NUTRITIONAL, PHYSIOLOGICAL and ENVIRONMENTAL EFFECTS of FEEDING DISTILLER's GRAINS PLUS SOLUBLE to FEEDLOT CATTLE a Thesis

NUTRITIONAL, PHYSIOLOGICAL AND ENVIRONMENTAL EFFECTS OF FEEDING DISTILLER’S GRAINS PLUS SOLUBLE TO FEEDLOT CATTLE

A Thesis

Presented to

The Faculty of Graduate Studies

The University of Guelph

Heba Saad Salim

In partial fulfillment of requirements

For the degree of

Doctor of Philosophy

September, 2011

ABSTRACT

NUTRITIONAL, PHYSIOLOGICAL AND ENVIRONMENTAL EFFECTS OF FEEDING DISTILLER’S GRAINS PLUS SOLUBLE TO FEEDLOT CATTLE

Heba Saad Salim Advisor: J. P. Cant

University of Guelph Co advisor: K. C. Swanson

In this study, four experiments were conducted to investigate the effect of inclusion level

of dry distillers grains plus solubles (DDGS) or modified wet distillers grains plus

solubles (MWDGS) (0, 16.7, 33.3, and 50% of ration DM) on performance, carcass characteristics, feeding behaviour, nutrient balance, nutrient excretion, and enzymatic activity using whole corn grain-based finishing diets. In experiment one, there were no effects (P > 0.05) of dietary treatment on final BW, ADG, days on feed, rumen pH at slaughter, dressing %, hot carcass weight, marbling score, lean yield, and lean color.

Liver abscess score was lower in steers fed DGS than steers fed the control. Visits of cattle to the feeder (VF) increased when cattle were fed up to 16.7% of DDGS or 33.3% of MWDSG. Number of meals (NM) and eating rate (ER) was greater and time per meal

(TM) was lower in cattle fed MWDGS compared to those fed DDGS. Also, increasing the distillers grains plus soluble (DGS) increased daily time at feeder (TF); however, ER decreased when cattle were fed up to33.3% of DGS and after that increased. In experiment two, total tract DM, OM, and starch digestibility decreased with increasing

DDGS up to 50%. Daily intake and total excretion of N, P, S, Mg, and K increased

linearly with increasing level of DDGS. Nitrogen retention did not change with level of

DDGS; however, P retention tended to increase and S retention increased with increasing

DDGS. The digestion and retention of Se, Mg, K, and Na did not differ among the

treatments. In experiment three, although the pancreatic protein concentration (mg/g)

increased linearly with increasing DGS levels, pancreatic mass (g and g/kg BW) did not change. Feeding DGS increased the pancreatic concentration of α-amylase and trypsin activity (U/g) compared to the control diet. Increasing the DGS level increased pancreatic concentration of trypsin activity (U/g). In experiment four, increasing DGS linearly increased kidney weight (g). Hepatic and renal glutathione peroxidases (GPX) activity was not influenced by inclusion level or form of DGS. However, renal GPX activity per kilogram of BW was affected by the form and linear effect interaction. Increasing inclusion level of DGS linearly increased carbamoyl phosphate synthetase (CPS) activity

(kU/liver, and U/kg of BW), argininosuccinate synthetase (AS) and ornithine transcarbamoylase (OTC) activity (U/g, kU/liver, and U/kg of BW).The results of these studies suggest that feeding DDGS or MWDGS up to 50% diet DM in whole corn grain- based finishing diets does not negatively affect animal performance, although animals appear to adapt by altering feeding behaviour and nutrient metabolism. However, environmental implications of manure should be considered in the feedlot.

ABBREVIATIONS

ADF, acid detergent fiber N, nitrogen ADG, average daily gain Na, sodium NADPH, nictotinamide adenine dinucleotide AS, argininosuccinate synthetase phosphate BF, back fat thickness NDF, neutral detergent fiber Ca, calcium NM, number of meals per day CDG, corn distillers grain OM, organic matter

CDS, condensed distiller's solubles OTC, ornithine transcarbamoylase

CP, crude protein P, phosphorus CPS, carbamoyl phosphate synthetase PUFA, polyunsaturated fatty acids DDGS, dry distillers grains plus solubles PUN, plasma urea N DG, distillers grains S, sulphur DGS, distillers grain plus solubles Se, selenium DIP, ruminally degradable intake protein SFC, steam-flaked corn DM, dry matter SI, small intestine DRC, dry-rolled corn TF, time spent at feeder per day ER, eating rate TM, time per meal GPx, glutathione peroxidase activity TV, time per visit GSH, glutathione UFA, unsaturated fatty acids GSHpx, blood gluthathione peroxidase UIP, undegradable intake protein concentration GSSG, oxidized glutathione VF, daily visits to the feeder K, potassium WBSF, Warner- Bratzler shear force LMA, longissimus muscle area WDGS, wet distillers grain plus solubles MCF, modified corn fiber

Mg, magnesium

MS, meal size

MWDGS, modified wet distillers grains plus solubles

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ACKNOWLEDGEMENTS

I wish to express my appreciation to Dr. K. C. Swanson for his supervision, guidance, patience, encouragement and assistance during the progress of this research and for his advice and counsel during my PhD programe. Dr K. C. Swanson has his unique way to inspire the search for knowledge and science. I also would like to thank

Dr. John Cant for his appreciated supervision and advice to my research. I am also grateful to the other members of my advisory committee: Dr. S. P. Miller and Mr. P. L.

McEwen for their effort and for their critical review of this manuscript and careful reading.

I would like to thank Ontario Cattlemen’s Association (FIP program), Beef Cattle

Research Council, and Ontario Ministry of Agriculture Food and Rural Affairs for their financial support of these studies. I would like to thank Dr Margaret Quinton and Dr

Gord Vander for their statistical analysis help. I would like to thank Dr Vern Osborne for giving me his great vacuum. I wish also to express my thanks to Charlie Watson and all the staff at the Elora Beef Research Centre and Brian McDougall and all staff at

University of Guelph Meat Lab for their assistance and doing great work. I would like to thank my lab mates, Katie Wood, Simone Holligan, Laura Martin, Yuri Montanholi,

Nyree Kelly, Tim Caldwell, and Mohamed Abo-Ismail for their help and friendship.

I wish to express my deepest gratitude to my parents, and my sister (Rania Salim) for their prayers and inspiration. A very special thank to my brother (Hany Salim), or my twin soul for introducing me to the science world since I was young. Also, I would like to thank my dear husband (Ehab. El-haroun), and my three daughters (Nourhan, Salma and

Sarah) for their love, patience, support, and encouragement throughout my college career.

TABLE OF CONTENTS

CHAPTER 1 ...... 1 GENERAL INTRODUCTION ...... 1 CHAPTER 2 ...... 4 LITERATURE REVIEW ...... 4 2.1 INTRODUCTION ...... 4 2.2 DISTILLERS GRAIN PLUS SOLABLES AND GROWTH PERFORMANCE OF CATTLE ...... 7 2.2.1 DRY MATTER INTAKE ...... 7 2.2.2 AVERAGE DAILY GAIN ...... 8 2.2.3 FEED EFFICIENCY ...... 9 2.3 FEEDING BEHAVIOR ...... 12 2.4 DISTILLERS GRAIN PLUS SOLUBLES AND MEAT QUALITY OF CATTLE ...... 13 2.4.1 FAT THICKNESS AND MARBLING ...... 13 2.4.2 RETAIL DISPLAY ...... 14 2.4.3 TENDERNESS EVALUATION ...... 15 2.5 DISTILLERS GRAIN PLUS SOLUBLES, TOTAL TRACT DIGESTION AND NUTRIENT BALANCE ...... 16 2.5.1 TOTAL TRACT DIGESTION ...... 16 2.5.2 NITROGEN BALANCE ...... 18 2.5.3 PHOSPHORUS BALANCE ...... 19 2.5.4 CALCIUM BALANCE ...... 20 2.5.5 EFFECT OF SULPHUR ON SELENIUM BIOAVAILABILITY ...... 20 2.5.6 OTHER FACTORS AFFECT SELENIUM BIOAVAILABILITY ...... 21 2.5.7 MAGNESIUM, POTASIUM, AND SODIUM ...... 22 2.6 DISTILLERS GRAIN PLUS SOLUBLE AND ORGAN MASS ...... 23 2.7 DISTILLERS GRAIN PLUS SOLUBLES AND α-AMYLASE AND TRYPSIN ENZYMES ACTIVITY ...... 24 2.8 DISTILLERS GRAIN PLUS SOLUBLES AND GLUTATHIONE PEROXIDASE ENZYMES ACTIVITY ...... 26 2.10 RESEARCH HYPOTHESES AND OBJECTIVES ...... 33 CHAPTER 3 ...... 34

INFLUENCE OF FEEDING INCREASING LEVELS OF DRY OR MODIFIED WET CORN DISTILLERS GRAINS PLUS SOLUBLES IN WHOLE CORN GRAIN-BASED FINISHING DIETS ON PERFORMANCE AND CARCASS TRAITS IN FEEDLOT CATTLE ...... 34 3.1 ABSTRACT ...... 34 3.2 INTRODUCTION ...... 35 3.3 MATERIALS AND METHODS ...... 36 3.3.1 Animal Care and Experimental Design ...... 36 3.3.2 Feeding behavior traits measurements and sample collection ...... 37 3.3.3 Laboratory analyses ...... 38 3.3.4 Carcass measurements ...... 38 3.3.5 Statistical Analysis ...... 39 3.4 RESULTS ...... 40 3.5 DISCUSSION ...... 43 CHAPTER 4 ...... 55 INFLUENCE OF FEEDING INCREASING LEVELS OF DRY CORN DISTILLERS GRAINS PLUS SOLUBLES IN WHOLE CORN GRAIN-BASED FINISHING DIETS ON TOTAL TRACT DIGESTION, NUTRIENT BALANCE, AND EXCRETION IN BEEF STEERS ...... 55 4.1 ABSTRACT ...... 55 4.2 INTRODUCTION ...... 56 4.3 MATERIALS AND METHODS ...... 57 4.3.1 Animal Care and Experimental Design ...... 57 4.3.2 Laboratory Analysis ...... 58 4.3.3 Statistical Analysis ...... 59 4.4 RESULTS ...... 60 4.5 DISCUSSION ...... 65 CHAPTER 5 ...... 80 INFLUENCE OF FEEDING INCREASING LEVELS OF DRY OR MODIFIED WET CORN DISTILLERS GRAINS PLUS SOLUBLES IN WHOLE CORN GRAIN-BASED FINISHING DIETS ON PANCREATIC MASS, AND Α-AMYLASE AND TRYPSIN ACTIVITY IN FEEDLOT CATTLE ...... 80 5.1 ABSTRACT ...... 80 5.2 INTRODUCTION ...... 81 5.3 MATERIALS AND METHODS ...... 82 5.3.1. Animal Care and Experimental Design ...... 82 5.3.2 Feed intake measurements and sample collection ...... 83

5.3.3 Laboratory analyses ...... 83 5.3.4 Statistical Analysis ...... 84 5.4 RESULTS ...... 85 5.5 DISCUSSION ...... 85 CHAPTER 6 ...... 92 INFLUENCE OF FEEDING INCREASING LEVELS OF DRY OR MODIFIED WET CORN DISTILLERS GRAINS PLUS SOLUBLES IN WHOLE CORN GRAIN-BASED FINISHING DIETS ON HEPATIC AND RENAL MASS, AND GLUTATHIONE PEROXIDASE AND UREA CYCLE ENZYME ACTIVITIES IN FEEDLOT CATTLE...... 92 6.1 ABSTRACT ...... 92 6.2 INTRODUCTION ...... 93 6.3 MATERIALS AND METHODS ...... 95 6.3.1 Animal Care and Experimental Design ...... 95 6.3.2 Dietary analyses and sample collection ...... 96 6.3.3 Hepatic and Renal GPx Enzyme and Protein Analysis ...... 96 6.3.4 Hepatic Urea Cycle Enzyme Analysis ...... 98 6.3.5 Statistical Analysis ...... 99 6.4 RESULTS ...... 99 6.5 DISCUSSION ...... 101 CHAPTER 7 ...... 109 GENERAL DISCUSSION AND CONCLUSION ...... 109 7.1 GENERAL DISCUSSION ...... 109 7.2 CONCLUSION ...... 112 CHAPTER 8 ...... 114 REFERENCES ...... 114

vii

LIST OF FIGURES

Chapter 2

Figure 2.1. Schematic of the dry milling industry with the feed products 6 produced

Figure 2.2. The oxidation and subsequent reduction of glutathione (GSH) 27

viii

LIST OF TABLES

Chapter 2

Table 2.1. Influence of dietary inclusion of wet and dry distillers grain plus 11 solubles (WDGS and DDGS) on performance in finishing yearling steers.

Table 2.2. Effect of selenium on activity of erythrocyte GPx in cow and their 29 calves.

Chapter 3

Table 3.1. Dietary composition and analysis (DM basis). 48

Table 3.2. Influence of dietary source and level of DGS on growth performance 50 and rumen pH in feedlot cattle fed dry whole corn-based finishing diets.

Table 3.3. Influence of dietary source and level of DGS on rumen pH and liver 51 abscesses in feedlot cattle fed dry whole corn-based finishing diets.

Table 3.4. Influence of dietary source and level of DGS on carcass characteristics 52 in feedlot cattle fed dry whole corn-based finishing diets.

Table 3.5. Influence of dietary source and level of DGS on feeding behavior in 54 feedlot cattle fed dry whole corn-based finishing diets.

Chapter 4

Table 4.1. Dietary composition and analysis (DM basis). 69

Table 4.2. Influence of dietary level of DDGS on DM, OM, CP, ADF , NDF and 70 starch intake and apparent digestibility in feedlot cattle fed dry whole corn-based finishing diets.

Table 4.3. Influence of dietary level of DDGS on nitrogen balance in feedlot 71 cattle fed dry whole corn-based finishing diets.

Table 4.4. Influence of dietary level of DDGS on phosphours balance in feedlot 72 cattle fed dry whole corn-based finishing diets.

Table 4.5. Influence of dietary level of DDGS on calcium balance in feedlot cattle 73 fed dry whole corn-based finishing diets.

Table 4.6. Influence of dietary level of DDGS on magnesium balance in feedlot 74 cattle fed dry whole corn-based finishing diets.

Table 4.7. Influence of dietary level of DDGS on sulphur balance in feedlot cattle 75 fed dry whole corn-based finishing diets.

Table 4.8. Influence of dietary level of DDGS on selenium balance in feedlot 76 cattle fed dry whole corn-based finishing diets.

Table 4.9. Influence of dietary level of DDGS on potassium balance in feedlot 77 cattle fed dry whole corn-based finishing diets.

Table 4.10. Influence of dietary level of DDGS on sodium balance in feedlot 78 cattle fed dry whole corn-based finishing diets.

Table 4.11. Influence of dietary level of DDGS on nitrogen balance in feedlot 79 cattle fed dry whole corn-based finishing diets.

Chapter 5

Table 5.1. Effect of inclusion level and form of distillers grains plus solubles 90 (DGS) on pancreatic mass and protein concentration in feedlot cattle.

Table 5.2. Effect of inclusion level and form of distillers grains plus solubles 91 (DGS) on α-amylase, and trypsin activity in feedlot cattle.

Chapter 6

Table 6.1. Influence of dietary source and level of DGS on plasma glucose and 104 urea N concentrations in feedlot cattle fed dry whole corn-based finishing diets.

Table 6.2. Influence of dietary source and level of DGS on liver and kidney mass. 105

Table 6.3. Influence of dietary source and level of DGS on glutathione peroxidase 106 (GPx) activity in liver and kidney.

Table 6.4. Influence of dietary source and level of DGS on Carbamoyl phosphate 107 synthetase (CPS), Argininosuccinate synthetase (AS), and Ornithine transcarbamoylase (OTC) activity in liver.

CHAPTER 1

GENERAL INTRODUCTION

Distillers grains (DG) are a byproduct of the grain milling industry, which are formed after the fermentation of cereal grain by yeast (Weigel et al., 1997). Corn grain is the primary grain to produce ethanol fuel by the dry milling process (Peter et al., 2000). The dry milling process adds several beneficial characteristics to corn distillers grains in ruminant diets compared with whole grain. For example, the protein from corn distillers grain (CDG) is more resistant to degradation by rumen microorganisms (Mertens, 1977); because the heat process changes the structure of corn grain to decrease the degradation in the rumen (McDonald, 1954; Little et al., 1968). Also, the dry milling process can increase the digestibility of fiber due to the effect of yeast actions on cellulose degradation. Therefore, CDG could be a useful feed ingredient for growing ruminants and high producing dairy cows where significant bypass protein is required (Waller et al.,

1980). The distillers grains consist of 25-35% crude protein, and the undegradable intake protein concentration (UIP) is 50% or higher of the crude protein (Benton et al., 2006).

Moreover, corn wet distillers grain plus solubles (WDGS) can be greater in NEg than

corn grain (Larson et al., 1993; Ham et al., 1994). Ruminal acidosis might be decreased

by increasing DG because it contains high amounts of fiber instead of starch

(Schingoethe, 2006). Because the dry milling process converts the starch (about two-

thirds of the corn) to ethanol by fermentation, the other nutrients such as protein and

other components (fat, fiber, and P) become more concentrated by approximately 3 times

in the ethanol byproducts as compared to corn grain. Protein, fat, NDF, and P are

increased in DGS from approximately 10 to 30%, 4 to 12%, 12 to 36%, and 0.3 to 0.9%

of DM, respectively (Klopfenstein et al., 2008).

Recently, the concern about the supply and environmental impact of using fossil fuels,

and developing renewable biofuel sources has increased (Hill et al., 2006). In addition,

the price of corn has dramatically increased due to increased demand for production of

ethanol as a fuel additive and for corn sweeteners (Schrage et al., 1991). In Canada, the

size of ethanol plants range from approximately 12.5 million litres per year to 225 million

litres per year output. Corn is the feedstock for ethanol plants in eastern Canada (Ontario

and Quebec). Four hundred litres of ethanol can be produced from one tonne of corn,

Canadian Renewable Fuels Association (CRFA, 2010). This trend has resulted in an abundance of wet and dry distiller’s grains, which are a feasible economical alternative to feeding corn grain (Lodge et al., 1997a). This has stimulated recent researchers to study the utilization of ethanol byproducts as a feed source for livestock (Klopfenstein et al.,

2008). Although optimizing the use of distillers grain (DG) is becoming increasingly important as ethanol production increases (Rincker and Berger, 2003), distillers grain has certain limitations for ruminant diets. When DG is fed as an energy source over 15 to

20% of diet DM, the intake of phosphorous, protein and sulfur are higher than requirements; therefore, the efficiency of nutrient utilization is decreased and nutrient excretion is increased, which might negatively affect the environment. Furthermore, the high level of sulfur (0.6 to 1.0% or higher) might cause polioencephalomalacia, and decrease DMI, ADG, and liver Cu stores when DGS are used above 30 to 40% of diet

DM (Klopfenstein et al., 2008). Although, there have been studies conducted that demonstrated that wet distillers grains plus solubles (WDGS) and dry distillers grains

plus solubles (DDGS) did not have a negative effect on ruminant performance fed dry-

rolled, high moisture-, or steam–flaked corn based diets, there is a shortage of

information about how feeding DDG and modified wet distillers grains plus solubles

(MWDGS) influence performance of cattle fed dry whole corn grain. Therefore, the

objective of this research is to examine the effect of increasing inclusion levels of DDGS

(90% DM) and MWDGS (42-50% DM) in whole corn diets on cattle performance, meat

quality, carcass characteristics, feeding behaviour, nutrient utilization and pancreatic enzymatic activity to determine the optimal level of ethanol byproduct inclusion.

CHAPTER 2

LITERATURE REVIEW

2.1 INTRODUCTION Ethanol by-products can be produced from various sources, including grain (corn,

sorghum, barley, wheat, etc.), lignocellulosic-biomass (wheat straw, corn stover, switch

grass), or from other products such as sugar cane (Klopfenstein et al., 2008). Corn grain is the major feedstock used for ethanol production in North America because corn is more available than other grains, and it has starch that can be fermented efficiently. The corn kernels contain approximately 70 to 72% starch on a DM basis (Bothast and

Schlicher, 2005). There are two processes (wet and dry milling) to produce ethanol and ethanol byproducts. Wet milling needs more energy and more equipment to produce ethanol than dry milling; therefore, dry milling constitutes 82% of ethanol production whereas wet milling constitutes 18% of ethanol production (RFA, 2008). The main difference between wet and dry milling is that in the wet milling process, the starch, fiber, gluten, and germ must be removed from corn grain (Bothast and Schlicher, 2005).

Typically, the wet milling process is used to produce corn sweeteners such as dextrose and high fructose corn syrup and corn oil; however, starch can be fermented for ethanol production (Schingoethe, 2006). The corn oil is extracted from the germ after it is removed from the corn kernel. The corn gluten feed is formed by adding the remaining germ meal to the corn bran. Also, corn gluten meal could be formed from separated gluten, which is a high-protein animal feed. In this process, the ethanol is produced by fermentation of sugars after separating starch solution from the solids (Bothast and

Schlicher, 2005).

On the other hand, the first step in the dry milling process is grinding the corn into flour (meal). Water is added to this meal to form mash, and then the mash is cooked, after adding the enzymes, to convert starch to simple sugar (dextrose) and to decrease bacteria concentrations before the fermentation. Also, pH is controlled in mash by adding ammonia, which is also used as a nutrient for the yeast. Yeast is then added to cooled mash to promote fermentation of sugar, which converts the sugar to ethanol and carbon dioxide. The fermentation process continues about 40 to 50 hours, and the result of this process is a mixture of ethanol and solid (RFA, 2008). Then the mixture is distilled to produce ethanol. The other product of this process, which is separated liquid from the mash, is called sweet water or thin stillage. Thus, thin stillage could be dehydrated to obtain condensed distiller's solubles (CDS), or syrup. Wet distillers grain, which is the remaining solid fraction, might either be sold as cattle feed or dehydrated to produce dried distillers grain (DDG). Condensed distiller's solubles can be blended with distillers grain to form distiller's grains plus solubles (DGS), or sold as is for cattle feed. Finally, there are 3 forms of distiller's grains plus solubles that are marketed: 1) dry form (DDGS) approximately 90% DM, 2) wet form (WDGS) approximately 30% DM and 3) modified form (MWDGS) approximately 50% DM (Bothast and Schlicher., 2005). Although drying costs for DDG increase the total costs of DDG compared with WDG (Ham et al.,

1994), DDGS may be more practical and economical in certain situations to feed than

WDGS because of storage challenges and increased costs of transportation of WDGS.

Schingoethe (2006) reported that 100 kg of corn produces about 40.2 L of ethanol, 32.3 kg of DDGS, and 32.3 kg of carbon dioxide. These processes are shown in (Figure 2.1), as adapted from Bothast and Schlicher. (2005).

Corn

Grind, Wet, Cook

Fermentation

Yeast, Enzymes

Still

Ethanol

Stillage

Distillers Grains Distillers Solubles WDG, MWDG, DDG

WDGS, MWDGS, DDGS

Figure 2.1. Schematic of the dry milling industry with the feed products produced.

Adapted from Bothast and Schlicher (2005).

2.2 DISTILLERS GRAIN PLUS SOLABLES AND GROWTH PERFORMANCE

OF CATTLE

Uwituze et al (2010) demonstrated that growth performance of heifers fed 25%

DDGS (DM basis) did not differ compared with those fed steam-flaked corn diets containing alfalfa hay or corn silage. Also, increasing DGS levels from 20 to 40%, as alternatives to dry rolled corn, did not compromise growth performance of cattle (Lodge et al., 1997b). However, there have been many studies (Larson et al., 1993; Vander Pol et al., 2009; Klopfenstein et al., 2008) that have reported positive effects of DG on growth performance. Thus, the results of the effect of DG on growth performance are inconsistent. Therefore, the effects of DG on DMI, average daily gain, and feed efficiency will be examined in the following sections.

2.2.1 DRY MATTER INTAKE

In most studies, increasing distillers grains in feedlot diets did not influence dry matter intake (DMI) in ruminants, possibly because the amount of starch is less and the amount

of fiber is greater in DGS than grain. For example, digestibility of fiber is less efficient

than starch, which could increase DMI and to achieve a similar rate of growth. Feeding high starch diets can cause acidosis, which can result in decreased DMI (Owens et al.,

1998). Al-Suwaiegh et al., (2002) indicated that DMI was not altered by replacement of

dry-rolled corn (DRC) with sorghum and corn distiller’s grains (11.1 and 10.4 kg/d

respectively, DM basis) up to 30% of ration DM in finishing diets compared with 10.7

kg/d (DM basis) in the control. In another study by Peter et al., (2000), DMI was not

affected with DDG inclusion in heifers compared with those fed modified corn fiber

(MCF). In steam-flaked corn (SFC) diets, DMI was not affected when including 25% of

DM from DDGS (May et al., 2007). However, other research has suggested that

increasing WDG from 0 to 40% of the diet DM reduced DMI linearly (Larson et al.,

1993). This may be due to increasing fat concentrations and energy density with high

levels of DG or other reasons such as growth of mold on the surface of WDG, which

negatively influences DMI. Moreover, there was no effect of form or source of DG on

DMI (Al-Suwaiegh et al., 2002). Al-Suwaiegh et al. (2002) found that DMI was 25.4 and

25.5 kg/d in lactating dairy cattle fed corn DDG and WDG, respectively. Similarly, DMI was 10.4 and 11.1 kg/d in finishing yearling steers fed dry corn DGS and wet sorghum

DGS in dry-rolled corn-based diets.

2.2.2 AVERAGE DAILY GAIN

Distillers grain in ruminant diets may influence ADG because of the contents of protein, fat and fiber. Average daily gain has been reported to have a quadratic trend with increasing DG in several studies. There was no significant difference in ADG among the dietary treatments when WDGS increased from 0 to 10, 20, 30, 40, and 50% (Vander Pol et al., 2009). Larson et al., (1993) found that average daily gain was increased about 6% by increasing the inclusion of wet distiller’s grains from 0 to 40% of the diet DM. Al-

Suwaiegh et al., (2002) indicated that ADG was greater in steers fed 30% of diet DM from corn or sorghum wet distillers grains (WDG) than in steers fed dry-rolled corn.

Increasing ADG values when animals are fed DG might be because DG is an excellent source of bypass protein, which helps meet the metabolizable protein requirements or because the lipid fraction of DG provides additional energy for growth. However, when animals were fed greater than 40% of diet DM, ADG started to decrease possibly due to several reasons. These may include the high fat concentration that potentially reduces the

8 digestibility of other nutrients, and the excess protein of DG that decreases the available energy for growth by increasing the energy cost of certain organs that are responsible for dealing with excess protein. Klopfenstein et al., (2008), summarized 9 trials in cattle fed corn-based diets and reported that ADG was improved by feeding various concentrations of wet distillers grains (WDGS) with the greatest ADG observed between 20 and 30% of

DDGS inclusion. Moreover, average daily gain was not influenced by feeding different forms of DG such as WDGS and DDGS. However, there is a shortage of information about how feeding MWDG influences average daily gain of cattle fed dry whole corn grain.

2.2.3 FEED EFFICIENCY

Distillers grains may improve the efficiency of gain because of highly ruminally undegradable protein, fat fractions, and highly digestible fiber. The protein in corn distillers grain (CDG) is more resistant to degradation by rumen microorganisms

(Mertens, 1977); because the heat process changes the structure of protein in DGS to decrease the degradation in the rumen (McDonald, 1954; Little et al., 1968). In addition, the dry milling process increases the digestible fiber due to effects of enzymes and yeast on degradation of the fiber of DGS. Therefore, feeding DG at certain levels can increase the feed efficiency because of these advantages, however feeding over the optimal level of DGS can result in the G:F decreasing due to the excess protein and high fat concentration of DG, which might impact the digestibility and available energy for gain.

For example, Al-Suwaiegh et al., (2002) indicated that efficiency of gain was greater in steers fed 30% of diet DM from corn or sorghum wet distillers grains than in steers fed dry-rolled corn. In a study conducted by Larson et al., (1993) feed efficiency in the 40%

WDGS group was 14% greater than the control group. Vander Pol et al., (2009) reported

that the efficiency of gain (G:F) was greater than the control group when WDGS was fed

at 20, and 40% of diet DM. Klopfenstein et al. (2008), using a meta analysis with data

from 5 trials, reported that increasing DDGS from 0 to 40% resulted in a cubic response

in G:F. In addition, the greatest value for G:F was between 10 and 20% of DDGS (0.160,

0.159 respectively) and with increasing the percentage of DDGS, the G:F decreased. The

excess protein and high level of fat might be partly responsible for decreased G:F when

high levels of DG are fed. Moreover, G:F was improved by increasing concentrations of

wet distillers grains (WDGS) fed upto 50% WDGS in corn-based diets as compared to a control group in nine trials (Klopfenstein et al., 2008) as shown in (Table 2.1).

Table 2.1. Influence of dietary inclusion of wet and dry distillers grain plus solubles (WDGS and DDGS) on performance in finishing yearling steers. Dietary treatments

DDGS1 WDGS2 20 30 40 20 30 40 50

DMI, Kg.d-1 10.53 10.56 10.49 10.33 10.20 9.90 9.44

ADG, Kg 1.69 1.70 1.66 1.74 1.76 1.73 1.66

G:F 0.159 0.155 0.152 0.168 0.172 0.174 0.175

1Corn dry distillers grains plus soluble (% of diet DM). 2Corn wet distillers grains plus soluble (% of diet DM). Adapted from Klopfenstein et al. (2008) Dietary treatment levels (DM basis) of wet and dry distiller grain plus solubles (WDGS and DDGS).

Although several studies attempted to determine the optimal level of DG depending on

different parameters, it is not an easy issue due to interactions between different factors

that affect determinations of the optimum DG. Schingoethe (2006) recommended that

inclusion of DDGS from 40 to 50% of diet DM can be used successfully as an energy

source for finishing cattle. However, the optimal level of DDGS was around 15% in

crossbred yearling heifers fed steam-flaked corn diets (Klopfenstein et al., 2008).

Corrigan et al., (2009a) indicated that in a dry-rolled corn diet, the optimum level of

WDGS is approximately 40% of diet DM. Determination of the optimum level of DGS that could be used without negative effects on cattle performance depends on several factors, the type of basal ingredients in diets is one of these factors. For example, the optimal level of DG in dry rolled corn diets is higher than the optimal level of DGS in steam-flaked corn diets. There are many feedlots in Ontario that feed dry whole corn

grain-based diets. Also, research on including MWDGS in whole corn grain-based diets

is limited. Therefore, this research was carried out to study the effect of inclusion level of

DDGS and MWDGS on cattle performance fed whole corn grain-based diets.

2.3 FEEDING BEHAVIOR

In beef cattle, improving feed efficiency is important to reduce the cost of production

and nutrient excretion to the environment (Nkrumah et al., 2006; Crews, 2005). Feeding

behaviour may influence feed efficiency. Gut fill and chemostatic mechanism control feeding behaviour, which could be modulated by feeding strategy, health, and environment (Grant and Albright., 1995). In addition, feeding behavior could affect DMI, and consequently the animal’s performance (Grant and Albright., 1995). Most studies conducted on feeding behavior have reported that cattle with different feed efficiency had

12 different feeding behaviour (Richardson and Herd, 2004; Nkrumah et al., 2006; Golden et al., 2008). For example, in a study by Nkrumah et al. (2006), the high efficient steers spent less than 50% of the time at the feeder compared to low efficient steers. Also, increasing NDF and fat concentrations, and decreasing starch concentration in DGS diets compared to whole corn grain might affect feeding behaviour. However, more studies are necessary on feeding behaviour and feed efficiency when cattle are fed high DGS in whole corn grain.

2.4 DISTILLERS GRAIN PLUS SOLUBLES AND MEAT QUALITY OF

CATTLE

Although there are several experiments that have been conducted on the effect of distiller’s grain on cattle performance, there is still limited information about the effect of

MWDGS and DDGS in whole corn grain diets on meat quality characteristics (Gill et al.,

2008). Therefore, more studies are needed to investigate the effects of DG in cattle fed whole corn grain-based diets on meat quality through focusing on fat thickness, retail display, and tenderness evaluation.

2.4.1 FAT THICKNESS AND MARBLING

Klopfenstein et al., (2008) reported that finishing yearling steers fed different dietary inclusions of wet distiller’s grains plus solubles (WDGS) became fatter than those fed corn grain-based diets in the same period on feed. When steers were fed 20% WDGS, the highest 12th rib fat and marbling scores were achieved and then decreased when increasing inclusion above 20%. However, Al-Suwaiegh et al. (2002) reported that marbling score was similar when steers were fed 30% WDG compared with those that

were fed DRC-based control diets. Therefore, the effect of DG on fat thickness and

marbling needs more research.

2.4.2 RETAIL DISPLAY

Appearance of steaks in the display counter is important in determining value of meat.

Subjective color evaluation depends on trained panellists, who use the procedure outlined

by Lavelle et al., (1995) to assign scores to each steak for overall appearance, muscle

color, and surface discoloration at each evaluation time as prescribed by Hunt et al.,

(1991). Panellists characterize meat color (8 = extremely bright cherry red, to 1 =

extremely dark red), overall appearance (8 = extremely desirable, to 1 = extremely

undesirable), and surface discoloration (8 = no [0%] discoloration, to 1 = complete [76 to

100%] discoloration). However, objective color evaluation of steak is measured by using

a Hunter Lab Miniscan spectrophotometer equipped with a 6-mm aperture and using

illuminant D65 to determine CIE (1976) L* (measure of darkness to lightness), a*

(measure of redness), and b* (measure of yellowness) values. There is a positive relationship between a* (measure of redness) and overall appearance. For example,

Roeber et al., (2005) compared feeding a control whole grain corn diet and other diets containing 12.5% DDG plus urea, 25% DDG, 25% WDG, 50% DDG, or 50% WDG

(DM basis). The greatest a* values in steaks after 138 h was achieved when steers were fed 25% WDG compared to those fed other diets, except diet of 12.5% DDG. In addition, steaks from steers fed 25% WDG had the lowest percentage of “moderately unacceptable” steaks which was considered the point at which strip loin steaks would be discounted for quick sale at retail. Increasing DG levels increase the a* values. Roberson

et al., (2005) found that xanthophylls (yellow pigment) may be the reason feeding DG

improves meat color stability. For example, steaks from steers fed DG were redder than

steaks from steers fed corn-based diets (Roeber et al., 2005). Furthermore, Gordon et al.,

(2002) reported that tocopherols were found in distiller’s grains, which are antioxidant

agents and might be one of the other reasons that cause color stability of beef steak and

enhance shelf life. However, in another study by Roeber et al., (2005) color stability of

strip loins during retail display were negatively influenced by increasing distiller’s grains

from 40 to 50% of dietary DM in cattle finishing diets possibly due to high

concentrations of polyunsaturated fatty acids (PUFA); however, color stability could be

enhanced by using 10 to 25% of DG in finishing diets as compared to diets without DG.

These contradictory results suggest that different inclusion levels of DG could result in

different effects on color stability due to the different levels of antioxidants and fats.

2.4.3 TENDERNESS EVALUATION

Tenderness is one of the most important parameters of meat quality (Miller et al.,

1995). The Warner- Bratzler shear force (WBSF) instrument is used to measure the

tenderness of steaks cooked to 70°C. Roeber et al. (2005) conducted two trials with

Holstein steers and reported that increasing distillers grain levels up to 50% of dietary

DM did not influence sensory and tenderness of meat. In another study by Aldai et al.

(2010) where cattle were fed corn or wheat DDGS at 20 or 40% of DM, they found that

the meat from cattle fed corn DDGS was more tender compared to meat from those fed

the barley-based finishing diet (control). Overall, data suggest that DG may be used up to

50% of the diet without negative effects on tenderness.

2.5 DISTILLERS GRAIN PLUS SOLUBLES, TOTAL TRACT DIGESTION AND

NUTRIENT BALANCE

During the process of ethanol production, the concentration of CP, fat, fiber, and minerals, such as N, P, and S increase by approximately three times in DGS compared to whole corn grain (NRC, 1998). With feeding high levels of DGS in the diet, increased concentrations of these components could affect total tract digestibility of these and of other components of the diet (Zinn, 1989; Owens et al., 2010). Some studies focused on the effect of DGS in DRC- or SFC-corn-based diets on total tract digestibility of diets

(Peter et al., 2000; Depenbusch et al., 2009; and May et al., 2009). There is a lack of information about the effect of inclusion of DGS in whole corn grain diets on digestion.

In a study by Spiehs and Varel (2009), intake of N, P, and S were increased by feeding increasing concentrations (up to 60% of diet DM) of wet distillers grains plus solubles

(WDGS) in dry rolled corn (DRC) diets. The excretion of N, P, and S has increased in urine and feces (Cole et al., 2005; Archibeque et al., 2007; Luebbe et al., 2008).

However, there is limited information on the effect of increasing inclusion levels of DGS on the bioavailability of minerals.

2.5.1 TOTAL TRACT DIGESTION

In most studies, total tract digested DM and OM decreased with increasing levels of

DGS. Dry matter digestibility was decreased when animals were fed 20% corn DDGS

(DM basis) (Peter et al., 2000). Depenbusch et al., (2009) found that apparent total tract digested DM and OM were 2.8% lower in steers fed finishing diets containing 15% of

DGS with steam-flaked corn (DM basis) compared to those fed the control diet. In a study by May et al. (2009), apparent total tract digested DM and OM were lower in

Holstein steers fed 25% corn DDGS in steam-flaked corn (SFC) or dry rolled corn (DRC)

compared to those fed the control diet. This decrease in apparently digested DM and OM

may be because the concentration of starch decreased and the concentration of fiber

increased in the diet with increasing inclusion level of DDGS. In contrast, total tract

digested DM and OM were not altered when steers were fed 15% corn or sorghum

WDGS in diets containing steam-flaked corn compared to those fed the control diet (May

et al., 2010). Moreover, others (Peter et al., 2000, Mateo et al., 2004; and Leupp et al.,

2009) have reported that apparent total tract digestion of OM was not affected by

increasing the DDGS levels up to 20, 40, or 60%, respectively.

The processing and drying of DDG might affect the availability and digestibility of the

protein (Corrigan et al., 2009 b; May et al., 2010). Increasing the inclusion level of DG in the diet increases dietary CP. This might differently affect total tract digestion of CP depending on the level of DG in the diet. Apparent total tract digestion of CP was decreased by feeding 25% corn DGS in steers fed SFC-based diets (Uwituze et al., 2010),

and by feeding up to 15% DDGS to growing dairy heifers (Kalscheur et al., 2005).

However, in other studies, replacing corn with DDGS resulted in no adverse effects on

CP digestibility in steers fed 90 and 70% concentrate diets (Mateo et al., 2004, and Chen

et al., 1977, respectively). These observations suggest that the inclusion level of DGS,

form of DGS, the grain processing and type of grain in the diet may influence total tract

digested DM, OM, and CP.

In a metabolism study by Vander Pol et al. (2009), they evaluated WDGS as a fat

source with different sources of fat such as corn and corn oil. They found the fat from

WDGS improved the performance compared to corn oil. They reported that energy value

of WDGS was greater than corn grain due to increased intake and digestion of fat,

propionate production, and unsaturated fatty acids (UFA) reaching the duodenum in

animals fed WDGS than those fed a corn-based control diet. However in a study by May et al. (2009), total tract digestibility of ether extract was lower in animals fed 25% of

DDGS with DRC or SFC-based finishing diets compared to animals fed control diets without DDGS. There is a lack of data about the digestibility of nutrients when beef cattle

are fed DDGS with whole corn grain-based diets. With the increasing use of DGS in

feedlot cattle, the dietary intake of N, P, S, and protein increased, which likely causes

increased excretion of N, P, S. There is a need for better understanding of how we can

manage manure from beef cattle fed DGS (Hao et al., 2009). In addition, the information

about the absorption, digestion, and retention of these minerals is lacking when feeding

high level of DGS in whole corn grain diet in feedlot cattle.

2.5.2 NITROGEN BALANCE

Increasing N excretion resulting from increasing dietary CP intake could result in

increased ammonia emissions from feedlots (Cole et al., 2005; Archibeque et al., 2007).

Moreover, in an in vitro study by Archibeque et al. (2007), increasing the concentration

of CP in diets from 9.1 to 13.9% increased emissions of ammonia. Also increasing the

concentration of CP in the diet from 11.5 to 13% of DM increased ammonia emissions

from 60 to 200% (Cole et al., 2005). In another study, decreasing the CP concentration from 13 to 11.5% reduced the daily ammonia emissions from a simulated feedlot surface by 44% (Todd et al., 2006). Moreover, decreasing the dietary CP intake decreased significantly the concentration of N in manure and reduced volatilization of ammonia

(Satter et al., 2002; Maguire et al., 2007). These data suggest that increasing the inclusion

level of DGS could increase the ammonia emissions from feedlots due to increased N intake and N excretion. Similarly, Spiehs and Varel. (2009) reported that total N excretion and urinary N excretion increased as WDGS inclusion increased in diets.

Increasing inclusion level of wheat DDGS up to 60% of DM in the diet increased the

manure N loss (Hao et al., 2009), and soil salinization in the long term.

2.5.3 PHOSPHORUS BALANCE

In feedlot cattle, the requirement of P in the diet is approximately 0.30% of the diet

DM (NRC, 1996). However, because P concentration in DDGS is approximately 0.82%

of DM, excess P is typically fed when DGS is included in feedlot diets. Hao et al. (2009)

reported that total P in manure is positively correlated with increasing dietary P intake.

Increasing the total concentration of P in manure could increase the risk of soil P

saturation and surface water contamination (Hao et al. 2009). Spiehs and Varel. (2009),

reported that increasing corn WDGS levels in the diet linearly increased the P

concentration in serum and total P excretion in steers. Similarly, in other studies, total P

excretion in cattle fed DG was greater than those fed corn-based diets without DG

(Luebbe et al., 2008). Bremer et al. (2008) reported that the area of the land to manage

the increase in P in manure was increased two times when cattle are fed 40% DGS.

The feces is often the major route of P excretion with a small percentage excreted in

urine in ruminants (Horst, 1986; Betteridge et al., 1986; Morse et al., 1992). Gibb et al.

(2008) indicated that P excretion in feces was increased with increasing wheat DDGS.

Spiehs and Varel. (2009) reported that urinary P excretion was greater than the excretion

in feces and increased linearly as WDGS increased in the diet.

2.5.4 CALCIUM BALANCE

The feces is the primary path for Ca excretion (McDowell, 1992). The sources of Ca

in feces are unabsorbed dietary Ca and endogenous Ca from intestinal mucosal secretion;

therefore, the amount of Ca excreted in the feces is affected by any factor impacting the

absorption of Ca (Liesegang et al., 2007). Also Groff et al. (1995) reported that the

bioavailability and the absorption of Ca are decreased by increasing dietary P and fiber

concentration. Decreasing the bioavailability of Ca may be because of reducing the

availability of absorption time by non-fermentable fiber sources, or increasing the microbes, which bind the Ca with fermentable fiber and convert the free Ca to unavailable form for absorption. Hill et al. (2008) reported that feeding DDGS decreased fecal Ca of sows from 55.6, 51.4, and 47.1 mg/g of DM at 7, 14, and 18 d, respectively compared to the control group fed beet pulp. Theresa and Kumar (2007) indicated that Ca concentration in serum was decreased by increasing concentration of dietary P. However,

Spencer et al. (1965) and Heaney (2000) found that the absorption of Ca was not affected

by high dietary P. Thus, more research seems warranted on the effects of increasing P

intake from DGS on the utilization and balance of Ca.

2.5.5 EFFECT OF SULPHUR ON SELENIUM BIOAVAILABILITY

Increasing dietary S intake when feeding high levels of DGS can be a concern because of increased production of ruminal H2S (Gould et al., 1997) and increased incidence of

polioencephalomalacia, increased emission of H2S (Klopfenstein et al., 2008), and

potential negative effects on Se availability (Ivancic and Weiss, 2001).

Sulphur is antagonistic to Se (Spears. 2003) because of similar chemical and physical

properties. Spears (2003) reported that the bioavailability of selenium was decreased by

20 increasing dietary sulphur. The urinary Se excretion was greater in sheep fed 2.4 g of sulphur.kg-1 compared to those fed 0.5 g of sulphur.kg-1 (Pope et al., 1979). Moreover, the concentration of Se in blood was greater in sheep fed low dietary S (0.5 g of sulphur.kg-1) than those fed high dietary S (2.4 g of sulphur.kg-1) (Pope et al., 1979). In lactating dairy cows, plasma selenium was linearly reduced with increasing dietary S concentration from 2.1 to 4.0 or 7.0 g of sulphur.kg-1 of diet (Ivancic and Weiss., 2001).

In addition, increasing dietary S from 2.2 to 4.0 g of sulphur per kg diet, Se concentrations were decreased in liver and rumen bacteria (van Ryssen et al., 1998).

Although the concentration of sulphur in the diet is one of the important factors which affect the bioavailability of selenium, there are other factors such as Se forms, the type of diet and dietary Ca concentration that influence the bioavailability of selenium.

2.5.6 OTHER FACTORS AFFECT SELENIUM BIOAVAILABILITY

Many studies have been conducted to investigate the effect of Se forms, the type of diet and dietary Ca concentration on Se bioavailability (Aspila, 1991; Koenig et al., 1997;

Ehlig et al., 1967). Also, the excretion of Se in urine was greater in goats (Aspila, 1991) and lambs (Ehlig et al., 1967) fed an inorganic form of Se (selenite) than those fed an organic (selenomethionine) form of Se. However, in other studies, the efficiency of Se absorption did not differ between organic (selenomethionine) and inorganic (selenite) forms of Se (Koenig et al., 1997; Ehlig et al., 1967; Aspila., 1991). In addition, the bioavailability of selenium differs between concentrate and forage diets due to the differences in S concentration or cyanogenetic glycosides (Koenig et al., 1997). The absorption and retention of Se were greater in sheep fed a barley-based concentrate diet compared to an alfalfa hay-based forage diet (Koenig et al., 1997). The absorption of Se

might be decreased by feeding high or low dietary calcium (Spears. 2003). For example,

selenium absorption was greatest in dairy cows fed 8.0 g of Ca per kg diet DM as

compared to those fed fed 4.0 12.5 g Ca per kg diet DM (Harrison and Conrad., 1984).

However, in young calves, selenium absorption was not influenced by feeding high (23.5

g of calcium/kg of diet) or low (1.7 g of calcium/kg of diet) Ca in the diet (Alfaro et al.,

1978). Not only do these factors affect the Se bioavailability, but also they could affect

the activity of glutathione peroxidase. Erythrocyte glutathione peroxidase activity (GPx)

was increased two fold in selenium-deficient heifers fed organic Se compared to those fed inorganic Se (Pehrson et al., 1989). Therefore, GPx could be a good indicator of Se bioavailability. Thus, further research is needed to investigate the effect of increasing dietary S through feeding high level of DGS on the Se deposition and activity of glutathione peroxidase enzymes in tissue.

2.5.7 MAGNESIUM, POTASIUM, AND SODIUM

There is a lack of data in regards to Mg, K, and Na excretion and retention with increasing dietary distillers grains inclusion. These minerals are important for osmotic pressure regulation, acid-base balance, muscle contractions and enzymatic reactions, such as the activation of amylase by Na (NRC. 1996). In beef cattle, maximum tolerable dietary concentration of Mg is 0.4 % (NRC. 1996). The major site of Mg absorption is the rumen and the primary excretion of Mg is through urine. The availability of Mg in concentrates is greater than in forage. The concentration of Mg in cereal grain is 0.11-

0.17%. Other minerals might affect Mg availability. For example, high concentration of

N, Ca, P, and K in the diet may decrease the absorption of Mg (Greene et al., 1983;

Fontenot et al., 1989). However, soluble carbohydrates and supplementation of Na in the

22 diet increase the absorption of Mg (Fontenot et al., 1989; Martens et al., 1987). The maximum tolerable dietary concentration of K is 3% (NRC. 1996). The absorption of K is present in rumen, omasum, and SI. The urine is the major route of K excretion. The concentration of K in cereal grain is less than 0.5%; therefore, the supplementation of K in high concentrate diets is important (NRC, 1996). The maximum tolerable dietary concentration of NaCl is 9% in growing cattle (NRC, 1996). The concentration of Na in cereal grain is not enough for beef cattle; therefore, Na supplementation is necessary as sodium bicarbonate or sodium chloride. Increasing the Na concentration in the diet might decrease feed intake and reduce growth (NRC, 1996). Although studying the absorption and digestion of these minerals may be important for nutrient management when cattle are fed DGS, more studies are needed to know the effect of increasing DGS on the concentration of these minerals in urine and feces in feedlot cattle fed whole corn grain.

2.6 DISTILLERS GRAIN PLUS SOLUBLE AND ORGAN MASS

The efficiency of energy utilization in the whole body is largely dependent on energy expenditure in the visceral organs (Ferrell and Jenkins., 1985). Increasing inclusion level of DGS could increase dietary intake of minerals and protein, which may increase the organ mass due to increasing workload (Sainz and Bentley., 1997; Swanson et al., 1999;

Johnson et al., 1990; and Wang et al., 2009). In previous studies, the effect of feed intake, the forage to concentrate ratio, and level of dietary protein on visceral organ mass were studied (Swanson et al., 1999; Swanson et al., 2008; Wang et al., 2009). Liver weight increased with increasing feed intake (Fluharty and McClure, 1997). Sainz and Bentley

(1997) found liver mass in steers fed ad libitum of a low concentrate diet (5%) is greater than those fed a restricted diet (85% of concentrate). Moreover, organ weights increased

with increasing dietary protein level (Swanson et al., 1999). Increased organs weights

might be associated with the ability of the organ to adapt to excess CP, P, and S in the diet. Because feeding high levels of DGS results in increased dietary intake of CP, fat, fiber, and minerals, it is possible that differences in liver, kidney, and pancreas weights may occur. In adult sheep, fed a high protein diet, the liver adapts to increase urea synthesis (Payne and Morris., 1969). Plasma urea levels and liver, kidney, and pancreas weights increased when pigs were fed high concentrations of protein (Chen et al., 1999).

The increased weight of pancreas may be due to the increased demand of pancreatic

enzymes to digest the additional protein (Chen et al., 1999). Thus, increasing organ mass

could reduce the growth performance due to increasing their energy cost (Swanson et al.,

1999; Johnson et al., 1990; and Wang et al., 2009). Therefore, studying the effect of

increasing dietary inclusion of DGS on organ mass will be useful to understand the

controlling mechanism of energetic efficiency in beef cattle.

2.7 DISTILLERS GRAIN PLUS SOLUBLES AND α-AMYLASE AND TRYPSIN

ENZYMES ACTIVITY

The exocrine role of the pancreas is known to include secretion of digestive enzymes such as α-amylase and trypsin to digest nutrients flowing to the small intestine (SI).

Pancreatic α-amylase is responsible for the initial hydrolysis of starch in the SI (Wright,

1993). Pancreatic trypsin is partly responsible for the initial hydrolysis of dietary protein in the small intestine. Luminal effects of protein might mediate trypsin synthesis and secretion, through a messenger peptide (Harmon, 1993). Corn processing could influence

starch and protein digestion. For example, starch digestion is affected by corn processing

(Zinn et al., 2002). They found that the ruminal and postruminal starch digestion was

lower in animals fed dry-rolled corn (DRC) compared to those fed steam-flaked corn

(SFC). In addition, in a review by Harmon (1992) it was suggested that the quality and

quantity of dietary protein could affect pancreatic α-amylase concentration. Thus, the dry

milling process could affect nutrient content and quality which could influence these

pancreatic enzymes.

In early studies (Clary et al., 1969; Van Hellen, 1979; Janes et al., 1985), data

suggested that the concentration and secretion of pancreatic α-amylase increased with increasing levels of dietary starch. However, pancreatic α-amylase secretion was decreased by duodenal starch infusion (Chittenden et al., 1984, Kreikemeier et al., 1990;

Walker and Harmon, 1995; Swanson et al., 2002a). Swanson et al. (2004), reported that pancreatic α-amylase secretion was increased by dietary casein infusion. These data indicate the potential positive effect of small intestinal protein flow on pancreatic α- amylase secretion; however, the positive effect of protein was not maintained when

starch was infused. In addition, α-amylase concentration and secretion increased with

casein infusion in calves infused with corn starch (Richards et al., 2003). Further studies

are needed in cattle consuming practical diets (eg., DGS inclusion) on pancreatic

digestive enzymes activity.

In some studies, pancreatic trypsin secretion and concentration were not affected by

postruminal protein infusion in the presence of starch (Richards et al., 2003) or by

different dietary protein sources (Khorasani et al., 1990). Similarly, protease activity in

sheep was not altered by abomasal starch or starch and casein infusion (Wang and

Taniguchi., 1998). However, in another study, infusion of duodenal casein increased the

pancreatic trypsin activity in sheep (Ben-Ghedualia et al., 1982). In a study by Swanson

et al. (2008), concentration of pancreatic α-amylase and trypsin activity were increased linearly by decreasing dietary starch and increasing the dietary protein up to 15% of diet

DM. Feeding high levels of DGS, in grain-based feedlot diets, results in large shifts in dietary carbohydrate (starch vs. fiber) and increased dietary CP. This could result in differences in pancreatic exocrine function. Consequently, the digestion, absorption, and retention of nutrients might be affected, which influences growth performance.

2.8 DISTILLERS GRAIN PLUS SOLUBLES AND GLUTATHIONE

PEROXIDASE ENZYMES ACTIVITY

Some previous studies indicated that increasing dietary S might reduce the bioavailability of Se and GPx activity in cattle (Ivancic and Weiss, 2001). Feeding DGS at high level increases dietary S intake, which may influence the activity of GPx enzymes in liver and kidney. However, the relationship between high dietary S intake and GPx is not presently clear. Selenium functions, through selenoproteins, as an antioxidant, in thyroid hormone metabolism, and in helping to support reproduction and immune function (Rayman, 2000). Glutathione peroxidase (GPx) contains selenocysteine at the active site (Arthur et al., 2003). The GPx enzyme functions as an antioxidant through catalysis of the reduced form of glutathione (GSH) and decreases cellular reactive oxygen species (ROS). These ROS are primarily hydrogen peroxide (H2O2) and cellular

metabolism byproducts (Flohe, 1973). The reaction between these strong oxidants and

the macromolecules in cells can cause cellular damage (such as genomic mutations) or

cell death (Halliwell et al., 1992; Poot, 1991). Therefore, the catalytic enzyme GPx

converts these ROS to water (H2O) to protect the cell from damage. The GPx oxidizes

two molecules of glutathione and converts one molecule of H2O2 to two molecules of

H2O. Then oxidized glutathione (GSSG) is converted to the active reduced form by

nictotinamide adenine dinucleotide phosphate (NADPH) as a hydrogen donor and by the

enzyme glutathione reductase (GSSG-R) (Paglia and Valentine, 1967) (Figure 2.2).

2 GSH + H2O2 ----GPx 2 H2O + GSSG + NADPH --GSSG-R 2 GSH +

NADP-.

Figure 2.2. The oxidation and subsequent reduction of GSH, from Paglia and

Valentine (1967).

In cattle, Se deficiency is associated with high calf mortality, mastitis and white

muscle disease (Gerloff, 1992). In addition, Hu et al. (2011) reported that selenium status

and selenoprotein mRNA could be regulated by certain forms of dietary Se in human.

Therefore, increasing dietary S when high levels of DGS are fed might negatively affect

GPx activity.

In a study by Murphy and Quirke (1997), blood gluthathione peroxidase (GSHpx) activity was lower in Holstein steers grazing S fertilized pasture than those grazing unfertilized pasture. In addition, Ivancic and Weiss (2001) reported decreased true Se digestibility when Holstein cows were fed 0.4% S from a mix of calcium and magnesium sulfate in diets containing less than 0.3 mg of Se/kg of DM for an extended period. These results suggest that high dietary S might affect GPx activity because of antagonistic effects between Se and S. However, in a study by Gant et al. (1998), concentrations of Se in whole blood and plasma during early lactation were not affected by feeding 0.4% dietary S from magnesium sulphate (MgSO4) over the last 21 d of gestation in cows. In sheep, the concentration of Se in whole blood was not affected by feeding up to 0.57% S in diets containing 0.12 mg of Se.kg-1 of DM (Hidiroglou et al., 1977). Because of these

27 inconsistent responses of dietary sulphur on Se status, more work seems warranted to further study these relationships.

Gluthathione peroxidase activity is highly sensitive to the bioavailability of Se.

Therefore GPx is considered a good indicator for examining Se status (Lei et al., 2007).

Oxidation of haemoglobin was increased in erythrocytes of rats by feeding Se-deficient diets (Rotruck et al. 1973). In Hereford cows, the activity of erythrocyte GPx was greater

(24.61 EU/g Hb) in cows fed a diet supplemented with Se compared to cows not supplemented with Se (14.94 EU/g Hb; Rowntree et al., 2004). Erythrocyte GPx-1 activity was greater in cows fed 13 mg Se.d-1 at 15 d post-partum and approximately 54 d post-partum than in cows fed at 15 d pre-partum (Enjalbert et al., 1999). However, when cows were fed 32.5 mg Se.d-1, erythrocyte GPx-1 activity was 161.7 and 181.1 EU/g Hb at 15 d and approximately 54 d post-partum, respectively compared to RBC-GPx activity of 1.67 EU/g at 15 d pre-partum. In another experiment by Enjalbert et al. (1999), RBC-

GPx activity was 67.2, 146.4, and 150.9 EU/g Hb, when cows were fed 0, 13, or 32.5 mg

Se.d-1, respectively, at 15 d postpartum. These results suggest that RBC-GPx activity is increased with increasing dietary Se in cattle as shown in Table 2.2.

Table 2.2. Effect of selenium on activity of erythrocyte GPx in cow and their calves.

Erythrocyte GPx-1 Animal Type Animal's Statue Se Supplements Activity (EU. g-1HB ) Cow1 with Se 24.61 without Se 14.94 Cow2 at 15 d pre-partum 13 mg Se/d 2.33 Cow at 15 d post-partum 13 mg Se/d 122.4 Cow at 54 d post-partum 13 mg Se/d 127.2 their calves at 15 d post-partum 186.2 their calves at 54 d post-partum 129.3 Cow at 15 d pre-partum 1.67 Cow at 15 post-partum 32.5 mg Se/d 161.7 Cow at 54 d post-partum 32.5 mg Se/d 181.1 Cow at 15 d postpartum without Se 67.2 Cow at 15 d postpartum 13 mg Se/d 146.4 Cow at 15 d postpartum 32.5 mg Se/d 150.9 their calves at 101 d post-partum 130.0 their calves at 101 d post-partum 156.4 their calves at 101 d post-partum 177.1 1 Rowntree et al., 2004 2 Enjalbert et al., 1999

Liver has the highest activity of GPx-1 compared to other tissues (Behne and Wolters,

1983). Lawrence and Burk (1976) found that in rats fed diets containing 0.5 ppm Se, hepatic GPx activity was greater than rats fed a Se-deficient diet. In another study by

Whanger and Butler (1988), hepatic GPx activity was 700 ± 105 EU/g protein in rats supplemented with 0.2 ppm Se as sodium selenite compared to hepatic GPx activity of 8

± 4 EU/g protein in rats fed 0.02 ppm Se. Similarly, in growing pigs, hepatic GPx-1

activity was 50 and 210 EU/g protein when pigs were fed 0.1 or 0.3 ppm Se, respectively

as sodium selenite compared to approximately 12.5 EU/g protein of GPx-1 activity when pigs were fed 0.03 ppm Se (Lei et al., 1998). Thus, hepatic GPx-1 seems to be responsive to changes in dietary Se and the concentration of Se in liver (Knowles et al., 1999).

The kidney has the greatest concentration of Se compared to other tissue (Maag and

Glen., 1967), and is the main site of plasma GPx production (Stanczyk et al., 2005).

There is a lack of information on GPx activity in kidney when cattle are fed high dietary

S through high inclusion level of DGS. In 12 month old steers, Maag and Glen. (1967) found that renal Se concentration was 1.32 ppm in steers fed 0.11 ppm Se compared to

1.20 ppm of hepatic Se. Moreover, the concentration of renal Se was 1.37 ppm compared to 0.38 ppm of hepatic Se in steers fed 0.18 ppm of Se (Ullrey et al., 1977). In addition, the kidney might play an important role to protect the cell from oxidative stress damage due to high concentration of glutathione (GSH) (Stanczyk et al., 2005). Therefore, further research is needed to study renal GPx activity when cattle are fed high levels of DGS.

2.9 DISTILLERS GRAIN PLUS SOLUBLES AND UREA CYCLE ENZYMES

ACTIVITY

Feeding high levels of DDG result in cattle consuming amounts of CP greater than

their requirement (NRC, 1996). This results in the necessity to metabolize excess dietary

protein. Thompson and Riley (1980) reported that plasma urea N (PUN) in steers was

increased from 7.0 mg/100 ml to 19.1 mg/100 ml by increasing the dietary CP from 9%

to 15% of DM. Moreover, they found that ammonia in rumen (NH3) and PUN correlated

positively with dietary protein (0.60 and 0.74, respectively). In addition, in another study

by Boling et al. (1978), increasing dietary protein increased plasma urea-N (PUN)

concentrations. The concentrations of plasma urea changes in urea altered parallel the

urea cycle enzymes activity (Elsasser et al., 1996). Thus, the high concentrations of NH3

in rumen fluid due to excess ruminally degradable intake protein (DIP) increase the

absorption of NH3 into the portal blood resulting in increased NH3 flowing to the liver.

The liver has a great ability to convert NH3 to less toxic urea through ureagenesis.

However, excessive NH3 absorption could result in liver damage and peripheral hyperammonaemia which can result in infertility, coma and/or death. The activity of urea cycle enzymes is a good indicator of liver function and protein status in ruminants

(Overton et al., 1998). The urea cycle includes 5 reactions. Reaction 1 is synthesis of carbamoyl phosphate, which is catalyzed by carbamoyl phosphate synthase (CPS) and 2 mol of ATP. Reaction 2 is synthesis of citrulline from carbamoyl phosphate by L-

ornithine transcarbamoylase (OTC). Reaction 3 is synthesis of argininosuccinate from

aspartate and citrulline and consumes 1 mol ATP. Reaction 4 is synthesis of arginine and

fumarate from arginiosuccinate by argininosuccinase (AS). Reaction 5 is synthesis of

31 ornithine and urea from arginine by arginase. Overall the reactions consume 3 mol of

ATP (Murray et al., 1988). Takiguchi and Mori (1995) reported that urea cycle enzymes are regulated by diet and are affected by nutritional factors in rats. In a study by Elsasser et al. (1996) in growing cattle, arginase activity was greater in steers fed a diet containing

14% CP (DM basis) compared to those fed a diet containing 8% CP (DM basis) over an

84-d period. Similarly, increasing dietary protein increased hepatic arginase activity in cattle (Boling et al., 1978). Moreover, in adult sheep, increasing the dietary protein increased the activity of urea enzymes (Payne and Morris., 1969). Takagi et al. (2008) examined changes in the activity of urea cycle enzymes at 2, 13, and 19 wk of age in calves and found that the activities of CPS, OTC, and AS increased and Al decreased with age. Because feeding high levels of DGS results in CP intakes significantly greater than requirements, information on the effect of feeding DGS on urea cycle enzymes is warranted.

2.10 RESEARCH HYPOTHESES AND OBJECTIVES

Most of the studies have been conducted on the effect of inclusion level of distillers

grains with dry rolled, high moisture-, or steam–flaked corn based diets on animal

performance and carcass characteristics. However, there is a lack of information about

the effect of increasing the DDGS and MWDGS up to 50% in whole corn grain diets.

Therefore, this study aims to determine the effects of feeding increasing levels of modified wet or dry corn distillers grains plus solubles (MWDGS and DDGS, respectively) in whole corn-based finishing diets on

a) Feedlot performance, carcass characteristics, meat quality, and feeding behavior.

b) Total tract digestion, nutrient balance, and excretion with specific emphasis on nitrogen and mineral loads.

c) The activity of digestive enzymes (α amylase and trypsin).

d) The activity of glutathione peroxidase and urea cycle enzymes.

CHAPTER 3

INFLUENCE OF FEEDING INCREASING LEVELS OF DRY OR MODIFIED WET CORN DISTILLERS GRAINS PLUS SOLUBLES IN WHOLE CORN GRAIN-BASED FINISHING DIETS ON PERFORMANCE AND CARCASS TRAITS IN FEEDLOT CATTLE

3.1 ABSTRACT

One hundred and fourteen cross-bred steer calves and seventeen heifers (BW = 357.2

± 5.8 kg) were used in a completely randomized block design (2 × 3 factorial arrangement of treatments plus a control) to determine the effect of inclusion level and form of distillers grains plus solubles (DGS) on feedlot performance, carcass characteristics, and meat quality using whole corn grain-based finishing diets. The DGS were fed at 0 (control), 16.7, 33.3, and 50% of ration DM using dry (DDGS) or modified wet (MWDGS) product. All diets contained 10% haylage as a forage source, and were formulated to meet or exceed the estimated requirements for MP. Steers were fed until ultrasound backfat thickness reached 10 mm. Data were analyzed using GLM of SAS; treatment means were compared using contrast statements (control vs. others, DDGS vs.

MWDGS, inclusion levels of DGS (linear, quadratic), and interactions between form and linear and quadratic inclusion levels). There were no effects (P > 0.05) of dietary treatment on final BW, ADG, days on feed, rumen pH at slaughter, dressing %, hot carcass weight, marbling score, lean yield, and lean color. A form by quadratic effect of inclusion level interaction (P = 0.004) was observed for body fat as a percentage of total fat because it decreased when DGS inclusion increased from 0% to 33.3% and then increased when DDGS increased from 33.3% to 50% of diet DM. Liver abscess scores was lower (P = 0.05) in steers fed DGS than steers fed the control diet. Daily time at

feeder (TF; min/d) increased linearly (P = 0.02) with increasing level of DGS. Time per

meal (TM; min) was lower (P = 0.01) in cattle fed MWDGS than cattle fed DDGS.

Eating rate (ER; g DM/min) had a quadratic effect (P = 0.02), because ER decreased

when level of DGS increased from 0 to 33.3% of DM then increased when level of DGS

increased up to 50% of DM. These data indicate that feedlot performance and carcass

characteristics were not affected by feeding DDGS or MWDGS up to 50% diet DM in

whole corn grain-based finishing diets; however, feeding behaviour was affected by form

and level of DGS.

Key Words: beef cattle, performance, carcass traits, ethanol byproducts, distillers

grains

3.2 INTRODUCTION

Distillers grains (DG) are a byproduct of the grain milling industry, which are formed after the fermentation of cereal grain by yeast (Klopfenstein et al., 2008). Corn grain is

the primary feedstock used to produce ethanol fuel by the dry milling process (Peter et

al., 2000). Because the dry milling process converts the starch in grain to ethanol by

fermentation, the other nutrients such as protein and other components (fat, fiber, P and

S) become more concentrated by approximately 3 times in the ethanol byproducts as

compared to original grain (Klopfenstein et al., 2008).

The renewable biofuel industry has expanded over the last several years (Hill et al.,

2006; Schrage et al., 1991) the price of corn has also increased (Schrage et al., 1991).

This has increased the availability of ethanol byproducts, which are a viable alternative to feeding corn grain (Lodge et al., 1997a). This has stimulated recent researchers to study the utilization of ethanol byproducts as a protein and an energy source for livestock

(Klopfenstein et al., 2008). Optimizing the use of distillers grains (DG) is becoming

increasingly important as ethanol production increases (Rincker and Berger, 2003). When

DG is fed at moderate to high concentrations in the diet, the concentrations of protein, P

and S are higher than requirements; therefore, the efficiency of nutrient utilization is

decreased ( Salim et al., 2011). Furthermore, the high level of sulfur (0.6 to 1.0% or

higher) might cause decreased DMI, ADG, and liver Cu stores when DGS are used above

30 to 40% of diet DM (Klopfenstein et al., 2008). The drying process of DGS may reduce

the feeding and nutritional value of DGS (Weiss et al., 1989). Most of the studies on

DGS have been conducted in cattle fed dry-rolled, high-moisture, or steam–flaked corn-

based diets. There are limited studies comparing the effect of MWDGS and DDGS on

beef cattle fed whole-corn grain-based finishing diets. Also, there is limited information

on how feeding DGS influences feeding behaviour. Therefore, the objectives of this

research were to examine the effect of inclusion levels of MWDGS and DDGS in whole

corn diets on cattle performance and carcass characteristics.

3.3 MATERIALS AND METHODS

3.3.1 Animal Care and Experimental Design

This experiment followed the recommendations of the Canadian Council on Animal

Care (1993) and met the approval of the University of Guelph Animal Care Committee.

This experiment had been conducted at the Elora Beef Research Centre. One hundred and fourteen cross-bred steer calves and seventeen heifers weighing approximately 357.2 ±

5.8 kg were randomly assigned to 14 pens (each treatment had 19 calves; 15 in barn 1 and

4 in barn 2) were used in a completely randomized block design with a 2 × 3 factorial arrangement of treatments plus a control. Treatments (Table 3.1) consisted of control,

16.7%, 33.3%, and 50% of diet DM from dried distillers grains plus solubles (DDGS) or

modified wet distillers grains plus soluble (MWDGS). Analysis of DDGS and MWDGS

is located in appendix 1. Calves were fed diets as a total mixed diet once daily. All diets

contained 10% haylage as a forage source, and all diets were formulated to meet or

exceed the estimated requirements for CP, minerals and vitamins according to NRC

(1996). Calves were fed a corn silage/haylage-based diet for at least two weeks before

starting the experiment to adapt to facilities. Calves were adapted to dietary treatments

over the first 28 d on experiment. During 28 d of adaption period, calves fed 25, 50, 60,

80 % of concentrated at the first, second, third and fourth week respectively. At the

beginning and end of the trial, cattle were weighed twice in the morning over two

consecutive days. The animal's body weights were taken every 28 d.

3.3.2 Feeding behavior traits measurements and sample collection

For cattle in barn 1, radio frequency ID tags were placed in the right ear prior to the

experiment. There were four electronic feeding stations (Insentec, B.V., Marknesse, the

Netherlands) as described by Mader et al. (2009) and Montanholi et al. (2009) for each pen in barn 1 allowing for monitoring of individual feed intake to determine feeding behavior characteristics . In barn 2, Calan gates (American Calan, Inc., Northwood, NH,

USA) were used to measure individual feed intake and no feeding behavior data was collected. Feeding behavior characteristics were defined as follows: daily visits to the feeder (VF; events/d), total daily intake (TI; kg as fed/d), number of meals per day (NM; events/d), time spent at feeder per day (TF; min/d), meal size (MS; kg DM/meal), time per visit (TV; min/visit), time per meal (TM; min/meal), eating rate (ER; g DM/min), and these represent the average of each individual animal over the whole period of

experiment. The visits to the feeder were computed at 60 min intervals throughout the

day. Meal was defined as distinct eating periods, which could include short breaks, but

which are separated by intervals of no longer than seven min (Forbes, 1986).

3.3.3 Laboratory analyses

Diet samples were collected weekly and frozen at -20о C for further analysis at the

Agri-Food Laboratories Inc. (Guelph, ON, Canada), and then dried in a 55о C oven, ground to pass a 1- mm screen, and analyzed for DM and ash by standard procedures

(AOAC, 1990; Method 930.15). Diet nitrogen concentrations were determined using a

Leco N analyzer (Leco Corporation, St. Joseph, MI, USA) and percent CP was calculated by multiplying percent N х 6.25. Neutral detergent fiber (NDF) concentration was determined by the method of Robertson and Van Soest (1981) using an Ankom fiber analyzer (Ankom Technology Crop., Fairport, NY, USA). Phosphorus concentrations were determined as described by AOAC (1990).

3.3.4 Carcass measurements

Cattle were slaughtered when backfat thickness (BF; between the 12th and13th rib) reached approximately 10 mm. Backfat thickness was estimated using ultrasound (Aloka

SSD- 500 ultrasound unit; Corometrics Medical Systems, Wallingford, CT, USA). After

169 d on feed, the remaining cattle on feed were slaughtered based on the greatest BF thickness (n = 38). At this time, there were 26 of the remaining cattle (n = 38) with less

than 8 mm of BF. The calves were slaughtered at the University of Guelph Meat

Laboratory. The pH of rumen fluid was measured immediately after slaughtering after

straining through two layers of cheese-cloth. Hot-carcass weights were determined

- directly after slaughter. Furthermore, the incidence of liver abscesses were scored (A =

one or two small abscesses or abscess scars (less than ~2.5cm in diameter), A = two to four small well-organized abscesses under 2.5cm in diameter or one larger >2.5cm in

+ diameter, and A = more than 5 active small abscesses or more than one large active

abscess (Brink, et al., 1990). These scores were converted into a numerical score where

none = 0, A− = 1, A = 2 and A+ = 3. Grade fat (minimum fat depth in last quadrant over

the LM muscle), longissimus muscle (LM) area, marbling score, and rib composition

including lean, bone, and fat (based on 9th to12th rib section dissections as an indicator

of body composition), lean color, and fat color were recorded as described previously

(Laborde et al., 2002; Mandell et al., 1997; Mader et al., 2009). Carcass grade and yield

characteristics were determined by Canadian Beef Grading Agency graders according to

Livestock and Poultry Carcass Grading Regulations (Agriculture Canada, 1992). Average

daily gains were computed by subtracting initial live weight from final BW divided by

the days on feed.

3.3.5 Statistical Analysis

Data were analyzed as a completely randomized block design using the GLM

procedure of SAS. The model included the effects of block (barn), DGS form (dry vs.

modified wet), inclusion level (0, 16.7, 33.3, and 50%) and the interaction. For feeding

behavior, data was analyzed by using the Mixed procedure of SAS. Contrasts statements

were used to compare control vs. other treatments, DDGS vs. MWDGS, linear and

quadratic effects of DGS inclusion level, and the interaction between linear and quadratic

effects of inclusion levels and forms of DGS. Significance was declared at P ≤ 0.05 and a

tendency was reported if 0.05 < P ≤ 0.10.

3.4 RESULTS

Initial and final BW did not differ (P = 0.47 and P = 0.58, respectively) among dietary treatments (Table 3.2). Average daily gain (kg.d-1), DM intake (kg.d-1), and DM intake

(percentage of BW) were not affected (P = 0.19, P = 0.33 and P = 0.16, respectively) by

form, level, or the interactions between form and level of DGS. There was no difference

(P = 0.08 and P = 0.24) among treatments in G: F (kg* kg-1) and days on feed

respectively.

Rumen pH did not differ (P ≥ 0.38) among the dietary treatments. Liver abscess score

was lower (P = 0.05) in steers fed DGS than steers fed the control diet (Table 3.3).

However, liver abscess score was not affected (P ≥ 0.1) by form or level of DGS. Liver

and lung weights (actual and relative to BW) were not influenced (P ≥ 0.19) by form and

level of DGS.

Dressing percentage and hot carcass weight were not influenced (P ≥ 0.31) by form

and level of DGS (Table 3.4). There were no differences (P ≥ 0.11) in marbling score, grade fat, longissimus muscle area, and lean yield among dietary treatment. Lean and total fat weights (actual and as a percentage of rib weight) were not influenced (P ≥ 0.17) by dietary treatment. There was quadratic effect due to the interaction (P = 0.04) between forms and levels for bone weight (kg). Moreover, bone weight as a percentage of rib weight (kg.kg-1) was greater (P = 0.05) in steers fed DGS compared to the control group.

There was a tendency for a quadratic response (P = 0.09) for bone weight as a percentage of rib weight (kg.kg-1), which increased with increasing inclusion of DGS level from 0 to

16.7% and decreased from 16.7% to 50% of diet DM. Body fat (kg) did not differ (P ≥

0.24) among dietary treatments. Body fat as a percentage of total fat (kg.kg-1) was greater

(P = 0.05) in steers fed DDGS than steers fed MWDGS. There was quadratic effect due

to the interaction (P = 0.004) between forms and levels for body fat as a percentage of total fat (kg.kg-1). Body fat as a percentage of total fat (kg.kg-1) was quadratically

affected (P = 0.002) with increasing MWDGS inclusion as body fat increased with

increasing DDGS inclusion from 0% to 16.7% and then decreased when DDGS increased

from 16.7% to 50%; however, body fat decreased when MWDGS inclusion increased

from 0% to 33.3% and then increased when MWDGS increased from 33.3% to 50% of

diet DM. Subcutaneous and intermuscular fat weights (actual and percentage of total fat)

were not affected (P ≥ 0.25) by forms and increasing levels of DGS. Lean and fat color did not differ (P ≥ 0.20) among treatments.

Daily visits to the feeder (VF) were greater (P = 0.001) in cattle fed DGS than cattle fed whole corn grain (control) (Table 3.5). There was a quadratic effect (P = 0.004) in daily visits to the feeder, because VF increased when DDGS inclusion increased from 0 to 16.7% of DDGS and then decreased when DDGS increased from 16.7 to 50% of DM.

Moreover, VF increased when MWDGS increased from 0 to 33.3% of DM and then decreased when MWDGS increased from 33.3 to 50% of DM. There were no significant differences (P = 0.55) in number of daily meals (NM) between the cattle fed DGS and cattle fed the control diet. However, number of daily meals (NM) was greater (P = 0.002) in cattle fed MWDGS diets than cattle fed DDGS diets. There was an interaction (P =

0.002) in number of daily meals (NM) between forms and quadratic inclusion levels of

DGS. The number of daily meals (NM) decreased when inclusion level of DDGS increased from 0 to 16.7% then increased when DDGS level increased from 16.7 to 50%; however, the number of daily meals (NM) increased when inclusion level of MWDGS

increased from 0 to 16.7% then decreased when inclusion level of MWDGS increased

from 16.7 to 50% of DM. Daily time at feeder (TF; min/d) increased linearly (P = 0.02)

with increasing the inclusion level of DGS, the greatest value was observed at 50% of

DDGS and at 33.3% of MWDGS. Meal size (MS; g DM) did not differ (P ≥ 0.08) among

the dietary treatments. There was an interaction (P = 0.004) between form and quadratic

inclusion level of DGS in total daily intake. There was a quadratic effect (P < 0.001) for

time per visit (TV; min), as it decreased with increasing DDGS and MWDGS from 0 to

16.7%, then increased when DDGS and MWDGS increased up to 50% and 33%

respectively, and decreased again only when MWDGS increased from 33.3% to 50% of

DM. There was no significant difference (P = 0.20) in time per meal (TM; min) between cattle fed DGS and cattle fed whole corn grain (control). Time per meal (TM; min) was greater (P = 0.01) in cattle fed DDGS than cattle fed MWDGS. There was an interaction

(P = 0.007) between form and quadratic inclusion level of DGS in time per meal (TM; min). Time per meal (TM; min) increased when DDGS increased from 0 to 16.7 % of

DM then decreased when DDGS increased from 16.7 to 50% of DM; however, time per meal (TM; min) decreased when MWDGS increased from 0 to 16.7 % then increased when MWDGS increased from 16.7 to 33.3% then slightly decreased at 50% of

MWDGS. Eating rate (ER; g DM/min) was greater (P = 0.005) in cattle fed the control diet than in cattle fed DGS. Moreover, eating rate (ER; g DM/min) was greater (P =

0.0002) in cattle fed MWDGS than in cattle fed DDGS. There was a quadratic effect (P =

0.02) in eating rate (ER; g DM/min) because ER decreased when level of DGS increased

from 0 to 33.3% of DM then increased when level of DGS increased from 33.3 to 50% of

DM.

3.5 DISCUSSION

Most of the research on DGS inclusion in feedlot diets has been conducted in cattle fed dry

rolled-, high moisture-, or steam-flaked corn-based diets (Al-Suwaiegh et al., 2002; Corrigan et al.,

2009 a, b; Ham et al., 1994; Lodge et al., 1997a). Recent research has suggested that there may be

an interaction between corn processing method and WDGS inclusion in feedlot diets with a greater

response observed feeding WDGS in diets containing less processed corn (Corrigan et al., 2009a),

although dry whole corn was not tested. Also, some recent research suggests no added benefit of

feeding dry-rolled corn-based diets as compared to dry whole corn-based feedlot diets (Gorocica-

Buenfil and Loerch, 2005). Feeding MWDGS rather than DDGS may be more beneficial because

of increased drying costs for DDGS. Also, drying of DGS may decrease the feeding value (Ham

et al., 1994). However, because of costs associated with transporting and challenges in storing

WDGS, it may be more practical and economical for some feedlots to feed DDGS rather than

WDGS. The results of this experiment suggest that form or level of DGS did not greatly influence growth performance (ADG, DMI, and G:F) in finishing cattle fed whole corn- base finishing diets. The lack of a response in DMI due to increasing DGS inclusion is in agreement with other research that reported increasing levels of corn DDGS from 0 to

15% (May et al., 2010), or 0 to 75% (Gordon et al., 2002) and WDG up to 30% (Al-

Suwaiegh et al., 2002) in corn-based diets did not influence DMI. However, other research has suggested that DMI decreased linearly as WDG increased from 0 to 40% of the diet DM (Larson et al., 1993). The broad variations in nutritional value of DG might be the reason of inconsistencies between our result and these studies (Spiehs et al., 2002).

Because DDGS and MWDGS have relatively high UIP levels (see Appendix 1), predicted DIP balance is negative for all treatments except control (NRC, 1996).

However, dietary CP was provided well in excess of predicted requirements, especially

for higher inclusion levels, and ruminants have the ability to recycle urea-N to the rumen

(Bunting et al., 1987.), so there likely was not a true DIP deficiency. Moreover, other

factors that could influence DMI are fat, starch, and S concentrations in the diet, energy density of the diet, and mold content resulting from storage of WDG (Zinn et al., 1997).

The drying process decreases the feeding value of DGS (Huls et al., 2008). Others have

reported that the feeding value of MWDGS decreased with increasing inclusion level of

DG with the greatest improvement achieved when cattle were fed 20% (Huls et al.,

2008). Also, increasing DGS levels from 20 to 40%, as alternatives to DRC, did not

compromise growth performance of cattle (Lodge et al., 1997b). Thus the effect of DGS

on the performance could differ among studies depending on the inclusion level.

Klopfenstein et al., (2008) reported that finishing yearling steers fed diets containing

wet distiller’s grains plus solubles (WDGS) become fatter than those fed corn grain

during the same period. Additionally, steers fed 20% WDGS had the highest 12th rib fat and marbling scores with decreases as WDGS inclusion increased above 20%. However,

Al-Suwaiegh et al. (2002) reported that marbling score was similar among all treatments, when steers were fed 30% WDG compared with those that were fed dry rolled corn. In

our study, there were no differences in dressing percentage and hot carcass weight among dietary treatments. Marbling score, grade fat, and LMA did not differ among dietary treatments. Moreover, lean and total fat weights (actual and as a percentage of rib weight) were not influenced by feeding different forms and levels of DGS. Similar to our results,

there were no significant differences in marbling or LMA when steers were fed WDGS

up to 30% of DM diet (Al-Suwaiegh et al. 2002). In addition, Rincker and Berger (2003)

found that carcass composition was not affected by feeding up to 50% of DDG or WDG

in whole corn diets. In the current study, cattle were fed to a similar backfat thickness

endpoint which possibly could reduce potential differences in carcass characteristics

between treatments. However, the lack of response in growth performance, suggests that

even if cattle were fed for a common time on feed, carcass characteristics were likely not

greatly influenced by dietary treatment.

There was a lack of an effect of increasing inclusion level of DGS on rumen pH in the

current study. In contrast, Corrigan et al. (2009 b) reported that increasing WDGS up to

40% decreased rumen pH compared to 0% of WDGS. Also, they found differences between DRC, HMC, and SFC in the variance of rumen pH. Therefore, the difference

between these results and the current result might be because rumen pH is affected by the

processing and source of grain, and the ratio of forage to concentrate.

Liver abscess scores were lower in steers fed DGS than steers fed the control diet.

This may be the result of increased fiber relative to starch in diets containing DGS and the resulting effects on potentially reducing ruminal acidosis (Schingoethe, 2006).

However, in the current study, liver abscess scores were likely not high enough to greatly impact performance.

The characteristics of feeding behaviour might influence animal performance.

Research has suggested that feeding behaviour might differ among cattle with different

feed efficiencies (Richardson and Herd, 2004; Nkrumah et al., 2006; Golden et al., 2008).

The feeding behaviour data in the current study are similar to the results from others

(Gomez et al., 2007; and Montanholi et al., 2009). Montanholi et al. (2009) found a strong association between DMI and certain characteristics of feeding behavior (MS and

ER). Similarly, the capacity for DMI was associated with MS and ER in dairy cows

(Senn et al., 1995). In addition, there is a relationship between feed efficiency, as

measured by residual feed intake (RFI), and feeding behavior traits (Gomez et al. 2007;

Montanholi et al., 2009), with desirable RFI reported in steers with fewer visits (VF),

smaller meals (MS), or lower eating rate (ER). Also, more efficient steers had fewer daily

number of meals (NM) (Robinson and Oddy, 2004; Golden et al., 2008); however, in another study, daily number of meals (NM) was not correlated with ADG, DMI, and F:G

(Montanholi et al., 2009). As stated earlier, some studies have reported a relationship

between feeding behaviour and growth performance and ADG (Montanholi et al., 2009)

and correlated positively (Schwartzkopf-Genswein et al., 2003) with time at feeder (TF).

In the current study, cattle spent a longer time at the feeder per day (TF) with increasing

DGS. This might be because NDF increases with increasing levels of DGS. Cattle that

spent the longest time at the feeder had the greatest numerical ADG and G:F, which was

achieved when cattle were fed 50% of DDGS or 33.3% of MWDGS. Schwartzkopf-

Genswein et al. (2011) found that the cattle with the greatest ADG spent a longer time at

the feeder compared to cattle with low ADG over an entire feeding period; conversely,

high ADG cattle spent shorter time at feeder in the finishing period. These results suggest

that increasing the time at feeder might influence DMI when cattle are fed high levels of

DGS. Similarly, the total daily bunk attendance is correlated positively with DMI in

steers fed finishing diets (Gibb et al., 1998). The difference between the current result

and other studies might be likely because the data from pen could be covered the

variation among individual animals (Schwartzkopf-Genswein et al., 2003).

Eating rate was associated with improved animal performance, which has been

indicated in previous research (Church et al., 1980; Frisch and Vercoe, 1969; Prawl et al.,

1997). However, eating rate in the present research decreased with increasing level of

DGS up to 33.3% of DM, and then increased with increasing the level of DGS from up to

50%. The ER values were greatest in cattle fed control diet, which had the lowest

numerical value of G:F compared to cattle fed DGS diets. These results agree with

Montanholi et al. (2009), who reported that there is a positive correlation between ER and

RFI. However the current results disagree with Schwartzkopf-Genswein et al. (2011) and

Hickman et al. (2002) who reported that the animals with the greatest ADG and G:F had

a greater ER compared to animals with lower G:F. In general, increasing DGS level could

affect animal behaviour and it may be different depending on form of DGS.

These data indicate that feedlot performance and carcass traits were generally not

affected by feeding DDGS or MWDGS up to 50% diet DM in whole corn grain-based

finishing diets. However, liver abscess scores were reduced in steers fed DGS compared

to steers fed the control diet and feeding behaviour was influenced by form and level of

DGS.

Table 3.1. Dietary composition and analysis (DM basis). Dietary Treatment DDGS1 MWDGS2 Ingredient Control 16.7 33.3 50.0 16.7 33.3 50.0 Dry whole corn grain 80.2 68.3 51.7 35.0 68.3 51.7 35.0 DDGS - 16.7 33.3 50.0 - - - MWDGS - - - - 16.7 33.3 50.0 Alfalfa/grass haylage 10.0 10.0 10.0 10.0 10.0 10.0 10.0 Soybean meal 4.3 ------Urea 0.5 ------Mineral premix3 2.7 2.7 2.7 2.7 2.7 2.7 2.7 Vitamin/monensin premix4 2.3 2.3 2.3 2.3 2.3 2.3 2.3 Analysis5 DM 71.4 72.9 73.0 73.9 66.5 63.3 60.0 CP, % 16.0 15.8 19.3 21.9 15.7 18.7 22.1 DIP, % CP6 57.4 36.5 30.1 26.0 38.0 32.36 28.7 UIP, % CP7 42.6 63.5 69.9 74.1 62.0 67.6 71.3 SolP, % CP8 23.2 19.0 17.1 15.9 16.5 13.1 10.9 ADF, % 8.4 9.7 11.7 13.6 9.9 11.3 12.2 NDF, % 15.9 21.4 26.2 29.4 19.6 23.0 25.3 Starch, %9 55.7 46.8 36.0 25.2 47.05 36.5 26.0 Ca, % 1.16 1.19 1.31 1.14 1.09 1.16 1.21 P, % 0.36 0.44 0.53 0.60 0.45 0.55 0.66 K, % 0.77 0.79 0.92 0.98 0.83 0.95 1.09 Mg, % 0.19 0.21 0.25 0.27 0.22 0.26 0.30 Na, % 0.20 0.24 0.29 0.29 0.24 0.29 0.34 S, % 0.14 0.21 0.30 0.35 0.23 0.30 0.38 Se, ppm 0.36 0.35 0.36 0.37 0.35 0.35 0.36 1Corn dry distillers grains plus solubles. 2Corn modified wet distillers grains plus solubles. 3Contains 79.5% limestone, 16% salt, 4.5% CF Beef Cattle trace mineral mix (contains 153,013 mg/kg Zn, 122,445 mg/kg Mn, 30,598 mg/kg Cu, 27,100mg/kg Fe, 368 mg/kg Co, 1,531 mg/kg I, 235 mg/kg Se; DM basis).

4 Contains 93.63% dry ground corn, 5.66 % vitamin premix (4,400,000 IU/kg vitamin A, 1,100,000 IU/kg vitamin D, and 7,700 IU/kg vitamin E), 0.71% monensin premix (200 g monensin/kg), 512.5 mg/kg thiamine (98%); DM basis). 5Average of weekly diet samples. 6Degraded intake protein was calculated from ingredients analysis. 7Undegraded intake protein was calculated from ingredients analysis. 8Soluble intake protein as % of CP was calculated from ingredients analysis. 9Starch was calculated according to NRC (1996).

Table 3.2. Influence of dietary source and level of DGS on growth performance in feedlot cattle fed dry whole corn-based finishing diets. Dietary Treatment P-value3 DDGS1 MWDGS2

Item Control 16.7 33.3 50 16.7 33.3 50 SEM 1 2 3 4 5 6

Initial BW, 349 369 343 355 369 345 334 17.8 0.84 0.65 0.55 0.47 0.51 0.97 Kg Final BW, 584 558 604 559 580 580 573 14.6 0.59 0.71 0.58 0.62 0.81 0.96 Kg ADG, 1.65 1.74 1.70 1.91 1.86 1.96 1.80 0.132 0.22 0.40 0.21 0.55 0.63 0.19 kg/d DM intake, 10.8 10.7 10.7 10.9 10.5 11.5 10.3 0.32 0.99 0.91 0.97 0.53 0.88 0.33 kg/d DM intake, 1.90 1.94 1.80 1.97 1.83 2.01 1.83 0.033 0.88 0.61 0.81 0.68 0.50 0.16 % of BW Gain: Feed, 0.152 0.162 0.159 0.174 0.177 0.214 0.176 0.017 0.18 0.12 0.18 0.27 0.16 0.08 kg/kg Days on Feed4 152 115 160 116 118 120 140 14.6 0.14 0.72 0.37 0.30 0.95 0.24

Table 3.3. Influence of dietary source and level of DGS on rumen pH and liver abscesses in feedlot cattle fed dry whole corn-based finishing diets.

Dietary Treatment DDGS1 MWDGS2 P-value3

Item Control 16.7 33.3 50 16.7 33.3 50 SEM 1 2 3 4 5 6

Rumen pH4 5.5 5.6 5.2 5.7 5.6 5.4 5.6 0.19 0.87 0.91 0.70 0.38 0.99 0.69

Liver abscess score5 0.67 0.15 0.24 0.11 0.03 0.48 0.18 0.21 0.05 0.70 0.11 0.26 0.58 0.75

1Corn dry distillers grains plus soluble (% of diet DM). 2Corn modified wet distillers grains plus soluble (% of diet DM). 3Contrast P- values: 1- control vs others, 2- DDGS vs MWDGS, 3- linear level, 4- Quadratic level, 5- linear (forms Χ levels), 6- Quadratic (forms Χ levels). 4From rumen fluid sample collected at slaughter time. 5Presence of liver abscesses were scored using a 3 point scale (no abscess (O), one or two small (less than ~ 2.5 cm in diameter) abscesses or abscess scars (1), two to four active abscesses under 2.5 cm in diameter or one larger (> 2.5 cm in diameter) active abscess (2), more than 5 active small abscesses or more than one large active abscess (3)).

Table 3.4. Influence of dietary source and level of DGS on carcass characteristics in feedlot cattle fed dry whole corn-based finishing diets.

Dietary Treatment DDGS1 MWDG2 P-value3

Items Control 16.7 33.3 50 16.7 33.3 50 SEM 1 2 3 4 5 6

Hot Carcass, kg 347 325 358 324 340 337 345 9.09 0.34 0.52 0.46 0.92 0.47 0.74 Dressing, % 59.5 58 59 58 59 58 60 0.87 0.31 0.55 0.57 0.28 0.33 0.59 Marbling score4 5.4 5.0 5.4 5.1 5.0 5.2 4.8 0.22 0.16 0.31 0.20 0.76 0.20 0.65 Grad-Fat mm 9.9 8.0 9.5 9.0 9.1 9.3 7.9 0.90 0.24 0.95 0.29 0.74 0.58 0.59 Longissimus Muscle 155.8 136.2 130.4 121.0 95.0 102.1 129.9 19.87 0.11 0.23 0.23 0.13 0.48 0.12 Area, cm2 Lean yield, % 5 59.6 60.1 60.32 59.6 60.3 58.9 60.9 0.62 0.45 0.93 0.46 0.96 0.64 0.34 Rib sample Wt, kg 4.2 3.9 4.4 3.9 4.0 4.0 4.1 0.13 0.30 0.70 0.45 0.88 0.84 0.28 Lean, kg 2.3 2.3 2.4 2.2 2.3 2.2 2.4 0.09 0.69 0.61 0.65 0.96 0.44 0.53 Lean, % of rib wt 55.2 57.4 55.6 55.6 58.8 55.3 58.5 1.75 0.37 0.35 0.64 0.64 0.29 0.75 Bone, kg 0.8 0.8 0.9 0.8 0.8 0.8 0.7 0.01 0.03 0.01 0.08 0.01 0.004 0.04 Bone, % of rib wt 18.1 19.7 19.7 20.4 19.8 19.4 18.4 0.64 0.05 0.21 0.13 0.09 0.09 0.94 Total fat, kg 1.1 0.9 1.1 0.9 0.9 1.0 0.9 0.10 0.21 0.72 0.38 0.54 0.72 0.70 Total Fat, % of rib wt 26.7 22.9 24.8 24.0 22.3 25.2 23.0 2.02 0.17 0.82 0.33 0.39 0.80 0.98 Body Fat, kg6 0.2 0.1 0.2 0.1 0.1 0.1 0.1 0.02 0.28 0.31 0.49 0.39 0.39 0.24 Body Fat, % of total 0.00 15.1 15.9 15.3 15.7 15.0 13.9 16.6 0.28 0.33 0.05 0.06 0.02 0.39 fat 4 Sub Fat, kg 0.4 0.4 0.5 0.4 0.4 0.4 0.4 0.03 0.37 0.74 0.52 0.88 0.79 0.67

Sub Fat, % of total fat 39.7 43.2 43.0 41.5 42.7 43.4 41.4 2.72 0.33 0.97 0.58 0.25 0.99 0.98 Inter Fat, kg 0.5 0.4 0.5 0.4 0.4 0.4 0.4 0.05 0.27 0.72 0.52 0.54 0.64 0.79 Inter Fat, % of total 41.7 38.6 41.9 41.0 40.7 41.0 39.7 1.78 0.52 0.97 0.73 0.72 0.68 0.74 fat Lean color7 4.0 3.9 4.0 4.0 4.0 4.0 4.0 0.028 0.78 0.27 0.76 0.54 0.59 0.20 Fat color8 4.0 4.0 3.9 4.0 4.0 4.0 4.0 0.025 0.70 0.35 0.74 0.44 0.36 0.27

1Corn dry distillers grains plus soluble (% of diet DM). 2Corn modified wet distillers grains plus soluble (% of diet DM). 3Contrast P- values: 1- control vs others, 2- DDGS vs MWDGS, 3- linear level, 4- Quadratic level, 5- linear (forms Χ levels), 6- Quadratic (forms Χ levels). 4Longissimus muscle scored subjectively for marbling using a 10-point scale (1= devoid of marbling to 10 = abundant marbling). 5 Grade fat included as a covariate in the model (P<0.001) for lean yield.

6Percentage dissected fat component (Sub = subcutaneous, Inter = intermuscular, body = body cavity fat) in the 10th to 12th rib.

7Lean color: longissimus muscle scored subjectively for lean color using a 5-point scale (1 = very dark red to 5 = very light red).

8Fat color: carcass fat scored subjectively for fat color using a 5-point scale (1 = bright yellow to 5 = white).

Table 3.5. Influence of dietary source and level of DGS on feeding behavior in feedlot cattle fed dry whole corn-based finishing diets1.

Dietary Treatment P-value4 DDGS2 MWDGS3 Item Control 16.7 33.3 50 16.7 33.3 50 SEM 1 2 3 4 5 6 Visits to feeder 40.3 56.3 47.8 46.9 45.9 50.7 49.8 2.63 0.001 0.45 0.02 0.004 0.76 0.14 (VF; events/ d) Number of meals 8.07 7.03 7.56 7.82 8.33 8.25 8.20 0.329 0.55 0.002 0.96 0.32 0.02 0.002 (NM; events/ d) Time at feeder 110 106 117 123 112 137 117 5.6 0.16 0.16 0.02 0.53 0.38 0.02 (TF; min/d) Total daily intake 19.3 16.7 16.3 16.4 16.6 20.2 18.8 0.67 0.01 <0.001 0.14 0.05 <0.001 0.004 (TI; Kg as fed/ d) Meal size 2.63 2.76 2.33 2.23 2.16 2.59 2.43 0.134 0.14 0.66 0.08 0.86 0.43 0.18 (MS; Kg DM) Time per visit 3.07 2.12 2.74 3.21 2.67 2.91 2.70 0.144 0.02 0.53 0.86 0.0003 0.37 0.01 (TV; min/visit) Time per meal 15.1 20.9 18.9 17.0 14.0 17.5 15.4 1.62 0.20 0.01 0.50 0.08 0.07 0.007 (TM; min/meal) Eating rate 178 151 129 134 156 152 160 8.5 0.0006 0.007 0.0009 0.02 0.003 0.09 (ER; g DM/min) 1Number of animals (n = 104). 2Corn dry distillers grains plus soluble (% of diet DM). 3Corn modified wet distillers grains plus soluble (% of diet DM). 4Contrast P- values: 1- control vs others, 2- DDGS vs MWDGS, 3- linear level, 4- Quadratic level, 5- linear (forms Χ levels), 6- Quadratic (forms Χ levels).

CHAPTER 4

INFLUENCE OF FEEDING INCREASING LEVELS OF DRY CORN DISTILLERS GRAINS PLUS SOLUBLES IN WHOLE CORN GRAIN-BASED FINISHING DIETS ON TOTAL TRACT DIGESTION, NUTRIENT BALANCE, AND EXCRETION IN BEEF STEERS

4.1 ABSTRACT

Four cross-bred steers (average BW = 478.1 ± 33 kg) were used in a 4 × 4 Latin

Square design to determine the effects of dietary inclusion level of dry corn distillers grains plus solubles (DDGS) on total tract digestion, nutrient balance, and nutrient excretion. The DDGS were fed at 0 (control), 16.7, 33.3, and 50% of diet DM. All diets contained 10% haylage as a forage source (DM basis), and were formulated to meet or exceed the estimated requirements for CP. Steers were fed experimental diets for ad libitum intake for a 14-d adaptation period followed by 5 d of fecal and urine collection.

Data were analyzed using GLM of SAS; treatment means were compared using contrast statements (control vs. others, and linear and quadratic effects of DDGS inclusion level).

Increasing inclusion level of DDGS in diets from 0% to 50% of diet DM linearly decreased (P < 0.05) total tract DM, OM, and starch digestibility (from 77.8 to 72.9%,

79.1 to 73.7%, and 89.2 to 81.5%, respectively). Daily N and P intakes linearly increased

(P = 0.06 and P = 0.01, respectively) by increasing DDGS inclusion level. Fecal and urinary N, P, S, Mg, and K excretion linearly increased (P < 0.05) when DDGS inclusion level increased. However, Se and Na excretion did not change (P > 0.05) among treatments. Retention (g/d; intake – urinary and fecal excretion) of N did not differ (P >

0.05) among treatments. Retention of P tended (P = 0.07) to linearly increase and

retention of S (g/d) linearly increased (P = 0.004) with increasing DDGS inclusion level.

There were no effects (P > 0.05) of dietary treatment on digestion and retention of Se,

Mg, K, and Na. Plasma P and S concentrations increased (P = 0.03 and 0.01, respectively) with increasing DDGS inclusion level. These data indicate that feeding

DDGS up to 50% diet DM in whole corn grain-based finishing diets does not have a negative effect on nutrient retention. However, total excretion of N, P, S, Mg, and K increased as DDGS inclusion level increased.

Key Words: beef cattle, digestion, distillers grains, nutrient balance

4.2 INTRODUCTION

In recent years the ethanol industry has expanded and inclusion level of distillers

grains plus solubles (DGS) in beef finishing diets has increased. Corn distillers dried

grains with solubles (DDGS) can be fed as a replacement for corn grain as a protein and energy source in beef cattle diets (Klopfenstein et al., 2008). Most research on DGS inclusion in feedlot diets has been conducted with diets containing processed corn (dry- rolled, steam-flaked, and high moisture; Klopfenstein et al., 2008). Less is known about the impact of DGS inclusion in whole corn grain-based diets.

During ethanol production, starch is converted to ethanol and the concentration of CP, fat, fiber, and minerals such as N, P, and S increase by approximately threefold in DDGS compared to the whole corn grain (NRC, 1998). Feeding wet distillers grains plus solubles (WDGS) at 20, 40, and 60% as a replacement for dry rolled corn (DRC) increased linearly the intake of N, P, and S (Spiehs and Varel, 2009). Consequently, urinary and fecal N, P, and S excretion were increased (Cole et al., 2005; Archibeque et

56 al., 2007; Luebbe et al., 2008) potentially increasing the nutrient load to the environment.

Furthermore, the bioavailability of some trace minerals in ruminants might be influenced by binding of minerals to undigested fiber (Spears, 2003) or antagonistic effects of other minerals. Thus, feeding high levels of DDGS could alter dietary bioavailability of minerals in ruminants. There is a lack of information on how the inclusion levels of DDGS affect the bioavailability of these minerals.

Our goal was to examine the effect of feeding increasing inclusion levels of DDGS in whole corn-based finishing diets on total tract digestion, nutrient balance, and excretion with specific emphasis on nitrogen and minerals.

4.3 MATERIALS AND METHODS

4.3.1 Animal Care and Experimental Design

This experiment followed the recommendations of the Canadian Council on Animal

Care (1993) and met the approval of the University of Guelph Animal Care Committee.

Four Angus/ Simmental cross-bred steers (478.1 ± 33 kg) were used in a 4 × 4 Latin

Square design to determine the effects of DDGS inclusion level on total tract digestion, nutrient balance, and nutrient excretion. Dietary treatments were dietary inclusion of 0,

16.5, 33.3 and 50% of diet DM (Table 4.1). All diets contained 10% forage (grass/legume haylage) and were balanced to meet or exceed the estimated requirements for CP, vitamins, and minerals (NRC, 1996). Before the experiment, steers were trained to lead by halter and adapted to a high grain diet over approximately 28 d. Experimental periods were 19 d: 14 d for adaptation to experimental diet and 5 d for fecal and urine collection.

Steers were housed in indoor, individual pens (1.17 × 1.17 m). During the collection

periods, steers were tethered and fitted with fecal collection bags and rubber urine collection funnels. The fecal collection bags and urine collection funnels were attached with adjustable belting to allow for comfortable movement of the steers. Steers had ad libitum access to feed and water. Diets were fed at approximately 120% of ad libitum intake. Feed and orts samples were collected daily during the collection period. Total feces were collected, weighed and a sub-sample (5% of weight; wet basis) obtained twice daily. Urine was collected into acidified containers using a vacuum system and urine was

weighed and a sample collected daily. Containers contained 200 mL of 6 N HCl to

maintain urine pH < 3. Daily fecal, urine, feed, and orts samples were weighed and

composited within animal across sampling period and stored at -20°C until analysis.

Between collection periods, stanchion panels were removed from one side of the pens to

allow enough space for exercise. Blood samples were also collected via jugular

venipuncture on the final day of each collection period before feeding in the morning.

Plasma and serum were harvested by centrifugation (3000 × g) and stored at -20 C until

analysis for plasma Ca, P, K, Na, S, and Se concentrations and for serum glucose and

urea N concentrations.

4.3.2 Laboratory Analysis

Diet, orts and fecal samples were dried in a 55º C oven, ground to pass a 1-mm

screen, and analyzed for DM and ash by standard procedures (AOAC, 1990). Diet, orts

and fecal samples were analyzed for NDF and ADF concentrations as described by

Robertson and Van Soest (1981) using an Ankom fiber analyzer (Ankom Technology

Corp., Fairport, NY). Diet, orts and fecal samples were analyzed for starch

concentrations as described by Hall (2000). Diet, orts, fecal and urinary nitrogen

concentrations were measured by using a modified Kjeldahl digest and Technicon Auto

Analyzer (Thomas et al., 1967). For mineral analysis, feed, orts, fecal and urine samples

were digested and brought to volume with deionized water (Anderson, 1999).

Concentrations of Ca, K, Mg, Na, P, and S were measured in the clear extract supernatant

by inductively coupled plasma optical emission spectrometry (ICP-OES) without further

processing (Anderson, 1999). Selenium in feed, orts, and fecal samples was determined

by spectrofluorimetric analysis (method 974.15; AOAC, 1995). Intake, urine and fecal

excretion, and digestibility were determined for DM, OM, ADF, NDF, starch, N, and P

and nutrient balance (including digestibility) was determined for N, P, Ca, Mg, S, Se, K,

and Na. Nutrient retention was calculated by difference (feed intake – urinary and fecal

excretion).

Serum was analyzed for urea N (Sampson et al., 1980) and glucose (Trinder, 1969) concentrations using kits from Roche Diagnostics (Indianapolis, IN). Plasma was analyzed for plasma Ca, P, K, Na, and S concentrations using ICP-OES as described by

Anderson (1999). For plasma selenium analysis, plasma was diluted with 1% HNO3/ 1%

propanol/ 0.01% TritonX-100/ 0.01% EDTA (1:20) and quantified against a multi- element standard curve by inductively coupled plasma mass spectrometry (ICP-MS).

4.3.3 Statistical Analysis

Data were analyzed as a 4 × 4 Latin Square design using the GLM procedure of SAS.

The model included the effect of animal, period, and DDGS inclusion level (0, 16.7, 33.3, and 50%). Contrast statements were used to determine control vs. other treatments, linear,

and quadratic effects of feeding increasing inclusion levels of DDGS. Significance was

declared at P ≤ 0.05 and a tendency was reported if 0.05 < P ≤ 0.10.

4.4 RESULTS

Daily DM intake (kg/d) did not differ among the treatments (Table 4.2). Fecal excretion of DM (g/d) tended (P = 0.07) to be greater in steers fed DDGS diets compared to controls. Total daily excretion of urine (g/d) increased (P = 0.03) linearly with increasing DDGS inclusion level. Apparently digested DM (g/d) tended (P = 0.09) to linearly decrease with increasing DDGS inclusion level and apparently digested OM

(g/d) decreased linearly (P = 0.05) with increasing inclusion level of DDGS. Apparently digested NDF (g/d) and ADF (g/d) were linearly increased (P = 0.02 and P = 0.03, respectively) with increasing inclusion level of DDGS. A quadratic response (P = 0.04) in apparently digested starch (g/d) was observed because of a greater decrease in starch digestion between treatments as DDGS inclusion increased. Apparently digested DM and

OM (percentage of intake) was greater (P = 0.01) in steers fed the control diet compared to steers fed DDGS diets. Apparently digested DM and OM (percentage of intake) decreased linearly (P = 0.01 and P = 0.02, respectively) with increasing DDGS inclusion.

A linear decrease (P = 0.04) in starch digestibility (percentage of intake) was observed with DDGS addition. Digestibility of NDF and ADF were not influenced (P > 0.05) by increasing DDGS inclusion levels in diets.

Daily intake of N tended linearly increased (P = 0.06) with increasing DDGS inclusion level (Table 4.3). Increasing inclusion level of DDGS in diets linearly increased (P = 0.02) urinary N excretion (g/d). Fecal N excretion (g/d) increased (P =

0.04) in steers fed DDGS compared to controls, and tended (P = 0.07) to increase linearly

60 with increasing DDGS levels in the diets. Total N excretion was increased linearly (P =

0.03) with increasing level of DDGS in diets. There was a quadratic response (P = 0.01) in urinary and fecal N excretion (percentage of N excreted), as urinary and fecal N excretion (percentage of N excreted) decreased and increased, respectively, when DDGS levels increased from 0% to 16.7% of DM diet and then increased and decreased, respectively, when DDGS levels increased from 16.7 to 50% of diet DM. Fecal N excretion (% of N intake) was quadratically affected (P = 0.05) by increasing level of

DDGS and urinary N excretion (% of N intake) tended to have a quadratic effect (P =

0.07). Apparently digested N (g/d) tended (P = 0.06) to linearly increase as DDGS inclusion level increased. Apparently digested N (% of intake) was quadratically affected

(P = 0.05) by increasing inclusion level of DDGS. Retention of N (g/d, % of N intake, and % of digested N) was not influenced by increasing DDGS inclusion level.

Daily P intake was greater (P = 0.04) in steers fed DDGS diets compared to control steers (Table 4.4). Total P intake linearly increased (P = 0.01) in steers fed DDGS diets.

Urinary and fecal P excretion were greater in steers fed DDGS (P = 0.05 and P = 0.04, respectively) and increased linearly (P = 0.01 and P = 0.01, respectively) with increasing

DDGS inclusion level. Urinary and fecal P excretion (% of total P excreted) tended (P =

0.08) to linearly increase and decrease, respectively, with increasing inclusion of DDGS.

However, urinary and fecal P excretion (percentage of P intake) did not differ among treatments. Apparently digested P (g/d) was lower (P = 0.05) in steers fed the control diet than in steers fed DDGS diets and increased linearly (P = 0.02) with increasing level of

DDGS. Apparently digested P (percentage of P intake) was not affected (P > 0.05) by increasing inclusion level of DDGS. Retention of P (g/d) did not differ between steers

feed DDGS and controls but tended to be linearly increase (P = 0.07) with increasing

inclusion level of DDGS. Retention of P as a percentage of P intake and as a percentage

of digested P were not influenced by increasing DDGS inclusion level.

Daily Ca intake did not differ among treatments (Table 4.5). Urinary and fecal Ca

excretion (mg/d) was greater and lesser (P = 0.04 and P = 0.01, respectively),

respectively, in steers fed DDGS compared to controls. Urinary and fecal Ca excretion

(mg/d) increased and decreased linearly (P = 0.01 and P = 0.02, respectively) with

increasing DDGS inclusion level. Urinary and fecal Ca excretion (percentage of total Ca

excreted) tended (P = 0.06) to increase and decrease linearly, respectively, with

increasing inclusion level of DDGS. However, increasing DDGS inclusion level in diets

did not affect urinary and fecal Ca excretion (percentage of Ca intake), apparently

digested Ca (percentage of Ca intake), retention of Ca (g/d), and retention of Ca as a

percentage of Ca intake and as a percentage of digested Ca.

Daily Mg intake (g/d) tended to be greater (P = 0.06) and fecal Mg excretion (g/d) was

greater (P < 0.001) in steers fed DDGS compared to controls (Table 4.6). Daily Mg intake (g/d) and fecal Mg excretion (g/d) was linearly increased (P = 0.02 and P < 0.001,

respectively) with increasing inclusion level of DDGS. There was a quadratic response (P

= 0.04) in urinary Mg excretion (g/d), as urinary Mg excretion increased when DDGS

levels increased from 0% to 16.7% and then decreased when DDGS levels increased

from 16.7 to 50% of diet DM. Urinary and fecal Mg excretion (percentage of Mg

excreted) was linearly decreased and increased (P = 0.001), respectively, with increasing

DDGS inclusion level. There was a quadratic response (P = 0.05) in urinary Mg excretion

(percentage of Mg intake), as excretion increased when DDGS levels increased from 0%

62 to 16.7% and then decreased when DDGS levels increased from 16.7 to 50% of diet DM.

Apparently digested Mg (g/d and percentage of Mg intake) and retention of Mg (g/d, percentage of Mg intake, and as a percentage of digested Mg) were not influenced by increasing DDGS inclusion level.

Daily S intake increased linearly (P < 0.001) as level of DDGS increased (Table 4.7).

Urinary and fecal S excretion (g/d) in steers fed DDGS were greater (P < 0.001 and P =

0.03, respectively) compared to controls. Also, urinary and fecal S excretion (g/d) increased linearly (P < 0.001 and P = 0.01, respectively) with increasing inclusion level of DDGS. Urinary and fecal S excretion (percentage of S excreted) increased and decreased linearly (P < 0.001), respectively, as level of DDGS increased. Moreover, urinary and fecal S excretion (percentage of S intake) increased and decreased linearly (P

= 0.05 and P = 0.001, respectively) with increasing inclusion of DDGS. Increasing inclusion of DDGS in diets linearly increased (P < 0.001 and P = 0.001, respectively) apparently digested S (g/d) and digested S (percentage of S intake). Retention of S (g/d) linearly increased (P = 0.004) by increasing DDGS inclusion level. However, retention of

S as a percentage of S intake and as a percentage of digested S were not affected by increasing inclusion level of DDGS.

Daily Se intake, urinary and fecal Se excretion (g/d), apparently digested Se (g/d), and retention of Se (g/d) were not influenced by increasing DDGS inclusion level in diets

(Table 4.8).

Daily K intake (g/d) was not affected by increasing inclusion level of DDGS (Table

4.9). Fecal K excretion (g/d) was greater (P = 0.004) in steers fed DDGS compared to steers fed the control diet. There was a quadratic response (P = 0.02) in fecal K excretion

(g/d), as fecal K excretion increased as DDGS level increased from 0% to 33.3% and

then decreased when DDGS level increased from 33.3 to 50% of diet DM. Urinary and

fecal K excretion (percentage of total K excreted) was lesser and greater (P = 0.02),

respectively, in steers fed DDGS than steers fed controls. Urinary K excretion

(percentage of total K excreted) was quadratically affected (P = 0.01) with increasing

DDGS inclusion level with the lowest excretion observed in steers fed 33.3% of DDGS.

Fecal K excretion (percentage of K excretion) tended to have a quadratic effect (P = 0.01) with increasing inclusion level of DDGS, as fecal K excretion (percentage of K excretion) increased when DDGS levels increased from 0% to 33.3% of DM increased fecal K excretion (percentage of K excretion) and then decreased when DDGS increased from 33.3% to 50% of diet DM. Apparent K digested (percentage of K intake) tended to decrease when DDGS increased from 0% to 33.3% of DM and then tended to increase

(P = 0.08) when DDGS increased from 33.3% to 50% of DM diet. However, retention of

K (g/d) was not influenced by DDGS inclusion level.

Daily Na intake (g/d), total Na excretion (g/d), and retention of Na (g/d) were not influenced (P > 0.10) by increasing inclusion level of DDGS (Table 4.10). Fecal Na excretion (percentage of Na intake) tended to be greater (P = 0.06) in steers fed the control diet than those fed DDGS diets and was linearly decreased (P = 0.05) by increasing inclusion level of DDGS. Apparently digested Na (percentage of Na intake) tended to be greater (P = 0.06) in steers fed DDGS compared to controls and was linearly increased (P = 0.05) with increasing DDGS inclusion level.

Plasma P and S concentrations (µg/g) were lower in the control group (P = 0.04 and P

= 0.02, respectively) than DDGS groups and increased linearly (P = 0.03 and P = 0.01,

respectively) with increasing DDGS inclusion level (Table 4.11). Plasma Ca (ug/g), Na

(ug/g), and Se (ug/ml) concentrations did not differ among the treatments (P > 0.05).

Serum urea N concentrations (mmol/l) were greater (P = 0.002) in steers fed DDGS diets compared to controls. Serum urea N concentrations (mmol/l) increased linearly (P =

0.0001) with increasing DDGS inclusion level. Serum glucose concentration (mmol/l) was not affected by increasing inclusion level of DDGS in the diets.

4.5 DISCUSSION

The observed linear decrease in apparently digested DM and OM (g/d) with increasing

DDGS inclusion could be because of the increased fat, fiber and /or sulphur and decreased starch concentrations in the diet and the differential digestibilities of these specific components or the effect on digestibility of other components of the diet (Zinn,

1989; Owens et al., 2010). These observations are similar to those of Peter et al. (2000)

who found that inclusion of 20% corn DDGS (DM basis) reduced DM digestibility. In

steers fed finishing diets containing 15% DGS with steam-flaked corn (DM basis),

apparent total tract digested DM and OM were 2.8% lower than the controls (Depenbusch

et al., 2009). Moreover, feeding 25% corn DDGS in steam-flaked corn (SFC) or dry-

rolled corn (DRC) diets reduced apparent total tract digested DM and OM compared to control diets in Holstein steers (May et al., 2009). The reduction in starch digestibility with DDGS inclusion could be due to a change in the ruminal microbial community

(Callaway et al., 2010) because of a change in substrate (fiber vs. starch) available for fermentation.

Similar to our results, others (Spiehs and Varel, 2009; Luebbe et al., 2008) have reported increased N intake and excretion when feeding distillers grains at increased

levels in feedlot diets. Increasing N excretion due to increasing dietary CP concentration

could result in increased ammonia emissions from feedlots (Cole et al., 2005; Archibeque

et al., 2007).

In agreement with our work, others (Luebbe et al., 2008; Spiehs and Varel, 2009) have

reported increased P excretion with increasing DGS inclusion level in feedlot diets. This

could be a concern if not managed correctly because of the risk of soil P saturation and

surface water contamination (Hao et al., 2009). The increased P was primarily in feces,

which agrees with Gibb et al. (2008). In addition, plasma P concentration (µg/g) was

greater in DDGS groups and increased linearly with increasing DDGS inclusion level.

These data emphasize that P excretion increases when feeding high levels of DDGS. It is

not clear why there was a tendency for an increase in P retention with increasing DDGS

inclusion as P supply well exceeded estimated requirements (NRC, 1996).

There is a lack of information on how increased P intake from DGS can influence Ca

utilization and balance. Urinary Ca excretion increased as DDGS increased in the diet

even though Ca intake did not differ between treatments. Reabsorption of Ca in the

kidney might be affected by increasing dietary P or ratio of Ca:P. Others have reported that the bioavailability and the absorption of Ca were decreased by increasing dietary P and fiber concentration (Groff et al., 1995). However, in this study, the total Ca excretion decreased while Ca retention was not influenced as dietary P increased. Overall this suggests that high dietary P in this study did not affect Ca absorption, which supports the results from Spencer et al. (1965) and Heaney (2000).

There may be concerns with feeding high levels of DGS in feedlot diets because of

high S intake and the effects on excess ruminal H2S production and

polioencephalomalacia (Spiehs and Varel, 2009) and a potential reduction in true Se digestibility (Ivancic and Weiss, 2001) which could result in Se deficiencies. Others have reported that bioavailability of selenium was decreased by increasing dietary sulphur

(Pope et al., 1979; van Ryssen et al., 1998; Ivancic and Weiss, 2001; Spears, 2003) because S is antagonistic to Se as they have similar chemical and physical properties.

However, our study indicates that S concentrations likely were not high enough in diets or Se intakes were high enough to avoid any anatagonistic effects of S on Se utilization.

Interestingly, retention of S (g/d) linearly increased by increasing DDGS inclusion level suggesting that S deposition may have increased. However, H2S emissions were not

measured. This may have partly contributed to the linear increase in estimated S retention

as retention was calculated by difference (feed intake – urinary and fecal excretion). It is

not known the physiological impact of an increase in S retention, although it could result

in decreased anti-oxidant activity of glutathione peroxidase enzymes (Murphy and

Quirke, 1997) because high dietary S might decrease Se levels in blood and tissue

(although not observed in the current experiment). Consequently, activity of glutathione

peroxidase enzymes and animal health might be affected by decreased availability of Se,

which may influence cellular antioxidant systems (Ivancic and Weiss, 2001). Further

research on the relationship of high dietary S and Se deposition and activity of

glutathione peroxidase enzymes in tissue is needed.

There is a lack of data in regards to Mg, K, and Na excretion and retention with

increasing distillers grains inclusion in feedlot diets. Total excretion of Mg and K

increased as DDGS inclusion level increased. However, Na excretion did not change

among treatments. There were no effects of dietary treatment on digestion and retention

of Mg, K, and Na. These data might yield useful information about nutrient management

of Mg, K, and Na when increasing DGS is fed in feedlot diets.

Plasma P and S concentrations and serum urea N concentrations increased linearly

with increasing DDGS inclusion level due to increasing the dietary P, S, N intake. This is

likely a suitable indicator of increasing P, S, and N intake and excretion. On the other

hand, plasma Ca, Na, and Se concentrations did not differ among the treatments.

Similarly, Khan et al. (1987) found that plasma Se concentrations did not change when

calves were fed 0.2% or 0.75% S in the diet.

In conclusion, increasing DDGS inclusion levels up to 50% diet DM in whole corn

grain-based finishing diets decreased DM, OM, and starch digestibility (%). On the other

hand, it increased N, P, and S intake and excretion, which would increase mineral loads

in manure and increase ammonia and H2S emissions from feedlots. Increasing DDGS inclusion level increased total excretion of Mg and K but did not influence Na excretion.

Digestion and retention of Mg, K, and Na did not differ among treatments. In addition,

increasing DDGS inclusion level increased concentrations of P, S, and N in blood, which

could be good indicators of increased intake and excretion of these minerals in feedlot

cattle fed high level of DDGS. These findings might help to better understand nutrient

management of these minerals. Moreover, environmental implications of manure should

be considered when cattle are fed high levels of DDGS in feedlot cattle.

Table 4.1. Dietary composition and analysis (DM basis). Dietary Treatment DDGS,% of DM1 Ingredient Control 16.7 33.3 50.0 Dry whole corn grain 80.2 68.3 51.7 35.0 DDGS - 16.7 33.3 50.0 Alfalfa/grass haylage 10.0 10.0 10.0 10.0 Soybean meal 4.3 - - - Urea 0.5 - - - Mineral premix2 2.7 2.7 2.7 2.7 Vitamin/monensin premix3 2.3 2.3 2.3 2.3 Analysis4 DM 73.3 75.7 75.9 77.5 CP, % 13.3 13.4 16.3 20.6 DIP, % CP 57.4 36.5 30.1 26.0 UIP, % CP 42.6 63.5 69.9 74.1 SolP, % CP 23.2 19.0 17.1 15.9 ADF, % 6.9 8.6 10.2 13.8 NDF, % 13.7 17.0 20.7 25.5 Starch, % 52.0 46.0 38.3 25.1 Ash, % 4.9 5.3 5.2 5.9 Ca, % 0.96 0.97 0.90 0.97 P, % 0.30 0.35 0.43 0.53 K, % 0.70 0.69 0.79 0.90 Mg, % 0.13 0.15 0.18 0.21 Na, % 0.18 0.21 0.19 0.20 Se, ppm 0.30 0.35 0.32 0.37 S, % 0.13 0.23 0.33 0.45 1Corn dry distillers grains plus solubles. 2Contains 79.5% limestone, 16% salt, 4.5% trace mineral mix (contains 153,013 mg/kg Zn, 122,445 mg/kg Mn, 30,598 mg/kg Cu, 27,100 mg/kg Fe, 368 mg/kg Co, 1,531 mg/kg I, 235 mg/kg Se; DM basis). 3Contains 93.63% dry ground corn, 5.66% vitamin premix (4,400,000 IU/kg vitamin A, 1,100,000 IU/kg vitamin D, and 7,700 IU/kg vitamin E), 0.71% monensin premix (200 g monensin/kg), and 512.5 mg/kg thiamine (98%; DM basis). 4Average of diet samples as % of DM basis except Se concentration as ppm.

Table 4.2. Influence of dietary level of DDGS on DM, OM, CP, ADF , NDF and starch intake and apparent digestibility in feedlot cattle fed dry whole corn-based finishing diets. Dietary Treatment Contrast P-value DDGS1 Item Control 16.7 33.3 50 SEM Control vs. Linear Quadratic levels others levels DM Intake, kg/d 10.9 11.5 10.6 10.2 540.1 0.90 0.28 0.38 DM Feces ,g/d 2407.7 3053.7 2928.4 2752.5 198.35 0.07 0.35 0.08 Total Urine/d 7436 7781 8573 11643 1072 0.18 0.03 0.25 Apparent digested, g/d DM 8298.8 8233.9 7055.9 7424.0 413.07 0.18 0.09 0.62 OM 8202.0 8190.5 7394.3 7139.7 361.45 0.18 0.05 0.75 ADF 407.7 545.8 515.2 965.6 124.98 0.11 0.03 0.26 NDF 789.5 946.4 1123.7 1586.9 192.14 0.10 0.02 0.46 Starch 5133.5 4649.5 3716.3 2157.4 203.62 0.01 <0.001 0.04 Digestibility, % DM 77.8 73.6 72.3 72.9 1.07 0.01 0.01 0.07 OM 79.1 74.6 73.3 73.7 1.17 0.01 0.02 0.08 ADF 52.5 56.1 49.1 63.0 3.64 0.43 0.18 0.21 NDF 52.2 48.6 52.1 58.8 3.76 0.83 0.22 0.22 Starch 89.2 85.3 84.4 81.5 2.09 0.06 0.04 0.81 1Corn dry distillers grains plus soluble (% of diet DM).

Table 4.3. Influence of dietary level of DDGS on nitrogen balance in feedlot cattle fed dry whole corn-based finishing diets. Dietary Treatment DDGS1 Contrast P-value Control Linear Quadratic Control 16.7 33.3 50 Item SEM vs. others levels levels N intake, g/d 193 223 225 273 23.19 0.13 0.06 0.70 N excretion, g/d Feces 63.0 74.0 82.6 76.2 4.86 0.04 0.07 0.12 Urine 46.3 38.7 51.1 69.8 5.98 0.35 0.02 0.07 Total 109.3 112.7 133.6 145.9 9.99 0.11 0.03 0.67 N excretion, % of total N excretion Feces 58.9 67.0 64.2 52.8 2.48 0.44 0.11 0.01 Urine 41.1 33.0 35.8 47.2 2.48 0.44 0.11 0.01 N excretion, % of N intake Feces 32.7 34.0 39.5 28.2 2.58 0.69 0.53 0.05 Urine 23.7 17.8 22.3 26.2 2.25 0.57 0.28 0.07 Apparent N digested g/d 130.2 149.2 142.0 197.2 18.74 0.18 0.06 0.37 % of N intake 67.3 66.0 60.5 71.8 2.58 0.69 0.53 0.05 N retained g/d 84.0 110.5 90.9 127.4 15.69 0.21 0.17 0.76 % of N intake 43.7 48.2 38.1 45.6 4.03 0.95 0.82 0.73 % of digested N 64.8 72.6 61.9 63.4 4.47 0.83 0.48 0.51 1Corn dry distillers grains plus soluble (% of diet DM).

Table 4.4. Influence of dietary level of DDGS on phosphorus balance in feedlot cattle fed dry whole corn-based finishing diets. Dietary Treatment Contrast P-value DDGS1 Item Control Linear Control 16.7 33.3 50 SEM Quadratic levels vs. others levels P intake, g/d 31.3 38.4 42.2 54.5 4.44 0.04 0.01 0.57 P excretion, g/d Feces 16.3 18.3 20.8 24.2 1.57 0.04 0.01 0.67 Urine 2.6 3.6 4.9 7.3 0.91 0.05 0.01 0.44 Total 18.9 22.0 25.6 31.6 1.55 0.01 0.001 0.39 P excretion, % of total P excretion Feces 86.1 82.0 80.5 75.8 3.42 0.14 0.08 0.93 Urine 13.9 18.0 19.5 24.2 3.42 0.14 0.08 0.93 P excretion, % of P intake Feces 52.7 51.6 52.5 43.9 4.24 0.52 0.23 0.41 Urine 9.2 10.0 10.7 14.5 2.49 0.42 0.19 0.56 Apparent P digested g/d 15.1 20.1 21.4 30.3 3.17 0.05 0.02 0.56 % of P intake 47.3 48.4 47.5 56.1 4.24 0.52 0.23 0.41 P retained g/d 12.4 16.5 16.5 23.0 3.16 0.14 0.07 0.72 % of P intake 38.1 38.4 36.8 41.5 5.27 0.90 0.73 0.69 % of digested P 78.8 67.6 78.0 73.9 9.14 0.61 0.92 0.71 1Corn dry distillers grains plus soluble (% of diet DM).

Table 4.5. Influence of dietary level of DDGS on calcium balance in feedlot cattle fed dry whole corn-based finishing diets. Dietary Treatment Contrast P-value DDGS1 Item Control vs. Linear Quadrati Control 16.7 33.3 50 SEM Others levels c levels Ca intake, g/d 106 107 86 83 12.19 0.32 0.15 0.88 Ca excretion, mg/d Feces 75351 65577 61014 56651 2467 0.01 0.008 0.29 Urine 35 409 1103 1749 309 0.04 0.02 0.65 Total 75386 65986 62117 58401 2594 0.01 0.01 0.30 Ca excretion, % of total Ca excretion Feces 100 99 98 97 1.01 0.15 0.06 0.62 Urine 0.0 0.7 1.7 3.3 1.01 0.15 0.06 0.62 Ca excretion, % of Ca intake Feces 70.0 59.9 73.6 69.5 9.12 0.81 0.76 0.73 Urine -0.1 0.4 1.3 2.6 0.96 0.20 0.09 0.70 Apparent Ca digested mg/d 30643 40967 24566 25892 11291 0.99 0.53 0.68 % of Ca intake 30.0 40.1 26.4 30.5 9.12 0.81 0.76 0.73 Ca retained mg/d 30608 40558 23463 24143 11539 0.92 0.47 0.67 % of Ca intake 30.1 39.7 25.1 28.0 10.04 0.94 0.63 0.73 % of digested Ca 101.1 99.5 92.5 90 5.64 0.29 0.16 0.94 1Corn dry distillers grains plus soluble (% of diet DM).

Table 4.6. Influence of dietary level of DDGS on magnesium balance in feedlot cattle fed dry whole corn-based finishing diets.

Dietary Treatment Contrast P-value DDGS1 Control vs. Linear Quadratic Item Control 16.7 33.3 50 SEM Others levels levels Mg intake, g/d 14.0 16.9 18.4 23.8 2.13 0.06 0.02 0.58 Mg excretion, g/d Feces 8.4 10.5 12.8 16.7 0.70 0.001 <0.001 0.24 Urine 1.2 1.9 1.0 0.9 0.16 0.59 0.06 0.04 Total 9.5 12.3 13.8 17.6 0.74 0.001 <0.001 0.53 Mg excretion, % of total Mg excretion Feces 87.3 84.3 93.0 94.7 1.22 0.05 0.001 0.10 Urine 12.7 15.7 7.0 5.3 1.22 0.05 0.001 0.10 Mg excretion, % of Mg intake Feces 62.1 62.8 70.4 71.4 5.79 0.40 0.22 0.97 Urine 8.9 12.5 5.4 4.0 0.99 0.21 0.003 0.05 Apparent Mg digested g/d 5.6 6.4 5.6 7.1 1.66 0.69 0.63 0.85 % of Mg 37.9 37.2 29.6 28.6 5.79 0.40 0.22 0.97 intake Mg retained g/d 4.4 4.5 4.6 6.2 1.58 0.72 0.47 0.65 % of Mg intake 29.0 24.8 24.3 24.6 6.34 0.56 0.64 0.73 % of digested Mg 73.9 64.4 81.7 83.4 5.93 0.72 0.14 0.38 1Corn dry distillers grains plus soluble (% of diet DM).

Table 4.7. Influence of dietary level of DDGS on sulphur balance in feedlot cattle fed dry whole corn-based finishing diets. Dietary Treatment Contrast P-value DDGS1 Control vs. Linear Quadratic Item Control 16.7 33.3 50 SEM others levels levels S intake, g/d 13.44 25.62 30.32 45.47 3.61 0.003 <0.001 0.70 S excretion, g/d Feces 5.09 5.89 6.85 8.30 0.61 0.03 0.01 0.61 Urine 2.85 6.18 9.01 14.17 0.91 <0.001 <0.001 0.35 Total 7.94 12.07 15.86 22.46 1.44 0.002 <0.001 0.42 S excretion, % of total S excretion Feces 65.3 50.3 45.7 38.0 2.25 <0.001 <0.001 0.15 Urine 34.7 49.7 54.3 62.0 2.25 <0.001 <0.001 0.15 S excretion, % of S intake Feces 38.8 25.9 22.9 18.6 2.40 0.001 0.001 0.12 Urine 21.4 28.0 29.7 32.9 3.29 0.06 0.05 0.62 Apparent S digested g/d 8.4 19.7 23.5 37.2 3.1 0.002 <0.001 0.72 % of S intake 61.2 74.1 77.1 81.5 2.4 0.001 0.001 0.12 S retained g/d 5.5 13.6 14.5 23.0 2.63 0.009 0.004 0.93 % of S intake 39.7 46.0 47.4 48.6 5.3 0.26 0.28 0.65 % of digested S 64.0 59.0 61.5 59.4 6.6 0.62 0.72 0.83 1Corn dry distillers grains plus soluble (% of diet DM).

Table 4.8. Influence of dietary level of DDGS on selenium balance in feedlot cattle fed dry whole corn-based finishing diets. Dietary Treatment Contrast P-value DDGS1 Item Control 16.7 33.3 50 SEM Control vs. others Linear levels Quadratic levels Se intake, mg/d 2.9 3.7 3.0 3.5 0.57 0.48 0.64 0.83 Se excretion, mg/d Feces 1.3 1.3 1.2 1.4 0.12 0.66 0.40 0.52 Urine 0.4 0.5 0.3 0.5 0.09 0.93 0.97 0.61 Total 1.7 1.8 1.6 1.9 0.19 0.75 0.58 0.52 Se excretion, % of total Se excretion Feces 73.8 74.5 79.4 77.2 3.24 0.42 0.34 0.67 Urine 26.2 25.5 20.6 22.8 3.24 0.42 0.34 0.67 Se excretion, % of Se intake Feces 46.8 37.1 42.4 41.3 6.09 0.39 0.70 0.51 Urine 16.5 14.4 11.1 15.0 3.74 0.51 0.66 0.45 Apparent Se digested mg/d 1.7 2.4 1.8 2.1 0.53 0.50 0.76 0.71 % of Se intake 53.2 62.9 57.6 58.7 6.09 0.39 0.70 0.51 Se retained mg/d 1.2 1.9 1.5 1.6 0.56 0.54 0.78 0.67 % of Se intake 36.7 48.5 46.5 43.7 9.48 0.42 0.67 0.47 % of digested Se 66.1 75.9 81.1 71.0 8.85 0.37 0.63 0.30 1Corn dry distillers grains plus soluble (% of diet DM).

Table 4.9. Influence of dietary level of DDGS on potassium balance in feedlot cattle fed dry whole corn-based finishing diets.

Dietary Treatment Contrast P-value DDGS1 Control vs. Linear Quadrat Item Control 16.7 33.3 50 SEM others levels ic levels K intake, g/d 73.6 77.9 78.2 93.6 8.11 0.34 0.15 0.52 K excretion, g/d Feces 8.8 12.7 13.9 12.6 0.84 0.004 0.02 0.02 Urine 22.8 22.0 21.4 29.9 2.39 0.57 0.10 0.10 Total 31.6 34.7 35.3 42.5 2.65 0.10 0.03 0.48 K excretion, % of total K excretion Feces 28.4 36.7 40.9 30.5 2.18 0.02 0.33 0.01 Urine 71.6 63.3 59.1 69.5 2.18 0.02 0.33 0.01 K excretion, % of K intake Feces 12.0 18.6 20.0 13.2 3.21 0.20 0.74 0.08 Urine 31.3 28.9 27.2 32.9 2.21 0.54 0.77 0.11 Apparent K digested g/d 64.8 65.2 64.3 81.0 8.49 0.61 0.26 0.37 % of K intake 88.0 81.4 80.0 86.8 3.21 0.20 0.74 0.08 K retained g/d 42.0 43.1 42.9 51.1 6.97 0.66 0.42 0.63 % of K intake 56.7 52.5 52.8 53.9 4.60 0.52 0.71 0.59 % of digested K 64.3 62.3 65.0 62.3 3.87 0.82 0.86 0.93

1Corn dry distillers grains plus soluble (% of diet DM).

Table 4.10. Influence of dietary level of DDGS on sodium balance in feedlot cattle fed dry whole corn-based finishing diets.

Dietary Treatment Contrast P-value DDGS1 Item Control 16.7 33.3 50 SEM Control vs. others Linear levels Quadratic levels Na intake, g/d 18.6 23.0 18.1 18.8 2.74 0.67 0.75 0.53 Na excretion, g/d Feces 3.9 3.2 3.4 2.9 0.54 0.28 0.27 0.93 Urine 6.4 5.7 6.2 7.9 1.28 0.88 0.41 0.39 Total 10.3 8.9 9.7 10.7 1.45 0.77 0.75 0.43 Na excretion, % of total Na excretion Feces 39.3 35.3 39.3 26.8 5.48 0.42 0.22 0.47 Urine 60.7 64.7 60.7 73.2 5.48 0.42 0.22 0.47 Na excretion, % of Na intake Feces 23.0 18.2 20.2 15.3 1.93 0.06 0.05 0.97 Urine 33.7 28.0 32.4 42.6 6.11 0.93 0.30 0.24 Apparent Na digested g/d 14.7 19.8 14.6 16.0 2.31 0.45 0.92 0.44 % of Na intake 77.0 81.8 79.8 84.7 1.93 0.06 0.05 0.97 Na retained g/d 8.3 14.1 8.4 8.1 1.99 0.43 0.51 0.17 % of Na intake 43.3 53.8 47.4 42.1 6.11 0.55 0.73 0.24 % of digested Na 56.4 61.3 59.6 49.9 8.91 0.96 0.61 0.44 1Corn dry distillers grains plus soluble (% of diet DM).

Table 4.11. Influence of dietary level of DDGS on nitrogen balance in feedlot cattle fed dry whole corn-based finishing diets. Dietary Treatment DDGS1 Contrast P-value Control vs. Linear Quadratic Control 16.7 33.3 50 Item SEM others levels levels Plasma Ca, µg/g 92 94 93 95 1.4 0.39 0.39 0.96 P, µg/g 116 134 129 146 7 0.04 0.03 0.99 K, µg/g 125 135 130 134 3 0.05 0.15 0.32 Na, µg/g 3138 3100 3088 3113 31 0.34 0.55 0.36 S, µg/g 888 915 924 940 10 0.02 0.01 0.60 Se, µg/mL 0.089 0.085 0.085 0.095 0.004 0.93 0.29 0.12 Serum Glucose, mmol/L 4.15 4.19 4.50 4.29 0.21 0.50 0.47 0.57 Urea N, mmol/L 3.09 3.10 3.83 5.05 0.15 0.002 <0.001 0.01 1Corn dry distillers grains plus soluble (% of diet DM).

CHAPTER 5

INFLUENCE OF FEEDING INCREASING LEVELS OF DRY OR MODIFIED WET CORN DISTILLERS GRAINS PLUS SOLUBLES IN WHOLE CORN GRAIN-BASED FINISHING DIETS ON PANCREATIC MASS, AND Α- AMYLASE AND TRYPSIN ACTIVITY IN FEEDLOT CATTLE

5.1 ABSTRACT

One hundred and fourteen cross-bred steer calves and seventeen heifers (BW = 357.2

(control), 16.7, 33.3, and 50% of ration DM using dry (DDGS) or modified wet (50%

DM; MWDGS) product. Data were analyzed using GLM of SAS; treatment means were compared using contrast statements (control vs. other treatments, DDGS vs. MWDGS, inclusion levels of DGS (linear, quadratic), and interactions between form and linear and quadratic inclusion levels). There were no effects (P ≥ 0.27) of dietary treatment on pancreatic mass (g and g/kg BW), however pancreatic protein concentration (mg/g) tended to increase linearly (P = 0.06) with increasing inclusion level of DGS. Pancreatic concentration of α-amylase activity (U/g) tended to increase (P = 0.09) in cattle receiving

DGS vs. control. Pancreatic concentration of trypsin activity (U/g) tended to be greater (P

= 0.06) in cattle receiving DGS vs. control and tended to be greater (P = 0.09) in cattle fed MWDGS than in cattle fed DDGS. Moreover, pancreatic concentration of trypsin activity (U/g) increased linearly (P = 0.01) with increasing inclusion of DGS. These data

indicate that pancreatic concentration of α-amylase and trypsin activity may be influenced by the form and inclusion level of DGS in whole corn grain-based finishing diets.

Key Words: beef cattle, pancreas, α-amylase, trypsin, distillers grains

5.2 INTRODUCTION

The utilization of ethanol byproducts in beef cattle diets has increased (Lodge et al.,

1997a). During the ethanol production process, the starch is fermented to ethanol resulting in concentrating of other nutrients (Klopfenstein et al., 2008). Thus, increasing inclusion level of DGS in the diet increases the dietary protein and decreases the dietary starch. However, there is lack of information on how these changes affect pancreatic digestive enzyme activity.

The function of the exocrine pancreas is to partially hydrolyse digesta in the small intestine (SI). For example, pancreatic α-amylase and trypsin are responsible for partial initial hydrolysis of starch and protein, respectively (Swanson and Harmon, 2002). There is lack of information on effects of DGS on pancreatic enzymes. Corn processing methods can affect starch digestion. For example, feeding steam-flaked (SFC) increased the ruminal and postruminal starch digestion compared to dry-rolled corn (DRC); moreover, feeding SFC reduced postruminal digestibility when ruminal starch digestibility increased compared to DRC (Zinn et al., 2002). The activity of pancreatic enzymes may respond differently based on the absence or presence of starch (Swanson et al., 2002a, 2004). Pancreatic α-amylase concentration might be influenced by quality and quantity of dietary protein (Harmon, 1992; Swanson et al., 2008). Pancreatic trypsin secretion was not affected by different dietary protein sources (Khorasani et al., 1990), or

postruminal casein infusion (Richards et al., 2003; Swanson et al., 2004). However,

pancreatic trypsin activity increased linearly with increasing dietary CP concentration up

to 15% of DM (Swanson et al., 2008). The response of pancreatic trypsin activity might

be different when animals are fed practical diets above the protein requirement. Therefore

the objectives were to determine the effect of inclusion level and form of distillers grains plus solubles (DGS) on pancreatic mass, α-amylase, and trypsin activity in feedlot cattle fed whole corn grain-based finishing diets.

5.3 MATERIALS AND METHODS 5.3.1. Animal Care and Experimental Design

This experiment followed the recommendations of the Canadian Council on Animal

Care (1993) and met the approval of the University of Guelph Animal Care Committee.

One hundred and fourteen cross-bred steer calves and seventeen heifers weighing approximately 357.2 ± 5.8 kg were randomly assigned to 14 pens (each treatment had 19 calves – 15 barn 1, 4 barn 2), and used in a completely randomized block design with a 2

× 3 factorial arrangement of treatments plus a control to determine the effect of distillers grain plus solubles form and level on feedlot performance, carcass characteristics, and meat quality. Treatments (Table 3.1) consisted of control, 16.7%, 33.3%, and 50% of DM from dried distillers grains plus solubles (DDGS), and 16.7%, 33.3%, 50% of DM from modified wet distillers grains plus soluble (MWDGS). Calves were fed diets as a total mixed diet once daily. All diets contained 10% haylage as a forage source, and all diets were formulated to meet or exceed the estimated requirements for CP, minerals and vitamins according to NRC (1996). Calves were fed a corn silage/haylage-based diet for at least two weeks before starting the experiment to adapt to facilities. Calves were

adapted to dietary treatments over the first 28 d on experiment. At the end of the trial,

cattle were weighed in the morning over two consecutive days.

5.3.2 Feed intake measurements and sample collection

For cattle in barn 1, radio frequency ID tags were placed in the right ear prior to the

experiment. There were four electronic feeding stations (Insentec,B.V., Marknesse, the

Netherlands) as described by Mader et al. (2009) for each pen allowing for monitoring of

individual feed intake. In barn 2, Calan gates (American Calan, Inc., Northwood, NH,

USA) were used to measure individual feed intake. The animal's body weights were

taken every 28 d.

The slaughter day was based on when the backfat thickness (BF; between the 12th

and13th rib) reached approximately 10 mm as assessed through real time ultrasound

(Aloka SSD-500 ultrasound unit; Cormetrics Medical Systems, Wallingford, CT, USA).

The calves were slaughtered at the University of Guelph Meat Laboratory. Pancreas

Weight was recorded at slaughter. Pancreatic tissues were collected directly after

slaughter, rinsed in ice-cold saline, snap-frozen in liquid N2, and stored at -80°C until

analysis.

5.3.3 Laboratory analyses

Diet samples were collected weekly and frozen at -20о C until further analysis at the

Agri-Food Laboratories Inc. (Guelph, ON, Canada), and then dried in a 55о C oven, ground to pass a 1- mm screen, and analyzed for DM and ash by standard procedures

(Association of Official Analytical Chemists guidelines (AOAC, 1990; Method 930.15).

Diet nitrogen concentrations were determined using a Leco N analyzer (Leco

Corporation, St. Joseph, MI, USA) and percent crude protein was calculated by

multiplying percent N × 6.25. Neutral detergent fiber (NDF) concentration was

determined by the method of Robertson and Van Soest (1981) using an Ankom fiber

analyzer (Ankom Technology Crop., Fairport, NY, USA). Starch was calculated

according to NRC (1996). Phosphorus concentrations were determined as described by

AOAC (1990). Pancreatic tissue (1 g) was homogenized in 0.9% NaCl (9 mL) using a polytron (Brinkmann Instruments Inc, Westbury, NY). Activity of α-amylase was determined using the procedure of Wallenfels et al., (1978) using a kit from Teco

Diagnostics (Anaheim, CA). Trypsin activity was assayed by using the method described by Geiger and Fritz, (1986) after activation with 100 units (U)/L enterokinase (Glazer and

Steer., 1977; Swanson et al., 2002a). One U of enzyme activity equals 1 μmole product

produced per min. Enzyme activity data are expressed as unit per gram of pancreas, unit per gram protein, kilo unit per pancreas, and unit per kilogram of body weight. Analyses were adapted for use on a PowerWave XS microplate spectrophotometer (BioTek

Instruments Inc., Winooski, VT, USA).

5.3.4 Statistical Analysis

Data were analyzed as a completely randomized block design using the GLM procedure of SAS. The model included the effects of block (barn), distillers grain plus solubles form (dry vs. modified wet), inclusion level (0, 16.7, 33.3, and 50%) and the

interaction. Orthogonal contrasts statements were used to determine control vs. other treatments, DDGS vs. MWDGS, linear and quadratic effects of inclusion level DGS, and the interaction between linear and quadratic effects of inclusion level and forms of DGS.

Significance was declared at P ≤ 0.05 and a tendency was reported if 0.05 < P ≤ 0.10.

5.4 RESULTS

Pancreatic weight (g and g/kg BW) did not differ (P ≥ 0.27) among dietary

treatments (Table 5.1). Pancreatic protein (mg/g) tended to be greater (P = 0.10) in cattle

fed DGS compared to cattle fed the control diet. Pancreatic protein (mg/g) tended to

increase linearly (P = 0.06) with increasing DGS inclusion levels in the diets. However,

pancreatic protein (g/pancreas, and mg/ Kg of BW) did not differ (P ≥ 0.52) among dietary treatments. Pancreatic α-amylase activity (U/g) tended to be greater (P = 0.09) in cattle fed DGS than cattle fed the control diet (Table 5.2). Pancreatic α-amylase activity

(U/g of protein, kU/pancreas, and U/kg of BW) did not differ (P ≥ 0.27) among dietary treatments. Pancreatic trypsin activity (U/g) tended to be greater (P = 0.06) in cattle fed

DGS compared to cattle fed the control diet. Pancreatic trypsin activity (U/g) tended to be greater (P = 0.09) in cattle fed MWDGS than in cattle fed DDGS. There was a linear increase (P = 0.01) in pancreatic trypsin activity (U/g) with increasing inclusion level of

DGS. However, pancreatic trypsin activity (U/g of protein, kU/pancreas, and U/kg of

BW) did not differ (P ≥ 0.16) among dietary treatments. The ratio of pancreatic α- amylase: trypsin did not differ (P ≥ 0.12) among dietary treatments.

5.5 DISCUSSION

Pancreatic weight (g, and g/kg BW) did not differ among dietary treatments, which agrees with Swanson et al. (2008) who reported increasing dietary protein from 8.8 up to15.4% CP did not affect the pancreatic mass in beef steers. However, in non-ruminants,

Hashimoto and Hara (2004) found that feeding high dietary protein increased pancreatic mass in rats compared to those fed low dietary protein. The reason we did not see an

effect may be because the dietary protein was over the suggested requirements (NRC,

1996) for all treatments.

In previous research, feeding a diet containing high quality protein (19% casein)

increased synthesis of pancreatic α-amylase compared to a diet containing poor-quality

protein (zein) in rats (Johnson et al., 1977). In addition, the concentration of α-amylase was increased by feeding diets high in non-branched-chain AA compared to a low dietary

protein (Hashimoto and Hara., 2004). In the current study, the quality of protein was not

the reason of greater concentration of pancreatic α-amylase activity (U/g) in cattle fed

DGS than cattle fed the control diet. In addition, the dietary protein of the 16.7% DGS

diet was similar to the dietary protein in the control; however, pancreatic α-amylase

activity (U/g) was greater in 16.7% of DGS compared to the control diet. In addition,

corn processing plays an important role in determination of postruminal starch flow,

which could result in different responses in pancreatic α-amylase activity. For example,

the proportion of dietary starch passing to the SI in dry-rolled and whole corn diets was

greater than when high-moisture corn diets were fed (Owens et al., 1986). Therefore,

these results could be due to the differences in SI starch flow between the control (whole

corn grain) and 16.7% DGS diet.

In this study, although the nutrients flowing to the small intestine (SI) were not

measured, there likely was a decrease in intestinal starch flow with increasing DGS

inclusion. These results support the findings from previous studies (Kreikemeier et

al.,1990; Walker and Harmon, 1995; Swanson et al., 2002a) where decreasing starch flow

to the small intestine resulted in increased pancreatic α-amylase concentration (U/g) or

secretion (U/hour). Similarly, in a study by Swanson et al. (2008), α-amylase

concentration increased linearly with decreasing dietary starch. Also, increasing the

dietary protein increased pancreatic α-amylase activity (U/g, U/g of protein, U/pancreas,

and U/kg of BW) in feedlot steers fed 8.8, 11.0, 13.2, and 15.4% CP (Swanson et al.,

2008). Past research also has indicated that increasing intestinal protein flow may increase pancreatic α-amylase concentration or secretion (Richards et al., 2003).

Although dietary protein increased with increasing DGS inclusion level in the current experiment, there were no significant differences in pancreatic α-amylase activity among the animals fed DGS. This might be because pancreatic α-amylase activity could not achieve any further improvement when the dietary protein concentration increases above requirements (NRC, 1996). These results suggest that the concentration of the dietary protein might affect the pancreatic α-amylase concentration. Moreover, there was no need for further improvement in pancreatic α-amylase activity when dietary starch decreased with high levels of DGS.

Pancreatic trypsin activity (U/g) was greater in cattle fed MWDGS than in cattle fed

DDGS. This might be because drying the DGS may influence protein digestion and the signalling pathways involved in regulating pancreatic exocrine function. In addition, heat treatment of protein increases ruminal escape protein (Cleale et al., 1987) and potentially decreases microbial protein flow. In a study by Nakamura et al. (1994), heat damage increased ruminal escape protein from 63.2 to 67.8% in DDG. Also, availability of some of AA in escape protein was decreased by heat damage due to cross-links formation between peptide chains (Mauron, 1981). Therefore feeding high levels of DDG decreased lysine concentration in duodenal digesta (Willms et al., 1991). Lysine was decreased from 2.13 to 1.69% in DDG by heat damage and became the first limiting AA. Thus, the

form of DGS and the balance of AA might affect the concentration of pancreatic trypsin

activity. In the current study, the activity of pancreatic trypsin increased with increasing

inclusion level of DGS. These data support the results reported by Swanson et al. (2008)

who found that increasing dietary protein to 14.3% of the diet DM linearly increased

pancreatic trypsin concentration. Similarly, the activity of trypsin increased when casein

was infused into the duodenum of sheep (Ben-Ghedualia et al., 1982). However, postruminal protein infusion did not affect trypsin secretion in the presence of starch

(Richards et al., 2003). Moreover, Walker and Harrmon (1995) reported that protease concentrations decreased when starch was infused in steers fed a 98% fescue-hay diet (12 and 12.5% CP). In sheep, abomasal starch or starch and casein infusion did not influence protease activities (Wang and Taniguchi., 1998). Therefore, decreasing dietary starch with increasing DGS inclusion levels might decrease the negative effect of starch on pancreatic trypsin activity. However, in the current study, there were no observed

differences in the content of pancreatic trypsin activity (kU/pancreas, and U/kg of BW)

suggesting that feeding DGS may have minimal effects on the capacity to produce and

secrete trypsin activity into the small intestine as well as pancreatic tissue mass.

In conclusion, although feeding DDGS and MWDGS up to 50% of diet DM did not

affect pancreatic mass, increasing DGS inclusion level increased linearly the

concentration of pancreatic protein. Feeding DGS diets increased the concentration of

pancreatic α-amylase activity (U/g) and trypsin activity (U/g) compared to the control

diet. In addition, the concentration of trypsin activity (U/g) was greater in cattle fed

MWDGS compared to those fed DDGS. Moreover, increasing the inclusion level of DGS

linearly increased the concentration of pancreatic trypsin activity (U/g). Therefore, these

88 results indicate that pancreatic concentration of α-amylase and trypsin activity may be affected by form and inclusion level of DGS in whole corn grain-based finishing diets.

However, total tissue content of α-amylase and trypsin activity was not influenced by

DGS inclusion.

Table 5.1. Effect of inclusion level and form of distillers grains plus solubles (DGS) on pancreatic mass and protein concentration in feedlot cattle. Dietary Treatment P-value3 DDGS1 MWDGS2 Item Control 16.7 33.3 50 16.7 33.3 50 SEM 1 2 3 4 5 6 Pancreatic weight g 527 452 536 481 517 479 487 29 0.27 0.84 0.33 0.66 0.83 0.88 g/kg BW 0.907 0.812 0.892 0.869 0.890 0.829 0.850 0.047 0.31 0.97 0.35 0.33 0.66 0.87 Pancreatic protein mg /g 108 110 114 114 112 117 114 2.8 0.10 0.46 0.06 0.33 0.66 0.77 tissue g/pancreas 57.3 50.3 61.1 55.5 58.2 56.4 56.9 4.16 0.82 0.65 0.94 0.93 0.73 0.94 mg/kg of 97.6 88.9 102 100 100 97.5 97.2 7.37 0.99 0.86 0.73 0.86 0.52 0.95 BW 1Corn dry distillers grains plus solubles. 2Corn modified wet distillers grains plus solubles. 3Contrast P- values: 1- control vs others, 2- DDGS vs MWDGS, 3- linear level, 4- Quadratic level, 5- linear (forms Χ levels), 6-Quadratic (forms Χ levels).

Table 5.2. Effect of inclusion level and form of distillers grains plus solubles (DGS) on α-amylase, and trypsin activity in feedlot cattle. Dietary Treatment P-value3 DDGS1 MWDGS2 Item Control 16.7 33.3 50 16.7 33.3 50 SEM 1 2 3 4 5 6 α-Amylase U/g tissue 223 273 261 260 240 273 254 19.0 0.09 0.57 0.13 0.16 0.47 0.35 U /g of Protein 2065 2663 2271 2218 2149 2340 2299 226.3 0.28 0.51 0.55 0.27 0.91 0.35 kU/pancreas 119 124 140 126 126 133 125 11.9 0.42 0.83 0.47 0.36 0.49 0.51 U /Kg of BW 204 220 237 229 216 227 218 23.0 0.39 0.65 0.39 0.52 0.38 0.49 Trypsin U/g 5.4 5.7 5.2 6.1 5.5 6.3 6.3 0.2 0.06 0.09 0.01 0.50 0.72 0.93 U/g of protein 50 55 45 53 49 54 60 4.2 0.58 0.39 0.30 0.44 0.20 0.93 kU/pancreas 2.8 2.6 2.8 2.9 2.9 3.0 3.0 0.2 0.70 0.25 0.36 0.71 0.84 0.70 U/kg of BW 4.9 4.6 4.8 5.4 5.0 5.2 5.4 0.3 0.58 0.36 0.16 0.34 0.81 0.85 Amylase:trypsin 43 49 60 48 49 44 41 5.9 0.32 0.15 0.62 0.16 0.12 0.22 1Corn dry distillers grains plus solubles. 2Corn modified wet distillers grains plus solubles. 3Contrast P- values: 1- control vs others, 2- DDGS vs MWDGS, 3- linear level, 4- Quadratic level, 5- linear (forms Χ levels), 6- Quadratic (forms Χ levels).

CHAPTER 6

INFLUENCE OF FEEDING INCREASING LEVELS OF DRY OR MODIFIED WET CORN DISTILLERS GRAINS PLUS SOLUBLES IN WHOLE CORN GRAIN-BASED FINISHING DIETS ON HEPATIC AND RENAL MASS, AND GLUTATHIONE PEROXIDASE AND UREA CYCLE ENZYME ACTIVITIES IN FEEDLOT CATTLE.

6.1 ABSTRACT

Forty-two cross-bred steers (BW = 357 ± 5.8 kg) fed whole corn grain-based finishing diets were used in a completely

randomized design (2 × 3 factorial arrangement of treatments plus a control) to determine the effect of inclusion level and form

of distillers grains plus solubles (DGS) on hepatic and renal mass, and glutathione peroxidase (GPx) and urea cycle enzyme

activities. The DGS were fed at 0 (control), 16.7, 33.3, and 50% of diet DM using dry (DDGS) or modified wet (MWDGS)

product. Data were analyzed using the Mixed procedure of SAS; treatment means were compared using contrast statements

(control vs. other treatments, DDGS vs. MWDGS, inclusion levels of DGS (linear, quadratic), and interactions between form

and linear and quadratic inclusion levels). Kidney weight (g) increased linearly (P = 0.01) with increasing inclusion levels of

DGS. Renal protein concentration (mg/g) did not differ (P ≥ 0.43) among treatments. There were no effects (P ≥ 0.11) of

dietary treatment on hepatic and renal GPx activity (U/g, U/mg of protein, and kU/liver). A form by linear effect of inclusion

level interaction (P = 0.04) was observed for renal GPx activity (U/kg of BW). The interaction likely occurred because renal

GPx activity (U/kg of BW) tended to increase linearly (P = 0.12) with increasing DDGS inclusion level and decreased

numerically (P = 0.65) with increasing MWDGS inclusion levels. Hepatic carbamoyl phosphate synthetase (CPS) activity

(U/g) did not differ (P = 0.13) among treatments. Hepatic CPS activity (kU/liver, and U/kg of BW) tended to linearly increase

(P = 0.06 and P = 0.08, respectively) with increasing inclusion level of DGS. Hepatic argininosuccinate synthetase (AS) and

ornithine transcarbamoylase (OTC) activity (kU/liver, and U/kg of BW) increased linearly (P ≤ 0.05) with increasing inclusion

levels of DGS in diet. These data indicate that hepatic and renal GPx activity generally was not influenced and hepatic CPS,

AS, and OTC activity were tended to increase or linearly increased with increasing inclusion level of DGS in whole corn

grain-based finishing diets.

Key Words: beef cattle, glutathione peroxidases, urea cycle enzymes, distillers grains

6.2 INTRODUCTION

Ethanol by-products are a good alternative feed source for cattle (Klopfenstein et al., 2008) and have increased in use

because of increased ethanol production and feed costs. However, there is still unknown information about the effects of

excess dietary sulphur and protein by feeding high levels of distillers grains plus solubles (DGS) on metabolism in cattle. For

example, increasing dietary S might decrease the bioavailability of Se resulting in decreased glutathione peroxidase (GPx)

activity (Ivancic and Weiss., 2001). Moreover, increasing dietary protein might change the activity of enzymes of ureagenesis

to detoxify excess ammonia from the blood (Takiguchi and Mori, 1995).

Selenium is necessary to achieve an effective and efficient immune system in both animals and humans (Arthur et al.,

2003). Biological functions of Se, in antioxidant reactions, thyroid hormone metabolism, reproduction and immune function, occur through selenoproteins, which include GPx (Rayman, 2000). Gerloff, (1992) reported that a deficiency in Se is associated with high calf mortality, mastitis and white muscle disease in cattle. Increasing dietary sulphur in cattle might reduce the GPx activity in cattle through decreasing Se bioavailability (Ivancic and Weiss., 2001; Rocher et al 1992). The activity of GPx in tissue can be used as an indicator of Se bioavailability in the diet (Lei et al., 2007). Increasing dietary protein causes increased ammonia in portal blood. Detoxification of NH3 is one of the important functions of the liver in ruminants.

The liver is efficient at converting NH3 to less toxic urea through ureagenesis under normal conditions. However, the liver may not completely extract NH3 in cases of excessive NH3 absorption or liver damage, which causes peripheral hyperammonaemia

(infertility, coma and death). The activities of urea cycle enzymes provide indices of protein status in ruminants (Overton et al.,

1998). The urea cycle consists of key enzymes: carbamoyl phosphate synthetase (CPS, EC 6.3.4.16), ornithine transcarbamoylase (OTC, EC 2.1.3.3), argininosuccinate synthetase (AS, EC 6.3.5.4) which are regulated by diet in rats

(Takiguchi and Mori, 1995). There is a lack of information about urea cycle enzyme activities when cattle are fed high levels of DGS. Therefore, this study was conducted to examine how feeding increasing levels of dry or modified wet corn distillers grains plus solubles in whole corn grain-based finishing diets affects hepatic and renal mass, GPx activity and hepatic urea cycle enzyme activity in feedlot cattle.

6.3 MATERIALS AND METHODS

6.3.1 Animal Care and Experimental Design

This experiment followed the recommendations of the Canadian Council on Animal Care (1993) and met the approval of

the University of Guelph Animal Care Committee. A subset of 42 cross-bred steers from a larger feedlot study (n = 114 steers

and 17 heifers), weighing 357 ± 5.8 kg were randomly assigned to 7 pens (n = 6 steers per treatment), and used in a completely

randomized design with a 2 × 3 factorial arrangement of treatments plus a control to determine the effect of DGS form and

level on activities of hepatic and renal GPx and hepatic urea cycle enzymes. Treatments (Table 3.1) consisted of 0% (control),

and 16.7%, 33.3%, and 50% of DM from dried distillers grains plus solubles (DDGS) or modified wet distillers grains plus

solubles (MWDGS). Steers were fed diets as a total mixed diet once daily. All diets contained 10% haylage as a forage source,

and all diets were formulated to meet or exceed the estimated requirements for CP, minerals and vitamins according to NRC

(1996). Calves were fed a corn silage/haylage-based diet for at least two weeks before starting the experiment to adapt to

facilities. Radio frequency tags were placed in the right ear of each steer and steers were fed for individual intake using

electronic feeders (Insentec, B. V., Marknesse, the Netherlands) as described by Mader et al. (2009). Steers were adapted to

dietary treatments over the first 28 d on experiment. At the beginning and end of the trial, cattle were weighed twice in the morning over two consecutive days. Steers were slaughtered at 60, 120, 180 d on feed. At each selected day of days on feed,

tissue samples were collected from two steers per dietary treatment. All slaughtered steers reached approximately 10 mm of

backfat thickness (between the 12th and 13th rib) as estimated using ultrasound (Aloka SSD-500 ultrasound unit; Cormetrics

Medical Systems, Wallingford, CT, USA).

6.3.2 Dietary analyses and sample collection

Diet samples were collected weekly and frozen at -20оC for further analysis at the Agri-Food Laboratories Inc. (Guelph,

ON, Canada). Diet samples were dried in a 55оC oven, ground to pass a 1-mm screen, and analyzed for DM and ash by

standard procedures (AOAC, 1990; Method 930.15). Diet nitrogen concentrations were determined using a Leco N analyzer

(Leco Corporation, St. Joseph, MI, USA) and percent crude protein was calculated by multiplying percent N х 6.25. Neutral

detergent fiber (NDF) concentration was determined by the method of Robertson and Van Soest (1981) using an Ankom fiber

analyzer (Ankom Technology Crop., Fairport, NY, USA). Blood samples were collected on d 56 via jugular venipuncture.

Plasma was isolated by centrifugation at 3000 × g for 20 min and stored at – 20°C until analysis for glucose and urea N

concentrations. Moreover, plasma was analyzed for urea N (Sampson et al., 1980) and glucose (Trinder, 1969) concentrations

using kits from Teco Diagnostics (Anaheim., CA, USA). Analyses were adapted for use on a PowerWave XS microplate

spectrophotometer (BioTek Instruments Inc., Winooski, VT, USA). Liver and kidney weights were recorded at slaughter.

Samples of liver (mid-lobe) and kidney (cortex) were collected, rinsed in ice-cold saline, snap-frozen in liquid N2, and stored at

-80°C until analysis.

6.3.3 Hepatic and Renal GPx Enzyme and Protein Analysis

Liver samples (0.5g) were homogenized in 5 mL of cold buffer (50 nM Tris-HCl (pH 7.5), 5 mM EDTA, and 1 mM DTT) using a polytron (Brinkmann Instruments Inc.,Westbury, NY). The homogenate was then centrifuged at 10,000 x g for 15 min at 4° C and the supernatant was stored at -80° C until analysis. Glutathione peroxidase activity was measured by using a kit from Cayman Chemical Corporation (Ann Arbor, MI) and according to principles described by Paglia and Valentine (1967).

The sample (20 μL) was pipetted into a 96-well plate and mixed with 100 μL of assay buffer (50 mM Tris-HCl, pH 7.6, containing 5 mM EDTA), 50 μL of co-substrate mixture (a lyophilized powder consisting of NADPH, glutathione, and glutathione reductase), and 20 μL of cumene hydroperoxide to initiate the reaction. Analyses were adapted for use on a

PowerWave XS microplate spectrophotometer (BioTek Instruments Inc., Winooski, VT, USA). The plate reader was set at 25°

C and at 340 nm. The activity of GPx was calculated by using the following formula:

GPx activity = (|ΔA340/min| / 0.00373 μM-1) × (0.19 ml / 0.02 ml) = nmol/min/mL,

where |ΔA340/min| was the absolute value of the change in absorbance at 340 nm per min. One enzyme unit was defined as the amount of enzyme that will cause the oxidation of 1 nmol NADPH per min. The activity of GPx was expressed as enzyme units per gram of wet tissue, units per milligram of protein, kilounits per liver or kidney, and units per kilogram of BW. Protein concentrations of tissues were determined by using a bicinchoninic acid assay kit (Pierce, Rockford, IL), with BSA as the standard.

6.3.4 Hepatic Urea Cycle Enzyme Analysis

Liver tissue samples were homogenized and extracted according to a modified method described by Kharbuli et al. (2006).

Hepatic tissue (0.5 g) was homogenized in buffer containing 100 mM Tris-HCl buffer (pH 7.5), 50 mM KCl, 1 mM EDTA, 1

mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 500 kIU/mL of aprotinin, using a polytron. For CPS and OTC

analyses, mild sonication (for 10s, with a 2-s interval for 5 times) was used to break the mitochondrial membrane. The homogenate was centrifuged at 10,000 × g for 10 min. The entire process was carried out on ice. The supernatant was stored at

-80°C until assayed. Activities of the hepatic urea cycle enzymes were determined according to a modified method described by Takagi et al. (2008). For analysis of CPS activity, supernatant (0.2 ml) was added to 0.8 ml of assay mixture, which contained 50 µmol of potassium phosphate buffer (pH 7.5), 50 µmol of ammonium chloride, 50 µmol of sodium bicarbonate,

10 µmol of ATP, 10 µmol of L-ornithine, 15 µmol of MgSO4, and 5 µmol of N-acetyl-L-glutamate. The reaction mixture was

incubated for 60 min at 37 ºC. For analysis of AS activity, supernatant (0.2 ml) was added to 0.8 ml of assay mixture, which

contained 50 µmol of potassium phosphate buffer (pH 7.0), 3 µmol of L citrulline, 5 µmol of L-aspartate, 10 µmol of ATP, 10

µmol of MgSO4, and 20 units of urease. The reaction mixture was incubated for 60 min at 37 ºC. For analysis of OTC activity, supernatant (0.1 ml) was added to 1.9 ml of assay mixture, which contained 90 µmol of glycylglycine buffer (pH 8.3), 20 µmol

of L-ornithine, and 20 µmol of carbamoyl phosphate. The reaction mixture was incubated for 15 min at 37 ºC. Perchloric acid

(10%) was used to stop the reaction after the incubation time for all urea enzyme assays. Then all tubes were centrifuged to

remove precipitated protein. The enzyme activity was measured by determining the change in citrulline between 0 and 60 min

for CPS and AS or 0 and 15 min for OTC. Citrulline concentration in the supernatant was measured by the method of Boyde

and Rahmatullah (1980) as modified for the PowerWave XS microplate spectrophotometer. One unit (U) of enzyme activity equals 1 µmol of citrulline produced or consumed/h. The urea cycle enzyme activity data are expressed as U per g of wet tissue, U per mg of protein, kilounits (kU) per liver, and U per kg of BW.

6.3.5 Statistical Analysis

Data were analyzed as a completely randomized design using the Mixed procedure of SAS. The model included the effects of treatment, days on feed (DOF), and treatment × DOF. Orthogonal contrasts statements were used to determine control vs. other treatments, DDGS vs. MWDGS, linear and quadratic effects of inclusion level DGS, and the interaction between linear and quadratic effects of inclusion levels and forms of DGS. Significance was declared at P ≤ 0.05 and a tendency was reported if 0.05 < P ≤ 0.10.

6.4 RESULTS

On d 56, plasma glucose concentration did not differ (P ≥ 0.10) among dietary treatment (Table 6.1). On d 56, plasma urea

concentration (mg.dl-1) was greater (P = 0.0007) in steers fed DGS compared to controls and was greater (P = 0.02) in steers

fed MWDGS than steers fed DDGS. Moreover, plasma urea concentration (mg.dl-1) increased linearly (P < 0.0001) with

increasing inclusion level of DGS.

Liver weight (gram and gram per kilogram of BW) did not differ (P ≥ 0.23) among treatments (Table 6.2). Kidney weight

(g) was greater (P = 0.03) in steers fed DGS than steers fed the control diet. Kidney weight (gram and gram per kilogram of

BW) increased linearly (P = 0.01 and P = 0.004 respectively) with increasing inclusion level of DGS.

Hepatic GPx activity (unit per gram, unit per milligram of protein, kilo unit per liver, and unit per kilogram of BW) did not

differ (P ≥ 0.28) among treatments (Table 6.3). Renal GPx activity (unit per gram, unit per milligram of protein, and kilounit per kidney) did not differ (P ≥ 0.11) among treatments. Renal GPx activity per kilogram of BW tended to be greater (P = 0.07)

in the DDGS group compared to the MWDGS group. There was an interaction (P = 0.04) between the linear effect of levels

and form for renal GPx activity per kilogram of BW. The interaction likely occurred because renal GPX activity per kilogram

of BW tended to increase linearly (P = 0.12) with increasing DDGS inclusion level and decreased numerically with increased

inclusion level of MWDGS (data not shown).

Hepatic CPS activity (unit per gram) did not differ (P = 0.13) among treatments (Table 6.4). Hepatic CPS activity (kilo unit

per liver, and unit per kilogram of BW) tended to increase linearly (P = 0.06, and P = 0.08, respectively) with increasing

inclusion level of DGS. Hepatic AS activity per gram (P = 0.13) did not differ among treatments. Hepatic AS activity kilounit per liver tended to be greater (P = 0.10) in steers fed DGS compared to controls. Hepatic AS activity per gram, unit per milligram of protein, kilo unit per liver, and unit per kilogram of BW (U/g, U/mg of protein, kU/liver, and U/kg of BW) tended to be increase linearly (P ≤ 0.09) with increasing inclusion levels of DGS in diet. Hepatic OTC activity per gram, kilounit per

100

liver, and unit per kilogram of BW tended to increase linearly (P ≤ 0.06) with increasing inclusion level of DGS. Hepatic OTC activity kilo unit per liver tended to be greater (P = 0.08) in the DGS group compared to the control group.

6.5 DISCUSSION

The use of DGS in feedlot diets has increased. This has resulted in increased intake of several minerals and CP, often above

recommended intake levels (NRC, 1996), because of the greater concentrations in DGS as compared to corn grain. The

physiological consequences of increased mineral and CP intake resulting from feeding high levels of distillers grains are

largely unknown.

Tissues can adapt to changes in diet by altering tissue mass or metabolism per g tissue (Wang et al., 2009). Although liver

weight was not influenced by dietary treatment, the observed increase in kidney weight with increasing inclusion of DGS may

have been in response to increased mineral and/or CP intake resulting in increased workload (Sainz and Bentley., 1997;

Swanson et al., 1999; Johnson et al., 1990; and Wang et al., 2009). The lack of a difference in liver weight with increasing

inclusion of DGS is evidence that the liver has an ability to adapt when animals are fed diets with high protein levels.

Similarly, in adult sheep, fed a high protein diet, the liver adapts to increase urea synthesis (Payne and Morris., 1969).

The kidney is the major organ of clearance of ammonia and minerals. In the current study, the concentrations of plasma urea

and kidney weight increased linearly with increasing inclusion levels of DGS. Similarly, in pigs, plasma urea levels and kidney

weight increased when fed high concentrations of protein (Chen et al., 1999).

101

Increasing levels of DDGS linearly increased dietary S intake and retention in steers (Salim et al., 2011). This increase may affect the biological function of Se by decreasing the bioavailability of Se and the activity of GPx (Murphy and Quirke, 1997;

Rayman, 2000; Ivancic and Weiss., 2001). Although liver is commonly sampled to assay Se status, the kidney has the greatest concentration of Se compared to other tissues (Maag and Glen., 1967; Ullrey et al., 1977) and is the main site of plasma GPx production (Avissar et al., 1994; Stanczyk et al., 2005). We also observed that GPx activity (U/mg protein) was higher in kidney than liver tissue. However, there is a lack of information about how high dietary S affects hepatic and renal GPx activity when steers are fed high inclusion levels of DDGS and MWDGS. In this study, neither hepatic nor renal GPx activity was reduced by increasing inclusion level of DGS. These results suggest that increasing dietary S up to 0.38% of diet (DM basis) was not enough to negatively impact the availability of Se to tissues. This is consistent with the lack of effect on dietary

Se retention when DDGS inclusion increased (Salim et al., 2011). Also, feeding 0.4% of dietary S from MgSO4, over the last

21 d of gestation in cows, did not affect the concentrations of Se in whole blood or plasma during early lactation (Gant et al.,

1998). Hidiroglou et al. (1977) found that feeding up to 0.57% S in sheep diets containing 0.12 mg of Se/ kg of DM, did not influence the concentration of Se in whole blood, but decreased true Se digestibility. Moreover, when cows were fed 0.4% added S with less than 0.3 mg of Se/kg of DM for an extended period, Se status was compromised due to decreased Se absorption from the gut (Ivancic and Weiss., 2001). Our results indicate that feeding up to 50% of DDGS or MWDGS did not affect hepatic GPx activity in feedlot cattle fed whole corn grain-based diets, suggesting that dietary Se (0.3 ppm) in this study was adequate to maintain GPx activity.

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Increasing dietary protein increases plasma urea-N (PUN) concentrations (Boling et al., 1978) which was also observed in

the current study. The urea cycle (and the enzymes within) is necessary to convert ammonia to urea, which is less toxic to the

animal. Urea cycle enzymes are influenced by several nutritional factors (Takiguchi and Mori., 1995). Boling et al. (1978)

reported that the bovine arginase activity in liver increased due to the increase of dietary protein. In developing Holstein

calves, the activity of the urea cycle enzymes (CPS, OTC, and AS) was associated with increasing PUN concentration (Takagi

et al., 2008). The activity of urea cycle enzymes has been shown to have a positive correlation with urea biosynthesis rates

(Rattenbury et al., 1980). In our research, the activity of urea enzymes increased linearly with increasing inclusion level of

DDGS and MWDGS from 0% to 50% of diet DM. This is likely due to excess N intake and ammonia- and amino acid N-

absorption, resulting in increased PUN production. Others also have reported, in adult sheep, increased activity of urea enzymes when high dietary protein was fed (Payne and Morris., 1969). Moreover, in another study by Elsasser et al. (1996) in growing cattle, urea cycle enzymes increased due to feeding high dietary protein diets greater than 18% CP (DM basis).

Therefore, steers fed up to 50% of DGS appear to adapt to excess N intake by increasing activity of urea cycle enzymes.

In conclusion, increasing the inclusion level of DDGS and MWDGS up to 50% of diet DM did not decrease hepatic and renal glutathione peroxidase (GPx) activity (U/g, U/mg of protein, and kU/liver). Increasing the inclusion level of DGS up to

50% of diet DM increased the activity of hepatic CPS, AS and OTC. These data indicate that although feeding up to 50% DGS might cause an increase in kidney mass, steers can adapt to moderately high intake of dietary S and protein when fed in diets containing up to 50% DGS.

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Table 6.1. Influence of dietary source and level of DGS on plasma glucose and urea N concentrations in feedlot cattle fed dry whole corn-based finishing diets.

Dietary Treatment P-value3 DDGS1 MWDGS2 Item Control 16.7 33.3 50 16.7 33.3 50 SEM 1 2 3 4 5 6

Glucose, mg/dl 89.8 88.5 96.0 89.5 101 90.7 92.3 2.78 0.29 0.14 0.87 0.10 0.47 0.19 Urea N, 10.4 9.9 15.0 17.1 12.6 17.6 18.5 1.24 0.0007 0.02 <0.0001 0.70 0.06 0.03 mg/dl

1Corn dry distillers grains plus solubles (% of diet DM). 2Corn modified wet distillers grains plus solubles (% of diet DM). 3Contrast P- values: 1- control vs others, 2- DDGS vs MWDGS, 3- linear level, 4- Quadratic level, 5- linear (forms Χ levels), 6-Quadratic (forms Χ levels).

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Table 6.2. Influence of dietary source and level of DGS on liver and kidney mass.

Dietary Treatment P- Value3 DDGS1 MWDGS2 Item Control 16.7 33.3 50 16.7 33.3 50 SEM 1 2 3 4 5 6

Final body 546 598 578 547 596 624 582 28.5 0.19 0.27 0.58 0.07 0.21 0.44 weight, kg Liver weight g 7038 7454 7847 7631 7440 7620 7790 490 0.28 0.95 0.23 0.58 0.99 0.81 g/kg BW 12.9 12.6 13.6 14.2 12.5 12.3 13.6 0.64 0.77 0.22 0.20 0.22 0.20 0.30 Kidney weight g 1066 1167 1248 1300 1190 1242 1281 67 0.03 0.98 0.01 0.53 0.89 0.91 g/kg BW 1.96 1.95 2.19 2.43 2.02 1.99 2.24 0.101 0.11 0.20 0.004 0.20 0.10 0.52 1Corn dry distillers grains plus solubles (% of diet DM). 2Corn modified wet distillers grains plus solubles (% of diet DM). 3Contrast P- values: 1- control vs others, 2- DDGS vs MWDGS, 3- linear level, 4- Quadratic level, 5- linear (forms Χ levels), 6- Quadratic (forms Χ levels).

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Table 6.3. Influence of dietary source and level of DGS on glutathione peroxidase (GPx) activity in liver and kidney.

Dietary Treatment P- value3 DDGS1 MWDGS2

Item Control 16.7 33.3 50 16.7 33.3 50 SEM 1 2 3 4 5 6

Hepatic GPx activity4 unit/g tissue 8.19 8.43 9.03 8.65 9.45 10.09 7.36 1.304 0.65 0.81 0.99 0.28 0.92 0.43 unit/mg protein 0.062 0.067 0.068 0.062 0.067 0.073 0.050 0.0134 0.84 0.83 0.81 0.37 0.71 0.85 kU/liver 57.4 62.8 70.0 69.1 70.6 76.7 58.4 11.87 0.42 0.89 0.58 0.33 0.87 0.55 U/kg BW 106 105 122 123 119 125 100 18.4 0.65 0.89 0.70 0.56 0.62 0.65 Renal GPx activity unit/g tissue 9.04 9.31 8.91 9.32 8.48 8.74 7.22 0.85 0.69 0.15 0.47 0.76 0.11 0.56 unit/mg protein 0.117 0.102 0.110 0.093 0.077 0.106 0.090 0.0184 0.31 0.49 0.43 0.69 0.68 0.45 kU/kidney 9.6 11.1 11.1 12.3 10.1 10.9 9.2 1.35 0.43 0.21 0.46 0.58 0.15 0.65 U /kg BW 17.7 18.4 19.5 22.7 16.9 17.5 16.1 2.19 0.74 0.07 0.49 0.78 0.04 0.44 1Corn dry distillers grains plus solubles (% of diet DM). 2Corn modified wet distillers grains plus solubles (% of diet DM). 3Contrast P- values: 1- control vs others, 2- DDGS vs MWDGS, 3- linear level, 4- Quadratic level, 5- linear (forms Χ levels), 6-Quadratic (forms Χ levels). 4one enzyme unit defined as the amount of enzyme that will cause the oxidation of 1 nmol NADPH per min.

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Table 6.4. Influence of dietary source and level of DGS on Carbamoyl phosphate synthetase (CPS), Argininosuccinate synthetase (AS), and Ornithine transcarbamoylase (OTC) activity in liver.

Dietary Treatment P- value3 DDGS1 MWDGS2 Item Control 16.7 33.3 50 16.7 33.3 50 SEM 1 2 3 4 5 6 CPS activity4 0.23 U/g tissue 1.25 1.38 1.69 1.68 1.40 1.50 1.62 0.25 0.69 0.13 0.81 0.67 0.71 2 0.00 U/mg protein 0.012 0.012 0.015 0.015 0.012 0.014 0.014 0.39 0.65 0.19 0.96 0.62 0.73 21 kU /liver 8.68 10.2 13.8 12.5 10.2 11.3 12.7 1.89 0.14 0.65 0.06 0.63 0.68 0.53 U /kg BW 15.8 17.3 23.3 23.3 17.4 18.3 22.5 3.49 0.24 0.52 0.08 0.93 0.52 0.50 AS activity3 U/g tissue 20.5 26.9 31.9 30.5 25.9 24.6 34.7 5.02 0.13 0.74 0.06 0.84 0.91 0.42 0.04 U/mg protein 0.196 0.241 0.285 0.276 0.244 0.225 0.320 0.19 0.91 0.09 0.97 0.95 0.55 66 kU AS /Liver 145 203 256 223 195 187 274 41.4 0.10 0.81 0.05 0.69 0.97 0.37 U AS /Kg of 267 348 439 415 321 295 468 69.4 0.14 0.49 0.05 0.95 0.68 0.23 BW OTC activity5 U/g tissue 4519 4913 4895 5311 4611 5465 5325 375 0.18 0.76 0.06 0.86 0.66 0.72 U/mg protein 43.3 44.2 43.6 48.5 42.5 49.8 49.0 3.40 0.43 0.56 0.14 0.72 0.51 0.52 4134 kU OTC /Liver 32155 36545 38338 40542 33906 41137 3248 0.08 0.90 0.02 0.69 0.77 0.96 8 6659 U OTC /Kg of BW 59134 62813 66066 76033 57751 72788 5515 0.20 0.57 0.02 0.44 0.64 0.69 1 1Corn dry distillers grains plus soluble (% of diet DM). 2Corn modified wet distillers grains plus soluble (% of diet DM).

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3Contrast P- values: 1- control vs others, 2- DDGS vs MWDGS, 3- linear level, 4- Quadratic level, 5- linear (forms Χ levels), 6- Quadratic (forms Χ levels). 4One unit (U) of enzyme activity equals 1 µmol of citrulline produced/h. 5One unit (U) of enzyme activity equals 1 µmol of citrulline produced/15 min.

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CHAPTER 7

GENERAL DISCUSSION AND CONCLUSION 7.1 GENERAL DISCUSSION

Increasing feed costs is a major concern in beef production. Increasing the inclusion

level of distillers grains plus solubles (DGS) has been necessary with increasing ethanol

production and decreasing availability of corn grain. Most studies on DGS inclusion in

feedlot diets have been conducted with dry-rolled, steam-flaked, and high moisture corn-

based diets (Klopfenstein et al., 2008). There is a shortage of information on the effect of

DGS in whole corn grain-based diets. Therefore, this thesis investigated the effect of

increasing inclusion level of DDGS or MWDGS on growth performance, carcass

characteristics, feeding behaviour, total tract digestion, nutrient balance, nutrient

excretion, and digestive enzyme activities, GPx activity, and urea cycle enzyme

activities.

In experiment one, increasing the inclusion level of two forms of DGS (DDGS and

MWDGS) up to 50% of DM did not influence final BW, ADG, days on feed, rumen pH

at slaughter, dressing %, hot carcass weight, marbling score, lean yield, and lean color.

Feeding DGS decreased liver abscess scores compared to cattle fed whole corn grain

(control). Visits of cattle to the feeder (VF) increased when cattle were fed up to 16.7%

DDGS or 33.3% MWDSG. Number of meal (NM) and eating rate (ER) was greater and

time per meal (TM) was lower in cattle fed MWDGS compared to those fed DDGS.

Also, daily time at feeder (TF) increased with increasing level of DGS; however, ER

decreased when cattle were fed up to 33.3% DGS and after that increased. This indicates

109 that DGS could affect feeding behaviour differently depending on form of DGS. These data from experiment one suggest that feeding DDGS or MWDGS up to 50% diet DM did not influence negatively feedlot performance and carcass traits in whole corn grain- based finishing diets even though there were feeding behavior responses. The data from feeding behaviour might be useful to improve feed efficiency in feedlot cattle. Further research is needed on environmental and physiological effects when cattle are fed high level of DGS.

In experiment two, dry corn distillers grains plus solubles (DDGS) were fed at 0

(control), 16.7, 33.3, and 50% of diet DM. Total tract digestion of DM, OM, and starch was decreased with increasing DDGS inclusion level up to 50 % of DM. Moreover, increasing the inclusion level of DDGS increased fecal and urinary excretion of N, P, S,

Mg, and K due to increasing daily intake of these minerals. However increasing the inclusion level of DDGS did affect the excretion of Se and Na. Also, retention of N was not influenced by inclusion level of DDGS. On the other hand, increasing the inclusion level of DDGS tended to increase P retention and linearly increased S retention.

Digestion and retention of Se, Mg, K, and Na was not changed with increasing inclusion level of DDGS. The concentration of N, P and S increased in blood with increasing inclusion level of DDGS, could be good indicators for excretion of these minerals. These results indicated that increasing replacement of whole corn grain with up to 50% DDGS in feedlot finishing diets does not affect negatively nutrient retention. However, increasing the inclusion level of DDGS increased total excretion of N, P, S, Mg, and K, which could result in increased concentration of P in manure and increased emissions of ammonia and H2S from feedlots. These data might help to better understand the dietary

110

bioavailability of some minerals, and to determine the tolerance level of dietary S. Also,

these results could help to develop suitable nutrient management plans for these minerals and to better understand environmental implications of manure when feedlot cattle are fed high levels of DDGS.

In experiment three, pancreatic mass was measured and pancreatic tissue collected from all the animals in experiment one. Pancreatic mass was not influenced by increasing inclusion level of DDGS or MWDGS; however increasing DGS increased linearly pancreatic protein concentration (mg/g). Feeding DGS diets increased pancreatic concentration of α-amylase and trypsin activity (U/g) compared to whole corn grain

(control). Feeding cattle MWDGS increased pancreatic concentration of trypsin activity

(U/g) compared to those fed DDGS diets. In addition, pancreatic concentration of trypsin activity (U/g) increased linearly when the inclusion level of DGS increased. This is likely due to increasing dietary CP and decreasing dietary starch with increasing DGS. These results indicate that feeding different forms and levels of DGS could influence the activity of digestive enzymes such as α-amylase and trypsin. This might help to explain differences in nutrient digestion when cattle are fed high levels of DGS in whole corn grain-based finishing diets.

In experiment four, hepatic and renal mass were taken and samples collected from forty-two steers. Steers were fed 0 (control), 16.7, 33.3, and 50% of DDGS or MWDGS.

Although there was no liver weight response to the dietary treatments, increasing the inclusion level of DGS increased linearly the kidney weight (g). Renal protein concentration (mg/g) and hepatic and renal glutathione peroxidases (GPx) activity (U/g,

U/mg of protein, and kU/liver) were not affected by form and level of DGS in the diets.

111

Renal GPx activity as kilogram of BW was affected by the interaction between the form

and the effect of linear inclusion level of DGS because increasing DDGS inclusion level

tended to increase renal GPx activity as kilogram of BW; however, increasing MWDGS

inclusion levels numerically decreased renal GPx activity as kilogram of BW. In general,

increasing the inclusion level of DGS did not decrease the activity of hepatic and renal

GPx. This is likely consistent with the lack of an effect on dietary Se retention when

steers were fed high level of DDGS in experiment two. Increasing inclusion level of DGS

linearly increased urea N (mg/dl) in plasma. Moreover, increasing inclusion level of DGS linearly increased hepatic CPS activity (kU/liver, and U/kg of BW), hepatic

argininosuccinate synthetase (AS), and ornithine transcarbamoylase (OTC) activity (U/g,

kU/liver, and U/kg of BW). These data indicate that kidney and liver have the ability to

adapt to excess dietary S and CP when steers are fed up to 50% DDGS or MWDGS. This

information might improve our understanding on the effect of high dietary S on animal

health through immune function of Se and effect of high dietary CP on urea cycle

enzyme activities when cattle are fed high levels of DDGS or MWDGS.

7.2 CONCLUSION

Overall, this study investigated the effect of increasing inclusion level of DDGS or

MWDGS on growth performance, carcass characteristics, feeding behaviour, digestion,

nutrient balance, nutrient excretion, and digestive, immune, and urea cycle enzyme

activities. Our findings suggest that although feeding DDGS or MWDGS up to 50% of

DM did not affect growth performance and carcass characteristics, there were feeding

behaviour responses to increasing inclusion level of DGS. Moreover, feeding up to 50%

DDGS generally does not influence the retention of nutrients; however, it increases total

112 excretion of N, P, S, Mg, and K. Increasing the dietary CP and decreasing dietary starch when cattle are fed up to 50% of DGS influences the activities of α-amylase and trypsin.

Although liver mass was not influenced by form and level of DGS, kidney mass increased potentially because of the increasing load of minerals when cattle are fed high levels of DGS, which might indicate an adaptation response.

These data provide needed knowledge about the response of digestive enzymes and urea cycle enzymes to increasing dietary CP. Also, these data give more explanation about the relationship between high dietary S and the bioavailability of Se and its role in immune function through the activity of hepatic and renal GPx. Thus, this information could increase our understanding of the nutritional, physiological and environmental effects of increasing the inclusion level of DDGS or MWDGS in feedlot diets.

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CHAPTER 8

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APPENDICES APPENDIX 1

Nutrient analysis, % (DM basis) Whole Item corn DDGS1 MWDGS2 Dry matter 88.9 89.84 51.37 Protein%(DM Basis) 8.53 29.37 28.76 DIP, % CP 41.2 16.92 20.38 UIP, % CP 58.8 83.08 79.62 SolP, % CP 12 10.79 4.03 ADF 3.5 17.8 14.5 NDF 12.2 38.1 33.3 TDN% 86.85 79.66 82.67 NE( Lac)MCAL/Kg 2.01 1.61 1.66 NE( gain)MCAL/Kg 1.66 1.44 1.53 NE( maint)MCAL/Kg 2.37 2.11 2.22 P% 0.28 0.88 0.93 K% 0.38 1.16 1.24 Mg% 0.12 0.35 0.38 Na% 0.01 0.25 0.30 S% 0.04 0.53 0.50

1Corn dry distillers grains plus solubles. 2Corn modified wet distillers grains plus solubles.

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