LEVEL OF DISTILLER’S GRAIN WITH SOLUBLE EFFECTS ON GAS EMISSIONS FROM
GROWING STEERS
By
Landon Drew Cross

A THESIS
Submitted to
Michigan State University
In partial fulfillment of the requirements
For the degree of
MASTER OF SCIENCE
Animal Science
2011

ABSTRACT
LEVEL OF DISTILLER’S GRAIN WITH SOLUBLE EFFECTS ON GAS EMISSIONS FROM
GROWING STEERS
By
Landon Drew Cross
A rising concern with feeding high levels of distiller’s grain with soluble (DGS) is its
high S and N content and the effects it might have on S- and N-containing emissions from gas
produced in the rumen and manure. Two trials were conducted with 12 Holstein steers housed in
individual environmentally-controlled rooms to monitor gas emissions. Three dietary treatments
were fed in trial 1; containing 0% (control), 40%, and 60% DDGS. In trial 2, treatments were
the same except the 60% DDGS dietary treatment was replaced with a 40% DDGS diet fortified
with 8 ppm Mo and 90 ppm Cu, which will be referred to as 40% DDGS+. The 40% DDGS+
diet served as a potential strategy to mitigate S-containing gas emissions. Each trial was divided
into 2 phases; phase 1 of each trial monitored emissions when urine and feces were collected in
the same vessel. Phase 2 of each trial monitored emissions while steers were fitted with fecal
bags to separate feces from urine. Distiller’s grain with soluble increased H2S and NH3
production (P < 0.05) and these emissions were decreased to undetectable levels during phase 2
of each trial compared to emissions generated in phase 1 (P < 0.01). Addition of Mo and Cu in
trial 2 tended to decrease H2S emissions when adjusted for S-intake (P = 0.08). In trial 2, the
40% DDGS+ treatment decreased CO2 emissions (P ≤ 0.05) and tended to generate less CH4
emissions compared to the control and the traditional 40% DDGS diet.

ACKNOWLEDGEMENTS
I would like to first express my deepest appreciation to my major advisor, Dr. Steven
Rust, for providing me an opportunity at Michigan State University to pursue my Masters
degree. He has truly been a great teacher and mentor to me and had an important role in my
development as a student and researcher. I would also like to thank Dr. Wendy Powers; without
her guidance, the current research would have not been possible. Furthermore, I would like to
extend my gratitude to my remaining committee members, Dr. Tom Herdt and Dr. Mike
VandeHaar. Their insight during meetings and in conversations has contributed crucial ideas to
this study. I would also like to thank Dr. Steve Bursian for always having his door open and
expressing genuine care and concern for myself and the other graduate students.
I am very grateful to have worked with so many great people through Beef Cattle
Teaching and Research Center, Animal Air Quality Research Facility, and several technicians in
the Animal Science department at Michigan State. It was truly a pleasure to work with Ken
Metz, Phil Summer, and Fred Openlander at the BCTRC. I would also like to individually thank
Andy Fogiel for taking the time to train me on the equipment at the AAQRF and assist my
understanding of the air data. I am grateful for how reliable Jolene Roth was throughout my
study at AAQRF by managing the student help and making sure everything was being done
correctly. Additionally, I would like to express my gratitude to Mark Schilling for constructing
the stalls to allow us to house the steers within the chamber rooms and for providing further
assistance in managing students and the overall operations at AAQRF. It was also a privilege to
have such great student help including Natalie Palumbo, Kylie Thompson, Tony Wernette,
Bryan Yeip, along with others that were hired later. I would have been lost in the lab had it not
been for the assistance of Dave Main, Cara Robison, Lei Zhang, and Jane Link. Their guidance

iii

and direction was crucial to the success of my research. Furthermore, I am sincerely grateful to
Jim Liesman for his statistical help as well as being a friend and mentor throughout this process.
I also want to express my gratitude to my fellow graduate students for making my experience
here very pleasant and providing memories that will last a life time.
Finally, I would not be where I am today if it were not for my parents Greg and Sandy
Cross along with other family members and friends. They have always believed in me and served
as an inspiration to many of my achievements. I am forever grateful for their unwavering love
and support.

iv

TABLE OF CONTENTS

LIST OF TABLES ......................................................................................................................... ix
LIST OF FIGURES ...................................................................................................................... xii
LIST OF ABBREVIATIONS ..................................................................................................... xvii
CHAPTER 1: REVIEW OF LITERATURE .................................................................................. 1
BACKGROUND ON DISTILLER’S GRAIN WITH SOLUBLE ............................................. 1
Dry Milling Process ................................................................................................................. 1
DGS Composition ................................................................................................................... 2
Feeding distiller’s grain with soluble to feedlot cattle ................................................................ 4
Inclusion levels for DGS ......................................................................................................... 4
Variability of DGS................................................................................................................... 5
Effects of feeding wet vs. dry DGS ......................................................................................... 6
Effects on manure .................................................................................................................... 6
Roles of sulfur in the body .......................................................................................................... 8
Importance of dietary S ........................................................................................................... 8
Sulfate-reducing bacteria ......................................................................................................... 8
Hydrogen sulfide production in the rumen .............................................................................. 9
Feeding high sulfur diets ........................................................................................................... 11
Effects of high S diets on performance and health ................................................................ 11
Sulfur toxicity ........................................................................................................................ 11
Polioencephalomalacia .......................................................................................................... 12
Risks associated with hydrogen sulfide emissions ................................................................ 13
Methods to measure sulfur and hydrogen sulfide emissions..................................................... 15
Portable field analyzers ......................................................................................................... 15
Ruminal gas cap..................................................................................................................... 16
In vitro sulfide determination ................................................................................................ 17
Animal air chambers .............................................................................................................. 17
Strategies to mitigate hydrogen sulfide production................................................................... 19
Biocides ................................................................................................................................. 19
Ionophores ............................................................................................................................. 19
Effect of feeding urea on S and N balance ............................................................................ 20
v

Sulfur, molybdenum, and copper compound......................................................................... 21
Iron binding effects with sulfides .......................................................................................... 23
Sulfide inhibition by 9,10 anthraquinone .............................................................................. 24
Review of ruminant gas emissions ............................................................................................ 25
CHAPTER 2: AN INTRODUCTION AND OBJECTIVE STATEMENT ................................. 26
INTRODUCTION ..................................................................................................................... 26
OBJECTIVE STATEMENT ..................................................................................................... 28
CHAPTER 3: LEVELS OF DISTILLER’S GRAIN WITH SOLUBLE AND STRATEGIES TO
MITIGATE SULFUR-CONTAINING EMISSIONS .................................................................. 29
SUMMARY .............................................................................................................................. 29
INTRODUCTION ..................................................................................................................... 30
MATERIALS AND METHODS .............................................................................................. 32
Gas collection ........................................................................................................................ 32
Feed, feces and urine collection ............................................................................................ 38
Feces and urine segregation using fecal bags ........................................................................ 38
Collections and mineral analysis ........................................................................................... 39
Statistical analysis ................................................................................................................. 40
RESULTS.................................................................................................................................. 42
Trial 1 .................................................................................................................................... 42
Trial 2 .................................................................................................................................... 50
DISCUSSION ........................................................................................................................... 58
CHAPTER 4: EFFECTS OF DISTILLER’S GRAIN WITH SOLUBLE AND
SUPPLEMENTAL COPPER AND MOLYBDENUM ON NITROGENOUS EMISSIONS AND
NITROGEN RETENTION ........................................................................................................... 62
SUMMARY .............................................................................................................................. 62
INTRODUCTION ..................................................................................................................... 64
MATERIALS AND METHODS .............................................................................................. 66
Gas collection ........................................................................................................................ 66
Feed, feces and urine collection ............................................................................................ 72
Feces and urine segregation using fecal bags ........................................................................ 72
Collections and mineral analysis ........................................................................................... 73
Statistical analysis ................................................................................................................. 74
RESULTS.................................................................................................................................. 76
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Trial 1 .................................................................................................................................... 76
Trial 2 .................................................................................................................................... 84
DISCUSSION ........................................................................................................................... 92
CHAPTER 5: CARBON FOOTPRINT AND ENERGY PARTITION FROM CATTLE FED
VARIOUS LEVELS OF DISTILLER’S GRAIN WITH SOLUBLE .......................................... 96
SUMMARY .............................................................................................................................. 96
INTRODUCTION ..................................................................................................................... 97
MATERIALS AND METHODS .............................................................................................. 99
Gas collection ........................................................................................................................ 99
Feed, feces and urine collection .......................................................................................... 103
Feces and urine segregation using fecal bags ...................................................................... 103
Energy balance calculations ................................................................................................ 104
Collections and mineral analyses ........................................................................................ 105
Statistical analysis ............................................................................................................... 106
RESULTS................................................................................................................................ 109
Trial 1 .................................................................................................................................. 109
Trial 2 .................................................................................................................................. 117
DISCUSSION ......................................................................................................................... 124
CHAPTER 6: CONCLUSIONS ................................................................................................. 128
APPENDICES ............................................................................................................................ 131
APPENDIX A ......................................................................................................................... 132
Trial 1 .................................................................................................................................. 132
Trial 2 .................................................................................................................................. 141
APPENDIX B ......................................................................................................................... 150
Trial 1 .................................................................................................................................. 150
Trial 2 .................................................................................................................................. 163
APPENDIX C ......................................................................................................................... 173
Trial 1 .................................................................................................................................. 173
Trial 2 .................................................................................................................................. 181
APPENDIX D: DETERMINATION OF COPPER AND MOLYBDENUM LEVELS IN
FERMENTATION VESSELS TO MITIGATE SULFIDE GAS PRODUCTION. ............... 189
SUMMARY ............................................................................................................................ 189
INTRODUCTION ................................................................................................................... 190
vii

MATERIALS AND METHODS ............................................................................................ 192
Experiment 1........................................................................................................................ 192
Experiment 2........................................................................................................................ 200
Statistical analysis ............................................................................................................... 202
RESULTS................................................................................................................................ 203
Experiment 1........................................................................................................................ 203
Experiment 2........................................................................................................................ 206
DISCUSSSION ....................................................................................................................... 211
SUPPLEMENTAL TABLES AND FIGURES ...................................................................... 214
LITERATURE CITED ............................................................................................................... 223

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LIST OF TABLES

Table 3.1 Dietary ingredients and composition for trial 1 and 2 .................................................. 34
Table 3.2 Gas unit conversion chart ............................................................................................. 37
Table 3.3 Effects of dry distiller’s grain with soluble on performance in trial 1 .......................... 43
Table 3.4 Effects of distiller’s grain with soluble levels on sulfur-containing emissions for phase
1 and 2 during trial 1 ..................................................................................................................... 44
Table 3.5 Effects of distiller’s grain with soluble on total sulfur balance for trial 1 during phase 2
....................................................................................................................................................... 48
Table 3.6 Effects of feeding distiller’s grain with soluble on excreta pH and output during trial 1
....................................................................................................................................................... 49
Table 3.7 Effects of dry distiller’s grain with soluble fortified with copper and molybdenum on
performance in trial 2 .................................................................................................................... 51
Table 3.8 Effects of dry distiller’s grain with soluble fortified with copper and molybdenum
sulfur-containing emissions for phase 1 and 2 during trial 2 ........................................................ 52
Table 3.9 Effect of distiller’s grain with soluble fortified with copper and molybdenum on total
sulfur balance for trial 2 ................................................................................................................ 56
Table 3.10 Effects of feeding distiller’s grain with soluble on excreta pH and output during trial
2..................................................................................................................................................... 57
Table 3.11 Hydrogen sulfide gas emitted per gram of sulfur intake during phase 1 of each trial 57
Table 4.1 Dietary ingredients and composition for trial 1 ............................................................ 68
Table 4.2 Gas unit conversion chart ............................................................................................. 71
Table 4.3 Effects of dry distiller’s grain with soluble on performance in trial 1 .......................... 77
Table 4.4 Effects of distiller’s grain with soluble levels on nitrogenous emissions for phase 1 and
2 during trial 1 ............................................................................................................................... 78
Table 4.5 Effects of distiller’s grain with soluble on total nitrogen balance for trial 1 ................ 82
Table 4.6 Effects of feeding distiller’s grain with soluble on excreta pH and output during trial 1
....................................................................................................................................................... 83

ix

Table 4.7 Effects of dry distiller’s grain with soluble fortified with copper and molybdenum on
performance in trial 2 .................................................................................................................... 85
Table 4.8. Effects of dry distiller’s grain with soluble fortified with copper and molybdenum on
nitrogenous emissions for phase 1 and 2 during trial 2 ............................................................... 86
Table 4.9 Effects of distiller’s grain with soluble on total nitrogen balance for trial 2 ................ 90
Table 4.10 Effects of feeding distiller’s grain with soluble on excreta pH and output during trial
2..................................................................................................................................................... 91
Table 5.1 Dietary ingredients and composition for trial 1 and 2 ................................................ 101
Table 5.2 Effects of dry distiller’s grain with soluble on performance in trial 1 ........................ 110
Table 5.3 Effects of distiller’s grain with soluble levels on carbon emissions and respiration for
phase 1 and 2 during trial 1 ......................................................................................................... 111
Table 5.4 Global warming potential gram equivalents per day from greenhouse gas emissions
from cattle fed various levels of distiller’s grain with soluble during trial 1. Values in parentheses
are percentages of the total ......................................................................................................... 114
Table 5.5 Effects of distiller’s grain with soluble on energy partition during trial 1 (MJ/d)...... 115
Table 5.6 Effects of dry distiller’s grain with soluble fortified with copper and molybdenum on
performance in trial 2 .................................................................................................................. 118
Table 5.7 Effects of distiller’s grain with soluble levels on carbon emissions and respiration for
phase 1 and 2 during trial 2 ......................................................................................................... 119
Table 5.8 Effects of distiller’s grain with soluble on energy partition during trial 2 (MJ/d)...... 123
Table A.1 Comparison of unfiltered raw data to filtered data during trial 1 .............................. 132
Table A.2 Probability of day effects for sulfur-containing emissions during trial 1 .................. 136
Table A.3 Comparison of unfiltered raw data to filtered data during trial 2 .............................. 141
Table A.4 Probability of day effects for sulfur-containing emissions during trial 2 .................. 145
Table B.1 Comparison of unfiltered raw data to filtered data during trial 1 .............................. 150
Table B.2 Probability of day affects on nitrogenous emissions during trial 1 ............................ 156
Table B.3 Comparison of unfiltered raw data to filtered data during trial 2 .............................. 163

x

Table C.1 Comparison of unfiltered raw data to filtered data during trial 1 .............................. 173
Table C.2 Probabilities of day effects for carbon emissions and oxygen consumption during trial
1................................................................................................................................................... 177
Table C.3 Comparison of unfiltered raw data to filtered data during trial 2 .............................. 181
Table C.4 Probability of day effects for carbon emissions and oxygen consumption during trial 2
..................................................................................................................................................... 185
Table D.1 Composition of distiller’s grain with soluble, and the Van Soest buffer media ........ 194
Table D.2 Effects of molybdenum and copper levels on the fermentation of rumen fluid using
distiller’s grain with soluble as a substrate during experiment 1 ................................................ 205
Table D.3 Effects of molybdenum and copper levels on the fermentation of rumen fluid using
distiller’s grain with soluble as a substrate during experiment 2 ................................................ 208
Table D.4 Dilutions of copper sulfate solution during experiment 1 and 2 ................................ 214
Table D.5 Dilutions of copper chloride solution during experiment 1 and 2 ............................ 215
Table D.6 Dilutions of sodium molybdate solution during experiment 1 and 2........................ 216
Table D.7 Fermentation vessel arrangement for molybdenum and copper concentrations during
experiment 2................................................................................................................................ 217
Table D.8 Probability of an interaction affects between Mo and Cu levels with Cu source during
experiment 1 and 2 ...................................................................................................................... 218
Table D.9 Preliminary results from an experiment with similar treatment levels as experiment 1
..................................................................................................................................................... 222

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LIST OF FIGURES
Figure 5.1 Predicted energy retention from cattle fed dried distiller’s grain with soluble estimated
from trial 1 results ....................................................................................................................... 116
Figure A.1 Daily hydrogen sulfide emissions output during trial 1 ........................................... 133
Figure A.2 Daily hydrogen sulfide emissions adjusted on sulfur intake during trial 1 .............. 133
Figure A.3 Daily hydrogen sulfide emissions adjusted on dry matter intake during trial 1 ....... 134
Figure A.4 Daily sulfur dioxide emissions output during trial 1 ................................................ 135
Figure A.5 Daily sulfur dioxide emissions adjusted on sulfur intake during trial 1 ................... 135
Figure A.6 Daily sulfur dioxide emissions adjusted on dry matter intake during trial 1 ............ 136
Figure A.7 Mean hydrogen sulfide emissions output during trial 1 ........................................... 137
Figure A.8 Mean hydrogen sulfide emissions adjusted on sulfur intake during trial 1 .............. 137
Figure A.9 Mean hydrogen sulfide emissions adjusted on dry matter intake during trial 1 ....... 138
Figure A.10 Mean sulfur dioxide emissions output during trial 1 .............................................. 139
Figure A.11 Mean sulfur dioxide emissions adjusted on sulfur intake during trial 1 ................. 139
Figure A.12 Mean sulfur dioxide emissions adjusted on dry matter intake during trial 1 ......... 140
Figure A.13 Daily hydrogen sulfide emissions output during trial 2 ......................................... 142
Figure A.14 Daily hydrogen sulfide emissions adjusted on sulfur intake during trial 2 ............ 142
Figure A.15 Daily hydrogen sulfide emissions adjusted on dry matter intake during trial 2 ..... 143
Figure A.16 Daily sulfur dioxide emissions output during trial 2 .............................................. 144
Figure A.17 Daily sulfur dioxide emissions adjusted on sulfur intake during trial 2 ................. 144
Figure A.18 Daily sulfur dioxide emissions adjusted on dry matter intake during trial 2 .......... 145
Figure A.19 Mean hydrogen sulfide emissions output during trial 2 ......................................... 146
Figure A.20 Mean hydrogen sulfide emissions adjusted on sulfur intake during trial 2 ............ 146

xii

Figure A.21 Mean hydrogen sulfide emissions adjusted on dry matter intake during trial 2 ..... 147
Figure A.22 Mean sulfur dioxide emissions output during trial 2 .............................................. 148
Figure A.23 Mean sulfur dioxide emissions adjusted on sulfur intake during trial 2 ................. 148
Figure A.24 Mean sulfur dioxide emissions adjusted on dry matter intake during trial 2 ......... 149
Figure B.1 Daily ammonia emissions output during trial 1 ........................................................ 151
Figure B.2 Daily ammonia emissions adjusted for nitrogen intake during trial 1 ...................... 151
Figure B.3 Daily ammonia emissions adjusted for dry matter intake during trial 1 ................... 152
Figure B.4 Daily nitrogen oxide emissions output during trial 1 ............................................... 153
Figure B.5 Daily nitrogen oxide emissions adjusted for nitrogen intake during trial 1.............. 153
Figure B.6 Daily nitrogen oxide emissions adjusted for dry matter intake during trial 1 .......... 154
Figure B.7 Daily nitrous oxide emissions output during trial 1 .................................................. 155
Figure B.8 Daily nitrous oxide emissions adjusted for nitrogen intake during trial 1 ................ 155
Figure B.9 Daily nitrous oxide emissions adjusted for dry matter intake during trial 1............. 156
Figure B.10 Mean ammonia emissions output during trial 1...................................................... 157
Figure B.11 Mean ammonia emissions adjusted for nitrogen intake during trial 1 .................... 157
Figure B.12 Mean ammonia emissions adjusted for dry matter intake during trial 1 ................ 158
Figure B.13 Mean nitrogen oxide emission output during trial 1 ............................................... 159
Figure B.14 Mean nitrogen oxide emissions adjusted for nitrogen intake during trial 1 ........... 159
Figure B.15 Mean nitrogen oxide emissions adjusted for dry matter intake during trial 1 ........ 160
Figure B.16 Mean nitrous oxide emissions output during trial 1 ............................................... 161
Figure B.17 Mean nitrous oxide emissions adjusted for nitrogen intake during trial 1.............. 161
Figure B.18 Mean nitrous oxide emissions adjusted for nitrogen intake during trial 1.............. 162
Figure B.19 Daily ammonia emissions output during trial 2 ...................................................... 164

xiii

Figure B.20 Daily ammonia emissions adjusted for nitrogen intake during trial 2 .................... 164
Figure B.21 Daily ammonia emissions adjusted for dry matter intake during trial 2 ................. 165
Figure B.22 Daily nitrogen oxide emissions output during trial 2 ............................................. 166
Figure B.23 Mean ammonia emission output during trial 2 ....................................................... 167
Figure B.24 Mean ammonia emissions adjusted for nitrogen intake during trial 2 .................... 167
Figure B.25 Mean ammonia emissions adjusted for dry matter intake during trial 2 ................ 168
Figure B.26 Mean nitrogen oxide emissions output during trial 2 ............................................. 169
Figure B.27 Mean nitrogen oxide emissions adjusted for nitrogen intake during trial 2 ........... 169
Figure B.28 Mean nitrogen oxide emissions adjusted for dry matter intake during trial 2 ........ 170
Figure B.29 Mean nitrous oxide emissions output during trial 2 ............................................... 171
Figure B.30 Mean nitrous oxide emissions adjusted for nitrogen intake during trial 2.............. 171
Figure B.31 Mean nitrous oxide emissions adjusted for dry matter intake during trial 2 .......... 172
Figure C.1 Daily methane emissions output during trial 1 ......................................................... 174
Figure C.2 Daily methane emissions adjusted for dry matter intake during trial 1 .................... 174
Figure C.3 Daily non-methane total hydrocarbon emissions output during trial 1 .................... 175
Figure C.4 Daily non-methane total hydrocarbon emissions adjusted for dry matter intake during
trial 1 ........................................................................................................................................... 175
Figure C.5 Daily carbon dioxide emissions output during trial 1 ............................................... 176
Figure C.6 Daily carbon dioxide emissions output during trial 1 ............................................... 176
Figure C.7 Daily oxygen consumption during trial 1 ................................................................. 177
Figure C.8 Mean methane emissions output during trial 1 ......................................................... 178
Figure C.9 Mean methane emissions adjusted for dry matter intake during trial 1 .................... 178
Figure C.10 Mean non-methane total hydrocarbon emissions output during trial 1 .................. 179

xiv

Figure C.11 Mean non-methane total hydrocarbon emissions adjusted for dry matter intake
during trial 1 ................................................................................................................................ 179
Figure C.12 Mean carbon dioxide emissions output during trial 1............................................. 180
Figure C.13 Mean carbon dioxide emissions adjusted for dry matter intake during trial 1 ....... 180
Figure C.14 Daily methane emissions output during trial 2 ....................................................... 182
Figure C.15 Daily methane emissions adjusted for dry matter intake during trial 2 .................. 182
Figure C.16 Daily non-methane total hydrocarbon emissions output during trial 2 ................. 183
Figure C.17 Daily non-methane total hydrocarbon emissions adjusted for dry matter intake
during trial 2 ................................................................................................................................ 183
Figure C.18 Daily carbon dioxide emissions output during trial 2 ............................................. 184
Figure C.19 Daily carbon dioxide emissions adjusted for dry matter intake during trial 2 ........ 184
Figure C.20 Daily oxygen consumption during trial 2 ............................................................... 185
Figure C.21 Mean methane emissions output during trial 2 ....................................................... 186
Figure C.22 Mean methane emissions adjusted for dry matter intake during trial 2 .................. 186
Figure C.23 Mean non-methane total hydrocarbon emissions output during trial 2 .................. 187
Figure C.24 Mean non-methane total hydrocarbon emissions adjusted for dry matter intake
during trial 2 ................................................................................................................................ 187
Figure C.25 Mean carbon dioxide emissions output during trial 2............................................. 188
Figure C.26 Mean carbon dioxide emission output during trial 2 .............................................. 188
Figure D.1 Image illustrating the removal of gas from the head space of the fermentation vessels
and methods for sub-sampling gas.............................................................................................. 198
Figure D.2 Standard curve for all treatment combinations using both copper sulfate and copper
chloride as copper sources in experiment 1 ................................................................................ 199
Figure D.3 Standard curve for all treatment combinations using both copper sulfate and copper
chloride as copper sources in experiment 2 ................................................................................ 201
Figure D.4 Two-way interaction of molybdenum level by copper level for hydrogen sulfide
adjusted for dry matter degradation during experiment 2 ........................................................... 209
xv

Figure D.5 Comparison of hydrogen sulfide adjusted for dry matter degradation with the
treatment levels from experiment 1 to the elevated levels in experiment 2................................ 210
Figure D.6 Three-way interaction plot between copper source, copper level, and molybdenum
level for in-vitro dry matter disappearance during experiment 1 ............................................... 219
Figure D.7 Two-way interaction plot between copper source and molybdenum level for in-vitro
dry matter disappearance during experiment 1 ........................................................................... 219
Figure D.8 Two-way interaction plot between copper source and molybdenum level for pH
during experiment 1 .................................................................................................................... 220
Figure D.9 Two-way interaction plot between copper level and molybdenum level for pH during
experiment 1................................................................................................................................ 220
Figure D.10 Two-way interaction plot between copper level and molybdenum level for hydrogen
sulfide during experiment 2 ........................................................................................................ 221

xvi

LIST OF ABBREVIATIONS
AAQRF…………………………Animal Air Quality Research Facility
ACH…………………………….air changes per hour
ADG…………………….………average daily gain
BCTRC……………………….…Beef Cattle Teaching Research Facility
BW………………………………body weight
CDS……………………………..condensed distiller’s with soluble
CGM……………………….……corn gluten meal
CP……………………………….crude protein
CS……………………………….corn silage
DE………………………….…...digestible energy
DGS……………………….……distiller’s grain with soluble
DDGS………………………..…dried distiller’s grain with soluble
DM……………………………...dry matter
DMI……………………………..dry matter intake
DPD……………………………..N, N-dimethyl-p-phenylenediamine
DTRC……………………….…..Dairy Teaching Research Facility
FE………………………………fecal energy
G:F……………………………...gain to feed ratio
GI…………………………...…..gastro-intestinal
GHG…………………….………greenhouse gas
GWP……………………….…....global warming potential
HCl……………………………...hydrochloric acid
xvii

HE……………………………....heat energy
HMC……………………………high moisture corn
IE……………………………….gross energy intake
IVDMD………………………...in-vitro dry matter disappearance
MB……………………………...methylene blue
MS……………………………....mineral supplement
MWDGS………………………..modified wet distiller’s grain with soluble
NEg……………………………..net energy gained or retained energy
NI……………………………….nitrogen intake
Nt…………………………….….total nitrogen
NMTHC………………………...non-methane total hydrocarbon
PEM…………………………….polioencephalomalacia
PUFA…………………………...polyunsaturated fatty acids
SI………………………………..sulfur intake
SBM…………………………….soybean meal
SRB……………………………..sulfate-reducing bacteria
TCA…………………………….tri-carboxylic acid
TMR…………………………….total mixed ration
TPP……………………………...thiamine pyrophosphate
TRS………………………….….total reduced sulfur
TRT……………………………..treatment
UE………………………………urine energy
UV………………………………ultraviolet
xviii

VFA……………………………..volatile fatty acid
VOC…………………………….volatile organic compound
WDG…………………………....wet distiller’s grain
WDGS…………………………..wet distiller’s grain with soluble

xix

CHAPTER 1: REVIEW OF LITERATURE
BACKGROUND ON DISTILLER’S GRAIN WITH SOLUBLE
For centuries, fermentation of cereal grains has been used to produce beverage alcohol
(Klopfenstein, 2008). A co-product from the fermentation process, distiller’s grains plus soluble
th

(DGS) has been used as a feedstuff in beef cattle diets as early as the 19 century (Henry, 1900;
Klopfenstein, 2008). Little was known about the effectiveness and nutrient composition of the
co-product feed, DGS when it was first being used as a feedstuff in beef cattle diets. Recently,
increased production of ethanol has led to a surplus of DGS. The Renewable Fuels Association
(2006) reported that ethanol production in the United States alone was expected to increase by
nearly 25% over the previous year, reaching 4.9 billion gallons. At the current rate, production
has already exceeded the 2012 target of 7.5 billion gallons per year set forth by the Energy
Policy Act of 2005 (Renewable Fuels Association, 2006; Depenbusch, 2008). According to the
Renewable Fuels Association (2010), ethanol production is currently greater than 13 billion
gallons per year. The availability and cost benefits of feeding DGS have made it a very popular
energy and protein replacement over traditional corn and soybean meal (SBM) in diets.
Extensive research has provided a better understanding of nutrient composition and feeding
strategies of DGS.
Dry Milling Process
To produce the co-product DGS, corn or other cereal grains are used for ethanol
production. In the typical dry milling process, the entire corn kernel is used and the starch is
converted into ethanol during the fermentation process (Bothast and Schilicher, 2005). The basic
steps involved in the dry-grind milling process include cleaning and grinding corn to a flour or
fine grind meal. The combination of the resulting flour and water form a mash. Enzymes are
1

added to the mash to breakdown the starch in the kernel to glucose molecules. The mash is then
cooked to reduce undesirable bacteria. After the mash is cooked it is then allowed time to cool,
during this process yeasts are added to the mash to aid the conversion of glucose to ethanol and
carbon dioxide. Next, a distillation process extracts the ethanol and the remaining water and
solids are referred to as whole stillage. Typically whole stillage is centrifuged to separate the
liquid from the solids. The liquid portion is considered distiller’s soluble or thin stillage. The thin
stillage can be concentrated in an evaporator to become condensed distiller’s with soluble
(CDS), also referred to as syrup. The solid portion separated out is considered wet distiller’s
grains (WDG). The combination of CDS and WDG form wet distiller’s grains with soluble
(WDGS) and can be dried to produce dried distiller’s grain with soluble (DDGS) (Kalscheur and
Garcia, 2008).
DGS Composition
Distiller’s grain with soluble is an excellent source of protein, fat, digestible fiber, and
minerals, all of which can be utilized in ruminant diets (Botheast and Schlicher, 2005). The beef
NRC (2001) lists crude protein (CP) at 29.7% for DDGS. Kalscheur and Garcia (2008) report
studies illustrating that CP ranges from 27 to 34%. Roughly 2/3 of the corn kernel is starch,
which is removed during the dry milling process. The result of this process causes DGS to be
roughly 3-fold higher in protein, fat, fiber, and mineral concentrations. Protein increases from 10
to 30%, fat from 4 to 12%, NDF from 12 to 36%, and P from 0.3 to 0.9% of DM (Stock et al.,
2000). The solids fraction of DGS is greater in CP and crude fiber compared to the CDS fraction,
but the CDS fraction is greater in fat and minerals in comparison to the solids fraction (Rausch
and Belyea, 2006; Kalsheur et al., 2008). Sulfuric acid is added during the fermentation process
to regulate pH, which increases the amount and concentration of S in DGS as well (Vannes et al.,

2

2009; Kelzer et al., 2010). Buckner et al. (2008) reported S concentrations in DGS ranging from
0.44% to as high as 1.5%. Sulfur concentration should be closely monitored with mid to high
levels of DGS added to cattle diets as the NRC (2000) lists maximum tolerable concentration in
feedlot cattle rations at 0.4%.

3

FEEDING DISTILLER’S GRAIN WITH SOLUBLE TO FEEDLOT CATTLE
Inclusion levels for DGS
Increased levels of DGS in cattle diets decrease starch intake and in most cases increase
levels of CP and fat in feedlot diets. Feeding elevated levels of DGS to finishing cattle is thought
to negatively affect feedlot performance and carcass quality (Gunn et al., 2009). Decreased
dietary starch content may lead to decreased carcass yield grade and marbling score (Smith and
Crouse, 1984). Data from a study conducted by Gunn et al (2009) indicated that live
performance, marbling scores, quality grades, and color stability of ground product during retail
display were negatively affected when DDGS increased from 25 to 50% of the diet (DM basis).
Gunn and others (2009) concluded that this response was related not only to increased CP or fat
concentrations within the diet of steers fed elevated levels of DDGS, but also to a combination
effect of both CP and fat within these diets. Larson et al. (1993), Ham et al. (1994), Jones (2007),
and Depenbusch (2009) reported that moderate levels of DGS in feedlot diets improved ADG,
DMI, and G:F. However, all of these studies were conducted with inclusion of either WDGS or
DDGS at or below 40% (DM basis). Very little research has evaluated DGS levels above 40%
(DM basis) and the effects on growth performance and carcass characteristics (Farlin, 1981;
Vander Pol et al., 2006). Gordon et al. (2002) reported a decrease in performance of finishing
heifers when DDGS inclusion levels exceeded 45% of dietary DM. Few studies have looked at
effects of distillers grains on meat quality (Kroger et al., 2004; Roeber et al., 2005; Gill et al.,
2008). A report from Corah and McCully (2006) indicated a decrease in marbling scores when
DGS were fed at greater than 30% of DM and an increase in yield grade at all inclusion levels. A
study conducted by Roeber et al. (2005) determined that steaks from Holstein steers fed either
WDGS or DDGS at 40% dietary DM had less acceptable retail display compared to steers fed

4

lower concentrations of DGS. Steaks from Holstein steers fed moderate inclusion levels of DGS,
around 25% of DM basis had greater redness than steaks from steers fed 0 or 50% DGS (Roeber
et al., 2005). Depenbusch et al. (2009) reported that increasing levels of DGS from 0 to 75%
increased concentrations of linoleic acid (18:2n-6cis), total n-6 fatty acids, and total
polyunsaturated fatty acids (PUFA) in cooked ribeye. In addition, myofibrillar tenderness and
overall tenderness increased linearly with the inclusion of DGS from 0 to 75% of the diet DM
(Depenbusch et al., 2009).
Variability of DGS
Variability in nutrient content that exists among different sources of DGS is one of the
challenges for utilization in livestock diets (Spiehs et al., 2002). Studies from Holt and Pritchard
(2004) and Kaiser (2005) would suggest that the composition of WDGS can have considerable
variance. There are several factors that contribute to this variability but the ratio of distiller’s
grain and CDS used to manufacture commercial DGS is thought to be the major contributor to
nutrient variability among different ethanol plants (Kleinschmit et al., 2007; Cao et al., 2009).
Distiller’s grain with soluble is comprised of 2 co-products, the spent grain or solids fraction and
the CDS fraction. When more of the CDS fraction is added back to the grain fraction to form
DGS, fat and mineral concentrations are increased. In comparison, DGS that has a higher grain
fraction with less CDS added back will likely be higher in CP and crude fiber (Rausch and
Belyea, 2006; Kalcheur et al., 2008; Cao et al., 2009). In addition to distiller’s grain to CDS
ratio, processing methods can influence variability of nutrients in DGS as well. Sulfur
concentration of DGS is highly variable within and among ethanol plants. Variability of S
concentration (0.44 to 1.5%; Buckner et al., 2008) is most likely influenced by the inclusion of

5

sulfuric acid (Vannes et al., 2009; Kelzer et al., 2010) to control pH during fermentation and for
use as a cleaning agent.
Effects of feeding wet vs. dry DGS
Dried distiller’s grain with soluble is a more costly co-product compared to WDGS, due
to the additional expense associated with the drying process. Traditional WDGS contains 3035% DM and is similar in nutrient content on a DM basis to DDGS (Kalscheur and Garcia,
2008). However, DDGS may result in a decrease of protein and AA availability due to possible
protein damage from heat during the drying process (Kleinschmit et al., 2007; Kalscheur and
Garcia, 2008). Wet distiller’s grain with soluble is often lower in price on a DM basis compared
to DDGS; however, producers must have proper facilities to store and handle WDGS. Methods
to store and handle WDGS are challenges producers face (Kalscheur and Garcia, 2008). Wet
distiller’s grain with soluble has other advantages aside from cost which include higher
palatability and usage as a ration conditioner to dry diets with smaller particle sizes. Total mixed
rations (TMR) that contain 10-20% WDGS maintain greater homogeneity as dry particles stick
together which results in less particle separation and less sorting by cattle. Wet distiller’s grain
with soluble can be partially dried to bring the DM up to between 45-55%. This product is called
modified wet distiller’s grain with soluble (MWDGS). Nutrient composition is usually similar to
that of WDGS and DDGS (Kalscheur and Garcia, 2008).
Effects on manure
The increase in nutrient concentrations in WDGS or DDGS has led to further challenges
in managing excreta. Wet distiller’s grain with soluble in feedlot diets increases N, P, and S
contents relative to conventional feedlot diets. Elevated levels of CP and fats in DGS diets create
challenges managing manure as well. Undigested organic residues such as proteins,

6

carbohydrates, and fats compose livestock excreta. Volatile organic compounds (VOC),
ammonia (NH3), volatile fatty acids (VFA), S- compounds, and aromatic compounds are formed
by aerobic and anaerobic digestion of organic residues in manure by bacteria (Mackie et al.,
1998; Spiehs and Varel, 2009). Volatile organic compounds present in livestock diets contribute
largely to negative odor perceived by humans (Zahn et al., 1997; Powers et al., 1999; Zahn et al.,
2001). Cattle fed diets with DGS ranging from 20 to 40% of the diet DM will have elevated
levels of CP, fat, and minerals particularly P and S. These excess nutrients can potentially
contribute to environmental pollution from increased N-emissions, H2S emissions, P runoff, and
greater odor production (Varel et al., 2008). Studies conducted by Koziel et al. (2006) reported
that the odorant p-cresol cause much of the overall odor impact on swine and cattle operations.
Cresol originates from amino acids tyrosine, tryptophan (Mackie et al., 1998), and phenylalanine
(Mohammed et al., 2003). Distiller’s grain with soluble is approximately three times higher in
these 3 AA’s as well as others due to the greater concentration of CP compared to traditional
corn fed diets (NRC, 1998; Stein et al., 2006; Varel et al., 2008). Greater concentrations of other
AA’s such as methionine and cysteine along with increased concentrations of sulfates in diets
high in either WDGS or DDGS lead to an increase in reduced S or sulfides that can potentially
form elevated levels of emitted H2S from the manure compared with conventional corn-base
diets (Shurson et al., 1998; Varel et al., 2008).

7

ROLES OF SULFUR IN THE BODY
Importance of dietary S
Many macro and micro minerals are required by ruminal bacteria and the host ruminant
animal. Sulfur is among these mineral requirements as it is a necessary component of amino
acids cysteine and methionine (Kung, 2008), as well as the B-vitamins biotin and thiamine along
with other organic compounds (Crawford, 2007). Adequate levels of S in ruminant diets are also
essential for healthy rumen micro-flora and animal performance (Loneragan et al., 2001). Sulfur
deficiency can lead to decreased microbial protein synthesis, decreased microbial population,
decreased OM digestibility, and depressed lactate utilization (Whanger and Matrone, 1970;
Rumsey, 1978). Cattle fed diets deficient in S often suffer anorexia, weight loss, signs of
depression, lethargy, and death (Starks et al., 1953; Slyter et al., 1988; NRC, 1996). Concern
about S consumption in the feed is becoming a more popular issue due to feeding high levels of
DGS, which has nearly a 3-fold greater concentration of S compared to corn. Distiller’s grain
with soluble creates other challenges because of the variability of S and minerals found in the coproduct between and within processing facilities.
Sulfate-reducing bacteria
The NRC (2005) reports that dietary S for ruminant animals should be between 0.18 and
0.24% of DM to support microbial growth; and to provide an adequate level of S-containing
compounds for the host animal (Crawford, 2007). Ruminant animals are capable of utilizing both
organic and inorganic forms of S due to sulfate-reducing bacteria (SRB) that exist within the
rumen via anaerobic respiration (Liamleam and Annechhatre, 2007). Sulfate-reducing bacteria
-

-

2-

are capable of reducing inorganic S compounds, particularly sulfate (SO4 ) to sulfides (HS , S ,
-

S°, or HSO3 ) as part of a process to form microbial protein or for absorption and oxidation to
8

sulfate within the liver (Anderson, 1956; Kandylis, 1983; Fron et al., 1990). Inorganic S can be
found in the form of ammonium sulfate ((NH4)2SO4), copper sulfate (CuSO4), potassium
sulfate (K2SO4), calcium sulfate (CaSO4), and sodium sulfate (Na2SO4) along with others
(Kung, 2008). The site of digestion for organic compounds synthesized by microbes usually
takes place in the abomasum and small intestine. The organic compounds are absorbed for
utilization by the host animal (Block et al., 1952; Anderson, 1956; Fron et al., 1990).
The SRB are grouped according to their different mechanisms used to reduce S to
sulfides. The two different mechanisms used to reduce S are assimilatory processes or
dissimilatory processes. Typically, a dissimilatory process is used for energy production, while
-

an assimilatory process is used to reduce SO4 to form biological compounds necessary for cell
survival (Odom and Singleton, 1993; Kung, 2008). Both forms of SRB exist within the rumen
-

-

2-

but dissimilatory groups are responsible for the reduction of SO4 to HS and S to allow
protonation of S to form H2S. Many forms of bacteria are capable of forming sulfides; however,
Desulfovibrio organisms are the main SRB in the rumen (Cumming et al., 1995; Kung, 2008).
Studies from Cumming and others (1995) showed that SRB did not increase proportionally with
the increase of S concentration in the diet. However, adaptation to high levels of S was noted
after 10 to 12 d as ruminal organisms began to have a greater capacity to produce sulfides. The
rate of sulfate reduction by ruminal bacteria increased with high dietary S according to studies
conducted by Oliveira et al. (1997).
Hydrogen sulfide production in the rumen
The amount of S-containing compounds found inside the rumen will determine the extent
of dissimilatory sulfate reduction from ruminal microbes (Kung, 2008). According to Odom and
9

Singleton (1993), the sulfide compounds that are predominantly formed in the dissimilatory
2-

-

-

process are S , S°, HS , or HSO3 . These reduced forms of sulfide have a pKa value near 7 and
are readily protonated inside a rumen that typically has a pH range of 5.5 to 7.2. This protonation
contributes to most of the H2S produced in the rumen. A small amount remains in the liquid
phase in a variety of S-containing compounds (Kung, 2008). The increase of S in diets from
feeding high levels of DGS not only contributes to a possible increase in H2S production, but
could negatively impact animal performance and health.

10

FEEDING HIGH SULFUR DIETS
Effects of high S diets on performance and health
Several studies suggest that high levels of dietary S lead to decreased feed intake and
feed efficiency. Bouchard and Conrad (1974) reported lower DM dietary intake in dairy cows
when S was included in the diet at concentrations of 0.35% or greater. Bolsen et al. (1973)
reported a 32% reduction in feed intake when cattle were fed 0.41% S with high concentrate
diets. A study from Rumsey (1978) showed similar results, as the addition of high S levels in
feedlot diets decreased feed intake and weight of steers fed high concentrate diets. The addition
of 0.5% S (as calcium sulfate or sodium sulfate) in lambs decreased growth rate, depressed feed
intake, and adversely influenced feed conversion (Johnson et al., 1968; Kandylis, 1983). A more
recent study from Zinn et al. (1997) reported cattle diets with moderately excessive levels of S
(0.25% of DM) had detrimental effects on ADG, DMI, G:F ratio, dietary NE, and longissimus
muscle area. In addition to decreasing animal performance; high dietary S can also lead to Stoxicity, polioencephalomalacia (PEM), decreased liver Cu stores (Smart et al., 1986), diarrhea,
increased H2S emissions, and death (Bird, 1972; Bulgin et al., 1996; Loneragan et al., 2001).
Sulfur toxicity
Ingestion of large amounts of S in ruminant animals can lead to acute S-toxicosis and
even death. Although S can be tolerated at high levels in monogastrics, ruminal bacteria are
-

2-

capable of reducing S usually in the form of SO4 to S , which is readily absorbed into the
blood stream through the rumen wall and lungs. Once S is absorbed in the bloodstream, it can
cause several metabolic and respiratory problems to the animal (Bray, 1969; Kung, 2008).
During the process of eructation and inhalation, ruminant animals absorb H2S gas in the lungs

11

where H2S enters the blood stream (Bird, 1972; Gould, 1998). The immediate signs associated
with S-toxicosis include thrashing, kicking at the stomach, staggering, and moaning followed by
death within 48 h. This short time frame suggests that ruminal bacteria have a high capacity to
produce sulfide without the need for adaptation (Kung, 2008). High levels of sulfide in ruminal
gas have been reported to cause respiratory distress, decreased feed intake, and decreased
ruminal motility (Bird, 1972; McAllister et al., 1992).
Oxidative metabolism and production of ATP are negatively affected by excess S through
inhibition of: carbonic anhydrase, an enzyme that forms bicarbonate; dopa oxidase, which
produces melanin; along with catalase and peroxidase enzymes that breakdown hydrogen
peroxides (H2O2) to water (H2O) and oxygen (O2). Additionally, dehydrogenases and
dipeptidases are affected, decreasing hydrolysis of AA in the GI tract (Short and Edwards, 1989).
In addition, sulfides are also thought to block the enzyme cytochrome c oxidase, which can cause
several metabolic disorders (Kung, 2008). Sulfides in the blood stream may also hinder oxygen
transport to tissues by binding to hemoglobin creating sulfhemoglobin. Respiration may also be
effected by sulfides through inhibition of normal sensory functions of the carotid body
(chemoreceptors), which help regulate and maintain partial pressure of O2 and carbon dioxide
(CO2) (Bulgin et al., 1996; Kung, 2008).
Polioencephalomalacia
Polioencephalomalacia is a disorder of the central nervous system that softens the grey
matter of the brain in ruminant animals and is often associated with a deficiency in thiamine or
consumption of plants containing high levels of thiaminase (Goonerate et al., 1989; Olkowski et
al., 1992). Excess S has an antagonist relationship with thiamine, rendering thiamine biologically
12

unavailable to the animal and subsequently putting the ruminant animal at risk of PEM. Clinical
symptoms attributed with PEM include blindness, head pressing, and circling. If PEM goes
untreated; symptoms progress to lameness, convulsions, and death (Merck, 1991; Kung, 2008).
Polioencephalomalacia can be easily determined by necropsy of the brain and heart. Thiamine is
a cofactor of thiamine pyrophosphate (TPP) for the TCA cycle and the pentose shunt, and
lesions will be present in the brain and heart when thiamine is deficient (Morck, 1993).
Symptoms of PEM have been reported in cattle fed diets between 0.4 to 0.5% S (Gould et al.,
1991), but in some cases cattle have been fed up to 1.5% S without signs of PEM or S-toxicosis
(Slyter el al., 1986). This is evidence that a likely interaction does occur with S and other
micronutrients within the rumen that might determine the effect and rate of S that becomes
reduced and absorbed either through the rumen wall or lungs (Kung, 2008).
Risks associated with hydrogen sulfide emissions
High levels of sulfates and other forms of S in diets are reduced in the rumen by SRB to
produce H2S that are not only hazardous to the animal but can cause potential air quality issues
by contributing to environmental concentration of H2S gas (Powers et al., 2006). Excess
generation of H2S in the rumen will depress normal rumen function and could lead to respiratory
problems (Kandylis, 1983), since nearly 60% of belched gas can be inhaled into the lungs
(Bulgin et al., 1996). Hydrogen sulfide gas is also very dangerous for humans to inhale.
Exposure to H2S at concentrations between 200 to 500 ppm has resulted in sudden onset of
hemorrhagic pulmonary edema that can be fatal in humans. At concentrations greater than 2000
ppm of H2S, respiratory paralysis occurs after only 1 or 2 breaths and is followed by convulsions
and death within minutes (Osweiler et al., 1985; Gerber et al., 1991). Even at lower
13

concentrations (50 to 200 ppm), H2S could cause respiratory irritation in humans (Green et al.,
1991). Long term exposure to low levels of H2S can cause chronic respiratory problems and
ocular irritation (Hays et al., 1972). Increased sulfides in the air and rain can be corrosive to
structures, particularly to iron and steel (Odom and Singleton, 1993). Excess H2S in ambient air
can have a detrimental impact to the environment, such as acid rain and eutrophication (Guanghui et al., 2006).
Manure pits beneath animal confinement facilities and slatted floor feedlots allow
decomposition of manure, which is a main contributor to H2S production. Hydrogen sulfide has
a low solubility in water and therefore will mostly remain trapped in bubbles in the manure
(Pickrell, 1991; Hooser et al., 2000). Agitation of the manure during removal can cause a rapid
release of H2S at lethal concentrations within and surrounding confinement facilities and/or
feedlots (Osweiler et al., 1985; Donham et al., 1988). Increasing levels of protein or other Scontaining organic matter in animal diets may lead to an increase of H2S in the manure and a
greater potential risk during manure handling and removal to both the humans and animals
(Hooser et al., 2000). These potential dangers and implications associated with total reduced
sulfur (TRS) have led to stricter regulations on confinement and feedlot operations to control and
monitor concentrations of TRS (Koelsch et al., 2004).

14

METHODS TO MEASURE SULFUR AND HYDROGEN SULFIDE EMISSIONS
Portable field analyzers
Efforts to improve soil and air quality along with industry concern to regulate
concentrations of dietary S have influenced the development and methods for measuring S. This
allows better diet formulation for ruminant animals along with offering better strategies for
proper waste management to decrease environmental effects. Measuring S and sulfides can be
difficult due to the volatile nature of S-containing compounds, particularly H2S (Shurson et al.,
1999). Reduced forms of S are very reactive in the air which causes a rapid inter-conversion of
various S forms (Spoelstra, 1980). Methods to measure sulfates and sulfide compounds were
originally developed for soil tests. Gravimetric and turbidimetric procedures are common
methods used to measure sulfates in wastewater (Sawyer and McCarty, 1978). Methods to
measure sulfides include colorimetric or volumetric methods. Both methods pose limitations in
separating inorganic from organic S, as well as differentiating elemental S from sulfate to
determine percentage of sulfide (Shurson et al., 1999). A study was conducted at the University
of Nebraska to determine H2S concentrations near beef cattle feedlots. The study used two
Jerome 631-S analyzers with memory to survey TRS concentrations at 15-minute intervals with
the analyzers placed approximately one meter from the ground surface (Koelsch et al., 2004).
The placement of analyzers was important because H2S gas is heavier than air and tends to be at
highest concentrations near the surface of manure storage (Shurson et al., 1999). Jerome 631
analyzers were developed by Arizona Instrument Corporation, Jerome Instrument Division
(Tchobanoglous and Burton, 1991). Similar portable H2S analyzer by ASTM (1996) can

15

continuously measure H2S as low as 1 ppb by measuring the rate of change of reflectance. The
cost associated with these instruments may limit their use in the field.
Ruminal gas cap
Sulfate-reducing bacteria in the rumen convert S compounds into H2S, which
accumulates in the area between the surface of ruminal fluid and the ruminal wall. Ruminal gas
caps have been used extensively as a way to measure H2S concentrations within the rumen of
cattle. A common method for the gas cap procedure is to make an incision on the left paralumber
fossa. An 18 gauge 89 mm spinal needle is inserted through the prepared body wall into the
ruminal gas cap. The needle is then attached to a calibrated H2S detector tube. A volumetric gas
sampling pump draws the gas from the ruminal gas cap into the detector; usually 50-300 ml of
gas is collected to obtain H2S readings (Gould et al., 1997 and Loneragan et al., 1998). Ruminal
gas caps are effective in determining enteric H2S production within the rumen but does not
account for gas produced further down the GI tract or from excreta. Another disadvantage
associated with using ruminal gas caps is several gases cannot be recorded at a time, making it
difficult to determine total emissions. Vannes et al. (2009) conducted a study at University
Nebraska to measure H2S levels post feeding. The study used cannulated cattle and a gas
collection device was inserted directly inside the rumen of each steer. Hydrogen sulfide levels
within the rumen were then determined by pulling samples from the gas collection device and
performing an in vitro assay to measure sulfide.

16

In vitro sulfide determination
Acidic conversion of sulfide to methylene blue is very specific and potentially the most
sensitive assay available to accurately measure H2S. This method is conducted by trapping H2S
gas in water with a pH 8 or greater followed by the addition to 2 reagents, first the addition of 7.2
N hydrochloric acid (HCl) with 0.02 M N,N-dimethyl-p-phenylenediamine (DPD) then the
addition of 1.2 N HCl with 0.03 M ferric chloride is immediately followed. Samples are quickly
vortexed and placed in the dark to allow proper formation of methylene blue (MB). Greater
concentrations of H2S in solution will produce a darker blue color. Samples can then be plated
and ran through a spectrophotometer with a wavelength near 650 nm to determine absorption.
Absorption levels are then compared to a standard curve to determine concentration of H2S in
each solution (Siegel, 1964). Methylene blue color is formed as the DPD reagent is oxidized by
3+

iron (Fe ), which reacts with H2S (Moneras et al., 2010).
Animal air chambers
Recent research has been conducted by housing livestock animals in open circuit
respirometers. Much of the data currently has been reported from studies at Iowa State
University and Michigan State University. The Animal Air Quality Research Facility (AAQRF)
at Michigan State University measures incoming or ambient air gas concentrations of O2, CO2,
methane (CH4), non-methane total hydrocarbons (NMTHC), H2S, sulfur dioxide (SO2),
-

ammonia (NH3), nitrous oxide (N2O), and nitrogen oxides (NOx; NO2 and NO ). Expelled air
from animal chamber rooms is then adjusted for incoming air gas concentrations, temperature
(C∘), and air-flow (m /min). Sulfide containing gas concentrations are measured using a TEI
3

17

450i Pulsed Fluorescence SO2-H2S-CS Analyzer (Thermo Fisher Scientific, Franklin, MA,
USA). The 450i analyzer operates by H2S converting to SO2 by exciting molecules under
different wavelengths using ultraviolet (UV) light (Thermo Fisher Scientific, Franklin, MA,
USA). Ammonia and NOx gas concentrations were measured using a TEI 17C
Chemiluminescence Analyzer (Thermo Fisher Scientific, Waltham, MA, USA) while N2O is
measured using an Innova 1412 Photoacoustic Field Gas Monitor. Methane and NMTHC gas
concentrations were measured using a TEI 55C Gas Chromatographic Analyzer and O2 and CO2
were measured using a BINOS 100 2M Dual-Channel Infrared Gas Analyzer.

18

STRATEGIES TO MITIGATE HYDROGEN SULFIDE PRODUCTION
Biocides
Biocides such as hypochlorite (Odom and Singleton, 1993), methylenebis thiocyanate (Zou and
King, 1995), and gentamicin (Tanimoto et al., 1989) have been used in industrial situations to
control sulfide production. Although biocides may be able to limit SRB and reduce production of
S-containing emissions, they are not recommended for use in ruminant diets because of their
broad anti-microbial spectrum which can negatively impact ruminal fermentation and can be
highly toxic to the animal (Kung, 2008).
Ionophores
Ionophores have been considered as a strategy to manipulate production of desirable end
products such as VFAs and microbial proteins and minimize production of undesirable
greenhouse gases (Kung, 2008), such as CH4, CO2, and N2O. RumensinTM (monensin) is
commonly used as a feed additive in ruminant diets to improve feed efficiency. Monensin is an
ionophore capable of altering ruminal fermentation by inhibition of hydrogen-producing bacteria
that have a gram-positive cell wall structure. Antibiotics and ionophores are typically
administered for resistance against pathogenic gram-positive bacteria (Nagaraja, 1995). Sulfatereducing bacteria are gram-negative, so ionophores would not have a direct effect on inhibiting
sulfide production. Indirect effects are thought to occur when monensin and other ionophores are
fed. A possible example would be that monensin selects against hydrogen and formate producing
bacteria that could result in the decrease of CH4 and H2S production (Kung, 2008).
Consequently, more propionate and less acetate and butyrate are produced by selecting against
formate producing bacteria (Van Nevel and Demeyer, 1996). Propionate is a VFA that is a
product commonly found in ruminants fed high concentrate diets and yields 34 ATP/mol of
19

glucose. Acetate and butyrate are other major VFA’s that are end products of digestion in
ruminants but only yield 20 and 25 ATP/mol of glucose, respectively.
Both methanogens and SRB are dependent on hydrogen as a substrate to form CH4 and
H2S, repectively. By inhibiting production of hydrogen, methanogens and SRB would be
depleted, so theoretically ionophores would decrease CH4 and H2S emissions (Van Nevel and
Demeyer, 1996). An in vitro study conducted by Quinn et al. (2009) indicated that the inclusion
of 3 different ionophores (lasalocid sodium and monensin sodium at 5 mg/L or laidlomycin
propionate at 1.65 mg/L) and 2 antibiotics (chlortetracycline hydrochloride at 5 mg/L and tylosin
tartarate at 1.25 mg/L) in the presence of S had similar acetate, propionate, and
acetate:propionate as the control culture. In addition, when S was approximately 0.42% of
substrate DM, the 3 ionophores and 2 antibiotics did not affect production of H2S gas in an in
vitro rumen culture system. Even if the inclusion of ionophores did increase propionate and
decreasing greenhouse gas (GHG) production, there is concern that adaptation may occur in the
ruminal microbiota and inhibition of H-dependent bacteria may be deminished over time (Van
Nevel and Demeyer, 1996). More studies should be conducted to determinine if ionophores and
antibiotics are effective at enhancing greater propionate production and decreasing CH4 and H2S
emissions.
Effect of feeding urea on S and N balance
Protein rich feeds are major contributors to S concentration in ruminant diets. One
consideration to decrease H2S emissions is to feed a ration with decreased levels of dietary S
while maintaining high levels of N within the diet. The use of urea as a non-protein N (NPN)

20

supplement in ruminant rations can dramatically reduce dietary S. In many rations where urea is
fed, supplementation of S may be required to meet dietary requirements. Sulfur deficiencies can
limit NPN utilization in purified diets but supplementation with inorganic sulfate can negate
issues associated with S deficiencies (Thomas et al., 1951). In order to feed urea as a source of
NPN and to lower dietary S as a strategy to decrease H2S emissions, adequate S and N balance
must be met to maximize utilization. Elemental S has been used in ruminant diets deficient in S
but is poorly utilized compared to Na2SO4 and methionine (Spais et al., 1968; Johnson et al.,
1971). Some forms of supplemental methionine (methionine hydroxyl) have shown negative
effects on DMI and milk protein production in dairy cattle (Goodrich and Tillman, 1966; Bray
and Hemsley, 1969; Rosser et al., 1971; Salsbury and Chandler, 1971). A study conducted by
Starks et al. (1953) suggested that lambs retained more N and S when elemental S was added to
diets supplemented with urea. The study compared 2 homo-nitrogenous diets at 2.46 and 2.47%
of dietary DM and two S levels (0.062% versus 0.705% of DMI).
Studies on urea with S and N balance are important on establishing that a relationship
between N and S does exist for retention rates and to maximize utilization of NPN. However, in
the case of feeding DGS where high concentrations of CP and S are present, feeding urea in most
cases would not be practical because high levels of N would be present in moderate to high
levels of DGS diets. To utilize DGS, other strategies should be considered to decrease H2S
emissions.
Sulfur, molybdenum, and copper compound
Molybdate (MoO4) has been identified as a compound that is capable of inhibiting SRB.
It is thought that MoO4 works as an analog of sulfate, blocking the sulfate activation step that is
21

catalyzed by ATP sulfurylase (Oremland and Capone, 1988). Conflicting research has been
reported on whether MoO4 specifically inhibits SRB or if it has inhibitory affects on other
ruminal bacteria as well. Jones et al., (1982) concluded that methanogenesis is inhibited by
inclusion of MoO4. Whereas a study from Westerman and Ahring (1987) showed that low levels
of MoO4 (1 mM) actually increased CH4 production. A study from Kung (2008) demonstrated
MoO4 was a specific inhibitor of SRB and did not have any effect on CH4 or hydrogen
production. Sodium molybdate decreased H2S concentrations in ruminal gas caps with cattle fed
high S diets, however results were not consistent among cattle (Loneragan et al., 1998). In
addition, the study conducted by Loneragan et al. (1998) demonstrated that inclusion of MoO4
dramatically decreased liver Cu stores in cattle.
Molybdenum (Mo) and S react to form tetrathiomolybdates that then react with copper
(Cu) and particulate matter in the rumen, forming highly stable compounds that cannot be
digested and absorbed (Allen and Gawthorne, 1987; Suttle, 1991). When Mo intake exceeds 1
mg/kg of DMI, a reduction in Cu absorption in ruminant animals usually results (Suttle and
Field, 1983; Suttle et al., 1984). The synergetic effect between Mo and S begins with the
substitution of S for oxygen in the MoO4
2-

2-

ion to ultimately yield tetrathiomolybdate (MoS4

2-

2-

2-

MoO4  MoO3S  MoO2S2  MoOS3  MoS4

2-

2-

Tetrothiomolybdate has the potential to bind ruminal Cu ions to form MoS4Cu, rendering the
entire complex biologically unavailable to the animal. The compound formed between S, Mo,

22

).

and Cu molecules in plasma is highly stable and usually is a result of excess Mo in the diet,
exceeding 10 mg/kg of DMI (Mills et al., 1978; Mills, 1980).
Molybdenum toxicity and Cu deficiency are often areas of concern when the MoS4Cu
compound forms. Symptoms that are often expressed by Mo toxicity include scouring,
achromotrichia, anemia, and weight loss (Lesperance et al, 1985). Lesperance et al. (1985) found
that feeding Mo as high as 100 ppm in the diet led to high retention rates of Mo (105 mg/day)
and elevated plasma Cu levels. The authors recommended plasma and urinary Mo were both
effective indicators of Mo intake. Another study from Marcilese et al. (1970) also indicated
feeding Mo increased Cu excretion in the urine of sheep. The increase in Cu excretion is thought
to be caused by thiomolybdates binding to albumin to cause a conformational change in the
protein, and thus increases the affinity of albumin for Cu (Woods and Mason, 1987). Increasing
levels of Mo in ruminant diets requires an increase in dietary Cu to prevent symptoms of
molybdenosis. Ingestion of high levels of Mo over time may also cause hypocupremia
(Underwood, 1977). Because of the interrelationship between S, Mo, and Cu; supplemental Mo
and Cu may serve as a strategy to prevent reduction of dietary S and therefore restrict production
of H2S gas in ruminant animals.
Iron binding effects with sulfides
Iron (Fe) may inhibit Cu reserves in ruminant diets at inclusion levels as low as 250 mg
Fe/kg of DMI (Mason, 1979). Also, Fe

2+

may bind S

2-

to form an insoluble ferrous sulfide
2-

(FeS) complex. Although not well understood, it is thought the S is donated by the Cu binding
2-

transport proteins in the mucosa which are rich in sulfhydryl groups. With the loss of S , the
formation of the mercaptide bonds are blocked, which are necessary for binding Cu ions (Mason,
23

1979). Ferrous sulfide complex mechanism is not well understood, but it is thought that FeS is
formed in the rumen and becomes soluble in the abomasum where the sulfide may dissociate and
form insoluble complexes with Cu (Gangelback et al., 1994; Suttle et al., 1984).
Sulfide inhibition by 9,10 anthraquinone
Another compound- 9,10 anthraquinone (AQ) has been reported to decrease CH4
production in laboratory in vitro studies (Garcia-Lopez et al., 1996) and inhibit reduction of
sulfate in the rumen (Hession et al., 1995; Kung et al., 1996; Bracht and Kung, 1997; Kung et al.,
1998). Anthraquinone decreased sulfide production in a diet containing 1.09% S to levels below
that found in a diet with only 0.29% S (Kung, 2008). Inhibition of sulfide production from AQ is
caused by possible uncoupling of the electron transport chain due to the redox potential of
anthraquinones. Without sufficient energy in the form of ATP, SRB cannot continue to reduce
ruminal S to produce sulfides. By restricting production of sulfides, H2S formation would be
limited (Cooling et al., 1996; Kung, 2008).

24

REVIEW OF RUMINANT GAS EMISSIONS
Little research has been conducted in animal air chamber rooms with feedlot cattle fed
high levels of S and the effects on H2S emissions. Furthermore, limited research is available on
strategies to mitigate H2S emissions in ruminant animals using in vivo forms of measurement.
Feedlot rations with high levels of DGS typically have high concentrations of S. Increased
demand for ethanol and growing numbers of ethanol plants in the USA has provided greater
production and availability of DGS for implementation in animal diets. Although DGS may
serve as an economical feed alternative in place of corn and SBM, feeding DGS does present
challenges due to the variability of the feed and the high dietary mineral concentrations. Among
these challenges, ruminant animals are often put at risk for possible health issues related to
mineral toxicities associated with DGS diets. In addition, feeding DGS increases environment
risks with manure handling, GHG, and other hazardous emissions.
Continued research is vital to better understand effects DGS may have on emissions in
ruminant animals. The use of animal air chambers to measure emissions could offer new
information in the area of studying in vivo fermentation and gas emissions from ruminant
animals. Additionally, use of mineral compounds may be the most advantageous method to
decrease emissions while limiting negative effects on ruminal fermentation and animal health.

25

CHAPTER 2: AN INTRODUCTION AND OBJECTIVE STATEMENT
INTRODUCTION
The rapid growth in the ethanol industry over the last decade has led to a surplus of
distiller’s grain with soluble (DGS). Ethanol production is currently the second largest
component of corn demand in the U.S., accounting for 30% of the total gross corn utilization
during the 2008/09 marketing season (Renewable Fuels Association, 2010). Feeding DGS has
become a popular energy and protein replacement relative to corn and soybean meal in livestock
diets. According to the USDA Livestock & Grain Market News (July 15, 2011), dried distiller’s
grain with soluble (DDGS) has consistently offered a favorable price compared to corn over the
last 2 years. The increased availability and reasonable price has encouraged feedlot managers to
increase the levels of DGS in cattle diets. However, high inclusion levels of DGS can have
negative effects on animal health and the environment. During the dry milling process for
production of ethanol, starch is removed from the corn kernel, leaving fat, crude protein (CP),
fiber, and other minerals such as sulfur (S) and phosphorus (P) 3-fold more concentrated within
DGS compared to corn (Klopfenstein et al., 2008). Additionally, sulfuric acid is added during the
fermentation process as a cleaning agent and to regulate pH. This further contributes to the
increase of S in DGS (Vannes et al., 2009; Kelzer et al., 2010). Buckner et al. (2008) reported S
concentrations in DGS varying from 0.44% to as high as 1.5%. This increase in mineral
concentrations in DGS can compromise the animal’s health, create challenges in waste
management, as well as increase the concentrations of hazardous gas emissions from manure
storage. Part of the risk associated with feeding S to ruminant animals is the production of H2S
in the rumen from sulfate-reducing bacteria. Loneragan et al. (1998) reported H2S concentrations
in the ruminal gas cap as high as 18.77 mg/L or 13,500 ppm in cattle fed high S diets. Ruminal
26

H2S is inhaled into the lungs where it is absorbed and enters the bloodstream (NRC, 2005;
Crawford, 2007). It has been reported that 60% of the eructated H2S is absorbed in the lungs
(Bulgin et al., 1996). Therefore, cattle producing high concentrations of ruminal H2S from high
S diets should eructate approximately 40% of the enteric H2S produced into the environment.
Studies have reported that molybdate (MoO4) is capable of inhibiting sulfate-reducing
bacteria and decreasing H2S production in ruminal gas caps (Oremland and Capone, 1988;
Loneragan et al., 1998; Kung, 2008). Supplemental MoO4 may also inhibit methanogens,
resulting in a decrease of enteric CH4 production (Jones et al., 1982). Additionally, Molybdenum
(Mo) and S react to form tetrathiomolybdates that then react with copper (Cu) and particulate
matter in the rumen, forming highly stable compounds that cannot be digested and absorbed
(Allen and Gawthorne, 1987; Suttle, 1991). Because of the interrelationship between S, Mo, and
Cu; supplemental Mo and Cu may serve as a strategy to prevent reduction of dietary S and
therefore restrict production of H2S gas in ruminant animals.
Cattle diets with increased levels of DGS also offer elevated levels of nitrogen (N), which
may contribute to greater excreted N and emitted nitrogenous gases from the manure (Mackie et
al., 1998; Spiehs and Varel, 2009). Nitrogenous gases produce strong odors and can cause acid
rain from the elevated levels of environmental NH3 and NOx emissions, as well as contribute to
greenhouse gas (GHG) emissions by producing N2O gas (Pollution Prevention and Abatement
Handbook, 1998; Intergovernmental Panel on Climate Change, 2007).

27

OBJECTIVE STATEMENT
The objective of trial 1 was to determine what affects DDGS fed at 0, 40, and 60% of the
dietary DM would have on gas emissions generated from growing steers. We hypothesized that
S- and N-emissions would be greater as the level of DDGS increased in the cattle diet. Based on
other reports, we expect the steers to emit H2S gas from ruminal fermentation. The objective for
trial 2 was to determine whether supplementation of 8 ppm Mo and 90 ppm Cu into a 40%
DDGS diet would decrease H2S emissions from growing steers as well as determine how Mo
and Cu may affect other emissions. The use of Mo and Cu as a dietary treatment at 8 and 90
ppm, respectively, was hypothesized to mitigate H2S and possibly CH4 gas by either inhibitory
effects or by forming an insoluble compounds (MoS4Cu) in the rumen and manure. Lastly, the
third objective was to determine the relative amounts of gas emissions that originates directly
from the animal (ruminal and enteric) versus short-term manure storage.

28

CHAPTER 3: LEVELS OF DISTILLER’S GRAIN WITH SOLUBLE AND STRATEGIES TO
MITIGATE SULFUR-CONTAINING EMISSIONS
L.D. Cross, W.J. Powers, J.S. Liesman, and S.R. Rust
Michigan State University, East Lansing 48824
SUMMARY
A rising concern with feeding high levels of DGS is its high S content and the effects it
might have on S-containing emissions from gas produced in the rumen and excreted feces. Two
trials were conducted with 12 Holstein steers randomly assigned individual environmentallycontrolled rooms to monitor gas production. In trial 1, steers (3 treatments, 4 steers/treatment)
were assigned to diets containing either 0 (control), 40, or 60% DDGS. In trial 2, treatments were
the same except the 60% DDGS dietary treatment was replaced with a 40% DDGS diet as in trial
1 fortified with 8 ppm Mo and 90 ppm Cu (40% DDGS+) to potentially mitigate S-containing
gas emissions. Fecal bags were placed on steers at the end of each trial to determine the effects
of separating urine and feces on gas emissions. In trial 1, feeding DDGS increased H2S mass
output (P = 0.05) and output adjusted for DMI (P = 0.04) compared to emissions produced from
the control diet. In trial 2, the 40% DDGS+ diet tended to decrease H2S emissions when adjusted
for S-intake (P = 0.08). In both trials, placement of fecal bags to separate urine and feces nearly
abolished H2S emissions (P < 0.01), indicating that H2S gas was not directly emitted from
enteric ruminal fermentation in the animal. Supplementing Mo and Cu in cattle diets high in S
may be an effective strategy to decrease H2S emissions generated by cattle excreta.
Key words: distiller’s grain with soluble, hydrogen sulfide, sulfur dioxide, molybdenum, copper

29

INTRODUCTION
Growth in the ethanol industry has increased the availability of distiller’s grain with
soluble (DGS). The increased availability and reasonable price have encouraged feedlot
managers to include more DGS in cattle diets. During the dry milling process used to produce
ethanol and DGS, starch is removed from the corn kernel, leaving fat, crude protein (CP), fiber,
and other minerals such as sulfur (S) and phosphorus (P) nearly three times more concentrated
within DGS compared to corn (Klopfenstein et al., 2008). Sulfuric acid is added during the
fermentation process as a cleaning agent and to regulate pH, this increases the amount of S in
DGS as well (Vannes et al., 2009; Kelzer et al., 2010). Buckner et al. (2008) reported S
concentrations in DGS as high as 1.5%. High levels of S can compromise animal health and
increase the risk of S toxicity or Polioencephalomalacia (PEM). These conditions can severely
depress animal performance, and if untreated, even cause death. Another potential concern with
feeding higher levels of DGS is environmental pollution from increased hydrogen sulfide (H2S)
emissions (Varel et al., 2008). One possible strategy to mitigate H2S emissions is to add
supplemental copper (Cu) and molybdenum (Mo) to the diet to form a biologically unavailable
compound between Cu-Mo-S before dietary sulfates (SO4) can be reduced to H2S within the
rumen.
2-

Molybdenum and S react to form tetrathiomolybdates (MoS4 ) or (TM) that then react
with Cu and particulate matter in the rumen, forming a highly stable compound that is very
difficult for the animal to digest and absorb (Gould et al., 2002). The formation of this compound
is hypothesized to bind dietary sulfates before sulfate-reducing bacteria (SRB) within the rumen
can reduce the sulfate to H2S gas (Allen and Gawthorne, 1987; Suttle, 1991). However, feeding
30

excess Mo (>10 ppm) may put the animal at risk of Mo toxicity as well as Cu deficiency (Mills
et al., 1978; Mills, 1980).
In trial 1, cattle were fed dry distiller’s grain with soluble (DDGS) at levels of 0, 40, and
60% to determine the effects on S-containing emissions. Feeding high levels of DGS (dried or
wet) in livestock diets increases the concentrations of CP, S, and P in the diet. Increased S
concentrations cause an increase of H2S production within the ruminal gas cap (Lonergan et al.,
1998) and in theory would cause an increase of H2S emissions from the animal by eructation or
flatulation. In trial 2, cattle were fed diets containing levels of DDGS at 0, 40 and 40% with the
inclusion of 8 ppm Mo and 90 ppm Cu. Supplementing additional Mo and Cu should reduce
available dietary S and thereby mitigate H2S emissions. The purpose of both trials was to
determine if increased concentrations of dietary S through increased inclusion levels of DDGS
would cause increased emission of H2S gas and sulfur dioxide (SO2) gas, and to determine the
potential for supplemental Cu and Mo to reduce S-containing emissions.

31

MATERIALS AND METHODS
Gas collection
Two studies were conducted at Michigan State University at the Animal Air Quality
Research Facility (AAQRF) to address concerns associated with high inclusion levels of
distiller’s grain with soluble. Approval for this study was provided by the Michigan State
University Animal Care and Use Committee (AUF # 07/09-110-00). Individual
Environmentally-controlled rooms at AAQRF monitor incoming and outgoing air from the 12
rooms for concentrations of oxygen (O2), carbon dioxide (CO2), methane (CH4), non-methane
total hydrocarbons (NMTHC), H2S, sulfur dioxide (SO2), ammonia (NH3), nitrous oxide
-

(N2O), and nitrogen oxides (NOx; NO2 plus NO ). Before arrival to AAQRF, steers were held at
Michigan State University’s Beef Cattle Teaching and Research Center (BCTRC) where they
were weighed and vaccinated for prevention of clostridial infections using Ultrabac-7 (Pfizer,
New York, NY) and respiratory infection with Bovi-Shield GOLD® 5 (Pfizer, New York, NY).

At trial initiation; a two week adjustment time was allotted before steers were transported to
AAQRF. During this time, steers (n = 12) were started on a corn-based concentrate diet (control
diet). After the first week, steers (n = 8) were randomly adjusted to a 40% DDGS diet. The
control diet consisted of 81% high moisture corn (HMC), 10% corn silage (CS), 5% soybean
meal (SBM), and 4% mineral supplement. The 40% DDGS diet consisted of 40% DDGS, 46%
HMC, 10% CS, and 4% mineral supplement. Steers were halter trained to lead for safe handling
and to limit stress on the animal during the study at AAQRF. Four days prior to arrival at
AAQRF, steers were housed in metabolism stalls at BCTRC to adapt them to living conditions
that were similar to the individual, environmentally-controlled rooms at AAQRF. Steer weights
32

were taken prior to feeding the last 2 d before the steers were transported to AAQRF, animal
weights were taken post-study as well. After 10 d of being housed in individual environmentallycontrolled rooms during trial 1, 4 steers were randomly switched from the 40% DDGS diet to a
60% DDGS diet; establishing the 3 dietary treatments containing 0, 40, and 60% DDGS.
In both trials, 12 Holstein steers were placed into individual environmentally-controlled
rooms with four steers per dietary treatment. The dietary treatments for trials 1 and 2 are shown
in Table 3.1. Three levels of DGS (0, 40, and 60%) replaced corn in a finish diet in trial 1. All
diets in trial 1 were top dressed with thiamine (200 mg/d) for each animal as a preventive step
against PEM. Daily records were kept on eye and ear twitch count over 1 min during feeding
shift to monitor physical symptoms associated with PEM. Visual symptoms of PEM were not
detected in either trial 1 or trial 2. On d 5 of trial 1, a steer on the control diet had to be pulled off
the study due to poor feed intake and formation of hematoma near the left hook bone. In trial 2,
two levels of distiller’s grain with soluble were fed (0 and 40%). A third treatment was identical
to the 40% distiller’s grain with soluble but it was fortified with 90 ppm Cu and 8 ppm Mo. The
latter treatment was called 40% DDGS+. The source of Mo was sodium molybdate (Na2MoO4)
and the Cu source was copper chloride (CuCl2). Copper chloride was selected to minimize the
amount of additional S in the diet.

33

Table 3.1 Dietary ingredients and composition for trial 1 and 2
Trial 1
Ingredient, % of DM
1

DDGS
High moisture corn
Corn silage
Soybean meal
DGS supplement
BFS50 supplement
Total

Trial 2

40

60

Control

40

40+

81
10
5
4
100

40
46
10
4
100

60
26
10
4
100

81
10
5
4
100

40
46
10
4
100

60
26
10
4
100

73.6

74.5

6.4
7.2
19.9
33.5
3.9
19.1
4.5

7.1
7.0
19.6
33.3
3.9
18.8
5.0

4.7

4.7

Diet composition
76.0
68.6
% of DM
8.0
4.4
8.8
4.2
23.2
11.7
58.6
22.1
2.2
5.5
24.6
12.9
6.6
1.3

Dry matter, %

67.7

74.8

Ash
ADF
NDF
Starch
Ether extract
Crude protein
3
ADIP
Gross energy, Mcal/kg

5.6
5.9
11.8
60.3
2.9
12.9
1.5

7.4
7.1
19.0
33.5
4.6
21.5
5.5

4.0

4.3
4.4
BFS50 supplement
1.4
24.9
48.3
0.3
9.6
0.2
9.6
5.1
0.7
100

Ingredient, % of DM
Akey TM premix # 4TM*
Limestone
Soybean meal, 48% N
RumensinTM 80
TM salt
Vitamin E, 5%
Urea, 45% N
Potassium chloride
Selenium 90
Total
1
2

2

Control

4.4

DGS supplement
2.4
71.5
0.4
18.0
0.1
7.6
100

DDGS- dry distiller’s grain with soluble
40% + is fortified with supplemental 8 ppm molybdenum and 90 ppm copper

3

Acid detergent insoluble proteins (ADIP) represent a portion of undegradable intake protein
(UIP) that is completely indigestible to the cattle
* Akey TM premix # 4 composition: 9% Mg, 4% S, 0.02% Co, 1% Cu, 0.09% I, 2% Fe, 4% Mn,
0.03% Se, 4% Zn, 4,400,000 IU vitamin A, 550,000 IU vitamin D, and 5,500 IU vitamin E/kg
(Akey Inc., Lewisburg, OH)

34

An adaption period was provided for cattle assigned to the control and 40% DDGS diets
prior to their entry in the environmentally-controlled rooms. Steers assigned to the 60% DDGS
diet were adjusted from 40 to 60% DDGS during the first 10 d in the chamber during trial 1.
Cattle were allocated 1 d to adapt to the chamber rooms during trial 2. After the adjustment
period emissions were recorded over a 14 d period in trial 1 and 23 d period in trial 2, using the
last 4 d to determine mean emissions within treatment for both trials, which will be referred to as
phase 1. The last 4 d were used to represent mean emissions at or near a steady state.
Temperature was maintained near 16° C and air flow was closely regulated within a range of 17
3

m /min or 40 air changes per hour (ACH) while gaseous emissions were sampled throughout
each d. Humidity in each chamber room was recorded but was not regulated. Each chamber,
including ambient air recordings, were sampled for 15 min with the first 9.5 min used as a purge
period, leaving the last 5.5 min of sampling as the recorded data. Sampling cycle of the 12 rooms
plus the background air would take 3 h 15 min which allowed for 7 to 8 observations per room
per d. Exhausted air was sampled within an aluminum duct in each room where Teflon coated
sample lines would draw air to the gas analyzers by positive static pressure from each
individually-sealed chamber room. Each room was also fitted with an in-house manufactured
type-J thermocouple, and a Campbell Scientific

TM

HMP45C (Logan, Utah, Campbell Scientific,

Inc.) temperature and relative humidity probe. The gas analyzer used to measure S-containing
emissions was a pulsed fluorescence TEI 450i analyzer (Thermo Fisher Scientific, Franklin, MA,
USA) for H2S, and SO2. Pulsed fluorescence operates under the assumption that H2S can be
converted to SO2. Sulfur dioxide molecules absorb the ultraviolet (UV) light from a source lamp
within the analyzer. Ultraviolet light is pulsed to increase optical intensity and provide greater
35

UV energy. This allows for better detection of low concentrations of SO2. Sulfur dioxide
molecules decay to lower energy states, emitting UV light at different wavelengths (Thermo
Fisher Scientific, Franklin, MA, USA). Gas sampling and data collection were all computer
controlled by LabView 8.2.1 software and the FieldPoint system (National Instruments, Austin,
TX, USA). Analyzers reported gas in units of ppm; data were transposed into units of mg/min
(Table 3.2) as output emissions. The units were then adjusted to 24 h and expressed as mass unit
output, output adjusted for S intake (SI), and output adjusted for dry matter intake (DMI).

36

Table 3.2 Gas unit conversion chart
1. STP
2. m3/min

STP × air flow, m /min

3. L/min

(Air flow @ STP, m /min × 1000 L/ m )

1. ppm

A. Air Flow @ STP

(((temp,° C) + 273° K) × 0.967 atm)

Expired, ppm – incoming, ppm = emitted, ppm

B. Emitted gas
2.
C.

Volume of ideal gas @
0°C & 760 mm Hg

Units
cancel

1. mg/L

Adjusted equation for all gases

mg/min

3

3

3

Emitted gas, mg/kg × 0.000001 kg/mg
MW, g/mol/(22.414 L/mol) ×1000 mg/g
(A.3) × (B.2) × (C.1)

37

Feed, feces and urine collection
During each gas collection period for trial 1 and trial 2 (phase 1), daily samples of total
mixed ration (TMR) and feed weigh-back (orts) from each steer were collected and immediately
placed in a freezer for storage. Orts were monitored daily to adjust the amount of TMR offered to
110% of the previous day’s consumption. All feed samples were later freeze-dried and ground
through a Wiley Mill (Thomas Scientific) to a 1 mm particle size or smaller. A manure pan was
positioned behind each stall, and the stalls were set at a small slope to allow urine to flow back
for collection of urine and feces in the manure pan. Once feces and urine mixture exceeded a 5
cm depth inside the pan, manure was mixed and partial removal was performed to maintain a
minimum 5 cm depth. The removed excreta were weighed to calculate total excretion. An
approximate 0.5 kg sample of manure mixture was collected during each removal and
immediately stored in a freezer for later analysis. Excreta removal was conducted during the
morning shift, whereas feeding along with TMR and ort collections were conducted in the
afternoon. During each shift, chamber rooms and stalls were thoroughly cleaned and chamber
rooms were only entered if they were 5 chambers or more ahead of the chamber room being
sampled for emissions.
Feces and urine segregation using fecal bags
On d 15 of trial 1 and d 24 of trial 2, all manure and urine was removed and weighed
from each fecal pan. Fecal bags were placed on each steer and clean pans were put back in place
to collect urine only. Fecal and urine segregation took place during the last 3 d of trial 1 and the
last 6 d of trial 2. In trial 1, only the last 2 d were used to record mean emissions and the last 4 d
were used in trial 2. This duration of each trial will be referred to as phase 2. Urine pans were
pulled twice daily and filtered through a screen before urine weight and volume were recorded.

38

Two 1% aliquots were collected of the composited daily urine collected, one aliquot was treated
with 6 N HCl until urine pH reached 4 determined by a litmus paper test and the second sample
was left untreated. All urine samples were stored in a freezer for later analysis. Fecal bags were
removed from each steer and feces was weighed daily during the evening feeding. Mixed fecal
sub-samples were collected at 5% of daily fecal weight and immediately placed in a freezer.
Total mixed ration and orts samples continued to be collected during phase 2 and emissions were
recorded for later comparison with phase 1 emissions.
Collections and mineral analysis
Feed samples including TMR and orts were freeze-dried and ground through a 1 mm
screen in a Wiley Mill (Thomas Scientific) prior to mineral analysis. All samples excluding urine
samples were prepared in a microwave digestion system by adding 10 ml of nitric acid to 0.5g
feed and fecal samples. Samples in the 10 ml of nitric acid were covered in Saran

TM

wrap and

left in a ventilated hood over night. The samples were then transported in a pressurized Teflonlined digestion vessel and placed in the microwave digester under 1200W at 100% power with a
30 min ramp time, max PSI of 180, at 190°C for a 10 min hold time (Gengelback et al, 1994).
Post digestion vessels were allowed time to cool for 5 min and then 2 ml of 30% hydrogen
peroxide was added to each vessel and let sit unsealed for 15-30 min to allow more time to cool
down for handling. Each digested sample was then poured into a separate 25 ml volumetric flask
and vessels were rinsed with ddH2O. The rinsed water was then added to the volumetric flask
and additional ddH2O was included to bring the total volume within the volumetric flasks to 25
ml (CEM Corporation, 1999). Each 200 µl digest and urine sample were pipetted and diluted
with 5 ml of a solution containing 0.5% EDTA and Triton X-100, 1% ammonia hydroxide, 2%

39

propanol and 20 ppb of Sc, Rh, In, and Bi as internal standards. Samples were then analyzed for
mineral content using an Agilent 7500ce Inductively Coupled Plasma- Mass Spectrometer (ICPMS). Three modes were used to minimize spectral interferences for mineral analysis. Sulfur and
Cu were analyzed using He-mode and Mo was analyzed in non-gas mode. Urine and water
samples were also analyzed for S, Cu, and Mo using an Agilent 7500ce Inductively Coupled
Plasma-Mass Spectrometer (ICP-MS). Concentrations of feed, feces, water and urine were
determined using an Olympus AU 640e (Olympis America Inc., Center Valley, PA). The
+

Olympus ISE module uses ether membrane electrodes for cations sodium (Na ) and potassium
+

-

(K ) and a molecular oriented PVC membrane electrode for chloride (Cl ). Specific cations
+

+

-

(Na , K ) and anions (Cl ) develop an electrical potential with ions of interest according to the
Nernst Equation. The electrical potential is then compared to the Internal Reference Solution
(Block Scientific Inc.) and translated into voltage and then into the ion concentration of the
sample.
Water usage by the 12 chambers was measured daily. Daily water consumption per steer
was calculated by multiplying daily total usage by the ratio of individual chamber urine output to
total urine output for the 12 chambers.
Statistical analysis
Statistical analysis was performed using PROC MIXED sub-routine of SAS 9.2 (SAS
Inst., Inc., Cary, NC). Air flow (cfm) and H2S gas data (ppm) were passed through filters using
SAS 9.2 to eliminate any possible erroneous data that may have been caused from equipment
malfunction or from accidental entry of calibration data. Data mean output from unfiltered
compared to filtered data is provided in the appendices in Table A.1. Air flow that equaled zero
40

(n = 1430) and H2S input data greater than 0.10 ppm (n = 6) was filtered out of the data set
before analysis. The independent variables or class statements considered for analysis was phase,
dietary treatment (TRT), date, and chamber. Each model statement included TRT, phase, and the
phase by TRT interaction. Date and date by TRT were analyzed separately to determine day
effects for H2S and SO2 to help determine the appropriate days to represent mean emission
within a steady state. Chamber within treatment was used as the random effect for both phase
and day analysis. Both S-containing gases were analyzed as output mass units (mg/d), output per
S intake, and as output per DMI. Mean emissions (H2S and SO2), DMI, SI, and pH were all
tested for normal distribution using Shapiro-Wilk’s test and for homogeneous variance using a
Levene’s and Brown Forsythe’s test. In trial 1, mean emissions for all units of H2S and SO2
were natural log transformed to reduce the likelihood of homoscedasticity and normality.
Contrast statements were used to determine treatment differences within each phase. Statistical
analysis on performance data was determined using TRT as the independent variable and
average daily gain (ADG), DMI, body weight (BW), and gain to feed ratio (G:F) as the
dependent variables. Sulfur balance was analyzed similar to performance data only changing
independent variables to S in water, feed, urine, feces, gas, S retained, and percent S retained.
Statistical significance was declared at a P-value at or below 0.05 and trends at P-value at or
below 0.10.

41

RESULTS
Trial 1
In trial 1, cattle tended to gain less weight per d with the 60% DDGS diet (P = 0.06;
Table 3) compared to cattle on the 40% DDGS diet. Daily weight gain on the 40% and 60%
DDGS treatments were similar to control treatments. Dry matter intakes and feed to G:F were
similar among treatments. Cattle on 60% DDGS diet had numerically lower DMI and G:F than
the other treatments. Cattle fed the 40% DDGS diet tended to have lower digestibility compared
to cattle the fed 60% DDGS diet (P < 0.10). Performance data provided in Table 3 was
determined from the days post arrival up to the day cattle departed the air quality facility. Cattle
fed all treatments were in positive energy balance and had a rate of gain typical of cattle in
metabolism units (Depenbusch et al., 2009; Gunn et al., 2009). Due to natural log
transformations, upper confidence limits (UCL) were back transformed by taking the exponent of
the untranformed means plus the SEM and lower confidence limts (LCL) by taking the exponent
of the untranformed means minus the SEM (Table 4).

42

Table 3.3 Effects of dry distiller’s grain with soluble on performance in trial 1
Diet
Control

40% DDGS

Initial BW, kg

252

245

Final BW, kg

288

DMI, kg/d

6.07

Item

60% DDGS

1

SEM

P- value

246

22

0.97

287

274

20

0.84

6.36

5.74

0.84

0.84

ADG, kg

0.91

1.06

0.71

b

0.10

0.06

Gain: feed

0.153

0.183

0.125

0.03

0.28

b

2.46

0.10

b

2.48

0.09

DM digestibility, %
OM digestibility, %
1

ab

1

ab

69.26

ab

70.15

a

a

71.35

a

71.30

63.97
63.89

DDGS- dry distiller’s grain with soluble

a, b

Means without a common superscript within a row tend to differ (P ≤ 0.10)

43

Table 3.4 Effects of distiller’s grain with soluble levels on sulfur-containing emissions for phase 1 and 2 during trial 1
Phase 1
Item

0
‡

40
a

Phase 2
60

b

P- value

0
ab

40

60

TRT

Phase

TRT × Phase

0.20

< 0.01

0.28

UCL*

12.28
34.92

124.45
171.65

60.88
92.24

-5.25
12.26

9.91
28.58

3.92
21.10

LCL*

-5.23

86.65

35.77

-18.79

-5.03

-9.83

0.99
1.89
0.20

3.38
4.44
2.44

1.73
2.59
0.96

-0.46
0.24
-1.08

0.2
1.23
-0.13

0.20
0.87
-0.41

0.24

< 0.01

0.72

H2S, mg/d

H2S, mg/g SI
UCL
LCL
†‡

2.15
5.49
-0.47

19.02
25.83
13.51

9.76
14.40
6.01

ab

-0.95
1.54
-2.90

2.43
5.35
0.07

0.97
3.55
-1.11

0.16

< 0.01

0.29

SO2, mg/d
UCL
LCL

3.75
25.56
-12.15

-4.83
10.94
-16.83

11.77
32.76
-4.20

-19.4
-6.19
-29.04

-6.66
10.60
-19.37

-15.5
-3.10
-24.96

0.95

0.08

0.46

SO2, mg/g SI
UCL
LCL

0.26
1.24
-0.50

-0.11
0.63
-0.70

0.29
1.14
-0.38

-1.01
-0.26
-1.56

-0.09
0.66
-0.69

-0.10
0.64
-0.69

0.81

0.29

0.57

SO2, mg/kg DMI
UCL
LCL

0.56
4.86
-2.35

-0.80
2.28
-3.00

1.76
5.86
-1.17

-3.68
-1.39
-5.23

1.72
6.49
-1.54

-2.74
-0.44
-4.39

0.79

0.25

0.30

H2S, mg/kg DMI
UCL
LCL

a, b
†
‡

a

b

Means without common superscripts within a phase differ (P ≤ 0.05)

Means tend to show a linear effect within a phase (P ≤ 0.10)
Means tend to show a quadratic effect within a phase (P ≤ 0.10)

* Upper and lower confidence limits (UCL, LCL) express reliability of the estimated mean emissions. The mean emissions were all
back-transformed from a natural log transformation to satisfy normal distribution and equal variance
44

Phase 1
Mean emissions reported for phase 1 were determined using data from d 11 to d 14 which
represent steady state emissions (Table 3.4). Day effects were highly significant on S-containing
emissions during phase 1 (Table A.2). Emissions across treatments had similar daily variability
with no significant day by treatment interaction observed. Days selected to report means is
further illustrated in the appendices in Figures A.1-A.6.
The phase by treatment interactions were not significant for all parameters measured.
Consequently, the main effects of treatment and phase will be discussed separately. Treatment
differences are shown graphically in Figures A.7-A.12. Hydrogen sulfide emissions expressed in
mass units and per unit of DMI were greater for DGS diets as compared to the control (Table
3.4). There was a tendency (P < 0.10) for a quadratic response with the 40% DDGS diet having
the highest H2S emissions. Hydrogen sulfide expressed in mass units per unit of DMI also
expressed tendency (P < 0.10) for a linear response. Sulfur dioxide was similar among
treatments.
Phase 2
Mean emissions were reported (Table 3.4) from data collected on the last 2 d of the trial
(d 16 and 17) to best report emissions within a steady state (Figures A.1-A.9). A day effect was
reported for H2S emissions during phase 2 but treatment by day interaction was not significant
(Table A.2).
Separation of urine and feces significantly decreased H2S emissions (P < 0.01) when
expressed in mass units per d or as a function of SI and DMI. This suggests that most of the H2S
generated in phase 1 originated from the manure mixture and very little from enteric
45

fermentation in the animal. Feed, water, feces, and urine collections along with gas data during
the last 2 d of trial 1 were used to estimate total sulfur balance by comparing sulfur intake (SI) in
feed and water to sulfur expelled in the form of feces and urine. Sulfur intake increased (P =
0.01) with greater inclusion levels of DDGS (Table 3.5). Water intake was calculated as a
function of urine output to predict the contribution of water SI to total SI. Sulfur levels in feed
were greater in the 40% and 60% DDGS diets compared to control diets (P < 0.01). Expelled S
in urine and feces showed a similar trend as S intake increased as levels of DDGS increased.
Cattle fed 40% and 60% DDGS diets had greater sulfur in urine (P < 0.01) as compared to the
control diets. The amount of S retained on a mass unit basis as well as expressed as a percentage
was similar among treatments. A study conducted from Bouchard and Conrad (1972) found that
feeding dietary S above 0.3% of the diet DM using various types of sulfates resulted in greater S
retention. Feeding S at levels of 0.24% using K2SO4 + MgSO4 and Na2SO4 as sources of
supplemental sulfates resulted in retention levels in tissure near 40% and 30% in dairy cattle,
respectively (Bouchard and Conrad, 1972). Our results offer similar retention levels in cattle fed
high S diets; however, we did not see differences in the percent of S retained between low and
high S diets. Increasing levels of DDGS in cattle diets showed a numeric reduction in the percent
of S retained. The control diets had the lowest apparent digestibilty (53.47%) compared to the
40% and 60% DDGS diets, 74.94% and 72.75% respectively (P = 0.02). Our results for the
percent of apparent S digested agree with other studies. Boila and Golfman (1991) reported
apparent digestibility of S to be around 75% for diets with both low and high levels of S, 1.3 and
3.9 g/kg DM respectively. Previous work from Bouchard and Conrad (1973) reported apparent S
digestibility in the range of 70% to 80% for cattle fed 0.18% to 0.24% dietary S. Within the

46

current study, cattle on the control diet were fed 0.22% dietary S but only had a S digestibility of
53.47%.
The acidity of manure (feces and urine mixture) in phase 1 increased (P = 0.03) as the
level of dietary DDGS increased (Table 3.6). In phase 2, pH of fecese tended to be greater for the
40% DDGS treatment than for the control or 60% DDGS diet (P = 0.08). Urine pH was similar
among treatments. Aerobic and anaerobic digestion of organic residues in manure by bacteria
may result in sulfate reduction, contributing to emissions H2S and along with other S compounds
(Mackie et al., 1998; Spiehs and Varel, 2009). Inside the rumen, sulfides are readily protonated
within a pH range of 5.5 to 7.2 (Kung, 2008). It would be reasonable to presume that forms of
SRB in manure would favor environments within or near the same pH range. Manure collected
during phase 1 was the main source that contributed to H2S emissions. However, manure pH was
near or above pH 8. This pH increase compared to feces collected during phase 2 may be a result
of sulfate reduction and S protonation.

47

Table 3.5 Effects of distiller’s grain with soluble on total sulfur balance for trial 1 during phase 2
Diet
Item

Control

Feed S intake, g/d
1

a

13.25

a

40% DDGS
ab

34.90

ab

60% DDGS

SEM

P- value

Linear

Quadratic

44.47

b

6.08

0.01

< 0.01

0.90

b

0.02

0.02

< 0.01

0.53

b

6.08

0.01

< 0.01

0.90

b

2.69

< 0.01

< 0.01

0.72

Water S intake , g/d

0.07

Total S intake, g/d

13.34

Urinary S, g/d

1.73

Fecal S, g/d

6.20

7.12

11.52

b

1.15

0.01

0.01

0.07

-0.0125

0.0053

0.0031

0.017

0.72

0.47

0.70

c

2.56

< 0.01

< 0.01

0.22

ab

5.21

0.02

0.01

0.15

a

a
a

2

S emissions , g/d
Total S excreted, g/d
3

S digestibility , %

a

7.92

a

0.16

Contrast

ab

35.06

b

12.52

a

b

19.65

b

0.19

44.64
19.54

31.07

53.47

75.94

S retained, g/d

5.43

15.41

13.58

4.25

0.24

0.14

0.35

S retained, %

40.64

37.35

29.55

9.26

0.65

0.43

0.70

1
2
3

72.75

Water S intake was calculated as a function of urine output
Sulfur emissions during phase 2 were not different from zero
Digestibility calculated by 1- (fecal S/total S intake) × 100

a, b, c

Means without common superscripts within a row differ

* Sulfur balance was determined from data collected during phase 2 (2 d)

48

Table 3.6 Effects of feeding distiller’s grain with soluble on excreta pH and output during trial 1
Diet
Item

0% DDGS
1

a

Manure pH

7.73

2

a

Feces pH

60% DDGS

2

Dry fecal output , kg/d
2

Urine output , L/d

Linear

Quadratic

0.24

0.03

0.01

0.89

0.20

0.08

0.34

0.04

8.76

b

6.64

6.15

7.42

6.84

0.62

0.14

0.05

0.97

1.98

1.80

2.11

0.23

0.57

0.82

0.32

2.42

5.26

6.69

1.40

0.13

0.05

0.99

Manure- collected during phase 1; mixture of feces and urine
Feces and urine- collected separately during phase 2

a, b

P- value

ab

8.46

SEM

b

ab

8.67

2

2

40% DDGS

6.00

Urine pH

1

Contrast

Treatment means within a row without a common superscript differ

49

Trial 2
The purpose of trial 2 was to introduce appropriate levels of Mo and Cu into the diet to
bind dietary S before reduction to hydrogen sulfide occurred from ruminal SRB. Molybdenum
was added in the form of Na2MoO4 and Cu was provided through supplemental CuCl2 in the
40% DDGS+ diets. Sodium molybdate and CuCl2 were included to provide an addition 6 ppm
Mo and 60 ppm Cu, respectively. However, TMR analyses would indicate that 8 ppm Mo and 90
ppm Cu were included in the 40% DDGS+ diet. The effects of dietary treatments on animal
performance are shown in Table 3.7. Cattle were in positive energy balance and consuming
more than 2% of their BW. Growth and feed conversion efficiency were similar among
treatments. Molybdenum and Cu levels were greater (P < 0.01) for the DDGS+ diet. Dry matter
digestibility (P = 0.07) tended to be greater for cattle fed the control diet compared to the cattle
fed 40% DDGS+ diet.
Sulfur emissions were measured as mass units (mg/d), mass units per unit of SI (mg/g
SI), and as mass units per unit of DMI (mg/kg DMI). For all measures of H2S and SO2,
separation of feces and urine in phase 2 significantly decreased emissions (P < 0.01; Table 3.8).
As shown in trial 1, very little of the H2S emitted came directly from the animal but originates
from the manure in the fecal pans.

50

Table 3.7 Effects of dry distiller’s grain with soluble fortified with copper and molybdenum on
performance in trial 2
Diet
Control

40% DDGS

Initial BW, kg

300

Final BW, kg

342

DMI, kg/d

1

313

Item

40% DDGS+

2

SEM

P- value

312

16

0.82

351

339

16

0.86

8.07

7.12

7.49

0.47

0.40

ADG, kg

1.08

0.96

0.70

0.16

0.30

Gain: feed

0.132

0.131

0.091

0.02

0.20

DM digestibility, %

73.90

b

2.23

0.07

OM digestibility, %

74.41

66.66

b

2.35

0.11

b

0.40

< 0.01

4.84

< 0.01

a
a

a

Mo, ppm

1.63

Cu, ppm

12.89

1
2

a

ab

68.34

ab

69.57

a

1.38

65.49

9.49

a

b

22.29

111.51

DDGS- dry distiller’s grain with soluble
40 % DDGS+ is supplemented with 8 ppm Mo and 90 ppm Cu

a, b

Means without a common superscript within a row tended to differ

51

Table 3.8 Effects of dry distiller’s grain with soluble fortified with copper and molybdenum sulfur-containing emissions for phase
1 and 2 during trial 2
Phase 1
Item

0

40

40 +

0

40

40 +

SEM

TRT

Phase

TRT × Phase

< 0.01

< 0.01

< 0.01

58.25

514.84

419.36

b

-30.25

-24.56

-2.65

-

UCL*

88.50

570.02

470.20

-10.32

-3.93

20.47

-

LCL*

29.36

462.02

370.87

-47.83

-42.84

-23.42

-

H2S, mg/g SI

3.56

b

-2.54

-0.58

-0.05

1.14

< 0.01

< 0.01

0.03

H2S, mg/kg DMI

6.76

65.48

51.07

b

-5.22

-3.26

-0.72

-

< 0.01

< 0.01

< 0.01

UCL
LCL

10.24
3.55

72.22
58.74

57.18
44.96

-2.94
-7.22

-0.74
-5.50

2.07
-3.24

-

SO2, mg/d

3.80

15.71

20.55

-14.64

-7.42

-18.88

6.95

0.46

< 0.01

0.11

SO2, mg/g SI

0.19

0.34

0.39

-1.06

-0.23

-0.44

c

0.20

0.05

< 0.01

0.32

SO2, mg/kg DMI

0.40

1.98

2.40

-2.13

-1.17

-2.49

0.84

0.43

< 0.01

0.06

a

a

b

P- value

H2S, mg/d

a, b

a

Phase 2

b

11.78

b

8.53

b

c

Means without common superscripts within a phase differ (P ≤ 0.05)

* Upper and lower confidence limits (UCL, LCL); exponent of untransformed mean ± 1 SEM, express reliability of the estimated
mean emissions. The mean emissions were all back-transformed from a square root transformation to satisfy normal distribution and
equal variance. Standard error of the mean (SEM) is expressed for all untransformed means

52

Phase 1
Emissions during trial 2 for phase 1 were determined using a 4 d measurement interval (d
20 to d 23) which represented a pseudo-steady state condition (Table 3.8). Day variability was
highly significant in all measures of emissions for both H2S and SO2 (data not shown), however
the treatment by day interaction was non-significant for all expressed measurements. Daily
emission plots for both H2S and SO2 are provided in the appendices (Figures A.13-A.18) with
days used for each phase reported. Bar graphs are provided in the appendices for visual
comparisons of treatment means within each phase (Figures A.19-A.24).
A treatment by phase interaction was evident for H2S expressed in mass units (P < 0.01),
mass unit per SI (P = 0.03), and mass unit per DMI (P < 0.01; Table 3.8). Addition of DDGS
increased H2S emissions for all units measured (P < 0.01) compared to emissions from cattle fed
the control diet. Comparing an orthogonal contrast between 40% DDGS and 40% DDGS+
treatment would indicate a trend (P = 0.08) for a 27% reduction in H2S emissions with the
addition supplemental Cu and Mo. A 22% reduction in H2S emissions was also seen when mass
unts were adjusted for DMI with the 40% DDGS+ treatment; however, this difference was not
significant.
Phase 2
Emissions reported during trial 2 for phase 2 were determined from data collected on d
26 to d 29 (Table 3.8). Both H2S and SO2 produced negative emissions during phase 2. This is
likely caused by a greater in-air gas concentration compared to gas concentrations ouput from the
53

chambers. Lower concentrations within the chamber rooms compared to in-air concentrations
may be a result of emissions being near or below the detection limit of the analyzer. It is possible
that some H2S was being removed from the air via inhalation and absorption within the animal.
Sulfur dioxide emissions were less (P < 0.05) for the 40% DDGS diets compared to the control
diets on a mass unit per SI basis. However, caution should be observed when interpreting these
results as treatment means were all negative.
Feed, water, feces, and urine collections along with gas data during the last 4 d of trial 2
were used to estimate total S balance by comparing SI in feed and water to S excreted in the
form of feces, urine, and gas. Sulfur loss as gas was not different from zero during phase 2.
Dietary sulfur intake was greater for the 40% DDGS diets compared to S intake from steers on
the control diet (P < 0.01; Table 3.9). Similarly, excreted sulfur in urine (P < 0.01) and feces (P =
0.03) were also greater for both 40% DDGS diets compared to control diets. Sulfur in gas form
was emitted at low levels and contributed little to total expelled S. Amount of retained S was
determined by all unaccounted sulfur. Both DDGS diets had greater retained S (g/d) compared to
control diets (P < 0.01). Addition of Cu and Mo to the 40% DDGS diet did not lower S excreted
in feces or urine. It did slightly increase S emissions (P = 0.03) but the magnitude was very small
(not different from zero) relative to the amount S consumed and excreted in feces and urine.
Sulfur retention was similar among 40% DDGS treatments but differed the control diets on mass
unit basis (P < 0.01) and a percent of S retained (P = 0.03). Sulfur digestibility was greater (P <
0.01) for both DDGS diets as compared to the control diets as well. One can infer from this
observation the S in DDGS is more available than S in the basal or control diet.

54

Fecal (P = 0.01) and manure (P = 0.02) pH were greater for both 40% DDGS diets
compared to control (Table 3.10). Similar to the results from trial 1, the manure mixture had a
greater pH compared to the pH of feces or urine.

55

Table 3.9 Effect of distiller’s grain with soluble fortified with copper and molybdenum on
total sulfur balance for trial 2
Diet
Item

Control
a

40% DDGS
b

40% DDGS+
b

SEM

P- value

Feed S intake, g/d

11.51

35.41

38.91

2.11

< 0.01

Water S intake, g/d

0.18

0.19

0.22

0.03

0.55

Total S intake, g/d

11.69

b

2.12

< 0.01

Urinary S, g/d

3.35

19.80

b

1.70

< 0.01

Fecal S, g/d

7.40

b

0.47

0.03

b

0.005

0.03

b

1.86

< 0.01

75.94

b

3.28

< 0.01

b

1.38

< 0.01

b

4.0

0.03

a

a

b

19.85

a

1

b

9.12
a

S emissions , g/d

-0.036

Total S expelled, g/d

10.71

2

a

a

S digestibility , %

34.94

S retained, g/d

0.95

S retained, %
1
2

a

8.1

a

b

35.59

39.14

9.37

ab

-0.027

b

28.94

b

74.25

b

6.63

-0.011
29.16

9.96

ab

18.6

25.5

Sulfur emissions were not different from zero, despite apparent treatment differences
Digestibility calculated by 1- (fecal S/total S intake) × 100

a, b

Means without common superscripts within a row differ

* Sulfur balance was determined from data collected during phase 2 (4 d)

56

Table 3.10 Effects of feeding distiller’s grain with soluble on excreta pH and output during trial 2
Diet
Item

0% DDGS
1

a

Manure pH

7.23

2

a

8.21

b

40% DDGS+

SEM

P- value

b

0.20

0.01

b

8.09

2

Dry fecal output , kg/d
2

Urine output , L/d

5.70

5.56

0.16

0.02

8.05

2

Urine pH

2

b

4.92

Feces pH

1

40% DDGS

6.64

6.63

0.52

0.14

2.22

2.29

2.63

0.20

0.35

3.81

3.99

4.14

0.73

0.95

Manure- collected during phase 1; mixture of feces and urine
Feces and urine- collected separately during phase 2

a, b

Treatment means within a row without a common superscript differ

Table 3.11 Hydrogen sulfide gas emitted per gram of sulfur intake during phase 1 of each trial
Trial 1
Item

Trial 2

Control

40% DDGS

60% DDGS

Control

40% DDGS

40% DDGS+

H2S, mg/d

12.28

124.45

60.88

58.25

514.84

419.36

S Intake, mg/d

12711

38971

46165

17163

43278

50215

0.10

0.32

0.13

0.34

1.19

0.84

H2S per S intake, %

57

DISCUSSION
In trial 1, the objective was to determine if S-containing gases such as H2S and SO2
would increase from cattle due to greater inclusion levels of DDGS within the diet. Feeding
increased levels of DDGS would provide greater levels of protein or other S-containing organic
matter in cattle diets compared to the cattle fed the control diet. Greater dietary S could elevate
levels of emitted H2S in the manure, which creates a greater risk during manure handling and
removal to both humans and animals (Hooser et al., 2000). Additionally, work from Bull and
Vandersall (1973) and Spears et al. (1977) suggests that ruminal digestion may be affected by S
levels within the diet. Within phase 1 of trial 1, dietary SI increased from 13 g/d for the control
diet, to 39 g/d for the 40% DDGS diet, and 46 g/d for the 60% diet, respectively (Table 3.11).
However, increasing levels of dieatary S did not increase S-containing emissions when
comparing 40% DDGS diets to 60% DDGS diets. This may suggest a limited capacity for S
reduction in the rumen which was exceeded with the 60% DDGS diet. Cattle fed 40% DDGS
diets had the greatest percent of SI converted to H2S gas. Feeding DDGS diets above 40% may
not increase S-containing gas production due to affects on ruminal fermentation and reduction in
DMI. Comparison of daily H2S emissions from trials 1 and 2 (Table 3.11) show a much greater
H2S emission per unit of SI, which suggests that ruminal adaptation may be occurring. It is
possible the SRB are increased in number, efficiency, or both as the length of exposure to high S
diets increases. It is also reasonable to speculate the length of time with exposure to low pH
generated in high concentrate fed animals may facilitate sulfate reduction. This may also explain
the declined performance measures from cattle fed 60% DDGS.

58

Majority of S-containing emissions were contributed from H2S gas, while sulfur dioxide
emissions were not different from zero for both phase 1 and 2. The significant decrease of H2S
emissions when urine and feces were collected separately inside the chamber (phase 2) would
suggest that very little, if any, S-containing gases formed inside the rumen escape by eructation;
and that mixture of urine and feces is the main source of S-containing emissions, particularly
H2S gas. Daily variance of H2S emissions may have been an effect of agitation of manure inside
the collection vessel from cattle defecating or stepping back into the collection vessel. Hydrogen
sulfide has a low solubility in water and therefore will mostly remain trapped in bubbles in the
manure (Pickrell, 1991; Hooser et al., 2000); however, H2S escapes rapidly when manure is
agitated (Osweiler et al., 1985; Donham et al., 1988). When looking at emissions within each
day, H2S spikes were consistantly recorded around 6:00am (data not shown), the time at which
manure was removed to maintain a 2 in residual base in the pan.
The use of 8 ppm Mo and 90 ppm Cu as a supplemental feed additive to decrease Scontaining gases emitted from the animal suggested this strategy may be effective for reduction
of H2S emissions, particularly decreasing the proportion of SI that is converted to H2S gas.
When H2S emissions were adjusted on SI, a 27% decraese was detected between emissions from
cattle on 40% DDGS diets compared to cattle on 40% DDGS+ diets supplemented with an
additional Mo and Cu (P = 0.08). These results provide evidence that a reaction with dietary S
2-

may be forming tetrathiomolybdate (MoS4 ), which readily reacts with Cu to form an insoluble
compound, MoS4Cu (Hamsell et al., 2010).

59

In other studies that measured H2S in the ruminal gas cap, increased concentrations were
detected in cattle fed diets high S diets (Gould et al., 1997; Loneragan et al., 1998). Loneragan et
al. (1998) reported H2S concentrations in the ruminal gas cap as high as 18.77 mg/L or 13,500
ppm in cattle fed high S diets. Inhalation of eructated gas into the lungs is the likely route of H2S
absorption from cattle as protonated sulfides are unable to be absorbed through the rumen wall
(NRC, 2005; Crawford, 2007). Other reports have approximated 60% of eructated gases are
inhaled and enter the respiratory tract (Bulgin et al., 1996). In phase 2 of both trials, H2S gas was
decreased to undetectable levels. These results would suggest that almost all H2S emissions
recorded in phase 1 resulted from the mixture of urine and feces in the collection pan and not by
eructation from the cattle.
Maintaining a resident population of SRB in the fecal pan contributed to the elevated
emissions of H2S gas. The collection system used during phase 1 of each trial may more
accurately portray emissions from liquid manure handling and storage systems. Manure (feces
and urine) mixture during phase 1 was the main source of H2S emissions recorded within the
-

2-

chamber rooms. Molecular H2S begins to dissociate to HS or S

in liquid at pH 8 or higher. As

more sulfide is protonated and released as H2S gas, manure pH increases (Thistlethwayte, 1972;
Xue et al., 1998). Results from both trials agree with these reports that elevated manure pH
increases as manure S is protonated and emitted in the form of H2S. Additionally, temperature
also influences protonation of sulfides similar to pH; a temperature increase of 1° C has the same
effect as increasing the pH by 0.15 (Thistlethwayte, 1972; Xue et al., 1998). In the current study,
60

the averge room temperature had a set point around 16° C and did not deviate much from the set
point temperature. Therefore, temperature had likely little effect to the higher H2S emissions
reported and greater percent of dietary S emitted as H2S in trial 2 compared to trial 1 (Table 11).
Sulfur balance for trial 1 and trial 2 provided different retention levels in mass units and
as a percent of total S intake. Bioavailability of dietary S is reported to be dependent on total S
intake and form of S (Fron et al., 1990). Furthermore, available nitrogen (N) in the diet may be
metabolized resulting in greater levels of S excreted as sulfate and total S concentration in urine
(Salsbury et al., 1971; Fron et al., 1990). Increased levels of DDGS would have increased levels
of N in the diet, which could influence S retention. Bouchard and Conrad (1972) reported the
greatest S retention levels in cattle that were fed a lower N:S ratio. This fits with the retention
differences reported in trial 2. The calculated N:S was 11.1 for the control diet compared to 6.1
for the 40% DDGS diet and 6.6 for the 40% DDGS+ diet (data not shown).
Further studies would be beneficial to confirm the impact Mo and Cu have on cattle diets
with high levels of DDGS, and if they do in fact reduce H2S emissions. Results from our study
suggest that manure contributes much of the S-containing emissions. More research on the
source of emissions could serve as useful information for future research to help determine
whether it would be better to treat the animal diet or if possible application into manure storage
systems would be a better strategy for overall reduction of S-containing emissions.

61

CHAPTER 4: EFFECTS OF DISTILLER’S GRAIN WITH SOLUBLE AND
SUPPLEMENTAL COPPER AND MOLYBDENUM ON NITROGENOUS EMISSIONS AND
NITROGEN RETENTION
L.D. Cross, W.J. Powers, J.S. Liesman, and S.R. Rust
Michigan State University, East Lansing, MI. 48823, USA

SUMMARY
When moderate to high levels of DGS are fed, dietary CP is elevated, which may
contribute to environmental pollution from increased nitrogenous emissions. A study was
conducted to evaluate the affects of DDGS on NH3 and other N-emissions. Two trials were
conducted within this study using 12 Holstein steers housed in individual environmentallycontrolled rooms to monitor gas production. Three dietary treatments were fed in trial 1;
containing 0% (control), 40%, and 60% DDGS. In trial 2, treatments were the same except the
60% DDGS dietary treatment was replaced with a 40% DDGS diet fortified with 8 ppm Mo and
90 ppm Cu, which will be referred to as 40% DDGS+. Each trial was divided into 2 phases;
phase 1 of each trial monitored emissions when urine and feces were collected in the same vessel
(manure mixture). Phase 2 of trial 1 monitored emissions for 2 d while phase 2 of trial 2
monitored emissions for 4 d while steers were fitted with fecal bags to separate feces from urine.
In trial 1, N2O showed a linear reduction as inclusion of DDGS increased from 0, 40, to 60% of
the DM diet (P = 0.02) during phase 1. In trial 2, NH3 emissions tended to increase with
inclusion of 40% DDGS compared to the control diet when expressed as mass units (P = 0.03),
mass units per DMI (P = 0.06), and mass units per N intake (P = 0.10). Separation of feces and
urine during phase 2 of both trials significantly decreased NH3 emissions (P < 0.01) while
increasing NOx emissions (P < 0.01). Nitrogen balance for both trial 1 and trial 2 shows that
62

DDGS tends to improve N digestibility compared to the control diet (P < 0.05). Molybdenum
and Cu treatment did not have an effect on N-emissions, N digestibility, or N retention.
Key words: distiller’s grain with soluble, ammonia, nitrogen, molybdenum, copper

63

INTRODUCTION
Distiller’s grain with soluble in feedlot diets increases crude protein (CP) levels along
with other nutrients relative to conventional feedlot diets. Distiller’s grain with soluble is nearly
3-fold more concentrated in protein, fat, fiber, and other minerals (Stock et al., 2000). This
increase in nutrient concentration in DGS is the result of removing the starch from the corn
kernel during the dry milling process for ethanol production (Klopfenstein et al., 2008). The
greater concentration of protein in diets containing high levels of DGS may contribute to an
increase in ammonia (NH3) and other aromatic compounds that are formed by aerobic and
anaerobic digestion of organic residues by bacteria (Mackie et al., 1998; Spiehs and Varel,
2009). Typically, increasing dietary N will increase urinary N excretion (Gueye et al., 2003;
McBride et al., 2003). The amount of urinary N excretion (Erickson et al., 2001) and the urine
pH are determinants of the amount of N-emissions (Luebes et al., 1974; Cole et al., 2005).
In addition to increasing dietary N, DGS also increases levels of S in cattle diets. A study
from Rumsey (1978) suggests that increasing S content may reduce ruminal pH and ruminal
concentration of NH3. Introducing supplemental Mo and Cu to high S diets may limit the
availability of S in the rumen. Dietary S and S in protein react with Mo to form
2-

tetrathiomolybdates (MoS4 ) that then react with Cu and particulate matter in the rumen,
forming a highly stable compound that is very difficult for the animal to digest and absorb
(Gould et al., 2002).
In trial 1, cattle were fed dry distiller’s grain with soluble (DDGS) at 0, 40, and 60% to
determine the effects on NH3 and other N-emissions. The purpose of trial 1 was to determine if
increased concentrations of dietary N through increased inclusion levels of DDGS would cause
64

increased emissions of NH3 and other nitrogenous gases such as nitrous oxide (N2O), and
-

nitrogen oxides (NOx; includes NO2 and NO ).
In trial 2, dietary treatment of 60% DDGS was replaced with another 40% DDGS diet
containing 8 ppm molybdenum and 90 ppm copper. Molybdenum and Cu bind only to degraded
S from protein and inorganic S from the diet or saliva. Therefore, degradable N may be
influenced by the availability of S in the rumen (Suttle, 1991). Including 8 ppm Mo and 90 ppm
Cu with 40% DDGS in cattle diets may influence NH3 and other N-emissions.

65

MATERIALS AND METHODS
Gas collection
Two studies were conducted at Michigan State University at the Animal Air Quality
Research Facility (AAQRF) to address concerns associated with high inclusion levels of DDGS.
Approval for this study was provided by the Michigan State University Animal Care and Use
Committee (AUF # 07/09-110-00). Individual Environmentally-controlled rooms at AAQRF
monitor incoming and outgoing air from 12 rooms for concentrations of NH3, N2O, and NOx
gases. Steers were housed in chamber rooms to determine whether increased DDGS in the diet
would increase N- emissions. Before arrival to AAQRF, steers were held at Michigan State
University’s Beef Cattle Teaching and Research Center (BCTRC) where they were weighed and
vaccinated for prevention of clostridial disease using Ultrabac-7 (Pfizer, New York, NY) and
respiratory infections (Bovi-Shield GOLD® 5 Pfizer, New York, NY). At trial initiation; a 2 wk

adjustment time was allotted before steers were transported to AAQRF. During this time, steers
(n = 12) were started on a corn-based concentrate diet (control diet). After the first week, steers
(n = 8) were randomly assigned to a 40% DDGS diet. The control diet consisted of 81% high
moisture corn (HMC), 10% corn silage (CS), 5% soybean meal (SBM), and 4% mineral
supplement. The 40% DDGS diet consisted of 40% DDGS, 46% HMC, 10% CS, and 4%
mineral supplement. Steers were broke to lead to insure safe handling and limit stress on the
animal during the study at AAQRF. Four days prior to arrival at AAQRF, steers were housed in
metabolism stalls at BCTRC to adapt them to similar living conditions that they would have in
the individual, environmentally-controlled rooms at AAQRF. Steer weights were taken before
feeding on 2 consecutive days before transportation to AAQRF. Weights were also taken on 2

66

consecutive days upon return to the feedlot facility. Steers (n=12) were randomly assigned to 3
dietary treatments prior to arrival at AAQRF.
Two different trials were conducted at AAQRF to determine the effects of increasing
DDGS on animal performance and N emissions. In both trials, 12 Holstein steers were placed
into environmentally-controlled rooms with 4 steers per dietary treatment. The dietary treatments
for trials 1 and 2 are shown in Table 4.1. In trial 1, 3 levels of DDGS were fed (0, 40, and 60%)
and 2 levels in trial 2 (0 and 40%). In trial 2, the third treatment was 40% DDGS fortified with 8
ppm Mo and 90 ppm Cu. The latter treatment was called 40% DDGS+ (Table 2). The source of
Mo was sodium molybdate (Na2MoO4) and the Cu source was copper chloride (CuCl2). Copper
chloride was selected to minimize the amount of additional S in the diet. All diets were top
dressed daily with thiamine (200 mg) as a preventive step against S toxicity. A 1 min eye and ear
twitch count was recorded daily to monitor physical symptoms associated with PEM. Recorded
twitches for each steer were unchanged during both studies. On d 5 of trial 1, a steer on the
control diet had to be pulled off the study due to poor feed intake and formation of hematoma
near the left hook bone.

67

Table 4.1 Dietary ingredients and composition for trial 1
Trial 1

Trial 2

40

60

Control

40

40 +

81
10
5
4
100

1

DDGS
High moisture corn
Corn silage
Soybean meal
DGS supplement
BFS50 supplement
Total

40
46
10
4
100

60
26
10
4
100

81
10
5
4
100

40
46
10
4
100

60
26
10
4
100

73.6

74.5

6.4
7.2
19.9
33.5
3.9
19.1
4.5

7.1
7.0
19.6
33.3
3.9
18.8
5.0

4.7

4.7

Dry matter, %

67.7

74.8

Ash
ADF
NDF
Starch
Ether extract
Crude protein
3
ADIP
Gross energy, Mcal/Kg

5.6
5.9
11.8
60.3
2.9
12.9
1.5

7.4
7.1
19.0
33.5
4.6
21.5
5.5

Diet composition
76.0
68.6
% of DM
8.0
4.4
8.8
4.2
23.2
11.7
58.6
22.1
2.2
5.5
24.6
12.9
6.6
1.3

4.0

4.3

4.4

Ingredient, % of DM
TM

Akey TM premix # 4
Limestone
Soybean meal, 48% N
RumensinTM 80
TM salt
Vitamin E, 5%
Urea, 45% N
Potassium chloride
Selenium 90
Total
1
2

2

Control

Ingredient, % of DM

*

BFS50 supplement
1.4
24.9
48.3
0.3
9.6
0.2
9.6
5.1
0.7
100

4.4

DGS supplement
2.4
71.5
0.4
18.0
0.1
7.6
100

DDGS- dry distiller’s grain with soluble
40% + is fortified with supplemental 8 ppm molybdenum and 90 ppm copper

3

Acid detergent insoluble proteins (ADIP) represent a portion of undegradable intake protein
(UIP) that is completely indigestible to the cattle
* Akey TM premix # 4 composition: 9% Mg, 4% S, 0.02% Co, 1% Cu, 0.09% I, 2% Fe, 4% Mn,
0.03% Se, 4% Zn, 4,400,000 IU vitamin A, 550,000 IU vitamin D, and 5,500 IU vitamin E/kg
(Akey Inc., Lewisburg, OH)
68

Steers were adapted to diets and chamber rooms for 10 d (trial 1) prior to collecting
emissions data and1 d in trial 2. Emissions were recorded over 14 d in trial 1 and 22 d in trial 2.
Emissions data from the last 4 d were summarized and reported as emissions during phase 1.
Temperature in the chambers was maintained near 16° C and air flow was closely regulated near
3

17 m /min or 40 air changes per hour. Humidity in each chamber room was recorded but was not
regulated. Sampling cycle of the 12 rooms plus the background air would take 3 h 15 min and
allowed for 7 to 8 observations per room per d. During the 15 min sampling cycle, the first 9.5
min were used as a purging period and gaseous content of air was recorded the last 5.5 min.
Exhausted air was sampled within an aluminum duct in each room where Teflon coated sample
lines would draw air to the gas analyzers by positive static pressure from each individually
sealed chamber room. Each room was also fitted with an in-house manufactured type-J
thermocouple, and a Campbell Scientific

TM

HMP45C (Logan, Utah, Campbell Scientific, Inc.)

temperature and relative humidity probe. Ammonia along with NOx emissions were measured
using a TEI17C chemiluminescence analyzer (Thermo Fisher Scientific, Waltham, MA, USA).
-

Chemiluminescence is dependent on the reaction of NO and ozone (O3) to form NO2 and O2 as
a product. The reaction produces infrared light that is proportional to the concentration of NO.
-

To determine NOx gases, NO2 must be transformed to NO using a Mo-converter heated at
approximately 325° C. Finally, total nitrogen (Nt) is measured by converting NOx and NH3
-

gases to NO within a steel converter heated to 825° C. Once NOx and Nt concentrations are
measured, NOx can then be subtracted from Nt to provide a concentration for NH3 gas (TEI17C
operator’s manual). Nitrous oxide gas was monitored using an Innova Photoacoustic Field Gas69

Monitor 1412 (LumaSense Technologies, Ballerup, DK). Photoacoustics measures the amplitude
of the acoustic wave. Electrical signals are generated from the microphones and amplified before
being sent to an analogue-to-digital converter. The digitized signal is then converted to a gas
concentration using appropriate calibration factors stored in the analyzer (Innova Airtech
Instruments A/S, Ballerup, DK). Gas sampling and data collection were all computer controlled
by LabView 8.2.1 software and the FieldPoint system (National Instruments, Austin, TX, USA).
Analyzers output concentration was measured in ppm and was transposed into units of mg/min
(Table 4.2). The units were then adjusted to 24 h and expressed as a mass output, output adjusted
for N intake (NI), and output adjusted for dry matter intake (DMI).

70

Table 4.2 Gas unit conversion chart
1. STP
2. m3/min

STP × air flow, m /min

3. L/min

(Air flow @ STP, m /min × 1000 L/ m )

1. ppm

A. Air Flow @ STP

(((temp,° C) + 273° K) × 0.967 atm)

Expired, ppm – incoming, ppm = emitted, ppm

B. Emitted gas
2.
C.

Volume of ideal gas @
0° C & 760 mm Hg

Units
cancel

1. mg/L

Adjusted equation for all gases

mg/min

3

3

3

Emitted gas, mg/kg × 0.000001 kg/mg
MW, g/mol/(22.414 L/mol) × 1000 mg/g
(A.3) × (B.2) × (C.1)

71

Feed, feces and urine collection
During each gas collection period for trial 1 and 2, daily samples of total mixed ration
(TMR) and feed weigh-back (orts) from each steer were collected and immediately placed in a
freezer for storage. Orts were monitored daily to adjust the amount of TMR offered to 110% of
the previous d composition. All feed samples and orts were later freeze-dried and ground through
a Wiley Mill (Thomas Scientific) fitted with a 2 mm screen followed by a 1 mm screen. Stalls
were set on an incline to allow urine to flow back and be collected in the manure pan as well as
excreted feces. The feces and urine mixture was maintained at a 5 cm depth inside the pan during
phase 1 of each trial. Partial removal of excreta was performed daily to maintain a 5 cm depth in
the pan. The removed excreta were weighed to calculate total excretion. A 0.5 kg mixed sample
of excreta was collected during each removal and immediately stored in a freezer for later
analysis. Excreta removal was collected during the morning shift, whereas feeding along with
TMR and ort collections were conducted in the afternoon. During each shift, chamber rooms and
stalls were thoroughly cleaned and chamber rooms were only entered if they were 5 chambers or
more ahead of gas sampling.
Feces and urine segregation using fecal bags
On d 15 of trial 1 and d 23 of trial 2, all manure and urine was removed and weighed
from each fecal pan. Fecal bags were placed on each steer and clean pans were put back in place
to collect urine only. Fecal and urine segregation took place during the last 2 d of trial 1 and the
last 4 d of trial 2. This period of each trial will be referred to as phase 2. Urine pans were pulled
twice daily and filtered through a screen before urine weight and volume were recorded. Two 1%
aliquots were taken of the composited daily urine sample, one aliquot was treated with 6 N HCl
until urine pH reached 4 determined by a litmus paper test and the second sample was left

72

untreated. All urine samples were stored in a freezer for later analysis. Fecal bags were removed
from each steer and weighed daily during the evening feeding. A 5% aliquot of feces were
collected daily and immediately placed in a freezer. Total mixed ration, orts, fecal, and urine
samples were analyzed for CP using a LECO FP-2000 analyzer (3000 Lakeview Ave., St.
Joseph, MI) to estimate total N balance.
Collections and mineral analysis
Feed samples including TMR and orts were freeze-dried and ground through a 1 mm
screen in a Wiley Mill (Thomas Scientific) prior to mineral analysis. All samples excluding urine
samples were prepared in a microwave digestion system by adding 10 ml of nitric acid to 0.5g
feed and fecal samples. Samples in the 10 ml of nitric acid were covered in Saran

TM

wrap and

left in a ventilated hood over night. The samples were then transported in a pressurized Teflonlined digestion vessel and placed in the microwave digester under 1200W at 100% power with a
30 min ramp time, max PSI of 180, at 190° C for a 10 min hold time (Gengelback et al, 1994).
Post digestion vessels were allowed time to cool for 5 min and then 2 ml of 30% hydrogen
peroxide was added to each vessel and let sit unsealed for 15-30 min to allow more time to cool
down for handling. Each digested sample was then poured into a separate 25 ml volumetric flask
and vessels were rinsed with ddH2O. The rinsed water was then added to the volumetric flask
and additional ddH2O was included to bring the total volume within the volumetric flasks to 25
ml (CEM Corporation, 1999). Each 200 µl digest and urine sample were pipetted and diluted
with 5 ml of a solution containing 0.5% EDTA and Triton X-100, 1% ammonia hydroxide, 2%
propanol and 20 ppb of Sc, Rh, In, and Bi as internal standards. Samples were then analyzed for
mineral content using an Agilent 7500ce Inductively Coupled Plasma- Mass Spectrometer (ICPMS). Three modes were used to minimize spectral interferences for mineral analysis. Sulfur and
73

Cu were analyzed using He-mode and Mo was analyzed in non-gas mode. Urine and water
samples were also analyzed for S, Cu, and Mo using an Agilent 7500ce Inductively Coupled
Plasma- Mass Spectrometer (ICP-MS). Concentrations of feed, feces, water and urine were
determined using an Olympus AU 640e (Olympis America Inc., Center Valley, PA). The
+

Olympus ISE module uses ether membrane electrodes for cations sodium (Na ) and potassium
+

-

(K ) and a molecular oriented PVC membrane electrode for chloride (Cl ). Specific cations
+

+

-

(Na , K ) and anions (Cl ) develop an electrical potential with ions of interest according to the
Nernst Equation. The electrical potential is then compared to the Internal Reference Solution
(Block Scientific Inc.) and translated into voltage and then into the ion concentration of the
sample.
Water usage by the 12 chambers was measured daily. Daily water consumption per steer
was calculated by multiplying daily usage by the rates of individual chamber urine output to total
urine output for the 12 chambers. Samples were analyzed for the percent of DM, OM, ADF,
NDF, and ADIP. Vessels were hot weighed empty before 1 g samples were added to each vessel
and weighed prior to being heated over night in a 105° C oven. Vessels including the sample
were then heated in a 500° C oven to determine ash content to calculate OM. Neutral and acid
detergent fibers were determined using ANKOM

220

Fiber Analyzer. Remaining contents from

each filter bag that had undergone ADF analysis was then weighed and analyzed for protein
content using a LECO analyzer to determine the percent of ADIP.
Statistical analysis
Statistical analysis was performed using PROC MIXED sub-routine of SAS 9.2 (SAS
Inst., Inc., Cary, NC). Air flow (cfm) and NOx gas data (ppm) were passed through filters using
74

SAS 9.2 to eliminate any possible erroneous data that may have been caused from equipment
malfunction or from accidental entry of calibration data. Mean output from unfiltered compared
to filtered data is provided in the appendices in Table B.1 and Table B.3 for trial 1 and 2,
respectively. Air flow that equaled zero and NOx input data greater than 0.35 ppm was filtered
out of the data set before analysis. The independent variables or class statements considered for
analysis was phase, dietary treatment (TRT), date, and chamber. Each model statement included
TRT, phase, and the phase by TRT interaction. Date and date by TRT were analyzed separately
to determine day effects for NH3, N2O, and NOx emissions to help determine the appropriate
days to represent mean emission within a steady state. Chamber within treatment was used as the
random effect for both phase and day analysis. All nitrogenous gases were analyzed as mass
units output, output per NI, and as output per DMI. Mean emissions (NH3, N2O, and NOx),
DMI, NI, and pH were all tested for normal distribution using Shapiro-Wilk’s test and for
homogeneous variance using a Levene’s test. Ammonia emissions in trial 2 were natural log
transformed to satisfy equal variance and normal distribution. Contrast statements were used to
determine treatment differences within each phase. Performance data, nitrogen balance, and
excreta pH were analyzed using PROC GLM sub-routine of SAS 9.2 (SAS Inst., Inc., Cary, NC)
as there was no defined random effect for these analyses. Pair-wise comparisons were analyzed
using Tukey-Kramer’s test. Statistical analysis on performance data was determined using TRT
as the independent variable and average daily gain (ADG), DMI, body weight (BW), and gain to
feed ratio (G:F) as the dependent variables. Nitrogen balance was analyzed similar to
performance data only changing independent variables to N in feed, urine, feces, gas, N retained,
and percent N retained. Statistical significance was declared at a P-value at or below 0.05 and
trends at P-value at or below 0.10.
75

RESULTS
Trial 1
In trial 1, cattle tended to gain less weight with the 60% DDGS diet (P = 0.06; Table 4.3)
compared to cattle on the 40% DDGS diet. Performance data provided in Table 4.3 was
determined from the days post arrival up to the day cattle departed the air quality facility. Cattle
on all treatments were in positive energy balance and had a rate of gain typical of cattle in
metabolism units (Depenbusch et al., 2009; Gunn et al., 2009). Daily weight gain on the 40%
and 60% DDGS treatments were similar to control treatments. Dry matter intakes and F:G ratio
were similar among treatments. Although not significant, cattle on 60% DDGS diet had
numerically lower DMI and G:F than the other treatments. Cattle fed the 60% DDGS diet tended
to have lower DM and OM digestibility compared to cattle the fed 60% DDGS diet (P < 0.10).
The lower performance with the 60% DDGS diet is supported by the low digestibility’s
observed.

76

Table 4.3 Effects of dry distiller’s grain with soluble on performance in trial 1
Diet
Control

40% DDGS

Initial BW, kg

252

Final BW, kg

288

DMI, kg/d

6.07

1

245

Item

1

SEM

P- value

246

22

0.97

287

274

20

0.84

6.36

5.74

0.84

0.84

ADG, kg

0.91

1.06

0.71

b

0.10

0.06

Gain: feed

0.153

0.183

0.125

0.03

0.28

b

2.46

0.10

b

2.48

0.09

DM digestibility, %
OM digestibility, %
1

ab

60% DDGS

ab

69.26

ab

70.15

a

a

71.35

a

71.30

63.97
63.89

DDGS- dry distiller’s grain with soluble

a, b

Means without a common superscript within a row differ

77

Table 4.4 Effects of distiller’s grain with soluble levels on nitrogenous emissions for phase 1 and 2 during trial 1
Phase 1
Item

Phase 2

P- value

0

40

60

0

40

60

SEM

TRT

Phase

TRT × Phase

10.57
17.13

16.85
27.30

1.26
2.20

2.39
3.07

1.49
2.42

-

0.88

< 0.01

0.37

UCL*

11.08
19.34

LCL*

6.35

6.52

10.40

0.72

1.48

0.92

-

NH3, mg/g NI

95.68

60.96

83.04

16.34

14.31

9.70

12.84

0.55

< 0.01

0.31

NH3, g/kg DMI

1.90

1.94

2.94

0.41

0.62

0.48

0.44

0.62

< 0.01

0.16

69.17

60.21

25.31

458.46

311.94

49.63

0.15

< 0.01

0.30

0.62

0.37

0.09

3.59

1.68

1.34

b

0.42

0.03

< 0.01

0.10

NOx, mg/kg DMI

11.39

12.19

4.96

92.97

72.68

62.50

16.04

0.50

< 0.01

0.71

N2O, g/d

1.85

1.04

1.37

1.70

1.36

1.55

0.31

0.23

0.65

0.74

b

NH3, g/d

NOx, mg/d

†

NOx, mg/g NI

N2O, mg/g NI

†

†

N2O, g/kg DMI
a, b
†

a

b

a

a

b

283.48
b

ab

16.02

6.88

6.19

12.95

8.37

6.79

2.59

0.06

0.85

0.52

303.68

195.05

221.85

317.89

373.76

332.14

95.72

0.94

0.18

0.65

Means without common superscripts within a phase differ (P ≤ 0.05)

Means with superscripts show a linear effect between treatments in a phase (P ≤ 0.05)

* Upper and lower confidence limits (exponent of untransformed mean ± 1 SEM) express reliability of the estimated mean
emissions. Emissions expressed as mass units were back-transformed from a natural log transformation to satisfy normal distribution
and equal variance

78

Phase 1
Mean emissions reported for phase 1 were determined using data from d 11 to d 14 which
represent emissions from cattle adjusted to the facility and their assigned diets. Day-to-day
variation were highly significant (P < 0.01) for NH3 emissions expressed as mass units per DMI
and all units of measure for NOx emissions during phase 1 of trial 1 as shown in appendices
(Table B.2). Day by treatment interactions were detected for NOx expressed as mass units (P =
0.05) and mass units per DMI (P = 0.06). Days selected to represent phase 1 of trial 1 are further
illustrated within the appendices in Figures B.1-B.9.
Ammonia and NOx emissions were not different among treatments in phase 1 (Table
4.4). Nitrous oxide emissions expressed as mass units per NI were decreased in the DDGS diets
compared to the control diets (P = 0.02). Furthermore, N2O emission expressed as mass units per
NI display a linear decrease in emissions as levels of dietary DDGS increase (P ≤ 0.05).
Treatment comparisons are shown graphically in B.10-B.19 in the appendices.
Phase 2
Mean emissions were reported from data collected on the last 2 d of the trial (d 16 and
17) to best report emissions while steers were fitted with fecal bags. Day by treatment
interactions were not observed in phase 2 (Figures B.1-B.9). Emissions on d 15 were not used to
allow a 1 d adjustment between phase 1 and phase 2. A day effect was reported for NOx
emissions when expressed as mass units per DMI during phase 2 (Table B.2).
Ammonia emissions were significantly decreased across all treatments as a result of
separating urine and feces in phase 2 (P < 0.01; Table 4.4) compared to phase 1. This suggests
that most of the NH3 generated in phase 1 originated from the manure mixture and very little
79

from enteric fermentation in the animal. In contrast, NOx emissions showed a significant
increase during phase 2 compared to phase 1 (P < 0.01). The pH and salinity of feces may favor
chemolithotrophic along with other types of bacteria that oxidize N compounds (Jones and Hood,
1980). Another possible explanation may involve the separation of bacteria in feces and the
soluble N compounds in urine which likely limit ureolytic activity (Schmidt et al., 2002).
Distiller’s grain with soluble diets decreased NOx gas expressed as mass units (P = 0.03) during
phase 2 compared to the emissions from cattle on the control diet. Emissions from NOx showed
a linear decrease (P = 0.04) as DDGS in the diet increased. Ammonia and N2O emissions were
similar between treatments for all parameters measured during phase 2.
Feed, feces, and urine collections along with gas data during the last 2 d of trial 1 were
used to estimate total N balance by comparing NI from feed to N expelled in the form of feces,
urine, and gas. Cattle fed 60% DDGS tended to have a greater feed NI compared to the cattle on
the control diet (P = 0.08; Table 4.5). Nitrogen intake increased linearly (P = 0.03) as levels of
DDGS increased in the diet. Expelled N from urine (P < 0.01) and feces (P = 0.09) showed a
linear increase as N in the diet increased. Cattle fed 60% DDGS diets had greater N in urine (P <
0.01) as compared to the control. Urine N from cattle fed 40% DDGS diets was intermediate.
Nitrogen content in feces and N loss from gas was similar between all diets. Total expelled N
was greater in the 60% DDGS diet compared to the control (P = 0.01). Total expelled N showed
a linear increase as levels of DDGS in the diet increased (P < 0.01). Digestible N was greater in
the 40 and 60% DDGS diet compared to the control (P = 0.04). Similar to NI and N expelled, a
linear increase was observed (P = 0.01) for N digestibility as DDGS increased in the diet. The
amount of N retained on a mass unit basis as well as expressed as a percentage of N intake was
similar among treatments. The percent of N retained agrees with other studies as Koeln and
80

Paterson (1986) reported a N retention from calves fed corn gluten meal (CGM), SBM, and
toasted soybean meal (TSBM) at 44-45% of the NI across all dietary treatments. Additionally,
Chen et al. (1977) conducted a study with varying levels of processed distillers soluble and DGS
added to diets of beef cattle and reported dietary effects on N balance. Percent N retained from
the various diets in this study fell with a range of 25-40%, which are within a similar range of the
N retention levels within the current study.
The pH of manure (feces and urine mixture) linearly increased (P = 0.01) as the level of
dietary DDGS increased (Table 4.6). Manure from cattle on the control diet a lower pH
compared to the manure from cattle fed 60% DDGS diet (P = 0.03). When pH from feces was
measured, there was a tendency (P = 0.08) for the control and 60% DDGS diet to be more acidic
compared to the 40% DDGS diet. All dietary treatments tended to be more acidic when feces
was measured alone compared to the feces and urine mixture. A quadratic effect was present for
fecal pH (P = 0.04). In contrast to the linear effect on manure pH, urine pH decreased (P = 0.05)
as the level of dietary DDGS increased. Urine output also linearly increased (P = 0.05) as the
level DDGS increased in the cattle diets.

81

Table 4.5 Effects of distiller’s grain with soluble on total nitrogen balance for trial 1
Diet
Item

Control
a

40% DDGS
ab

Contrast
60% DDGS

SEM

P- value

Linear

Quadratic

238.98

b

30.98

0.08

0.03

0.91

N intake, g/d

131.23

Urinary N, g/d

30.74

59.37

89.61

c

8.44

< 0.01

< 0.01

0.28

Fecal N, g/d

47.65

53.43

61.52

5.23

0.19

0.09

0.56

N emissions, g/d

3.13

3.95

2.91

1.27

0.82

0.99

0.55

154.04

b

13.22

0.01

< 0.01

0.33

Total N expelled, g/d
1

a

a

81.51

b

ab

114.95

N digestibility , %

63.69

74.21

74.26

b

2.52

0.04

0.01

0.56

N retained, g/d

49.72

92.19

84.94

22.22

0.36

0.22

0.46

N retained, %

28.33

41.98

34.88

5.14

0.55

0.76

0.32

1

a

207.14

b

Digestibility calculated by 1- (fecal N/N intake) ×100

a, b, c

Means without common superscripts within a row differ

* Nitrogen balance was determined from data collected during phase 2 (2 d)

82

Table 4.6 Effects of feeding distiller’s grain with soluble on excreta pH and output during trial 1
Diet
Item

0% DDGS
1

a

Manure pH

7.73

2

a

60% DDGS

8.46

2

Dry fecal output , kg/d
2

Urine output , L/d

P- value

Linear

Quadratic

0.24

0.03

0.01

0.89

8.76

6.64

b

6.15

a

0.20

0.08

0.34

0.04

7.42

6.84

0.62

0.14

0.05

0.97

1.98

1.80

2.11

0.23

0.57

0.82

0.32

2.42

5.26

6.69

1.40

0.13

0.05

0.99

Manure- collected during phase 1; mixture of feces and urine
Feces and urine- collected separately during phase 2

a, b

SEM

b

ab

8.67

2

Urine pH

2

40% DDGS

6.00

Feces pH

1

Contrast

Treatment means within a row without a common superscript differ

83

Trial 2
Molybdenum was added in the form of Na2MoO4 and Cu was provided through
supplemental CuCl2 in the 40% DDGS+ diet. Sodium molybdate and CuCl2 were intended to
provide an addition 6 ppm Mo and 60 ppm Cu; however, TMR analyses determined 8 ppm Mo
and 90 ppm Cu. The effects of dietary treatments on animal performance are shown in Table 4.7.
Cattle were in positive energy balance and consuming more than 2% of their BW. Growth and
feed conversion efficiency were similar among treatments. Molybdenum and Cu levels were
greater (P < 0.01) for the 40% DDGS+ diet. Dry matter digestibility (P = 0.07) and OM
digestibility (P = 0.11) tended to be greater for cattle fed the control diet compared to the cattle
fed 40% DDGS+ diet.
Nitrogen emissions were measured as a mass unit output, mass units per unit of NI, and
as mass units per unit of DMI. Separation of feces and urine in phase 2 significantly decreased
NH3 emissions (P < 0.01; Table 4.8). As shown in trial 1, very little of the NH3 emitted came
directly from the animal but originates from the manure collected in the fecal pans.

84

Table 4.7 Effects of dry distiller’s grain with soluble fortified with copper and molybdenum on
performance in trial 2
Diet
Control

40% DDGS

Initial BW, kg

300

Final BW, kg

342

DMI, kg/d

1

313

Item

40% DDGS+

2

SEM

P- value

312

16

0.82

351

339

16

0.86

8.07

7.12

7.49

0.47

0.40

ADG, kg

1.08

0.96

0.70

0.16

0.30

Gain: feed

0.132

0.131

0.091

0.02

0.20

DM digestibility, %

73.90

b

2.23

0.07

OM digestibility, %

74.41

b

2.35

0.11

b

0.40

< 0.01

4.84

< 0.01

Mo, ppm
Cu, ppm
1
2

a
a

a

1.63

12.89

a

ab

68.34

ab

69.57

a

1.38

65.49
66.66
9.49

a

22.29

111.51

DDGS- dry distiller’s grain with soluble
40 % DDGS+ is supplemented with 8 ppm Mo and 90 ppm Cu

a, b

Means without a common superscript within a row differ

85

b

Table 4.8. Effects of dry distiller’s grain with soluble fortified with copper and molybdenum on nitrogenous emissions for phase 1
and 2 during trial 2
P- value
Phase 1
Phase 2
TRT
Phase TRT × Phase
Item
0
40
40 +
0
40
40+
SEM
18.79
25.73

b

UCL*

8.56
11.72

a

21.23
29.07

1.86
2.55

1.07
1.46

1.34
0.78

-

LCL*

6.25

13.72

15.50

1.36

1.83

0.98

-

49.8
65.5
35.8

87.6
115.2
66.6

95.5
125.6
72.6

14.7
19.0
11.2

5.0
6.5
3.8

6.4
8.4
4.9

b

b

NH3, g/d

NH3, mg/g NI
UCL
LCL

2

a

b

0.83

< 0.01

< 0.01

-

0.69

< 0.01

< 0.01

1

0.90

< 0.01

< 0.01

NOx, mg/d
NOx, mg/g NI

2

NOx, mg/kg DMI

1.02
1.36
0.76

2.44
3.26
1.83

2.58
3.45
1.93

0.30
0.40
0.22

0.15
0.20
0.11

0.19
0.25
0.14

-

704

NH3, g/kg DMI
UCL
LCL

476

273

823

785

747

141

0.57

0.97

0.45

4.12

2.24

1.54

6.94

3.68

3.73

1.04

0.15

0.21

0.70

86.0

60.2

39.1

138

109

108

22.9

0.64

0.08

0.97

a

a

N2O, g/d
N2O, mg/g NI

ab

-2.52
1, 2

N2O, mg/kg DMI

a

-14.6

-303

b

ab

-2.35

b

-11.0

-311

b

-2.07

b

-9.5

-254

a

b

a

-1.83

a

-14.1
-283

a

a

b

-1.89

-1.45

b

0.16

0.05

< 0.01

0.49

b

b

0.89

< 0.01

0.13

0.29

b

22.9

0.04

0.15

0.71

-8.8

a

-268

1

-200

0% DDGS differs from combined 40% DDGS diets in phase 1 (P ≤ 0.05)

2

-6.9

0% DDGS differs from combined 40% DDGS diets in phase 2 (P ≤ 0.05)

a, b

Emissions without a common superscript within a row tend to differ (P ≤ 0.10)

* Upper and lower confidence limits (exponent of untransformed mean ± 1 SEM) express reliability of the estimated mean
emissions
86

Phase 1
Emissions during trial 2 for phase 1 were determined using a 4 d measurement interval (d
20 to d 23). Day variability was highly significant (P < 0.01) for NOx and N2O emissions but
was non-significant for NH3 emissions (data not shown). Treatment by day interaction was nonsignificant for all expressed measurements. Daily emission plots for all nitrogenous gases are
provided in the appendices (Figures B.19-B.27) with days used for each phase reported. Bar
graphs are provided in the appendices for visual comparisons of treatment means within each
phase (Figures B.28-B.36).
Addition of DDGS in phase 1 at 40% of the DM increased NH3 emissions compared to
cattle fed the control diet when emissions were expressed as mass units per DMI (P < 0.05) as
illustrated in Table 4.8. Ammonia emissions expressed as mass units (P < 0.10) and mass units
per NI (P = 0.10) tended to show a similar increase in the 40% DDGS diets compared to the
control diet. Conversely, NOx emission tended to decrease in the 40% DDGS diets compared to
the control (P < 0.10). No differences were determined among treatments when mass units of
NOx were adjusted for DMI or NI. Nitrous oxide was significantly lower in the control diet
compared to the 40% DDGS diets when mass units were adjusted for NI (P < 0.01). Nitrous
oxide emissions for both phase 1 and phase 2 generated negative emissions across all treatments.
Because of the magnitude of the emissions from zero, these negative emissions may be a result
of a detection error for the incoming N2O gas as chamber emissions are calculated by subtracting
the incoming air from the output air of each chamber.

87

Phase 2
Emissions reported during trial 2 for phase 2 were determined from data collected on d
26 to d 29. Separation of feces from urine in phase 2 decreased NH3 emissions, whereas NOx
and N2O emissions increased (P < 0.01; Table 4.8) compared to phase 1. This inverse
relationship may be an affect of decreasing free hydrogen when separating feces from urine,
allowing a greater chance for N to undergo oxidation to form NOx gases or N2O. Ammonia and
NOx emissions were greater (P < 0.05) in the control diet compared to the 40% DDGS diets
when expressed as mass units per NI. Cattle fed 40% DDGS diet fortified with 8 ppm Cu and 90
ppm Mo tended to increase (P < 0.10) N2O emissions compared to 40% DDGS diets without the
additional Mo and Cu when emissions were expressed as mass units and mass units per DMI.
Feed, water, feces, and urine collections along with gas data during the last 4 d of trial 2
were used to estimate total N balance by comparing N in feed to N expelled in the form of feces,
urine, and gas. Nitrogen loss from gas may be an under-estimate as negative N2O emissions
were included in the calculation. Dietary NI from feed was greater for the 40% DDGS diets
compared to NI from steers on the control diet (P < 0.01; Table 4.9). Similarly, excreted N in
urine (P < 0.01) was also greater for both 40% DDGS diets compared to control diets. Nitrogen
in gas was emitted at low levels and contributed less than 0.8% and 0.7% of expelled N and N
intake, respectively. Total expelled N was greater in both 40% DDGS diets compared to the
control (P = 0.03). The 40% DDGS+ diet differed from control when comparing mass N retained
(P = 0.03). The percent of digestible N was greater in the 40% DDGS diets compared to the
control diet (P < 0.01). This would suggest that N is more available to the animal in the DDGS
diets compared to the basal or control diet. The percent of N retained tended to increase in the
88

40% DDGS diet (P = 0.09) compared to the control and 40% DDGS+ diet. Addition of Cu and
Mo to the 40% DDGS diet did not lower N excreted in feces or urine compared to the 40%
DDGS diet.
Fecal (P = 0.01) and manure (P = 0.02) pH were greater for both 40% DDGS diets
compared to control (Table 4.10). Similar to the results from trial 1, the manure mixture had a
greater pH compared to the individual pH of feces or urine.

89

Table 4.9 Effects of distiller’s grain with soluble on total nitrogen balance for trial 2
Diet
Item

Control
a

40% DDGS

40% DDGS+

b

SEM

P- value

212.40

b

16.45

< 0.01

N intake, g/d

129.83

Urinary N, g/d

43.15

78.89

77.26

b

6.72

< 0.01

Fecal N, g/d

62.73

66.40

76.35

5.36

0.23

N emissions, g/d

0.85

-0.01

0.79

0.39

0.27

154.41

b

11.32

0.03

b

3.22

< 0.01

ab

11.00

0.03

ab

4.52

0.09

Total N expelled, g/d
1

a

a

106.72

a

N digestibility , %

50.52

N retained, g/d

23.11

N retained, %

16.85

1

a
a

215.86

b

b

145.29

b

69.36

b

70.56

b

32.85

63.31
57.99
26.15

Digestibility calculated by 1- (fecal N/N intake) × 100

a, b

Means without common superscripts within a row differ

* Nitrogen balance was determined from data collected during phase 2 (4 d)

90

Table 4.10 Effects of feeding distiller’s grain with soluble on excreta pH and output during trial 2
Diet
Item

0% DDGS
1

a

Manure pH

7.23

2

a

8.21

2

Dry fecal output , kg/d
2

Urine output , L/d

b

40% DDGS+

SEM

P- value

b

0.20

0.01

8.09

5.70

5.56

b

0.16

0.02

8.05

2

Urine pH

2

b

4.92

Feces pH

1

40% DDGS

6.64

6.63

0.52

0.14

2.22

2.29

2.63

0.20

0.35

3.81

3.99

4.14

0.73

0.95

Manure- collected during phase 1; mixture of feces and urine
Feces and urine- collected separately during phase 2

a, b

Treatment means within a row without a common superscript differ

91

DISCUSSION
Elevated N-emissions from cattle may be influenced by several factors including an
increase in urinary N excretion (Erickson et al., 2001) as well as lowering urinary pH (Luebes et
al., 1974; Cole et al., 2005). Urine contributes a large majority of its N content in the form of
urea. The urea from the urine is hydrolyzed by urease in the feces when mixed, which
contributes a large portion of emitted NH3 (Misselbrook et al., 2005). Within our study, results
from both trials show a decrease of NH3 in phase 2 when feces and urine were collected
separately compared to the NH3 emissions recorded in phase 1. This provides further evidence
that feces and urine mixture contribute a large majority of NH3 emissions. In accord with
previous reports (Luebes et al., 1974; Cole et al., 2005), NH3 emissions increased during phase 1
of both trials as DDGS level and manure pH increased. Acidic urine increases pH when it comes
into contact with feces, liberating more NH3 (Cole et al., 2005; Misselbrook et al., 2005). An
inverse relationship seems to exist with NH3 and NOx emissions, as emitted NOx increased in
phase 2 compared to phase 1 in both trials. A study measuring N-emissions from coal
combustion reported that an increase in NH3 production decreased the conversion of fuel N to
NOx (Kambara et al., 1995). An increase of volatile N may limit the N available to undergo
oxidation. Emissions from NOx also decreased as dietary N increased from greater levels of
DDGS in the diet. In trial 1, NOx and N2O linearly decreased as levels of dietary DDGS
increased. In trial 2, 40% DDGS diets emitted greater levels of NH3 for all parameters measured
compared to the control diets. Nitrous oxide levels decreased as the DDGS level increased in
92

trial 1when expressed as mass units per NI, while the opposite seemed to occur in trial 2.
However, since estimates were negative in trial 2 the reader should be cautious of the results.
In trial 1, NI linearly increased as the level of dietary DDGS increased (P = 0.03).
Urinary N (P < 0.01), fecal N (P = 0.09), and total N expelled (P < 0.01) all linearly increased as
levels of feed N increased. Digestible N was greater in the DDGS diets compared to the control
diet as shown in both trials. These results are surprising as DGS is reported to offer a greater
fraction of ruminally undegradable intake protein (UIP) and lower degradable intake protein
(DIP) in cattle diets (Bothast and Schlicher, 2005). However, a study from Salisbury et al.
(2004) reported no differences in N digestibility and N retention in wethers fed low and high
levels of UIP. The percent of N retained from both trials show a greater N utilization in DDGS
diets compared to the control diet. Fron et al. (1990) reported that cattle diets containing greater
levels of sulfur (S) tend to utilize dietary N more efficiently. The present study supports the
literature as dietary S intake increased from approximately 13 g/d in the control diet to 35 and 45
g/d in the 40 and 60% DDGS diets, respectively (data not shown).
In trial 2, the dietary treatment of 40% DDGS+, contained 8 ppm Mo and 90 ppm Cu.
The purpose of this treatment was to mitigate the amount of available S in the cattle’s diet that
could be reduced by sulfate-reducing bacteria (SRB) in the rumen and manure. However,
limiting available S in ruminant diets may indirectly influence N-emissions. Bouchard and
Conrad (1972) reported that a N:S ratio exists; as dietary S decreases so would the percent of N
retained, causing an increase in excreted N (Rumsey 1978). Based on the results from trial 2,
NH3 emissions tended to show a linear increase from the control, 40% DDGS, and 40% DDGS+
diet for all parameters measured (P ≤ 0.10). Averaging dietary treatments in phase 1, NH3 gas
contributed 90% of the total N-emissions.
93

The collection system used during phase 1 of each trial more accurately portrays
emissions from liquid manure handling and storage systems. Manure (feces and urine) mixture
during phase 1 was the main source of NH3 emissions recorded within the chamber rooms.
As mentioned previously, excreta pH (Luebes et al., 1974; Cole et al., 2005) and mixture of urine
and feces (Misselbrook et al., 2005) contribute to an increase in NH3 emissions. During trial 2 of
the current study, an increase in NH3 emissions did tend to correlate to diets that produced
greater manure pH. Distiller’s grain with soluble diets had increased levels of dietary S and it has
been shown that urine pH will decrease with high sulfide contents (Thistlethwayte, 1972; Xue et
al., 1998). Conversely, manure pH increases as reduced forms of S and N react with free
hydrogen and are emitted from the manure as H2S and NH3, respectively. Results from both
trials agrees with previous reports (Thistlethwayte, 1972; Xue et al., 1998) by offering further
evidence that an increase in manure pH will emitt greater levels of NH3 along with H2S from the
manure.
Feeding DDGS at 60% of the diet DM did not contribute to elevated N-emissions
compared to 40% DDGS diets despite increasing urinary N excretion and decreasing urinary pH.
Continued research would be beneficial to provide a better understanding of the relationship of
available S in the diet with N retention and N-emissions. Furthermore, other studies suggest that
reducing CP in the diet by at least 5 percentage units in the diet composition can drastically
reduce total N excretion by 30% (Misselbrook et al., 2005) to 66% (Castillo et al., 2000). Our
data falls within the range of the results from Misselbrook et al. (2005) and Castillo et al. (2000)
as the dietary CP was decreased from 24.6 (60 % DDGS) to 12.9 % of the dietary DM (control),
N excretion was decreased 47%. The current study suggests adding DDGS to cattle diets does
94

contribue to an increase in NH3 emissions compared to traditional corn-base diets (control).
Because of the growing availability to cattle producers along with lower costs to feed to cattle as
an energy and protein source, it may be advantageous to determine a strategy that could mitigate
N loss as gas. Furthermore, manure contains plant available N that can be used as a fertilizer for
corn production to improve soil quality and corn yield (Sawyer, 2001). Therefore, manure high
in N may provide an aconomic incentive for use as a fertilizer for corn producers. Further
research should be conducted to determine a strategy to decrease N-emissions from manure
associated with cattle being fed diets containing high levels of CP, particularly with dry and wet
forms of DGS.

95

CHAPTER 5: CARBON FOOTPRINT AND ENERGY PARTITION FROM CATTLE FED
VARIOUS LEVELS OF DISTILLER’S GRAIN WITH SOLUBLE

L.D. Cross, W.J. Powers, J.S. Liesman, and S.R. Rust
Michigan State University, East Lansing 48824

SUMMARY
A study was conducted to evaluate the effects of DDGS on C-containing gases, GHG
footprint, and energy retention. Two trials were conducted within this study. Both trials were
conducted with 12 Holstein steers housed in individual environmentally-controlled rooms to
monitor gas production. Three dietary treatments were fed in trial 1; containing 0% (control),
40%, and 60% DDGS. In trial 2, treatments were the same except the 60% DDGS dietary
treatment was replaced with a 40% DDGS diet fortified with 8 ppm Mo and 90 ppm copper Cu
(40% DDGS+). Each trial was divided into two phases; phase 1 of each trial monitored
emissions when urine and feces were collected in the same vessel. Phase 2 of trial 1 monitored
emissions for 2 d while phase 2 of trial 2 monitored emissions for 4 d while steers were fitted
with fecal bags to separate feces from urine. In trial 1, the 40% DDGS diet emitted 23% less
GHG (N2O, CH4, and CO2) compared to the control diets. In trial 2, the 40% DDGS+ treatment
decreased CO2 emissions (P ≤ 0.05) and tended to also reduce CH4 emissions (P = 0.08)
compared to the control and the traditional 40% DDGS diet. An energy balance was calculated
from phase 2 of each trial. In trial 1, UE linearly increased as the level of DDGS increased (P =
0.02). In trial 2, the 40% DDGS diets had a greater retained energy or NEg compared to the
control (P = 0.02).
Key words: distiller’s grain with soluble, methane, carbon dioxide, greenhouse gas, energy

96

INTRODUCTION
Distiller’s grain with soluble (DGS) is nearly 3-fold more concentrated in protein, fat,
fiber, and other minerals (Stock et al., 2000). This increase in nutrient concentration in DGS is
the result of removing the starch from the corn kernel during the dry milling process for ethanol
production (Klopfenstein et al., 2008). The availability and cost benefits of feeding DGS have
made it a very popular energy and protein replacement over the traditional use of corn and
soybean meal (SBM) in diets. A fraction of the dietary gross intake energy (IE) is lost as
methane (CH4) gas in cattle due to fermentation of carbohydrates by ruminal methanogens.
Methane production typically accounts for 5.5 to 6.5% of the IE (Johnson and Ward, 1996;
Kurihara et al., 1998), but has been reported to account for as high as 12% of the loss from feed
IE (Behlke et al., 2007). Aside from CH4 production accounting for energy loss, it is also a
potent greenhouse gas and has been associated with global warming. Of the total CH4 emitted
from all sources each year, 58 million tons/yr or 73% was contributed by livestock species (US
Environmental Protection Agency, 1994). In a more recent report, livestock contributed about
40% of the total CH4 (FAO, 2006). Based on the unit of dry matter intake (DMI), forage-based
diets produce more CH4 than cereal grain diets when fed to ruminant animals (Behlke et al.,
2007). However, limited research has been conducted to determine available energy from dry
distiller’s grain with soluble (DDGS) and what impact feeding DDGS to feedlot cattle may have
on CH4 production. Two trials were conducted; the objective of the first trial was to address this
issue by determining if increasing dietary DDGS would affect CH4 emissions and the total
greenhouse gas (GHG) footprint, along with the level of retained or net energy (NEg) available
97

to the animal. In trial 2, supplemental copper (Cu) and molybdenum (Mo) were included to
mitigate S-containing emissions at 90 and 8 ppm, respectively. However, it was also important to
determine what affects additional Cu and Mo may have on performance, energy retention, and
CH4 production.

98

MATERIALS AND METHODS
Gas collection
Two studies were conducted at Michigan State University at the Animal Air Quality
Research Facility (AAQRF) to address concerns associated with high inclusion levels of DDGS.
Approval for this study was provided by the Michigan State University Animal Care and Use
Committee (AUF # 07/09-110-00). Environmentally-controlled rooms at AAQRF monitor
incoming ambient and outgoing air from each of the 12 rooms for concentrations of CH4, nonmethane volatile organic compounds (VOCs), nitrous oxide (N2O), as well as respired carbon
dioxide (CO2) and oxygen (O2) consumption. Before arrival to AAQRF, steers were held at
Michigan State University’s Beef Cattle Teaching and Research Center (BCTRC) where they
were weighed and vaccinated for prevention of clostridial disease using Ultrabac-7 (Pfizer, New
York, NY) and respiratory infections (Bovi-Shield GOLD® 5 Pfizer, New York, NY). At trial

initiation; a 2 wk adjustment time was allotted at BCTRC before steers were transported to
AAQRF. During this time, all 12 steers were started on a corn-based concentrate diet (control
diet). After the first week, 8 steers were randomly assigned and adjusted to a 40% DDGS diet.
The control diet consisted of 81% high moisture corn (HMC), 10% corn silage (CS), 5% SBM,
and 4% mineral supplement. The 40% DDGS diet consisted of 40% DDGS, 46% HMC, 10%
CS, and 4% mineral supplement. Steers were broke to lead to ensure safe handling and limit
stress on the animal during the study at AAQRF. Four days prior to arrival at AAQRF, steers
were housed in metabolism stalls at BCTRC to allow adaptation to similar living conditions that
they would have in the individual, environmentally-controlled rooms at AAQRF. Steer weights
were taken before feeding on 2 consecutive days before transportation to AAQRF. Weights
99

were also taken on 2 consecutive days upon return to the feedlot facility. Steers (n=12) were
randomly assigned to three dietary treatments prior to arrival at AAQRF. In trial 1, 4 steers from
the 40% DDGS diet on d 11 of being housed in environmentally controlled rooms were
randomly switched to a 60% DDGS diet.
Two different trials were conducted at AAQRF to determine the effects of increasing
DDGS on animal performance, C-containing emissions and total greenhouse footprint, and
energy retention. The dietary treatments for trials 1 and 2 are shown in Table 5.1. In trial 1, 3
levels of DDGS were fed (0, 40, and 60%) and 2 levels in trial 2 (0 and 40%) with 4 steers per
treatment. In trial 2, the third treatment was 40% DDGS fortified with 8 ppm Mo and 90 ppm
Cu. The latter treatment was called 40% DDGS+. The source of Mo was sodium molybdate
(Na2MoO4) and the Cu source was copper chloride (CuCl2). Copper chloride was selected to
minimize the amount of additional S in the diet. All diets during trial 1 were top dressed daily
with thiamine (200 mg) as a preventive step against S toxicity. Eye and ear twitching was
recorded daily to monitor physical symptoms associated with PEM. Recorded twitches for each
steer were unchanged during both studies. On d 5 of trial 1, a steer on the control diet had to be
pulled off the study due to poor feed intake and formation of hematoma near the left hook bone.

100

Table 5.1 Dietary ingredients and composition for trial 1 and 2
Trial 1
Ingredient, % of DM
1

DDGS
High moisture corn
Corn silage
Soybean meal
DGS supplement
BFS50 supplement
Total

Trial 2
2

Control

40

60

Control

40

40 +

81
10
5
4
100

40
46
10
4
100

60
26
10
4
100

81
10
5
4
100

40
46
10
4
100

60
26
10
4
100

73.6

74.5

6.4
7.2
19.9
33.5
3.9
19.1
4.5

7.1
7.0
19.6
33.3
3.9
18.8
5.0

4.4

4.7

4.7

Diet composition
Dry matter, %

67.7

74.8

Ash
ADF
NDF
Starch
Ether extract
Crude protein
3
ADIP
Gross energy, Mcal/kg

5.6
5.9
11.8
60.3
2.9
12.9
1.5

7.4
7.1
19.0
33.5
4.6
21.5
5.5

68.6
% of DM
8.0
4.4
8.8
4.2
23.2
11.7
58.6
22.1
2.2
5.5
24.6
12.9
6.6
1.3

4.0

4.3

4.4

Ingredients, % of DM
Akey TM premix # 4*
Limestone
Soybean meal, 48% N
TM
Rumensin
80
TM salt
Vitamin E, 5%
Urea, 45% N
Potassium chloride
Selenium 90
Total
1
2
3

76.0

BFS50 supplement
1.4
24.9
48.3
0.3
9.6
0.2
9.6
5.1
0.7
100

DGS supplement
2.4
71.5
0.4
18.0
0.1
7.6
100

DDGS- dry distiller’s grain with soluble
40 + is fortified with supplemental 8 ppm molybdenum and 90 ppm copper
ADIP- acid detergent insoluble proteins

* Akey TM premix # 4 composition: 9% Mg, 4% S, 0.02% Co, 1% Cu, 0.09% I, 2% Fe, 4% Mn,
0.03% Se, 4% Zn, 4,400,000 IU vitamin A, 550,000 IU vitamin D, and 5,500 IU vitamin E/kg
(Akey Inc., Lewisburg, OH)
101

Steers were adapted to diets and chamber rooms for 10 d (trial 1) prior to collecting
emissions data and1 d in trial 2. Emissions were recorded over 14 d in trial 1 and 22 d in trial 2.
Emissions data from the last 4 d were summarized and reported as emissions during phase 1.
Temperature in the chambers was maintained near 16° C and air flow was closely regulated near
3

17 m /min or 40 air changes per hour. Humidity in each chamber room was recorded but was not
regulated. Sampling cycle of the 12 rooms plus the background air would take 3 h 15 min, which
allowed 7 to 8 observations per room per d. During the 15 min sampling cycle, the first 9.5 min
were used as a purging period and gaseous content of air was recorded the last 5.5 min.
Exhausted air was sampled within an aluminum duct in each room where Teflon coated sample
lines would draw air to the gas analyzers by positive static pressure from each individually
sealed chamber room. Each room was also fitted with an in-house manufactured type-J
thermocouple, and a Campbell Scientific

TM

HMP45C temperature and relative humidity probe

(Logan, Utah, Campbell Scientific, Inc.). Methane and VOCs were measured using a TEI 55C
Direct Methane, Non-methane Hydrocarbon Analyzer (Thermo Fisher Scientific, Waltham, MA,
USA) by back-flushed chromatography. Carbon dioxide and O2 were measured using a
BINOS® 100 2M Dual-Channel Gas Analyzer (Rosemount Analytical Inc., Orrville, OH, USA)

by applying a combination of infrared sensors, thermo conductivity, electrochemical and
paramagnetic oxygen sensors. Gas sampling and data collection were all computer controlled by
LabView 8.2.1 software and the FieldPoint system (National Instruments, Austin, TX, USA).
Analyzers output concentration was measured in ppm and was transposed into units of mg/min.
The units were then adjusted to 24 h and expressed as mass unit output, output adjusted for DMI,
and output adjusted for DE intake.
102

Feed, feces and urine collection
During each gas collection period for trial 1 and trial 2, daily samples of total mixed
ration (TMR) and feed weigh-back (orts) from each steer were collected and immediately
placed in a freezer for storage. Orts were monitored daily to adjust the amount of TMR offered to
110% of the previous d delivery. All feed samples and orts were later freeze-dried and ground
through a Wiley Mill (Thomas Scientific) fitted with a 2 mm screen followed by a 1 mm screen.
Stalls were set on an incline to allow urine to flow back and be collected in the manure pan as
well as excreted feces. The feces and urine mixture was maintained at a 5 cm depth inside the
pan during phase 1 of each trial. Partial removal of excreta was performed daily to maintain a 5
cm depth in the pan. The removed excreta were weighed to calculate total excretion. A 0.5 kg
sample of excreta was collected during each removal and immediately stored in a freezer for
later analysis. Excreta removal was collected during the morning shift, whereas feeding along
with TMR and ort collections were conducted in the afternoon. During each shift, chamber
rooms and stalls were thoroughly cleaned and chamber rooms were only entered if they were 5
chambers or more ahead of gas sampling.
Feces and urine segregation using fecal bags
On d 15 of trial 1 and d 23 of trial 2, all manure and urine was removed and weighed
from each fecal pan. Fecal bags were placed on each steer and clean pans were put back in place
to collect urine only. Fecal and urine segregation took place during the last 2 d of trial 1 and the
last 4 d of trial 2. This period of each trial will be referred to as phase 2. Urine pans were pulled
twice daily and filtered through a screen before urine weight and volume were recorded. A 2%
aliquot was collected daily of the composited urine sample. All urine samples were stored in a
freezer for later analysis. Combined water usage of the 12 chambers was measured daily. Daily

103

water consumption per steer was calculated by multiplying daily total usage by the rates of
individual chamber urine output to total urine output for the 12 chambers. Fecal bags were
removed from each steer and weighed daily during the evening feeding. A 5% aliquot of feces
were collected daily and immediately placed in a freezer. Total mixed ration, orts, fecal, and
urine samples were analyzed for IE using a Parr Oxygen Bomb Calorimeter (Parr Instrument
Company, Moline, IL, USA) to estimate total energy partition for each diet within trial 1 and
trial 2. Urine samples were freeze-dried and the DM fraction was used to calculate urine energy.
Energy balance calculations
Energy values expressed in MJ/d for TMR, orts, feces, and urine were determined from
samples collected during phase 2 of trial 1 and 2 when feces and urine were collected separately.
Gross intake energy (IE) was determined by subtracting the ort’s total energy from the total
energy offered within the TMR for each steer. The total loss is the sum of energy loss in feces,
urine, and CH4 gas. Digestible energy (DE) was calculated as the difference of fecal energy (FE)
from IE. Metabolizable energy (ME) was calculated by subtracting urine (UE) and CH4 gaseous
energy (GE) from DE. Energy from CH4 gas was calculated by using conversion factors 39.54
KJ/L and 0.716 g/L (Brouwer, 1965; Kurihara et al., 1998). Heat energy (HE) was estimated
using the equation: HE = 16.26 KJ × O2 (L/d) + 5.02 KJ × CO2 (L/d) – 2.17 KJ × CH4 (L/d) –
5.99 KJ × N (g/d) (Brouwer, 1965; Ferrell et al., 1988; Kurihara et al., 1998). Methane emissions
were analyzed as g/d and was converted to L/d by dividing the gases mass output (g/d) by its
molecular weight (g/mol) and then multiplying the conversion factor 22.42 (L/mol). Net energy
(NEg) represents the difference of HE from ME.

104

Collections and mineral analyses
Feed samples including TMR and orts were freeze-dried and ground through a 1 mm
screen in a Wiley Mill (Thomas Scientific) prior to mineral analysis. All samples excluding urine
samples were prepared in a microwave digestion system by adding 10 ml of nitric acid to 0.5g
feed and fecal samples. Samples in the 10 ml of nitric acid were covered in Saran

TM

wrap and

left in a ventilated hood over night. The samples were then transported in a pressurized Teflonlined digestion vessel and placed in the microwave digester under 1200W at 100% power with a
30 min ramp time, max PSI of 180, at 190° C for a 10 min hold time (Gengelback et al, 1994).
Post digestion vessels were allowed time to cool for 5 min and then 2 ml of 30% hydrogen
peroxide was added to each vessel and let sit unsealed for 15-30 min to allow more time to cool
down for handling. Each digested sample was then poured into a separate 25 ml volumetric flask
and vessels were rinsed with ddH2O. The rinsed water was then added to the volumetric flask
and additional ddH2O was included to bring the total volume within the volumetric flasks to 25
ml (CEM Corporation, 1999). Each 200 µl digest and urine sample were pipette and diluted with
5 ml of a solution containing 0.5% EDTA and Triton X-100, 1% ammonia hydroxide, 2%
propanol and 20 ppb of Sc, Rh, In, and Bi as internal standards. Samples were then analyzed for
mineral content using an Agilent 7500ce Inductively Coupled Plasma-Mass Spectrometer (ICPMS). Three modes were used to minimize spectral interferences for mineral analysis. Sulfur and
Cu were analyzed using He-mode and Mo was analyzed in non-gas mode. Urine and water
samples were also analyzed for S, Cu, and Mo using an Agilent 7500ce Inductively Coupled
Plasma- Mass Spectrometer (ICP-MS). Concentrations of feed, feces, water and urine were
determined using an Olympus AU 640e (Olympis America Inc., Center Valley, PA). The

105

+

Olympus ISE module uses ether membrane electrodes for cations sodium (Na ) and potassium
+

-

(K ) and a molecular oriented PVC membrane electrode for chloride (Cl ). Specific cations
+

+

-

(Na , K ) and anions (Cl ) develop an electrical potential with ions of interest according to the
Nernst Equation. The electrical potential is then compared to the Internal Reference Solution
(Block Scientific Inc.) and translated into voltage and then into the ion concentration of the
sample.
Feed, fecal and urine samples were analyzed for the percent of DM, OM, ADF, NDF,
starch, ether extract, CP, ADIP, and gross energy. Vessels were hot weighed empty before 1 g
samples were added to each vessel and weighed prior to being heated over night in a 105° C
oven for DM determination. Vessels including the sample were then placed in a 500° C oven to
determine ash content to calculate OM. Neutral and acid detergent fibers were determined using
ANKOM

220

Fiber Analyzer (Ankom Technology, Macedon, NY) following the procedure

outlined by Ankom Technology (1998). Remaining contents from each filter bag that had
undergone ADF analysis was then weighed and analyzed for protein content using a LECO FP2000 analyzer (Leco Corp., St. Joesph, MI) to determine the percent of ADIP. Crude protein was
also determined on feed, fecal, and urine samples using a LECO analyzer. Ether extractable fat
was determined with a modified soxhlet extraction procedure (Association of Analytical
Chemists, 1990). Starch and free sugar analysis using a plate reader (Karkalas, 1985) was
performed following Karkalas (1985) procedure.
Statistical analysis
Statistical analysis was performed using PROC MIXED sub-routine of SAS 9.2 (SAS
Inst., Inc., Cary, NC). Air flow (cfm), CO2 (ppm) and O2 (%) were passed through filters using
106

SAS 9.2 to eliminate any possible erroneous data that may have been caused from equipment
malfunction or from accidental entry of calibration data. Mean output from unfiltered compared
to filtered data is provided in the appendices in Table A.1. Air flow that equaled zero, CO2
greater than 1200 ppm, and O2 greater than 21.4% was filtered out of the data set before
analysis. The independent variables or class statements considered for emissions analysis were
phase, dietary treatment (TRT), date, and chamber. Each model statement included TRT, phase,
and the phase by TRT interaction. Date and date by TRT were analyzed separately to determine
day effects for CH4, VOC, CO2, and O2 emissions to help determine the appropriate days to
represent mean emission within a steady state. Chamber within treatment was used as the
random effect for both phase and day analysis. Methane, VOC, and CO2 gases were analyzed as
output mass units per d, output per kg of DMI, and as output per Mcal of DE. Carbon dioxide,
CH4, and N2O were adjusted for global warming potential (GWP) and analyzed using the same
PROC MIXED procedure as the other dependent emission variables. Mean emissions were all
tested for normal distribution using Shapiro-Wilk’s test and for homogeneous variance using a
Levene’s test. Contrast statements were designed for pair-wise treatment comparison, along with
linear and quadratic contrasts in trial 1. In trial 2, orthogonal comparisons of the control vs. the 2
DDGS diets were considered.
Performance data and energy partition were analyzed using PROC GLM sub-routine of
SAS 9.2 (SAS Inst., Inc., Cary, NC) as there was no defined random effect for these analyses.
Pair-wise comparisons were analyzed using Tukey-Kramer’s test. Statistical analysis on
performance data was determined using TRT as the independent variable and average daily gain
(ADG), DMI, body weight (BW), and gain to feed ratio (G:F) as the dependent variables.
107

Energy partition was analyzed similar to performance data only changing independent variables
to IE, FE, UE, GE, total energy loss, DE, ME, HE, and energy retention or NEg. Statistical
significance was declared at a P-value at or below 0.05 and trends at P-value at or below 0.10.

108

RESULTS
Trial 1
In trial 1, cattle tended to gain less weight per d when fed the 60% DDGS diet (P = 0.06;
Table 4.2) compared to cattle on the 40% DDGS diet. Daily weight gain for cattle fed the control
diet was similar to both 40 and 60% DDGS diets. Dry matter intakes and G:F were similar
among treatments. Cattle fed the 60% DDGS diet had numerically lower DMI and G:F than
cattle fed the other treatments. Cattle fed the 40% DDGS diet tended to have greater digestibility
compared to cattle the fed 60% DDGS diet (P = 0.10). Performance data provided in Table 4.2
was determined from the time period between entry and departure from AAQRF (22d). Cattle
fed all treatments were in positive energy balance and had a rate of gain typical of cattle in
metabolism units (Depenbusch et al., 2009; Gunn et al., 2009).

109

Table 5.2 Effects of dry distiller’s grain with soluble on performance in trial 1
Diet
Control

40% DDGS

Initial BW, kg

252

Final BW, kg
DMI, kg/d

Items

1

60% DDGS

SEM

P- value

245

246

22

0.97

288

287

274

20

0.84

6.07

6.36

5.74

0.84

0.84

ADG, kg

0.91

1.06

0.71

b

0.10

0.06

Gain: feed

0.153

0.183

0.125

0.03

0.28

b

2.46

0.10

b

2.48

0.09

DM digestibility, %
OM digestibility, %
1

ab

ab

69.26

ab

70.15

a

a

71.35

a

71.30

63.97
63.89

DDGS- dry distiller’s grain with soluble

a, b

Means without a common superscript within a row differ (P ≤ 0.10)

110

Table 5.3 Effects of distiller’s grain with soluble levels on carbon emissions and respiration for phase 1 and 2 during trial 1
Methane emissions
Phase 1

Phase 2

P- value

Item

0

40

60

0

40

60

SEM

TRT

Phase

Phase × TRT

g/d

45.12

32.91

42.60

43.22

31.30

39.68

6.30

0.34

0.50

0.98

g/kg DMI
g/Mcal DE

7.33

5.13

6.91

8.58

6.76

8.73

1.08

2.28

0.04

0.92

3.63

2.88

3.37

3.46

2.47

3.12

0.54

0.47

0.37

0.94

0.69

0.75

0.41

0.28

0.45

< 0.01

0.22

264.09

b

140.97

143.98

95.06

35.71

0.27

< 0.01

0.47

b

38.31

38.05

22.46

13.29

0.24

< 0.01

0.46

Non-methane VOC emissions
g/d

2.27

mg/kg DMI

†

mg/Mcal DE

†

2.00
a

361.94

a

126.02

1.64
ab

306.55

ab

116.44

88.36

Respiration
O2, kg/d

-6.39

-6.10

-6.40

-4.51

-4.37

-4.80

0.78

0.94

< 0.01

0.76

CO2, kg/d

4.42

3.58

3.80

3.96

3.44

3.46

0.37

0.41

< 0.01

0.33

CO2, g/kg DMI

715

568

611

779

822

760

100

0.85

0.03

0.47

CO2, g/Mcal DE

246

220

208

220

211

190

36.3

0.78

< 0.01

0.40

a, b
†

Means without common superscripts within a row for each phase differ (P ≤ 0.10)

Means within a phase tend to show a linear effect (P ≤ 0.10)

111

Phase 1
Emission data from d 11 to 14 were selected to represent mean emissions at a stable
condition for phase 1 (Table 5.3). During phase 1, only O2 emissions (P = 0.02) and CO2
adjusted for DMI (P = 0.05) had a significant day effect (data not shown). Methane and
respiratory gases (CO2 and O2) were similar among treatments during phase 1. Non-methane
VOC emissions tended to show a linear reduction in emissions as DDGS increased in the diet (P
< 0.10) when adjusted for DMI or DE (Table 5.3).
The total GHG footprint was estimated using a global warming potential (GWP) factor
fixed over a 100 yr period (Table 4.4). Cattle fed the 40% DDGS diet tended to have the lowest
GHG footprint compared to the control and 60% DDGS diets, however differences were nonsignificant. The 3 major GHGs recorded were; CO2, which contributes around 66-70%, CH4
accounted for 23-27%, and N2O 6-8% gram equivalents. According to Lashof and Ahuja (1990),
CO2 makes up approximately 70% of the total gases that contribute to the GWP within the
atmosphere. This is similar to the percent emitted from the cattle within the current study.
Phase 2
Mean emissions that represent phase 2 were selected the last 2 d of the trial (d 16 and 17)
to allow a 1 d adjustment before reporting emissions data while cattle were fitted with fecal bags.
Methane adjusted for DMI (P < 0.01), both measures for non-methane VOC (P < 0.01), CO2
adjusted for DMI (P = 0.03), and O2 (P < 0.01) had significant day effects (data not shown). All
of the gases that showed d variability had similar 2 d averages. The differences between days
suggest that more than a 2 d collection period is needed. Separation of feces and urine in the
112

chamber rooms did not affect CH4 emissions mass output when comparing phase 1 and 2.
However, phase 2 emissions were elevated compared to phase 1 when CH4 was adjusted for
DMI (P = 0.04) due to a reduction in DMI when cattle were fitted with fecal bags during phase
2. All parameters of non-methane VOC emissions were significantly decreased in phase 2
compared to phase 1 (P < 0.01). Oxygen consumption and emitted CO2 also declined during
phase 2 compared to phase 1 (P < 0.01).
The estimated GHG footprint for all dietary treatments during phase 2 was lower (P <
0.01) than phase 1, which can be attributed to less CO2 emissions (Table 5.4). Carbon dioxide
was emitted within a range of 64-69% of the total GHG footprint. The contribution to the total
GHG from CH4 and N2O were similar to the levels indicated from phase 1, producing 22-27%
and 8-9%, respectively.
Gross intake energy (IE) was similar among dietary treatments within phase 2 during trial
1 (Table 5.5). Energy loss in urine linearly increased as inclusion levels of DDGS increased in
the diet (P = 0.02). This is likely the result of spillage of N compounds into the urine as the level
of DDGS increased. However, no treatment differences were determined with FE, GE, and total
energy loss. Digestible energy, ME, HE, and NEg were similar among dietary treatments as well.
Based on the NE values from trial 1 at 0, 40, and 60% DDGS diets; predicted values were
determined for 80 and 100% DDGS (Figure 5.1). A polynomial line equation was determined
using the actual values (0, 40, and 60% DDGS) to generate the predicted NEg for 80 and 100%
DDGS.

113

Table 5.4 Global warming potential gram equivalents per day from greenhouse gas emissions from cattle fed various levels of
distiller’s grain with soluble during trial 1. Values in parentheses are percentages of the total
Phase 1
Item

GWP100

1

Contrast

Control

40 % DDGS

60% DDGS

SEM

P- value

Linear

Quadratic

CO2***

1

4417.48 (67.0)

3575.51 (70.5)

3801.90 (66.2)

373.44

0.31

0.19

0.37

CH4

25

1624.14 (24.6)

1184.91 (23.4)

1533.46 (26.7)

226.90

0.39

0.58

0.21

N2O

298

552.70 (8.4)

310.14 (6.1)

407.87 (7.1)

91.39

0.24

0.19

0.23

Total

-

6594.32 (100)

5070.56 (100)

5743.23 (100)

456.13

0.19

0.17

0.17

Phase 2
CO2

1

3961.93 (65.8)

3438.77 (69.2)

3464.90 (64.7)

373.44

0.55

0.31

0.68

CH4

25

1555.95 (25.8)

1126.88 (22.7)

1428.31 (26.7)

226.90

0.43

0.52

0.25

N2O

298

507.78 (8.4)

404.18 (8.1)

461.74 (8.6)

91.39

0.73

0.63

0.53

Total

-

6025.66 (100)

4969.83 (100)

5354.94 (100)

456.13

0.40

0.29

0.37

1

GWP100 indicates global warming potential within a fixed 100 yr period from the Intergovernmental Panel on Climate Change (2007)

a, b

Means without common superscripts within row differ

*** Phase 1 differs from phase 2 (P < 0.01)

114

Table 5.5 Effects of distiller’s grain with soluble on energy partition during trial 1 (MJ/d)
Diet

Contrast

Item

Control

40% DDGS

60% DDGS

SEM

P- value

Linear

Quadratic

Intake energy

107.31

112.33

114.36

14.84

0.94

0.73

0.99

Feces

32.36

34.15

36.68

4.21

0.74

0.49

0.84

Urine

2.04

4.08

b

0.48

0.04

0.02

0.79

Methane

2.39

1.73

2.19

0.36

0.45

0.53

0.26

Total loss

36.78

39.11

42.95

4.48

0.64

0.40

0.77

Digestible energy

74.95

78.18

77.67

11.63

0.98

0.85

0.92

Metabolizable energy

70.53

73.21

68.00

11.08

0.98

0.93

0.88

Heat energy

40.93

40.47

45.11

8.83

0.91

0.77

0.77

Net energy (gain)

29.60

32.75

26.30

9.91

0.89

0.87

0.67

a

1

1

ab

3.23

HE = 16.26 KJ × O2 (L/d) + 5.02 KJ × CO2 (L/d) – 2.17 KJ × CH4 (L/d) – 5.99 KJ × N (g/d); Brouwer (1965)

a, b

Means within a row without common superscripts differ

115

Figure 5.1 Predicted energy retention from cattle fed dried distiller’s grain with soluble
estimated from trial 1 results

♦ = actual measured values for NEg
■ = actual measured values for IE
× = predicted values

116

Trial 2
Molybdenum was added in the form of Na2MoO4 and Cu was provided through
supplemental CuCl2 in the 40% DDGS+ diets. Sodium molybdate and CuCl2 were included to
provide additional Mo and Cu. The actual intake of Mo and Cu to the 40% DDGS+ diet was 8
and 90 ppm, respectively. The effects of dietary treatments on animal performance are shown in
Table 5.6. Cattle were in positive energy balance and consuming more than 2% of their BW.
Growth and feed conversion efficiency were similar among treatments. Dry matter digestibility
(P = 0.07) tended to be greater in cattle fed the control diet compared to the cattle fed 40%
DDGS+ diet. Dietary levels of Mo and Cu were greater (P < 0.01) for the DDGS+ diet compared
to the control and traditonal 40% DDGS diet.

117

Table 5.6 Effects of dry distiller’s grain with soluble fortified with copper and molybdenum on
performance in trial 2
Diet
Control

40% DDGS

Initial BW, kg

300

Final BW, kg

342

DMI, kg/d

1

313

Items

40% DDGS+

2

SEM

P- value

312

16

0.82

351

339

16

0.86

8.07

7.12

7.49

0.47

0.40

ADG, kg

1.08

0.96

0.70

0.16

0.30

Gain: feed

0.132

0.131

0.091

0.02

0.20

DM digestibility, %

73.90

65.49

b

2.23

0.07

OM digestibility, %

74.41

66.66

2.35

0.11

Mo, ppm

1.63

b

0.40

< 0.01

Cu, ppm

12.89

4.84

< 0.01

1
2

a

a
a

ab

68.34

69.57
a

1.38

9.49

a

22.29

b

111.51

DDGS- dry distiller’s grain with soluble
40 % DDGS+- supplemented to provide 8 ppm Mo and 90 ppm Cu

a, b

Means without a common superscript within a row differ (P ≤ 0.10)

118

Table 5.7 Effects of distiller’s grain with soluble levels on carbon emissions and respiration for phase 1 and 2 during trial 2
Methane emission
Phase 1
Item

0

40
a

g/d

61.22

g/kg DMI

7.22

g/Mcal DE

3.22

ab
A

Phase 2
40 +

P- value

60.68

a

7.90

0

40

40 +

SEM

TRT

Phase

Phase × TRT

40.85

b

51.05

46.90

53.07

6.74

0.56

0.28

0.03

b

a

9.36

7.56

8.90

0.98

0.67

< 0.01

0.02

B

2.67

2.01

2.44

0.33

0.19

0.33

0.03

B

0.15

< 0.01

0.58

0.82

B

< 0.01

0.04

0.29

-

-

-

5.34

AB

2.53

1.87

Non-methane VOC emission
A

g/d

1.50

mg/kg DMI

168

A

B

0.82

B

B

0.87

B

A

1.36

A

B

0.74

B

0.90

UCL*

188

120

116

267

121

152

-

LCL*

149

95.1

91.4

211

95.4

120

-

-

-

-

31.8

40.6

B

6.52

< 0.01

0.62

0.80

mg/Mcal DE

A

77.8

107

90.6

B

34.6

B

38.8

238

A

71.0

107

135

B

Respiration
O2, kg/d

-5.18

CO2, kg/d

5.92

CO2, g/kg DMI
CO2, g/ Mcal DE
a, b

A

681
308

A

-6.18
AB

5.39

730
237

B

-6.17

-6.37

-5.95

-4.88

0.73

0.77

0.86

0.28

5.08

B

5.18

5.09

4.81

0.20

0.12

< 0.01

0.12

649

921

831

797

53.0

0.54

< 0.01

0.06

268

221

221

20.2

0.09

< 0.01

0.11

233

B

Means without common superscripts within a row for each phase differ (P ≤ 0.10)

A, B

Means without common superscripts within a row for each phase differ (P ≤ 0.05)

* Upper and lower confidence limits (exponent of untransformed mean ± 1 SEM) express reliability of the estimated mean
emissions. Emissions adjusted for DMI were back-transformed from a natural log transformation to satisfy normal distribution and
equal variance
119

Phase 1
Mean emissions reported were collected from d 12 to 15 (Table 5.7). The TEI 55C
analyzer that detects CH4 and VOC emissions had a malfunction on d 17 to 21. Only CH4
adjusted for DMI tended to show a treatment × day interaction (P = 0.08; data not shown). Nonmethane VOC emissions expressed as a mass output and adjusted for DMI both had a highly
significant day effect (P < 0.01) and a tendency for a treatment difference (P < 0.10; data not
shown). Carbon dioxide and O2 consumption also had significant day effects (P = 0.02 and P =
0.04, respectively). The daily variation suggests longer measurement periods are needed.
No differences were determined in CH4 emissions between the control and 40% DDGS
diets (Table 4.7). However, the addition of Mo and Cu at 8 and 80 ppm, respectively, tended to
decrease CH4 emissions compared to the control and 40% DDGS diets when expressed as a
mass output and when adjusted for DMI (P ≤ 0.10). Methane emissions adjusted for DE were
also decreased as a result of feeding the 40% DDGS+ diet compared to the control diet (P ≤
0.05). Non-methane VOC emissions were lower in both DDGS diets compared to the control
when expressed as mass output, adjusted for DMI, and when adjusted for DE (P ≤ 0.05). Nonmethane VOC adjusted for DMI had to be log transformed to satisfy the assumptions of
normality and homoscedasticity, which is why upper and lower confidence intervals are reported
rather than reporting the standard error of the mean. Carbon dioxide was decreased in the 40%
DDGS+ diet compared to the control when expressed as a mass output and adjusted for DE (P ≤
0.05).

120

Phase 2
The mean emissions reported in phase 2 (Table 5.7) represent d 26 to 29 when cattle were
fitted with fecal bags. Fecal bags were placed on the steers on d 24, allowing a 2 d adjustment
period prior to days used for analysis. Methane reported as mass output had a significant
treatment × day interaction (P = 0.01; data not shown) and non-methane VOC adjusted for DMI
showed a tendency for a treatment × day interaction (P = 0.09; data not shown). Methane
adjusted for DMI, VOC mass output, VOC output/DMI, CO2 mass output, and O2 consumption
all had significant day effects (P < 0.01; data not shown).
Methane emissions adjusted for DMI were significantly different in phase 2 compared to
phase 1 (P < 0.01; Table 5.7). Methane mass output, CH4 adjusted for DMI, and DE had a
significant phase × treatment interaction (P < 0.05). All parameters of VOC emissions showed a
significant treatment difference (P < 0.01) with VOC emissions showing a phase difference
when adjusted for DMI (P = 0.04). Similar to phase 1, VOC emissions were decreased in the
40% DDGS diets compared to the control (P ≤ 0.05). All parameters of CO2 were significantly
different in phase 2 compared to phase 1 (P < 0.01). However, CO2 mass output and CO2
adjusted for DE were decreased in phase 2, whereas CO2 adjusted for DMI increased in phase 2
compared to phase 1. Carbon dioxide adjusted for DMI also had a tendency for a phase ×
treatment interaction (P = 0.06).
In trial 2, all the N2O data was recorded as a negative number. This problem has
previously occurred during winter months. Perhaps the relative humidity or gases from the
heating unit were responsible. Feed, fecal, and urine samples collected in phase 2 during trial 2

121

were used to calculate an energy balance (Table 5.8). Intake energy and energy losses were
similar among treatments. However, the 40% DDGS diets did have a greater NEg compared the
control diet (P < 0.01).

122

Table 5.8 Effects of distiller’s grain with soluble on energy partition during trial 2 (MJ/d)
Diet

Contrast

Item

Control

40% DDGS

40% DDGS+

SEM

P-value

Cont vs. DDGS

40 vs. 40 +

Gross energy intake

122.81

143.32

142.95

10.68

0.34

0.15

0.98

Feces

41.52

43.50

50.51

3.40

0.20

0.22

0.18

Urine

2.98

3.29

3.38

0.32

0.68

0.40

0.87

Methane

2.72

2.75

2.97

0.37

0.88

0.78

0.69

Total loss

47.23

49.54

56.86

3.72

0.22

0.22

0.20

Digestible energy

81.28

99.82

92.44

8.22

0.32

0.17

0.54

Metabolizable energy

75.58

93.78

86.10

7.93

0.31

0.17

0.51

Heat energy

63.97

58.46

46.65

7.22

0.27

0.23

0.28

Net energy

11.61

b

5.97

0.02

< 0.01

0.64

1

1

a

b

35.32

39.45

HE = 16.26 KJ × O2 (L/d) + 5.02 KJ × CO2 (L/d) – 2.17 KJ × CH4 (L/d) – 5.99 KJ × N (g/d); Brouwer (1965)

a, b

Means within a row without common superscripts differ

123

DISCUSSION
Methane emissions were similar in phase 1 and 2 when cattle were fitted with fecal bags
to separate urine and feces. This would suggest that CH4 production is enteric and emitted
through eructation and flatulation. Compared to the control and 60% DDGS diets, steers fed the
40% DDGS diet tended to perform better based on ADG and DM and OM digestibilities (P <
0.10) while emitting the least amount of greenhouse gases (P = 0.08). Cattle fed the 40% DDGS
diet emitted the least amount of GHG and numerically lowest CH4 production in both phase 1
and 2 during trial 1. One explanation for a decrease in GHG and CH4 in the 40% DDGS diet
may be that CH4 acts as an energy sink, collecting H from rumen microorganisms (Fahey and
Berger, 1988). The DDGS diets were greater in dietary N and S, which may provide competing
H sink sources in the ruminal gas cap; resulting in less H to yield formate (HCOOH), which
actively converts to CH4 by methanogens (Fahey and Berger, 1988). Hydrogen sulfides that are
formed in the ruminal gas cap are likely absorbed by inhalation into the lungs (NRC, 2005;
Crawford, 2007) where H2S molecules enter the bloodstream and are eventually excreted as
sulfates in the urine. Therefore, NH3 and H2S gases may still be produced at high enough
concentrations in the rumen to compete with HCOOH for protons and limit formation of CH4
gas, despite enteric sources contributing very little NH3 and H2S emissions from eructation.
Other studies support this argument, as Loneragan et al. (1998) reported H2S in the ruminal gas
cap as high as 19 mg/L or 13,500 ppm in cattle fed high S diets. Additionally, volatile fatty acid
(VFA) production from ruminal microorganisms are likely to influence the amount of CH4
124

produced. An in-vitro study conducted by Behlke et al. (2007) reported a linear reduction of CH4
produced per g of digested DM when replacing brome hay with various levels of DDGS at: 0,
25, 50, 75, and 100%. Decreasing the ratio of forage:concentrate also decreases the ratio of
acetate:propionate and CH4 production (Annison et al., 1970; Van Soest, 1982; Fahey and
Berger, 1988; Behlke et al., 2007).
The 40% DDGS+ diet in trial 2 showed a tendency to reduce CH4 and CO2 emissions.
This may be the result of the sensitivity of methanogenic bacteria to dietary conditions. Increased
passage rate, increased fermentation, and decreased pH often improve animal performance as the
C and H lost in CH4 production is decreased and retained in propionate, increasing ME in the
diet (Fahey and Berger, 1988). Within trial 2 of the current study, the 40% DDGS diets had a
numerically greater ME and showed a significant increase in NE compared to the control (P =
0.02). In trial 1 and 2, fecal loss accounted for approximately 30-35% of the gross IE. A study
conducted by Tyrrell et al. (1988) reported loss from FE to be around 35% in diets fed to
lactating cows. Kurihara et al. (1999) reported a FE loss near 30% when feeding a high grain diet
to Brahman heifers. Other reports suggest that of the total IE, approximately 45% is lost as heat,
40% lost as feces, 10% lost as urine and gas, leaving only 5% of the energy retained for growth
(Ferrell, 1988). In both trials, energy lost as heat accounted for 33 to 52% of the total IE. Energy
loss from CH4 gas is low within the current study compared to others. However, the previous
studies fed diets containing a greater amount of forage compared to the diets fed in this study.
Consequently, NE in the current study is greater in comparison to other reports. Estimation of the
net energy value of DDGS was 0.93 Mcal/kg DMI when subtracting the estimated tabular values
for CS and SBM (Figure 5.1). The improved performance when feeding the 40% DDGS diet
125

compared to the control and 60% DDGS diets infers a large positive associative effect. This may
be reflected in improved ruminal fermentation, but this was not measured in the current study.
Furthermore, Figure 5.1 would suggest that feeding DDGS in moderate levels, between 25 to
40% shows the greatest efficiency in converting IE to NEg.
All livestock species are reported to emit 58 million tons/yr or 73% of the total annual
emitted CH4 each year (US Environmental Protection Agency, 1994). The U.S. Environmental
Protection Agency (2000) reported that the average passenger car emits 95 g/d of hydrocarbons
and over 14 kg/d of CO2. Additionally, a small truck emits 133 g/d of hydrocarbons and just
under 20 kg/d of CO2. In comparison, steers from our study that weighed around 300 kg and
consumed about 7 kg DMI/d concentrate diet, emitted approximately 30 to 60 g/d of
hydrocarbons (CH4 and VOC) and 3.5 to 6.0 kg/d of CO2 per steer. Based on these results, a
passenger car emits 2- fold the hydrocarbons and a small truck emits nearly 3- fold the
hydrocarbons compared to the emissions generated from steers within the current study. Carbon
dioxide emitted from a passenger car and small truck is nearly 3 and 4- fold greater, respectively,
than the CO2 emitted by the steers within our study.
Based on our results, steers fed concentrate diets consisting of either corn or DDGS
produce less CH4 compared to estimates from cattle fed forage-based diet and therefore have a
greater NE. This energy efficiency results in less overall GHG as well. However, globally the
majority of cattle are fed in a grazing system. In order to mitigate the GHG footprint from
livestock globally, further research should consider strategies to reduce GHG in livestock reliant
on forage-based feeds without sacrificing the health and performance of the animal. Within more

126

intensive feeding systems or feedlots, further research could provide stronger evidence that
feeding moderate levels of DDGS (20 to 40%) generates lower GHG compared to other
concentrate diets. Furthermore, more research should be conducted on the effects of
supplementing Cu and Mo may have on ruminant health and reduction of CH4 emissions.

127

CHAPTER 6: CONCLUSIONS
The current study provides evidence that manure is the main source of H2S and Nemissions. The H2S gas produced in the rumen was likely absorbed in the lungs or lower GI tract
as protonated sulfides are unable to be absorbed through the rumen epithelium (NRC, 2005;
Crawford, 2007). Hydrogen sulfide and NH3 emissions from the manure (urine and feces) did
not linearly increase as dietary inclusion of DDGS increased from 0 to 60%. Hydrogen sulfide
emissions tended to demonstrate a quadratic effect, as H2S emissions increased with the 40%
DDGS diet and decreased at 60%. No dietary differences were determined for NH3 emissions or
greenhouse gases (GHG; CH4, CO2, N2O). Cattle fed the 60% DDGS diet tended to have lower
ADG and DM digestibility. This suggests that feeding DDGS at 60% inclusion or greater may
negatively affect ruminal fermentation, and also explain the decreased H2S emissions in the 60%
DDGS diet compared to the 40% DDGS diet. Others have reported diminishing performance
when DGS inclusion levels exceed 45% of the dietary DM (Gordon et al., 2002; Gunn et al.,
2009). Further research looking at mitigation strategies for these gases should consider waste
treatment strategies rather than dietary treatments to the animal.
Including Mo and Cu in the diet tended to decrease manure H2S emissions. The decrease
of H2S emissions could be an effect of insoluble compounds (MoS4Cu) forming in the manure
(Hamsell et al., 2010). Another possible explanation is Mo and Cu have inhibitory effects on
sulfate-reducing bacteria (SRB), decreasing H2S emissions from the manure. Molybdate has
been reported to decrease H2S production in ruminal gas caps by inhibition of SRB (Oremland
128

and Capone, 1988; Loneragan et al., 1998; Kung, 2008). Therefore, it is reasonable to consider
this inhibitory effect may occur in the manure as well. The tendency for Mo and Cu to decrease
H2S emissions should be further researched with more animals per treatment. Additionally,
measurements of tetrathiomolybdates in the blood and feces could help determine if Mo and Cu
are forming insoluble compounds. Studies could be performed as well to test the potential
inhibitory effects of Mo and Cu on SRB in the rumen and feces.
The use of Mo and Cu with a 40% DDGS diet also showed tendencies to decrease CH4
emissions. However, further research should consider using a treatment with high forage diets
that produce greater levels of CH4 gas than the high concentrate diets used in the current study.
Additionally, decreasing energy loss from CH4 production could improve animal performance
and offer a financial incentive to producers. Methane emissions may have decreased as a result
of rumen microbial sensitivity to an increase in dietary Mo and Cu (Jones et al., 1982). It is
unclear how Mo and Cu may be decreasing CH4 gas. Future studies should consider looking at
the impact of Mo and Cu on methanogens and ruminal CH4 gas production.
The rate of excreted S and N linearly increased with greater concentrations of DDGS in
cattle diets. In both trials, the 40% DDGS diets increased S and N digestibility compared to the
control diet. Sulfur and N retention were also increased in the 40% DDGS diets in trial 2.
However, S and N retention were similar in trial 1. Increasing levels of DDGS fed to cattle
provides greater concentrations of dietary methionine and cysteine. Some of the increased
excretion of S may be explained by the greater levels of S-containning amino acids the the
DDGS diets. Metabolism of methionine and cysteine results in elevated S excretion as sulfates in

129

urine (Fron et al.,1990). Gross intake energy and total energy loss did not differ from increasing
concentrations of DDGS in cattle diets. Urine energy decreased with the inclusion of DDGS in
trial 1 but did not differ in trial 2. Conversely, net energy gain was greater in the 40% DDGS
diets compared to the control diet in trial 2 but was similar in trial 1. Overall, feeding DDGS at
moderate levels does increase H2S and NH3 emissions compared to traditional corn-based diets.
The use of Mo and Cu may decrease manure H2S emissions and ruminal CH4 gas production.

130

APPENDICES

131

APPENDIX A

Trial 1
Table A.1 Comparison of unfiltered raw data to filtered data during trial 1
Unfiltered raw data*
Variable

N

Mean

Standard Deviation

Minimum

Maximum

Temperature, ºF

18547

58.863

3.944

49.108

68.569

Humidity, %

18547

49.663

11.234

24.896

104.000

Air flow, cfm

18547

500.968

188.588

0.000

654.083

H2S, ppm

18547

0.005

0.008

0.000

0.135

SO2, ppm

18547

0.001

0.001

0.000

0.018

Filtered raw data*
Variable

N

Mean

Standard Deviation

Minimum

Maximum

Temperature, ºF

18547

58.863

3.944

49.108

68.569

Humidity, %

18547

49.663

11.234

24.896

104.000

Air flow, cfm

17117

542.821

125.766

106.843

654.083

H2S, ppm

18541

0.005

0.007

0.000

0.098

SO2, ppm

18547

0.001

0.001

0.000

0.018

* Raw data represents all of the days cattle were housed within environmentally-controlled
rooms

132

Figure A.1 Daily hydrogen sulfide emissions output during trial 1
Phase 1

Phase 2

* Days 11-14 represent steady state mean emissions during phase 1. Days 16 and 17 represent
means for phase 2. Error bars for phase 1 and phase 2 represent the SEM

Figure A.2 Daily hydrogen sulfide emissions adjusted on sulfur intake during trial 1
Phase 1

Phase 2

* Days 11-14 represent steady state mean emissions during phase 1. Days 16 and 17 represent
means for phase 2. Error bars for phase 1 and phase 2 represent the SEM
133

Figure A.3 Daily hydrogen sulfide emissions adjusted on dry matter intake during trial 1
Phase 1

Phase 2

* Days 11-14 represent steady state mean emissions during phase 1. Days 16 and 17 represent
means for phase 2. Error bars for phase 1 and phase 2 represent the SEM

134

Figure A.4 Daily sulfur dioxide emissions output during trial 1
Phase 1
se 1

Phase 2
ase 2

* Days 11-14 represent steady state mean emissions during phase 1. Days 16 and 17 represent
means for phase 2. Error bars for phase 1 and phase 2 represent the SEM

Figure A.5 Daily sulfur dioxide emissions adjusted on sulfur intake during trial 1
Phase 1
ase 1

Phase
hase 2 2

* Days 11-14 represent steady state mean emissions during phase 1. Days 16 and 17 represent
means for phase 2. Error bars for phase 1 and phase 2 represent the SEM
135

Figure A.6 Daily sulfur dioxide emissions adjusted on dry matter intake during trial 1
Phase 2

Phase 1

* Days 11-14 represent steady state mean emissions during phase 1. Days 16 and 17 represent
means for phase 2. Error bars for phase 1 and phase 2 represent the SEM

Table A.2 Probability of day effects for sulfur-containing emissions during trial 1
Phase 1

Phase 2

Variable

TRT

Day

TRT × Day

TRT

Day

TRT × Day

H2S, mg/d

0.36

< 0.01

0.52

0.26

0.02

0.25

H2S, mg/g SI

0.51

< 0.01

0.68

0.10

0.07

0.59

H2S, mg/kg DMI

0.41

0.01

0.69

0.31

0.02

0.40

SO2, mg/d

0.59

< 0.01

0.50

0.81

0.88

0.73

SO2, mg/g SI

0.61

< 0.01

0.39

0.48

0.73

0.40

SO2, mg/kg DMI

0.66

< 0.01

0.44

0.92

0.36

0.98

136

Figure A.7 Mean hydrogen sulfide emissions output during trial 1

Figure A.8 Mean hydrogen sulfide emissions adjusted on sulfur intake during trial 1

137

Figure A.9 Mean hydrogen sulfide emissions adjusted on dry matter intake during trial 1

138

Figure A.10 Mean sulfur dioxide emissions output during trial 1

Figure A.11 Mean sulfur dioxide emissions adjusted on sulfur intake during trial 1

139

Figure A.12 Mean sulfur dioxide emissions adjusted on dry matter intake during trial 1

140

Trial 2
Table A.3 Comparison of unfiltered raw data to filtered data during trial 2
Unfiltered raw data*
Variable

N

Mean

Standard Deviation

Minimum

Maximum

Temperature, ºF

28870

60.367

1.813

48.688

65.353

Humidity, %

28870

36.041

11.308

16.109

104.000

Air flow, cfm

28870

545.111

179.405

0.000

659.829

H2S, ppm

28870

0.010

0.014

-0.003

0.405

SO2, ppm

28869

0.003

0.001

-0.003

0.009

Filtered raw data*
Variable

N

Mean

Standard Deviation

Minimum

Maximum

Temperature, ºF

28870

60.367

1.813

48.688

65.353

Humidity, %

28870

36.041

11.308

16.109

104.000

Air flow, cfm

26609

591.430

86.750

97.005

659.829

H2S, ppm

28791

0.010

0.012

-0.003

0.100

SO2, ppm

28869

0.003

0.001

-0.003

0.009

* Raw data represents all of the days cattle were housed within environmentally-controlled
rooms

141

Figure A.13 Daily hydrogen sulfide emissions output during trial 2
Phase 1

Phase 2

* Days 20-23 represent steady state mean emissions during phase 1. Days 26-29 represent means
for phase 2. Error bars for phase 1 and phase 2 represent the SEM

Figure A.14 Daily hydrogen sulfide emissions adjusted on sulfur intake during trial 2
Phase 1

Phase 2
22

* Days 20-23 represent steady state mean emissions during phase 1. Days 26-29 represent means
for phase 2. Error bars for phase 1 and phase 2 represent the SEM
142

Figure A.15 Daily hydrogen sulfide emissions adjusted on dry matter intake during trial 2
Phase 1
hase

Phase 2
22

* Days 20-23 represent steady state mean emissions during phase 1. Days 26-29 represent means
for phase 2. Error bars for phase 1 and phase 2 represent the SEM

143

Figure A.16 Daily sulfur dioxide emissions output during trial 2
Phase 1

Phase 2
22

* Days 20-23 represent steady state mean emissions during phase 1. Days 26-29 represent means
for phase 2. Error bars for phase 1 and phase 2 represent the SEM

Figure A.17 Daily sulfur dioxide emissions adjusted on sulfur intake during trial 2
Phase 1

Phase 2
22

* Days 20-23 represent steady state mean emissions during phase 1. Days 26-29 represent means
for phase 2. Error bars for phase 1 and phase 2 represent the SEM
144

Figure A.18 Daily sulfur dioxide emissions adjusted on dry matter intake during trial 2
Phase 1

Phase 2
22

* Days 20-23 represent steady state mean emissions during phase 1. Days 26-29 represent means
for phase 2. Error bars for phase 1 and phase 2 represent the SEM

Table A.4 Probability of day effects for sulfur-containing emissions during trial 2
Phase 1

Phase 2

Variable

TRT

Day

TRT × Day

TRT

Day

TRT × Day

H2S, mg/d

0.99

< 0.01

0.38

0.47

0.21

0.64

H2S, mg/g SI

0.99

< 0.01

0.95

0.73

0.78

0.63

H2S, mg/kg DMI

0.92

< 0.01

0.61

0.56

0.53

0.87

SO2, mg/d

0.55

< 0.01

0.35

0.14

< 0.01

0.99

SO2, mg/g SI

0.39

0.01

0.71

0.53

< 0.01

0.90

SO2, mg/kg DMI

0.55

< 0.01

0.42

0.07

< 0.01

0.89

145

Figure A.19 Mean hydrogen sulfide emissions output during trial 2

Figure A.20 Mean hydrogen sulfide emissions adjusted on sulfur intake during trial 2

146

Figure A.21 Mean hydrogen sulfide emissions adjusted on dry matter intake during trial 2

147

Figure A.22 Mean sulfur dioxide emissions output during trial 2

Figure A.23 Mean sulfur dioxide emissions adjusted on sulfur intake during trial 2

148

Figure A.24 Mean sulfur dioxide emissions adjusted on dry matter intake during trial 2

149

APPENDIX B

Trial 1
Table B.1 Comparison of unfiltered raw data to filtered data during trial 1
Unfiltered raw data*
Item

N

Mean

SD

Minimum

Maximum

Temperature, ºF

18547

58.863

3.944

49.108

68.569

Humidity, %

18547

49.663

11.234

24.896

104.000

Air flow, cfm

18547

500.968

188.588

0.000

654.083

Ammonia, ppm

18547

0.871

0.664

0.000

4.891

Nitric oxide, ppm

18547

0.017

0.014

0.012

0.216

Nitrogen dioxide, ppm

18547

0.064

0.028

0.017

0.270

Nitrous oxide, ppm

8064

0.650

0.251

0.096

1.313

Filtered raw data*
Item

N

Mean

SD

Minimum

Maximum

Temperature, ºF

18547

58.863

3.944

49.108

68.569

Humidity, %

18547

49.663

11.234

24.896

104.000

Air flow, cfm

17117

542.821

125.766

106.843

654.083

Ammonia, ppm

18547

0.871

0.664

0.000

4.891

Nitric oxide, ppm

18547

0.017

0.014

0.012

0.216

Nitrogen dioxide, ppm

18547

0.064

0.028

0.017

0.270

Nitrous oxide, ppm

8064

0.650

0.251

0.096

1.313

* Raw data represents all of the days cattle were housed within environmentally-controlled
rooms

150

Figure B.1 Daily ammonia emissions output during trial 1
Phase 1

Phase 2

* Days 11-14 represent steady state mean emissions during phase 1. Days 16 and 17 represent
means for phase 2. Error bars for phase 1 and phase 2 represent the SEM

Figure B.2 Daily ammonia emissions adjusted for nitrogen intake during trial 1
Phase 1

Phase 2

* Days 11-14 represent steady state mean emissions during phase 1. Days 16 and 17 represent
means for phase 2. Error bars for phase 1 and phase 2 represent the SEM
151

Figure B.3 Daily ammonia emissions adjusted for dry matter intake during trial 1
Phase 1

Phase 2

* Days 11-14 represent steady state mean emissions during phase 1. Days 16 and 17 represent
means for phase 2. Error bars for phase 1 and phase 2 represent the SEM

152

Figure B.4 Daily nitrogen oxide emissions output during trial 1
Phase 1

Phase 2

* Days 11-14 represent steady state mean emissions during phase 1. Days 16 and 17 represent
means for phase 2. Error bars for phase 1 and phase 2 represent the SEM

Figure B.5 Daily nitrogen oxide emissions adjusted for nitrogen intake during trial 1
Phase 1

Phase 2

* Days 11-14 represent steady state mean emissions during phase 1. Days 16 and 17 represent
means for phase 2. Error bars for phase 1 and phase 2 represent the SEM

153

Figure B.6 Daily nitrogen oxide emissions adjusted for dry matter intake during trial 1
Phase 1

Phase 2

* Days 11-14 represent steady state mean emissions during phase 1. Days 16 and 17 represent
means for phase 2. Error bars for phase 1 and phase 2 represent the SEM

154

Figure B.7 Daily nitrous oxide emissions output during trial 1
Phase 1

Phase 2

* Days 11-14 represent steady state mean emissions during phase 1. Days 16 and 17 represent
means for phase 2. Error bars for phase 1 and phase 2 represent the SEM

Figure B.8 Daily nitrous oxide emissions adjusted for nitrogen intake during trial 1
Phase 1

Phase 2

* Days 11-14 represent steady state mean emissions during phase 1. Days 16 and 17 represent
means for phase 2. Error bars for phase 1 and phase 2 represent the SEM

155

Figure B.9 Daily nitrous oxide emissions adjusted for dry matter intake during trial 1
Phase 1

Phase 2

* Days 11-14 represent steady state mean emissions during phase 1. Days 16 and 17 represent
means for phase 2. Error bars for phase 1 and phase 2 represent the SEM

Table B.2 Probability of day affects on nitrogenous emissions during trial 1
Phase 1

Phase 2

Item

TRT

Day

TRT × Day

TRT

Day

TRT × Day

Ammonia, g/d

0.66

0.13

0.22

0.80

0.73

0.23

Ammonia, mg/g NI

0.44

0.13

0.31

0.71

0.84

0.25

Ammonia, g/kg DMI

0.42

< 0.01

0.20

0.82

0.06

0.25

Nitrogen oxide, mg/d

0.49

< 0.01

0.05

0.33

0.79

0.22

Nitrogen oxide, mg/g NI

0.47

< 0.01

0.26

0.20

0.81

0.28

Nitrogen oxide, g/kg DMI

0.52

< 0.01

0.06

0.45

0.61

0.75

Nitrous oxide, g/d

0.29

0.42

0.75

0.40

0.86

0.18

Nitrous oxide, mg/g NI

0.18

0.24

0.72

0.37

0.57

0.15

Nitrous oxide, g/kg DMI

0.34

0.17

0.73

0.59

0.91

0.94

156

Figure B.10 Mean ammonia emissions output during trial 1

Figure B.11 Mean ammonia emissions adjusted for nitrogen intake during trial 1

157

Figure B.12 Mean ammonia emissions adjusted for dry matter intake during trial 1

158

Figure B.13 Mean nitrogen oxide emission output during trial 1

1

-

Nitrogen oxide includes both NO and NO2 gas

Figure B.14 Mean nitrogen oxide emissions adjusted for nitrogen intake during trial 1

1

-

Nitrogen oxide includes both NO and NO2 gas
159

Figure B.15 Mean nitrogen oxide emissions adjusted for dry matter intake during trial 1

1

-

Nitrogen oxide includes both NO and NO2 gas

160

Figure B.16 Mean nitrous oxide emissions output during trial 1

Figure B.17 Mean nitrous oxide emissions adjusted for nitrogen intake during trial 1

161

Figure B.18 Mean nitrous oxide emissions adjusted for nitrogen intake during trial 1

162

Trial 2
Table B.3 Comparison of unfiltered raw data to filtered data during trial 2
Unfiltered raw data*
Item

N

Mean

SD

Minimum

Maximum

Temperature, ºF

28870

60.367

1.813

48.688

65.353

Humidity, %

28870

36.041

11.308

16.109

104.000

Air flow, cfm

28870

545.111

179.405

0.000

659.829

Ammonia, ppm

28870

0.870

0.575

-0.017

5.771

Nitric oxide, ppm

28870

0.086

0.072

-0.020

3.540

Nitrogen dioxide, ppm

28870

0.131

0.118

-0.026

6.684

Nitrous oxide, ppm

12897

-0.019

0.064

-0.253

0.238

Filtered raw data*
Item

N

Mean

SD

Minimum

Maximum

Temperature, ºF

28870

60.367

1.813

48.688

65.353

Humidity, %

28870

36.041

11.308

16.109

104.000

Air flow, cfm

26609

591.430

86.750

97.005

659.829

Ammonia, ppm

28870

0.870

0.575

-0.017

5.771

Nitric oxide, ppm

28860

0.086

0.061

-0.020

0.335

Nitrogen dioxide, ppm

28853

0.129

0.047

-0.026

0.344

Nitrous oxide, ppm

12897

-0.019

0.064

-0.253

0.238

* Raw data represents all of the days cattle were housed within environmentally-controlled
rooms

163

Figure B.19 Daily ammonia emissions output during trial 2
Phase 1

Phase 2

* Days 20-23 represent steady state mean emissions during phase 1. Days 26-29 represent means
for phase 2. Error bars for phase 1 and phase 2 represent the SEM

Figure B.20 Daily ammonia emissions adjusted for nitrogen intake during trial 2
Phase 1

Phase 2

* Days 20-23 represent steady state mean emissions during phase 1. Days 26-29 represent means
for phase 2. Error bars for phase 1 and phase 2 represent the SEM
164

Figure B.21 Daily ammonia emissions adjusted for dry matter intake during trial 2
Phase 1

Phase 2

* Days 20-23 represent steady state mean emissions during phase 1. Days 26-29 represent means
for phase 2. Error bars for phase 1 and phase 2 represent the SEM

165

Figure B.22 Daily nitrogen oxide emissions output during trial 2

* Days 20-23 represent mean emissions during phase 1. Days 26-29 represent means for phase 2

Figure B.23 Daily nitrous oxide emissions output during trial 2

* Days 20-23 represent mean emissions during phase 1. Days 26-29 represent means for phase 2
166

Figure B.23 Mean ammonia emission output during trial 2

Figure B.24 Mean ammonia emissions adjusted for nitrogen intake during trial 2

167

Figure B.25 Mean ammonia emissions adjusted for dry matter intake during trial 2

168

Figure B.26 Mean nitrogen oxide emissions output during trial 2

Figure B.27 Mean nitrogen oxide emissions adjusted for nitrogen intake during trial 2

169

Figure B.28 Mean nitrogen oxide emissions adjusted for dry matter intake during trial 2

170

Figure B.29 Mean nitrous oxide emissions output during trial 2

Figure B.30 Mean nitrous oxide emissions adjusted for nitrogen intake during trial 2

171

Figure B.31 Mean nitrous oxide emissions adjusted for dry matter intake during trial 2

172

APPENDIX C

Trial 1
Table C.1 Comparison of unfiltered raw data to filtered data during trial 1
Unfiltered raw data
Variable

N

Mean

SD

Minimum

Maximum

Temperature,º F

18547

58.863

3.944

49.108

68.569

Humidity, %

18547

49.663

11.234

24.896

104.000

Air flow, cfm

18547

500.968

188.588

0.000

654.083

CH4, ppm

18547

5.111

2.057

2.056

29.609

NMTHC, ppm

18547

0.055

0.038

0.000

0.291

CO2, ppm

18547

619.836

99.749

392.140

1355.350

O2, %

18547

20.811

0.199

20.627

26.545

Filtered raw data
Variable

N

Mean

SD

Minimum

Maximum

Temperature,º F

18547

58.863

3.944

49.108

68.569

Humidity, %

18547

49.663

11.234

24.896

104.000

Air flow, cfm

17117

542.821

125.766

106.843

654.083

CH4, ppm

18547

5.111

2.057

2.056

29.609

NMTHC, ppm

18547

0.055

0.038

0.000

0.291

CO2, ppm

18520

618.899

96.740

392.140

1198.020

O2, %

18518

20.804

0.063

20.627

21.387

173

Figure C.1 Daily methane emissions output during trial 1
Phase 1

Phase 2

* Days 11-14 represent steady state mean emissions during phase 1. Days 16 and 17 represent
means for phase 2. Error bars for phase 1 and phase 2 represent the SEM

Figure C.2 Daily methane emissions adjusted for dry matter intake during trial 1
Phase 1

Phase 2

* Days 11-14 represent steady state mean emissions during phase 1. Days 16 and 17 represent
means for phase 2. Error bars for phase 1 and phase 2 represent the SEM
174

Figure C.3 Daily non-methane total hydrocarbon emissions output during trial 1
Phase 1

Phase 2

* Days 11-14 represent steady state mean emissions during phase 1. Days 16 and 17 represent
means for phase 2. Error bars for phase 1 and phase 2 represent the SEM

Figure C.4 Daily non-methane total hydrocarbon emissions adjusted for dry matter intake during
trial 1
Phase 1

Phase 2

* Days 11-14 represent steady state mean emissions during phase 1. Days 16 and 17 represent
means for phase 2. Error bars for phase 1 and phase 2 represent the SEM
175

Figure C.5 Daily carbon dioxide emissions output during trial 1

Phase 1

Phase 2

* Days 11-14 represent steady state mean emissions during phase 1. Days 16 and 17 represent
means for phase 2. Error bars for phase 1 and phase 2 represent the SEM

Figure C.6 Daily carbon dioxide emissions output during trial 1
Phase 1

Phase 2

* Days 11-14 represent steady state mean emissions during phase 1. Days 16 and 17 represent
means for phase 2. Error bars for phase 1 and phase 2 represent the SEM
176

Figure C.7 Daily oxygen consumption during trial 1
Phase 1

Phase 2

* Days 11-14 represent steady state mean consumption during phase 1. Days 16 and 17 represent
means for phase 2. Error bars for phase 1 and phase 2 represent the SEM

Table C.2 Probabilities of day effects for carbon emissions and oxygen consumption during
trial 1
Phase 1

Phase 2

Variable

TRT

Day

TRT × Day

TRT

Day

TRT × Day

CH4, g/d

0.38

0.99

0.84

0.44

0.11

0.70

CH4, g/kg DMI

0.31

0.85

0.80

0.73

< 0.01

0.23

VOC, g/d

0.39

0.80

0.61

0.50

< 0.01

0.68

VOC, mg/kg DMI

0.15

0.81

0.83

0.57

< 0.01

0.73

CO2, g/d

0.20

0.36

0.62

0.63

0.12

0.89

CO2, g/kg DMI

0.10

0.05

0.94

0.94

0.03

0.49

O2, g/d

0.94

0.02

0.79

0.93

< 0.01

0.14

177

Figure C.8 Mean methane emissions output during trial 1

Figure C.9 Mean methane emissions adjusted for dry matter intake during trial 1

178

Figure C.10 Mean non-methane total hydrocarbon emissions output during trial 1

Figure C.11 Mean non-methane total hydrocarbon emissions adjusted for dry matter intake
during trial 1

179

Figure C.12 Mean carbon dioxide emissions output during trial 1

Figure C.13 Mean carbon dioxide emissions adjusted for dry matter intake during trial 1

180

Trial 2
Table C.3 Comparison of unfiltered raw data to filtered data during trial 2
Unfiltered raw data
Variable

N

Mean

SD

Minimum

Maximum

Temperature,º F

28870

60.36718

1.812508

48.68827

65.35302

Humidity, %

28870

36.04142

11.30774

16.10901

104

Air flow, cfm

28870

545.1113

179.4046

0

659.829

CH4, ppm

28870

2.134286

588.5773

-100000

9.999296

NMTHC, ppm

28870

-3.37049

588.5415

-100000

10.13834

CO2, ppm

28870

944.197

185.3528

-7.13237

1783.55

O2, %

28870

20.76054

0.535329

-0.02451

26.4835

Filtered raw data
Variable

N

Mean

SD

Minimum

Maximum

Temperature,º F

28870

60.36718

1.812508

48.68827

65.35302

Humidity, %

28870

36.04142

11.30774

16.10901

104

Air flow, cfm

26609

591.4301

86.75048

97.00471

659.829

CH4, ppm

28870

2.134286

588.5773

-100000

9.999296

NMTHC, ppm

28870

-3.37049

588.5415

-100000

10.13834

CO2, ppm

28870

944.197

185.3528

-7.13237

1783.55

O2, %

28870

20.76054

0.535329

-0.02451

26.4835

181

Figure C.14 Daily methane emissions output during trial 2
Phase 1

Phase 2

* Days 12-15 represent steady state mean emissions during phase 1. Days 26-29 represent means
for phase 2. Error bars for phase 1 and phase 2 represent the SEM

Figure C.15 Daily methane emissions adjusted for dry matter intake during trial 2
Phase 1

Phase 2

* Days 12-15 represent steady state mean emissions during phase 1. Days 26-29 represent means
for phase 2. Error bars for phase 1 and phase 2 represent the SEM
182

Figure C.16 Daily non-methane total hydrocarbon emissions output during trial 2
Phase 1

Phase 2

* Days 12-15 represent steady state mean emissions during phase 1. Days 26-29 represent means
for phase 2. Error bars for phase 1 and phase 2 represent the SEM

Figure C.17 Daily non-methane total hydrocarbon emissions adjusted for dry matter intake
during trial 2
Phase 1

Phase 2

* Days 12-15 represent steady state mean emissions during phase 1. Days 26-29 represent means
for phase 2. Error bars for phase 1 and phase 2 represent the SEM
183

Figure C.18 Daily carbon dioxide emissions output during trial 2
Phase 1

Phase 2

* Days 12-15 represent steady state mean emissions during phase 1. Days 26-29 represent means
for phase 2. Error bars for phase 1 and phase 2 represent the SEM

Figure C.19 Daily carbon dioxide emissions adjusted for dry matter intake during trial 2
Phase 1

Phase 2

* Days 12-15 represent steady state mean emissions during phase 1. Days 26-29 represent means
for phase 2. Error bars for phase 1 and phase 2 represent the SEM
184

Figure C.20 Daily oxygen consumption during trial 2
Phase 1

Phase 2

* Days 12-15 represent steady state mean consumption during phase 1. Days 26-29 represent
means for phase 2. Error bars for phase 1 and phase 2 represent the SEM

Table C.4 Probability of day effects for carbon emissions and oxygen consumption during trial 2
Phase 1

Phase 2

Variable

TRT

Day

TRT × Day

TRT

Day

TRT × Day

CH4, g/d

0.12

0.32

0.20

0.78

0.37

0.01

CH4, g/kg DMI

0.21

0.59

0.08

0.45

< 0.01

0.42

VOC, g/d

0.08

< 0.01

0.72

< 0.01

< 0.01

0.24

VOC, mg/kg DMI

0.06

< 0.01

0.58

< 0.01

0.02

0.09

CO2, g/d

0.03

0.02

0.30

0.55

< 0.01

0.74

CO2, g/kg DMI

0.61

0.05

0.47

0.25

< 0.01

0.62

O2, g/d

0.66

0.04

0.58

0.23

< 0.01

0.77

185

Figure C.21 Mean methane emissions output during trial 2

Figure C.22 Mean methane emissions adjusted for dry matter intake during trial 2

186

Figure C.23 Mean non-methane total hydrocarbon emissions output during trial 2

Figure C.24 Mean non-methane total hydrocarbon emissions adjusted for dry matter intake
during trial 2

187

Figure C.25 Mean carbon dioxide emissions output during trial 2

Figure C.26 Mean carbon dioxide emission output during trial 2

188

APPENDIX D: DETERMINATION OF COPPER AND MOLYBDENUM LEVELS IN
FERMENTATION VESSELS TO MITIGATE SULFIDE GAS PRODUCTION.
L.D. Cross, W.J. Powers, J.S. Liesman, and S.R. Rust
Michigan State University, East Lansing 48824
SUMMARY
Laboratory experiments were conducted to determine the effects of different or ranging
levels of Mo and Cu in high concentrate diets as a strategy to mitigate H2S emissions. Two
experiments were conducted; experiment 1 used 125 ml fermentation vessels with 10 ml of
rumen fluid, 10 ml of Van Soest media without reducing agent, 0.5 g of DDGS as a substrate,
along with various combinations of Mo and Cu in solution added to each vessel. Concentrations
included; 0, 3, 6, and 9 ppm Mo in combination with 0, 30, 60, and 90 ppm Cu using 2 Cu
sources, CuCl2 and CuSO4. In experiment 2, Mo and Cu concentrations increased 10-fold, using
0, 30, 60, and 90 ppm Mo in combination with 0, 300, 600, and 900 ppm Cu. Vessels for each
experiment were tested in duplicate and incubated at 39° C for 16 h. Results from experiment 1
demonstrated a quadratic effect for H2S production from Cu treatments only (P < 0.01). In
experiment 2, both Mo and Cu linearly decreased H2S emissions as treatment levels increased (P
< 0.01). In both experiments, lower emitted levels of H2S gas were observed when CuCl2 was
added (P < 0.01) compared to CuSO4. Treatment with Mo and Cu did not affect microbial
fermentation based on overall gas production, IVDMD, and pH.
Key words: Copper, molybdenum, distiller’s grain with soluble, in vitro, hydrogen sulfide

189

INTRODUCTION
Distiller’s grain with soluble (DGS) is a co-product from ethanol production that has
become a popular feed source to livestock, particularly feedlot cattle. During production of DGS,
sulfuric acid is added during the fermentation process to regulate pH and as a cleaning agent, and
this increases the amount and concentration of S in DGS (Vannes et al., 2009a; Kelzer et al.,
2010). High levels of sulfates and other forms of dietary S are reduced in the rumen by sulfatereducing bacteria (SRB) to produce hydrogen sulfide (H2S) and other ionic forms that are not
only hazardous to the animal but can cause potential air quality issues by increased H2S
emissions. Excess generation of H2S in the rumen will depress normal rumen function and could
lead to respiratory problems (Kandylis, 1983), as the majority of belched gas is inhaled into the
lungs (Bulgin et al., 1996). Hydrogen sulfide gas is dangerous for humans to inhale (Osweiler et
al., 1985; Gerber et al., 1991). Excess H2S in ambient air can have a detrimental impact to the
environment, such as acid rain, and eutrophication (Guang-hui et al., 2007).
Molybdate (MoO4) inhibits SRB by working as an analog of sulfate, blocking the sulfate
activation step that is catalyzed by ATP sulfurylase (Oremland and Capone, 1988). Sodium
molybdate decreases H2S production in cattle fed high S diets, although results were not
consistent among cattle (Loneragan et al., 1998). Additionally, the study from Loneragan et al.
(1998) showed that inclusion of MoO4 dramatically decreased liver Cu stores in cattle.
Molybdenum (Mo) and S react to form tetrathiomolybdates that react with copper (Cu) and
particulate matter in the rumen. This results in the formation of highly stable compounds that
cannot be digested and absorbed (Allen and Gawthorne, 1987; Suttle, 1991). The synergetic
190

effect between Mo and S begins with the substitution of S for oxygen in the MoO4

2-

ion to

2-

ultimately yield tetrathiomolybdate (MoS4 ).
2-

2-

MoO4  MoO3S  MoO2S2

2-

2-

 MoOS3  MoS4

2-

Tetrathiomolybdate has the potential to bind ruminal Cu ions, rendering the entire complex
biologically unavailable to the animal.
The objective of these experiments was to determine if various combinations of Cu and
Mo would decrease H2S gas productions within an in vitro system. In experiment 1,
concentrations were chosen based on maximum tolerable concentrations set by the NRC (2000).
Concentrations selected were 0, 30, 60, and 90 ppm Cu from 2 different Cu sources and 0, 3, 6, 9
ppm Mo. In the 2nd experiment, the concentrations selected were greater than experiment 1 so we
could determine if 10-fold greater concentrations would alter sulfide gas production as well as on
microbial viability. A report from Huber et al. (1971) indicated feeding Mo up to 100 ppm over 6
months showed no signs of toxicity. However, inclusion levels as high as 100 ppm may not be
necessary in order to mitigate sulfide gas production as more recent research has shown sulfide
gas production to be decreased by greater than 70% using 25 ppm Mo of the liquid within
fermentation vessels (Kung et al., 2000).

191

MATERIALS AND METHODS
Experiment 1
The experiment was conducted using in vitro batch fermentations to determine the
appropriate concentrations of Mo and Cu to potentially reduce H2S production. Approval for this
study was provided by the Michigan State University Animal Care and Use Committee (AUF #
07/09-110-00). A 4 × 4 × 2 factorial arrangement of treatments were used as our experimental
design with 4 levels of Mo (0, 3, 6, and 9 ppm) and 4 levels of Cu (0, 30, 60, and 90 ppm) and 2
Cu sources. Sodium molybdate (Na2MoO4) was the compound used as the Mo source and the 2
Cu sources were copper chloride (CuCl2) and copper sulfate (CuSO4). Potential solubility
differences between the Cu sources may impact the formation of the indigestible triad
compound. Calculations for dilutions are shown in Tables D.4-D.6. The amount of Cu and Mo
added to each fermentation vessel were calculated based on the amount of substrate (0.5 g).
Serum bottles (125 ml) served as fermentation vessels to which the dried distiller’s grain with
soluble (DDGS) substrate (Table D.1), 10 ml Van Soest buffer media without the reducing agent
(Table D.1), 10 ml of strained rumen fluid, and 0.2 ml of various mineral treatments were added.
Two fermentation vessels containing buffer and rumen fluid served as negative controls.
Rumen fluid collected for in-vitro fermentation procedures was obtained from a
ruminally cannulated cow housed at Michigan State University Dairy Teaching and Research
Center (DTRC). The cow was fed a diet containing 50% alfalfa hay, 30% DDGS, 16.5% dry
ground corn, and 3.5% vitamin and mineral pre-mix. The cow was adjusted to the DDGS
concentrate diet and was allotted a minimum of 28 d to acclimate before rumen fluid was
collected. Rumen fluid was collected taking 2 samples near the rumen-reticulo opening, 1 within

192

the ventral sac, and 2 near the posterior blind-sac to provide a representative sample of SRB
population throughout the rumen (Lyford and Church, 1988).

193

Table D.1 Composition of distiller’s grain with soluble, and the Van Soest buffer media
DDGS compostition

% DM

Dry matter

87.0

Crude protein

27.0

Crude fat

7.5

Crude fiber

13.0

Sulfur

0.7

Van Soest media*

ml/vessel

Distilled water
®

4.99
1

0.02

Trypticase peptone

2

0.001

3

2.49

Macromineral solution

4

2.49

Resazurin

0.001

Total

9.99

Micromineral solution

Rumen buffer solution

1

®

Trypticase peptone used was a pancreatic digest of casein (Becton Dickinson BBL
#4311921
2
Micromineral solution contained 13.2 g CaCl2 × 2H2O, 10.0 g MnCl2 × 4H2O, 1.0 g CoCl2
× 6H2O, and 8.0 g FeCl3 × 6H2O to 100 ml of distilled water
3

Rumen buffer solution contained 4.0 g ammonium bicarbonate and 35 g of sodium
bicarbonate to 1 L of distilled water
4
Macromineral solution contained 5.7 g Na2HPO4 anhydrous, 6.2 g KH2PO4 anhydrous, and
0.6 g MgSO4 × 7H2O to 1 L of distilled water

* Van Soest media was prepared in the ingredient order listed from top to bottom

194

Bottles were capped with rubber stoppers and crimped to create a closed system and
placed in an orbital shaker incubator (Forma Scientific) at 39° C for 16 h. Bottles were covered
to allow in vitro fermentation to occur in the dark to best simulate conditions during ruminal
fermentation. After 16 h, bottles were removed from the incubator at approximately 1 m 45 s
intervals in the same order they were set in the incubator to allow homogenous incubation time
between all serum bottles. Sixteen h incubation time was selected to represent approximate
retention time in the rumen (Phillip et al., 1980). An in vitro study conducted by Kahlon et al.
(1975) suggested 6 to 18 h incubation time was required for SRB to synthesize various Scontaining sources into microbial protein. Once the fermentation vessel was removed from the
incubator, a syringe needle attached to a digital pressure gauge (MediaGauge

TM

, SSI

Technologies, Inc.) was inserted through the rubber stopper and pressure (psi) was measured and
recorded. Pressure (psi) and known head space was then used to calculate total gas volume (ml)
using Boyle’s Gas Law at STP, which states:
Gp = Vh/Pt × Pa
or
Vh = Gp/Pa × Pt
where Gp is the total gas volume of the head space (ml), Pa is the atmospheric pressure (psi) and
Pt is the reading from the digital pressure gauge (Mauricio et al., 1999). The pressure gauge
needle was then removed and a second needle connected to a 100 cc glass syringe (B-D YALE,
Becton, Dickerson & Co., U.S.A.) was inserted to obtain a sample of the gas. The gas pressure
within the fermentation vessel forced gas into the glass syringe until the system reached
equilibrium pressure between the serum bottle and syringe. When the system was at equilibrium,
195

the tubing connecting the needle to the syringe was clamped to trap the gas in the glass syringe.
A 10 ml sub-sample was then extracted from the 100 cc glass syringe using a 20 ml glass syringe
(B-D YALE, Becton, Dickerson & Co., U.S.A.) that was attached to the tubing using a Yadapter. Tubing was crimped accordingly to prevent gas loss and direct the gas into the 20 ml
glass syringe (Figure D.1). Gas from each fermentation vessel collected in the syringe was then
bubbled in 10 ml of pH 10.00 water in 50 ml glass test tubes to trap S-containing gas in a liquid
state (Seigel, 1965; Kung, 1998). Gas was bubbled in a more alkaline solution using a pH of 10
compared to procedures described by Kung (1998) to reduce potential gas loss of sulfur. Below
pH 10, H2S can volatilize and escape from the solution as a gas (Cord-Ruwisch, 1985). This
process was repeated for all fermentation vessels.
Sulfide determination was performed using the methylene blue (MB) reaction; adding 1
ml of 0.02 M of N, N-dimethyl-p-phenylene diamine sulfate (DPD) in 7.2 N HCl and 1 ml 0.03
3+

M ferric chloride in 1.2 N HCl to each test tube. The DPD reagent is oxidized by Fe , which
reacts with H2S to form MB (Monares et al., 2010).
All test tubes were vortexed and placed in the dark for approximately 20 to 30 minutes to allow
complete color formation. A standard was prepared from sodium sulfide (Na2S∙9H2O) using
methods described by others (Siegel, 1965; Kung, 1998; Vanness et al., 2009b). Sulfur in
solution (µmol /ml) was determined by pipeting 200 µl of the standard solution in plate reader
wells and comparing absorbencies at a wavelength of 630 nm to the standard curve on a
spectrophotometer (Figure D.2). Plates were arranged by copper treatments; all CuCl2 with Mo

196

combinations were on a single plate, and all CuSO4 with Mo combinations were pipetted onto a
different plate.

197

Figure D.1 Image illustrating the removal of gas from the head space of the fermentation vessels
and methods for sub-sampling gas

100 cc glass syringe

Y-adapter

Crimper

Tubing detaches
20 cc glass syringe
125 ml vessel

* The rubber tubing for the 20 cc glass syringe was crimped before tubing was detached to
bubble sub-sample into alkaline water

198

Figure D.2 Standard curve for all treatment combinations using both copper sulfate and copper
chloride as copper sources in experiment 1

199

Experiment 2
A second experiment was conducted using a 10-fold greater Mo and Cu concentrations
than in experiment 1. A similar factorial arrangement of treatments was used as described in
experiment 1. The concentrations of Cu and Mo were (0, 300, 600, and 900 ppm) and (0, 30, 60,
90 ppm), respectively. Procedures were similar to those described in experiment 1, except for the
following changes. Gas was bubbled into pH 8 water as opposed to using pH 10 water in
experminent 1. Also, the entire gas extracted from each serum bottle was bubbled in the water
compared to only 10 ml being used in experiment 1. Testing within our laboratory determined
similar results from using either method. Hydrogen sulfide concentrations were calculated using
a similar procedure as experiment 1 by comparing the absorbency to a calibration curve (Figure
D.3).

200

Figure D.3 Standard curve for all treatment combinations using both copper sulfate and copper
chloride as copper sources in experiment 2

201

Statistical analysis
Statistical analysis was performed using PROC MIXED sub-routine of SAS 9.2 (SAS
Inst., Inc., Cary, NC). Data was analyzed as a randomized complete block design with a 4 × 4 ×
2 factorial arrangement of treatments. Analysis was run using concentrations of Mo, Cu, and Cu
source as the independent variables for experiments 1 and 2. The dependent variables reported
were molar units of H2S produced, H2S produced per g of degraded substrate DM, in-vitro dry
matter disappearance (IVDMD), and pH. Main effects of Mo and Cu level were analyzed to
determine differences across treatment levels. The molar unit of gas produced in the fermentation
vessels were corrected for the molar unit of gas produced in the blanks (vessels containing no
DDGS substrate). Interaction effects between Cu level, Mo level, and Cu source are provided in
Table D.8. Each experiment was analyzed separately. All dependent variables were tested for
homogeneous variance using Bartlett’s test and for normal distribution using Shapiro Wilk’s test.
Statistical significance was declared at a P-value at or below 0.05 and trends at P-value at or
below 0.10.

202

RESULTS
Experiment 1
2 or 3-way interactions between Cu level, Cu source, and Mo level for H2S production
were not significant (Table D.8). Copper levels at 30 and 60 ppm from both CuSO4 and CuCl2
had similar nmols of H2S produced, percent of IVDMD, and pH. However, the decrease in
nmols of H2S produced (P < 0.01), H2S per g of degraded substrate (P = 0.01), and pH (P <
0.01) was determined when comparing 90 ppm with 30 and 60 ppm Cu from the CuCl2 source
only (Table D.2). Treatment of Cu demonstrated a quadratic effect in the nmols of H2S produced
and H2S adjusted for DM degradation across Cu levels (P < 0.01) for both Cu sources. However,
CuSO4 also showed a linear increase (P < 0.01) as Cu treatments increased H2S production
compared to the 0 ppm Cu vessels.
The pH using CuCl2 (P < 0.01) linearly decreased and a quadratic effect was identified
using CuSO4 (P = 0.04; Table D.2).The pH of the fermented solution was more acidic at 9 ppm
Mo compared to 6 ppm Mo (P = 0.04), but similar to the 0 and 3 ppm Mo treatments. The pH of
Mo showed a quadratic effect (P = 0.04), as the 3 and 6 ppm Mo treatments had a greater pH
compared to the 0 and 9 ppm Mo treatments. Additionally, the percent of IVDMD tended to
show a quadratic effect for Mo levels (P = 0.05) and Cu levels from CuSO4 (P < 0.01).
There was a tendency for a 3-way interaction between Cu level, Cu source, and Mo level
when considering the percent of IVDMD (P = 0.06; Table D.8). Only IVDMD (P = 0.09) and pH
(P = 0.06) showed a tendency for a 2-way interaction between Mo level and Cu source. Finally,
203

only pH expressed a tendency (P = 0.04) for a 2-way interaction between Mo level and Cu level.
However, the statistical differences in pH between copper sources are unlikely of any biological
significance as the pH of all Cu levels between each Cu source is within a ± 0.05 pH range. No
significant differences were expressed for the 2-way interaction between Cu level and Cu source;
however, Cu source as a main affect did differ between nmols of H2S, H2S adjusted on DM
degradation, and pH (P < 0.01). Copper sources also tended to cause different IVDMD (P =
0.06).

204

Table D.2 Effects of molybdenum and copper levels on the fermentation of rumen fluid using distiller’s grain with soluble as a
substrate during experiment 1
Molybdenum, ppm
Contrast
Item
0
3
6
9
SEM
P-value
Linear
Quadratic
735.2
759.2
759.0
759.3
27.7
0.84
0.48
0.88
H2S, nmol
H2S, µmol/g
IVDMD, %
pH

1

Item

3.18
44.73
ab
5.69

3.18
46.03
ab
5.70

3.10
46.51
a
5.70

3.25
45.37
b
5.68

0.13
0.68
0.004

0.88
0.29
0.01

0.82
0.43
0.07

0.57
0.08
0.04

90

SEM
33.9

P-value
< 0.01

Linear
0.48

Quadratic
< 0.01

Copper from CuCl2, ppm
30
60

0
a

b

b

a

H2S, nmol***

555.8

712.4

698.5

H2S, µmol/g***

a

2.53
43.89

b

3.17
45.22

b

3.13
44.95

2.58
46.21

0.17

0.01

0.88

< 0.01

0.90

0.35

0.11

0.97

a

a

a

b

0.005

< 0.01

< 0.01

0.17

IVDMD, %*
pH***

5.70

5.69

a

5.66

Copper from CuSO4, ppm

H2S, nmol

555.8

838.2

854.6

820.4

b

33.9

< 0.01

< 0.01

< 0.01

H2S, µmol/g
IVDMD, %
pH

2.53

a

b

b

b

0.17

< 0.01

< 0.01

< 0.01

b

0.90
0.005

0.04
0.08

.046
0.16

< 0.01
0.04

1

a

5.69

594.9

43.89
5.70

a

b

3.54

47.48
5.71

b

b

3.65

b

46.70
5.70

3.64

45.17
5.69

Represents µmol of H2S per g of degraded DDGS on a DM basis

a, b

Means without common superscripts within a row differ

*** Copper source differ (P ≤ 0.01); *Copper source tend to differ (P ≤ 0.10)

205

Experiment 2
Greater treatment differences were demonstrated when Mo and Cu treatments were
increased 10-fold compared to experiment 1 (Table D.3). Treatment of Mo and Cu from both
CuCl2 and CuSO4 showed a significant linear reduction in the µmols of H2S produced and H2S
emissions adjusted for DM degradation as the treatment inclusion level increased from 0, 300,
600, to 900 ppm (P < 0.01). A quadratic effect was also shown in the µmols of H2S produced (P
= 0.04) and H2S emissions adjusted for DM degradation (P < 0.01) when CuCl2 was used as the
Cu source (Table D.3). Copper sulfate also demonstrated a tendency for a quadratic effect (P =
0.09) for µmols of H2S produced. When comparing treatment differences for CuCl2, 300 ppm
differed from 600 and 900 ppm for the µmols of H2S produced (P < 0.05). For CuSO4, each
level of Cu treatment differed in µmols of H2S produced (P < 0.01) with 900 ppm Cu emitting
the lowest levels of H2S.
Hydrogen sulfide production was mitigated by increasing Cu and Mo inclusion levels.
Furthermore, it is interesting that the treatment of Mo and Cu at these levels did not seem to
affect the percent of IVDMD and pH; suggesting Cu and Mo concentration up to 900 and 90
ppm, respectively, does not alter in-vitro microbial fermentation. Copper sulfate expressed linear
increase in pH (P < 0.01) as Cu levels increased. The 300 ppm Cu treatment differed (P = 0.01)
from the 0, 600, and 900 ppm Cu levels from CuSO4. Copper treatment from CuCl2 increased
pH compared to vessels without Cu treatment (P < 0.01; Table D.3). Despite statistical
differences in pH, it is unlikely that pH had any biological effect on microbial viability as the pH
levels for all treatments were within typical pH range (5.5-7.2) for ruminal micro-organisms
206

(Kung, 2008). Copper chloride treatment had a lower reduction in µmols of H2S produced (P <
0.01), while producing a greater average pH (P < 0.01) compared to Cu treatments from CuSO4.
Additionally, a significant 2-way interaction was present with Cu level by Mo level for µmols of
H2S produced (P = 0.02) as reported in Table D.8. The 2-way interaction for µmols of H2S
produced is further illustrated in Figure D.5.

207

Table D.3 Effects of molybdenum and copper levels on the fermentation of rumen fluid using distiller’s grain with soluble as a
substrate during experiment 2
Molybdenum, ppm
Contrast
Item
0
30
60
90
SEM
P-value
Linear
Quadratic
††
a
ab
ab
b
0.09
< 0.01
< 0.01
0.58
H2S, μmol
2.39
2.09
2.13
1.73
H2S, µmol/g
IVDMD, %
pH

1, ††

Item

a

b

11.10
43.2
5.88

9.41
43.36
5.87

c

7.86
43.98
5.90

0.42
0.57
0.03

< 0.01
0.72
0.56

< 0.01
0.47
0.40

0.81
0.86
0.74

900

SEM
0.13

P-value
< 0.01

Linear
< 0.01

Quadratic
0.04

4.46
44.07
b
6.04

c

0.57
0.78
0.04

< 0.01
0.48
< 0.01

< 0.01
0.69
< 0.01

< 0.01
0.55
< 0.01

d

0.13

< 0.01

< 0.01

0.09

d

0.57
0.78
0.04

< 0.01
0.75
0.01

< 0.01
0.20
< 0.01

0.15
0.75
0.08

Copper from CuCl2, ppm
300
600

0
a

H2S, μmol***
H2S, µmol/g***
IVDMD, %
pH***

b

4.03

18.21
43.99
a
5.86

2.26

a

a

H2S, μmol
H2S, µmol/g
IVDMD, %
pH

b

10.45
43.08
b
6.06

c

1.30

c

5.90
44.11
b
6.08

c

0.99

Copper from CuSO4, ppm

4..27

18.52
45.76
a
5.86

1

ab

9.75
43.25
5.92

a

b

2.93

b

13.34
43.85
b
5.70

c

1.86

c

8.79
42.68
ab
5.75

1.19

5.53
43.02
ab
5.75

Represents µmol of H2S per g of degraded DDGS on a DM basis

a, b, c
††

Means without common superscripts within a row differ

Molybdenum level has a significant interaction with Cu level (P ≤ 0.05)

*** Copper sources differ (P ≤ 0.01)

208

Figure D.4 Two-way interaction of molybdenum level by copper level for hydrogen sulfide adjusted for dry matter degradation
during experiment 2

* Copper level represents an average from both Cu sources (CuCl2 and CuSO4)

209

Figure D.5 Comparison of hydrogen sulfide adjusted for dry matter degradation with the treatment levels from experiment 1 to the
elevated levels in experiment 2

* Experiment 1 treatments were 0, 3, 6, and 9 ppm Mo from Na2MoO4 and 0, 30, 60, and 90 Cu from CuCl2 and CuSO4; experiment
2 treatments were increased 10-fold

210

DISCUSSSION
In-vitro laboratory work was conducted to determine appropriate combination of Mo and
Cu that could be added to diets containing high levels of DDGS as a strategy to mitigate H2S
emissions without depressing ruminal function. Hydrogen sulfide concentrations showed high
variance between experiments when comparing untreated vessels (Figure D.6). This may be an
artifact of time that the rumen fluid was extracted from the cannulated cow. Kung et al. (2000)
reported that volatile-fatty acid (VFA) concentrations and S production may be dependent on
time of ruminal fluid collection relative to the last feeding. The pH within the rumen may offer
further evidence of H2S production as collection for experiment 2 was conducted within 4 h of
feeding, which had a rumen fluid pH of 5.63. Whereas in experiment 1, rumen fluid was
collected approximately 8 h post feeding and had a rumen fluid pH of 6.24. Another explanation
why H2S production was low in experiment 1 for untreated vessels compared to experiment 2
may possibly be due to the different methods used to bubble gas into alkaline water. In
experiment 1, a 1:1 ratio using 10 ml of gas to 10 ml of pH 10 water was used; whereas in
experiment 2, total gas withdrawn from the vessels were bubbled into 10 ml of pH 8 water.
Similar studies by others used a 1:1 ratio of gas to pH 8 water (Siegal, 1965; Kung et al., 2000;
Vanness et al., 2009). According to Cord-Ruwisch (1985) H2S is still capable of volitilization in
solution below a pH of 10. Within our lab, a comparison between gas bubbled in pH 8 water and
pH 12 water was conducted with blank vessels, vessels containing 0.5 g of DDGS subsrate, and
vessels with subsrate including treatment of 1000 ppm of Mn. The pH 12 water increased H2S
production 43.6% in the blank vessels, 47.8% with the DDGS substrate, and 39.2% with
substrate treated with 1000 ppm Mn (data not shown).
211

In experiment 1, levels of emitted H2S were low relative to other studies. A study from
Kelzer et al. (2010) conducted an experiment using aproximately 0.7 g DM substrate consisting
of 81% DGS that was incubated for 24 h at 39° C, producing H2S at 2.23 and 1.13 µg/ml or 69.7
and 35.3 nmol/ml concentration of sulfide, respectively. These results are in accord to
experiment 2 of the current study. A study conducted by Kung et al. (2000) used a 375 mg
substrate containing either 1.09% S or 0.29% S incubated for 24 h at 40° C and reported a
production of H2S at 4.3 and 2.0 µmol, respectively. Within the current study, the control
(untreated) vessels for experiment 1 had the lowest levels of emitted H2S at 0.55 µmol and the
amount of H2S produced from the control vessels for experiment 2 was 4.03 µmol. Smith et al.
(2010) reported H2S gas to be in the range of 27.6 and 35.9 µmol per g of fermented DM when
0.8% S in substrate was added to in-vitro fermentation vessels. This is greater than the untreated
vessels from experiment 2 of the current study, as H2S levels adjusted for DM degradation was
18.21 µmol/g. However, the experiments within the current study were incubated for 16 h
compared to 24 h used from Kung et al. (2000), Kelzer et al. (2010), and Smith et al. (2010). The
amount of S added as substrate within our study is between the levels reported from Kung et al.
(2000) and similar to the 0.8% S in substrate level from Smith et al (2010) as our DDGS
substrate contanied 0.7% S. Furthermore, Kung et al. (2000) reported similar differences in pH
when using Mo as a inhibitory sulfide treatment and Kelzer et al. (2010) also reported a similar
pH range within treatment using Mn at levels ranging from 500 to 2500 ppm. Smith et al. (2010)
reported IVDMD to be approximately 70% when S was included at 0.2, 0.4, and 0.8% of the

212

substrate. In-vitro dry matter disappearance within our study was between 43-48% for both
experiments.
The combination of Cu and Mo at elevated levels did mitigate H2S emissions in-vitro
from experiment 2 within the current study. However, these concentrations were above the
dietary levels recommended by the NRC (2000) to feed to finishing cattle. When Mo was added
at concentration < 10 ppm, no differences were seen compared to the untreated vessels. Copper
treatment from both CuCl2 and CuSO4 < 100 ppm increased H2S emissions in-vitro as indicated
from experiment 1 of the current study. A live animal study will offer further insight on how Mo
and Cu interact with S in the rumen or excreta from growing steers fed various levels of DGS
that in-vitro work may not be able to determine at lower concentrations. Furthermore, use of
environmentally controlled rooms will offer the opportunity to monitor several gases at once and
determine if the use of Cu and Mo to reduce H2S emissions may affect other hazardous gases as
well.

213

SUPPLEMENTAL TABLES AND FIGURES

Table D.4 Dilutions of copper sulfate solution during experiment 1 and 2
Copper determination in 1 L stock solution
600 ppm:

[(Cu = 63.55 g-mol)/ (CuSO4 + 5H2O = 249.68 g-mol)] = 0.2543 Cu
-1

-1

-1

(600 mg∙L )/ (0.2543 Cu)/ (1000 mg∙g ) = 2.36 g∙L

Concentration

Equation steps
-1

-4

(90 mg∙kg )∙(5∙10
90 ppm:

-1

-5

-4

-4

0.05

kg DDGS*) = 0.015 mg Cu per vessel
-1

-5

1

0 ppm :

L per vessel

-1

-5

(0.015 mg)/ 600 mg∙L ) = 2.5∙10
2. (2.5∙10

2

-5

L)∙(1000 ml∙L ) =

1. (30 mg∙kg )∙(5∙10

1

kg DDGS*) = 0.03 mg Cu per vessel

-1

-1

30 ppm:

0.075

(0.03 mg)/ 600 mg∙L ) = 5.0∙10
-5

L per vessel

-1

(60 mg∙kg )∙(5∙10

1. (5.0 ∙10

-5

L)∙(1000 ml∙L ) =
-1

60 ppm:

kg DDGS*) = 0.045 mg Cu per vessel

(0.045 mg)/ (600 mg∙L ) = 7.5∙10
(7.5∙10

2

Volume , ml

L per vessel

-1

L)∙(1000 ml∙L ) =

0.025
-

0 ppm includes 0.1 ml of water to equal the volume of the treated serum bottles
Total volume equaled 0.1 ml. Water was added to adjust for the difference

* DDGS = substrate

214

-

Table D.5 Dilutions of copper chloride solution during experiment 1 and 2
Copper determination in a 1 L solution
[(Cu = 63.55 g-mol)/ (CuCl2 + 2H2O = 170.48 g-mol)] = 0.3728 Cu
-1

-1

-1

(600 mg∙L )/ (0.3728 Cu)/ (1000 mg∙g ) = 1.61 g∙L

Concentration

Equation steps
-1

-4

(90 mg∙kg )∙(5∙10
90 ppm:

-1

-5

-4

-4

0.05

kg DDGS*) = 0.015 mg Cu per vessel
-1

-5

1

0 ppm :

L per vessel

-1

-5

(0.015 mg)/ 600 mg∙L ) = 2.5∙10
4. (2.5∙10

2

-5

L)∙(1000 ml∙L ) =

3. (30 mg∙kg )∙(5∙10

1

kg DDGS*) = 0.03 mg Cu per vessel

-1

-1

30 ppm:

0.075

(0.03 mg)/ 600 mg∙L ) = 5.0∙10
-5

L per vessel

-1

(60 mg∙kg )∙(5∙10

2. (5.0 ∙10

-5

L)∙(1000 ml∙L ) =
-1

60 ppm:

kg DDGS*) = 0.045 mg Cu per vessel

(0.045 mg)/ (600 mg∙L ) = 7.5∙10
(7.5∙10

2

Volume , ml

L per vessel

-1

L)∙(1000 ml∙L ) =

0.025
-

0 ppm includes 0.1 ml of water to equal the volume of the treated serum bottles
Total volume equaled 0.1 ml. Water was added to adjust for the difference

* DDGS = substrate

215

-

Table D.6 Dilutions of sodium molybdate solution during experiment 1 and 2
Molybdenum determination in a 1 L solution
[(Mo = 95.94 g/mol)/ (Na2MoO4 + 2H2O = 241.95 g/mol)] = 0.3965 Mo
-1
-1
-1
(60 mg∙L )/ (0.3965 Mo)/ (1000 mg∙g ) = 0.15 g∙L
Concentration

Equation steps
-1

-4

(9 mg∙kg )∙(5∙10
9 ppm:

-1

-5

-4

-5

-1

-4

0.05

kg DDGS*) = 0.0015 mg Cu per vessel
-1

-5

(0.0015 mg)/ 60 mg∙L ) = 2.5∙10
-5

6. (2.5∙10
1

0 ppm

L per vessel

L)∙(1000 ml∙L ) =

5. (3 mg∙kg )∙(5∙10

2

kg DDGS*) = 0.003 mg Cu per vessel
-1

-1

1

0.075

(0.003 mg)/ 60 mg∙L ) = 5.0∙10
-5

3 ppm:

L per vessel

-1

(6 mg∙kg )∙(5∙10

3. (5.0 ∙10

-5

L)∙(1000 ml∙L ) =
-1

6 ppm:

kg DDGS*) = 0.0045 mg Cu per vessel

(0.0045 mg)/ (60 mg∙L ) = 7.5∙10
(7.5∙10

2

Volume , ml

L per vessel

-1

L)∙(1000 ml∙L ) =

0.025
-

0 ppm includes 0.1 ml of water to equal the volume of the treated serum bottles
Total volume equaled 0.1 ml. Water was added to adjust for the difference

* DDGS = substrate

216

-

Table D.7 Fermentation vessel arrangement for molybdenum and copper concentrations during
experiment 2
1

0 ppm Cu (CuSO4) 

Bottle 1 & 2

Bottle 3 & 4

Bottle 5 & 6

Bottle 7 & 8

30 ppm Cu (CuSO4) 

Bottle 9 & 10

Bottle 11 & 12

Bottle 13 & 14

Bottle 15 & 16

60 ppm Cu (CuSO4) 

Bottle 17 & 18

Bottle 19 & 20

Bottle 21 & 22

Bottle 23 & 24

90 ppm Cu (CuSO4) 

Bottle 25 & 26

Bottle 27 & 28

Bottle 29 & 30

Bottle 31 & 32

0 ppm Cu (CuCl2) 

Bottle 33 & 34

Bottle 35 & 36

Bottle 37 & 38

Bottle 39 & 40

30 ppm Cu (CuCl2) 

Bottle 41 & 42

Bottle 43 & 44

Bottle 45 & 46

Bottle 47 & 48

60 ppm Cu (CuCl2) 

Bottle 49 & 50

Bottle 51 & 52

Bottle 53 & 54

Bottle 55 & 56

90 ppm Cu (CuCl2) 

Bottle 57 & 58

Bottle 59 & 60

Bottle 61 & 62

Bottle 63 & 64

1
2

9 ppm Mo

1

3 ppm Mo

2

6 ppm Mo

1

0 ppm Mo

Inclusion Levels

Molybdenum was added using Na2MoO4
Experiment 2 had a total of 4 untreated bottles and experiment 1 was conducted with 2

* Duplicate blanks would be bottles 65 & 66

217

Table D.8 Probability of an interaction affects between Mo and Cu levels with Cu source during experiment 1 and 2
Experiment 1

Mo level

Mo level ×
Cu level

Mo level ×
Cu source

Mo level ×
Cu level ×
Cu source

Cu level

Cu source

Cu level ×
Cu source

H2S , nmol/ml

< 0.01

< 0.01

0.37

0.80

0.36

0.42

0.57

H2S, nmol

< 0.01

< 0.01

0.31

0.84

0.30

0.42

0.53

H2S, µmol/g degraded DM

< 0.01

< 0.01

0.12

0.88

0.58

0.29

0.91

Total gas volume, ml

0.04

< 0.01

0.23

< 0.01

0.16

0.12

0.05

IVDMD, %

0.19

0.06

0.16

0.29

0.85

0.09

0.06

< 0.01

< 0.01

0.26

0.01

0.04

0.06

0.20

Mo level ×
Cu source

Mo level ×
Cu level ×
Cu source

Item
1

pH

Experiment 2

Cu level

Cu source

Cu level ×
Cu source

H2S, nmol/ml

< 0.01

0.03

0.52

< 0.01

< 0.01

0.71

0.95

H2S, µmol

< 0.01

< 0.01

0.19

< 0.01

0.02

0.40

0.98

H2S, µmol/g degraded DM

< 0.01

< 0.01

0.20

< 0.01

0.03

0.19

0.91

Total gas volume, ml

< 0.01

< 0.01

0.75

< 0.01

< 0.01

< 0.01

0.01

IVDMD, %

0.88

0.50

0.33

0.72

0.51

0.52

0.99

pH

0.55

< 0.01

0.71

0.56

0.97

0.13

0.97

Item

1

Mo level

Mo level ×
Cu level

Hydrogen sulfide represents the concentration of the total sulfide gas trapped in alkaline solution

218

Figure D.6 Three-way interaction plot between copper source, copper level, and molybdenum
level for in-vitro dry matter disappearance during experiment 1

Figure D.7 Two-way interaction plot between copper source and molybdenum level for in-vitro
dry matter disappearance during experiment 1

219

Figure D.8 Two-way interaction plot between copper source and molybdenum level for pH
during experiment 1

Figure D.9 Two-way interaction plot between copper level and molybdenum level for pH during
experiment 1

220

Figure D.10 Two-way interaction plot between copper level and molybdenum level for
hydrogen sulfide during experiment 2

221

Table D.9 Preliminary results from an experiment with similar treatment levels as experiment 1
Molybdenum, ppm
Item
0
3
6
9
SEM
P-value
†
714.0
678.4
747.8
645.4
37.5
0.26
H2S, nmol
a

a

H2S, nmol/g

4.21

IVDMD, %

35.58

34.08

a

a

†

†

pH

4.09

a

a

5.93

Item

5.91

ab

b

0.24

< 0.01

< 0.01

0.17

40.01

40.90

b

0.59

< 0.01

< 0.01

0.05

b

b

0.012

< 0.01

< 0.01

0.61

SEM
51.5

P-value
< 0.01

Linear
< 0.01

Quadratic
< 0.01

b

0.32

< 0.01

< 0.01

< 0.01

bc

0.86

< 0.01

< 0.01

< 0.01

6.04

b

0.016

< 0.01

< 0.01

< 0.01

714.6

61.2

0.15

0.25

0.07

b

0.32

< 0.01

< 0.01

0.03

3.87

b

6.00

a

b

b

H2S, nmol***

814.8

536.9

516.4

H2S, nmol/g***

a

5.96
29.36a

b

b

IVDMD, %***
pH***

3.33

b

H2S, nmol/g
IVDMD, %

†

36.14

b

6.06

6.08

Copper from CuSO4, ppm
821.4
885.7

5.96

a, b, c

c

b

5.80

814.8

2.92

32.50

a

H2S, nmol

pH

3.09
6.00

Copper from CuCl2, ppm
30
60

0

a
a

b

3.82

b

3.96

b

585.2
3.51

34.25

3.22

43.08

44.11

44.07

b

0.81

< 0.01

< 0.01

< 0.01

a

a

b

b

0.016

< 0.01

< 0.01

0.01

5.85

b

90

29.36
5.80

b

Contrast
Linear
Quadratic
0.42
0.38

5.96

5.93

Means without common superscripts within a row differ (P < 0.05)

Molybdenum level tends to express an interaction with Cu level (P < 0.10)

*** Copper sources differ (P < 0.01)

222

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