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This is to certify that the
thesis entitled

PHOSPHORUS DIGESTABILITY IN MATURE HORSES

presented by

TARA ERIN LAVIN

has been accepted towards fulﬁllment
of the requirements for the

Master of degree in Animal Science
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PHOSPHORUS DIGESTABILITY IN MATURE HORSES
BY

TARA ERIN LAVIN

A THESIS
Submitted to
Michigan StateUniversity
in partial fulﬁllment of the requirements
for the degree of
MASTER OF SCIENCE
Animal Science

2009

ABSTRACT
PHOSPHORUS DIGESATBILITY IN MATURE HORSES
By

Tara Erin Lavin

Experiments were conducted to increase the knowledge of phosphorus
digestibility in mature horses as much of the available research has focused on young and
growing animals or utilized ponies as a model. Concern is growing over the
environmental impact of phosphorus entering bodies of water due to the application of
animal manure to the land, it is likely that a greater percentage of the manure
management practices of horse owners in the United States will fall under governmental
regulation. The ﬁrst study fed 6 mature horses diets of alfalfa hay, grass hay, or grass hay
and oats. No differences were found in phosphorus output in the urine or feces resulting
in no differences in apparent digestibility regardless of intake level. All horses
maintained a near zero phosphorus balance. In the second study mature horses were fed
six diets with three levels of phosphorus intake, each diet with a corresponding phytase
supplemented diet. There was no effect of phytase supplementation on phosphorus output
in the urine or feces resulting in no differences in phosphorus apparent digestibility.
Analysis of the feed and feces for phytate revealed a 93% average disappearance rate of
phytate, suggesting that horses are already highly capable of degrading phytate and that
phytase supplementation would not be cost effective if effects are to be seen. The results
of these two studies show that mature horses are highly capable of maintaining a near
zero phosphorus balance, and that increased intakes only result in increased potentially

detrimental outputs to the environment.

ACKNOWLEDGMENTS

I would like to thank the members of my committee (Dr. Nielsen, Dr. Hill, Dr.
Orth, and Dr. Herdt) for their guidance and support. And also for allowing me to pick
their brains and bounce ideas off them; it was a great opportunity to work with such
talented and thought provoking people.

I would like to thank the people who helped so much in getting the work done,
including Adrienne Woodward, Justine Zingsheim, Jane Link and Jim Leismann. I am
forever in debt to my good friend Cara O’Connor-Robison whose countless hours of
assistance kept me going throughout the past two years, not to mention the incredible
Piﬁa—Coladas. I would also like to thank all the other Animal Science graduate students
who lent their hilarious anecdotes and great advice to aid in the maintenance of my
sanity.

I would like to thank Jason Shelton and Cargill Animal Nutrition for their support
of this research. I would like to thank the University of Wisconsin-River Falls faculty
who encouraged me to pursue graduate school. And the Trussell and Bedner families who
have adopted me and given me access to a wide range of horses and equine-experiences.

I would like to thank my family (Mom, Grandma, Chris, and Kelley) who shaped
who I am today and have always been there to support me. Mom, thank you for
encouraging us to chase our dreams and making it possible to do so. And, of course, I’d
like to thank my ever-faithful Sheepdog, Pemberly, who not only guarded us and the
research horses every night, but also continuously found great new ways to make me
smile.

Thanks to all of you it was a great two years!

iii

TABLE OF CONTENTS

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

List of Tables vi
Chapter 1
Introduction 1
Chapter 2
Literature Review 2
Phosphorus: Functions 2
The Equine GI Tract 3
Endogenous Losses 6
Determining Requirements 7
Requirements of/Recommendations for the Horse 10
Inﬂuence of Dietary P on Fecal/Urinary/Plasma Levels ,,,,,,,,,,,,,,,,,,,,, 11
Dietary P Physical Forms 12
Inorganic 12
Organic: Phytic Acid 12
Phytase/Alkaline Phosphatases 15
Availability of Phytate-P to Ruminants and Other Mongastrics ,,,,,,,,, 17
Availability of Phytate-P to Horses 19
Strategies to Improve Phytate Digestibility 22
Other Factors That Affect P Absorption 23
Minerals and Vitamins ' 23
Management Considerations 28
Passage Rate 29
Other Losses 31
Environmental Impact 31
Conclusion 37
Chapter 3
Phosphorus balance in mature horses fed alfalfa hay, grass hay, and grass
hay plus oats 39
Introduction 39
Materials and Methods 40
Results 44
Discussion 47
Chapter 4
Effects of Phytase Supplementation in Mature Horses Fed Alfalfa
Hay and Pelleted Concentrate Diets 52
Introduction 52
Materials and Methods 53
Results 59

 

Discussion 64

iv

Chapter 5
Summary and Conclusions

 

Appendix A
Introduction

69

72
72

 

Materials and Methods
Results and Discussion

Appendix B

72

 

 

74

78

 

Introduction

 

Materials and Methods
Results

 

78
78
79

 

Discussion

Literature Cited

80

82

LIST OF TABLES

Table 1: Total P, phytic acid-P, and phytase activity in some common feedstuffsm, p. 15

Table 2: Feed analysis by diet for phosphorus, calcium and magnesium as
percentage of total diet p. 45

Table 3: Intake of hay and oats on a DM basis, fecal and urine total output per
day, and total P content of excreta per day t). 46

Table 4: Phosphorus, calcium and magnesium intake, output, balance, apparent
digestibility and body weights for each of the six diets; P-value for the
contrast of all alfalfa diets versus all grass hay diets. p. 46

Table 5: Phosphorus, calcium and magnesium serum values 1). 47

Table 6: Percent of total diet as—fed of dietary ingredients including concentrate and
forage for each of the six experimental diets, and total dietary percent of P,
phytate-P, and Ca p. 60

Table 7: Analyzed dry matter, phytate, phosphorus, calcium, magnesium, and
phytase activity of the concentrates (C, C+P, H, H+P, F+P) and forage used
for the six diets p. 61

Table 8: Intake of hay and oats on a DM basis, fecal and urine total output per
day, and total P content of excreta per day 9. 61
Table 9: Phosphorus intake, output, balance, apparent digestibility, estimated true
digestibility, and body weights for each of the six diets; P-values for the
contrasts of all phytase diets versus all non-phytase diets (P vs. NP) and
the two high forage diets versus the four lower forage diets p. 62
Table 10: Phytate intake output, balance, and apparent digestibility for each of the
six diets; P-values for the contrasts of all phytase diets versus all non-phytase
diets (P vs. NP) and the two high forage diets versus the four lower forage
diets n. 63

Table 11: Phosphorus, calcium, and magnesium serum values 1). 64
Table 1A: Results of phosphorus analysis from ICP-MS utilizing open-cell digests

(A), ICP-MS utilizing microwave digests (B), and colorimetric method

utilizing microwave digests (C). Coefficients of variation between A and
B,AandC,andBandC p.75

vi

Table 2A: Comparison of serum phosphorus analysis between ICP-MS and the

colorimetric method

Table 18: Estimated true digestibilities for the six diets from both reported

studies

Table 23: Estimated endogenous losses for the six diets from both reported

studies

Table 38: Estimated true digestibilities and endogenous losses suggested by the

NRC (2007)

vii

p. 77

p. 79

p. 80

p. 80

CHAPTER 1
INTRODUCTION

Concern is growing over the environmental impact of phosphorus entering bodies
of water due to application of animal manure to the land. Many livestock operations are
under regulation as to the amount of phosphorus they can emit from their operations,
including some of the large equine facilities in the United States. The manure
management practices of smaller equine facilities may soon fall under governmental
regulation. It is therefore necessary to be able to estimate the amount of phosphorus that
the average horse excretes in its manure, and to ﬁnd ways to reduce phosphorous output.
To accomplish this goal more information is needed on the digestibility of phosphorus in
the wide variety of feedstuffs commonly used in the equine industry. Information is also
needed on the diﬂ‘erences in digestibility of phosphorus seen in all age groups and
exercise statuses. Much of the available research has centered on the young growing
animal, or animals entering training. The purpose of the following studies was to examine
phosphorous digestibility in some common feedstuffs fed to mature horses that were
either lightly exercised or at maintenance. And also to determine the efficacy of phytase
supplementation to diets of mature maintenance horses to reduce phosphorus
concentrations in the manure, as this is a common practice in both the swine and poultry

industries as a method to reduce fecal P.

CHAPTER 2
LITERATURE REVIEW
Phosphorus: Functions
Phosphorus has more known functions than any other mineral element (NRC,
2001) and is second only to Ca in abundance within the body (Gropper et al., 2005).
Eighty percent of the P in a horse is found in the skeleton (bones and teeth), 16% in the
muscle, and the rest is distributed between the organs, blood and skin (Grace et al., 1999).
Phosphorus forms various calcium-phosphate complexes, such as hydroxyapatite, which
are the major constituents of bone (Gropper et al., 2005). The ratio of Ca:P in normal
bone of horses is 2:1, while the whole body ratio is approximately 1.7 :1 (Frape, 2004),
this is because P is also distributed among the soft tissues for a variety of essential
functions. All energy-utilizing processes in the animal eventually involve high-energy
phosphate bonds such as those in adenosine triphosphate (ATP) or creatine phosphate in
the muscles. All cells utilize P for the formation of DNA, RNA, and cell membranes. As
part of several basic compounds, P inﬂuences the release of oxygen into the muscle tissue
(2,3-diphosphogcerate), glycogen synthesis (UDP-glucose) and degradation (glycogen
phosphorylase), insulin/glucagon release (CAMP), and muscle contractions (1P3).
Phosphorus is the main intracellular buffer (Gropper et al., 2005), and phosphate is an
important buffer in the gastrointestinal (GI) tract.
When there is not enough P available in the diet, horses will have symptoms
similar to those seen in humans suffering from rickets which include lameness, delayed
growth, weak bones, and skeletal deformations. When there is too much P in the diet, it

can interfere with Ca absorption and cause nutritional secondary hyperparathyroidism

(N SH), also known as “big head” syndrome due to the ﬁbrous tissue that invades the Ca
deprived bones and enlarges them (Ramirez and Seahorn, 1997). This most oﬁen results
from inverted Ca:P ratios, or the use of feedstuffs that decrease Ca absorption such as
those high in oxalates (Ramirez and Seahorn, 1997); NSH has become rare in the United
States as Ca supplementation has become common (Hintz, 1997). Feeding imbalanced
Ca:P ratios and excess magnesium (Mg) has been linked to enterolith formation in horses
(Bray, 1995), which can be lethal if they become large enough to block the intestine. The
maximum tolerable concentration of P in equine diets is estimated at 1%, assuming

adequate Ca:P ratio (NRC, 2005).

The Equine GI Tract

Horses are non-ruminant herbivores and hindgut fermentors. While the stomach
and small intestine are similar to other monogastrics, such as the pig, the ceacum and
large intestine of the hindgut are similar to the ruminant animal’s rumen. The majority of
the horse’s digestive tract is maintained at or near neutrality, except for the stomach,
which can range in pH from 1.6-6.0 (Alexander, 1962), this varies with the fed or fasted
state, type of feedstuff, and region of the stomach (Ellis and Hill, 2005). The low pH in
the stomach helps to initiate the digestive process; digesta usually remains in the stomach
for 2 hours or less (F rape, 2004) and then it enters the small intestine (pH range of 6.7 to
6.9), followed by the small colon, where pH ranges from 6.5-7.5 (Alexander, 1962) and is
maintained by a buffering system including bicarbonate and phosphate from salivary,
pancreatic and biliary secretions. It is essential that this buffering takes place, as large

amounts of volatile fatty acids (V FA), lactate and hydrogen ions are produced during the

process of fermentation. This process occurs to some degree in the small intestine, but
mainly in the large intestine. Passage through the small intestine typically proceeds at a
rate of 30 cm/min in the 20 to 25 m long tube (F rape, 2004). The sacculated structure of
the ceacum and large intestine serve to slow down the rate of passage so that the
microbial population can convert structural carbohydrates from feeds into usable VF A for
the horse. Feed spends most of its time in the ceacum and large intestine (36 to 72 h) and
then moves onto the small colon, which is primarily involved in absorbing water and
forming fecal balls, which move through the rectum and to the anus for excretion

The inorganic phosphate concentration of the stomach of horses is reportedly
small (8 mEq/L), increases in the jejunum to 22 mEq/L, and decreases in the small
intestine (Alexander, 1962). There is another, larger increase in inorganic phosphate
concentrate in the ventral colon (31 mEq/L), which is doubled in the dorsal colon (63
mEq/L) and decreased in the small colon (59 mEq/L) (Alexander, 1962). While the
author did not report how horses had been housed or fed prior to the experiment, the
higher concentration of phosphate in the large intestine raised the question of where the
primary absorption of P actually occurs in the horse. In the ruminant (Khorasani, 1997,
Reinhardt et al., 1988; Pfeffer et al., 1970), swine (Moore and Tyler, 1955a,b), and
human (Gropper et al., 2005) the main site is the small intestine. Schryver et a1. (1972)
determined that the main site of P absorption in the horse is the dorsal large colon and
small colon. They used ceacal and oral doses of 32F and an un-absorbable tracer
(chromium oxide) to map the net ﬂow of P in the digestive tract. There is an initial
increase from dietary intake in the ﬁrst half of the small intestine and a net removal from

the second half. There is also a large net ﬂow into the ceacum and net removal from the

dorsal colon and ﬁ'om the small colon. Their ﬁndings supported those of Alexander
(1962), as phosphorous concentrations were signiﬁcantly higher in the large intestine
than in the small intestine, presumably due to the various secretions associated with
digestion. This higher concentration may serve as the primary buffer in the dorsal colon
for the relatively larger proportion of VF A produced in the hindgut than in the stomach
and small intestine, while bicarbonate is the primary buffer system in the ceacum and
ventral colon (Alexander, 1963). Additionally, Matusi et al. (1999) observed the same
pattern of phosphorous secretion and absorption as Schryver et al. (1972). Horses are
therefore similar to ruminants, swine, dogs, and humans in that there is net absorption of
P in the small intestine; however, they differ in that the major site of P absorption in the
horse is the large intestine.

The walls of the small intestine secrete a variety of digestive enzymes responsible
for degradation of soluble carbohydrates, proteins, and fats. Calcium, Mg, and P are
absorbed here. There are two mechanisms of P absorption: a saturable Na and Vitamin D-
dependent active transporter, which is separate from the Ca-transporter (Horst, 1986), and
a gradient-dependant facilitated diffusion process (Gropper et al., 2005). At high dietary
concentrations, diffusion is the primary method of P absorption (Wasserrnan and Taylor,
1976). Parathyroid hormone (PTH) is secreted from the parathyroid gland when plasma
Ca is low, which in turn increases production of calcitriol, the active from of Vitamin D,
from the kidney. Calcitriol increases the amount of Ca reabsorbed from the kidney, and
increases Ca and P uptake from the intestine. Parathyroid hormone also causes Ca and P
to be released from bone. Once serum Ca is in the normal range, these processes are

reversed (Gropper et al., 2005). Very low serum P concentrations can also cause calcitriol

to be released, and thus cause increased P absorption (Horst, 1986), although P serum
concentrations are not as tightly regulated as Ca (Frape, 2004). In the normal healthy
horse, serum P values range ﬁom 2.3-5.4 mg/dl, while serum Ca ranges ﬁ'om 10.4-13.4
mg/dl (Merck, 1998).

Various anaerobic organisms are found in the stomach and small intestine of the
horse. In the small intestine, the number of anaerobic bacteria range from 106-109 cfu/ml
small intestinal content, which includes various lactate users, entercocci, streptococci,
and enterobacteria (Julliand, 2005). The majority of digestion that occurs in the large
intestine is dependant upon the ﬂora and fauna formd there, as the walls of the large
intestine secrete only mucus, no digestive enzymes (F rape, 2004). Therefore, the hindgut
contains more yeast, fungi, protoza, and bacteria (Julliand, 2005). The microbes in the
hindgut of the horse utilize the insoluble carbohydrates (cellulose, hemicellulose, pectin)
and undigested fats, proteins, and soluble carbohydrates from the horse’s diet to produce
VFA, which serve as a major energy source for the horse and are readily absorbed from

the large intestine.

Endogenous Losses

The ﬂow of P into the digestive tract is much greater than the intake from the diet.
Salivary, pancreatic, and biliary secretions contain P and are secreted into the upper
gastro-intestinal tract. Approximately 10 to 12 L of saliva are secreted daily in a normal
horse (F rape, 2004), which has been estimated to contain 0.25 mEq HPO42'/liter and 50-
mE/liter bicarbonate (F rape, 2004). The ﬂow of pancreatic secretions in horses is

continuous, and increases when the horse consumes food. Approximately 10 to 12 L can

be secreted in 24 h (Frape, 2004). The constant biliary ﬂow in the horse is estimated at 4—
L per day (Alexander and Hickson, 1970). The portions of these secretions that are not
reabsorbed contribute to the endogenous losses seen in the feces. Other sources of
endogenous loss include microbial cell loss and intestinal cell sloughing. The endogenous
losses of P in ponies were estimated to be 10 mg/kg BW/d (Schryver et al., 1971a), when
four 2 to 2.5 yr old geldings where given 32P IV or IM injections. In a review of 30
studies, where nutrient balance data was collected, Pagan (1998b) estimated endogenous
losses of P to be 8.5 mg/kg BW/d. These values are based on the Lucas regression
technique of plotting retention of P (g/d) against multiple intake concentrations of P (g/d).
Actual endogenous losses result in a negative Y-intercept, which represents the amount
of P digested at an intake of zero. The slope of the regression line is representative of the
estimated true digestibility. These studies were done in horses weighing between 500 and
600 kg and the estimated true P absorption was 25% (Pagan, 1998a). However, the actual
calculated requirement using these values was only slightly higher than those using the
older numbers of 10 mg/g BW/d and 45% true absorption efficiency, and therefore the
NRC (2007) chose to stay with the lower estimate to minimize any environmental impact

of feeding excess P.

Determining Requirements

The amount of P consumed is either available for absorption or unavailable,
regardless of time in the digestive tract. The unavailable P will be excreted in the feces.
The P that is absorbed enters the bloodstream and is available to body tissues and organs.

As previously noted, bone serves as a storage form of P that can be released into the

bloodstream to augment dietary supply as needed. To determine requirements we must
know the efficiency of absorption of a mineral from the intestine which maybe
determined by injecting the animal with labeled P, allowing the injected P to equilibrate
with the P pool of the bloodstream, followed by a balance study where the amount of P
the animal is fed and the amount of P in the urine and feces is determined. The unlabeled
P in the feces is presumably dietary P that could not be utilized or did not become
labeled, and the labeled P is ﬁ'om endogenous sources that became labeled after
equilibration. The fecal excretion can be thought of in three fractions, ﬁrst P of dietary
origin that is not available for absorption, second P of endogenous origin that is secreted
into the feces such as P of microbial or intestinal cell origin or from secretions that are
not reabsorbed, and ﬁnally P of endogenous origin that is secreted to maintain blood
levels (NRC, 2001).

Balance data can be corrected for endogenous losses of P, and estimates of P
absorption can be made by dividing (intake - fecal P + estimated endogenous P losses) by
intake, the NRC (2007) calls this the “estimated true digestibility”. Estimates of
endogenous losses and digestibility of feedstuffs often must be made using less intensive
methods than using labeled P. In these cases, balance studies are performed using a wide
range of P concentrations in the diets. The amount of mineral retained (intake-fecal
output) is than plotted against the levels of intake. This is called 3 Lucas regression, in
which the Y-intercept will represent the estimated endogenous losses, or losses that
would occur when intake was zero, while the slope represents estimated true digestibility
(Pagan, 1998a; NRC 2007). This method is less than optimal since P digestibility is likely

increased as amount of P in the diet decreases to zero. This relationship is not a straight

line. However, when a better model is not available, this serves as a good estimator
(Schryver et al. 1971a). When estimates of endogenous losses are inadequate, studies
often report apparent digestibilities which are [(intake — fecal output)/intake]* 100. These
may underestimate the actual digestibility of the mineral, as they do not account for
endogenous P utilization and losses (NRC, 2007). For example, in ponies 6 mo to 2.5 yr
of age, apparent P digestibility was 18.5%, while estimated true digestibility was 45%
(Schryver et al., 1971b). Often studies simply report the fecal and urinary losses and an
apparent absorption or retention value. Absorption and retention are often used
interchangeably when discussing P metabolism in the horse, as urinary contributions are
so low that they rarely affect the value. Therefore, absorption and/or retention often
represent intake - fecal output and sometimes intake - fecal and urinary output.

For balance studies to be accurate, many factors must be controlled including feed
monitoring, complete collection of excreta, and use of identical proportions of all samples
(i.e. saving a 10% fecal/mine sample each time) unless complete collections are utilized.
Equally important is the quality of laboratory analysis. As water intake inﬂuences the
DM content of the feces, and dilutes the amount of mineral present, determining DM for
sample mixing is ideal. Water intake often varies between horses, and between days
within a horse. Therefore, feces should be pooled based on DM. The amount of exercise
all of the animals receive must also be standardized. If they are to receive a period of
turnout time during the balance study, they should be together in the same pen so they all
receive the same stimuli for activity. Also, adult horses must have a constant energy

balance so that results are not confounded by weight gain or loss. Because of the tedious

nature of balance studies, and previous lack of concern for P impact of the environment,

there is a lack of data on P metabolism in the horse.

Requirements of/Recommendations for the Horse

The NRC (1989) states that true P absorption for horses ranges from 30 to 55%,
depending on the age and type of horse and the source and concentration of P fed. In
determining requirements, the NRC (2007) assumed a 35% absorption efﬁciency for
sedentary, gestating, and/or working horses with the assumption that diets for this group
of horses are primarily composed of plant sources. For lactating and growing horses,
whose diets are often supplemented with inorganic P, they used an absorption efficiency
of 45%. To determine the amount of nutrient the horse needs to absorb each day, one
must know biological need, endogenous losses and nutrient requirement for all processes
including maintenance, growth, pregnancy, and lactation. As already discussed, the NRC
(2007) has chosen to stay with the endogenous P less estimate of 10 mg/kg BW/d
(Schryver et al., 1971a) for all horses.

To determine requirements for growth, mineral content was measured in 4-mo, 1-
yr, and 2-yr old mixed breed horses. Horses of this age range contain 8 g P/kg BW,
therefore a growing foal would require 8 g P/kg BW gained per day (Schryver et al.,
1974a). In growing Quarter Horses, the estimated endogenous loss was 18 mg/kg BW/d
(Cymbaluk et al., 1989), suggesting that growing horses have a higher requirement than
previously believed if breed and activity are not considered. This was supported by
Furtado et al. (2000) who demonstrated that endogenous losses in 10 month old Brazilian

horses was 10.3 mg P/kg BW/d. Growing horses likely have a greater need and thus may

10

have a higher true absorption efficiency. Grace et al. (1999b) estimated true absorption
efficiency to be 50% in growing 200-kg Thoroughbred horses gaining 1 kg/d. Growing
horses are adding muscle mass which requires P for normal tissue growth. There is no
evidence that mature exercising horses have an increased P requirement (NRC 2007), and
it was assumed that any increased requirement that growing horses may have due to

exercise would be met by the increased requirements established for growth.

Influence of Dietary P on F ecal/ Urinary/Plasma Levels

In four studies using mature horses or ponies, fecal P as a percentage of intake
ranged from 82.2 to 87% (Hth and Schryver, 1973; Patterson et al., 2002; van Doorn et
al., 2004; Morris-Stoker et al., 2001). This is in contrast to two studies using yearling
horses and growing ponies whose fecal P output as a percent of intake ranged from 53.5
to 67.2% (Hainze et al., 2004; Schryver et al. 1971b). These studies demonstrate that
there are differences in P digestibility by age, dietary composition, physiological state,
and frame size.

When horses of various ages were fed 133% or 275% of the NRC (1989)
recommendations for Ca and P, fecal P excretion followed intake, so horses receiving
more P excreted more in the feces (28.48 vs. 53.79 mg/kg BW/d). This is to be expected
since the feces is the primary mode of P excretion. Urinary excretion ranged ﬁ'om 0.23 to
0.28 mg/kg BW/d (Buchholz-Bryant et al., 2001). Schryver et al. (1971a) reported that
higher P diets increased urinary and fecal excretion of P and raised plasma P
concentrations in 2-yr old ponies. Fecal, but not endogenous losses were related to
dietary intake. They also reported that urinary excretion of 32P tracer, given IV or IM,

increased directly with intake. It was therefore concluded that renal excretion is the

11

primary mechanism of regulation of P serum concentrations of horses, but the levels of P
are so low in the urine that the primary losses are fecal. Fecal excretion of P in the horse
is dependant on intake, diet composition, P source, and age, and physiological state,
which makes it hard to determine average output values for horses. More studies are
needed with diets similar to those fed in the industry, and with a wide population of

horses before useful estimates can be made.

Dietary Phosphorus Physical Forms
Inorganic

There are two main types of P in animal diets: inorganic and organic, with the
most common organic source being phytate (myoinositol l,2,3,4,5,6-hexakis dihydrogen
phosphate). Forms of inorganic P utilized in diets are monocalcium phosphate, dicalcium
phosphate, monosodium phosphate, deﬂuoronated rock phosphates and animal by-
product ingredients (which can also be organic in nature). The digestibility of P from
monosodium phosphate is estimated to be 52.7% (Hintz et al., 1973), while the
availability of P in steamed bone meal, dicalcium phosphate and monosodium phosphate
is estimated to be 58% (Hintz and Schryver, 1972a). The majority of P in forages is in

the inorganic form.

Organic: Phytic Acid
Phytic acid is the primary storage form of P in the seeds of plants. It is liberated
during germination to provide energy (ATP), and inositol phosphate intermediates for

transport of materials into cells and to serve as secondary messengers such as inositol

12

triphosphate (1P3) (V ohra and Satyanarayana, 2003). Its structure consists of an inositol
ring surrounded by six phosphate groups. Each group has two alcohol groups. Phytic acid
is a strong acid with the six most easily displaceable protons of the molecule having a
pKa of 1.84. It also has two protons in the weak acid range (pKa 5.7 to 6.3), and three to
four with very weak acid range (pKa 9.7 to 12.0) (Costello et al., 1976). This gives phytic
acid the potential to bind with cations in a wide pH range (Reddy et al., 1989). Salts of
phytic acid are called “phytates” and the Ca/Mg salt is called “phytin” (V ohra and
Satyanarayana, 2003). At neutral pH, each phytic acid molecule has one or two
negatively charged oxygen atoms to which positive cations can bind strongly between
two phosphate groups of the same molecule to from insoluble salts (Erdman, 1979).
Phytic acid is a strong chelating agent and binds to Ca, Mg, Zn, Cu, Fe, Ni, Mn, K, and
Mo (Reddy et al., 1989). Once insoluble salts are formed, the availability of the P and
cation for absorption in the animal is drastically decreased (Maga, 1982; Sandberg et al.,
1993; Davies and Nightingale, 1975). Phytate can be considered an antinutritional factor
by decreasing availability of minerals. It reduces the availability of proteins (Dvorakova,
1998; Saio et al., 1967; Cosgrove, 1980), starches (Deshpande and Cherya, 1984;
Knuckles and Betschart, 1987), and lipids. There also appears to be a negative effect on
the activity of some enzymes such as amylase (Thompson and Yoon, 1984; Harland and
Morris, 1995), pepsin (Deshpande and Cheryan, 1984), and trypsin (Singh and Krikorian,
1982; Caldwell, 1992). These deleterious effects are most likely due to binding between
phytic acid and the molecules or binding of a phytic acid-cation complex to one of these

molecules via the cation (Reddy et al., 1989).

13

Phytate is very abundant in cereals and certain legumes and can be found in the
aleurone, or pericarp of the seed in cereals and the cotyledons of legumes (Reddy et al.,
1989). Phytate content increases throughout ripening and peaks at seed maturity
(Abemethy et al., 1973; Nahapetian and Bassiri, 1975; Welch and House, 1982; Welch et
al., 1974). In corn, about 80% of the phytate is found in the germ, while in rice and wheat
80 to 87% is found in the pericarp or aleurone layer (Reddy et al., 1989). Phytate P
accounts for more than 80% of the total P in cereals and cereal products, and 50 to 70%
of the P in soybeans (Reddy et al., 1989). Table 1 lists the total P, phytate P and %

phytate P of some common feedstuffs used in the equine industry.

14

 

Table 1: Total P, phytic acid-P, and phytase activity in some common feedstuffs.
(Eekhout and de Paepe, 1994; NRC, 2007, 2001 .)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

F l ff Total P, % of DM Phytic acid-P, % of Phytase*, unit/kg
Total P
Wheat Middlings 1.02 66 4,381
Wheat Bran 1.16 84 2,957
Wheat 0.33 67 1,193
Barley 0.37 60 582
Corn distillers 0.90 21 385
Rice Bran 1.78 64 122
Alfalfa (dehydrated 0.23 0 60
pellets)
Soybeans (heated) 0.57 46 55
Oats 0.36 59 42
Soybean Meal,
44% CP 0.66 53 40
Sorghum 0.27 70 24
Corn grain 0.28 68 15
Beet pulp 0.10 0 3
Legume Hay 0.28-0.31 - -
Grass Hay 0.26-0.34 - -
Bermudagrass 0.27 - -
Blood meal 0.30 - -
Fish meal 2.69 - -
Meat and Bone 4.73 _ _

Meal

 

 

 

 

 

*: Phytase unit deﬁned as the amount of inorganic P released, umol/min, from a
0.0015 M Na-phytate solution at pH 5.5 and 37 °C
-: Indicates value not determined for that feedstuff

 

Phytase/A lkaline Phosphatases

Phytase enzymes (myo-inositol hexaphosphate phosphorohydrolases) catalyze the

removal of inorganic orthophosphates from the phytic acid molecule (N ayni and

Markalds, 1986). Various cations, proteins, and starches that were bound to phytic acid

may also be released. The Nomenclature Committee of the International Union of

Biochemistry and Molecular Biology (NCIUBMB) lists phytases in two categories: 3-

15

 

phytase (myo-inositol hexakis phosphate 3-phosphorohydrolase; EC 3.1.3.8) and 6-
phytase (myo-inositol hexakis phosphate 6-phosphorohydrolase; EC 3.1.3.26). The
number is based on the carbon position of the inositol ring from which the enzyme
hydrolyzes a phosphate from ﬁrst; so 3-phytase ﬁrst removes phosphate form the 3-C
position (V ohra and Satyanarayana, 2003).

Phytase activity has been identiﬁed in the intestine of the rat, chicken, calf, and
man (Bitar and Reinhold, 1972; Patwardhan, 1937; Spitzer and Phillips, 1945). Since it is
currently unknown exactly how horses are able to utilize phytate-P, it might be a good
idea to include alkaline phosphatase in the scope of research. Alkaline phosphatase,
which is capable of hydrolyzing phosphate esters in an alkaline medium at the brush
border, has activity in the small intestine of the horse (163.9 to 244.5 U/min/mg tissue)
(Bertone, 1990). There is approximately a 25-fold decrease in activity between the small
and large intestines. The levels of alkaline phosphatase found in the hindgut of the horse
are much higher than those of the cat, dog or man, indicating uniquely high digestive and
absorptive action occurring in the hindgut of the horse (Frape, 2004). Alkaline
phosphatase is found throughout the body and is responsible for the dephosphorylation of
many compounds.

The microbes of ruminants have high phytase activity and are able to free phytate-
bound P almost 100% (Raun et al., 1956; Vohra and Satyanarayana, 2003). Many
microbes have extracellular phytase activities (V ohra and Satyanarayana, 2003). The
fungi, Aspergillus niger, was found to be highly active in phytase production, and has
been used for various supplements such as Natuphos (BASF, Inc.) and Allzyme (Alltech,

Inc). Other microbes have also been used for supplements, such as the fungi Trichoderma

l6

reesei used to produce Finase (Alko, Ltd.). Thermal stability is ideal in a supplemental
phytase, as it must often endure the pelleting process where temperatures can reach 80° C
or more. Ronozyme (Roche Novo Nordisk, Inc.) is advertised to be more resistant to heat.
More information is needed on the inherent intestinal phytase activity and microbial
phytase activity in the horse.

A ﬁnal source of phytase is that which is part of the feedstuffs provided. Eeckhout
and Depepe (1994) reported that rye (5,130 units/kg), triticale (1,688 units/kg), wheat
(1,193 units/kg), and barley (582 units/kg) had high activity levels of phytase. Wheat
middlings also contain high phytase activity (Underwood and Suttle, 1999). Maize, oats,
sorghum, and oilseeds had little or no phytase activity. High temperatures during
pelleting can also inactivate the innate phytase in the feedstuffs (Sirnons et al., 1990).
Interestingly, soaking some supplemental phytase in water can enhance the activity in

diets fed to swine (Liu et al., 1997).

Availability of Phytate-P to Ruminants and Other Monogastrics

Phytate is quickly hydrolyzed in the rumen (Reid et al., 1947) due to the high
phytase activity of the microbes present (Raun et al., 1956). Therefore, ruminants are not
dependant on the phytase activity of the feedstuff. Phytate-P is considered almost
completely available to dairy cattle (Morse et al., 1992; NRC, 2001), while sheep are
reported to absorb 78 to 81% of the phytate-P in cereal and vegetable protein sources
(Field et al., 1984).

Under normal conditions, the P in phytate is relatively unavailable to

monogastrics because they produce little phytase in the intestine (V ohra and

17

Satyanarayana 2003; Taylor, 1965; Nelson, 1967). In comparison to highly available
inorganic P sources such as sodium and potassium phosphates, the P of cereals and
vegetable proteins is only about 20 to 45% as absorbable in pigs and 25 to 50% in
chickens (Scares, 1995). In pigs, apparent absorption of P from wheat and barley is
higher than in com (47 and 39 vs. 17%) even though they have similar proportions of
phytate—P (70.7 and 63.6 vs. 65.6% of total P) (Jongbloed and Kemme, 1990).
Digestibility is not only a function of the amount of phytate-P in the feedstuff, but also
the inherent phytase activity of the feedstuff (Underwood and Suttle, 1999; Pointillart et
al., 1987). In an effort to improve phytate digestibility in monogastrics and decrease
output to the environment, phytase supplementation of the diet is widely utilized in the
United States. Supplementation of pigs with phytase can eliminate the need for adding
inorganic P to the diets of finishing pigs (Murry et al., 1997; Liu et al., 1997; O’Quinn et
al., 1997). In broilers, phytase supplementation can improve P availability by 20 to 40%,
depending on the type of diet and amount of phytase added (Sirnons et al., 1990;
Komegay et al., 1996; Yi et al., 1996). About 50% of the phytate-P in the diet was
released when chicks were fed phytase (N atuphos) at 850 U (activity not
reported)(Schoner et al., 1993). In pigs fed phytase (derived ﬁ'om Aspergillus niger) at
400 U (activity not reported) (Hoppe et al., 1993) and 246 U (microbial phytase EC
3.1.3.26, one U is the quantity of enzyme which liberates l-umol inorganic P/min from
5.1 mM-sodium phytate at pH 5.5 and 37°C) (Komegay and Qian, 1996), phytase
demonstrated similar results.

The availability of phytate-P is not simply related to the proportion of phytate, but

also the Ca and P concentrations and ratio in the diet, the age and physiological state of

18

the animal (Underwood and Suttle, 1999), and as previously stated, the inherent phytase
activity of the feedstuff (Pointillart et al., 1987). The availability of P from phytate-P can
be improved when the Ca:P ratio is kept low, 0.8 to 1.4 vs. 2.0 in chicks (Edwards and
Veltrnann, 1983) and 1.5 to 1.7 vs. 2.9 to 3.0 or 1.2 vs. 2.0 in pigs (Lei et al., 1994; Qian
et al., 1996). Phytase supplementation in swine diets (Komegay and Qian, 1996; Liu et
al., 1997; O’Quinn et al., 1997) and poultry diets (Simons et al., 1990) increases Ca
digestibility. The utilization of phytate-P has also been improved by the addition of 5 or
10-ug/kg diet of Vitamin D (l,25-dihydroxycholecalciferol) in chickens (Edwards,
1993), but this may have only been a secondary effect to an increased absorption of Ca
and P from the gut (Biehl and Baker, 1997). Diets can be formulated to contain
appropriate amounts of Ca and P in the correct ratio to meet animals needs without the
use of phytase, it’s primary purpose is to reduce the cost of feeding excess P sources and

to decrease output of P by the animals by increasing their utilization of phytate-P sources.

Availability of Phytate-P to Horses

The ﬁrst equine studies investigating phytate-P availability to ponies fed pelleted
diets of grass hay, beet pulp, corn and soybean meal with sodium phytate demonstrated
no difference in estimated true P absorption (44%) from those fed the same diet but
containing monosodium phosphate (45% estimated true P absorption) (Schryver et al.,
1971a). When mature horses were fed hay and mixed grain diets with or without phytase
(Allzyme, Alltech Inc.) added at 200 U/g (activity deﬁnition not reported), no effect on
apparent P digestibility was observed. They reported apparent digestibility ﬁ'om 5.77 to

8.13 mg P/kg BW/d (Morris-Stoker et al., 2001). When mature horses were fed 300, 600,

19

or 900 U/kg (one U is the quantity of enzyme that liberates 1- umol of inorganic P per
min from 5.1 mM Na-phytate at pH 5.5 and 37°C) phytase (Natuphos 600, BASF,
Wyandotte, WI) on top of a grass hay and grain diet of corn, oats, and soybean meal, no
effect of enzyme supplementation was seen on P digestibility (Patterson et al., 2002). In
yearling horses fed grass hay and various concentrates including whole oats, sweet feed,
a pelleted concentrate, and alfalfa cubes, phytase (Allzyme, Alltech, Inc., Nocholasville,
KY) at 1500 U/g (one U is the quantity of enzyme that releases l-umol of inorganic P per
min from 0.15 mM Na-phytate at pH 5.5 and 37°C) supplementation was found to have
no effect on P digestibility (Hainze et al., 2004). In all of these studies, the amount of
phytate-P in the feedstuffs and feces was not determined. Therefore, it is unknown how
much of the phytate-P may have been liberated either by supplemental or microbial
enzyme activity.

When mature horses were fed oat, corn, wheat bran, and rice bran diets that
contained 55% of the total P as phytate-P the apparent digestibility of phytate-P was 60%
(van Doorn, 2003). This was increased to 71% (P<0.02) by the addition of 740 U/kg (one
U is the quantity of enzyme that releases l-umol of inorganic P per min from 0.5 mM
Na-phytate at pH 5.5 and 37 °C) phytase (N atuphos 5,000 Liquid, DSM Food Specialties,
Delft, The Netherlands) (van Doorn, 2003). These researchers found no difference in the
apparent P digestibility when a phytate-rich diet, containing large amounts of wheat and
rice bran, was compared to a diet containing inorganic P from monocalcium phosphate
(van Doom et al., 2004). In contrast, Hintz et al. (1973) found that P ﬁom wheat bran
(high in phytate-P) is approximately half as available as P from an inorganic source

(N aHzPO4). Van Doom et al. (2004a) found that the Ca digestibility on the phytate-P

20

diet was increased when the diet was supplemented with phytase (31.5 vs. 26.4%), but
both phytate diets (supplemented and not) had lower Ca absorption than the control and
inorganic P diets (47.7% and 42.35%). Additionally, Hintz et al. (1973) found that the
apparent digestibility of Ca was lower when wheat bran was fed to ponies. This is likely
because phy tate binds Ca into insoluble complexes that are excreted via the feces, and
phytase activity releases the Ca from binding. No differences in Mg retention or apparent
digestibility were reported between any of the treatment diets (van Doom et al., 2004a).
This concurs with Hintz and Schryver (1972a) who reported that sodium phytate or
wheat bran did not affect Mg metabolism in ponies.

To more closely examine the digestion of phytate-P in the horse, Matsui et al.

(1999) fed 2 yr old horses either a low or high phytate diet. The low diet was primarily
timothy hay, alfalfa hay, and inorganic P supplied by sodium dihydrogenphosphate
providing 21.1 g P/d that was 26% phytate-P, and a Ca:P ratio of 1.1. The high phytate
diet consisted of timothy hay, oats, corn, soybean meal, wheat bran, and a much smaller
amount of inorganic P to make the total P concentrations of the two diets equal. The high
phytate diet had a Ca:P ratio of 1.2, and horses consumed 59% of the 22 g P/d as

phy tate-P. All horses received CrzO3 in the feed as an un-absorbable marker. Horses were
fed their respective diets for 5 days, and 3 hours after the last feeding horses were
sacriﬁced. The daily passage of P in an intestinal segment was then calculated as: daily Cr
intake x P in digesta divided by Cr in digesta. There was an 11 and 28% reduction of
phytate-P on the low and high phytate diets, respectively, between the amount of intake

and the concentration of the digesta in the upper small intestine, with no differences in

21

phytate-P content in the upper and lower small intestine. However, there was a decrease
in phy tate-P in the lower large intestine (rectum) as compared to the upper large intestine
(0.8 m caudal to the ileocaecal valve) when horses were fed the high phytate diet.
Similarly, on the low phytate-diet, the amount of phytate-P in the lower large intestine,
but not the upper large intestine, was signiﬁcantly less than that in the lower small
intestine. There was no difference between diets in the total amount of phytate-P present
in the lower large intestine (2.6 g phytate-P). These results suggest that phytate-P is
partially digested in the small intestine, but digested to a larger extent in the lower large
intestine. Digestion in the large intestine would be most likely attributed to microbial
phytase activity. On the high-phytate diet, 82% of the dietary phytate-P was degraded by
the time it was sampled at the rectum. Similarly, 47% was digested when a low phytate
diet was fed. These results indicate that 47 to 82% of phytate-P is available to the horse

without phytase supplementation.

Strategies to Improve Phytate Digestibility

The phytate content of feedstuffs is inﬂuenced by a variety of factors including:
environmental conditions, location, irrigation, soil, fertilizer application, and year of
growth (Reddy et al., 1989). When incubated with water at 55° C for 24 h, phytate in
wheat, rye, and barley was reduced by 46-77%, and was reduced by 84-99% when
incubated with acetate buffer (pH 4.8) at the same temperature and time. In oats soaked
and ground, phytate was reduced by 8-26%; in dehulled oats it was reduced by 72-77%.
(Fredlund et al., 1997). Decreasing the phytate content of feeds would create less P waste

once fed to the animal.

22

Other options to decrease P waste include feeding supplemental phytase, however
it will increase cost of the diet. Besides reducing phytate in the feeds and supplementing
diets with phytase, researchers have now developed transgenic pigs that secrete phytase
in their saliva (Golovan et al., 2001). Thus diets could be formulated to include high
phytate-P feeds and eliminate the use of inorganic P sources and decrease P losses to the
environment if the production values of these animals could be maintained at a level that

was acceptable to producers and consumers.

Other Factors that Eﬂect P Absorption
Minerals and Vitamins

There are many factors that affect the absorption, requirement, and excretion of P
that further complicate the determination of daily P output by horses. These include other
minerals, phytate/oxalate content of diet, vitamin D, age of the horse, exercise, dietary
composition including ﬁber content, and passage rate. Phosphorus is also lost through
various excretions including sweat, hair loss and hoof growth.

There is some controversy as to the effects of dietary Ca on P metabolism. The
majority of horse studies have found that high dietary concentrations of Ca do not affect
P metabolism when the Ca:P ratios are 1.4 vs. 3.2 (Highfill et al., 2005), 1.2 vs. 4.1
(Whitlock et al., 1970), and 0.4, 1.9, or 4.3 (Schryver et al., 1970). However, van Doom
et al. (2004b) reported that when feeding ponies varying concentrations of Ca and the
same concentration of P resulting in Ca:P ratios of 1.1, 2.6, and 4.3, there was a
depression in apparent P digestibility from the low Ca diet compared to the intermediate

and high (24.8 vs. 10.8 and 13.3%), and a similar pattern was seen in P retention from the

23

low to intermediate and high diets (25.8 vs. 11.5 and 16.1 mg P/kg BW/d). Apparent
digestibility of Ca was also lower on the intermediate and high Ca diets versus the low
(27.9 and 27.4 vs. 42.2%), while Ca retention was highest on the high Ca diet (65.6 vs.
13.5 and 27.0 mg Ca/kg BW/d). While the high Ca diet had a wider Ca:P ratio than those
tested in the previous studies, there was no difference in P metabolism between the high
and intermediate concentrations of Ca supplementation. Another interesting note is that
all of these studies were done in relatively young horses and ponies. Van Doom et al.
(2004b) used the oldest ponies at 4 yr of age. The other studies used horses from 2 to 5
months of age (Whitlock et al., 1970) and ponies ﬁom 6 to 14 months (Schryver et al.,
1970). Growing animals are accruing muscle and bone tissue, therefore these results may
not be applicable to a maintenance animal.

A review by Pagan (1998a) summarizing 30 balance studies regressed P
digestibility against Ca intake and Ca:P ratio and found no correlation between them. The
reviewed studies had an average Ca:P ratio of 2.3 and used primarily Thoroughbreds,
with an average weight of 500 to 600 kg. These results might therefore be more
applicable to the average horse. The studies also incorporated a wide range of feedstuffs:
alfalfa hay, sweet feed and fescue hay, and pelleted concentrates with timothy hay.
Perhaps Ca is more likely to have an effect on P digestibility when phytate-P is present,
since high dietary concentrations of Ca inhibit phytase activity (Taylor and Coleman,
1979; Nelson and Kirby, 1979). Additional studies need to be conducted with animals
representing other age and physiological states fed constant Ca:P ratios while varying

concentrations of Ca.

24

While high Ca may or may not alter P metabolism, high dietary P, without
proportional increases in Ca, does decrease Ca metabolism in horses (Schryver et al.,
1971b; Frape, 2004; Ramirez and Seahorn, 1997). When dietary P was increased from
0.2 to 1.2% of diet while Ca was maintained at 0.4% of diet in young ponies, resulting in
Ca:P ratios of 2 and 0.3, Ca absorption was decreased by more than 50% (Schryver et al.,
1971b). Pagan (1998a) noted that Ca digestibility was negatively correlated to P and
NDF, so that as the percentage of P in the diet increased, true digestibility of Ca
decreased. The slope of this line was -0.65 with R2=0.35. The depressive effects of excess
P on Ca absorption is one of the reasons that it is recommended horses should receive a
Ca:P ratio in the diet of no less than 2:1. Ratios below this may put the horse at risk for
inadequate Ca absorption, which has been correlated with NSH (Ramirez and Seahorn,
1997), and various malformations of bone.

Toxicity studies of vitamin D in horses by Breidenback et al. (1998) have shown
that horses have an odd reaction to pharmacological doses (40,0001U Vitamin D3/kg
BW/d). Toxic doses led to 2 and 20 fold increases in Ca and P excretion respectively.
Apparent digestibility of P and plasma P increased signiﬁcantly, but serum Ca was
unchanged, and Ca apparent digestibility tended to decline. Horses have a lower normal
plasma concentration of calcitriol (20—40 pmol/L) than other mammals. In other
mammals, high doses of Vitamin D primarily lead to increased apparent digestibility and
renal excretion of calcium as well as Ca deposition in soft tissue (Boass and Toverud,
1996; Hodgkinson et al., 1979; Bordeau et al., 1986; Smother et al., 1986). More research
is needed on Vitamin D metabolism in horses, especially for horses that are continuously

stalled and receive little sunlight

25

Varying concentrations of Mg and K have no effect on P metabolism (Hintz and
Schryver, 1973; Hintz and Schryver, 1976). This maybe because the major site of
absorption for Ca, Mg, and K is the small intestine (Hintz et al., 1978; Hintz and
Schryver; 19723, 1973, 1976), while P is likely absorbed to a large extent in the hindgut
of the horse as previously discussed. Adequate absorption of the other minerals is of
more concern in high P diets, particularly if those diets are high in phytate-P. Binding of
phytate with Zn decreases availability of this mineral (Reddy et al., 1989). Phytic acid
may also reduce the availability of Fe via a similar mechanism (Reddy et al., 1989).

In a study conducted using 2-yr old ponies that remained in positive P balance
throughout the study, indicating that they were not being fed above their requirement,
high P diets (1.19% P) increased the retention of P in comparison to low (0.2% P) diets,
but as a percent of intake differences were not signiﬁcant. The high P diet increased
plasma P in comparison to the low P diet (Schryver et al., 1971a). Buchholz—Bryant et al.
(2001) studied young, mature, and aged horses. The young and mature horses were fed
66.7 mg P/kg BW on the high P diet or 33.25 mg P/kg BW on the low P diet; while aged
horses were fed 47.8 mg P/kg BW on the high P diet, and 25.3 mg P/kg BW on the low P
diet. All three age groups maintained a near zero P balance on average throughout the
168-d trial, as the increases in P retention between the high and low P diets were not great
enough to move them far ﬁom a zero balance. Therefore feeding P above the amount
necessary to maintain a zero P balance will not enhance P absorption, but is likely to
depress it. The addition of 5% sodium chloride versus only 1% sodium chloride to the
diets of ponies increases P absorption, from 28% for diets with 1% sodium chloride to

40% with 5 % sodium chloride in the diets (Schryver et al., 1987).

26

Much like Ca, there have been varied results relative to Al impact on P
metabolism. When mature ponies were fed high concentrations of A] (4,500 ppm) in a
0.3% P, 0.7% Ca diet, P absorption was decreased and the ponies went into a negative P
balance, with decreased plasma P (Schryver et al., 1986). Researchers found that there
was high bone turnover when ponies were fed the high Al diet. Al depresses P absorption
(Schryver et al., 1986). However, a more recent study found that when horses were fed
931 ppm Ang feed, or 12 mg Al/kg BW/d, there was no effect on P metabolism (Roose
et al. 2001). This study utilized 23-d diet adaptations and 5-d total collections, the diets
contained Ca:P ratios of 3.1 on the non-supplemented diet and 2.6 on the A1-
supplemented diet. The higher P content of the Al-supplemented diet may have masked
some of the effects of Al on P metabolism, or it is possible that horses have a dose
response to Al, and 931 ppm was not enough to elicit negative effects on P metabolism.

The dietary content of phytate and oxalate can also alter P absorption. The
digestibility of P from phytate was previously discussed. Rats have shown an adaptive
increase in microbial phytase activity after a period of P deprivation (Moore and Veum,
1983). This idea may be applicable to horses that are deprived of P by poor nutrition, or
those fed a low P diet, or diet with mainly phytate-P, especially with too much Ca.
Oxalate in the diet causes decreased Ca absorption and increased endogenous losses of P,
thus decreasing P retention (McKenzie et al., 1981; Blaney et al., 1981). Horses
consuming diets that contain oxalates and poor Ca sources, such as wheat bran or poor
quality grass hay, particularly tropical grasses, are at the greatest risk for developing NSH

(Ramirez and Seahorn, 1997).

27

Management Considerations

The age of the horse is an important consideration for mineral metabolism as the
true digestibility of both Ca and P decline (71 to 42% and 52 to 6%) between the ages of
6 and 24 months (Cymbaluk et al., 1989). When the animal is actively laying down bone
and remodeling bone to deal with new stresses, such as those induced by training, there is
likely an increased demand for P and Ca or more efficient absorption. Exercise affects P
requirements largely in relation to bone metabolism. Yearlings entering training have an
initial decrease in P absorption and an increase in P output with the onset of bone
demineralization that often accompanies typical training practices (Nielsen et al., 1998a;
Stephens et al., 2004). After the initial bone loss that accompanies the early stages of
training, an increase in bone mineral occurs as speed is introduced and this results in an
increase in Ca and P demand. Ca and P needs must be met to provide for adequate bone
formation. However, as long as the Ca:P ratio is maintained there is no beneﬁt to feeding
horses entering race training P above 0.24% of the diet (Nielsen et al. 1998b).

Horses who have been on stall rest and are being returned to activity will likely
face the same challenges as the young horse entering training. During stall rest, little
force is applied to their bones. Since bone responds to the forces put on it, horses on stall
rest will likely have some demineralization of the bone. As animals return to work, it is
important that diets be formulated to meet their needs for bone remodeling to deal with
the stresses of work. The mature horse may have a different response to exercise. A
review of 15 studies with P intakes ranging from 19 to 200 mg P/d resulted in an average

P retention of 4.7 mg/kg BW/d; this was not different if the horses were sedentary or

28

exercised (Lawrence et al., 2003). This may indicate that exercising mature horses do not

have an increased P requirement over their sedentary counterparts.

Passage Rate

The rate of passage through the equine digestive tract is faster than that of the
ruminant (F rape, 2004). This is most likely due to their evolutionary development to
consume large quantities of low quality forage. Thus, it would be essential to be able to
quickly process the material and derive as many nutrients as possible from it so that more
could be consumed. The passage rate of feeds in the horse is dependant upon physical
form such that a pelleted diet would pass more quickly than a primarily forage diet
(Frape, 2004). A horse with proper dentition usually chews forage to particles less than
1.6 mm (F rape, 2004), so chopping that does not reduce particle size below 1.6 mm may
have no effect. The amount of water consumed would also affect passage rate.

Studies using markers to identify passage rates have varying results. Vander Noot
et al. (1967) reported almost 100% recovery rates of Cr203 at 4 days which is similar to
rates observed by Olsson et al. (1949). A study that utilized yellow Styrofoam particles
and pelleted or non-pelleted hay and grain diets had complete marker recovery at 63 h
(Hintz and Loy, 1966). Two studies, one using carbon granules and the other Cr203,
reported total collection of the marker by 48 h (Alexander, 1946; Haenlein et al., 1966).
These studies did not report differences in passage rates between different feed materials,
however most studies fed relatively similar ﬁber types. Horses on pasture have a much
higher passage rate than those fed other types of roughage. This is most likely due to the

higher water content of pasture; but high ﬁber meals have a faster transit time than low

29

ﬁber diets of the same particle size (Frape, 2004). Transit time of meals is also much
faster following a 12-h fast (Frape, 2004).

The ratio of soluble to insoluble ﬁber entering the stomach alters the viscosity of
digesta, the water holding capacity of the feeds, and potentially the rate of passage (Bach-
Knudsen, 2001). Increased dietary ﬁber reduces the apparent absorption of P in the large
intestine of swine (Partridge et al., 1986). In rats, high ﬁber supplementation increases
fecal excretion of Ca, Mg, Fe, Mn, Zn, and Cu (Gralack, 1996). The digestibility of P in
horses is negatively correlated with NDF in the diet, so that as NDF was increased from
35 to 60%, true P digestibility decreased from 40% to 10 to 20% (Pagan, 1998a). In
horses, larger particles seem to increase passage rates through the small intestine, and
ceacum (Drogoul et al., 1996). Rapid ﬂow of digesta could potentially cause decreased
fermentation which may be a protective mechanism in the horse, as the horse is unable to
handle large amounts of gas production (Ellis and Hill, 2005). Cattle, who are capable of
eructation, have an average fecal particle size of 830 um, while horses average 1,600-
1,630 um (Uden and Van Soest, 1982). Ruminants may have a higher digestibility of
phytate-P as feed is processed to a smaller size, theoretically being exposed to phytase
enzymes for longer periods of time than in the horse.

An interaction of passage rate and/or ﬁber type might have an inﬂuence on
phytate-P digestibility as the increase in passage rate through the hindgut or interference
with enzyme activity such as binding with ﬁbers would decrease availability of P. Also,
since a large part of phytate-P availability to horses is thought to lie with the phytase
activity of the microbes of the hindgut, it is important to note that large starch meals or

quick changes in the diet drastically affect the microbial population in the hindgut and

30

would hinder phytate-P utilization. Seven hours after a meal of hay, concentrate +
minimal hay, or fasted, the total bacteria per (m1 x 10 '7) in the ceacum and ventral colon
of horses averaged: 500, 800, and 5, respectively (F rape, 2004). The availability of
soluble carbohydrates will also drastically affect microbial populations, particularly if

large amounts of them reach the hindgut and are fermented.

Other Losses

Besides fecal and urinary losses, the horse also expends P in sweat, hair, and hoof
growth. These inputs of P are eventually lost and unavailable to tissues. However, they
are considered to have a very minimal impact of overall P balance. Equine saliva has
been reported to contain 0.19-2.15 mmol P/L (Eckersall et al., 1985; Eckersall, 1984).
When sweat was collected for 20 minutes from polo horses, it contained 11-17 mg P
(Schryver etal., 1978), suggesting losses of about 73-113 mg P/kg BW/h. Hoof structures
are estimated to contain 181.5 ppm P. This concentration varies depending on age, diet,
and which part of the hoof is sampled (Evans, 1992). Samples cf mane hair contain 294
to 324 pg P/g hair (Asano et al., 2002; Asano et al., 2005). Horse body hair contains 0.35

to 0.39 mg P/g hair (Wysocki and Klett, 1971).

Environmental Impacts

Phosphorus posses an environmental hazard because, as it is introduced to bodies
of water, it causes an increase in algae and weed growth that, at ﬁrst, is unsightly and can
decrease the recreational value of the body of water. Eventually, if algae and weed

growth go unchecked, decomposition of the organic matter utilizes the dissolved oxygen

31

in the water. This is detrimental to ﬁsh and other aquatic life, and can result in growth
and reproductive problems and ultimately death (Knowlton and Cobb, 2006). Agriculture
has been identiﬁed as the primary non-point source of pollutants that decrease water
quality of ponds, streams, rivers, lakes, and reservoirs (EPA, 2000). The main
constituents of manure that can cause problems include P, nitrates, ammonia, salts, heavy
metals, and organic solids (Waskom and Davis, 1999). Concern is growing about animal
wastes relative to the environment. The United States Environmental Protection Agency
has set new standards for large livestock operations, called concentrated animal feeding
operations or CAFO’s (EPA, 2003). These regulations are only a minimum standard and
states can choose to legislate more stringent requirements. This is particularly relevant in
Michigan, where a large portion of the economy revolves around the natural water
resources of the state. Michigan law already prohibits any direct or indirect discharge of
nutrients that are or may become injurious to the designated uses of the lakes (MDE,
2007). The cost of treating water to remove pollutants is usually far more expensive and
ineffective than simply preventing contamination in the ﬁrst place (EPA, 2000).

The EPA bases its regulations of animal operations on the number of animal units
they house. The amount of regulation the operation is subject to is based on whether they
are considered a small, medium, or large CAFO. Small operations generally have less
than 250 animal units while large operations have 1,000 animal units. The EPA deﬁnes
an animal unit as 454.5 kg live weight. Beef cattle, average weight 454.5 kg, are therefore
1 unit; while dairy cows, average weight 636.4 kg are considered 1.4 units. Horses on the
other hand are considered 2 units, although few light horses reach 909 kg. Race tracks

account for 96% of all the horse operations in the United States that fall under the large

32

CAFO designation, as they are one of the few places were 500 or more horses are housed
at one time (Warren, 2003). Much smaller facilities housing 20 horses or less account for
96% of all equine operations and 73% of the equine population in the United States
(NAHMS, 1998). While these small facilities may not currently be under strict
regulations, it is very likely that the state and federal governments will begin to regulate
even the smallest contributors as concern for environmental contamination rises.

The average horse produces 2.8 kg manure/ 100 kg BW a day, or 5,110 kg of
manure a year for a 500-kg horse (Warren, 2003). The manure from even one horse can
have an impact on the environment when it is compiled throughout the years. Soiled stall
bedding that is disposed of with manure may also contribute. Bedding can add 3 to 7 kg
of waste a day (Warren, 2003). The concern over additional waste due to bedding is one
of the reasons that horses are considered 2 animal units. However, more research is
needed to determine the actual contribution of the various bedding types. Research
conducted on 6 common bedding types found a wide range of removal rates (10.5 to 21.1
kg bedding/day). The P concentration of all bedding types was 1.1 to 3.1 g P/kg bedding
(Woodward et al., 2008). Therefore, the P contribution due to bedding is likely dependant
upon removal rate, and could be modiﬁed through management considerations. Composts
with large amounts of bedding reduce the water-soluble P content of the waste (Gagnon
et al., 1999), while other studies have found no difference in P concentration after
composting with bedding materials (Airaksinen et al., 2001).

Besides additional waste due to bedding, horses are often fed P, and many other
nutrients, in great excess of their needs, resulting in higher P in manure. This also adds to

the argument for deﬁning horses as 2 animal units. The majority of feed companies do

33

not limit the amount of P included in their products, and there has been no maximum P
concentration determined. In a review of nutrient balances performed in horses,
Lawrence et al. (2003 a,b) found a positive correlation between intake and excretion of
N, P, and K. If dietary concentrations of P can be decreased, fecal P will also be
decreased if P is fed above the requirement. The third argument for horses contributing
more P to the land is that horses do not produce a product that removes nutrients ﬁ'om the
farm, such as milk, eggs, or meat.

Over 80% of horse facilities in the United States dispose of manure on site
(NAHMS, 1998), which means that more P stays on the farm and only continues to
stockpile instead of being exported from the farm. Over half of the farms that retain their
manure report spreading it on pastures and ﬁelds as the primary method of disposal
(NAHMS, 1998). The increasing amounts of feed that are imported rather than grown on
site further exacerbates this imbalance of P inputs and exports. This may lead to P
buildups in the soil. Also, since plants require a N:P ratio of 8:1, applying manure with a
ratio of 2.1 to 5.6:1 N:P to meet nitrogen needs means applying large excesses of P that
will not be used to support plant growth (Hainze et al., 2004). The National Research
Council (1993) estimates that only 30% of the P imported onto farms (such as feeds and
fertilizers) is exported (such as crops and animal products). The surplus 70% remains on
the farm and potentially causes excessive soil P concentrations (Sturgul and Bundy,
2004). This is often seen in horse management systems where some portion of
concentrate, forage, or both is imported. Larger operations are generally limited in the
acres of land upon which they are able to apply manure on; which often results in

excessive amounts of P being applied (Sturgul and Bundy, 2004). Limited distribution of

34

nutrients and export of nutrient-rich product ﬁom the farm both contribute to excessive P
loads in soils of horse farms.

The EPA (2003) recognizes three primary ways that P from manure can enter a
body of water. First, horses can be allowed direct access to a water source or there can be
intentional dumping of manure into a water source. Second, sm'face runoff and erosion
account for a large amount of the P that makes its way into bodies of water.
Contamination via runoff is caused by water passing over soils, collecting nutrients,
including P, and eventually dumping them into streams, lakes, and rivers. Drainage tiles
used in ﬁelds can be a direct line for P to Max waters (MDE, 2007). Finally, P can
enter bodies of water via leaching into soil and ground water. Leaching is when P moves,
often without the aid of water, through the soil until it reaches groundwater; this process
is especially prevalent in solids that are highly saturated with P, sandy soils, ﬁelds with
high organic matter, or when water tables are very high (Sims et al., 1998). Once the P is
in the groundwater, it can travel to surface water. In a P budget prepared for the state of
Wisconsin, the major inputs of P to cropland were animal manure and commercial
fertilizer, while the major P removals were crop uptake and losses in runoff and erosion
(Bundy, 1998). The estimated losses of P due to runoff and erosion represented 1.6% of
the P applied to croplands in WI each year (Bandy, 1998).

Two primary physical forms of P in manure affect water quality. The water-
soluble fraction, which contains mainly inorganic P, and an insoluble or organic fraction,
that would account for P bound to soils or phytate. Inorganic P is more readily available
and utilized by plants during the growing season than organic P (Hainze et al., 2004).

However, both create environmental concerns as potential contaminates of ground and

35

surface water (Sharpley et al., 1994). The water soluble-inorganic ﬁaction is more prone
to runoff in ﬁeld management systems that do not incorporate manure or fertilizer. This
would be seen in a pasture-based horse management system where P runoff is most
prevalent during heavy rains (Sharpley et al., 1994). The insoluble or organic P is lost
along with soil particles and other organic matter through erosion. This insoluble fraction
can account for 60 to 90% of the P lost from tilled agricultural ﬁelds (Sharpley et al.,
1994)

Horse manure is unique in that 60 to 70% of total P in manure is in the insoluble
fraction and only 30 to 40% in the soluble fraction (Hainze et al., 2004). Other livestock
species have a greater proportion of fecal P in the soluble fraction (Barnett, 1994b). In
cattle, 62% of total P is inorganic or soluble while 44% in poultry manure is inorganic or
soluble (Dou et al., 2000). Therefore, as horse manure contains a higher proportion of this
insoluble or organic P, it might be a higher potential contaminant when used in a tilled
farming system (Hainze et al., 2004) as the insoluble portion of P is the greatest
contributor of P contamination during erosion or runoff ﬁ'om ﬁelds. However, horse
manure in a traditional pasture setting would be less prone to runoff as it contains less
inorganic or water soluble P (Hainze et al., 2004). When runoff reaches a body of water,
the smaller soluble portion can have an immediate effect on the algae and weed growth
(Sharpley et al.,1996). The insoluble portion generally settles to the bottom of the body of
water, and overtime the P is released into the water (Sturgul and Bundy, 2004).

Variations in diet formulation can alter the amount of P a horse excretes. In a
study conducted using three commercially available equine feeds, researchers found

variability in the P concentrations and the apparent digestibility of P (Wilson et al.,

36

2003). Many commercial concentrates are available, and they could result in a wide
range of fecal P concentrations. Increasing P concentrations in the diet by addition of
varying concentrates to a grass hay diet increases fecal P outputs of horses (Hainze et al.,
2004). Additionally, digestibility of forage can be increased by the addition of
concentrates (Kienzle et al., 2002). Forages can vary in P content and digestibility as
much as concentrate diets. A study using Arabian geldings found that alfalfa had a higher
apparent digestibility than two grass hays (tall fescue and Caucasian bluestem), and that
the alfalfa alone exceeded P requirements for the horses (Crozier et al., 1997).
Phosphorus concentration and digestibility within the same general species of hay can
vary depending on growth and harvesting conditions. Even diets with the same amount of
total P can result in different amounts of fecal P due to variations in feedstuff
digestibility. The P content of the hay and concentrate portions of equine diets vary
widely, therefore more research is needed on complete diets to begin to quantify an

average production of P in horse manure.

Conclusions

The horse derives no beneﬁt from excess P concentrations in the diet, and high P
concentrations can be detrimental to the metabolism of other minerals. When P is fed
above the true requirement fecal P concentrations increase. Contamination by P
originating from horse feces can be detrimental to bodies of water, decreasing their
ability to sustain aquatic life and their usefulness for leisure activities. It is therefore
important to formulate diets that meet, but do not exceed, the horses true requirement.

Studies are needed to deﬁne this true requirement in a wide range of horses and to

37

determine the digestibility of P from the many feedstuffs used commonly in the industry.
The investigation of the efficacy of supplemental phytase to decreasing P output in
equine feces may prove useful if diets can be formulated to contain P at levels low
enough so that any decrease in P output due to increased availability of P from phytate-P

would be environmentally signiﬁcant.

38

CHAPTER 3
PHOSPHOROUS BALANCE IN MATURE HORSES FED ALFALFA HAY, GRASS
HAY, AND GRASS HAY PLUS OATS
Introduction
Phosphorus (P) is the second most abundant mineral in the animal body. As an

essential component of equine diets, it is involved with, or can be found in, bone
formation, high-energy phosphates, phospholipids, and genetic material. Due to the
nature of common equine feedstuffs (Eeckhout and De Paepe, 1994; NRC, 2007) P is
often fed in excess; when applied to the land, such as spreading of manure on ﬁelds, it is
susceptible to runoff into ponds, streams, and lakes. Increasing levels of P leads to
overproduction of algae, which can drastically decrease the oxygen supply of the water
available to ﬁsh and other aquatic life (Knowlton and Cobb, 2006). The United States
Environmental Protection Agency has recently set standards for large livestock
operations, which primarily affect equine operations that house 500+ horses (EPA, 2003).
While this might not seem like a concern to the average horse owner, the EPA has only
set minimum standards, and states are capable of enacting legislation to impose higher
regulations on a greater percentage of the equine industry. Therefore, many more horse
owners’ manure management systems may soon fall under state and federal regulation to
protect water quality (Warren, 2003). In order to formulate equine diets to maintain P-
balance while minimizing output to the environment, it is important to understand how
the horse utilizes the P from speciﬁc feedstuffs incorporated into the diet. This requires

balance studies. Unforttmately, due to their intensive nature, such information is limited.

39

As there are known interactions between P, Ca and Mg that could affect absorption Ca
and Mg balance were also studied.

This study aims to determine the P-balance in mature horses when fed a variety of
common equine diets. Our hypothesis was that P digestibility varies with total P, and that

overall digestibility of P will decrease as dietary P increases.

Materials and Methods
Animal Management

The Michigan State University Institutional Animal Care and Use Committee
approved all methods. Six mature Arabian geldings (16.0 d: 0.3 yr, 452 i 11 kg) were
used in a 6x6 Latin square design study, such that during each period each diet was fed
and that by the end of the study each horse had received all diets. Prior to the start of the
experiment, all animals were housed in large mixed grass pastures at the Michigan State
University Horse Teaching and Research Center with ﬁ'ee access to grass hay, water, and
a mineral block. On the ﬁrst day of the study, animals were m6ved into 3x3 m stalls
bedded with wood shavings and the horses were housed in the stalls while on the study.
The ﬁrst 6 weeks were conducted October 8‘”, 2007 through November 18th, 2007.
Horses were returned to pasture and fed regularly for a 2-mo washout period, then the
second 6 weeks ran from January 21“, 2008 through March 3”, 2008. Horses received
light exercise 5 days a week as beginning equitation mounts. On non-collection
weekends, horses were turned out in a dry lot for approximately 5 horns on Saturday and
Sunday. The study consisted of six 14-d feeding periods, each with 11 days of diet

adaptation and 3 days of total urine and fecal collection, similar collection protocols have

40

been previously used by other researchers (Patterson et al., 2002; Hainze et al., 2004;
O’Connor et al., 2008). Horses were weighed twice before the start of each new period;
the average of the two weights was used for feed calculations. The body condition score
(BCS) was determined using the Henneke system (Henneke et al., 1983) before the start
of each new period; observations from multiple researchers were averaged to obtain the
most accurate score. Hay was fed at 1.6% (as fed) of each horse’s adjusted body weight.
To prevent overfeeding, the horses’ BWs were adjusted by BCS using the following
formula: Adjusted Weight=BW- [(BCS-5)*0.04*BW]. This was derived from the NRC
(2007) statements that for an average 500-kg horse, the amount of weight to be gained or
lost to move one BCS unit is 16 to 20 kg. Thus, 20 kg represents 4% of 500 kg so the BW
was adjusted by 4% for each BCS unit greater than 5.
Diets

All hay bales were core-sarnpled prior to feeding and cats were sampled
throughout the experiment. The diets included ﬁrst cutting alfalfa hay (A1), second
cutting alfalfa hay (A2), third cutting alfalfa hay (A3), timothy grass hay (GI-I), timothy
grass hay and 1 kg oats/d (GH+1), and timothy grass hay and 2 kg oats/d (GH+2). All
alfalfa hay was taken off the same ﬁeld, and the timothy grass hay was all from the same
cutting. Feed samples for each period were pooled and analyzed separately. Horses were
fed at 700 and 1600; these hours best accommodated the horses’ other duties as
instructional mounts at the University farm. Orts were collected before the morning

feeding and weighed; feed refusals were rare. Horses had continuous access to water.

41

Total Collections

At the start of each collection period, total collection devices (TCD; Equisan
Marketing, Melbourne, Australia) were ﬁtted to each animal. The TCDs were
completely emptied of feces and urine every 8 hours into plastic containers. If the horse
refused to use the TCD to urinate, the TCD was removed and a researcher collected the
urine in a pitcher. Total volume of the urine was measured and recorded; a 10% sample
of urine was kept in tightly capped Nalgene bottles. Feces were weighed, recorded, mixed
and a 10% sample of feces was kept in a plastic bag. All samples were frozen at -20° C
immediately after collection. After the completion of the collection period, all samples
for a horse were pooled, thoroughly mixed, and a representative sample of urine and
feces was retained for analysis. A fasting 10—ml blood sample was obtained from each
horse via jugular venipuncture into a glass tube (BD Vacutainer, BD Frankline Labs, NJ)
prior to the start of each collection. All blood samples were kept on ice for
approximately 20 min. Samples were centrifuged (GS-6KR Centrifuge, Beckman,
Fullerton, CA) for 15 min. Serum was pipetted off the top, placed into duplicate micro-
centrifuge tubes, and then serum was ﬁ'ozen at -20° C. Frozen fecal samples were oven-
dried (Thelco, Precision Scientiﬁc, Winchester, VA), then fecal and feed samples were
ground through a 1-mm screen (Cyclotec 1093 Sample Mill, Foss, Eden Prairie, MN).
Mineral Analysis

A portion of urine was acidiﬁed at a rate of 25 ul per ml of urine with 12 M HCl
(EMD Chemicals, Inc., Gibbstown, NJ) to insure all precipitate was in solution
(O’Connor et al., 2006); acidiﬁed urine was used for Ca and Mg analysis, while non-

acidiﬁed urine was used for P analysis

42

Fecal and feed samples were microwave digested as described by Shaw et a1.
(2002). Brieﬂy, approximately 0.400 g of fecal or feed sample was measured into a
Teﬂon-lined digestion vessel, 10-ml of 70% nitric acid (70% trace-metal grade; Fisher
Scientiﬁc, Pittsburgh, PA) was added to each sample, and then samples were allowed to
digest for 24 h lightly covered at room temperature. Vessels were placed in the
microwave assembly and inserted into the microwave accelerated reaction system
(MARS-5, CEM Corp., Matthews, NC). Samples were run at 1,200 W, 100% power, 200
PSI max, 190° C, with a ramp time of 30 min and a hold time of 10 min. Samples were
allowed to cool in a hood for 5 min and then 2 ml of 30% hydrogen peroxide (J. T. Baker,
Phillipsburg, NJ) was added. After 15 min, samples were brought to volume with ddI-IzO,
and samples were stored at room temperature. All glassware used in the mineral analyses
was washed in 30% nitric acid and rinsed with double-deionized water.

Samples were vortexed thoroughly before being used for assays. Digested fecal
and feed samples were all diluted 10x, while mine was run with no dilution. Serum
samples were ﬁrst precipitated with 12.5% Trichloroacetic acid (TCA) to remove
interfering proteins for P assay, samples were then centrifuged and the supernatant was
used for the assay; samples were run at a 4x dilution. Water from the spigot ﬁom which
horses were watered was sampled and assayed. For all samples, 50 pl was pipetted, in
duplicate, into well plates, then 250 pl of Molybdate-Sulfuric (MS) solution and 25 pl of
p-Methylaminophenol (Elon) solution were added; samples were vortexed and incubated
for 45 min. The liquid digests, urine and serum samples were analyzed for P using a

spectrophotometer at 700 nm (Gomori, 1942).

43

Calcium and Mg concentrations in feeds, feces, urine, and serum were determined
by atomic absorption spectroscopy (U nicam 989, Thermo Electron Corp, Franklin, MA).
A bovine liver standard (1577b, National Institute of Standards and Technology,
Gaitherburg, MD) was digested and analyzed with the samples against the same
inorganic standard for each element (atomic absorption standards: CertiPUR, EMD
Chemicals Inc., Gibbstown, NJ; P standard: LabChem Inc., Pittsburgh, PA).
Calculations

Mineral concentrations were used to calculate average mineral intake per day, as
well as urinary and fecal mineral excreted per day. As urinary P was negligible (0.03 to
0.04 g/d), P balance was calculated as:

P Balance= (Intake of P)—(Output of P in Feces)

Ca and Mg balance were calculated as:

Ca (or Mg) Balance= (Intake of Mineral)—(Output of Mineral in Feces and Urine)

The percent AD of P was calculated as:

P Balance x 100

 

Apparent Digestibility, % =
Total Intake of Mineral

The percent AD of Ca and Mg was calculated as:

Ca (or Mg) Balance x 100

 

Apparent Digestibility, % =
Total Intake of Mineral

Statistical Analysis
Data were analyzed using the NﬂXED method procedure of SAS software

(Version 9.1, SAS Inst, Inc., Cary, NC), with period and horse set as random variables.

44

Results are reported on a DM basis as LS mean i SEM. The GLM procedure of SAS

software and orthogonal contrasts were used to examine the effect of hay type.

Results

The GH had the lowest P content of 2.9 d: 0.3 g/kg DM. The P content of the three
cuttings of alfalfa hay was: A1 = 5.0 :l: 0.1 g/kg, A2 = 4.6 i 0.1 g/kg, and A3= 4.3 :t 0.1
g/kg. The cats had an average P concentration of 5.3 i 0.4 g P/kg. The percent
composition of each diet of P, Ca, and Mg are presented in Table 2, along with the Ca:P
ratios for each diet. Average feed intake, urine and fecal output, and total P concentration
of the excreta are presented in Table 3. The different P concentrations of the feedstuffs
resulted in differences in P intake between diets. The P intake, output, balance, and
apparent digestibilities are presented in Table 4. Contrary to our hypothesis, there was no
difference in the P-balance or AD between any of the diets. The water was found to

contain 0.21 to 0.41 mg P/L. This value was so low that it was not considered in any of

 

 

 

 

 

 

 

 

the balance calculations.
Table 2: Feed analysis by diet for P, Ca and Mg as percentage of total diet.
Diet

Percent of Total Diet Al A2 A3 GH GH+1 GH+2
% P 0.5 0.5 0.4 0.3 0.4 0.4
% Ca 1.9 1.6 2.0 0.5 0.5 0.4
Ca:P 3.8 3.5 4.7 1.5 1.4 1.2
% Mg 0.4 0.3 0.3 0.2 0.2 0.2

 

 

 

 

 

 

 

 

45

 

Table 3: Intake of hay and cats on a DM basis, fecal and urine total output per day, and total P content of

 

 

 

 

 

 

 

 

excreta per day.
Diet
Item A1 A2 A3 GH GH+1 GH+2 SEM
Hay Intake, kg DM/d 6.2 6.0 6.2 6.2 6.2 6.2 0.2
Oat Intake, kg DM/d 09" 1.33 0.01
Fecal Output, kg dm/d 2.9c 2.9c 2.6c 3.3b 3.4b 3.7a 0-2
Urine Output, L/d 8.5a 7.1;3 10.5al 3.3b 3.4b 4.8b 1-2
Total P Output, g PM 29.5 27.0 26.3 24.2 25.0 30.5 1.9

 

 

 

 

 

 

 

 

a, b, 0: Values within the same row with differing superscripts differ significantly (P<0.05)

 

 

 

Table 4: Phosphorus, Calcium and Magnesium intake, output, balance, apparent digestibility and body
weights for each of the six diets; P-value for the contrast of all alfalfa hay diets versus all grass hay diets (A

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

vs. G).
Diet A vs G
Item A1 A2 A3 GH GH+1 GH+2 SEM v :18 wine
Weight 452 456 458 459 460 464 12 0.05 -
P
Intake, mg - kg BW'1 - (1'1 69a 61b 57b 45 55b 65ab 4 0.001 0.02
Urine, mg - kg BW‘1 -d" 0.2 0.1 0.1 0.1 0.1 0.1 0.0 0.54 0.16
Feces, mg - kg BW‘1 -d" 65 59 58 53 54 66 4 0.12 0.34
Balance, mg - kg BwT- d" 4 2 -1 -8 1 l 6 0.40 0.35
Apparent Digestibility, % 5 2 -l -23 0 -8 11 0.14 0.17
Ca
Intake, mg - kg BW" . d‘I 2653 2115 2703l 696 71° 71° 7 0.001 0.001
Urine, mg - kg BW'l -d" 19a 16° 18" 4b 4b 3b 3 0.006 0.001
Feces, mg - kg BW'1 - (1'1 100a 100a 130a 39b 35b 33B 14 0.001 0.001
Balance, mg ° kg BW'1 ° d—l 145a 93b 112ab 26 32c 25c 15 0.001 0.001
Apparent Digestibility, % 5 5a 4221b 4231) 37B 36 35b 8 0.05 0.21
Mg
Intake, mg - kg BW" - d" 43° 41" 42b 26 29 31° 1 0.001 0.001
Urine, mg - kg BW" -d“ 3" 9a 10° 3b 3b 5b 1 0.001 0.001
Feces, mg - kg BW" -d“ 263 23ab zlbc 17d 13 13 2 0.004 0.001
Balance, mg - kg BW" - d'r 14 9 ll 6 8 9 2 0.20 0.09
Apparent Digestibility, % 28 21 25 24 27 28 5 0.80 0.74

 

 

a, b, c, (1: Values within the same row with differing superscripts differ significantly (P<0.05)

 

46

 

Intake of Ca was higher on the alfalfa diets than on the grass hay diets (Table 4).
This resulted in higher outputs of Ca in the mine and feces and higher Ca balances on the
alfalfa diets. However, there were no differences in Ca AD between diets. Intake of Mg
was also higher on the alfalfa diets than on the grass hay diets, which resulted in higher
Mg output in the urine and feces (Table 4). However, there were no differences in Mg
balance or AD between diets. There was a trend for the alfalfa diets to result in a higher
Mg balance than the grass hay diets.

As expected there were no differences in serum P, Ca, or Mg concentrations

between diets (Table 5). All horses maintained serum concentrations within reference

 

 

 

 

 

 

 

 

range.
Table 5: Phosphorus, Calcium, and Magnesium serum values.
Diet
Item A1 A2 A3 GH GH+1 GH+2 SEM P-value
Serum P, mg/dL 3.6 3.7 3.3 3.5 4.2 3.8 0.3 0.23
Serum Ca, mg/dL 12.2 10.1 11.3 11.8 11.9 11.6 0.8 0.49
Serum Mg, mg/dL 1.5 1.8 1.7 1.6 1.7 1.6 0.1 0.26

 

 

 

 

 

 

 

 

 

Discussion

The requirement for horses of the age, BW, and exercise status used in this study
is approximately 16 g P/d (NRC, 2007). Using very commonly fed equine diets, P intake
exceeded the horses’ daily P requirements. However, horses in this study were still able
to maintain a near zero P-balance, which one would expect for mature horses at
maintenance. Feeding horses above their true requirement for P seems to have no
apparent beneﬁt in the mature maintenance horse and can result in large detrimental

outputs of P to the environment. That being said, it must again be emphasized that these

47

were very common equine diets with little to no concentrate fed. It may be impractical for
the industry to formulate diets that contain lower amounts of P as the contribution of P
from forages fed at standard rates can exceed P requirements.

The near zero or negative apparent digestibilities of P in these diets may be
explained, in part, by the fact that these were mature animals receiving only light
exercise. These data suggest that horses of this class are very capable of maintaining P
homeostasis. Crozier et al. (1997) reported a low AD of P from alfalfa of 8% and from
Caucasian bluestem of -9% when fed to 2 to 6 year old Arabian horses. These are fairly
close to our average P-AD for the alfalfa (2%) and grass hay (-10%) diets. Sturgeon et al.
(2000) reported P-AD of 24, 6, and -1% for diets of alfalfa hay, matua grass hay, or
Bermuda grass hay, respectively, fed to mature horses. The higher P-AD for alfalfa seen
on that study may have been due to lower levels of intake (32 mg P - kg BW‘1 - d'l), as
compared to the current study (57-69 mg P - kg BW'- d"), eliciting higher absorption.
Buchholz-Bryant et al. (2001) also reported negative P-AD in mature stock type horses
fed coastal Bermuda grass hay and a pelleted concentrate.

Our P-AD values are lower than those obtained utilizing ponies as a model with
additional concentrate portions of the diet. The P-AD of mature ponies fed alfalfa bays
and oats ranged from 22-24% (Hintz et al. 1984), the intake levels (34-37 mg P-kg BW‘
l~cl") were lower than the current study (57—69 mg P°kg BW'l-d’l). Additionally, the diets
in this study consisted primarily of long-stem forages. Other studies have used diets
containing greater amounts of concentrate or utilized chopped or ground roughage. Such
as Hintz and Schryver (1973) who reported P-AD of 15-18% in mature ponies fed

pelleted diets of timothy hay, cellulose, corn, soybean meal, calcium carbonate, and

48

varying levels of magnesium oxide. And in 2 yr old ponies fed diets containing 38%
ground grass hay and 59% of beet pulp, ground corn and soybean meal P-AD was 19%;
while there were concentrates added to this diet the intake levels (34—49 mg P - kg BW'1 -
d") were similar to intakes of the current study on the grass hay diets (45-65 mg P - kg
BW‘1 - d’l). Hypothetically, the coarseness of the current study’s diet could have
contributed to greater endogenous losses of P from the gastrointestinal tract.
Phosphorus balance, and therefore the AD, could also have been affected by the
average weight loss of nearly 22 kg between periods 1 and 6 (464 :t 8 vs. 443 d: 11 kg)
associated with calorie restriction to prevent horses from reaching BCS that were
considered hazardous. Horses were already being fed above maintenance levels.
Decreases in total body mass depress the maintenance requirement for P, and therefore
likely results in less P being absorbed and excess P being expelled into the feces.
Horses maintained a positive Ca balance on all diets. This is similar to other
studies utilizing mature horses. Higher intake levels associated with the alfalfa diets did
elicit higher Ca balance. However there was only a trend (P = 0.054) for a treatment
difference for AD with the A1 diet being greater than the GH and GH+2 diets. The Ca-
AD of 46% for alfalfa hay diets is lower than the 72% Ca-AD reported when mature
horses were fed alfalfa hay diets with intakes of 248 mg Ca ' kg BW‘1 - d", which is
similar to the 211 to 270 mg Ca - kg BW'1 - (1'1 horses received on our alfalfa hay diets
(Sturgeon et al. 2000). On the same study when horses were fed either matua or Bermuda
grass hay with Ca intakes of 92 or 54 mg Ca - kg BW'l - d'l, Ca-AD were 57 and 40%
respectively which is again higher than the 30% Ca-AD seen on our grass hay diets. In

this case intake of Ca was higher on the two grass hay diets than on our grass hay diets,

49

which may have lead to the higher Ca—AD. The Ca-AD for alfalfa from the current study
closely matches the 46% reported by Crozier et al. (1997) when mature horses were fed
alfalfa, and the Ca-AD reported by Hintz et al. (1984) of 44-48% when mature ponies
were fed alfalfa hays and oats.

Horses are different from ruminants in that they absorb a greater percentage of
dietary Ca; this is regulated by large urinary outputs of Ca that are not seen in ruminants
(Schryver et al. 1983). The fact that Ca and P are not absorbed in a 2:1 ratio is partially
explained by this very high absorption and excretion of Ca seen in the horse. Therefore
the large urinary Ca outputs and relatively higher Ca-AD than P-AD were expected.

Horses remained in a positive Mg balance on all diets. In mature horses fed
alfalfa, matua grass hay, or Bermuda grass hay Mg-AD were 58, 48, and 49%
respectively (Sturgeon et al. 2000). These are higher than the 25 and 26% Mg-AD from
our alfalfa and grass hay diets. Intakes on the study by Sturgeon et al. (2000) were 72, 33,
and 26 mg ° kg BW‘1 - d"’ on the alfalfa, matua grass hay, or Bermuda grass hay diets
respectively; compared to 41 to 48 mg - kg BW.1 - d'1 and 26 to 31 mg - kg BW'1 - d'I of
Mg intake on the current study on the alfalfa or grass hay diets. In mature horses fed
alfalfa hay Mg-AD was 27%, and 29 or 32% when those horses were fed Caucasian
bluestem or tall fescue (Crozier et al., 1997). Higher values were reported by Hintz et al.
(1984) for Mg-AD of 50-53% when mature ponies where fed alfalfa hays and oats.

Also of note, horse owners have long suggested a correlation between feeding
alfalfa and increased water consumption. As Table 3 demonstrates, horses on alfalfa hay
had a 43% increase in urine output per day. While this did not correlate with an increase

in total P output, it would cause increased bedding waste.

50

These data suggest that any P intake above the amount required for the mature
lightly-exercised horse is either not absorbed or is excreted with no evident beneﬁt to the
horse and potential negative effects on the environment. There was no difference in
apparent P digestibility between alfalfa or grass hay diets; however, alfalfa diets resulted
in much higher urine outputs and would increase bedding waste. It is possible to exceed a
mature horse’s P requirement with forage alone when fed at levels near industry
standards; formulating diets that do not exceed the horses true requirement may prove
impractical. More studies are needed to more precisely deﬁne the minimum P
requirement of horses at various life stages and to investigate dietary manipulations to

decrease excess P output to the environment.

51

CHAPTER 4
EFFECTS OF PHYTASE SUPPLEMENTATION IN MATURE HORSES FED

ALFALF A HAY AND PELLETED CONCENTRATE DIETS

Introduction

Phytic acid (myoinositol l,2,3,4,5,6-hexakis dihydrogen phosphate), the primary
storage form of P in the seeds of plants, is very abundant in cereals and some legumes.
Phytase (myo-inositol hexaphosphate phosphorohydrolase) catalyzes the removal of
inorganic orthophosphates from the phytic acid molecule (N ayni and Markalds, 1986).
The P from phytic acid, or phytate, is highly available to ruminants (Morse et al., 1992;
Field et al., 1984), but, under normal conditions, it is relatively unavailable to
monogastrics who lack the microbial population housed in the rumen (Scares, 1995).
Supplemental phytase from microbial sources has proven highly beneﬁcial in increasing
phytate-P availability in swine (Murry et al., 1997; Liu et al., 1997; O’Quinn et al., 1997)
and poultry (Sirnons et al., 1990; Komegay et al., 1996; Yi et al., 1996) and, therefore,
decreasing P output into the environment. When P is applied to ﬁelds via manure, it is
susceptible to runoff into ponds, rivers, and lakes. Increasing levels of P cause
eutrophication of the waters, increased algae growth, decreased oxygen levels, and many
detrimental effects to ﬁsh and other aquatic life (Knowlton and Cobb, 2006).

Previous studies in horses have shown no effect of phytase supplementation on P
digestibility (Patterson et al., 2002; Hainze et al., 2004; van Doorn et al., 2004a), few
have reported phytate content of the diet and feces. Deterrrrirring the phytate balance may
provide a better idea as to the efﬁcacy of phytase supplementation to equine diets. The

purpose of this study was to determine if phytase supplementation increased P

52

digestibility in diets where P amounts were low comparable to those used commonly in
the industry, and to determine phytate digestibility in a fairly customary diet. Also, there
are known interactions of P, Ca, and Mg that may affect absorption, and phytase has been
shown to affect Ca and Mg availability, therefore Ca and Mg balance was studies. Our
hypothesis was that feeding supplemental phytase would increase phytate and P

digestibility, as well as decrease fecal P.

Materials and Methods
Animal Management

The Michigan State University Institutional Animal Care and Use Committee
approved all methods. Six mature Arabian geldings (16.0 :I: 0.3 yr, 478 :l: 14 kg) were
used in a 6x6 Latin square design study, such that during each period each diet was fed
and that by the end of the study each horse had received all diets. Prior to the start of the
experiment, all animals were housed in large mixed-grass pastures at the Michigan State
University Horse Teaching and Research Center with free access to grass hay, water, and
a mineral block. On the ﬁrst day of the study, animals were moved into 3x3-m stalls
bedded with wood shavings. Horses were housed in the stalls while on the study from
June 9th through September 13’, 2008. Horses were turned out during the day between
feeding times in a dry lot with free access to water. The study consisted of six 14-d
feeding periods, each with 11 days of diet adaptation and 3 days of total urine and fecal
collection; similar collection protocols have been previously used by other researchers
(Patterson et al., 2002; Hainze et al., 2004; O’Connor et al., 2008). Horses were weighed

twice before the start of each new period; the average of the two weights was used for

53

feed calculations. The body condition score (BCS) was determined using the Henneke
system (Henneke et al., 1983) before the start of each new period; observations from
multiple researchers were averaged to obtain the most accurate score.
Diets

Second-cutting alfalfa hay was included in all diets; hay bales were core-sampled
prior to the start of the study, and mineral values were used to formulate the concentrate
portion of the diets. The diets included: control diet (C), control diet plus phytase
supplement (C+P), high P diet (H), high P diet plus phytase supplement (H+P), forage
only (F), and forage plus phytase supplement (F +P). Phytase (Optiphos 4000 PF, JBS
United, Sheridan, IN) was added to the pelleted concentrates before pelleting. The
product used is considered very stable in normal pelleting temperatures, therefore
signiﬁcant loss of activity was not expected due to pelleting. All diets were initially fed at
1.5% of each horse’s BW with intake being altered in three horses to prevent BCS
changes greater than 1 unit and to comply with farm requirements. The ﬁrst four diets
consisted of a 40:60 concentrate to hay ratio on a weight basis. The F+P diet was fed
forage at 1.498% BW and concentrate at 0.002% BW, while the F diet was completely
forage. Concentrates were sampled throughout the study for analysis. Horses were fed at
700 and 1600. Orts were collected before the morning feeding and weighed; feed refusals
were rare. Horses had continuous access to water.
Total Collections

At the start of each collection period, total collection devices (TCD; Equisan
Marketing, Melbourne, Australia) were ﬁtted to each animal. The TCDs were

completely emptied of feces and urine every 8 horn‘s into plastic containers. If the horse

54

refused to use the TCD to urinate, the TCD was removed and a researcher collected the
urine in a pitcher. Total volume of the urine was measured and recorded; a 10% sample
of urine was kept in tightly capped Nalgene bottles. Feces were weighed, recorded, mixed
and a 10% sample of feces was kept in a plastic bag. Output results are summarized in
Table 8. All samples were frozen at -20° C immediately after collection. After the
completion of the collection period, all samples for a horse were pooled, thoroughly
mixed, and a representative sample of urine and feces was retained for analysis. A fasting
10-ml blood sample was obtained from each horse via jugular venipuncture into a glass
tube (BD Vacutainer, BD F rankline Labs, NJ) prior to the start of each collection. All
blood samples were kept on ice for approximately 20 min. Samples were centrifuged
(GS-6KR Centrifuge, Beckman, Fullerton, CA) for 15 rrrin. Serum was pipetted off the
top, placed into duplicate micro—centrifuge tubes, and then serum was ﬁ'ozen at -20° C.
Frozen fecal samples were oven-dried (Thelco, Precision Scientiﬁc, Winchester, VA),
then fecal and feed samples were ground through a 1-mm screen (Cyclotec 1093 sample
mill, Foss, Eden Prairie, MN).
Mineral Analysis

A portion ofmine was acidiﬁed at a rate of25 pl per ml ofurine with 12 M HCl
(EMD Chemicals, Inc., Gibbstown, NJ) to insure all precipitate was in solution
(O’Connor et al., 2006); acidiﬁed urine was used for Ca and Mg analysis, while non-
acidiﬁed urine was used for P analysis.

Fecal and feed samples were microwave-digested as described by Shaw et al.
(2002). Brieﬂy, approximately 0.400 g of fecal or feed sample was measured into a

Teﬂon-lined digestion vessel, 10-ml of 70% nitric acid (70% trace-metal grade; Fisher

55

Scientiﬁc, Pittsburgh, PA) was added to each sample, and then samples were allowed to
digest for 24 h lightly covered at room temperature. Vessels were placed in the
microwave assembly and inserted into the microwave accelerated reaction system
(MARS-5, CEM Corp., Matthews, NC). Samples were nm at 1,200 W, 100% power, 200
PSI max, 190° C, with a ramp-time of 30 min and a hold-time of 10 min. Samples were
allowed to cool in a hood for 5 min and then 2 ml of 30% hydrogen peroxide (J. T. Baker,
Phillipsburg, NJ) was added. After 15 min, samples were brought to volume with ddH20,
and samples were stored at room temperature. All glassware used in the mineral analyses
was washed in 30% nitric acid and rinsed with double-deionized water.

Samples were vortexed thoroughly before being used for assays. Digested fecal
and feed samples were all diluted 10x, while urine was run with no dilution. Serum
samples were ﬁrst precipitated with 12.5% Trichloroacetic acid (TCA) to remove
interfering proteins for P assay, samples were then centrifuged and the supernatant was
used for the assay; samples were run at a 4x dilution. Water ﬁ'om the spigot used to
water the horses was sampled and assayed. For all samples, 50 pl was pipetted, in
duplicate, into well plates, then 250 pl of Molybdate—Sulfuric (MS) solution and 25 pl of
p-Methylaminophenol (Elon) solution were added; samples were vortexed and incubated
for 45 rrrin. The liquid digests, urine and serum samples were analyzed for P using a
spectrophotometer at 700 nm (Gomori, 1942).

Phytate concentration of feeds and feces was determined colormetrically as
described by Latta and Eskin (1980). Brieﬂy, 1 g of each sample was digested in
duplicate with 20 ml of 2.4% HCl for one hour. Samples were centrifuged and the

supernatant was used. For fecal samples, 6 ml of sample and 6 ml of ddH20 were mixed.

56

For feeds, 0.5 ml sample and 12 ml ddH20 were used; 10 ml of the diluted sample was
then pipetted onto an anion column (BioRad Resin, Anion, 200-400 Mesh AG 1-X8,
chloride form). The column was eluted with 15 ml of 0.1 M NaCl so that phytate alone
remained on the column. The column was then eluted with 10 ml of 0.7 M NaCl, and the
sample was collected. Wade’s reagent was added to the sample and centriﬁrged. Samples
were read on a spectrophotometer (DU 7400, Beckman-Coulter, Fullerton, CA) at 500
nm, with the Ca salt of phytic acid, Ca salt with 23.98% P (P9539, Sigrna-Aldrich, St.
Louis, MO), used to prepare the standard curve. The assay measures the number of
phosphate ester bonds, and assumes that a phytate molecule contains 6 phosphorous
molecules.

Calcium and Mg concentrations in feeds, feces, urine, and serum were determined
by atomic absorption spectroscopy (Unicam 989, Thermo Electron Corp., Franklin, MA).
A bovine liver standard (1577b, National Institute of Standards and Technology,
Gaitherburg, MD) was digested and analyzed with the samples against the same
inorganic standard for each element (atomic absorption standards: CertiPUR, EMD
Chemicals Inc., Gibbstown, NJ; P standard: LabChem Inc., Pittsburgh, PA).

Phytase Activity Analysis

Analysis of phytase activity for the C+P, H+P, and F+P concentrate portions of
their respective diets was conducted by Euroﬁns Scientiﬁc Inc. (Des Moines, IA)
utilizing the AOAC Phytase Method QD176. Feed samples were taken 84 d into the
study and frozen until analysis. The enzyme was incubated with a known concentration
of the sodium salt of phytic acid. Incubation was terminated by the addition of

molybdate/vanadate color stop reagent, forming a colored complex with the phytase-

57

liberated phosphate. The concentration of this colored complex was then determined
spectrophotometrically at 415 nm. The concentration is directly proportional to the
phytase activity of the sample.
Calculations

Mineral concentrations were used to calculate average mineral intake per day, as
well as urinary and fecal mineral excreted per day. As urinary P was negligible (0.03 to
0.05 g/d), P balance was calculated as:

P Balance= (Intake of P)-(Output of P in Feces)
Ca and Mg balance were calculated as:

Ca (or Mg) Balance= (Intake of Mineral)—(Output of Mineral in Feces and Urine)
The percent AD of P was calculated as:

P Balance x 100

 

Apparent Digestibility, % =
Total Intake of Mineral

The percent AD of Ca and Mg was calculated as:

Ca (or Mg) Balance x 100

 

Apparent Digestibility, % =
Total Intake of Mineral

Phytate balance was calculated in the same manner as the corresponding P values, except
phytate intake and output values were used. The difference in the amount of phytate from
intake to fecal output was termed “phytate disappearance”. The percent of phytate
disappearance was calculated as:

Phytate Balance x 100

 

Phytate Disappearance, % =
Total Intake of Mineral

58

Statistical Analysis

Data were analyzed using the MIXED method procedure of SAS software
(Version 9.1, SAS Inst., Inc., Cary, NC), with period and horse set as random variables.
Results are reported on a DM basis as least square mean :1: SEM. The GLM procedure of
SAS software and orthogonal contrasts were used to examine the effects of phytase

supplementation and higher forage inclusion rate in the diet.

Results

Percent of total diet as-fed of dietary ingredients including concentrate and forage
for each of the six experimental diets is summarized in Table 6. The diets contained 0.29
to 0.45% P and 0.04 to 0.08% phytate on a DM basis (Table 6). The contribution of each
dietary component is summarized in Table 7. The targeted phytase activity of 800
FTU/kg was slightly higher than the actual activity in the C+P and H+P concentrates
(Table 7). The actual activity of the F+P concentrate was also lower compared to targeted
activity of 8000 FTU/kg. Although there was a greater intake of hay on the F and F+P
diets than the other 4 diets, there were no differences between any of the diets in total
output of feces (kg DM/d), urine (L/d), or total P output of the excreta (g P/d) (Table 8).
As designed, intake of P differed between diets; it was highest on the H and H+P diets,
and lower on the C, C+P, F and F+P diets (Table 9). This did not correlate with any
signiﬁcant differences in P output, balance, and apparent digestibilities (Table 9). The
water was found to contain 0.21 to 0.41 mg P/L; this value was so low that it was not

considered in any of the balance calculations.

59

 

six experimental diets, and total dietary percent of P, phytate-P, and Ca.

Table 6: Percent of total diet as-fed of dietary ingredients including concentrate and forage for each of the

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Diet
C C+P H H+P F F+P
Concentrate, % as-fed of total diet
Corn, fine ground 6.34 6.34 6.34 6.34 - 0.15
m/falzg‘lldds By'Pm‘luct 27 t° 6.29 6.29 6.29 6.29 - -
Soybean Hulls 8.00 8.00 8.00 8.00 - -
Beet Pulp 5.72 5.72 5.72 5.72 - -
Rice-Broken 6.00 6.00 6.00 6.00 - -
Molasses-Cane 2.00 2.00 2.00 2.00 - -
Salt 0.40 0.40 0.40 0.40 - -
Calcium Carbonate 0.30 0.30 0.30 0.30 - -
Fat-Com Oil 0.50 0.50 0.50 0.50 - -
Bentonite 0.80 0.78 0.60 0.58 - -
Phosphate-Mono Dicalcium - - 0.40 0.40 - -
Soybean Protein Concentrate 1.17 1.17 1.17 1.17 - -
Corn Gluten Meal 60% Protein 2.00 2.00 2.00 2.00 - -
Quest Anise 4X 0.02 0.02 0.02 0.02 - -
776-Horse PX50981 0-04 0-04 0-04 0-04 - -
Maxi Bond 0.40 0.40 0.20 0.20 - -
51102 Trace Mineral Premix2 0-02 0-02 0-02 0-02 - -
phytase" - 0.02 - 0.02 - 0.001
Forage, % as-fed of total diet
Long stem alfalfa hay 60 60 60 60 100 99.85
Percent of Total Diet, dry matter
basis
% P 0.32 0.29 0.45 0.39 0.30 0.30
% Phytate 0.08 0.08 0.07 0.08 0.05 0.04
% Ca 1.39 1.39 1.47 1.47 1.81 1.64
Ca:P 4.3 4.8 3.3 3.8 6.0 5.5

 

mg/kg Thiamine

1:Provided the following per kilogram of diet: 13,216 IU/kg Vitamin A, 1,002 IU/kg Vitamin D, 99 IU/kg
Vitamin E, 0.001 mg/kg Vitamin BIZ, 0.11 mg/kg Biotin, 9 mg/kg Folic Acid, 8 mg/kg Riboﬂavin, ll

 

mg/kg Iron, 84 mg/kg Manganese, 0.3 mg/kg Selenium, 84 mg/kg Zinc

2:Provided the following per kilogram of diet: 1 mg/kg Cobalt, 18 mg/kg Copper, 1 mg/kg Iodine, 12

 

3: Optiphos 4000 PF, JBS United, Sheridan, IN

 

 

60

 

 

Table 7: Analyzed dry matter, phytate, phosphorus, calcium, magnesium, and phytase activity of
the concentrates (C, C+P, H, H+P, F+P) and forage used for the six diets.

 

 

 

 

 

 

 

 

 

 

 

Diet

C C+P H H+P F+P Forage
Analyzed Composition, DM
Basis
DM, % 90.29 89.37 89.14 88.83 88.71 88.90
P, g/kg 3.75 2.26 6.77 5.29 2.55 3.01
Phytate P g/kg 1.25 1.24 1.14 1.34 0.26 0.45
Ca, g/kg 7.67 7.75 9.75 9.65 0.65 18.06
Mg, g/kg 2.48 2.51 2.60 2.61 0.94 3.28
Phytase Activity, FTU/kg] - 440 - 530 3,400 -

 

 

 

 

 

 

1: One phytase unit (FTU) is deﬁned as the amount of enzyme which liberates 1 pmol inorganic
ortho-phosphate per min from 0.0051 mol/L Na-phytate at pH 5.5 and 37° C.

 

 

Table 8: Intake of hay and oats on a DM basis, fecal and urine total output per day, and total P

 

 

 

 

 

 

 

 

 

 

content of excreta per day.
Diet
Item C C+P H H+P F F+P SEM P-
Value
Hay Intake, kg DM/d 3.7 3.7 3.7 3.7 6.4 6.1 - -
Concentrate Intake, kg DM/d 2.5 2.5 2.5 2.5 - 0.6 - -
Fecal Output, kg DM/d 2.9 3.4 2.7 2.8 2.9 2.9 0.2 0.1
Urine Output, L/d 18.3 19.0 17.9 14.0 16.6 16.2 3.5 0.6
Total P Output, g PM 21.7 22.2 26.6 26.4 26.5 24.3 3.2 0.6

 

 

 

 

 

 

 

61

 

 

 

Table 9: Phosphorus, Calcium, and Magnesium intake, output, balance, apparent digestibility and body
weights for each of the six diets; P-values for the contrasts of all phytase diets versus all non-phytase diets
(P vs. NP) and the two high forage diets versus the four lower forage diets (H vs. L).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

P H
’ Diet vs. vs.
NP L
Item C C+P r1 H+P F F+P SEM V3116 P-Value
Weight 472 473 471 471 474 474 14 0.91 - -
P
Intake, mg-kg Bw‘l -d'1 43 39d 5921 52b 41 43c 1 0-01 0-01 0.01
Urine, mpg BW'Id'f 0.1 0.1 0.1 0.1 0.1 0.1 0.0 0.22 0.36 0.63
Feces, mgkg 3“]de 46 47 56 56 55 52 6 0.59 0.83 0.75
Balance, mgkg 3w" .d" -3 -8 3 .4 -14 -9 6 0.26 0.66 0.14
Apparent Digestibility, % -8 -21 4 -8 -34 -19 14 0.26 0.75 0.15
Ca
Intake, mg-kg BW'1 «1" 184b 184B 194b 1941’ 245a 236a 6 0-01 0.54 0.01
Urine, mpg Bw'l.d-1 25 21 18 11 17 13 7 0.50 0.41 0.57
Feces, mg-kg Bw"-d‘l 114 102 120 105 111 103 11 0.62 0.20 0.75
Balance, mg-kg Bw'I ~d'] 45c 61be 56bc 78b 117a 1208. 12 0.01 0.18 0-01
Apparent Digestibility, % 24c 32bc 29c 40 4831) 5]3 6 0.02 0.15 0.01
Mg
Intake, mg-kg BW‘l-d'l 26b 26b 265 26b 45“ 43a 1 0.01 0.46 0.01
Urine, mg-kg BW"-d“ ll 11 11 10 14 13 2 0.76 0.72 0.23
Feces, mg-kg Bw"-c1'l 29 29 32 27 27 29 2 0.36 0.51 0.39
Balance, mg-kg Bw"-d" -1 -1 -4 2 4 3 4 0.38 0.48 0.20
Apparent Digestibility, % -2 -3 -11 6 9 6 8 0.46 0.46 0.24

 

 

 

 

 

 

 

 

 

 

 

 

a, b, c, d: Values within the same row with differing superscripts differ signiﬁcantly (P<0.05)

 

Intake of Ca was greatest on the F and F+P diets (Table 9), but no difference was

seen between any of the diets in urine or fecal Ca output. The Ca balance was higher at

higher levels of intake and horses on all diets maintained a positive Ca balance. There

was no difference in Ca AD between the C, H, and F and their respective phytase added

diets. Intake of Mg was also greatest on the F and F+P diets (Table 9), but there were no

differences in urine Mg, fecal Mg, or Mg balance between all diets. Horses on all diets

62

 

maintained a near zero Mg balance, and there was no difference in AD between any of
the diets.

Phytate intake ranged from 6.2 to 10.7 mg phytate/kg BW/d (Table 10).
Differences in intake did not result in differences in phytate disappearance, which
averaged 93%. Phytase supplementation improved the phytate balance on the H diet from

9.0 to 10.0 mg - kg BW‘l - d'l, but there was no difference in the respective percentages

of disappearance.

 

Table 10: Phytate intake, output, balance, and percent disappearance for each of the six diets; P—values
for the contrasts of all phytase diets versus all non-phytase diets (P vs. NP) and the two high forage diets
versus the four lower forage diets (H vs. L).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

P H
Diet vs. vs.
NP L
Item c C+P H H+P F F+P SEM P‘ P-Value
Value
Phytate
Intake
’ ab b a c c
-l -l 0.3 0.001 0.14 0.01
mg-kg BW -d 10.3 10.2 9.6 10.7 6.2 6.3
Feces,
-l -l 0.4 0.7 0.6 0.7 0.5 0.6 0.1 0.55 0.13 0.58
mg-kg BW -d -
Balance
’ a ab b a c c
-l -l 0.3 0.001 0.47 0.01
mg-kg BW _ d 9.9 9.4 9.0 10.0 5.7 5.6
Disappearance,% 96 93 94 93 92 91 2 0.37 0.27 0.04

 

 

 

a, b, 0: Values within the same row with differing superscripts diﬁer signiﬁcantly (P<0.05)

 

As expected, there were no differences between diets in serum concentrations of
P, Ca, or Mg; all horses maintained serum concentrations within reference ranges

throughout the study (Table l 1).

63

 

 

 

 

 

 

 

 

 

Table 11: Phosphorus, Calcium, and Magnesium serum values.
Diet
Item C C+P H H+P F F+P SEM P—value
Serum P, mg/dL 3.1 2.9 3.4 3.2 2.9 3.0 0.2 0.37
Serum Ca, mg/dL 12.1 12.2 12.5 12.7 13.1 12.5 0.5 0.11
Serum Mg, mg/dL 1.9 1.9 2.0 1.9 1.9 2.0 0.1 0.82

 

 

 

 

 

 

 

 

 

Discussion

Horses maintained a near zero P balance. While 5 of the 6 diets resulted in
negative balances, those values are not signiﬁcantly different ﬁ'om the diet that was in
positive balance. These were mature maintenance animals, so a near zero balance is
expected. Negative balances are obtainable when animals are fed above their requirement
for a nutrient, and also have an endogenous loss of the same nutrient Horses were being
fed on average well above their suggested requirement of approximately 13 g P/d (NRC,
2007) and this may have contributed to the large fecal P losses. The negative P balances
and P-AD most likely indicate that the horses had a very low P requirement and where
therefore excreting a large portion of dietary P and their normal level of endogenous P
loss. Other researchers have reported low P-balances in mature horses. When horses
received 18 to 44 g P/d, P-balance ranged from 0.4 to 5.6 g/d (van Doorn et al., 2004a).
Which is similar to horses fed 28 to 33 g P/d (Patterson et al., 2002) or 20 g P/d (Morris-
Stoker et al., 2001) with reported P—balances of 3 to 6 g P/d or 3 to 4.2 g PM.

The digestibility of P was not affected by phytase supplementation. This may be
due to the diets containing relatively low amounts of phytate. Any additional P liberated
from phytate may not have had a large enough effect on overall P balance. The increase
in phytate balance ﬁ'om the H to the H+P diet may have been due to the higher phytate

intake on the H+P diet. The lack of effects on P balance could also be due to the level of

64

phytase activity in the diet, as perhaps higher levels of activity would result in differences
in P balance. However, Van Doom et al. (2004a) reported no differences in P balance
when mature horses were fed hay and a textured ration consisting of oat, corn, wheat
bran, and rice bran with and without supplementation of 740 U/kg phytase (one U is the
quantity of enzyme that releases l-umol of inorganic P per min from 0.5 mM Na-phytate
at pH 5.5 and 37 °C; Natuphos 5,000 Liquid, DSM Food Specialties, Delft, The
Netherlands). When mature horses were fed 300, 600, or 900 U/kg phytase (one U is the
quantity of enzyme that liberates l-umol of inorganic P per min from 5.1 mM Na-phytate
at pH 5.5 and 37°C; Natuphos 600, BASF, Wyandotte, WI) on top of a grass hay and
grain diet of corn, oats, and soybean meal, no effect of enzyme supplementation was seen
on P digestibility (Patterson et al., 2002). And in yearling horses fed grass hay and
various concentrates including whole oats, sweet feed, a pelleted concentrate, and alfalfa
cubes, phytase at 1500 U/g (one U is the quantity of enzyme that releases l-umol of
inorganic P per min from 0.15 mM Na—phytate at pH 5.5 and 37°C; Allzyme, Alltech,
Inc., Nocholasville, KY) supplementation was found to have no effect on P digestibility
(Hainze et al., 2004).

While these factors could play a role in whether phytase is effective in increasing
P availability in equine diets, a more probable explanation exists as to why most
researchers fail to detect beneﬁts from supplementing phytase to horses. The high
apparent digestibility of phytate in this study suggests that horses are capable of
degrading phytate, probably due to the microbial population in the hindgut. The hindgut
of the horse serves many similar functions to the rumen of the ruminant animal. Phytate

is quickly hydrolyzed in the rumen (Reid et al., 1947) due to the high phytase activity of

65

the microbes present (Raun et al., 1956). Phytate-P is considered almost completely
available to dairy cattle (Morse et al., 1992; NRC, 2001), while sheep are reported to
absorb 78 to 81% of the phytate-P in cereal and vegetable protein sources (Field et al.,
1984)

If P were liberated from phytate by the microbes in the hindgut, P absorption
would be improved — but only if P were absorbed in the hindgut of the horse. In the
ruminant (Khorasani, 1997, Reinhardt et al., 1988; Pfeffer et al., 1970), swine (Moore
and Tyler, 1955a,b), and human (Gropper et al., 2005), the main site of P absorption is
the small intestine. Schryver et al. (1972) determined that the main site of P absorption in
the horse is the dorsal large colon and small colon. Their ﬁndings supported those of
Alexander (1962), as phosphorous concentrations were signiﬁcantly higher in the large
intestine than in the small intestine, presumably due to the various secretions associated
with digestion. This higher concentration may serve as the primary buffer in the dorsal
colon for the relatively larger proportion of VFA produced in the hindgut than in the
stomach and small intestine, while bicarbonate is the primary buffer system in the
ceacum and ventral colon (Alexander, 1963). It would therefore make sense that a
mechanism would exist to re-absorb P from these secretions before they were expelled in
the feces. Additionally, Matusi et al. (1999) observed the same pattern of P secretion and
absorption as Schryver et al. (1972). Horses are therefore likely similar to ruminants,
swine, dogs, and humans in that there is net absorption of P in the small intestine;
however, they likely differ in that the major site of P absorption in the horse is the large
intestine. This is likely why there was no difference in P balance observed between

phytase supplemented and non-supplemented diets. Horses are already capable of

66

releasing P from phytate via the microbes in the hindgut, and this P is then available for
absorption in the hindgut.

Horses remained in positive Ca balance, and there was no effect of phytase
supplementation on Ca-AD. This is in contrast to van Doom et al. (2004a) in which
horses fed alfalfa meal, wheat bran, and rice bran had improved Ca-AD with phytase
supplementation. Phytate does form insoluble salts with Ca that reduce the availability of
both Ca and P, it is unclear why no effect of phytase supplementation was seen on Ca-
AD in the current study. It maybe due in part to the higher Ca intakes of 87 to 116 g/d on
the current study versus 69 g Ca/d fed by van Doorn et al. (2004a). Higher intake levels
could mask any effects of phytase releasing Ca from phytate. Horses are different ﬁ'om
ruminants in that they absorb a greater percentage of dietary Ca; this is regulated by large
urinary outputs of Ca that are not seen in ruminants (Schryver et al. 1983). The fact that
Ca and P are not absorbed in a 2:1 ratio is partially explained by this very high absorption
and excretion of Ca seen in the horse. Therefore the large urinary Ca outputs and
relatively higher Ca-AD than P-AD were expected.

. The horses maintained a near zero Mg balance on all diets, a near zero balance
was expected as these were mature maintenance animals. Horses were receiving 15 to 24
g Mg/d, which exceeds their requirement of 7 g/d (NRC, 2007). The low balances
resulted in an average Mg-AD of 0.8% which is much lower than that reported by Hintz
et al. (1984), of 50-53%, when mature ponies were fed alfalfa hay and oats. It is also
lower than that reported by Sturgeon et al. (2000) when mature horses were fed alfalfa
hay resulting in a Mg-AD of 58% at higher intake levels of 72 mg - kg BW“ - d"

compared to the 43 to 45 mg . kg BW'1 - cl'l horses received on the current study. It has

67

been shown that high P diets decrease Mg-AD in ponies from 50 to 36% (Hintz and
Schryver, 1972b). It is unclear why there were such low Mg-AD on the current study, it
most likely relates back to the horses being mature maintenance animals. There was no
effect of phytase supplementation on Mg digestibility, which agrees with previous studies
(van Doom et al., 2004a).

This study further demonstrates that feeding mature maintenance horses above
their requirement for P results in higher P outputs with no apparent beneﬁt to the horse.
There was no effect on P, phytate, Ca, or Mg output or their digestibilities due to phytase
supplementation. Theoretically, if phytase is to be a ﬁmctional feed additive for horses,
diets must contain low amounts of P and high amounts of phytate. Research is needed to
determine if P balance in horses receiving high cereal grain concentrate (greater than the
40% used in this study) and low forage diets would be affected by phytase
supplementation, as more phytate would be present in the diet. However, these data
suggest that horses are already capable of degrading phytate, and any additional beneﬁt to
be derived from phytase supplementation may not be cost effective. The most effective
way to decrease P output to the environment would therefore be to design low P diets, but

more research is needed to make these practical for the horse industry.

68

 

CHAPTER 5
SUMMARY AND CONCLUSIONS

Our research has shown that mature maintenance horses and horses receiving
light exercise maintain a near zero P balance, even when fed in excess of current
recommendations (NRC, 2007) for P intake. The mature horse derives no beneﬁt from
excess P concentrations in the diet, and high P concentrations can be detrimental to the
metabolism of other minerals. The ﬁrst study demonstrated that levels far above current
recommendations were easily obtained on forage-only diets. It might be impractical for
the industry to formulate diets that contain P that meets, but does not exceed, P
requirements as the contribution from forages fed at standard rates can exceed P
requirements of mature maintenance animals.

The P requirements of all processes including maintenance, growth, pregnancy,
and lactation need to be better deﬁned, as well as the endogenous losses from horses in
all stages of life and activity levels. Once the true requirement has been deﬁned, research
is needed to design diets that contain the lowest amounts of P possible so as to meet, but
not exceed, the true requirement, thereby reducing unnecessary output to the
environment. The most likely target of diet manipulation would be the concentrate
portion, as reductions in organic P supplementation, or reduction of high P feedstuff
content would decrease fecal output when P is already supplied at the required levels.
This may prove difficult as horses that are being fed large amounts of concentrate often
have a high energy requirement. Feedstuffs would need to be identiﬁed that can meet
these energy requirements at normal dry matter intakes while still addressing the problem

of P concentration. This will also require identiﬁcation of the true digestibility of the

69

 

wide range of feedstuffs utilized by the equine industry. Research will have to include
diets that address the various ratios of forage to concentrate that are fed, as differences in
ratio will affect digestibility.

As the second study demonstrates, phytase is not likely to be a useful feed
additive for the equine industry. Horses have a large microbial population in the hindgut
that functions much like the population housed in the rumen of ruminants. Ruminants are
capable of degrading phytate to liberate P due to their microbial population. Horses most
likely have similar microorganism that are responsible for the high apparent digestibility
of phytate seen in am research. As the horse relies heavily on the hindgut for digestion of
its largely forage diet, it makes inherent sense for a mechanism to exist for P absorption
near or after the location of feed digestion. As previously discussed, research has
supported the conclusion that P is absorbed to a signiﬁcant extent in both the small and
large intestine of the horse. As phytase is an enzyme, constructed of protein, it is likely
degraded in the stomach and small intestine. Any beneﬁt phytase may have would need
to be derived early on in digestion. Phytase enhances Ca absorption in other species by
liberating Ca from phytate. Our research did not ﬁnd similar results; however, this may
have been due to the low amount of phytate present in the diet. Higher phytate content
would increase the likelihood of binding to Ca, and phytase might therefore have an
appreciable effect.

Research is now needed to determine to what extent the P liberated from phytate
in the hindgut is absorbed by the horse. This level of absorption efﬁciency could then be
used in diet formulation to reduce the amount of inorganic P supplemented to the diet,

and ultimately the amount of P introduced to the environment. More research is also

70

 

needed to determine how high phytate diets affect the availability or function of other
minerals, such as Ca and Mg, starches, lipids, and proteins, in particular enzymes such as
trypsin, pepsin, and amylase in horses.

The activities of the microbes in the hindgut of the horse are not well understood.
Research is needed to deﬁne the population and its various functions and capabilities so
that they can be maximized upon by producers. The process of canulating horse’s
intestinal tracts needs to be reﬁned so that studies can be conducted over longer periods
of time utilizing larger sample sizes. This must also be accompanied by research into the
absorptive capacity of the hindgut of the horse. As a large portion of digestion occurs in
the hindgut it would seem evolutionarily beneﬁcial for the large intestine to have
mechanisms for absorption of many of the nutrients derived from microbial processing.

There is only so much that animal science will be able to accomplish in reducing
P emissions from livestock. Ultimately we will need to turn to crops and soil science to
develop manure and ﬁeld management strategies that minimize the possibilities of
erosion and runoff into waterways, and also to develop feeds with lower P

concentrations.

71

APPENDIX A

COMPARISON OF DATA OBTAINED FROM OPEN CELL DIGEST USING ICP—MS

AND DATA OBTAINED FROM MICROWAVE DIGEST USING ICP-MS OR

SPECTROPHOTOMETOR
Introduction
The study presented in Chapter 3 originally called for use of Inductively Coupled

Plasma Mass Spectrometry (ICP-MS) for mineral analysis of all samples. Phosphorus
data obtained from this analysis was then compared to data obtained via the colorimetric
method of Gomori (1942) utilizing microwave digested samples. To determine the effect

of digest method, the microwave digests were then analyzed using the ICP-MS and

results were compared.

Materials and Methods
Mineral Analysis-ICP-MS

Fecal samples were freeze dried and ground, feed samples were also ground.
Portions of all fecal and feed samples were retained for DM analysis. Ground samples
were kept at room temperature until needed for further analysis. Approximately 0.25 g of
fecal and feed samples were measured into digestion vials, 2.5-ml of 70% Nitric Acid
was added to each sample, they were then allowed to digest for 24 h at 86°C in a heating
oven. Double-deionized water was then used to bring the feed and fecal samples up to
approximately 12.5 g. Feed and fecal samples were centrifuged before 200 pl of each
sample was pipetted into an ICP-MS tube and then diluted with 5ml of ICP-MS Diluent
Internal Standard (2% Butanol, 0.05% Triton X-100, 0.05% EDTA, 1% Ammonium

Hydroxide, 10 ppb Scandium, 15 ppb Indium, 15 ppb Rhodium, 15 ppb Germanium, 15

72

ppb Bismuth). A portion of urine was acidiﬁed at a rate of 12 pl per ml of mine with
12M HCl to insure all precipitate was in solution (O’Connor, 2006). For mineral analysis,
200 pl of serum and acidiﬁed urine were pipetted into test tubes. All serum and urine
samples had 5 ml of ICP-MS Diluent Internal Standard added to them. All samples were
run in duplicate and vortexed shortly before being run through the ICP-MS (Agilent 7500
CE), located at the Michigan State University Diagnostic Center for Population and

Animal Health.

Mineral Analysis-Microwave Digests and Colorimetric Method

Fecal and feed samples were microwave digested as described by Shaw et al.
(2002). Brieﬂy, approximately 0.400 g of fecal or feed sample was measured into a
Teﬂon-lined digestion vessel, 10-ml of 70% nitric acid (70% trace-metal grade; Fisher
Scientiﬁc, Pittsburgh, PA) was added to each sample, and then samples were allowed to
digest for 24 h lightly covered at room temperature. Vessels were placed in the
microwave assembly and inserted into the microwave accelerated reaction system
(MARS-5, CEM Corp., Matthews, NC). Samples were run at 1,200 W, 100% power, 200
PSI max, 190° C, with a ramp time of 30 min and a hold time of 10 min. Samples were
allowed to cool in a hood for 5 min and then 2 ml of 30% hydrogen peroxide (J. T. Baker,
Phillipsburg, NJ) was added. After 15 min, samples were brought to volume with ddH20,
and samples were stored at room temperature. All glassware used in the mineral analyses
was washed in 30% nitric acid and rinsed with double-deionized water.

Samples were vortexed thoroughly before being used for assays. Digested fecal

and feed samples were all diluted 10x, while non-acidiﬁed urine was run with no dilution.

73

Serum samples were ﬁrst precipitated with 12.5% Trichloroacetic acid (TCA) to remove
interfering proteins for P assay, samples were then centrifuged and the supernatant was
used for the assay; samples were run at a 4x dilution. For all samples, 50 ul was pipetted,
in duplicate, into well plates, then 250 pl of Molybdate-Sulfuric (MS) solution and 25 ul
of p-Methylaminophenol (Elon) solution were added; samples were vortexed and
incubated for 45 min. The liquid digests, urine and serum samples were analyzed for P
using a spectrophotometer at 700 nm (Gomori, 1942).
Mineral Analysis-ICP-MS Utilizing Microwave Digest Samples

Microwave digest samples were run on the ICP-MS utilizing the same method
previously described. A P standard and corn bran standard (NIST corn Bran 8433) were
run at the same time for comparison.
Results and Discussion

Results obtained from the ICP-MS utilizing an open-cell digest did not closely
match those obtained utilizing the colorimetric method and microwave digests, or

utilizing the ICP-MS and microwave digests (Table 1A).

74

 

Table 1A: Results of phosphorus analysis from ICP-MS utilizing open-cell digests (A), ICP-MS
. utilizing microwave digests (B), and colorimetric method utilizing microwave digests (C). Coefficients
of variation between A and B, A and C, and B and C.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A B C
Sample Period Sample ICP-MS lCP-MS Colorimetric CV: A CV: A CV: B
Type Open Microwave Method vs. B vs. C vs. C
Cell Digest Microwave
Digest Digest
mg P/ g sample DM %

Hi 1 Fecal 3.9 3.4 3.0 10 19 9
Sand 1 Fecal 4.1 3.7 3.2 9 l7 9
Masq 2 Fecal 5.6 4.7 4.5 13 16 4
Geo 2 Fecal 8.1 6.7 5.7 13 25 12

Bo 3 Fecal 5.7 5.1 5.4 7 4 3
Cav 3 Fecal 6.8 5.8 4.9 1 1 23 12

Hi 3 Fecal 8.7 6.4 5.4 22 33 12

B0 4 Fecal 7.8 5.8 6.5 21 13 8
Masq 5 Fecal 10.9 8.9 8.8 14 15 l
Geo 5 Fecal 10.2 7.4 6.5 22 3| 9

Bo 6 Fecal 10.6 7.2 8.0 27 20 7

GH 1 Hay 1.9 1.9 1.5 l 16 16
GH 2 Hay 1.2 2.3 2.4 44 44 1
GH 3 Hay 1.4 2.4 2.4 35 35 1
GH 6 Hay 4.2 3.4 2.8 14 27 13

A1 2 Hay 4.1 3.9 3.3 4 15 11

A1 5 Hay 4.7 4.5 4.0 3 ll 8

A1 6 Hay 6.1 4.0 3.8 30 34 4

A2 1 Hay 4.0 3.7 3.0 5 20 15

A2 4 Hay 2.8 4.0 3.3 25 ll 14

A2 6 Hay 3.7 3.8 3.2 2 9 11

A3 2 Hay 1.9 3.4 3.1 40 35 6

A3 4 Hay 3.8 3.6 3.1 4 16 12

A3 6 Hay 2.5 3.4 3.5 21 23 2
Oats 4 Grain 5.6 4.5 4.3 15 18 4
Oats 6 Grain 3.0 5.4 4.9 41 34 8

Average: 17 22 8
Fecal 15 20 8
Hay I8 23 9
Grain 28 26 6

 

 

 

 

 

The standards run on the ICP-MS at the same time as the microwave digests returned

values that were within the expected range. It is unclear as to whether the discrepancy in

75

 

values is due to the digest or to some type of interference during sampling on the
machine. As the variation between the results obtained via the colorimetric method
utilizing microwave digests and the ICP-MS utilizing microwave digests was lower than
the variations between the colorimetric method and the ICP-MS utilizing open cell
digests, it is likely that the problem exists in the digest method. It appears that the open
cell digest might be better for fecal samples than feed samples. As the colorimetric
method is more standard in the industry, it was utilized for all phosphorus analysis in the
two projects.

The average coefficient of variation between ICP-MS and colorimetric P analysis
values for serum was 10%, with a range of 0 to 28% (Table 2A). This might be because
the colorimetric method called for precipitation with TCA prior to the assay to eliminate
interfering proteins. The level of P in urine was below the detection level of the ICP-MS,

and therefore that data is not presented here.

76

 

Table 2A: Comparison of serum phosphorus analysis between lCP-MS and
the colorimetric method.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ICP-MS Colorimetric CV
Horse Period mng/dl Serum %
Bo l 4.1 4.7 10
Masq l 3.0 3.4 8
Hi 1 3.4 2.9 10
Geo I 3.8 3.4 8
Cav l 3.6 3.9 5
Sand 1 2.8 4.0 26
B0 2 3.4 3.7 5
Masq 2 3.5 3.3 3
Hi 2 4.0 3.2 16
Geo 2 3.5 3.7 4
Cav 2 3.4 5.0 27
Sand 2 3.8 4.0 3
B0 3 3.8 4.0 3
Masq 3 3.4 4.1 13
Hi 3 3.6 3.1 9
Geo 3 3.6 3.6 1
Cav 3 3.2 4.8 28
Sand 3 3.6 3.6 0

 

 

 

 

 

77

 

APPENDIX B
ESTIMATED ENDOGENOUS LOSSES AND TRUE DIGESTABILITIES FROM
BOTH REPORTED STUDIES
Introduction
A regression technique was utilized to estimate the endogenous losses of P, Ca,

and Mg from the two reported studies. The studies were not designed to make this
estimate reliable, and therefore it was not presented with the study results. The values
may serve some purpose for comparisons with other work utilizing this technique and are

therefore reported here.

Materials and Methods
Calculations

Mineral concentrations were used to calculate average mineral intake per day, as
well as urinary and fecal mineral excreted per day. As urinary P was negligible (0.03 to

0.04 g/d), P balance was calculated as:

P Balance= (Intake of P)—(Output of P in Feces)
Ca and Mg balance were calculated as:

Ca (or Mg) Balance= (Intake of Mineral)-(Output of Mineral in Feces and Urine)

Estimated endogenous losses were calculated by plotting the mineral balance (Y-axis)
against the average mineral intake (X-axis), with the Y-intercept being used as the
amount of endogenous losses or the point where intake would be zero. Estimated true

mineral digestibility was then calculated as:

78

Est. True Dig, % = (Mineral Balance + Endogenous Fecal Losses) x 100

 

Total intake of Mineral

Statistical Analysis

Data were analyzed using the REG procedure of SAS software (Version 9.1, SAS

Inst., Inc., Cary, NC).

Results

The R2 values were less than desirable, with the possible exception of the Ca
estimates (Table 1B). The values of the estimated true digestibility and estimated
endogenous losses (Table 2B) did not closely match those suggested by the NRC (2007)

as shown in Table 3B.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 1B: Estimated true digestibilities for the six diets ﬁ'om both reported studies.
Estimated true digestibilities, % Estimate Regiaszlon
Diet SEM thl-ue R2 Vil-ue
Mineral Al A2 A3 GH GH+1 GH+2
P, % 39 41 40 32 44 29 8 0.60 0.3 0.002
Ca, % 60 48 47 54 60 52 8 0.22 0.7 0.001
Mg, % 37 31 35 40 42 41 5 0.48 0.3 0.001
C C+P H H+P F F+P

P, % 74 69 63 60 51 62 15 0.81 0.1 0.10
Ca, % 75 83 77 88 85 90 6 0.33 0.5 0.001
Mg, % 86 85 77 96 86 84 10 0.70 0.2 0.013

 

79

 

 

 

 

 

 

 

 

 

 

 

Table 23: Estimated endogenous losses for the six diets
from both reported studies.
Estimated Endogenous Losses
Mineral Study 1 Study 2
P, g/d 16.7 9.4
P, mg/kg BW/d 37.0 20
Ca, g/d 2.4 37
Ca, mg/kg BW/d 5.2 78
Mg, g/d 0.8 14
Mg, mg/kg BW/d 2 31

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 3B: Estimated true digestibilities and endogenous losses
suggested by the NRC (2007).
Mineral Estimated True Estimated Endogenous
Digestibility (%) Losses (mg/kg BW/d)
P 30 to 55 10
Ca 50 to 75 20 to 32
Mg 40 to 60 2 to 6
Discussion

The high estimated endogenous losses of Ca, P, and Mg may illustrate the
inherent problems with the regression technique utilized, particularly when dietary
intakes are far from zero. The technique functions on the principle that the y-intercept
represents losses when intake is zero. As intake approaches zero, absorption would be
increased and excretion would be decreased to continue to meet demand and maintain
blood levels, this could no longer be represented by a linear relationship. When intake
levels are far from zero, the discrepancy is likely magniﬁed, resulting in inﬂated
estimations of endogenous loss and estimated true digestibility. To illustrate the changes
in the estimation that wider intake ranges can have, values from both studies were
combined. The regression line of the combined data of intake versus apparent absorption

resulted in estimated endogenous losses of 7.92 g Ca/d (R2=0.55), which would represent

80

 

16.7 mg Ca - kg BW'1 - d'1 for a 473-kg horse. These values are actually lower than those
considered by the NRC (2007), which makes inherent sense, as these animals were
mature maintenance horses. When the same thing was done for P intake and balance of
the two studies, there was no real improvement in the estimate of endogenous P loss,
which was 15.7 g/d (R2=0.20), representing 33.1 mg - kg BW“ - d“ for a 473-kg horse,
which is about 3 times the amount suggested by the NRC (2007). The lack of difference
is likely because P intake was on average very similar between the two studies; intake on
the ﬁrst study was 12 to 36 g/d, while the second study ranged from 15 to 32 g/d of P
intake. Data was also combined for Mg intake and balance. Intake of Mg on the ﬁrst
study ranged from 11 to 23 g/d, the estimated endogenous losses were 1.02 g Mg/d
(R2=0.03) or 2.2 mg - kg BW'1 - d'l. The relatively small increase in range of Mg intake
improved estimation of Mg endogenous losses but a similar increase in P intake range did
not improve the estimation of P endogenous losses. This is likely because the range of P-
balance did not change between the ﬁrst and second studies (-22 to 7 g/d vs. -15 to 11
g/d), while the range of Mg-balance was different between the two studies (-13 to 18 g/d

vs. 0.2 to 13 g/d).

81

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