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DIETARY PROTEIN REQUIREMENT OF MATURE,
MODERATELY EXERCISED HORSES

presented by

Carissa Lee Wickens

has been accepted towards fulfillment
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6/01 cJCIFlC/DatoDuepGS-ots

DIETARY PROTEIN REQUIREMENT OF MATURE, MODERATELY EXERCISED
HORSES

By

Carissa Lee Wickens

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE

Department of Animal Science

2003

ABSTRACT

DIETARY PROTEIN REQUIREMENT OF MATURE, MODERATELY EXERCISED
HORSES

By

Carissa Lee Wickens

There is a dearth of empirical evidence demonstrating the degree to which
exercise increases dietary protein requirement of the horse relative to maintenance.
Knowledge of protein digestibility in diets fed to exercising horses is also essential to
meet the protein need of the animal. The objectives of this research were first, to
estimate dietary crude protein (CP) requirement using nitrogen (N) retention, serum urea-
N, and serum amino acid (AA) concentration as response criteria for moderately
exercised horses, and second, to determine apparent fecal N and AA digestibility of five
hayzconcentrate mix diets fed to horses performing moderate exercise. Five mature
Arabian geldings were used in a 5 x 5 Latin Square design. Nitrogen retention, basal and
post-feeding/exercise serum urea-N and serum AA results indicated that high (1016 g CP
per d) and very high (1 129 g CP per (1) protein diets exceeded CP requirement and that
very low (677 g CP per (1) and low (790 g CP per d) protein diets provided sufficient
amount of protein for a horse subjected to moderate exercise. Apparent fecal N and AA
digestibility increased linearly (P<O-05) as dietary CP intake increased. Also, inclusion
of soy bean meal to diet concentrates fed to exercising horses resulted in higher protein

quality and contributed to an increase in apparent fecal N and AA digestibility.

ACKNOWLEDGEMENTS

First, I would like to express my gratitude toward my advisor, Dr. Nathalie
Trottier, for giving me the opportunity to participate in her laboratory and to conduct
equine nutrition research. I wish to sincerely thank her for her wisdom, guidance, and
encouragement in developing my research and teaching skills. Dr. Trottier provided the
support necessary to succeed as a student and to learn what it means to become a
professional animal scientist. I would also like to thank my co-advisor, Dr. John Shelle,
for his involvement in my program and for his support of my project. Additionally, I
wish to thank my committee members, Dr. Christine Skelly, Dr. Hilary Clayton, and Dr.
Judith Marteniuk for the input of their time, energy, and valuable advice pertaining to my
research and to my overall program. I would like to thank Paula Hitzler, MSU Horse
Teaching and Research Facility manager, for providing the animals and facilities used to
complete this research. Thank you to Brian Story at the MSU Feed Mill for his help in
mixing our experimental diets. Sincerest thanks and appreciation to Julie Moore for her
assistance with my project, including her guidance and training in the laboratories.

Thank you also to Bob Burnett for his help in the laboratory including training and
maintenance of needed equipment. I would like to thank the many faculty members and
fellow graduate students who made my time at MSU fulfilling and memorable. Finally,
deepest thanks to my husband Edward for his love and support and for encouraging me to

pursue my degree.

iii

TABLE OF CONTENTS

LIST OF TABLES ...................................................... vi
LIST OF FIGURES ..................................................... vii
LIST OF ABBREVIATIONS ............................................. viii
CHAPTER 1: INTRODUCTION

Introduction ....................................................... 2

Exercise and nutrient metabolism ................................... 4

Defining exercise intensity .................................. 4

The role of nutrition in exercise performance .................... 6

Carbohydrate and fat ....................................... 7

Protein .................................................. 8

Methods for estimating protein requirement .......................... 9

Nitrogen retention ......................................... 9

Blood urea nitrogen ....................................... 11

Plasma free amino acid concentration ......................... 13

Indicator method: Direct and Indirect amino acid oxidation ......... 14

Methods for estimating nitrogen and amino acid digestibility ..... - ........ 15

Apparent fecal digestibility .................................. 15

Apparent ileal digestibility .................................. 16

Literature Cited .................................................... 18

CHAPTER 2: ESTIMATING DIETARY PROTEIN REQUIREMENT OF THE
EXERCISING ARABIAN HORSE

Abstract .......................................................... 24
Introduction ....................................................... 25
Materials and Methods .............................................. 26
Results ........................................................... 31
Discussion ........................................................ 34
Literature Cited .................................................... 62

CHAPTER 3: APPARENT FECAL NITROGEN AND AMINO ACID DIGESTIBILITY
OF DIETS FED TO EXERCISING HORSES

Abstract .......................................................... 66
Introduction ....................................................... 67
Materials and Methods .............................................. 68
Results and Discussion .............................................. 71
Literature Cited .................................................... 83

SUMMARY AND CONCLUSIONS
Summary and Conclusions ........................................... 86

iv

APPENDICES ......................................................... 89

Appendix A ....................................................... 90
Appendix B ....................................................... 91
Appendix C ....................................................... 93
Appendix D ....................................................... 95

LIST OF TABLES

Table 1. Ingredient and nutrient composition of concentrate diets (as-fed basis) ...... 40
Table 2. Horse performance data ........................................... 42
Table 3. Nitrogen balance in horses fed varying levels of protein .................. 43
Table 4. Effect of diet on urinary creatinine, urinary orotic acid, and orotic acid to
creatinine ratio ........................................................ 44
Table 5. Effect of diet on basal serum indispensable amino acid concentration ....... 45

Table 6. Effect of diet on post-feeding/exercise serum indispensable amino acid

concentration ........................................................... 46
Table 7. Ingredient and nutrient composition of concentrate diets (as-fed basis) ...... 76
Table 8. Forage to concentrate ratio (percent of total diet) ........................ 78

Table 9. Apparent nitrogen and amino acid digestibility of experimental diets, percent. 79

Table 10. Parameter estimates for crude protein, nitrogen and indispensable amino acid
digestibilities ........................................................... 81

vi

LIST OF FIGURES

Figure 1. Relationship between crude protein intake and nitrogen retention .......... 10
Figure 2. Relationship between crude protein intake and blood urea nitrogen ........ 12
Figure 3. Urea cycle ..................................................... 13
Figure 4. Relationship between daily total lysine intake and nitrogen retention ....... 48

Figure 5. Relationship between daily digestible lysine intake and nitrogen retention . . .50

Figure 6. Effect of dietary crude protein intake on basal and post-feeding/exercise serum
urea nitrogen concentration ................................................ 52

Figure 7a. Effect of dietary crude protein intake on basal and post-feeding/exercise serum
arginine concentration .................................................... 54

Figure 7b. Predicted post-feeding/exercise serum arginine concentration in response to
dietary crude protein intake ............................................... 54

Figure 8. Effect of dietary crude protein intake on basal and post-feeding/exercise serum
citrulline concentration ................................................... 56

Figure 9. Effect of dietary crude protein intake on basal and post-feeding/exercise serum
omithine concentration .................................................... 58

Figure 10. Effect of dietary crude protein intake on basal and post-feeding/exercise serum
glutamine concentration ................................................. ‘. 6O

vii

i

E

ADF
AMP
ATP
BC S
bpm
CP
DAAO
DE
FF A
H+
HR
IAAO

IMP

NADH
NDF
NH3

NFL;+

LIST OF ABBREVIATIONS
amino acid
amino acid intake
amino acid losses in feces
acid detergent fiber
adenosine monophosphate
adenosine triphosphate
body condition score
beats per minute
crude protein
direct amino acid oxidation
digestible energy
free fatty acids
hydrogen ion
heart rate
indirect amino acid oxidation
inosine monophosphate
nitrogen
nicotinamide adenine dinucleotide. reduced form
neutral detergent fiber
ammonia
ammonium

nitrogen intake

viii

Nr
NPN
SBM

TCA

nitrogen losses in feces
non-protein nitrogen
soy bean meal

tricarboxylic acid cycle

ix

The Chapters in this thesis are written according to

2003 British Journal of Nutrition format guidelines

CHAPTER 1

INTRODUCTION

Introduction

The horse industry places great emphasis on health and performance of the
exercising horse. Overall health and performance of the equine athlete depends primarily
upon nutrition, with two key nutrients of interest being energy and protein. Research in
the horse has shown that the requirement for digestible energy (DE) increases with
exercise (Anderson et al., 1983; Glade, 1983; Pagan and Hintz, 1986). There is a dearth
of empirical evidence demonstrating the degree to which exercise increases dietary
protein requirement relative to maintenance. In the current NRC (1989), protein
requirement for the moderately exercised horse is based on a crude protein (CP) to DE
ratio estimated for the mature horse at maintenance, resulting in a protein requirement 1-5
fold that of maintenance. It is not known whether a CF to DE ratio of 40 g to 1 Mcal is a
valid estimate of protein requirement for the exercising horse. We hypothesized that
moderate exercise in horses increases protein requirement relative to maintenance below
that recommended by NRC (1989). Interestingly, known methods used to estimate CP
requirement in other species have not been tested in the horse. Thus, in testing our
hypothesis. some of the common methods employed to estimate CP requirement were
used and presented in this thesis.

In addition to the importance of providing accurate levels of dietary protein, the
extent of protein availability in diets fed to horses is crucial to meeting the need of the
animal. Thus, knowledge of protein digestibility is essential. Exercising horses are
commonly fed diets consisting of increased concentrate relative to forage in order to meet
the increased energy demands associated with exercise (Lawrence, 1990). Earlier studies

provide estimates of protein digestibility of diets fed to horses and ponies at maintenance

Ix)

ranging from 35 up to 798% (Slade et al., 1970; Hintz et al., 1971; Glade, 1984). Protein
digestibility in diets fed to exercising horses has not been determined. Moreover,
ingredients used in diets tested in previous studies (Slade et al., 1970; Glade, 1984) are
not representative of those commonly used in formulating equine diets, especially in the
Midwestern United States. The CF to DE ratio in NRC (1989) has been determined from
horses consuming diets consisting solely of forage with a protein digestibility of 46%.
. However, total tract nitrogen (N) digestibility in ponies fed various types of forage
ranged from 57 to 74% (Gibbs et al., 1988). We hypothesized that apparent fecal N
digestibility would be higher than that reported in NRC (1989), and that apparent N and
amino acid (AA) digestibility would increase when soy bean meal (SBM) is added to the
concentrate portion of the diet.

The overall goal of this thesis is to provide further understanding of the protein
' requirement of the exercising horse. The specific objectives of this research were first, to
estimate dietary protein requirement using N retention, serum urea-N, and serum AA
concentration as response criteria in horses exposed to the same exercise regime and fed
graded levels of CP, and second, to determine apparent fecal N and AA digestibility of
five hayzconcentrate mix diets fed to horses performing moderate exercise. The first and
second objectives are addressed in chapters 2 and 3, respectively. The rationale for
hypothesizing protein requirement for exercise to be less than that estimated from a ratio
of 40 g CF to 1 Mcal DE is based on findings in the human literature. Thus, in the
following section, the reader will gain greater understanding and appreciation behind the
first research question. The rationale for the second hypothesis is based on evidence for

increased utilization of concentrate diet in exercising horses (NRC, 1989; Lawrence,

1990) and the superior protein digestibility of the ingredients used to formulate such
concentrates (Poultry NRC, 1994; Swine NRC, 1998). The study of nutrient digestibility
in the horse is limited by the horse’s extensive hind gut in itself and by the availability of
methods that do not require invasive interventions. The following section of this
introduction will allow the reader to understand some of the pitfalls associated with
determining protein digestibility in feed ingredients fed to horses.

Thus, the objective of this introduction is to provide the reader with current
knowledge on the impact of exercise on protein metabolism and with basic understanding
regarding methods commonly used to estimate dietary protein requirement, as well as
protein and AA digestibility.

1. Exercise and nutrient metabolism
1.1 Defining exercise intensity

For human athletes in particular, the level or intensity of exercise is usually
divided into three categories; low intensity, submaxirnal, or high intensity. Because
many other terms for these levels of exercise exist, defining them accurately and
consistently can be challenging. This is especially true for the human and equine athlete
since both species perform in many different disciplines, nonetheless some basic criteria
for each category of intensity are provided herein. During low intensity exercise,
subjects perform work at low Speeds with low muscle force production. Lower work
intensities are aerobic in nature and subjects usually perform at approximately 30-60%
(Lawrence, 1990) of maximal oxygen uptake (V02 max). The current NRC (1989) refers
to low intensity exercise in horses as light work which includes activities such as Western

and English pleasure. bridle path hack, and equitation. In subjects performing

submaximal exercise, energy is supplied by both aerobic and anaerobic catabolism of
available substrate (Miller-Graber et al., 1991a). Subjects performing submaximal
exercise engage in activity of longer duration and/or at increased speeds and will reach
approximately 75% of V02 max (Brooks, 1985). This type of exercise in horses is
defined as moderate work and includes ranch work, roping, cutting, barrel racing, and
jumping (NRC, 1989). During exercise trials designed to study electrolyte balance and
serum and muscle AA profiles in endurance exercised horses (Danielsen et al., 1995;
Trottier et al., 2002), horses were worked at medium speeds (2-8 m per sec), for longer
duration. This type of work load correlates well with the definitionof submaximal
exercise given above, thus shorter competitive endurance rides could also be included in
the moderate work category. Working heart rates in the study conducted by Danielsen et
a1. (1995) ranged from approximately 108 to 118 bpm. However, it should be recognized
that many endurance horses are often required to perform for much longer duration and at
greater speeds which would lead to further elevation in V02 max and heart rate.
Endurance exercise of this nature may not correspond to a submaximal workload making
it difficult to accurately classify endurance training or exercise in horses to one specific
category of intensity.

Finally, high intensity exercise involves sprint work (Lawrence, 1990), thus
higher speeds for shorter duration. This form of exercise is primarily anaerobic, and
muscle glycogen serves as the major fuel source (Katz etal., 1986). Humans performing
at high intensity have been reported. to reach 95 to 100% of V02 max (Katz et al., 1986).
In the current NRC (1989), high intensity exercise is defined as intense work and

examples include race training and polo. In the study described in this thesis, protein

requirement and digestibility are estimated in horses subjected to submaximal exercise
which will be referred to in the following chapters as moderate exercise.
1.2 The role of nutrition in exercise performance

During exercise, carbohydrate, fat, and protein can be used by working muscle as
a source of fuel. The extent to which each substrate is utilized during exercise depends
upon the intensity and duration of the exercise as well as the availability of each fuel
source. Therefore, when formulating diets for exercising humans or horses, the level of
exercise and the nutrient composition of the diet must be considered. The ultimate goal
in formulating diets for athletes, human or animal, is to meet the individual’s nutrient
requirements in order to maximize performance and delay the onset of fatigue. Fatigue
during exercise is brought on by substrate or fuel depletion and accumulation of end-
products associated with nutrient and muscle metabolism such as lactate, H+, and NH3.
Lactate and H+ accumulation in muscle tissue causes a decline in muscle pH which
diminishes ATP production and decreases force generation by muscle fibers (Lawrence,
1990). Ammonia production in muscle results primarily from the deamination of AMP to
1MP in the purine nucleotide cycle (Lowenstein, 1972). Ammonia accumulation in
muscle or blood may inhibit the TCA cycle enzyme isocitrate dehydrogenase, thus
decreasing a-ketoglutarate synthesis and resulting in a reduction in ATP and NADH
production (Miller and Lawrence, 1986). Fatigue in both submaximal and high intensity
exercise is related to accumulation of end-products; however, accumulation of end-
products and subsequent onset of fatigue occurs more rapidly at higher intensities
(Parkhouse and McKenzie, 1984). Fatigue during both submaximal and high intensity

exercise also results from depletion of carbohydrate (Lawrence, 1990), particularly

during work bouts or performances of longer duration. There is no evidence regarding
the extent to which dietary protein intake influences the onset of fatigue. Thus, a dietary
CP requirement to optimize performance is less than clear.

1.3 Carbohydrate and fat

Carbohydrate and fat are the primary energy sources during exercise (Mole and
Johnson, 1971; Dohm et al., 1977). During high intensity exercise, carbohydrate serves
as the primary fuel, while fat metabolism comprises a major source of energy in subjects
performing low intensity exercise (Miller-Graber et al., 1991a). Carbohydrate is supplied
to the working muscle primarily by the breakdown of muscle glycogen, but as muscle
glycogen is depleted, blood glucose from hepatic glycogenolysis and gluconeogenesis
becomes an important energy substrate (Winder, 1985; Bjorkman and Wahrcn, 1988).
Glucose is converted to pyruvate via glycolysis for ATP generation needed for muscle
contraction. At high intensities, oxidation of some glucose will be incomplete thus
forming lactic acid (Hermansen, 1981).

Oxidation of free fatty acids (FFA) and circulating and intramuscular triglycerides
can also supply energy to working muscle especially during submaximal exercise.
Lipolysis in adipose tissue results in the release of FF A which are transported in the
blood and taken up by the muscle for oxidation (Paul and Issekutz, 1967; Essen et al.,
1977; Terjung et al., 1983). Fat utilization as an energy source can have a sparing effect
on glycogen. Caloric intake composed of 55 to 65% carbohydrate, 20 to 30% fat, and 12
to 15% protein has been recommended in human athletes by Leaf and F risa (1989). In
studies conducted in humans and horses, it has been shown that exercise increases the

demand for energy (Edwards et al., 1935; Pagan and Hintz, 1986; Grandjean, 1989).

However, much controversy exists regarding an increased demand for dietary protein in
human and equine athletes.
1.4 Protein

The belief that protein intake could enhance athletic performance dates as far back
as 450 BC (Leaf and Frisa, 1989). It was theorized then that consumption of meat would
increase muscle strength. However, current researchers are still at odds regarding the
increased need for protein as a result of physical activity, particularly in human athletes
consuming adequate energy (Leaf and Frisa, 1989). Although selected AA, primarily
alanine. are used in gluconeogenesis during prolonged exercise (Lawrence, 1990), Young _
(1986) suggested that in exercising humans, the oxidation of protein accounts for less
than 5% of energy expenditure. Interestingly, in most species, it has been estimated that
muscle protein turnover accounts for approximately 25 to 30% of whole body protein
turnover (Young, 1986). In rats, endurance-type exercise results in decreased muscle
protein synthesis and increased rates of AA oxidation, particularly leucine (Young,
1986). A study conducted in human endurance athletes (Meredith et al., 1989) provided
evidence that protein requirement should be increased, as subjects attained N balance
when ingesting protein at a level approximately 17% above World Health Organization
recommendations (FAO/WHO/UNU, 1985).

Nitrogen retention increased in horses exposed to increasing workloads and fed
increasing levels of dietary protein and energy (Freeman et al., 1988) indicating that
protein requirement of the exercising horse may be increased relative to maintenance.
Conversely, Gontzea et a1. (1975) concluded from their N balance study, that habitual

physical activity in humans did not increase protein requirement. In addition, 25 to 40

min of treadmill exercise at approximately 65% of V02 max, did not affect nitrogen
balance in young adults (Kido et al., 1997), and it was concluded from this study that
exercise did not increase protein requirement relative to requirement of sedentary
individuals. In exercising human (Millward et al., 1994) and equine (Lawrence, 1990)
subjects, there is no known evidence that high protein intake relative to maintenance
enhances athletic performance. Yet, Hallebeek et al. (2000) reported digestible protein
intake in event horses to be 92% above protein requirement, thus suggesting critical
problems behind the lack of knowledge on CP requirement.

A limitation to estimating dietary protein requirement is that known methods
used to determine requirement in other species have not been tested in the horse.
Methods employed to assess protein status and requirement in exercising humans include
N balance, analysis of blood biochemistry, and isotopic tracer studies which measure AA
oxidation in response to exercise. These methods will be discussed in the sections that
follow.

2. Methods for estimating protein requirement
2.1 Nitrogen retention

Nitrogen retention has been used extensively in many species including humans
and swine (Rose. 1957; Hegsted, 1976; Young and Scrimshaw, 1978; King et al., 1993;
Otto et al., 2003) to estimate dietary protein requirement. Nitrogen retention is calculated
by subtracting fecal and urinary N losses from N intake and is a measure of N retained by
the animal for body protein synthesis and accretion. Figure 1 illustrates the relationship
between N intake and N retention. Below dietary protein requirement, N retention

increases linearly with increasing prorein intake. When protein intake exceeds the

animal’s potential for protein deposition, as estimated by N retention, N retention is

maximized.

 

CP
Requirement

E rJ-‘l

:?

.2

§ 0 Maintenance
2

E

0

§”

2

_\

 

 

Crude protein intake

Figure 1. Relationship between crude protein intake and nitrogen retention.

At maintenance. N balance or N equilibrium is achieved when N losses are equal to N
intake (Slade etal., 1970). Nitrogen requirements for horses and ponies at maintenance
have been obtained using N retention (Sladeet al., 1970; Hintz and Schryver, 1972; Prior
etal., 1974), but there is a paucity of studies that have employed N retention as a
response criterion to estimate protein requirement of the exercising horse fed graded
levels of protein.

The N containing compounds in urine include urea, ammonium, and trace
amounts of creatinine and AA including taurine and glycine. Most ammonium in urine
originates from glutamine synthesized in the perivenous hepatocytes as a secondary route

for ammonia detoxification (Boon et al., 1994). Glutamine is transported to the kidney

10

where it is deaminated and the ammonia formed excreted in the urine, a process known as
renal ammoniagenesis (Boon et al., 1994). Creatinine in urine originates from the
nonenzymatic dehydration of intracellular creatine, which, when phosphorylated, serves
as the primary energy store in working muscle (Wang etal., 1996). Creatinine is
proportional to skeletal muscle mass (Wang et al., 1996). In humans, muscle mass can be
estimated from 24-h urinary creatinine excretion, where 1 g creatinine is proportional to
21-8 kg skeletal muscle (Wang et al., 1996). Creatinine has also been used as a clinical
marker of renal function (Reyes et al., 1994).
2.2 Blood urea nitrogen

Urea is the major end product of N metabolism in mammals (Morris, 1985). Urea
is excreted by the kidney (Reyes et al., 1994) and originates from the deamination of AA
mobilized from body tissues or fed in excess of requirement. The urea cycle in the liver
is vital in maintaining low plasma ammonia concentration (Reyes et al., 1994). The
amino group generated as a result of AA catabolism is transferred to arginine by a series
of enzymatic reactions. Arginine is then cleaved to form omithine and urea by the
enzyme arginase (Reyes et al., 1994). When an animal is in N balance, urea synthesis is
a function of dietary protein level. In rats, pigs and humans, urea cycle activity has been
shown to increase with increasing dietary protein intake (Edmonds et al., 1987; Matthews
and Campbell, 1992; Bertolo et al., 2000). Blood urea-N has been used as a response
criterion to determine protein and amino acid requirements of lactating sows (Coma et
al., 1996). In exercising Quarter Horse mares, blood urea-N has been used to assess
protein status when a high protein diet (185% CP) was fed (Miller and Lawrence, 1988).

Figure 2 illustrates the hypothetical relationship between dietary protein intake and blood

11

urea-N. When balance between protein degradation and synthesis is achieved, blood

urea-N should be minimized. Thus, at protein intake exceeding requirement, blood urea-

N will increase exponentially.

Blood urea-N, mg/dL

 

  
 

CP
Requirement

I'Ll

  

 

‘7

Crude protein intake

Figure 2. Relationship between crude protein intake and blood urea-nitrogen.

In addition to urea, concentrations of other metabolites involved in the urea cycle

can be useful indicators of protein status. Figure 3 depicts the urea cycle beginning with

incorporation of C02 and amino-N, resulting from deamination of AA, into carbamoyl

phosphate inside the liver mitochondria. Omithine and carbamoyl phosphate combine to

form citrulline and the subsequent enzymatic reactions within the cytosol facilitate

production of urea. Thus, omithine and citrulline are key metabolites involved in the

formation of urea and drive the enzymatic reactions of the urea cycle forward (Morris,

1985). Decreases in plasma or serum omithine and citrulline concentration would

12

indicate impairment or saturation of urea cycle enzymatic activity (Reyes, 1994).

Orotic

acid is also a useful indicator of protein status. For instance, increased synthesis or

decreased utilization of carbamoyl phosphate into the urea cycle leads to an increase in

cytosolic orotic acid synthesis and subsequent urinary excretion (F ico et al., 1984).

 

Liver

C0 + NH,+
2 Mitochondria

2ATP

N-Acetyl-
glutamate

Carbamoyl
Phosphate
Synthetase

ZADP-l-Pi

 

xi

Carbamoyl
‘ phosphate

\

Ornithine

  
  

Orotic
Acid ‘

 

Ornithine
Transcarbamoylase

 

4e

 

 

~¥

Citrulline

 
  
 
 
 

Argininosuccinate

Aspartate Synthetase

ATP
AMP + PP;

Figure 3. Urea cycle.

2.3 Plasma fiee amino acid concentration

Urea

Arginase

Arginine

Fumarate

Argininosuccinase

Argininosuccinate

Liver Cytosol

Upon digestion of dietary protein, the majority of AA are transported in free form

in the blood to the liver where they will be used for hepatic protein synthesis, catabolized

9

and/or sent to peripheral tissues for protein synthesis and utilization. Serum AA

13

concentrations represent a balance between dietary AA intake and protein turnover
(Russell et al., 1986). Protein turnover refers to the continuous incorporation of AA into
protein synthesis or degradation of tissue protein and subsequent release of AA into
circulation. Concentration of AA in plasma can also be affected by the source and
availability of AA from dietary protein (Richardson et al., 1965). While interpretations
of changes in plasma AA concentrations can be difficult due to the multiple metabolic
processes involved in AA entry into andremoval from the plasma pool (Marchini et al.,
1993), change in serum AA pattern can provide some information on the adequacy of
dietary protein and AA intake. Plasma free AA measurement has been useful in swine
and humans for evaluating dietary protein quality and protein status, and for estimating
AA requirements (Puchal et al., 1962; Marchini et al., 1993).
2.4 Indicator method: Direct and Indirect amino acid oxidation

Other more recent techniques for determining protein status and AA requirements
include direct and indirect AA oxidation (DAAO and IAAO, respectively). The DAAO
method measures AA flux and oxidation in vivo by using amino acids labeled with stable
or radioactive isotopes. When an AA is consumed at or below requirement, the labeled
AA is conserved by the body and incorporated into protein synthesis, thus AA oxidation
to C02 is minimized (Zello etal., 1990). These techniques have been used successfully
in swine and humans to determine AA requirement (Kim etal., 1983; Zello et al., 1990;
Zello et al., 1993), but have not been used in horses. For the IAAO method to be
effective, the requirement of the labeled indicator AA must be known, as the indicator

AA is most sensitive to intake of the test AA when it is fed at its required level (Wilson et

14

al., 2000). To date, indispensable AA requirements for the mature horse have not been
determined, making it difficult to meet this criterion.
3. Methods for estimating nitrogen and amino acid digestibility
3.1 Apparent fecal digestibility

Digestion of protein and absorption of AA in the horse occurs in different
segments of the gastrointestinal tract and is influenced by the type of diet consumed.
Hintz et a1. (1971) reported the major site of protein digestion in ponies was prececal.
However, results of their study demonstrated significant disappearance of N from the
hind gut as well. The extent to which products of protein digestion in the cecum and
large intestine are absorbed and utilized by the horse remains unclear, and it is also not
understood to what extent microbial protein and AA synthesis in the lower gut
contributes to fecal N content in horses. Despite these uncertainties, several researchers
have provided estimates of apparent fecal N digestibility in ponies and horses at
maintenance (Slade and Robinson, 1970; Glade, 1984; Gibbs et al.. 1988). However,
apparent digestibility of AA in horse diets have not been investigated. Apparent fecal N
and AA digestibility is defined as the difference between N or AA intake and N or AA
losses in feces, expressed as a proportion of N or AA intake. While this method is
noninvasive and provides some information on the availability of N and AA from the
diet, it fails to differentiate between protein of dietary versus microbial protein origin,

thus leading to inaccurate measurements of digestibility (Darragh and Hodgkinson,

2000).

15

3.2 Apparent ileal digestibility

An alternative method to apparent fecal digestibility is apparent ileal digestibility,
which is believed to be a more sensitive and accurate technique (Darragh and
Hodgkinson, 2000). Apparent ileal digestibility is determined by subtracting the N and
AA measured in digesta collected at the distal ileum from the N and AA intake and
dividing the difference by N and AA intake. Collection of digesta from the distal ileum
at the ileal-cecal junction greatly reduces the confounding effect of microbial protein
metabolism in the large intestine and results in a more accurate estimate of protein and
AA digestibility. However, this method is more invasiveas it requires surgical insertion
of a cannula and can therefore be more expensive and labor intensive. This procedure
has not been routinely used in the horse, and it is unlikely to be readily adopted in horses
in the near future. In chapter 3 of this thesis, apparent fecal N and AA digestibility in

diets fed to moderately exercised horses were determined.

16

LITERATURE CITED

l7

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22

CHAPTER 2

NITROGEN BALANCE OVERESTIMATES DIETARY PROTEIN
REQUIREMENT OF THE EXERCISING HORSE

23

ABSTRACT

NITROGEN BALANCE OVERESTIMATES DIETARY PROTEIN

REQUIREMENT OF THE EXERCISING HORSE
By

Carissa Lee Wickens

Five mature Arabian geldings performing moderate exercise were randomly
assigned to 1 of 5 dietary treatments in a 5x5 Latin Square design to estimate dietary
crude protein (CP) requirement. Each period was 14 days in length and consisted of a
10-day diet adaptation followed by a 4-day total urine and fecal collection. All horses
consumed mixed grass hay containing 10% CP at 1% of their body weight. Concentrate
plus hay was fed to provide 677, 790, 903, 1016, and 1129 g CP daily corresponding to a
very low (V L), low (L), Control. high (H) and very high (V H) protein diet, respectively.
Nitrogen (N) retention, serum urea-N, and serum amino acid (AA) concentration were
used as response criteria. Nitrogen retention, basal and post-feeding/exercise serum urea-
N and serum AA concentration increased linearly (P<0-05) as‘daily CP intake increased.
Nitrogen retention increased (P<0-05) in horses fed H compared to Control and was not
different (P=1-0) compared to VH. Compared to Control, N retention was not different
(P>0'05) and was positive in horses fed VL and L diets. Post-feeding/exercising serum
urea-N increased in horses fed H compared to Control and was not different (P=0-43)
compared to VH. For a majority of the indispensable AA, post-feeding/exercise serum
concentration only differed (P<0-05) from Control in horses fed the VL protein diet.

These results indicate that H and VH protein diets exceeded CP requirement and that the

24

VL and L protein diets provided sufficient amount of protein for a horse subjected to
moderate exercise.
Key Words: Horse, Exercise, Protein, Nitrogen balance, Blood urea nitrogen
Introduction

There is a paucity of information on protein requirement of the exercising horse.
In the current NRC (1989), protein requirement for a horse performing moderate work is
based on a crude protein (CP) to digestible energy ratio estimated for the mature horse at
maintenance, resulting in protein requirement being 15 times higher than that of
maintenance. Protein catabolism comprises up to only 15% of energy production during
exercise, and protein is a relatively inefficient source of energy compared to that of
carbohydrate and fat (Lawrence, 1990). Moreover, feeding protein in excess of NRC
(1989) recommendations is a common practice in the horse industry (Hiney and Potter,
1996), but positive benefits associated with dietary protein supplementation have not
been documented in the horse (Lawrence, 1994). Thus, it is questionable whether the
relationship between dietary requirement for protein and energy is similar between
exercising and non-exercising horses. Excessive protein intakes are associated with
increased water demands, increased plasma urea concentration, higher energy costs
associated with nitrogen (N) excretion, and increased ammonia emission in barns (Meyer,
1987). In a study conducted by Freeman et al. (1988), N retention increased in horses
exposed to increasing workloads and fed increasing levels of dietary protein and energy,
indicating that exercise may increase the need for protein. However, to date, no
empirical studies have been designed to estimate CP requirement in response to graded

levels of dietary CP intake, thus the extent to which exercise increases dietary protein

25

requirement relative to maintenance remains unclear. We hypothesized that moderate
exercise in horses increases protein requirement below that recommended by NRC
(1989). The objective was to estimate dietary protein requirement using N retention,
serum urea-N, and sertun AA concentration as response criteria in horses exposed to the
same workload and fed graded levels of CP.
Materials and Methods
Animals. experimental design, and diets

Five mature Arabian geldings with an initial body weight of 473-3 i 164 kg were
selected and kept on clover-grass mix pasture during a pre-experiment exercise and
standardization period. Following conditioning, horses were randomly assigned to five
dietary treatments in a 5 x 5 Latin square design. Horses were housed individually in box
stalls (3'0 x 3-7 m) with free access to water. All horses consumed mixed grass hay
containing 10% CP (as fed basis) at 1% of their BW. Five diet concentrates were
formulated to achieve varying levels of crude protein intake. Ingredient and nutrient
composition of the concentrate diets are provided in Table l. A Control diet concentrate
was first formulated to meet NRC (1989) daily protein requirement estimate for moderate
exercise. A very low (VL) and a low (L) diet concentrate was formulated to provide 25%
and 125% lower protein respectively, relative to Control, and a high (H) and very high
(VH) diet concentrate was formulated to provide 125% and 25% higher protein
respectively. relative to Control. Thus, total diet, i.e., hay plus concentrate, was fed to
provide 677, 790, 903, 1016, and 1129 g CP daily corresponding to the very low (VL),
low (L), Control, high (H), and very high (VH) protein diet, respectively. The level of

protein intake in the VL, L, Control, H, and VH protein diets corresponded to a CP to

26

digestible energy (DE) ratio of 30, 35, 40, 45, and 50 g CP to 1 Meal DE, respectively.
To achieve a VL protein diet, com was used as the primary feed ingredient. Protein
concentration of the diet concentrates was increased by altering the com to oat ratio and
through the addition of soybean meal. Molasses was included in the concentrate mix as a
binder. Diet concentrates varied slightly in DE content and were fed to achieve the
desired protein intake levels. This resulted in minor energy deficiencies, thus corn oil
was top-dressed in small amounts to assure NRC (1989) energy requirement for moderate
exercise was met for each horse. Vitamin-mineral mix was top-dressed once daily to
provide NRC (1989) recommended levels of Ca, P, Vitamin A, Vitamin D, Vitamin E,
and Se. Meals were fed twice daily at 0700 and 1600. Acclimation of horses to
treatment diets consisted of feeding mixed grass hay at 1-2-1-5 % of each horse’s BW and
gradually introducing the Control diet concentrate during the last week of conditioning.
' Exercise Protocol

Prior to the start of the experiment, horses were subjected to a 6-week
standardization period. During the initial week, horses were trotted daily for 6
consecutive d on a mechanical walker (Free Flow Equineciser, Centaur Horse. Walkers,
Mira Loma, CA) at a speed of 3-6 m per sec for 10 min. Thereafter, workload was
increased each week by 10 min increments until horses were trotting 60 min per d.
During the experimental period, horses were exercised bi-directionally on the walker at
the trot at approximately 3-6 111 per sec, 60min per (1, 6 (1 per week. Exercise was
performed in the morning 2 h post-feeding. Heart rate (HR) monitors were used to assess
work load and to verify that pre-experiment fitness levels, based on resting and working

HR measured at the end of the standardization period, were maintained. Body weights

27

and body condition scores (BCS) were recorded every two weeks. Body condition score
was assessed according to NRC (1989) guidelines.

This study was approved by Michigan State University All University Committee
on Animal Use and Care.
Sample collection

The study consisted of five collection periods. Each period was 14 d in length
and consisted of a 10-d diet adaptation followed by a 4-d total fecal and urine collection.
Feces and urine were collected using gelding collection harnesses (Equisan Marketing,
Melbourne, Australia) and emptied every 5 h or more frequently as needed. At each
emptying, all feces were bagged and approximately 10% of the total urine volume was
sampled. Feces and urine were immediately stored at -20°C. At the end of each
collection period, daily fecal samples were thawed, pooled, weighed and homogenized
using a 136-kg mechanical mixer. Sub samples were collected (~500 g) and frozen at
-20°C. Daily urine samples were pooled, mixed, and a sample of pooled urine stored at
-20°C. During exercise, harnesses were removed. Horses did not urinate during this
time. If horses defecated, care was taken to collect and weigh the fecal matter. This fecal
matter was then discarded.

Blood samples (20 mL) were drawn from each horse via jugular venipuncture on
d 3 and 4 of the collection period. Blood samples were drawn prior to the moming meal
(15 h post-feeding) to obtain basal metabolite values. Basal values were obtained to
represent long-term metabolism. Horses were fed following blood collection and

exercised 2 h later. An additional blood sample was obtained within 10-20 min of

28

completion of exercise. Blood samples were centrifuged (Beckman, GS-6KR Centrifuge,
Fullerton, CA) at 3000 rpm for 15 min at 4°C and serum and plasma stored at -20°C.
Sample Analysis

For chemical analysis, fecal samples were freeze-dried (VirTis model 25-SRC,
VirTis Co., Gardiner, NY). Fecal and feed samples (hay and concentrate) were finely
ground using a cyclone mill (Foss Cyclotec sample mill 1093, Hoganas, Sweden) with a
1-mm mesh screen. Nitrogen concentration in feces, urine and feed was determined
using an automated N analyzer (Leco FP-2000, Leco Co., St. Joseph, MI; AOAC No.
9903). Dry matter of feed was determined following a 24-h drying period at 80°C using
a drying oven (Fisher Isotemp, Fisher Scientific, Hanover Park, IL). Amino acid analysis
was performed on feed samples using the Pico-Tag method (Waters Co., Milford, MA)
following a 24-h acid hydrolysis in 6N HCl at 113°C and 121 mm Hg. Norleucine was
used as an internal standard. Samples were derivatized with phenylisothiocyanate and
analyzed by high pressure liquid chromatography (HPLC) (Alliance 2690, Waters Co., .
Milford, MA) fitted with a 30-cm Pico-Tag column (Waters Co., Milford, MA). Amino
acid analysis was performed on basal and post-feeding/exercise serum samples using the
Pico-Tag method (Waters Co., Milford, MA). Briefly, protein was first precipitated by
mixing 200 11L serum with 1 mL Trifluoroacetic acidzmethanol (1:10) and centrifuging
(eppendorf centrifuge 5417R, Brinkmann Instruments, Westbury, NY) at 7000 rpm for 15
min at 4°C. The supernatant (155 11L) was removed and norleucine (125 mM) added as
an internal standard. This was followed by evaporation to dryness at 37°C with a
centrifuge evaporator (HETO Vacuum Concentration System, ATR, Laurel, MD).

Samples were derivatized with phenylisothiocyanate and analyzed by HPLC (Waters Co.,

29

Milford, MA) fitted with a 30-cm Pico-Tag column (Waters Co., Milford, MA).
Digestible lysine intake was calculated from the apparent fecal lysine digestibility
coefficient determined by Wickens et al. (unpublished).

Urea-N analysis was performed on serum samples using a commercially available
colorimetric assay (procedure no. 640-A, Sigma, St. Louis, MO). Absorbance was read
at 570 nm using a spectrophotometer (Beckman, DU 7400, Schaumburg, IL). For urinary
creatinine analysis, urine samples were diluted 1:10 and creatinine determined using a
commercially available colorimetric assay (procedure no. 555, Sigma, St. Louis, MO).
Absorbance was read at 500 nm using a spectrophotometer (Beckman, DU 7400,
Schaumburg, IL). Urinary orotic acid was analyzed using a procedure adopted from
Adachi et a1. (1963). Briefly, urine was acidified to pH 2-3 and 1 mL urine was added to
2 mL citric acid-potassium citrate buffer (02 M) and 05 mL saturated bromine water.
Samples stood for l min at room temperature followed by the addition of 1 mL ascorbic
acid (5%). Samples were then placed in a warm water bath (40°C) for 5 min, followed by
the addition of 2 mL p-dimethylaminobenzaldehyde (25%). Samples were then kept in
the warm water bath for 10 additional min, cooled under running water and the
absorbance read at 480 nm using a spectrophotometer (Beckman, DU 7400, Schaumburg,
IL).

Statistical Analysis

Data were subjected to ANOVA using the PROC MIXED procedure of SAS
(SAS Inst., Inc., Cary, NC). Effects of horse, period, and diet were included in all
statistical models. Horse was treated as a random effect. Differences between all pair-

wise comparisons were evaluated using the Tukey-Kramer test (Younger, 1998).

30

Relationships between dietary CP intake and N balance parameters, serum amino acid
concentration, serum urea-N concentration, urinary creatinine concentration, and urinary
orotic acid concentration were determined using contrast (linear and quadratic)
comparisons with orthogonal polynomials. Coefficients used for contrasts were based on
the calculated CP intake of 677, 790, 903, 1016, and 1129 g per d. Statistical
significance was based on an experiment-wise type-I error rate of 0-05. Regression
analysis was performed using the GLM procedure of SAS (SAS Inst, Inc., Cary, NC) to
determine the nature of the relationship between total and digestible lysine intake and N
retention. Using the PROC UNIVARIATE procedure of SAS (SAS Inst., Inc., Cary,
NC), total and digestible lysine intake versus N retention data were determined to be
normally distributed with homogeneous variance.
Results
Horse performance data

Horse performance data including body weight, BCS, resting and working HR, as
affected by period, are shown in Table 2. There was no effect of diet (P>0'05) on body
weight, BCS, resting or working HR. Pre-experiment body weight was not different
(P>0-05) compared to that in period 1. Body weight was higher (P<0-05) in period 5
compared to period 1, but was not different when compared to that in periods 2, 3, and 4.
Pre-experiment BCS was not different (P>0-05) compared to that in period 1. Body
condition score increased (P<0'05) from period 1 to 2, and there was no difference
(P>0-05) in BCS between periods 2, 3, 4, and 5. There was no difference (P>0-05) in
resting or working HR across periods, and HR throughout the trial did not change

(P>0'05) with respect to HR taken at the end of the standardization period.

31

Nitrogen balance

Nitrogen intake, losses, and retention data, as affected by dietary CP level are
presented in Table 3. Nitrogen intake, fecal N excretion, urinary volume, urinary N
excretion, and N retention increased linearly (P<0-05) as daily CP intake increased. A
quadratic relationship between these parameters and dietary CP intake was not found
(P>0'05). Compared to Control, fecal N was not different (P>O-05) in horses fed VL, L,
H and VH protein diets. Fecal N was higher (P<0-05) in horses fed the VH protein diet
compared to that in horses fed the VL diet. Compared to Control, urinary volume was
not different in horses fed VL, L, H, and VH protein diets. However, urinary volume was.
higher (P<0-05) in horses fed VH compared to that in horses fed VL. Compared to
Control, there was no difference in urinary N excretion in horses fed L or H protein diets.
Urinary N was higher (P<0105) in horses fed VH and lower (P<0-05) in horses fed VL
compared to that in horses fed Control. Compared to Control, there was no difference in
N retention in horses fed the VL (P=0-23) and L (P=0-80) protein diets. Compared to
Control, N retention increased (P<0-05) in horses fed H and was not different (P=1-0)
compared to VH. There was no quadratic relationship (P>0-05) between N retention and
increasing dietary CP intake. Relationship between N retention and daily total and
digestible lysine intake is presented in figure 4 and 5, respectively. Nitrogen retention
increased linearly (P<0-05) as daily total and digestible lysine intake increased. There
was no quadratic relationship (P>0-05) between N retention and increasing total or

digestible lysine intake.

32

Serum urea nitrogen, creatinine, and orotic acid

Basal and post-feeding/exercise serum urea-N, arginine, citrulline, omithine, and
glutamine concentration as affected by dietary CP level are presented in figures 6, 7, 8, 9,
and 10, respectively. Basal and post-feeding/exercise serum urea-N concentration
increased linearly (P<0-001) as dietary CP intake increased. Post-feeding/exercise serum
urea-N in horses fed VL tended to differ (P<0-06) compared to that in horses fed the L
protein diet but did not differ between horses fed L and Control (P=0-23). Post-
feeding/exercise serum urea-N increased (P<0-05) in horses fed VH and H protein diets
versus VL, L, or Control diets. Basal serum arginine, citrulline, and omithine
concentration were not different (P>0-05) among dietary treatments. A quadratic
relationship (P<0-05) was found between dietary CP and post-feeding/exercise serum
arginine concentration. Post-feeding/exercise serum arginine concentration increased
(P<0-05) as dietary C P intake increased, and reached plateau in horses fed the Control
diet. Post-feeding/exercise serum citrulline, omithine, and glutamine concentration
increased linearly (P<0-05) as dietary CP intake increased.

Urinary creatinine, urinary orotic acid, and orotic acid-to creatinine ratio data are -
presented in Table 4. Urinary creatinine and orotic acid concentration were not affected
(P>0-05) by dietary CP intake. Daily urinary orotic acid output increased linearly
(P<0-05) with increasing daily CP intake. Orotic acid to creatinine ratio was not different
. (P<0'05) between dietary treatments. There was no quadratic relationship (P>0-05)

between CP intake and the above parameters.

33

Serum amino acid concentration

Basal serum indispensable AA concentration data, as affected by dietary CP level
are presented in Table 5. For a majority of the indispensable AA, basal serum
concentration did not differ G’>0-05) among dietary treatments. Serum isoleucine and
valine concentration increased linearly (P<0‘05) as dietary CP intake increased. Post-
exercise serum indispensable AA concentration data are presented in Table 6. Post-
feeding/exercise serum concentration of all indispensable AA increased linearly (P<0-05)
as dietary CP intake increased. For a majority of the indispensable AA, post-
feeding/exercise serum concentration only differed (P<0105) from Control in horses fed
the VL protein diet. A quadratic relationship (P<0~05) was found for serum total sulfur
AA (TSAA) concentration. Total sulfur AA concentration increased (P<0-05) as CP
intake increased, and reached plateau in horses fed the Control diet.
Discussion

The current NRC (1989) crude protein (CP) requirement estimate for the
moderately exercised horse is derived from a CP to digestible energy (DE) ratio of 40 g
to 1 Meal determined for the mature horse at maintenance. Consequently, dietary CP
recommendation for the moderately exercised horse is 15 fold the recommended dietary
CP requirement for maintenance. We raised the question whether this ratio can also be
applied for the exercising horse. In this study, N retention, serum urea-N, and serum AA
concentration were used as response criteria to estimate protein requirement of Arabian
horses exposed to the same workload and fed varying levels of CP.

Freeman et a1. (1988) showed that N retention increased in exercising horses

exposed to increasing workloads and fed increasing level of dietary protein and energy.

34

In our study, in horses performing the same level of exercise, N retention increased as
dietary CP increased. Nitrogen retention ranged from 21 -0 g per (1 in horses fed the VL
protein diet up to 430 g per d in horses fed VH. Gibbs et a1. (1988) observed positive N
retention of 311 g per d in mature ponies at maintenance consuming alfalfa hay (18-1%
CP). Similarly, in a study conducted by Slade et a1. (1970), positive N retention as great
as 20 g per (1 was found in mature horses at maintenance consuming between 800 and
900 g CP per d. We had hypothesized protein requirement for moderate exercise to be
lower than that of NRC (1989). Surprisingly, N retention increased and was seemingly
maximized in horses fed 1016 g CP per (1, corresponding to a protein level of 125%
above that recommended by NRC (1989). Indeed, a linear rather than quadratic response
between N intake and retention was obtained, which precluded estimating protein
requirement using broken point analysis as performed by Dourmad and Etienne (2002).
Considering the difference in protein quality between the lower and higher protein diets,
the relationship between total or digestible lysine intake and N retention was examined
and found also to be linear. Jackson (1999) has reported that at higher intakes of protein,
measured N retention is consistently positive and suggested» this to be an artifact inherent
to N balance studies. Nitrogen retention at higher protein intake may be overestimated
due to an overestimation of N intake and underestimation of N losses (Jackson, 1999).
However, N retention has been used successfully to determine protein requirement in
other species including humans and swine (Rose, 1957.; Hegsted, 1976; Young and
Scrimshaw, 1978; King et al., 1993; Dourmad and Etienne, 2002; Otto et al., 2003). In
the current study, careful attention was given to collection and measurements of orts and

waste materials consistently across treatments. Estimated N losses during the 60-min

35

work bout were 6, 5, 6, 6, and 7 g per day for the VL, L, Control, H, and VH protein
diets, respectively. While this overestimated our N retention value, this overestimation
was uniform across treatments. Other reasons for overestimation of protein requirement
estimated from N balance studies include unmeasured N losses in sweat, skin
desquarnation, and moderate day to day changes in N metabolism (Hegsted, 1963; Young
and Scrimshaw, 1978; Garlick etal., 1999). In our study, urine and fecal collection was
performed over a 4-d period, thus accounting for possible daily fluctuations in N
metabolism. The extent to which N loss in sweat in particular and skin desquamation
contribute to the overall N losses in horses has not been addressed. In humans, N losses
associated with sweat and skin desquamation represents a small contribution to the
overall N losses (Jackson, 1999). Miller-Graber et al. (1991a) reported that in exercising
Quarter Horse mares fed a high protein (185%) diet, urea-N excretion in sweat increased.
Nitrogen losses in sweat were not accounted for in our study, and it is possible that sweat
may have been an important route of N excretion at higher protein intake thus resulting in
an overestimation of protein requirement.

Blood urea-N has been used successfully to determine protein and amino acid
requirements of the lactating sow (Coma et al., 1996). In this study, we have also
measured blood urea-N as a response criterion to estimate protein requirement. Basal and
post-feeding/exercise serum urea-N increased as daily CP intake increased indicating that
N retention was overestimated at higher protein intake. Our serum urea-N results are in
agreement with those obtained by Miller and Lawrence (.1988). In their study, feeding a
diet containing high protein concentration (185% CF) to exercising Quarter Horse mares

increased plasma urea-N, indicating that protein intake was in excess of protein

36

requirement (Miller and Lawrence, 1988). In the current study, the fact that senun urea-
N response to dietary CP intake was linear rather than exponential further indicates that
an additional route of N excretion, possibly via sweat or salvage by the hind gut
contributed to N losses at higher protein intake. In non-exercising humans consuming 74
g protein per (1, 40% of urea produced was salvaged via colonic microflora activity
(Danielsen and Jackson, 1992). Due to the high dependence on hind gut fermentation in
the horse, it is possible that substantial amounts of urea are utilized by the microbial
population.

Sermn concentration of arginine, the direct precursor of urea, was maximized in
horses fed the Control diet. Miller-Graber et al. (1991a) have suggested that in horses
consuming a high protein diet, urea cycle capacity in the liver may be exceeded.
However, in this study neither urinary orotic acid concentration nor orotic acid to

creatinine ratio changed with CP intake, and both citrulline and omithine concentration

_/

increased with increasing CP intake. Excretion of orotic acid and the orotic acid to
creatine ratio have been used to evaluate urea cycle activity in exercising horses (Miller-
Graber et al., 1991a; Miller-Graber et al., 1991b). Increased excretion of urinary orotic
acid has been observed in humans and rats as indicative of impaired urea cycle activity
(Fico et al., 1984; Reyes et al., 1994). In the current study, because urinary orotic acid
concentration remained similar across dietary treatments, it is unlikely that the enzymatic
capacity of the urea cycle was exceeded in horses consuming the high protein diets.
Rather, the fact that urea increased despite a plateau in serum arginine concentration may

have resulted from an increase in hepatic arginase activity in horses fed protein level

37

above requirement. Thus, arginine may be an important indicator of protein status in the
horse.

Glutamine may be an important route for N excretion in mammals (Remesy et al.,
1997), but has not been studied in horses. In this study, while basal serum glutamine
concentration decreased linearly with increasing CP intake, post-feeding/exercise
concentration increased linearly, indicating a reliance on glutamine for removal of N at
higher protein intake.

Plasma free AA concentration has been used extensively in many species to .
evaluate dietary protein quality and indispensable AA requirement (Richardson et al.,
1965; Young et al., 1971; Marchini et al., 1993). Although studies have been conducted
in horses to investigate the effect of exercise on plasma free AA concentration (Russell et
al., 1986; Poso et al., 1991; Trottier et al., 2002), no empirical studies have been
conducted to address the response of circulating indispensable AA to graded levels of
dietary CP intake in the exercised horse. In the current study, serum AA concentration
was used as a response criterion to estimate CP requirement in horses fed varying levels
of CP and exposed to moderate exercise. Except for histidine, post-feeding/exercise
serum concentration for all indispensable AA increased as dietary protein intake
increased. Mean serum concentration for the majority of indispensable AA, in particular
lysine, was higher in horses fed the Control diet compared to that in horses fed the VL
diet, indicating that these AA were in excess of requirement when fed in the Control diet.

The fact that response in N retention to increased CP intake was linear rather than
quadratic could be the result of compensatory increase in N retention in horses fed from a

low to higher protein diets as suggested by Slade et al. (1970). Increased lean body mass

38

or protein synthesis may explain in part the observed increase in N retention. Horse
performance and level of condition remained consistent throughout the experiment.

There were no differences (P>0-05) in body composition based on muscle mass
calculated from 24-h urinary creatinine excretion (Appendix D) across dietary treatments
or periods. However, from period 1 to period 5, there was a numerical increase in muscle
mass of 368 kg. This response parallels the observed increase in protein accretion of 131
and 269 g per d in horses fed the VL and VH protein diets, respectively. This represents
lean tissue accretion of 188 and 384 g per d in horses fed the VL and VH protein diets,
respectively.

In conclusion, while horses fed protein levels below that of NRC (1989)
maintained a positive N balance, N retention increased when horses were fed protein
above that of NRC (1989). Although feeding protein at 125% above NRC (1989) level
was necessary to achieve maximal N retention, this may not be indicative of requirement.
The blood urea-N (BUN) results challenge the N retention response as both long and
short term changes in BUN concentration indicated that protein intake was in excess of
requirement, when fed above NRC (1989). In addition to the BUN results, serum AA
concentrations were also elevated, thus casting doubt on the validity of the N retention
response. Arginine concentration plateaued in horses fed NRC (1989) recommended
protein level and may be an indicator of protein status in the horse. We had hypothesized
that protein requirement for the moderately exercised horse to be less than that of NRC
(1989). Our results indicate that protein requirement estimated from a ratio of 30 or 35 g

CP to 1 Meal DE is sufficient to meet the protein need for moderate exercise.

39

Table 1. Ingredient and nutrient composition of concentrate diets (as-fed basis)

 

 

 

Diett
VL L Control H VH
Ingredients
Com, yellow, (%) 94-0 50-0 40-0 34-0 28-0
Oats, (%) 0-0 44-0 48-0 46-0 44-0
Soybean meal, (%) 0-0 0-0 6-0 14-0 22-0
Molasses, (%) 6-0 6-0 6-0 6-0 6-0
Vitamin-mineral mix: + + + + +
Trace mineralized salt§ + + + + +
Corn 011' + + + + +
Nutrients
Calculated
Digestible energy, (kcal/kg) 3330-0 3100-0 3080-0 3090-0 3100-0
Crude protein, (%) 8-62 10-43 12-90 1589 18-88
Lysine, (%) 0-24 0-30 0-47 0-70 0-92
Analyzed
Dry matter, (%) 89-32 90-12 91-10 91-50 91-78
Gross energy, (kcal/kg) 3939-7 4069-3 41 l 19 4161 -0 4166-2
Crude protein, (%) 8-48 9-67 11-83 15-88 18-57
Acid detergent fibre, (%) 3-85 5-51 6-71 6-28 6-94
Neutral detergent fibre. (%) 7-48 15-05 17-55 15-97 16-41
Amino Acids
Indispensable
Arginine 0-41 0-58 0-78 1-12 137
Histidine 0-20 0-24 0-29 0-49 0-62
Isoleucine 0-30 0-36 0-47 0-69 0-83
Leucine 108 0-99 l-l 1 1-42 161
Lysine 0-17 0-25 0-40 0-71 0-93
Phenylalanine 0-36 0-43 0-53 0-77 0-92
Phenylalanine + Tyrosine 0-62 0-74 0-92 1-30 155
Threonine 0-24 0-28 0-35 0-48 0-59
Valine 0-39 0-48 0-59 0-82 0-97
Dispensable
Alanine 0-64 0-62 0-68 0-85 0-95
Aspartate 0-66 0-84 1 ~13 1-69 2-08

40

Table 1. Continued

Glutamate 1-80 2-13 2-54 3-29 3-80
Glycine 0-26 0-37 0-45 0-66 0-80
Proline 0-81 0-74 0-86 1-07 1-20
Serine 0-39 0-47 0-57 0-78 0-92
Tyrosine 0-26 0-31 0-38 0-53 0-63

 

' VL. L. Control, H and VH diets correspond to 677, 790, 903, 1016 and 1129 g of crude protein intake per
day, respectively.

‘Vitamin-mineral mix was top-dressed at 28 g per day to provide 15000 1U Vitamin A, 3125 IU Vitamin
D3, 375 IU Vitamin E, 4480 mg Ca, 4480 mg P and 0-84 mg Se.

§Trace mineralized salt was fed free choice in block form.

I Value of corn oil top dressed daily to meet NRC (1989) recommended digestible energy requirement for
moderate exercise varied based on body weight of horse and averaged 609, 375, 259, 351, and 305 g for
VL, L, Control, H, and VH protein diets respectively.

41

Table 2. Horse performance data

(Least squares mean values with standard errors)

 

Item Mean SEM

 

Body Weight. (kg)

Pre-experimentl 473 -3 16-4
Period 1 477-6” 17-5
Period 2 41115-4'b 17-5
Period 3 488-4” 17-5
Period 4 490-2” 17-5
Period 5 491 -4' 17-5
Body Condition Score
Pro-experiment 6-3 0-4
Period 1 ‘ 6-5" 04
Period 2 ' 7-1' 04
Period 3 7-1' 0-4
Period 4 7-0‘ 0-4
Period 5 7-0' 0-4
Resting Heart Rate. (bpm)
Pre-experiment 35-2 2-0
Period 1 330a 12
Period 2 32-4' 1-2
Period 3 32-9'I 13
Period 4 31-8‘I 1-2
Period 5 32-8a 1-2
Working Heart Rate, (bpm)
Pre-experiment 88-0 20
Period 1 87-2‘ 2-6
Period 2 90-4‘ 2-6
Period 3 90-7'I 28
Period 4 86-6‘I 26
Period 5 89-8' 2-6

 

‘b Least squares mean values within a column with unlike superscript letters differ (P<0-05).

42

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46

Figure 4. Relationship between daily total lysine intake and nitrogen retention. Response
between daily lysine intake and nitrogen retention was linear and described by

Y = 0-734x + 4763 (r2 = 0-42; P<O-05).

47

 

 

 

 

60p

Eu 603.800.. comoiz

I0
6
lo
5
lo
4
O
o
1a...
I
O
O
0 P n n P o
2
o o o o o o
5 4 3 2 1

Lysine intake, gld

48

Figure 5. Relationship between daily digestible lysine intake and nitrogen retention.
Response between daily digestible lysine intake and nitrogen retention was linear and

described by Y = 0823): + 1161‘) (r2 = 0-43; P<O-05).

49

Nitrogen retention, gld

60r-

50»

40-

20~

10-

 

10

15

I I I r

20 25 30 35
Digestible lysine intake, gld

50

 

4O

Figure 6. Effect of dietary crude protein intake on basal and post-feeding/exercise serum
urea-nitrogen concentration. Dietary crude protein level of 677, 790, 903, 1016, 1129 g
CP per d corresponds to VL, L, Control, H, and VB protein diets, respectively. Closed
(0) and opened (0) circle indicates basal and post-feeding/exercise serum urea-nitrogen
concentration, respectively. Linear response between dietary crude protein intake and
basal and post-feeding/exercise serum urea-nitrogen concentration was significant
(P<O'OS). Quadratic response between dietary crude protein intake and basal and post-

feeding/exercise serum urea-nitrogen concentration was not significant (P>O-05).

51

Urea nitrogen, mgIdL

N
N

N
O

.L
Q

.L
O)

.3
.5

.3
N

.5
O

 

   

. Basal
O Post-feedinglexercise

 

677 790 903 1016
Dietary crude protein, gld

52

1129

Figure 7a. Effect of dietary crude protein intake on basal and post-feeding/exercise serum
arginine concentration. Dietary crude protein level of 677, 790, 903, 1016, 1129 g CP
per (1 corresponds to VL, L, Control, H, and VH protein diets, respectively. Closed (0)
and opened (O) circle indicates basal and post-feeding/exercise serum arginine
concentration, respectively. Linear and quadratic response between dietary crude protein
intake and basal serum arginine concentration was not significant (P>0°05). Linear and
quadratic response between dietary crude protein intake and post-feeding/exercise serum

arginine concentration was significant (P<O'05).
Figure 7b. Effect of dietary crude protein intake on post-feeding/exercise serum arginine

concentration. Solid (—) line represents predicted post-feeding/exercise serum arginine

concentration in response to dietary CP intake. Response was quadratic (P<O-05).

53

Arginine, umollL

Arginine, umolIL

180 -

160 -

140 L

120 -

100 -

 

80

O Post-feedinglexercise

 

180 -

160 -

140 -

120 -

100 -

 

80

677

790 903 1016
Dietary crude protein, gld

1129

 

7

 

600

700

800 900 1000
Dietary crude protein, gld

54

1100

1200

Citrulline, umollL

120
110
100
90
80

70

 

Q Basal
O Post-feedinglexercise

 

677 790 903 101 6 1 1 29
Dietary crude protein, gld

56

Figure 8. Effect of dietary crude protein intake on basal and post-feeding/exercise serum
citrulline concentration. Dietary crude protein level of 677, 790, 903, 1016, 1129 g CP
per d corresponds to VL, L, Control, H, and VH protein diets, respectively. Closed (0)
and opened (O) circle indicates basal and post-feeding/exercise serum citrulline
concentration, respectively. Linear and quadratic response between dietary crude protein
intake and basal serum citrulline concentration was not significant (P>O'05). Linear
response between dietary crude protein and post-feeding/exercise serum citrulline
concentration was significant (P<O-05). Quadratic response for post-feeding/exercise

serum citrulline concentration was not significant (P>O-05).

55

Figure 9. Effect of dietary crude protein intake on basal and post-feeding/exercise serum
omithine concentration. Dietary crude protein level of 677, 790, 903, 1016, 1129 g CP
per (1 corresponds to VL, L, Control, H, and VH protein diets, respectively. Closed (0)
and opened (0) circle indicates basal and post-feeding/exercise serum omithine
concentration, respectively. Linear and quadratic response between dietary crude protein
intake and basal serum omithine concentration was not significant (P>O-05). Linear
response between dietary crude protein and post-feeding/exercise serum omithine
concentration was significant (P<0-05). Quadratic response for post-feeding/exercise

serum omithine concentration was not significant (P>0-05).

57

Ornithine, umollL

80

70

60

50

4O

30

 

O Post-feedinglexercise

 

 

677

790 903 1 016
Dietary crude protein, gld

58

1129

Figure 10. Effect of dietary crude protein intake on basal and post-feeding/exercise serum
glutamine concentration. Dietary crude protein level of 677, 790, 903, 1016, 1129 g CP
per (1 corresponds to VL, L, Control, H, and VH protein diets, respectively. Closed (0)
and opened (O) circle indicates basal and post-feeding/exercise serum glutamine
concentration, respectively. Linear response between dietary crude protein intake and
basal and post-feeding/exercise serum glutamine was significant (P<O-05). Quadratic
response between dietary crude protein intake and basal and post-feeding/exercise serum

glutamine was not significant (P>O-05).

59

Glutamine, umollL

400

350

300

250

200

150

 

 

Q Basal
o Post-feedinglexercise
677 790 903 1016 1 129

Dietary crude protein, gld

6O

LITERATURE CITED

61

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Coma J, Zimmerman DR, & Carrion D (1996) Lysine requirement of the lactating sow
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Danielsen M & Jackson AA (1992) Limits of adaptation to a diet low in protein in normal
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Dourmad JY & Etienne M (2002) Dietary lysine and threonine requirements of the
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F ico ME, Motyl T, & Milner JA (1984) Species Comparison of the influence of ammonia
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Lawrence LM (1990) Nutrition and fuel utilization in the athletic horse. Equine Practice
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Lawrence LM (1994) Nutrition in the athletic horse. In: The athletic horse: Principles
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Co., Philadelphia, 205-230.

Marchini J S, Cortiella J, Hiramatsu T, Chapman TE, & Young VR (1993) Requirements
for indispensable amino acids in adult humans: longer-term amino acid kinetic study
with support for the adequacy of the Massachusetts Institute of Technology amino acid
requirement pattern. Am J Clin Nutr 58, 670-683

Meyer H (1987) Nutrition of the equine athlete. In: Equine Exercise Physiology 2. Eds:
Gillespie JR & Robinson NE, ICEEP Publications, Davis, CA, 644-673.

Miller-Graber P, Lawrence L, Foreman J, Bump K, Fisher M, & Kurcz E (1991a) Effect
of dietary protein level on nitrogen metabolites in exercised Quarter Horses. In:
Equine Exercise Physiology 3. Eds: Persson SGB, Lindholm A, & Jeffcott LB, ICEEP
Publications, Davis, CA, 305-314.

Miller-Graber P, Lawrence L, Fisher M, Bump K, Foreman J, & Kurcz E (1991b)
Metabolic responses to ammonium acetate infusion in exercising horses. Cornell Vet
81, 397-410.

Miller PA & LM Lawrence (1988) The effect of dietary protein level on exercising
horses. J Anim Sci 66, 2185-2192.

National Research Council (1989) Nutrient requirements of horses. 5th rev ed. Natl
Acad Press, Washington, DC.

Otto ER, Yokoyama M, Ku PK, Ames NK, & Trottier NL (2003) Nitrogen balance and
ileal amino acid digestibility in growing pigs fed diets reduced in protein
concentration. J Anim Sci 81, in press.

Poso AR, Essen-Gustavsson B. Lindholm A, & Persson SGB (1991) Exercise induced
changes in muscle and plasma amino acid levels in the standardbred horse. In: Equine
Exercise Physiology 3. Eds: Persson SGB, Lindholm A, & Jeffcott LB, ICEEP
Publications, Davis, CA, 202-208.

Remesy C. Moundras C, Morand C, & Demigne C (1997) Glutamine or glutamate release

by the liver constitutes a major mechanism for nitrogen salvage. Am J Physiol 272,
G25 7-G264.

Reyes AA, Karl IE, & Klahr S (1994) Role of arginine in health and in renal disease. Am
J Physiol 36, F331-F346.

Richardson LR, Hale F, & Ritchie SJ (1965) Effect of fasting and level of dietary protein
on free amino acids in pig plasma. J Anim Sci 24, 368-372.

63

Rose WC (1957) The amino acid requirements of adult man. Nutr Abstr Rev 27, 631-647.

Russell MA, Rodiek AV, & Lawrence LM (1986) Effects of exercise, training and
sampling location on selected plasma free amino acids in horses. Can J Anim Sci 66,
827-831.

Trottier NL, Nielsen BD, Lang KJ, Ku PK, & Schott HC (2002) Equine endurance
exercise alters serum branched-chain amino acid and alanine concentrations. Equine
Vet J Suppl 34, 168-172.

Young VR, Hussein MA, Murray E, & Scrimshaw NS (1971) Plasma tryptophan
response curve and its relation to tryptophan requirements in young adult men. J Nutr
101, 45-60.

Young VR & Scrimshaw NS (1978) Nutritional evaluation of proteins and protein

requirements. In: Protein Resources and Technology. Eds: Milner M, Scrimshaw NS,
& Wang DIC, Avi, Westport, Connecticut, 136-173.

64

CHAPTER 3

APPARENT FECAL NITROGEN AND AMINO ACID DIGESTIBILITY OF
DIETS FED TO EXERCISING HORSES

65

ABSTRACT

APPARENT FECAL NITROGEN AND AMINO ACID DIGESTIBILITY OF DIETS
FED TO EXERCISING HORSES

By

Carissa Lee Wickens

Five mature Arabian geldings were used in a 5x5 Latin square design to
determine apparent nitrogen (N) and amino acid (AA) digestibility of five
hayzconcentrate mix diets. Horses were randomly assigned to l of 5 dietary treatments.
Each period consisted of a 10-day diet adaptation followed by a 4-day total urine and
fecal collection. All horses consumed mixed grass hay containing 10% crude protein
(CP) at 1% of their body weight. Total diet (hay plus concentrate) was formulated to
provide 677, 790, 903, 1016, and 1129 g CP daily corresponding to a very low (VL), low
(L), Control, high (H) and very high (V H) protein diet respectively. Diets were
composed of corn, oat, and SBM in varying proportions. Apparent fecal N and AA
digestibility increased linearly (P<O-05) as dietary CP intake increased. Compared to
Control, N digestibility was not different (P>O-OS) in horses fed L, H, and VH. Apparent
fecal lysine digestibility was not different (P>0-05) in horses fed VL and L compared to
that in horses fed Control and was higher (P<0-05) in horses fed H and VH. Inclusion of
SBM to the Control, H, and VH protein diet concentrates resulted in higher protein
quality and contributed to the increase in apparent fecal N and AA digestibility.

Key words: Horse, protein, amino acid, digestibility

66

Introduction

The current NRC (1989) protein requirement estimate for the exercising horse is
derived from a crude protein (CP) to digestible energy (DE) ratio of 40 g to 1 Meal
estimated for the horse at maintenance. This ratio was determined from horses
consuming a forage diet with a protein digestibility of 46%. Several studies have
reported protein digestibility to be higher than 46%. In ponies fed Coastal Bermuda
grass, low-protein alfalfa, or high-protein alfalfa hay, Gibbs et al. (1988) reported total
tract N digestibility of 57, 66, and 74%, respectively. Similarly, Slade et al. (1970), Hintz
et a1. (1971), and Glade (1984) have provided estimates of protein digestibility of diets
fed to horses and ponies at maintenance ranging from 35 up to 798%. However,
ingredients used in diets tested in earlier studies (Slade et al., 1970; Reitnour and Treece,
1971; Glade, 1984) are not representative of those routinely fed to horses.

Exercising horses are commonly fed a mixed ration consisting of increased level
of concentrate relative to forage in order to meet the increased energy demands
associated with physical activity. The concentrate portion is typically composed of
ingredients of higher protein quality than that found in most forages. Thus, it is expected
that protein digestion of a diet formulated for the exercising horse will be higher than that
reported by the current NRC (1989). Moreover, protein digestibility of diets fed to
exercising horses has not been determined. The objective of this study was to determine
apparent fecal N and AA digestibility of five hayzconcentrate mix diets fed to horses
performing moderate exercise. We hypothesized that apparent N digestibility would be
higher than values reported in NRC (1989) and that inclusion of soy bean meal (SBM)

increases apparent N and AA digestibility.

67

Materials and Methods
Animals, experimental design, and diets

As part of a N balance study conducted to estimate the protein requirement of the
moderately exercised horse (Wickens et al., unpublished), five mature Arabian geldings
with an initial body weight of 473-3 :1: 16-4 kg were randomly assigned to five dietary
treatments in a 5 x 5 Latin square design. Horses were housed individually in box stalls
(3-0 x 3-7 m) with free access to water. All horses consumed mixed grass hay containing
10% CP (as fed basis) at 1% of their BW. Five diet concentrates were formulated to
achieve varying levels of crude protein intake. Ingredient and nutrient composition of the
concentrate diets are provided in Table 7. A Control diet concentrate was first
formulated to meet NRC (1989) daily protein requirement estimate for moderate exercise.
A very low (VL) and a low (L) diet concentrate was formulated to provide 25% and
12-5% lower protein respectively, relative to Control, and a high (H) and very high (VH)
diet concentrate was formulated to provide 126% and 25% higher protein respectively,
relative to Control. Thus, total diet (hay plus concentrate) was formulated and fed to
provide 677, 790, 903, 1016, and 1129 g CP daily corresponding to the very low (VL),
low (L), Control, high (H), and very high (VH) protein diet, respectively. To achieve a
very low protein diet, corn was used as the primary ingredient. Protein concentrations of
the diet concentrates were increased by altering the corn to oat ratio and through the
addition of soybean meal to the Control, H, and VH protein concentrates. Molasses was
included in the concentrate mix as a binder. Diet concentrates varied slightly in DE
content and were fed to achieve the desired protein levels. This resulted in minor energy

deficiencies, thus corn oil was top-dressed in small amounts to assure NRC (1989) energy

68

requirement for moderate exercise was met for each horse. Vitamin-mineral mix was
top-dressed once daily to provide NRC (1989) recommended levels of Ca, P, Vitamin A,
Vitamin D, Vitamin E, and Se. Meals were fed twice daily at 0700 and 1600. Forage to
concentrate ratios of the total diet (hay plus concentrate) are shown in Table 8.

This study was approved by Michigan State University All University Committee
on Animal Use and Care.
Sample collection

The study consisted of five collection periods. Each period was 14 d in length
and consisted of a 10-d diet adaptation followed by a 4-d total fecal and urine collection.
Feces and urine were collected using gelding collection harnesses (Equisan Marketing,
Melbourne, Australia) and emptied every 5 h or more frequently as needed. At each
emptying, all feces were bagged, and immediately stored at -20°C. At the end of each
collection period, daily fecal samples were thawed, pooled, weighed, and homogenized
using a 136-kg mechanical mixer. Sub samples were collected (~500 g) and frozen at
-20°C for N and AA analysis.
Sample Analysis

For chemical analysis, fecal samples were freeze-dried (VirTis model 25-SRC,
VirTis Co., Gardiner, NY). Feed and fecal samples were finely ground using a cyclone
mill (Foss Cyclotec sample mill 1093, Hoganas, Sweden) with a l-mm mesh screen.
Nitrogen content in feed and feces was determined using an automated N analyzer (Leco
FP-ZOOO, Leco Co., St. Joseph, MI; AOAC No. 990-3). Dry matter of feed was
determined following a 24-h drying period at 80°C using a drying oven (Fisher Isotemp,

Fisher Scientific, Hanover Park, IL). Amino acid analysis was performed on feed and

69

 

‘3‘... '—

fecal samples using the Pico-Tag method (Waters Co., Milford, MA) following a 24-h
acid hydrolysis in 6N HCl at 113°C and 121 mm Hg. Norleucine was used as an internal
standard. Samples were derivatized with phenylisothiocyanate and analyzed by high
pressure liquid chromatography (Alliance 2690, Waters Co., Milford, MA) fitted with a
30-cm Pico-Tag column (Waters Co., Milford, MA).
Calculations

Apparent fecal N digestibility (AND) and AA digestibility (AAD) was calculated
in the following manner:

AND = (N,— Nf)/ N,
AAD = (AA, -— AA,)/ AA,

where N, and AA, are the total N and AA intake, and Nfand Man the total N and AA
losses in feces.
Statistical Analysis

Data were subjected to ANOVA using the PROC MIXED procedure of SAS
(SAS Inst., Inc., Cary, NC). Effects of horse, period, and diet were included in the
statistical model. Horse was treated as a random effect. Differences between all pair-
wise comparisons were evaluated using the Tukey-Kramer test (Younger, 1998).
Relationships between dietary CP intake and N and AA digestibility were determined
using contrast (linear and quadratic) comparisons with orthogonal polynomials.
Coefficients used for contrasts were based on the calculated CP intake of 677, 790, 903,
1016, and 1129 g per d. Statistical significance was based on an experiment-wise type-I
error rate of 0-05. Regression equations for apparent fecal CP, N, and AA digestibility

were calculated using the PROC REG procedure of SAS (SAS Inst, Inc., Cary, NC).

70

 

Results and Discussion

The forage to concentrate ratio for each diet is shown in Table 8. Ratios ranged
from 53:47 forage to concentrate to 63:37 forage to concentrate. These ratios correspond
to those currently recommended by the NRC (1989) for horses performing moderate
work. Apparent fecal N digestibility data, as affected by dietary CP intake are presented
in Table 9. Nitrogen digestibility increased linearly (P<0'001) as daily CP intake

increased. Slade et al. (1970) found similar results with increases in dietary CP in horses

 

fed at maintenance. Compared to Control, N digestibility was not different (P>O-05) in 4:
horses fed L, H and VH protein diets. Nitrogen digestibility was lower (P<O-05) in
horses fed the VL protein diet compared to that in horses fed Control. Slade ct al. (1970)
suggested that the increase in apparent N digestibility on higher protein intakes is due to a
reduction in the endogenous contribution to fecal N and an increased N availability for
microbial activity in the hind gut. The presence of many types of microflora in the large
intestine of the horse with proteolytic activity has been shown (Kern et al., 1973; Frape,
1975); however. the contribution of microbial protein and AA synthesis and absorption
across the wall of the large intestine to post-gut AA availability in the horse is unknown.
Hintz et al. (1971) reported significant disappearance of N fi'om the large intestine of the
horse. In humans and pigs, it is well recognized that some protein digestion and amino
acid absorption occurs in the large intestine (Metges, 2000). Thus it is possible that the
increased apparent digestibility of N in horses fed higher protein intakes is a result of
increased utilization by hind gut microflora. According to Frape (1975) this may also
partially explain increases in N retention in horses fed diets containing higher protein

concentration due to an expanded urea pool and a consequential utilization of NPN by the

71

microbial p0pulation in the large intestine. Indeed, we have found that N retention
increased as dietary CP increased above predicted requirement (Wickens et al.,
unpublished).

Protein digestibility is also affected by dietary fiber content. Glade (1984)
reported lower apparent N digestibility in horses consuming a diet containing straw and a
higher percent NDF. In our study, percent NDF of the total diet (hay plus concentrate)
was similar across all dietary treatments, thus it is unlikely that fiber content affected
apparent N digestibility.

In mature geldings fed various mixtures of feed ingredients, such as corn, SBM,
ground alfalfa, straw, and urea, apparent N digestibility ranged from 64-0 to 796%
(Glade, 1984). In our study, apparent N digestibility ranged fiom 58-8% to 689% when
diets containing feed ingredients such as mixed grass hay, corn, oat, and SBM where fed
to mature. moderately exercised horses. The current NRC (1989) provides equations for
estimating apparent protein digestibility of selected feed ingredients. These equations
were derived using horses fed diets consisting primarily of almond hulls, fishmeal, and
oat hay (Slade and Robinson, 1970). However, these feed ingredients are limited and
may not be representative of others commonly fed to horses such as corn, oat, and SBM.
Regression coefficients for apparent CP and N digestibility were calculated using our
feed ingredients including mixed grass hay, corn, oat, and SBM and are presented in
Table 10.

Individual AA requirements for the mature horse have not been determined, and
information on AA digestibility in feeds commonly fed to horses is not available. Thus,

in this study apparent‘fecal AA digestibility of the five hayzconcentrate mix diets was

72

measured. Apparent fecal AA digestibility data as affected by dietary CP intake are
presented in Table 9. For all indispensable AA, apparent digestibility increased linearly
(P<0~05) as dietary CP intake increased. With the exception of alanine, apparent
digestibility of dispensable AA also increased linearly (P<0-05) with increasing dietary
CP intake. Lysine has been shown to be the first limiting AA in the diet of young
growing horses (Breuer et al., 1971; Ott et al., 1979, 1981) and is most likely the first
limiting AA in diets fed to mature horses as well. In our study, apparent fecal
digestibility of lysine increased linearly (P<0-05) and was higher (P<O-05) in horses fed
the H and VH protein diets compared to that in horses fed Control, L, and VL.

Apparent fecal lysine digestibility was 57-0 and 572% in the VL and L protein diets,
respectively. The concentrate portion of these lower protein diets consisted of primarily
com or a corn:oat mixture. Thus, apparent lysine digestibility of a diet containing mixed
grass hay and corn is 570%, and the similar digestibility between the VL and L protein
diets indicates comparable digestibility between corn and oats. With soybean meal added
to the Control, H and VH‘protein diet concentrates, apparent lysine digestibility increased
(P<0-05) to 63-7, 73-2, and 738%, respectively. Lysine to protein ratio in SBM is 64 to
1 compared to 2-7 to 1 in corn, and lysine digestibility in SBM is higher when fed to
poultry (NRC, 1994) and swine (NRC, 1998) compared to that of corn. Thus, the
inclusion of SBM to the Control, H, and VH protein diet concentrates resulted in a
concentrate feed with higher protein quality. It may be favorable to use lysine
digestibility in formulating equine diets because it is likely to be the first limiting amino
acid in diets fed to mature horses and contributes to the improved N digestibility

observed in horses fed the Control, H and VH protein diets. Using CP digestibility in

73

diets containing SBM would lead to overestimation of protein need. Regression
coefficients have also been calculated for apparent AA digestibility in our study and are
presented in Table 10.

So far, the apparent fecal N and AA digestibility method has been used for
determining the availability of dietary N and AA in horses. In other species, in particular
swine (Donkoh and Moughan, 1994; Stein et al., 1999; Otto et al., 2003), AA recovered
at the distal ileum have been used to estimate AA digestibility and availability. The
major problem associated with apparent fecal digestibility is that it fails to account for the
confounding effect of microbial metabolism of dietary and endogenous protein (Darragh
and Hodgkinson, 2000). Microbial protein degradation can result in fecal N digestibility
coefficients which overestimate N and AA availability, and any net synthesis of protein
and AA by hind gut microbes could lead to underestimation of protein digestibility
(Darragh and Hodgkinson, 2000). Interestingly, for some indispensable AA including
lysine, histidine, isoleucine, leucine. and valine, Darragh and Hodgkinson (2000) found
no difference between apparent fecal and apparent ileal digestibility coefficients in
humans fed a meat, vegetable. cereal, or dairy product diet. It is not known whether this
is similar in the horse as microbial fermentation is of greater significance compared to
that in humans, and the extent to which microbial protein and AA synthesis in the large
intestine contributes to fecal N content remains unclear.

Collection of ileal content in horses has not been routinely performed. Recovery
of ileal content is more invasive and has only been reported on a small number of ponies
(Gibbs et al., 1988). It is unlikely that ileal collection to estimate pre-cecal N and AA

digestibility would be a readily acceptable and commonly used method in horses.

74

Although apparent fecal N and AA digestibility provide some indication of post-gut N
and AA availability, it is important to recognize the limitation associated with this
technique. New methods will need to be developed to assess pre-cecal N and AA
digestibility in horses, and to study hind-gut N and AA metabolism and the contribution
of microbial protein and AA synthesis to fecal N output.

In conclusion, results of this study provide apparent fecal N and AA digestibility
of diets containing varying ratios of feed ingredients typically used in the horse industry
for exercising horses including mixed grass hay, corn, oat, and SBM. Apparent fecal N
and AA digestibility increased as dietary CP increased, and apparent N digestibility in
our study was higher than values reported in the current NRC (1989). Apparent fecal
lysine digestibility was higher in diets containing SBM and thus contributed to the
improved apparent fecal N digestibility. Because lysine is likely to be the first limiting
amino acid in horse diets, it may be preferable to formulate equine diets using apparent
lysine digestibility. Using CP digestibility would overestimate total protein requirement

of the exercising horse in diets containing ingredients of high protein quality, such as

SBM.

75

Table 7. Ingredient and nutrient composition of concentrate diets (as-fed basis)

 

 

 

Dietl
VL L Control H VH
Ingredients
Corn, yellow, (%) 94-0 50-0 40-0 34-0 28-0
Oats, (%) 0-0 44-0 48-0 46-0 44-0
Soybean meal, (%) 0-0 0-0 6-0 14-0 22-0
Molasses, (%) 6-0 6-0 6-0 6-0 6-0
Vitamin-mineral mix: + + + + +
Trace mineralized salt§ + + + + +
Corn oilI + + + + +
Nutrients
Calculated
Digestible energy, (kcal/kg) 3330-0 3100-0 3080-0 3090-0 3100-0
Crude protein, (%) 8-62 10-43 12-90 15-89 18-88
Lysine. (%) 0-24 0-30 0-47 0-70 0-92
Analyzed
Dry matter, (%) 89-32 90-12 91 -10 91-50 91-78
Gross energy, (kcal/kg) 3939-7 4069-3 411 1-9 4161-0 4166-2
Crude protein. (%) 8-48 9-67 1 1-83 15-88 18-57
Acid detergent fibre, (%) 3-85 5-51 6-71 6-28 6-94
Neutral detergent fibre, (%) 7-48 15-05 17-55 15-97 16-41
Amino Acids
Indispensable
Arginine 0-41 0-58 0-78 1-12 1-37
Histidine 0-20 0-24 0-29 0-49 0-62
Isoleucine 0-30 0-36 0-47 0-69 0-83
Leucine 1-08 0-99 1 - l 1 1-42 1-61
Lysine O- l 7 0-25 0-40 0-71 0-93
Phenylalanine 0-36 0-43 0-53 0-77 0-92
Phenylalanine + Tyrosine 0-62 0-74 0-92 1-30 1-55
Threonine 0-24 0-28 0-35 0-48 0-59
Valine 0-39 0-48 0-59 0-82 0-97
Dispensable
Alanine 0-64 0-62 0-68 0-85 0-95
Aspartate 0-66 0-84 1 ~13 1-69 2-08

76

Table 7. Continued

Glutamate 1-80 2-13 2-54 3-29 3-80
Glycine 0-26 0-37 0-45 0-66 0-80
Proline 0-81 0-74 0-86 1-07 1-20
Serine 0-39 0-47 0-57 0-78 0-92
Tyrosine 0-26 0-31 0-38 0-53 0-63

 

' VL, L, Control, H and VH diets correspond to 677, 790,903, 1016 and 1129 g of crude protein intake per
day, respectively.

zVitamin-mineral mix was top-dressed at 28 g per day to provide 15000 1U Vitamin A, 3125 IU Vitamin
D3, 37-5 1U Vitamin E, 4480 mg Ca, 4480 mg P and 0-84 mg Se.

§Trace mineralized salt was fed free choice in block form.

I Value of corn oil top dressed daily to meet NRC (1989) recommended digestible energy requirement for
moderate exercise varied based on body weight of horse and averaged 609, 375, 259, 351, and 305 g for
VL, L, Control, H, and Vl-l protein diets respectively.

77

Table 8. Forage to concentrate ratio (percent of total diet)

 

 

 

Diet
VL L Control H VH
Forage 62-8 54-7 53-0 54-6 53-9
Concentrate 37-2 45-3 47-0 45-4 46-1

 

78

 

79

 

 

mz .. 00 .08 00 2...? .0 e0? 00 202 00 .08 8.8:?
m2 .. 0. .00.. 0. :08 0. £08 0. 33.0 0. .08 8.8m
02 .. 0. .00.. 0. 208 0. e08 0. .08 0. .08 88...
mz .. .0 .08 .0 .000 .0 £08 00 20.8 .0 .08 8.8.0
02 .. 0. .08 0. e000 0. 208 0. .08 0. .08 288.0
02 _. 00 .0 . w 00 :08 00 .08 .0 3000 00 80.8 258%..
02 m2 00 .000 00 .02 00 .08 00 .08 00 .08 8.82
0380:0005
mz .. 0.0 .08 00 2000 00 e. .2 00 308 0.0 .08 88>
mz .. 0.0 .0: 00 .0: 0.0 .800 00 .88 00 .08 8.82.:
mz .. .0 .0? .0 e0: .0 a... . 0 00 z. .8 .0 .08 ”08.88.5880
m2 _. 00 .08 00 .000 00 .08 00 .08 00 .08 8.8..
m2 .. 0. .08 0. e0: 0. 20.2 0. :03. 0. 100 8.80..
m2 _. .0 .000 00 a. . . 0 00 3.08 00 20.8 00 .08 8.80.8.
mz _. 0. .00.. 0. :08 0. 30.00 .0 2.02 0. .0 .0 8.8m...
02 .. 0. .08 3 008 0. s0... 0. 208 0. .80 8.50:.
0300:0006...
Eon OEE<
.mz ._ 00 .08 00 .08 00 2.0.8 0.0 30 .8 00 .08 88.8.2
08880 885 .28 88,. 20m 882 20m 88,. .200 88.. 20m 82>.
88>-.. are :> an... : 3.1.5 .8800 .85 .. Gus ..>

 

.05

 

8.0.00 08.0.88 :05 5.3 002.; 80:. 0.08.50 800,:

2008.. .306 8800:0898 .0 5.358%“. 28 05:8 0.8 =0wo.._: E0893 .0 038...

.5355? 8: n m2 .

.89.. .

.Amo.ov$ 5&6 28.2 6:889; BEE. 53. 3o. 0 55.3 mo:_a> :02: 8:33 831. 0......

88.8.5 .0 2......

80

Table 10. Parameter estimates for crude protein, nitrogen and indispensable amino acid digestibilities

 

 

 

Parameter
Bo P Value B. P Value R2

CP, (%Y 385 <o-001 2015 0-012 0-26
N, (g/d) 46-6 <0-OOl 0- 123 0017 0-23
Indispensable AA, (g/d)

Arginine 69-5 <O-001 0199 0001 0-41

Histidine 62-9 <0-001 0-541 0-002 035

Isoleucine 47-5 <O-001 0486 0-003 034

Leucine 65-5 <0-001 0-125 0099 0-12

Lysine 43-3 <O-001 0.577 0001 0-40

Phenylalanine 53-5 <O-001 0-409 0-002 O~36

Threonine 55-6 <O'001 0-537 0-009 027

Valine 52-3 <O-OOl 0345 0-009 027

 

* CP %, dry matter basis.

81

LITERATURE CITED

82

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Darragh AJ & Hodgkinson SM (2000) Quantifying the digestibility of dietary protein. J
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Donkoh A & Moughan PJ (1994) The effect of dietary crude protein content on apparent
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Frape DL (1975) Recent research into the nutrition of the horse. Equine Vet J 7, 120-129.

Glade MJ (1984) The influence of dietary fiber digestibility on the nitrogen requirements
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Gibbs PG, Potter GD, Schelling GT, Kreider JL, & Boyd CL (1988) Digestion of hay
protein in different segments of the equine digestive tract. J Anim Sci 66, 400-406.

Hintz HF, Hogue DE, Walker Jr. BF, Lowe JE, & Schryver HF (1971) Apparent
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83

Otto ER, Yokoyama M, Ku PK, Ames NK, & Trottier NL (2003) Nitrogen balance and
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concentration. J Anim Sci 81, in press.

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Slade LM & Robinson DW (1970) Nitrogen metabolism in nonruminant herbivores. [1.
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84

SUMMARY AND CONCLUSIONS

85

Summary and Conclusion

The horse industry prioritizes health and performance of the equine athlete, thus
great emphasis is placed on the nutritional needs of the exercising horse. Controversy
exists regarding the degree to which exercise increases the demand for dietary protein.
There is no known evidence that higher protein intake relative to maintenance enhances
athletic performance, yet surveys of feeding practices in the industry report horses are
often fed protein in excess of currently recommended levels. In addition, there is limited
information on the digestibility of protein in diets fed to exercising horses and
digestibility of individual amino acids is unavailable. Research designed to estimate
protein requirement for exercise and to determine the availability of protein and AA in
diets typically fed to exercising horses is critical. The overall goal of this study was to
provide further understanding of the protein requirement of the exercising horse. The
major findings of this experiment were as follows. First, N retention increased when
horses were fed protein 125% above NRC (1989). Although this level of dietary protein
was necessary to achieve maximal N retention, this response was paralleled by an
increase in serum urea-N. Nitrogen retention thus overestimated protein requirement.
This overestimation may have resulted from unmeasured nitrogen losses in sweat and
utilization of N by hind gut microflora at higher protein intake. In addition to serum
urea-N, serum AA concentration increased in horses fed Control relative to the low
protein diets indicating that protein intake was in excess when fed above NRC (1989).
The linear response in serum urea-N to graded level of protein intake for both long and
short-term metabolism suggests that protein level fed at a level 25% below NRC (1989)

may still have been in excess of protein requirement. However, it is important to

86

 

recognize that blood urea-N as a response criterion may be limited in the horse as serum
urea-nitrogen concentration could also be affected by alternative routes of nitrogen
excretion such as sweat or salvage by hind gut microflora. Concentration of serum
arginine, the direct precursor of urea, plateaued in horses fed NRC (1989) recommended
protein level and thus may be a useful indicator of protein status in the horse.

It was hypothesized that protein requirement reported in NRC (I 989)
overestimated the need for the moderately exercised horse. Response between nitrogen
retention, serum urea-N, the majority of serum AA and dietary protein intake was linear
rather than quadratic, thus precluding precise estimation of protein requirement using
break point analysis. Nonetheless, our results suggest that AA utilization for protein
synthesis and retention was not superior in horses fed NRC (1989) level compared to that
of horses fed 125 and 25% below NRC (I 989). Thus a protein to energy ratio as low as
30 g to 1 Meal may be appropriate to meet the demand for protein for the moderately
exercised horse.

Second. apparent fecal N and AA digestibility of diets containing varying ratios
of feed ingredients typically used in the horse industry for exercising horses including
mixed grass hay, corn, oat, and SBM were determined. Apparent fecal N and AA
digestibility increased as dietary crude protein increased, and apparent N digestibility was
higher than values reported in the current NRC (1989) ranging from 59 up to 69%.
Apparent fecal lysine digestibility was higher in diets containing SBM and ranged from
61 up to 76%, thus contributing to the improved apparent fecal N digestibility. These
results suggest protein digestibility is higher than currently reported in NRC (1989) in

diets formulated according to a ratio of 40 g CF to 1 Meal DE and to meet the DE

87

demand for exercise. Underestimation of protein digestibility contributes to feeding
protein in excess of requirement. In addition, using CP digestibility in diet formulation
rather than apparent lysine digestibility would overestimate total protein requirement of

the exercising horse in diets containing ingredients higher in protein quality, such as

SBM.

88

APPENDICES

89

Individual horse performance data
(Mean values with standard errors)

Appendix A

 

 

 

Horse
1 2 3 4 5 Mean SEM
Body Weight, (kg)
Pre-experiment 498 524 452 455 436 473 164
Period 1 505 536 456 455 436 478 175
Period 2 503 542 465 486 436 486 I75
Period 3 500 539 472 486 445 488 17-5
Period 4 507 546 467 486 445 490 I75
Period 5 503 552 471 486 445 491 175
Body Condition Score _
Pre-experiment 6-5 7 7 6 5 6-3 04
Period 1 7 7 7 6 5- 6-5 04
Period 2 8 8 7 6-5 6 7-1 04
Period 3 8 8 7 6-5 6 7-1 04
Period 4 7-5 8 7 6-5 6 7 0-4
Period 5 7-5 8 7 6'5 6 7 0-4
Resting Heart Rate, (bpm)
Pre-experiment 36 4O 28 35 37 35-2 20
Period 1 32 34 34 30 35 33 12
Period 2 32 36 32 27 35 324 l-2
Period 3 35 36 - 28 33 32-9 13
Period 4 29 35 3| 3] 33 31-8 12
Period 5 31 35 32 32 34 32-8 12
Working Heart Rate, (bpm)
Pre-experiment 87 93 88 90 82 88 20
Period 1 80 94 96 83 83 87-2 26
Period 2 90 98 89 87 88 904 26
Period 3 87 99 - 89 86 90-7 28
Period 4 8O 94 90 82 87 86-6 26
Period 5 82 99 89 88 9] 898 2-6

 

90

 

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94

Appendix D

Effect of diet and period on calculated muscle mass, kgf

(Least squares mean values with their standard errors)

 

 

Item Mean SEM
Diet
VL 155-9' 193
L 167-4' 21']
Control 1997' 21-1
H 1446' 19-3
VH 167-4' 193
Period
1 146-4‘| 193
2 1610' 21°]
3 171-7‘ 2H
4 172-8'I 19-3
5 183-2' 193

 

' Least squares mean values within a column with unlike superscripts differ (P<O-05).
' Muscle mass calculated from 24—h urinary creatinine excretion, where l g creatinine = 21 '8 kg skeletal

muscle (Wang et al.. 1996).

95