L aboratory analyses Supplement subsamples were weighed (0.5 g) in duplicate, dried in a forcedair oven at 100C overnight to calculate DM, and subsequently ashed in a muffle furnace at 650C for 6 h to calculate OM. To determine NDF concentration, samples were weighed (0.5 g) in duplicate in F57 filter bags and analyzed in an Ankom 200 Fiber Analyzer (Ankom Technology Corp., Macedon, NY) using sodium sulf i te and heat stable amylase. Samples were subsequently

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46 analyzed for ADF concentration as described by van Soest et al. (1991). Concentrations of CP 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) Protein supplements and bahiagrass hay were analyzed for nutrient composition by a commercial laboratory (Dairy One Forage Laboratory, Ithaca, NY.). Concentrations of VFA in ruminal fluid samples was determined in a wate r based solution using ethyl acetate extraction ( Ruiz Moreno et al., 2015). Samples were centrifuged for 10 min at 10,000 g. Ruminal fluid supernatant was mix ed with a meta phosphoric acid: crotonic acid (internal standard) solution at a 5:1 ratio and sam ples were frozen overnight, thawed and centrifuged for 10 min at 10,000 g. Supernatant was transferred into glass tubes and mixed with ethyl acetate in a 2:1 ratio of ethyl acetate to supernatant. After shaking tubes vigorously, the ethyl acetate fractio n (top layer) was transferred to vials. 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 CP7767, Varian Analyti cal Instruments, Walnut Creek, CA ). Column temperature was maintained at 110C, and injector and detector temperatures were 200 and 220 C, respectively. Concentration of NH3N was measured after centrifuging ruminal fluid samples at 10,000 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 (1 980) with the following modification: absorbance was read at 620 nm in flat bottom 96well plates using a plate reader (DU 500, Beckman Coulter Inc.). Plasma was analyzed for concentration of BUN using a quantitative colorimetric kit (B7551 120; Pointe Sci entific Inc., Canton, MI ). Plasma was

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47 analyzed for glucose using a quantitative colorimetric kit (G75211L; Pointe Scientific Inc., Canton, MI). Concentrations of iNDF in hay and feces were determined as described by Cole et al. (2011) with modifications proposed by Krizsan and Huhtanen (2013) Briefly, samples were weighed (0.5 g) into Ankom F57 filt er bags, and then incubated at 39C using a 4:1 ratio of McDougalls buffer:ruminal fluid in a DaisyII incubator (Ankom Technology Corp.) for 288 h to ensure complete digestion of potentially digestible NDF fraction After incubation, samples were rinsed a nd analyzed for NDF concentration as previously described. C alculations and s tatistical analysis Apparent total tract digestibility of DM, OM, CP, NDF, and ADF were calculated as follows: 100 100 Data were analyzed as a duplicated 4 4 Latin square design using the MI XED Procedure of SAS (SAS Institute Inc., Cary, NC). The model for intake and digestibility included fixed effects of treatment, square, and period, and the random effects of steer within square. Animal within period was the subject. Autoregressive was the best covariance structure based upon the smallest Akaike Information Criterion (AIC) values. Data repeated over time (ruminal pH, NH3N, VFA, and BUN) were analyzed as repeated measures using the MIXED p rocedure of SAS (SAS Inst. Inc.). The model included the fixed effects of treatment, time, the treatment x time interactions, square, and period; random effects included effects of steer within square, and steer within treatment, with animal as the experimental unit ( n = 8). Animal within period was the subject and the covariance structure used for all the parameters was unstructured, with the exception for total VFA, which was

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48 analyzed using compound symmetry. Unstructured and compound symmetry were the best covariance structures based upon the smallest AIC values. Differences between treatment means were identified by Tukeys least squares means comparison and significance was declared at P tendencies considered when 0.05 < P R esults and Discussion The chemical and nutrient composition of hay and protein supplements fed to steers is available in Table 3 1. Neither intake nor digestibility of nutrients was affected by protein supplementation, which was averaged over four periods (Table 3 2). Dry matter intake ( P = 0.49) for all treatments averaged between 6.2 and 7.2 kg d-1, with OMI ( P = 0.475) ranging from 5.8 to 6.7 kg d1. Intake of CP ( P = 0.47) was similar for all treatments ranging from 0.33 to 0.37 kg d1, confirming isonitrogenous supplementation of protein. Digestibility of DM ( P = 0.99) and OM ( P = 0.98) was similar across treatments with averaged values of approximately 51 and 53% for DM and OM, respectively. Bahiagrass ( Paspalum notatum ) is a common perennial grass grown in Florida and is often utilized for grazing beef cattle or production of hay (Chambliss and Sollenberger, 1991); however, bahiagrass hay is of poor quality and often requires additional supplementation (Moore et al., 1991). The nutritive value of the bahiagrass hay fed to steers in this study was poor as illu strated by the digestible organic matter ( DOM ) or TDN:CP ratio, however supplementation of protein would be expected to increase intake, thus potentially explaining the lack of differences observed in intake (Moore et al., 1995). An effect of treatment ( P = 0.65; Table 3 3) was not observed for ruminal pH, with averaged values ranging from 6.61 to 6.67, indicating a favorable ruminal environment for cellulolytic microorganisms activity (Russell and Wilson, 1996). An effect of time post feeding of supplement ation ( P < 0.001; Figure 31) was observed

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49 for pH however there was no treatment time interaction ( P = 0.37). Steers were provided ad libitum access to bahiagrass hay and water, and as a result the initial pH was greater at the 0 h, however upon consumption of the protein supplements, a decrease in pH was observed between 3 h and 9 h, stabilizing through 18 h and then increasing through 24 h. We speculate that the protein supplements decreased the pH partially due to other constituents of the supplements such as nonfiber carbohydrates (NFC) and the tendency of the steers to consume the protein supplements immediately upon arrival. The stabilization of pH may be related to an achieved balance after supplements were consumed, followed by an increase in p H due to consumption of bahiagrass hay alone coupled with rumination and subsequent buffering effects. Concentrations of ruminal NH3N ( P = 0.37) were not affected by treatments, however an effect of time ( P < 0.001; Figure 31) was observed, yet no treat ment time interaction ( P = 0.60) detected. Concentration of ruminal NH3N peaked at 3 h and steadily declined through 18 h, stabilizing between 21 and 24 h. Conversely to pH, the concentration of ruminal NH3N increased after consumption of protein suppl ements, which was expected and indicative of microbial degradation of provisional protein. Interestingly, Figure 31 illustrates the balance between pH and protein supplementation, i.e., upon consumption of protein supplements pH decreases and concentrati on of NH3N increases, yet as fermentation ensues a stability is reached until, presumably, protein has been either completely degraded or removed from the rumen. Concentrations of ruminal NH3N, ranging from 2.27 to 3.18 m M are lower than the value of 3.57 m M often quoted in reference to Satter and Slyter (1974) to maximize microbial protein synthesis H owever, Satter and Slyter (1974) suggest that the precise limiting concentration is perhaps closer to 20 mg NH3N/ L [1.43 m M ] but use of the higher value gives a slight margin of excess. Further more dietary requirements vary with age, stage of production, and size of cattle

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50 (NRC, 2016); however, despite apparent ly low concentrations of ruminal NH3N, th e values observed i n this study are within the sugg ested values to maximize microbial crude protein synthesis. An effect of treatment ( P < 0.001) was observed for concentrations of BUN, with steers supplemented with SBM having the greatest and DDGS having the least concentrations (10.86 and 6.85 mg dL1, respectively). Concentrations of r uminal NH3N and BUN are highly correlated and indicative of the energy to protein ratio in healthy cattle (Hammond, 1992). Supplementation of protein in s teers grazing bahiagrass and limpograss pastures resulted in concen trations of BUN between 9 and 12 mg dL1, thus indicating a transition range in which responding to protein supplementation, ADG was greater in steers with values below, and lesser in values above that range (Hammond, 1997). An increase in concentrations of BUN in steers supplemented with SBM in the current study may be a result of poor synchronization of energy and protein when feeding a low quality forage and a protein source rea dily degradable in the rumen. H owever, BCM and DDGS values are within the ra nge of 7 to 8 mg dL1 suggested by Preston et al. (1978) for finishing steers, and are therefore potentially more favorable in terms of ADG and decreased N loss. A tendency for a time effect ( P = 0.085) and treatment time interaction ( P = 0.085) was obse rved Concentrations of plasma glucose ( P = 0.37) were not different between treatments, however an effect of time ( P < 0.001; Figure 32) was observed, with no treatment time interaction ( P = 0.99) detected. Plasma glucose concentrations are tightly regulated, however an increase may result following a high carbohydrate meal or endogenous synthesis of glucose in the liver (Dukes et al., 1993). Moreover glucose is not readily absorbed, thus as NH3N decreases, an increase in glucose and subsequently ins ulin is observed (van Soest, 1994).

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51 Therefore, the increase in plasma glucose at 21 h may be related to the time effects observed in NH3N, in which gluconeogenesis within the liver is induced by precursors resulting from ruminal fermentation and digestion of the diet and subsequently detected in plasma (Reynolds, 2005). Treatment time interactions ( P < 0.001) were observed for molar proportions of acetate ( Table 34; Figure 33), propionate ( Figure 34), and butyrate ( Figure 3 5). Molar proportions of acetate were similar for all treatments ( P > 0.10) however a decrease at 3 h post feeding was observed in steers consuming DDGS, followed by a gradual increase until 12 h in which no differences were detected between DDGS, SBM, and BCM. M olar proportions of acetate increased in steers supp lemented with CSM at 6 h differ ing from all treatments except for 18 and 24 h. Consequently, an increase in molar proportions of propionate and butyrate at 3 h post feeding was observed for DDGS supplementation, compared wi th the remaining protein supplements. Molar proportions of propionate gradually declined in DDGS until 12 h, in which no further differences were detected compared with SBM and BCM. Molar proportions of propionate fluctuated throughout the 24 h observation with regards to CSM; nonetheless, molar proportions of propionate were decreased for CSM compared with other supplements. Despite the effect of supplementation over time, the increase in molar proportions of propionate did not affect the concentration of plasma glucose ( i.e., there was not an effect of treatment ) As previously stated endogenous secretions of glucose from the liver result from ruminal activity, with gluconeogenic precursors including propionate, amino acids glycerol, and lactate (Dukes et al., 1993); therefore, the increase in plasma glucose may be related to one of the other precursors, however this was not evaluated. Molar proportions of butyrate were similar to propionate with an increase observed in DDGS at 3 h and a gradual decline through 15 h in which no further differences were detected in treatments. Molar proportions of branchedchain

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52 volatile fatty acid (BCVFA) were greater ( P < 0.001) in SBM compared with CSM and DDGS, and gradually decreased post feeding ( P = 0.004; Figure 36) for all treatments until 12 h, however a treatment time interaction ( P = 0.30) was not observed. Production of BCVFA results from fermentation of branchedchain amino acid (BCAA), which are either used for AA resynthesis, or as growth factors for othe r microbial species (Allison, 1978); however, BCVFA production is mediated through the availability of glucose, depending upon microbial species. Thus, an increase in concentration of BCVFA in steers supplemented with SBM and BCM may indicate a greater availability of BCAA within the rumen. No treatment ( P = 0.39), time ( P = 0.55), or treatment time interaction ( P = 0.15) was observed for molar proportions of caproate. A treatment time interaction ( P < 0.001; Figure 3 7) was observed for molar proporti ons of valerate, with a peak at 3 h, and no further differences detected. Concentrations of total VF A was not affected by treatment ( P = 0.93), nor was a treatment time interaction ( P = 0.30) observed; however, concentrations of total VFA differed from 9 h compared with 18 and 24 h ( P < 0.001; Figure 38). Absorption rates of individual VFAs vary with concentrations of VFA s or changes in ruminal pH (Dijkstra, 1993), therefore the differences observed in concentration of total VFA may be related to the fluctuations observed in ruminal pH and subsequent absorption of VFA. Despite difference s observed in time post feeding concentration of total VFA did not differ between treatments indicating similar fermentation, and as the fermentation rate of feed is posi tively associated with microbial efficiency (van Soest, 1994), BCM performed similarly to commonly provided protein supplements. A treatment time interaction ( P < 0.001; Figure 39) was observed for A:P, which further reflects the relationship of DDGS an d CSM with regards to production of acetate and propionate.

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53 C onclusion Brassica carinata is not a new crop, however the residual meal remaining after oil extraction has not been extensively tested as a protein supplement for cattle. T he effects of suppleme nting B. carinata meal on ruminal fermentation parameters and blood profile in Angus crossbred steers were similar when compared with commonly used protein supplements, thus indicating its viability as a protein supplement for beef cattle.

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54 Table 3 1. An alyzed1 chemical and nutrient composition (DM basis) of hay and protein s upplements fed to ruminally cannulated Angus crossbred steers. Treatment 2 Item Bahiagrass hay 3 BCM CSM DDGS SBM DM, % 94.0 89.8 88.9 86.3 90.7 CP, % 7.2 43.3 49.2 32.8 52.9 NFC 4 % -5 21.7 13.2 20.2 28.7 NDF, % 71.4 23.5 28.6 30.7 10.2 ADF, % 41.8 12.8 18.7 14.3 8.4 TDN, % 56 80 67 83 79 S, % 0.35 1.75 ---1 Dairy One Forage Testing Laboratory, Ithaca, NY. 2BCM: Brassica carinata meal pellets (1.39 kg d1); CSM: co ttonseed meal ( 1.62 kg d1); D DGS: dry distillers grain plus solubles ( 2.15 kg d1); SBM: soybean meal ( 1.17 kg d1); composited over 4 periods. 3Bahiagrass hay ( Paspalum notatum ). 4NFC = nonfiber carbohydrates. 5-Indicates this item was not analyzed.

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55 Table 3 2. Effects of protein supplementation on nutrient intake and apparent total tract digestibility1 of ruminally cannulated Angus crossbred steers fed bahiagrass hay ad libitum with iNDF utilized as an internal marker. Treatment 2 P value 3 It em BCM CSM DDGS SBM SEM 4 TRT Intake, kg/d DM 6.83 6.87 6.23 7.19 0.771 0.49 OM 6.37 6.42 5.79 6.73 0.727 0.48 CP 0.37 0.37 0.33 0.37 0.053 0.47 NDF 5.08 5.06 4.53 5.27 0.555 0.48 ADF 2.54 2.51 2.28 2.63 0.246 0.46 Digestibility, % DM 51 .52 51.11 50.90 51.37 1.968 0.99 OM 53.59 53.22 52.74 53.42 1.939 0.98 CP 67.10 69.03 63.64 65.53 4.934 0.39 NDF 50.03 48.69 49.57 47.45 2.464 0.65 ADF 51.24 50.92 52.67 49.24 3.403 0.45 1 Hay and fecal samples were collected twice daily for 4 d; intak e of bahiagrass hay was measured using the GrowSafe System Ltd., Airdrie, Alberta, Canada. 2BCM: Brassica carinata meal pellets (1.39 kg d1); CSM: cottonseed meal (1.62 kg d1); DDGS: dry distillers grain plus solubles (2.15 kg d1); SBM: soybean meal (1. 17 kg d1); composited over 4 periods. 3Observed significance levels for treatment (TRT). 4Pooled standard error of treatment means, n = 8 steers/treatment.

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56 Table 3 3. Effects of protein supplementation on ruminal fermentation parameters and blood profile of ruminally cannulated Angus crossbred steers fed bahiagrass hay ad libitum. Treatment 1 P value 2 Item BCM CSM DDGS SBM SEM3 TRT TIME TRT TIME Ruminal pH 4 6.6 3 6.63 6.61 6.67 0.044 0.65 < 0. 00 1 0.3 8 NH 3 N 4 m M 2. 27 2.75 2.37 3.18 0.458 0.3 8 < 0.0 0 1 0.60 Glucose 4 m M 3. 6 7 3.63 3.63 3.72 0.04 5 0.37 < 0.0 0 1 0.99 BUN 4 mg/dL 8.87 b 9.21 b 6.85 c 10.86 a 0.532 < 0.001 0.085 0.085 a,b,c Within a row, means with different superscripts differ, P < 0.05 1BCM: Brassica carinata meal pellets (1.39 kg d1); CSM: cottonseed meal (1.62 kg d1); DDGS: dry distillers grain plus solubles (2.15 kg d1); SBM: soybean meal (1.17 kg d1); composited over 4 periods. 2Observed significance levels for treatment (TRT) and time postfeeding ( TIME ) effects, and fo r their interaction (TRT TIME). 3Pooled standard error of treatment means, n = 8 steers/treatment. 4Ruminal fluid and blood samples were collected every 3 h for 24 h; NH3N = ruminal ammonia nitrogen, glucose = plasma glucose, BUN = plasma urea nitrogen.

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57 Figure 31. Effects of protein supplementation post feeding in ruminal pH and concentrations of ruminal ammonia nitrogen ( P < 0.0001) in ruminally cannulated Angus crossbred steers fed bahiagrass hay ad libitum. 0 1 2 3 4 5 6 6.3 6.4 6.5 6.6 6.7 6.8 6.9 0 3 6 9 12 15 18 21 24NH3N, m MRuminal pHTime post feeding, h Ruminal pH NH3-N

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58 Figure 32. Effects of protei n supplementation post feeding in concentrations of plasma glucose ( P < 0.0001) in ruminally cannulated Angus crossbred steers fed bahiagrass hay ad libitum. 3 3.2 3.4 3.6 3.8 4 4.2 0 3 6 9 12 15 18 21 24Glucose, m MTime post feeding, h

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59 Table 3 4. Effects of protein supplementation on proportions of VFA (mol 100 mol1), total VFA concentrations (m M ) and acetate to propionate ratio (A : P ) in ruminally cannulated Angus crossbred steers fed bahiagrass hay ad libitum. Treatment 1 P value 2 Item BCM CSM DDGS SBM SEM 3 TRT TIME TRT TIME Acetate 74. 18 75.4 0 72.39 74. 25 0.3 16 < 0.0 0 1 < 0. 0 01 < 0. 0 01 Propionate 15. 75 15.0 3 16.2 3 15. 37 0.2 05 < 0.0 0 1 < 0. 0 01 < 0. 0 01 Butyrate 7 95 7. 57 9. 47 8.0 1 0.14 4 < 0. 0 01 < 0. 0 01 < 0. 0 01 BCVFA 4 1.3 4 ab 1.2 0 b 1.1 4 b 1. 57 a 0.0 63 < 0. 0 01 0.0 04 0.30 Valerate 0.6 3 0.59 0.60 0.62 0.016 0. 20 < 0. 0 01 < 0. 0 01 Caproate 0.1 3 0.18 0.15 0.16 0.016 0. 40 0.55 0.15 Total VFA 98.3 1 99.9 0 9 8.72 9 4 89 5.8 54 0.93 0.0 3 0. 0 25 A:P 4. 71 5.0 3 4. 49 4.8 4 0.08 4 < 0.0 0 1 < 0.0 0 1 < 0. 0 01 a,b Within a row, means with different superscripts diff er, P < 0.05. 1BCM: Brassica carinata meal pellets (1.39 kg d1); CSM: cottonseed meal (1.62 kg d1); DDGS: dry distillers grain plus solubles (2.15 kg d1); SBM: soybean meal (1.17 kg d1); composited over 4 periods. 2Observed significance levels for trea tment (TRT) and time effects, and for their interaction (TRT TIME). 3Pooled standard error of treatment means, n = 8 steers / treatment. 4 BCVFA = Branched chain volatile fatty acids: isobutyrate + isovalerate + 2 methylbutyrate.

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60 Figure 33. Effects of protein supplementation post feeding on molar proportions of acetate (mol 100 mol1) in ruminally cannulated Angus crossbred steers fed bahiagrass hay ad libitum. Treatment time interaction observed ( P < 0.0001). BCM: Brassica carinata meal pellets (1 .39 kg d1); CSM: cottonseed meal (1.62 kg d1); DDGS: dry distillers grain plus solubles (2.15 kg d1); SBM: soybean meal (1.17 kg d1). 62 64 66 68 70 72 74 76 78 0 3 6 9 12 15 18 21 24Acetate, mol 100 mol1Time post feeding, h BCM CSM DDGS SBM

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61 Figure 34. Effects of protein supplementation post feeding on molar proportions of propionate (mol 100 mol1) in ruminally cannulated Angus crossbred steers fed bahiagrass hay ad libitum. Treatment time interaction observed ( P < 0.0001). BCM: Brassica carinata meal pellets (1.39 kg d1); CSM: cottonseed meal (1.62 kg d1); DDGS: dry distillers grain plus solubl es (2.15 kg d1); SBM: soybean meal (1.17 kg d1). 13 14 15 16 17 18 19 0 3 6 9 12 15 18 21 24Propionate mol 100 mol1Time post feeding, h BCM CSM DDGS SBM

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62 Figure 35. Effects of protein supplementation post feeding on mol ar proportions of butyrate (mol 100 mol1) in ruminally cannulated Angus crossbred steers fed bahiagrass hay ad libitum. Treatment t ime interaction observed ( P < 0.0001). BCM: Brassica carinata meal pellets (1.39 kg d1); CSM: cottonseed meal (1.62 kg d1); DDGS: dry distillers grain plus solubles (2.15 kg d1); SBM: soybean meal (1.17 kg d1). 4 6 8 10 12 14 16 0 3 6 9 12 15 18 21 24Butyrate mol 100 mol1Time post feeding, h BCM CSM DDGS SBM

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63 Figure 36. Effects of protein s upplementation post feeding on molar proportions of BCVFA ( P = 0.004) in ruminally cannulated Angus crossbred steers fed bahiagrass hay ad libitum. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 0 3 6 9 12 15 18 21 24BCVFA mol 100 mol1Time post feeding, h

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64 Figure 37. Effects of protein supplementation post feeding on mol ar proportions of valerate (mol 100 mol1) in ruminally cannulated Angus crossbred steers fed bahiagrass hay ad libitum. Treatment time interaction observed ( P < 0.0001). BCM: Brassica carinata meal pellets (1.39 kg d1); CSM: cottonseed meal (1.62 kg d1); DDGS: dry distillers grain plus solubles (2.15 kg d1); SBM: soybean meal (1.17 kg d1). 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 0 3 6 9 12 15 18 21 24Valerate, mol 100 mol1Time post feeding, h BCM CSM DDGS SBM

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65 Figure 38. Effects of protein supplementation post feeding on concentrations of total VFA ( P = 0.025) in ruminally cannulated Angus crossbred steers fed bahiagrass hay ad libitum. 0 20 40 60 80 100 120 0 3 6 9 12 15 18 21 24Total VFA, m MTime post feeding, h

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66 Figure 39. Effects of protein supplementation post feeding on acetate to propionate ratio in ruminally cannulated Angus crossbred steers fed bahiagrass hay ad libitum. Treatment time interaction observed ( P < 0.0001). BCM: Brassica carinata meal pelle ts (1.39 kg d1); CSM: cottonseed meal (1.62 kg d1); DDGS: dry distillers grain plus solubles (2.15 kg d1); SBM: soybean meal (1.17 kg d1). 3 3.5 4 4.5 5 5.5 0 3 6 9 12 15 18 21 24A:PTime post feeding, h BCM CSM DDGS SBM

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67 CHAPTER 4 CHARACTERIZATION OF THE DIETARY PROTEIN IN BRASSICA CARINATA I ntroduction Brassica carinata is a nonfood oilseed crop with a favorable very long chain fatty acid composition for conversion to biofuel (Marillia et al., 2013). Oil extracted from the seed has been utilized as a 100% dropin jet biofuel, promoting the use of B. carinata as a renewable and potentially sustainable resource (AAFC, 2015). In the southeastern U.S., B. carinata would be an ideal candidate for use in crop rotation and cover crop due to its heat and drought tolerance, and cold and disease resistance (AAFC, 2015; Seepaul et al., 2016). A high protein meal (~40% CP) is obtained as a byproduct of oil extraction; however, this meal has not been extensively tested as a potential protein supplement for cattle. As the southeastern U.S. is typically comprised of cow calf operations (McBride and Matthews, 2011), cattle of various ages, stages of production and size often graze medium to poor quality pastures with limited protein content, thus high quality protein supplementation is necessary. Brassica carinata meal has been evaluated as a high quality source of crude protein for dry Holstein cows (Xin and Yu, 2014), when compared with canola meal. However, to our knowledge, B. carinata has not been evaluated compared with commonly used protein supplements in the southeastern U.S., or in beef ca ttle. Thus, the objective of this experiment was to characterize ruminal protein fractionation, and subsequent post ruminal degradation of protein in B. carinata compared with common protein supplements, and to determine the amino acid profile of B. carinata upon ruminal and post ruminal degradation.

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68 M aterials and Methods All procedures involving animals were approved by the Animal Care and Use Committee of the Institute of Food and Agricultural Sciences at the University of Florida, study # 201308011. E xperimental d esign and s ample c ollection The experiment was conducted at the University of Florida, Feed Efficiency Facility (FEF) in Marianna, FL, beginning in October, 2014. Eight ruminally cannulated Angus crossbred steers (473 119 kg of initial BW) we re used in a duplicated 4 4 Latin square design conducted over four consecutive 28d periods. Steers were randomly allocated to 8 pens, and within each period steers were randomly assigned to one of four treatments: 1.39 kg d1 B. carinata meal pellets ( BCM), 1.62 kg d1 cottonseed meal (CSM), 2.15 kg d1 dry distillers grains plus solubles (DDGS), or 1.17 kg d1 soybean meal (SBM), supplemented daily. Treatments were calculated to be isonitrogenous based on total N provided by supplementation of 1.39 kg d1 of BCM. On d 0, steers were shrunk weighed and housed individually in pens at the FEF with ad libitum access to water and bahiagrass hay ( Paspalum notatum ). Each pen at the FEF was equipped with two GrowSafe feed bunks (GrowSafe System Ltd., Airdrie, A lberta, Canada) to record hay intake by weight change measured to the nearest gram. Steers were acclimated to the facility, hay, and supplements from d 0 to d 14, and a ruminal in situ degradability procedure was conducted from d 21 to d 25, in which bags were placed in the rumen of supplement specific adapted steers for 0, 3, 6, 9, 12, 16, 24, 48, 72, and 96 h. Following ruminal incubation, the undegraded supplement after 16 h incubation was subjected to serial solutions simulating post ruminal digestion ( Calsamiglia and Stern, 1995; Gargallo et al. 2006), with subsequent analysis of concentration of CP and determination of the BCM AA profile. Ruminal in situ DM disappearance of treatments was determined using duplicate bags within steer. Supplement sample s were taken at the beginning of each period, dried for 48 h at

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69 55C, and weighed (5 g) into 10 20 cm Ankom in situ bags (R1020, Ankom Technology Corp., Macedon mg/cm2. In situ bags were heat sealed, placed in mesh laundry bags fitted with a zipper, and suspended in the ventral sac of the rumen from a nylon rope and carabiner attache d to a U bolt on the stopper of the cannula after soaking in warm (39C) water for 15 min. Bags were placed in the rumen altogether and incubated for 0, 3, 6, 9, 12, 16, 24, 48, 72, and 96 h, except for the 0 h bag, which was soaked in 39C water for 15 mi n to determine the A fraction of protein. Bags were removed at predetermined times, as previously mentioned, rinsed with cold running water to remove adherent particles and bacteria, and then rinsed with tap water 3 times and distilled water 5 times. Bag s were dried for 48 h at 55C and weighed. Residues from the in situ incubation were composited by incubation time within steer and composite samples were analyzed for determination of DM, OM, CP, NDF, and ADF. The 16 h bag was removed and analyzed separat ely to determine intestinally absorbable CP by the three step procedure (Calsamiglia and Stern, 1995; Gargallo et al. 2006). L aboratory analyses Samples were weighed (0.5 g) in duplicate, dried in a forcedair oven at 100C overnight to calculate DM, and subsequently ashed in a muffle furnace at 650C for 6 h to determine OM. To determine NDF concentration, samples were weighed (0.5 g) in duplicate in F57 filter bags and analyzed in an Ankom 200 Fiber Analyzer (Ankom Technology Corp., Macedon, NY) using s odium sulfate and heat stable amylase. Samples were subsequently analyzed for concentrations of ADF as described by van Soest et al. (1991). Concentrations of CP in feed and feces was determined by rapid combustion using a macro elemental N analyzer (Var io Max CN, Elementar Americas Inc., Mt. Laurel, NJ) following official method 992.15 (AOAC, 1995)

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70 Brassica carinata meal pellets and bahiagrass hay were analyzed for nutrient composition by a commercial laboratory (Dairy One Forage Laboratory, Ithaca, NY.). Determina tion of intestinally absorbable CP was analyzed according to the three step procedure (Calsamiglia and Stern, 1995; Gargallo et al. 2006), with modifications. Briefly, the 16 h bag was removed from the rumen, washed until runoff was clear, and dried in a f orced air oven at 55C for 48 h. Contents of ruminal in situ residue bags were composited and analyzed for DM, OM, CP, NDF, ADF, and AA profile ( University of Missouri, Experiment Station Chemical Laboratories, Columbia, MO ). Contents were then weighed int o 5 10 cm nylon bags (Ankom R510, pore size 50 m; Ankom, Fairport, NY; Boucher et al., 2009) in duplicate, heat sealed and suspended in a DaisyII incubator (Ankom, Fairport, NY) in a 2 L solution of pre warmed 0.1 N HCl solution (pH 1.8) containing 1 g L1 of pepsin (P 3000, Sigma, St. Louis, MO) at 39C for 1 h, under constant rotation. Nylon bags were removed from the incubator, rinsed with tap water until runoff was clear, and then further incubated in a 2 L pre warmed pancreatin solution (0.5 M KH2PO4 buffer, pH 7.7, containing 50 ppm of thymol and 3 g L1 of pancreatin; Sigma P 7545) for 24 h at 39C, under constant rotation. After incubation, bags were removed from solution, washed with tap water until runoff was clear, and dried in a forcedair ove n at 55C for 48 h. Contents from duplicate bags were composited, analyzed for DM and CP content (CP content was determined by rapid combustion using a macro elemental N analyzer; Vario Max CN, Elementar Americas Inc., Mt. Laurel, NJ), and sent for AA prof ile analysis ( University of Missouri, Experiment Station Chemical Laboratories, Columbia, MO ). C alculations and s tatistical analysis Residues from in situ incubations were fitted to a first order kinetic model according to rskov and McDonald (1979) using the nonlinear procedure of SAS (SAS Inst. Inc., Cary, NC). T he model used was:

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71 ( )= Undeg + D e ( ) Where R(t) = residue at each given incubation time (%); t = time incubated in the rumen (h); Undeg = undegradable fraction (%); D = potentially degradable fraction (%); e = 2.71828; Kd= degradation rate of D (% h1); and T0 = lag time (h). Effective rumen degradability (E) of DM, OM, NDF, and ADF was calculated according to the model: = + [ + ] Where, x = nutrient evaluated; SF = Soluble fraction, which is the proportion of material that washed out from the bags without rumen incubation (0 h); and Kp = fractional rate of passage, in this study assumed to be 5 % h1 (Foster et al., 2011). Effective rumen CP degradability representing RDP was determined by the equation (Mjoun et al., 2010): = + [ + ] Where, A = rapidly degradable CP that disappeared at 0 h after the rinsing procedure; B = potentially degradable CP; Kd and Kp are degradation constants described previously. Estimated RUP of feeds was calculated was determined as RUP intestinally digestible protein ( IDP ) T otal tract digestibility of CP was calculated as the sum of RDP and IADP. Contribution of RUP to intestinally absorbable AA ( disappearance in situ AA concentration in feed /10 (Mjoun et al., 2010). Pepsin pancreatin digestion (PPD) of protein was calculated using the model of Garga llo et al. (2006):

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72 = [ ( ) : ( ) ( ) ] Where: IS (N) = N content of the rumen exposed residue; P:P (N) = N content of the pepsinpancreatin residue; and S (N) = N content of the sample. In situ digestibility data were analyzed as a duplicated 4 4 Latin square using the MIXED procedure of SAS (SAS Inst. Inc.). The model included the fixed effects of treatment, square, period within square, and animal within square. Three step procedure data were analyzed using PROC MIXED of SAS (SAS Inst. Inc.). The model for protein characterization included fixed effects of treatment, and random effects of square, period, and steer within square. Differences between treatment means were identified by Tukeys least squares means comparison, significance was declared at P tendencies considered when 0.05 < P R esults and Discussion The chemical and nutrient composition of the hay and protein supplements provided to steers is available in Table 41. Concentrations of nutrient s for CSM SBM and DDGS were comparable with published values with exception to a slightly less DM in DDGS (NRC, 2016). Fractionation of ruminal protein differed ( P 42) between treatments with CSM and DDGS having nearly equivalent amounts of RDP (approximately 51% of CP), which differed from SBM and BCM having approximately 72% RDP as a percentage of CP. The RDP for CSM was lesser than published values reported in the NRC (2016) while the RDP for DDGS was greater than values reported in th e NRC (2016). Protein fractionation for SBM was similar to published values for RDP and RUP (NRC, 2016). Compared to DDGS, SBM had the greatest IDP ( P < 0.01), with CSM having the greatest IADP ( P < 0.01), and similar for BCM and SBM.

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73 T otal tract digestibility (TTDP; P < 0.01) of CP was greatest for SBM compared with CSM and DDGS. Metabolizable protein (MP) is defined as the true protein digested in the intestine, supplied by microbial protein and RUP (NRC, 2016). Though MP is the common nomenclature, TTDP has also been utilized in various studies; nonetheless, the concept is the same. The NRC (1996, 2001) assumed an 80 % digestibility of RUP as a result of insufficient information regarding digestibility; however, to accurately predict MP valid estimates a re necessary (NRC, 2016). Consequently, intestinal digestibility values for RUP (IDP) or MP are not available in the newest edition of the NRC (2016). Erasmus et al. (1994) observed an approximately 98% intestinal digestibility of RUP when SBM was fed to l actating dairy cows This value is similar to the IDP of 94.53 % observed in the current study for SBM but further illustrates the variability in digestibility of substrates R etention time of ruminal protein will affect estimates of RDP and RUP, i.e., a shorter retention time will result in an estimation of greater values for RUP and subsequent overestimates of MP (NRC, 2016). Estimates of RDP and RUP observed in the current study resulted from ruminal incubation for 16 h, considered to be the mean reside nce time of CP in the rumen ( Calsamiglia and Stern, 1995). Ruminal in situ degradation kinetics are presented in Table 43. The ruminal degradation rate s of DM, OM, and CP w ere greatest ( P for SBM The potentially degradable fraction of DM w as greater ( P for SBM and CSM compared with DDGS, despite a greater ( P soluble fraction of DM for DDGS compared with CSM. A delay ( P in lag time of DM was observed in CSM and SBM, compared with BCM M ore time was required ( P by CSM and SBM to degrade OM compared with DDGS despite a greater ( P = 0.02) undegradable fraction of OM in DDGS compared with SBM and a tendency

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74 ( P = 0.06) for the potentially degradable frac tion of OM to be increased in SBM While BCM was similar to other treatments in both lag time and the potentially degradable fraction of OM, BCM tended ( P = 0.07) to have a greater soluble fraction. Crude protein in BCM and SBM required less lag time ( P 0.01) than CSM and BCM had the greatest ( P soluble fraction; however, the potentially degradable fraction of CSM was greater ( P compared with BCM and DDGS. The undegradable fraction of DM ( P = 0.20 ) and CP ( P = 0.24) were not different bet ween tr eatments. Soybean meal is a more rapidly fermentable substrate in the rumen, as indicated by the increased degradation rate, despite greater lag times in DM and OM. As a protein supplement, SBM is often recommended as a source of RDP, with CSM and D DGS utilized as a source of RUP (Lee et al., 2016; NRC, 2016) supporting the data observed in the current study. Similar in proportions of RDP and RUP, BCM has a decreased rate of degradation compared to SBM, but a greater soluble fraction contributing to an increase in ruminally degradable protein. Determining the protein fractionation of supplements is important in formulating rations for cattle, however the availability of amino acids (AA) post ruminally is of greater interest as these will be availabl e as a portion of the MP (Merchen and Titgemeyer, 1992). The AA composition of BCM in the original feed sample, 16 h rumen sample, and post rumen residue is presented in Table 44. The total tract digestibility of individual AA, ruminally and post ruminall y, is presented in Table 45, with the contribution of RUP to intestinally absorbable AA (IAAA). Previous research on the fractionation and characterization of protein in B. carinata (Xin and Yu, 2014) resulted in values of RUP and TTDP (123 and 358 g/kg D M, respectively). Upon initial evaluation, these values may seem lesser than the current values, but the original concentration of CP in the diet used by Xin and Yu ( 2014) was not presented, dry Holstein

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75 ruminally cannulated cows were used, and a total mix ed ration (forage:concentrate = 78:22) was fed. The differences in experimental designs may be the main reason for the discrepancies in the values presented by Xin and Yu ( 2014) and those observed in this study Mjoun et al. (2010) compared fractionation of protein and subsequent AA profiles in distillers grains products in common soybean meal products, utilizing the in situ technique and the modified three step procedure described by Gargallo et al. (2006) The RDP and RUP values for SBM (67.7 and 32.3 % of CP) and DDGS (47.7 and 52.3 % of CP) in lactating Holstein cows reported by Mjoun et al. (2010) were similar to the estimates obtained in the current study Furthermore, the protein digestibility parameters (IDP, % of RUP; IADP and TTDP, % of CP) of SB M and DDGS were similar, confirming the values observed in the current study (Mjoun et al., 2010). The total tract digestibility of essential AA of B. carinata meal pellets and contribution of RUP to IAAA is presented in Table 46. As rumen microbes are able to synthesize all of the essential AAs (DMello, 2003), ruminants have no theoretical requirement for dietary pre formed protein or AA (Bach et al., 2005). P roduction of MCP alone, resulting from RDP, may be insufficient in supplying adequate amounts of AA for optimal production (Kung Jr. and Rode, 1996), especially during periods of rapid growth in cattle and high rates of production (Klopfenstein et al., 1978). Thus, limiting AAs, such as methionine and lysine, are of more concern and should therefore be supplied as RUP in order to meet the dietary requirements of ruminants. Depending upon the diet fed, the post ruminal AA supply will be altered ( i.e., in corn based diets lysine may be the limiting AA) differing from methionine as the limiting AA with barley fed diets (Fenderson and Bergen, 1975; Burris et al., 1976; Merchen and Titgemeyer, 1992). Therefore, defining the total tract composition, digestibility, and availability of AA in B.

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76 carinata is important in order to synchronize the supplementation of energy and protein when using a variety of feedstuff s C onclusion Brassica carinata is not a new crop, however the residual meal remaining after oil extraction has not been extensively tested as a protein supplement for cattle. Furthermore, Brassic a carinata meal has not been previously evaluated with regards to fractionation of protein, AA composition, or digestibility and subsequent absorption of AA which have been described in this study The evaluation of Brassica carinata meal as protein suppl emented for cattle consuming a forage based diet, resulted in a protein fraction comprised of 71.8% RDP and a total tract digestibility of dietary protein of 97%, thus indicating its viability as a highvalue protein supplement for beef cattle.

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77 Tabl e 41. Analyzed1 chemical and nutrient composition (DM basis) of hay and protein supplements fed to ruminally cannulated Angus crossbred steers. Treatment 2 Item Bahiagrass hay 3 BCM CSM DDGS SBM DM, % 94.0 89.8 88.9 86.3 90.7 CP, % 7.2 43.3 49.2 32.8 52.9 NFC 4 % -5 21.7 13.2 20.2 28.7 NDF, % 71.4 23.5 28.6 30.7 10.2 ADF, % 41.8 12.8 18.7 14.3 8.4 TDN, % 56 80 67 83 79 S, % 0.35 1.75 ---1 Dairy One Forage Testing Laboratory, Ithaca, NY. 2BCM: Brassica carinata meal pellets (1.39 kg d1); CSM: cottonseed meal (1.62 kg d1); DDGS: dry distillers grain plus solubles (2.15 kg d1); SBM: soybean meal (1.17 kg d1); composited over 4 periods. 3Bahiagrass hay ( Paspalum notatum ). 4NFC = nonfiber carbohydrates. 5-Indicates this item was not analyzed.

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78 Table 42. Characterization of protein supplements fed to ruminally cannulated Angus crossbred steers1 fed bahiagrass hay ad libitum. Treatment 2 P value 3 Item 4 BCM CSM DDGS SBM SEM 5 TRT RDP, % CP 71.79 a 47.80 b 5 5.05 b 72.30 a 3.298 < 0.001 RUP, % CP 28.20 b 52.19 a 44.94 a 27.69 b 3.298 < 0.001 IDP, % RUP 89.94 ab 89.91 ab 85.38 b 94.53 a 2.194 0.007 IADP, % CP 25.24 c 46.99 a 36.46 b 26.37 c 2.847 < 0.001 TTDP, % CP 97.06 ab 94.80 bc 93.74 bc 98.66 a 0.958 < 0.001 a,b,c Wi thin a row, means with different superscripts differ, P < 0.05 1Steers from Exp. 1. 2BCM: Brassica carinata meal pellets (1.39 kg d1); CSM: cottonseed meal (1.62 kg d1); DDGS: dry distillers grain plus solubles (2.15 kg d1); SBM: soybean meal (1.17 kg d1). 3Observed significance levels for treatment (TRT). 4Kd = rate of degradation of fraction D; IDP = estimated intestinal protein digestibility (Gargallo et al., 2006); RDP = A + B [Kd /(Kd + Kp)], where Kp is the rate of passage from the rumen, estimat ed to be 5% h1; RUP = 100 TTDP = total tract digestibility of dietary protein (TTDP = RDP + IADP); also MP. 5Pooled standard error of treatment means, n = 8 steers/treatment.

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79 Table 43. In situ digestion kinetics on DM, OM, and CP of protein supplements fed to ruminally cannulated Angus crossbred steers1 fed bahiagrass hay ad libitum. Treatment 2 P value 3 Item 4 BCM CSM DDGS SBM SEM 5 TRT DM K d % h 1 6. 6 1 b 2.85 c 5.16 bc 10.8 7 a 0.929 < 0.001 T0 h 0.53 b 2.87 a 1.20 ab 2.52 a 0.530 0.0094 SF, % 42.97 b 32.25 c 48.81 a 40.70 b 1.580 < 0.001 D, % 54.74 a b 59.78 a 45.83 b 59.11 a 3.223 0.0086 Undeg, % 2.38 7.98 5.36 0.12 3.063 0.2049 OM K d % h 1 6.71 b 2.54 c 5.31 bc 11.27 a 0.888 < 0.001 T0 h 0.99 ab 2.64 a 0.78 b 2.68 a 0.613 0.0096 SF, % 7.49 5.28 5.96 2.33 1.608 0.0674 D, % 92.48 94.68 93.95 97.65 1.604 0.0636 Undeg, % 0.03 ab 0.04 ab 0.09 a 0.01 b 0.017 0.0287 CP K d % h 1 7.59 b 3.86 c 4.6 8 bc 11.50 a 0.877 < 0.001 T0 h 0.87 b 8.89 a 3.44 a b 2.80 b 1.779 0.0066 SF, % 22.10 a 0.24 d 15.66 b 7.78 c 1.929 < 0.001 D, % 76.80 b c 98.70 a 74.67 c 88.60 a b 3.771 < 0.001 Undeg, % 0.69 1.06 9.67 3.94 3.876 0.2409 a,b,c ,d Within a row, means with different superscripts differ, P < 0.05 1Steers from Exp. 1. 2BCM: Brassica carinata meal pellets (1.39 kg d1); CS M: cottonseed meal (1.62 kg d1); DDGS: dry distillers grain plus solubles (2.15 kg d1); SBM: soybean meal (1.17 kg d1); composited over 4 periods. 3Observed significance levels for treatment (TRT). 4Kd = rate of degradation of fraction D, T0 = Lag time, SF = soluble fraction, D = potentially degradable fraction, and Undeg = undegradable fraction. 5 Pooled standard error of treatment means, n = 8 steers/treatment.

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80 Table 44. Amino acid composition of Brassica carinata meal pellets in original meal, ruminally incubated residue, and post ruminal residue. AA composition 1 (w/w %) AA BCM2 In situ 16 h residue 3 Post rumen residue 4 Taurine 0.10 0.15 0.13 Hydroxyproline 0.24 0.93 0.53 Aspartic Acid 2.39 0.78 2.95 Threonine 1.43 0.58 1.83 Serine 1.28 0. 51 1.59 Glutamic Acid 6.68 1.08 6.36 Proline 2.24 0.83 2.18 Glycine 1.80 0.57 1.98 Alanine 1.55 0.45 1.92 Cysteine 0.97 0.35 0.85 Valine 1.83 0.72 2.42 Methionine 0.70 0.15 0.80 Isoleucine 1.52 0.60 1.94 Leucine 2.58 0.72 3.10 Tyrosine 0.91 0.38 1.27 Phenylalanine 1.49 0.49 1.90 Hydroxylysine 0.05 0.03 0.03 Ornithine 0.02 0.01 0.04 Lysine 1.61 0.57 1.88 Histidine 0.98 0.19 0.95 Arginine 2.51 0.44 2.44 1 AA profiles analyzed by University of Missouri, Experiment Station Chemical Laboratories Columbia, MO. 2Original Brassica carinata meal pellets as supplied by Agrisoma Biosciences, Inc., Gatineau, Quebec. 3Rumen disappearance (%) at 16 h of incubation using in situ technique. 4 Post rumen disappearance (%) using modified three step procedure

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81 Table 45. T otal tract digestibility of amino acids from Brassica carinata meal pellets and contribution of RUP to intestinally absorbable amino acids. AA digestibility 1 Total AA composition In situ 16 h residue 2 (%) Post rumen residue 3 (%) Cont ribution of RUP to IAAA 4 (g/kg CP) Taurine 78.80 68.87 0.39 Hydroxyproline 67.99 50.75 1.07 Aspartic Acid 79.92 92.69 11.29 Threonine 79.26 91.22 6.89 Serine 79.99 91.21 5.95 Glutamic Acid 84.55 95.33 24.82 Proline 84.29 89.49 8.04 Glycine 82.12 9 2.19 7.54 Alanine 79.85 93.64 7.40 Cysteine 86.09 88.77 3.06 Valine 78.60 91.87 9.14 Methionine 81.56 94.84 3.09 Isoleucine 79.28 91.50 7.33 Leucine 80.52 93.73 11.91 Tyrosine 77.18 91.75 4.85 Phenylalanine 79.36 93.01 7.25 Hydroxylysine 91.80 60. 04 0.07 Ornithine 69.54 89.35 0.14 Lysine 80.92 91.55 7.16 Histidine 84.28 94.64 3.68 Arginine 84.19 95.15 9.53 Total 140.61 1

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82 Table 46. T otal tract digestibility of essential amino acids of Brassica carinata meal pellets and contribution of RUP to intestinally absorbable essential amino acids. AA digestibility 1 Essential AA composition In situ 16 h residue 2 (%) Post rumen residue 3 (%) Contribution of RUP to IAAA 4 (g/kg CP) Arginine 84.19 95.15 9.53 Histidine 84.28 94.64 3.68 Isoleuci ne 79.28 91.50 7.33 Leucine 80.52 93.73 11.91 Lysine 80.92 91.55 7.16 Methionine 81.56 94.84 3.09 Phenylalanine 79.36 93.01 7.25 Threonine 79.26 91.22 6.89 Valine 78.60 91.87 9.14 Total 65.98 1 Amino acid digestibility was calculated as [((initial

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83 CHAPTER 5 EVALUATION OF BRAS SICA CARINATA MEAL AS A PROTEIN SUPPLEMENT FOR GROWING BEEF HEIFERS I ntroduction Brassica carinata is a nonfood oilseed crop with a favorable very long chain fatty acid composition for conversion to biofuel (Marillia et al., 2013). Oil extracted from the seed has been utilized as a 100% dropin jet biofuel, promoting the use of B. carinata as a renewable and potentially sustainable resource (AAFC, 2015). In the southeastern U.S., B. carinata would be an ideal candidate for use in crop rotation and as a cov er crop due to its heat and drought tolerance, and cold and disease resistance (AAFC, 2015; Seepaul et al., 2016). A high protein meal (~40% CP) is obtained as a byproduct of oil extraction; however, this meal has not been extensively tested as a potential protein supplement for cattle. Analysis of the meal yields low concentrations of sinigrin and progoitrin, byproducts of ruminal degradation of glucosinolates (EFSA, 2008), which have been implicated in decreased intake, interference of thyroid hormone met abolism, and impaired fertility and reproductive performance in cattle. C attle in the southeastern U.S. often graze pastures of limited nutritive value which are not adequate to support high levels of production, especially during critical periods, necessi tating supplementation of protein (Hersom et al., 2011). Common protein supplements in this region result from byproducts of various industries and in conjunction with the poor quality hay available in winter, provide an opportunity to meet the nutritional requirements of growing heifers (Schulmeister et al., 2015) Brassica carinata meal has been evaluated as a high quality source of crude protein for ruminants utilizing an in situ procedure (Xin and Yu, 2014) however research in feeding B. carinata to cattle is limited Thus, the objective of this study was to determine the effects of supplementation with B. carinata meal on performance, attainment of puberty, and blood profile in growing Angus crossbred heifers consuming bermudagrass hay.

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84 M aterials and M ethods All procedures involving animals were approved by the Animal Care and Use Committee of the Institute of Food and Agricultural Sciences at the University of Florida, study # 201308011. E xperimental d esign and s ample c ollection The experiment was con ducted at the North Florida Research and Education Center in Marianna, FL. Sixty four Angus crossbred heifers (240 39 kg initial BW) were used in a generalized randomized block design. Heifers were stratified and blocked (2 blocks) by initial BW and randomly allocated to 18 pens over 2 consecutive years (10 pens in yr 1 and 8 pens in yr 2). Within block, pens were randomly assigned to one of two treatments: 0% BCM pellets (CTL) or 0.3% of BW d1 (as fed) of BCM pellets (BCM). Heifers were provided ad libi tum access to bermudagrass hay ( Cynodon dactylon) and water, and BCM pellets were supplemented daily in the pen. Initial BW was considered as the average of d 1 and 0 BW. Blood samples were collected on d 0, before feeding, for baseline analysis of initia l concentrations of thyroid hormones, progesterone, and acute phase proteins in plasma Body weight and blood samples were then collected every 7 d for the 70 d period, before the daily BCM supplementation. Blood was collected from jugular venipuncture every 7 d in the morning, before BCM supplementation, in 10mL evacuated tubes containing sodium heparin, placed on ice following collection, and subsequently centrifuged for 15 min at 4,000 g at 4C. After centrifugation, plasma was transferred into polypropylene vials (12 mm 75 mm; Fisherbrand; Thermo Fisher Scientific Inc., Waltham, MA) and stored at 20C for further analysis of concentrations of progesterone, triiodothyronine (T3), thyroxine (T4), haptoglobin (Hp), and ceruloplasmin (Cp). L aboratory analyses Hay samples were collected every 7 d, composited by pen within period and analyzed for DM, OM, CP, NDF, and ADF. Samples were weighed (0.5 g) into tared beakers, placed in an

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85 oven at 100C overnight to calculate DM, and subsequently placed in a m uffle furnace at 650C for 6 h to calculate OM. To determine concentration s of NDF, samples were weighed (0.5 g) into F57 filter bags and analyzed in an Ankom 200 Fiber Analyzer (Ankom Technology) using sodium sulfate and heat stable amylase. Samples wer e subsequently analyzed for concentration s of ADF. Concentrations of N in feed and feces was determined by rapid combustion using a macroelemental N analyzer (Vario Max CN, Elementar Americas Inc., Mt. Laurel, NJ) following official method 992.15 (AOAC, 1995) with C P calculated as concentration s of N multiplied by 6.25. Brassica carinata meal pellets were analyzed for nutrient composition by a commercial laboratory (Dairy One Forage Laboratory, Ithaca, NY.). Concentrations of progesterone were determined by an immunoassay (Immulite 1000, Siemens Health, Inc., Malvern, PA) according to manufacturers instructions. Females were considered to have attained puberty after the first increase in concentrations of progesterone in plasma samples exceed ing 1.0 ng mL1. Briefly, 200 uL of plasma was placed in a sample cup, loaded onto a conveyor belt with a kit specific (kit # LKPW1) test unit following, with samples and reagents then pipetted into the sample cup. After incubating for 30 min in a temperature controlled carousel, the unbound portion of sample and reagent was washed away, chemiluminescent substrate added and the signal read by a photomultiplier tube, in which the signal generated was proportional to the bound enzyme, which was then converted to concentration. Concentrations of T3 and T4 were analyzed similarly, using a solid phase, competitive chemiluminescent enzyme immunoassay (kit # LKT31 and LKT41, for T3 and T4, respectively). Plasma concentrations of Hp were determined using a biochemical assay measuring haptog lobinhemoglobin complex by the estimation of differences in peroxidase activity

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86 (Makimura and Suzuki, 1982). Results were obtained as arbitrary units resulting from the absorption reading at 450 nm. Quality control standards were analyzed by quantitative determination of bovine Hp in plasma (bovine haptoglobin ELISA test kit; Life Diagnostics, Inc., West Chester, PA). The ELISA standard curve was used to convert the arbitrary units obtained from the biochemical procedures into mg mL1 (Cooke and Arthington, 2013), with the lowest detectable value of 0.03 mg mL1. Inter and intra assay CV of Hp assays using the biochemical procedure were 3.65 and 3.02%, respectively. Plasma Cp oxidase activity was measured using the colorimetric procedures described by Dem etriou et al. (1974) and expressed as mg dL1 as described by King (1965). Inter and intra assay CV for Cp assays were 2.34 and 2.46%, respectively. Statistical analysis Data were analyzed as a generalized randomized block design using the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC). The model included fixed effects of treatment, week, treatment week interactions, block, and block treatment interactions, with the random effect of year. Repeated measures, with pen within year as subject, were used to analyze T3, T4, Cp, and Hp concentrations over time. A survival analysis was conducted using the LIFETEST procedure of SAS, to determine time to attainment of puberty. Differences between treatment means were identified by Tukeys least squares means comparison and significance was declared at P P R esults and Discussion The nutritional composition of bermudagrass hay and BCM pellets fed to heifers is presented in Table 5 1. Bermudagr ass hay used in this study had a CP concentration of 13.3% a nd depending on expected growth and performance of yearling heifers, it should be sufficient to meet their nutritional demands. However, the concentration of TDN in bermudagrass hay

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87 consumed in the current study was 55%, and could be considered limiting for achieving sufficient weight gains in developing heifers. Heifers supplemented with BCM at 0.3% of their BW for 70 d, had increased ADG ( P 1; Table 52). An increase in performance is expected when supplementing protein in hay based diets, due to an increase in the ruminal supply of substrate for microbial growth and fermentative activity, th us increasing MCP flow to the small intestine. Differences in initial BW (Table 52) were not observed for treatment or treatment block interactions ( P > 0.10); however, an effect of block was observed ( P < 0.001) for both initial and final BW. This was expected because of the initial stratification and blocking of heifers based on BW A tendency for a treatment effect was observed on final BW ( P = 0.088). Heifers in the second year of study weighed less at the initiation of the trial than those in the f irst year, which we speculate may account for the tendency of treatments to affect final BW, however this may be confounded by effect of block. Brassica carinata belongs to the mustard family, Brassicaceae, which contain high concentrations of glucosinola te s Lardy and Kerley (1994) suggested 90 to 140 mol g1 as high concentrations of glucosinolates in growing crossbred beef steers however the meal in the current study had 28 mol g1; nevertheless, it was imperative to evaluate their potential effects on growth performance, as upon digestion, bacterial myrosinases will degrade the stable, intact compound (Duncan and Milne, 1992). Sinigrin and progoitrin are glucosinolates relevant to carinata, and upon ruminal degradation, unstable compounds are produce d, resulting in formation of isothiocyanate and thiocyanate (EFSA, 2008). Thiocyanate and isothiocyanate are problematic with regards to fertility/reproductive impairment, thyroid metabolism, growth retardation, and inhibition of copper (EFSA, 2008) In the current study, the interval to

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88 attainment of puberty ( P = 0.67), was not affected by supplementation of BCM, compared with CTL heifers. An effect of block ( P < 0.001) was observed, indicating that light BW block heifers attained puberty earlier than thos e in the heavy BW. While this was unexpected similarly to the effects of treatment on final BW, we speculate that this may be due to observed differences in weight of heifers between the first and second year of study. No effect of treatment or block ( P > 0.05) on concentrations of T3 or T4 was observed, however, an effect of day ( P < 0.001) demonstrated an increase in T3 and T4 ( Figure 53), on d 7 which may be attributed to environmental factors such as cooler temperatures (Guyton, 1986; Figure 54). La rdy and Kerley (1994) observed a significant decrease in concentration s of T4 with increasing inclusion concentration s of glucosinolates. This was not observed in the current study, however there was a tendency ( P = 0.087) for heavy heifers to have an incr ease in plasma T4 and a subsequent tendency ( P = 0.077) for heavy CTL heifers to have an increased concentration s of plasma T4 compared with light CTL heifers. Concentration of plasma T3 observed for light lactating and non lactating cows (approx. 488 kg B W) was similar to heifers in the current study (128.9 to 109.2 ng mL1 vs. 122.7 to 128.7 ng mL1, respectively), however, Previous research indicates that as BW increases, plasma T4 and T3 decrease, which was observed in a study between light and heavy co ws (488 kg and 573 kg BW, respectively; Walker et al., 2015) A positive correlation between growth rate of calves and concentrations of plasma T3, has previously been reported, which may explain similar concentrations of plasma T3 between lighter cows and heifers in the present study, however thyroid hormones fluctuate depending on age, size, environment, and a host of other factors ( Tripathi et al. 2001) Thiocyanate has the potential to bind iodine, preventing trapping and uptake of iodine by the thyroi d gland (Barrett et al., 1997), however it is possible to alleviate the resulting deficiency

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89 by supplementing additional iodine. Plasma and serum T4 ha ve been used as indicator s for iodine status assessment in cattle (Hemingway et al., 2001 ; Takahashi et al., 2001), and it has been suggested that long term iodine deficiency can be diagnosed with concentrations of T4 below 1.56 g dL1 (Whittaker, 1999). Furthermore, thiocyanate has the potential to interfere with thyroid hormone synthesis (Guyton, 1986), in which case, additional supplementation of iodine is not effective. Concentrations of thyroid hormones are variable within blood and fluctuate with age, sex, and weight. Paulikova et al. (2011) assessed serum concentrations of T4 and T3 in apparently healt hy cattle at various ages, with concentrations of T4 in calves and heifers significantly different ( P < 0.05; 8.10 2.78; 9.15 3.67, g dL1; respectively) and concentrations of T3 similar ( P > 0.05; 1.91 0.65; 3.92 0.71, ng mL1; respectively). Cir culating thyroid hormones are positively correlated with energy balance, thus during negative energy balance, dairy cows responded with decreasing concentrations of T4 and T3 (McGuire et al., 1991), which has been implicated in fatty liver syndrome (Kapp e t al., 1978), hormonal imbalance, and potential reproduction disorders (Paulikova et al., 2011). Concentration of plasma Cp was decreased (Table 53; P < 0.001; 9.78 vs 11.47 mg dL1, respectively) in BCM supplemented heifers compared with CTL heifers. An effect of day was observed in concentration of plasma Cp ( P < 0.001; Figure 55), in which concentrations decreased from d 14 through 35, peaked at d 49, and stabilized for the duration of the study. Moriel and Arthington (2013) observed a peak of concent ration s of plasma positive APPs between d 8 and 14, which coincided with vaccinations, yet concentrations returned to baseline values between d 21 and 29. A similar pattern was observed in the latter part of the current study, however, concentrations were significantly decreased between d 14 and 35, despite treatments. Glucosinolates are sulfur containing moieties, and high concentrations of S may inhibit copper

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90 absorption (Yu and Benyen, 1996), subsequently affecting immune function, copper transport, and iron metabolism. Ceruloplasmin has been utilized as an indicator of nutritional Cu status in cattle as plasma Cu and Cp are highly correlated (Blakley and Hamilton, 1985). Additionally, Cp and Hp have been used as indicators of an acute phase response, whi ch is elicited during periods of stress, and in response to inflammation or disease, due to cytokine stimulation of hepatocytes to increase production of positive APPs (Carroll and Forsberg, 2007). Previous research indicates plasma concentration of Cp dec reases during periods of Cu deficiency (Mulhern and Koller, 1988) as Cp is a major transporter of plasma Cu (Cousins, 1985). D uring an immune challenge or in response to stress, protein deposition may be negatively affected as nutrients are partitioned to support immune function, thereby ensuring survival (Elasser et al., 2008). Therefore, heifers under an acute phase response would be expected to decrease intake and consequently weight gain, however, ADG was increased in heifers supplemented with BCM compa red with CTL heifers. Qiu et al. (2007) observed elevated concentrations of plasma Cp for heifer calves compared with steer calves after exposure to stressors ( P < 0.05; 20.1 vs 18.9 mg dL1, respectively), but concentrations were similar at weaning (11.08 mg dL1). Dietary concentrations of sulfur have been implicated in decreasing absorption of Cu leading to a Cu deficiency (Arthington et al., 1996), and subsequently a decrease in plasma concentration of Cp. Therefore, differences in plasma Cp may have resulted from dietary sulfur content, as BCM contains approximately 1.7%, whereas CTL heifers were not receiving additional sulfur. A ssessment of Cu status was not within the scope of this study, yet it may be of benefit to examine potential Cu deficiency resulting from BCM supplementation in future studies. Concentration of plasma Hp was not affected by supplementation of BCM ( P = 0.28; 0.08 and 0.04 mg mL1 for BCM and CTL, respectively). These results are similar to

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91 concentrations observed in healthy da iry cows (0.08 mg mL1), compared with cows infected with Theilera annulata, in which case plasma Hp ranged from 0.13 to 1.01 mg mL1, indicating a significant increase in Hp synthesis in response to infection (Nazifi et al., 2009). C onclusion Supplementa tion of BCM for 70 d in growing heifers consuming bermudagrass hay ad libitum, increased ADG by 0.28 kg d1 when compared with CTL, without altering the interval to attainment of puberty, or thyroid hormone metabolism. Supplementation of BCM led to variabl e results on plasma concentrations of APPs: 1) concentration of plasma Hp was not affected; 2) and concentration of plasma Cp was decreased in BCM heifers compared with CTL heifers. Additional research is necessary to understand the effects of supplementin g BCM on concentrations of plasma Cp, and to determine whether the decrease in plasma Cp was elicited by an acute phase response.

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92 Table 51. Analyzed1 chemical and nutrient composition (DM basis) of diet fed to growing Angus crossbred heifers. Item T reatment 2 BCM Bermudagrass hay DM, % 89.1 1.06 92.7 1.84 Glucosinolates 3 mol g 1 28.7 -4 CP, % 43.6 0.35 13.3 2.12 NFC 5 % 21.7 0.10 6 .0 6.58 NDF, % 23.6 0.14 71.2 8.13 ADF, % 13.2 0.57 38 .0 8.91 EE 6 % 2.5 0.10 -S, % 1.7 0.02 -TDN, % 76 5.66 55 2.83 1 Dairy One Forage Testing Laboratory, Ithaca, NY. 2BCM: Brassica carinata meal pellets; Bermudagrass hay ( Cynodon dactylon) fed ad libitum; values averaged over 2 years. 3Analyzed by Agrisoma Biosciences, Inc., Gatlineau, Quebec. 4-Indicates this item was not analyzed. 5NFC = nonfiber carbohydrates. 6EE = ether extract.

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93 Table 52. Effects of protein supplement ation on average daily gain, initial and final BW, and attainment of pubert y in Angus crossbred heifers fed bermudagrass hay ad libitum. Treatment 1 P value 2 Item BCM CTL SEM 3 TRT BLK TRT BLK ADG, kg 0.42 a 0.14 b 0. 101 < 0. 0 01 0.86 0.4 7 Initial BW, kg 243. 8 243. 2 34. 14 0.9 6 < 0.0 0 1 0. 80 Final BW, k g 272. 8 2 53.3 40. 88 0.0 88 < 0. 0 01 0.9 7 Puberty 4 d 430. 6 427. 5 5.8 4 0.6 8 < 0.0 0 1 0. 50 a,b Within a row, means with different superscripts differ, P < 0.05. 1BCM: Brassica carinata meal pellets; CTL: bermudagrass hay ( Cynodon dactylon) fed ad libitum; values averaged over 2 years. 2Observed significance levels for treatment (TRT) and block (BLK) effects, and for their interaction (TRT BLK). 3Pooled standard error of treatment means, n = 9 pens /treatment. 4Puberty is defined as concentrations of p1 over two consecutive 7 d measurements.

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94 Figure 51. Effects of block ( P < 0.0001) on initial and final BW of Angus crossbred heifers fed bermudagrass hay ad libitum over two consecutive years No effect of treatment ( P = 0.96) or treatment block interaction ( P = 0. 80 ) was observed for initial BW. No effect of treatment ( P = 0.088) or treatment block interaction ( P = 0. 97 ) was observed for final BW. 0 50 100 150 200 250 300 350 Light HeavyBW, kgBlock Initial Final

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95 Figure 52. Effects of block ( P < 0.0001) on days to attainment of puberty in Angus crossbred heifers fed bermudagrass hay ad libitum over two consecutive years No effect of treatment ( P = 0. 68 ) or treatment block interaction ( P = 0.49) was observed. 380 390 400 410 420 430 440 450 460 Light HeavyPuberty, dBlock

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96 Table 53. Effects of protein supplement ation on thyroid hormone1 metabolism and acute phase protein response in Angus crossbred heifers fed bermudagrass hay ad libitum. Treatment 2 P value 3 Item 4 BCM CTL SEM 5 TRT DAY TRT DAY BLK TRT BLK T 3 ng d L 1 128.6 4 122.69 3.98 2 0. 3 1 < 0.0 0 1 0.9 8 0.1 3 0. 60 T 4 g d L 1 4. 29 4.3 0 0.14 2 0.9 4 < 0.0 0 1 0.78 0.087 0.077 Haptoglobin, mg mL 1 0.08 0.04 0.019 0.28 0.4 4 0.3 9 0.3 8 0.37 Ceruloplasmin, mg d L 1 9. 7 8 b 11. 47 a 0.267 < 0.0 0 1 < 0.0 0 1 0.56 0.64 0.6 6 a,b Within a row, means with diffe rent superscripts differ, P < 0.05. 1T hyroid hormones: T3 = triiodothyronine; T4 = thyroxine. 2BCM: Brassica carinata meal pellets; CTL: bermudagrass hay ( Cynodon dactylon) fed ad libitum; values averaged over 2 years. 3Observed significance levels for tr eatment (TRT) and block (BLK) and for their interaction (TRT BLK). 4Concentrations of metabolites in plasma. 5 Pooled standard error of treatment means, n = 9 pens /treatment

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97 Figure 53. Effects of protein supplementation on thyroid hormones concen trations in plasma in Angus crossbred heifers fed bermudagrass hay ad libitum over two consecutive years No effect of treatment was observed for T3 ( P = 0. 31) or T4 ( P = 0.94), nor was an effect of treatment day observed for T3 ( P = 0.98 ) or T4 ( P =0.78). Day effect was observed ( P < 0.0001). 2.0 3.0 4.0 5.0 6.0 7.0 8.0 60 70 80 90 100 110 120 130 140 150 160 0 7 14 21 28 35 42 49 56 63 70T4, g/dLT3, ng/dLDay T3 T4

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98 Figure 54. Effects of protein supplementation on day ( P < 0.0001) on acute phase protein ceruloplasmin in Angus crossbred heifers fed bermudagrass hay ad libitum over two consecutive years An effect of treatment was observed ( P < 0.0001), however no treatment day interaction ( P = 0.56) was observed. 6 7 8 9 10 11 12 13 14 0 7 14 21 28 35 42 49 56 63 70Ceruloplasmin, mg/dLDay

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99 CHAPTER 6 SUMMARY Brassica carinata meal is a high protein residue obtained as a byproduct of oil extraction from seeds, which is then further refined to be used as highquality jet biofuel. This meal has not been extensively tested as a supplement for beef cattle, therefore three experiments were conducted to evaluate the effects of supplementation of B. carinata meal on animal performance and metabolism. Experiment 1 involved the assessment of ruminal fermentation parameters, nutrient digestibility, and blood profile in beef steers fed bahiagrass hay ( Paspalum notatum ), compared with frequently used protein supplements. Experiment 2 was designed to characteri ze ruminal protein fractionation, and subsequent post ruminal degradation of protein and amino acids. Experiment 3 assessed animal performance, attainment of puberty, and blood profile in growing beef heifers fed bermudagrass hay ( Cynodon dactylon) over tw o consecutive years. In Exp 1, a duplicated 4 4 Latin square design was used to determine the effects of supplementation with B. carinata meal on ruminal fermentation, digestibility, and blood profile in beef cattle consuming bahiagrass hay ( Paspalum notatum ), compared with frequently used protein supplements. Eight Angus crossbred steers (473 119 kg initial BW) were randomly allocated to 8 pens, over 4 periods of 28 d each. Within period, steers were assigned to one of four treatments: 1.62 kg d1 co ttonseed meal (CSM), 2.15 kg d1 dry distillers grains plus solubles (DDGS), 1.39 kg d1 B. carinata meal pellets (BCM), or 1.17 kg d1 soybean meal (SBM), supplemented daily, on an isonitrogenous basis. Steers had ad libitum access to bahiagrass hay and water. Intake was measured using the GrowSafe system. Following a 14 d adaptation, feed and fecal samples were collected twice daily for 4 d to determine apparent total tract nutrient digestibility using iNDF as an internal marker. Blood and ruminal fluid samples were collected every 3 h, during a 24 h period, to analyze blood urea nitrogen (BUN) and

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100 glucose in plasma, as well as pH, NH3N, and VFA concentrations in ruminal fluid. Data were analyzed using PROC MIXED of SAS with repeated measures. Model incl uded the fixed effects of treatment, time, treatment time, square, and period, and the random effects of steer(square) and steer(treatment). No effect of treatment ( P > 0.05) was observed for pH, NH3N, or glucose concentration. An effect of treatment ( P < 0.01) was observed for BUN, with steers receiving SBM having greater concentrations. There was no effect of treatment ( P > 0.05) on total VFA concentrations. Steers consuming CSM had greatest acetate molar proportion, and greater acetate to propionate r atio when compared with DDGS and BCM. Steers consuming DDGS had greatest molar proportions of butyrate and greater molar proportions of propionate compared with SBM and CSM. There was no effect of treatment ( P > 0.05) on DMI or apparent total tract digesti bility of DM, OM, CP, NDF, or ADF. In Exp 2, a ruminal in situ degradability study was conducted, utilizing steers from Exp.1, where the undegraded supplement remaining after 16 h of ruminal incubation was subjected to serial solutions simulating postru minal digestion, with subsequent analysis of CP content and determination of the BCM AA profile. An effect of treatment ( P observed for Kd of DM and CP with SBM having the greatest degradation rate. An effect of treatment ( P having nearly equivalent amounts of RDP and RUP (approximately 51 and 49%, respectively), which differed from SBM and BCM having approximately 72 and 28%, respectively. Compared to DDGS, SBM had the greatest IDP ( P < 0.01), with CSM having the greatest IADP ( P < 0.01), and similar for BCM and SBM. T otal tract digestibility of CP was greatest for SBM compared to CSM and DDGS.

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101 The objective of Exp 3 was to determine the effects of supplementation with B. carinata meal (BCM) on performance, attainment of puberty, and blood profile in growing beef heifers consuming bermuda grass hay ( Cynodon dactylon). Sixty four Angus crossbred heifers (240 39 kg initial BW) were stratified and blocked (2 blocks: light and heavy) by initial BW and randomly allocated into 18 pens over 2 consecutive yr (10 pens in yr 1 and 8 pens in yr 2). Within block, pens were randomly assigned to one of two treatments: 0 (CTL) or 0.3% of BW d1 (as fed) of BCM pellets, with both treatments having ad libitum access to bermudagrass hay and water, and BCM pellets supplemented daily. Blood samples and BW wer e collected weekly for 70 d, before daily supplementation. Plasma was analyzed for concentrations of progesterone, triiodothyronine (T3), thyroxine (T4), ceruloplasmin (Cp), and haptoglobin (Hp). An effect of treatment was observed ( P was no treatment or block ( P > 0.05) effect for plasma concentrations of T3, T4 or Hp; however, there was an effect of day ( P < 0.01) for T3, T4, and Cp. An effect of treatment ( P observed for concentrations of Cp, with CTL having greater concentrations compared with BCM. Time to attainment of puberty did not differ between treatments ( P = 0.68); however, an effect of block ( P < 0.01) indicated an earlier attainment of puberty in lig ht BW heifers. Brassica carinata meal performed similarly to co mmonly used protein supplements Increased ADG was observed in growing beef heifers when supplemented daily at 0.3% of BW d1, without affecting attainment of puberty thyroid hormone metaboli sm and acute phase protein synthesis As the residual meal has not been previously utilized as feed for cattle, the AA profile of the original meal, after 16 h in cubation in the rumen, and post ruminal residue has been determined in addition to the essential AA contribution of RUP Brassica carinata meal is a protein source with a CP fraction comprised of 71.8% RDP and a total dietary protein of 97%,

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102 thus indicating its viability as a high value protein supplement for beef cattle. Further research is necessary to evaluate the implications of feeding B. carinata on fetal development, and subsequent production traits, as well as assessing any potential negative effects of glucosinolates and subsequent byproducts related to copper deficiency.

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103 LITERATURE CITED AAFC. 2015. Carinata: An Oilseed Ready for Lift off Agriculture and Agri Food Canada (AAFC) ( Accessed 18 July 2017.) kerstedt, M., K. P. Waller, L. B. Larsen, L. Forsbck, and A. Sternesj. 2008. Relationship between haptoglobin and serum amyloid A in milk and milk quality. Int. Dairy J. 18:669674. doi:10.1016/j.idairyj.2008.01.002 Allison, M. J. 1978. Production of branchedchain volatile fatty acids by certain anaerobic bacteria. Appl. Environ. Microbiol. 35:872877. AOAC. 1995. Official methods of a nalysis. 16th ed. Assoc Off. Anal Chem ., Arlington, VA. ARA. 2017. ReadiJet | www.ara.com (Accessed 18 July 2017.) Arthington, J. D., L. R. Corah, and F. Blecha. 1996. The effect of molybdenum induced copper deficiency on acute phase protein concentrations, superoxide dismutase activity, leukocyte numbers, and lymphocyte proliferation in beef heifers inoculated with bovine herpesvirus 1. J. Anim. Sci. 74:211217. doi:10.2527/1996.741211x Arthington, J. D. and R. S. Kalmbacher. 2003. Effect of early weaning on performance of three year old, first cal f beef heifers and calves reared in the subtropics. J. Anim. Sci. 81:11361141. Bach, A., S. Calsamiglia, and M. D. Stern. 2005. Nitrogen metabolism in the rumen. J. Dairy Sci. 88: (E. Suppl. 1):E9E21. doi:10.3168/jds.S00220302(05)731337 Banta, J. P., M J. Hersom, J. W. Lehmkuhler, J. D. Rhinehart, and R. L. Stewart. 2016. An overview of cow calf production in the southeast: Forage systems, cow numbers, and calf marketing strategies. J. Anim. Sci. 94:60. (Abstr.) doi: 10.2527/ssasas2015124 Barrett, J. E., C. F. Klopfenstein, and H.W. Leipold. 1997. Detoxification of rapeseed meal by extrusion with an added basic salt. Cereal Chem. 74:168170. Barry, T. 2013. The feeding value of forage brassica plants for grazing ruminant livestock. Anim. Feed Sci. Tec hnol. 181:1525. doi:10.1016/j.anifeedsci.2013.01.012 Bellows, D., S. Ott, and R. Bellows. 2002. Review: Cost of r eproductive d iseases and c onditions in c attle. Prof. Anim. Sci. 18:2 632. doi:10.15232/S10807446(15)314807 Blakley, B. R. and D. L. Hamilton. 1985. Ceruloplasmin as an indicator of copper status in cattle and sheep. Can. J. Comp. Med. 49:405408.

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117 BIOGRAPHICAL SKETCH Tessa Marie Schulmeister was born at home, in the small town of Dothan, Alabama in 1983. When Tessa was five years old, she either wanted to become a teacher or a veterinarian due to her love of children and animals. Throughout her childhood Tessa w ould collect random animals and bring them home, much to her mothers dismay. At the age of sixteen, Tessa was employed in teaching children from the ages of three to five, preparing them for kindergarten, and working at the local mall, while attending hig h school. Tessa began college courses at the local junior college in 2001, however the terrorist attack of 911 compelled Tessa to join the U.S. Navy, where she enlisted for five years. After an honorable discharge from the U. S. Navy, she obtained a n asso ciates degree with emphasis in psychology from Blackhawk Junior College, and a bachelors degree in biology from Northern Illinois University where she was an undergraduate research assistant to Dr. Bethia King After graduation in 2012, Tessa moved to F lorida to be closer to family, and was employed by Dr. DiLorenzo in 2013. Under the employment of Dr. DiLorenzo, Tessa was encouraged to further her education, and began the pursuit of a masters degree from the University of Florida in Animal Sciences under the supervision of Dr. Nicolas DiLorenzo, Dr. Cliff Lamb, and Dr. Jose Dubeux. Tessas research was focused on the novel crop, Brassica carinata, and its potential use as a protein supplement in beef cattle. Tessa is also a full time mom to two wonderf ul not so little boys.