 

 

 

 

 

L University |

This is to certify that the

thesis entitled
ESTIMATION OF INEVITABLE PHOSPHORUS LOSSES AND
PREDICTING PHOSPHORUS EXCRETION OF DAIRY CATTLE FED A
RANGE OF DIETARY PHOSPHORUS CONCENTRATIONS

presented by

ZACHARY H. MYERS

has been accepted towards fulfillment
of the requirements for

MS . Animal Science
degree in

 

 

 

   
 

ajor professor

[hue February 12, 2003

 

0-7639 MS U is an Afﬁrmative Action/Equal Opportunity Institution

 

 

ESTIMATION OF INEVITABLE PHOSPHORUS LOSSES AND PREDICTING
PHOSPHORUS EXCRETION OF DAIRY CATTLE FED A RANGE OF DIETARY
PHOSPHORUS CONCENTRATIONS

By

Zachary H. Myers

A THESIS

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Animal Science

2003

 

ABSTRACT
ESTIMATION OF INEVITABLE PHOSPHORUS LOSSES AND PREDICTING

PHOSPHORUS EXCRETION OF DAIRY CATTLE FED A RANGE OF DIETARY
PHOSPHORUS CONCENTRATIONS

By
Zachary H. Myers

The ﬁrst objective of this research was to evaluate estimates of inevitable fecal
phosphorus (P) excretion as part of the maintenance requirement for P. Lactating cows
were fed a low P diet (0.26%) at three different average DMI rates (low, medium, and
high). Inevitable fecal P excretion is the fecal portion of the P maintenance requirement
of animals fed at or near their true maintenance requirement for P. Phosphorus balance
was 1.5 g/d for all three treatments and not different from zero. Regression analysis
deﬁned prediction of inevitable fecal P excretion at various DMI: (g/d) = [0.85 x DMI
(kg/d)] + 5.30 (g/d); (RMSE = 1.75 g P/kg DMI; R2 = 0.90).

The second and third objectives of this research were to collect P excretion data
from dairy animals fed varying dietary P concentrations to develop independent datasets
and to use these datasets to evaluate eight P excretion prediction models. Phosphorus
intake and excretion ranged from 12 to 121 g/d. Accuracy, bias, and precision were
evaluated together to determine the most appropriate models. Two models were more
approptiate than the other six: P excretion (g/d) = intake P — milk P (g/d) and P excretion
(g/d) = 5.92 + 0.741 x [intake P — milk P (g/d)]. These models are more useful to predict

P excretion than the other six models evaluated.

I dedicate this thesis to my wife, Sybil, who worked hard to support us while I achieved
this goal. You were always there when I needed you most.

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ACKNOWLEDGEMENTS

I would like to extend my gratitude to Dr. David Beede for allowing me the
opportunity to further my education. I have learned a lot from you and am indebted to
you for all that you have done for me. I also would like to thank my committee members
Drs. Michael Allen, Margaret Benson, and Tom Herdt for their valuable time, assistance.
and advice on this research.

Appreciation also is extended to Robert Kreft, Gordon Galloway, Randall
Bontranger, Robert Story, and the rest of the farm crew and feed mill staff at the
Michigan State University Dairy Teaching and Research Center for daily mixing,
feeding, and care of experimental animals. We also thank Dr. Michael Allen and Dave
Main for assisting and allowing us to use equipment in the Michigan State University
Dairy Forage Laboratory. Thanks also are extended to all the graduate students in the
Michigan State University Animal Science Department (dairy nutrition group) who
helped collect fecal and urine samples, and weigh cows. Tom Pilbeam provided
assistance with the Cr and urine P laboratory analyses, body condition scoring, and
helped with statistical analyses. Dr. Jill Davidson helped with assay trouble-shooting and
statistical analyses. Richard Longuski and Ashley Peterson body condition scored and
weighed cows. 1 would also like to thank all the undergraduate students that helped with
sample collections and with lab work. Your contributions to this research are much
appreciated.

I also would like to thank my parents, Dwayne and Barbara, and my siblings Chad
and Nickayla for their love and support. I also would like to thank Sybil’s family, Herb

and Linda MacDonald and B.D., Tiffany. Andrew, and Rebecca Reece for their love and

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support while Sybil and I were here in Michigan. I would also like to thank Sybil’s
family living in Michigan that were always fun to visit with and gave Sybil and I a touch
of family away from our immediate families.

Special thanks to my wife, Sybil, for her love and support while I earned my
degree. I could not have done this without you! I appreciate your patience and
understanding throughout the course of my MS program. I also appreciate the trips to the
dairy you made with me at all hours of the day and night to help and to keep me
company. I also thank you for the help you provided in the lab as well as tracking down
undergraduate students to help me in the lab. More important than this degree was the

relationship that you and I were able to develop with only each other to rely on.

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TABLE OF CONTENTS

LIST OF TABLES ........................................................................... ix
LIST OF FIGURES ........................................................................ xii
CHAPTER 1
INTRODUCTION ............................................................................ 1
CHAPTER 2
REVIEW OF LITERATURE
Physiological Roles of Phosphorus ................................................... 4
Partitioning of Phosphorus ............................................................ 6
Phosphorus Requirements ............................................................ 9
Inﬂuence of Dietary Phosphorus on Performance ................................ 12

Inﬂuence of Dietary Phosphorus on Fecal and Urinary Phosphorus

Excretion and Milk Phosphorus Secretion ......................................... 17

Inﬂuence of Other Factors on Fecal Phosphorus Excretion ..................... 19

Prediction of Phosphorus Excretion ................................................. 23
CHAPTER 3

EVALUATING ESTIMATES OF THE PHOSPHORUS MAINTENANCE
REQUIREMENT OF LACTATIN G HOLSTEIN COWS WITH VARYING
DRY MATTER INTAKES

Abstract ................................................................................. 28
Introduction ............................................................................. 29
Materials and Methods ............. . .................................................. 31
Conclusions ............................................................................. 43
Tables .................................................................................... 5 1

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Figures .................................................................................. 56

CHAPTER 4

INCREASING DIETARY PHOSPHORUS: UTILIZATION OF

PHOSPHORUS BY DAIRY CATTLE AT DIFFERENT POINTS
OF THE LACTATION CYCLE

Abstract ................................................................................. 57

Introduction ............................................................................. 59

Materials and Methods ................................................................ 60

Results and Discussion ................................................................ 75

Conclusions ............................................................................. 86

Tables .................................................................................... 89
CHAPTER 5

PREDICTING PHOSPHORUS EXCRETION OF DAIRY CATTLE FED A
RANGE OF DIETARY PHOSPHORUS CONCENTRATIONS

Abstract ................................................................................. 1 1 1
Introduction ............................................................................. 1 13
Materials and Methods ................................................................ 1 15
Results and Discussion ............................................................... 117
Conclusions .......................................................................... ,..122
Tables .................................................................................... 124
Figures ................................................................................... 131

CHAPTER 6

CONCLUSIONS AND IMPLICATIONS ................................................ 139

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REFERENCES ............................................................................... l 44

APPENDIX

A Digestion Procedure for Feed, Feces, and Milk Phosphorus .................... 149
B Procedure for Feed, Fecal, and Milk P Assay .................................... 151
C Digestion for Chromium Analysis .................................................. 153
D Urine Speciﬁc Gravity Analysis .................................................... 155
E Procedure for Urine Phosphorus Assay ............................................ 156

viii

LIST OF TABLES

CHAPTER 3

Table 1. Formulated ingredient and analyzed chemical composition
of the basal diet ................................................................................. 51

Table 2. Performance characteristics of cows as inﬂuenced by
experimental procedures ....................................................................... 52

Table 3. Characteristics of feces, urine, and milk as inﬂuenced by
experimental treatments ........................................................................ 53

Table 4. Phosphorus excretion and balance as inﬂuenced by

experimental treatments ....................................................................... 54
Table 5. Dietary P utilization as inﬂuenced by experimental treatments ................ 55
CHAPTER 4

Table 1. Formulated ingredient and analyzed chemical composition of the
basal diet (DM basis) for Experiments 1, 2, 3, and 4 ..................................... 89

Table 2. Formulated ingredient and analyzed chemical composition of the
basal diet for Experiments 5, 6, and 7 ...................................................... 90

Table 3. Description of all dependent variables used to evaluate
experimental objectives ....................................................................... 91

Table 4. Performance characteristics of nulliparous heifers (57 to

28 i 1.0 (1 prior to parturition) as inﬂuenced by experimental treatments .
(Experiment 1) ................................................................................. 93

Table 5. Phosphorus balance and characteristics of feces and urine for

nulliparous heifers (57 to 28 i 1.0 d prior to parturition) as inﬂuenced by
experimental treatments (Experiment 1) .................................................... 94

Table 6. Dietary P utilization for nulliparous heifers (57 to 28 i 1.0 (1
prior to parturition) as inﬂuenced by experimental treatments
(Experiment 1) ................................................................................. 95

Table 7. Performance characteristics of nulliparous heifers (38 to 9 i
1.0 (1 prior to parturition) as inﬂuenced by experimental treatments
(Experiment 2) ................................................................................. 96

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Table 8. Phosphorus balance and characteristics of feces and urine for

nulliparous heifers (38 to 9 i 1.0 (1 prior to parturition) as inﬂuenced by
experimental treatments (Experiment 2) .................................................... 97

Table 9. Dietary P utilization for nulliparous heifers (38 to 9 i 1.0 (1 prior
to parturition) as inﬂuenced by experimental treatments (Experiment 2) ............. 98

Table 10. Performance characteristics of non-lactating multiparous cows
(59 to 30 i 1.0 d prior to parturition) as inﬂuenced by experimental
treatments (Experiment 3) .................................................................... 99

Table 1]. Phosphorus balance and characteristics of feces and urine for
non-lactating multiparous cows (59 to 30 i 1.0 (1 prior to parturition) as
inﬂuenced by experimental treatments (Experiment 3) ................................. 100

Table 12. Dietary P utilization for non-lactating multiparous cows (59 to

30 i 1.0 d prior to parturition) as inﬂuenced by experimental treatments
(Experiment 3) ................................................................................. 101

Table 13. Performance characteristics of non-lactating multiparous cows
(40 to 11 i 1.0 d prior to parturition) as inﬂuenced by experimental
treatments (Experiment 4) .................................................................. 102

Table 14.‘ Phosphorus balance and characteristics of feces and urine for
non-lactating multiparous cows (40 to 11 i 1.0 d prior to parturition) as
inﬂuenced by experimental treatments (Experiment 4) ................................. 103

Table 15. Dietary P utilization for non-lactating multiparous cows (40 to
11 i 1.0 d prior to parturition) as inﬂuenced by experimental treatments
(Experiment 4) ................................................................................ 104

Table 16. Performance characteristics of early lactation cows (76 to 106 i .
10.0 DIM) as inﬂuenced by experimental treatments (Experiment 5) ................ 105

Table 17. Phosphorus balance and characteristics of feces, urine, and milk
of early lactation cows (76 to 106 i 10.0 DIM) as inﬂuenced by
experimental treatments (Experiment 5) .................................................. 106

Table 18. Dietary P utilization of early lactation cows (76 to 106 i 10.0
DIM) as inﬂuenced by experimental treatments (Experiment 5) ...................... 107

Table 19. Performance characteristics of mid lactation cows (162 to 192
i 6.2 DIM) as inﬂuenced by experimental treatments (Experiment 6) ............... 108

 

 

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Table 20. Phosphorus balance and characteristics of feces, urine, and milk
of mid lactation cows (162 to 192 i 6.2 DIM) as inﬂuenced by
experimental treatments (Experiment 6) ...................................................... 109

Table 21. Dietary P utilization of mid lactation cows (162 to 192 i 6.2
DIM) as inﬂuenced by experimental treatments (Experiment 6) .......................... 1 10

CHAPTER 5

Table 1. Phosphorus excretion models evaluated by the independent
Datasets ............................................................................................ 125

Table 2. Accuracy of prediction of measured P excretion (g/d) of lactating
and non-lactating dairy animals by various models using the full dataset ............... 126

Table 3. Bias of prediction of measured P excretion (g/d) of lactating and
non-lactating dairy animals by various models using the non-lactating
dataset ............................................................................................. 127

Table 4. Precision of various models to predict P excretion (g/d) by
lactating and non-lactating animals using the full dataset .................................. 128

Table 5. Accuracy of prediction of P excretion (g/d) of lactating dairy cows
by various models using the lactating cow dataset .......................................... 129

Table 6. Bias of prediction of measured P excretion (g/d) of lactating dairy
cows by various models using the non-lactating dataset .................................... 130

Table 7. Precision of various models to predict P excretion (g/d) by lactating
dairy cows using the lactating cow dataset ................................................... 131

Table 8. Accuracy of prediction of P,excretion (g/d) of non-lactating dairy
animals by various models using the non-lactating animal dataset .................... -. 132

Table 9. Bias of prediction of measured P excretion (g/d) of non-lactating
dairy animals by various models using the non-lactating dataset .......................... 133

Table 10. Precision of various models to predict P excretion (g/d) by non-
lactating using the non-lactating animal dataset ............................................. 134

xi

LIST OF FIGURES

CHAPTER 2
Figure 1. Partitioning and recycling of dietary P by ruminants .............................. 7
CHAPTER 3
Figure l. The relationship between endogenous fecal P and daily DMI .................. 56
CHAPTER 5

Figure 1. Predicted P excretion plotted against measured P excretion in the
full dataset for visualization of the precision of each model ............................... 135

Figure 2. Predicted P excretion plotted against measured P excretion in the
lactating cow dataset for visualization of the precision of each model ................... 136

Figure 3. Predicted P excretion plotted against measured P excretion in the
non-lactating animal dataset for visualization of the precision of each model ........... 137

Figure 4. Phosphorus prediction models developed using linear regression
of P excretion against intake P — milk P ...................................................... 138

xii

CHAPTER 1

INTRODUCTION

The Council of Agricultural Science and Technology (2002) reported that
producers feed 0.45 to 0.50% P. This range of dietary P concentration represents an
excess of 20 to 25% relative to the current NRC (2001) recommendation. Satter and Wu
(1999) stated that the trend to overfeed P may be the result of the Hignett and Hignett
(1951) study that reported low P diets decreased reproductive performance. Since that
study, however, there has been overwhelming evidence to suggest that low P diets do not
decrease reproductive performance in dairy cattle unless dietary P is low enough to
decrease DMI (Satter and Wu, 1999). If DMI is reduced, the resulting reduction in intake
of energy and other nutrients will increase infertility (Satter and Wu, 1999).

In general, a typical lactating cow‘s diet contains adequate P with little or no P
supplementation (NRC, 2001). Phosphorus supplementation costs $10 to 15/cow per
year (CAST, 2002), costing the U. S. dairy industry $100 million annually (Satter and
Wu, 1999). Fecal excretion is the main source of P loss (NRC, 2001) and P intake is
correlated positively with fecal P excretion (Wu et al., 2001; Knowlton and Herbein,
2002; Wu et al., 2000; Brintrup et a1, 1993; Morse et al., 1992; Martz et al., 1990). '
Therefore, overfeeding of P results in excess fecal P excretion (CAST, 2002; NRC,
2001). Wu et a1. (2000) reported that decreasing dietary P from 0.49 to 0.40 reduced
fecal P excretion by 23%. Decreasing dietary P by 20% can be achieved without
decreasing animal performance, resulting in a 25 to 30% decrease in P concentration of
manure and a similar reduction in the acreage needed for land application of manure

(CAST. 2002). Reducing excess dietary P will decrease both fecal P excretion and ration

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costs without deleterious effects to production. Most non-legume plants utilize N and P
from the soil in a ratio of 2-to-1. However, dairy manure N and P are present in a 1-to-2
ratio. Therefore, current land application practices (N -based) will lead to P build-up in
the soil and inevitable environmental P pollution caused by soil erosion and runoff. If
regulations shift land application of manure from N- to P-based, dairy producers must
have twice the amount of land to apply waste than that required by their current
Comprehensive Nutrient Management Plan (CNMP). As a result, more P in the form of
manure will have to be exported from the farm because the exportation of milk and meat
products, alone is not adequate to maintain a zero P balance (import P = export P).
Therefore, a decrease in imported P (i.e., purchased feedstuffs) and fecal P excretion .
must occur to maintain zero P balance.

Evaluation of Phosphorus Maintenance Requirement. It is important that the P
maintenance requirement be deﬁned as closely as possible to meet cows’ true P needs.
The NRC (2001) deﬁned the P maintenance requirement to be 1.0 g P/kg DMI plus 0.002
g P/kg BW to account for endogenous urine P excretion, rather than their previous (1989)
recommendation based on g P/kg of BW. This change was based on German research
that determined the P maintenance requirement to be 1.2 g/kg DMI for cows fed P at or
near their true requirement (Spiekers, 1993). Having P requirements more accurately
deﬁned ensures a closer estimation of P required (g/d) for optimum production. With this
information, nutritionists will be able to formulate diets to meet cows’ needs while
decreasing the amount of P that will be excreted.

Predcition of Phosphorus Excretion by Dairy Cattle. Finally, a better prediction

of the amount of P excreted is imperative to new and expanding dairies. Regulators

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develop CNMPs to serve as a guideline for how much land is required to support the
quantity of manure produced by a given dairy operation. However, in a review of P
literature, Davidson and Beede (1999) determined two of the models (ASAE 1980; 1996)
available to develop CNMPs were the most inaccurate and imprecise relative to four
other available models found in three studies (Morse et al., 1992; Van Horn etal.. 1994;
Davidson and Beede, 1999) for predicting P excretion. The two ASAE (1980; 1996)
models over-predicted P excretion by 68 and 106%, respectively. Neither model was
considered acceptable for the development of CNMPs. Of the remaining published
models reviewed by Davidson and Beede (1999), the suggestion (Van Horn et al., 1994)
to predict P excretion using a simple calculation P intake (g/d) minus milk P (g/d) was the
most accurate and precise model evaluated. This model over-predicted P excretion 39%
with a mean bias of 14.5%.

There were three objectives of this thesis research. Firstly, to determine the
inevitable maintenance requirement for P of lactating cows with varied DMI. Secondly,
to collect P excretion data from cows fed a wide range of dietary P concentrations.
Thirdly, the data collected for the second objective were used to evaluate eight models

for accuracy and precision in predicting measured fecal P excretion.

CHAPTER 2

LITERATURE REVIEW
This literature review provides information relating to the physiological roles of
phosphorus (P), P requirements, inﬂuence of varying dietary P on dairy cattle

performance, factors affecting P excretion, and prediction of P excretion.

Physiological Roles of Phosphorus

Phosphorus is an important mineral element that impacts the health and
productivity of dairy cows. There are more known biological functions for P than any
other element (NRC, 2001). Consequently, it has extremely important physiological
roles in an animal’s body. Phosphorus is important to the structural integrity of the body
because bones and teeth contain 80% of the P in the body (NRC, 2001). Ellenberger et
a1. (1950) determined that bovine bone contained 5% P. Therefore bone contained 89%
of the total P in the body of cattle in this experiment. In bones and teeth, P is present
mostly as apatite salts and calcium phosphate. Additionally, P is found in cell
membranes and cell contents as phospholipids, phosphoproteins, and nucleic acids.
Phosphorus is also important in energy metabolism. Energy compounds like adenosine
triphosphate (ATP), a primary product of the electron transport system in animals, store
energy in phosphate-to-phosphate bonds for bodily functions.

Because cattle are ruminants, they depend on ruminal microbes for energy and
protein. In order for microbes to survive they must have adequate P. However, prior to

modern feeding practices. cattle diets consisted mostly of forages with very little

 

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concentrate (grain). Because the highest concentration of P in a plant is contained in the
seed, diets typically were relatively low in P, although possibly not deﬁcient in many
cases relative to the animal’s P needs. However, Burroughs et a1. (1951) showed that
ruminal microbes have a P requirement in order to digest cellulose, and Breves and
Schroeder (1991) determined that microbes also require P to synthesize protein.
Therefore, a low P diet is a potential problem for cattle because of this mutualistic
relationship with microorganisms of the digestive tract.

However, cattle evolved a mechanism to recycle absorbed P to help meet the P
requirements of ruminal microorganisms. The salivary gland secretes large amounts of
inorganic P (absorbed from the bloodstream) into the rumen via saliva to meet the
microbes’ P requirements. It is recommended that available P in the rumen should be at
least 5 g/kg of OM digested in order to optimize plant cell wall degradation by microbes
(Durand and Komisarczuk, 1988). In vitro studies showed that cellulose digestion was
maximized when P was added at 20 to 60 ug/ml of media (Hall et al., 1961) and at 60
ug/ml of media (Chicco et al., 1965). Salivary P recycling is adequate to meet this
microbial P requirement when a cow receives adequate P from the diet to meet her bodily
needs (NRC, 2001).

In general, on a daily basis for animals consuming their dietary requirement,
saliva can contribute two times more P to the rumen than the diet (NRC, 2001 ). Salivary
P contributes 30 to 90 g P/d to the ruminal ecosystem (Reinhardt et al., 1988; Scott and
Buchan. 1988). Additionally, inorganic P from saliva can act as a buffer. Therefore, P in

saliva is involved in the ruminal buffering system (NRC, 2001). Phosphate ions act to

neutralize VFAs produced by microbial fermentation by serving as an electron acceptor

to bind protons.

Partitioning of Phosphorus

Because lactating cows have more than one requirement for P, it is important to
understand how P is partitioned once ingested and absorbed. These P requirements
include maintenance, milk, and pregnancy. Figure 1 shows a partitioning and recycling
schematic of dietary P by ruminants. A cow’s P intake (g/d) reﬂects the P content of the
feed. Phosphorus intake (g/d) can be divided into 2 fractions: 1) P (g/d) that is
unavailable for absorption regardless of how long it remains in the gastrointestinal tract;
and 2) P (g/d) that is potentially available for absorption. The unavailable P will
eventually be excreted in feces.

Furthermore, potentially available P can be subdivided into two more parts; a) P
(g/d) that is absorbed; and b) P (g/d) that is not absorbed. Fraction 3 enters the
exchangeable P pool (i.e., the bloodstream) and Fraction b is excreted in feces. The
phrase “exchangeable P pool” better describes the role that the bloodstream plays in P
metabolism. The bloodstream acts as a reservoir in which body tissues and organs can
remove P from or add to the exchangeable P pool depending on the animal’s P needs.

Besides the P demands already mentioned, some lactating cows have additional P
requirements that must be met. For example, in the case of ﬁrst lactation animals that are
still growing, there is a P requirement for growth. In the case of all lactating cows, once
P is absorbed in the small intestine it is available for transport to other parts of the body

to meet P demands. The mammary gland uses P from the exchangeable pool for milk

Figure l. Partitioning and recycling of dietary P by ruminants.

 

 

 

 

 

 

Endogenous Fecal P

 

 

 

 

WAC? = Absorption Coefﬁcient: the percentage of P supply absorbed.

production and secretion. If the cow is pregnant the placenta and developing fetus will
use P from the exchangeable pool. The NRC (2001) reported that the P demand for
pregnancy < 190 d of gestation is small and was set to zero in the model for determining
P requirements. Therefore, the approach taken by the NRC (2001) does not recognize a P
requirement for pregnancy until the cow is > 190 d of gestation. Once absorbed P is used
for milk or pregnancy, it leaves the exchangeable pool and is not available to meet other
P demands because it eventually will be removed from the body.

However, deposition of P into soft tissue and especially bone represent a storage
mechanism for situations when the demands for P are higher than what absorbed dietary
P can supply. Such is the case in early lactation. The onset of lactation increases the.
demand for P because of the additional P requirement for milk production. Early
lactation animals must use P stored in bones to meet the increased requirement
(Braithwaite, 1983). Therefore, P is mobilized from bone to increase the size of the
exchangeable pool. The mobilization of P in this way provides additional P to help the
mammary gland to meet the milk P requirement.

As previously discussed, cows have developed a mechanism to recycle absorbed
P via saliva. The salivary glands absorb P from the exchangeable P pool and secrete it
into the mouth via saliva. This inorganic P then reenters the rumen and small intestine
and is available for absorption again. This method of recycling gives the animal a second
chance at utilizing dietary P to meet its requirements. Phosphorus recycling is critical to
meeting P requirements when cows are consuming low P diets. Salivary P is completely

available for reabsorption. Any salivary P not absorbed is excreted in feces and

contributes to endogenous fecal P loss. Endogenous fecal P also includes P originating
from undigested microbial cells and intestinal cell sloughing.

Additionally, a relatively small portion of the exchangeable P pool is removed by
the kidney and excreted in urine. Urinary P loss can increase with high (relative to the
animal’s needs) P intake (Wu et al., 2001; Knowlton and Herbein, 2002; Morse et al.,
1992b). Therefore, total P excretion is equal to fecal P [unavailable P + absorbable P +
endogenous P] (g/d) plus urine P (g/d). The total absorbed P requirement for a lactating
cow is equal to the sum of all the requirements [absorbed P requirement (g/d) = milk P +

pregnancy P + growth P + maintenance P (g/d)].

Phosphorus Requirements

Elemental mineral requirements are usually determined factorially by summing
estimates of the true requirements for maintenance, growth, pregnancy, and lactation.
The true requirement is deﬁned as the actual P (g/d) needed to support an animal’s «
current P needs for maintenance, growth, pregnancy, and milk production, whereas the
absorbed P requirement is the actual P (g/d) absorbed to meet the animal’s day-to-day P
needs.
The dietary requirement for P is then determined by dividing the absorbed P requirement
by the true absorption coefﬁcient(s) in P from the diet. The absorption coefﬁcient is the
efﬁciency that P from a certain dietary source is absorbed. Each dietary ingredient has an
absorption coefﬁcient.

Endogenous Fecal Phosphorus Loss. The NRC (2001) stated that P excretion

consists of three fractions: 1) P of dietary origin that is not available for absorption or not

absorbed (unavailable P); 2) P of endogenous origin that is inevitably excreted in feces
(inevitable fecal loss); and, 3) P of endogenous origin that is excreted to maintain blood P
homeostasis (representing P absorbed by the small intestine in excess of what is needed
to maintain normal blood P concentration). Additionally, inevitable P loss (fraction 2
above) can be split into two parts: 1) P that has been absorbed by the small intestine,
secreted back into the digestive system via the saliva, and then excreted in feces; and, 2)
P associated with microbial cells of endogenous origin and sloughed intestinal cells that
are excreted in feces.

Phosphorus Maintenance Requirement. The P maintenance requirement by
deﬁnition is the inevitable fecal P loss when P supply is at or just below the true
requirement (NRC, 2001). There have been various attempts at assigning a value to the
true maintenance requirement. In the past, P maintenance requirement was expressed as
a function of BW based on fecal P excretion data extrapolated to zero P intake (NRC,
1989; ARC, 1980). However, the AF RC (1991) determined that this approach was
inadequate. The AF RC (1991) suggested that inevitable fecal P loss could be determined
more directly as a function of DMI and not live BW and would more accurately reﬂect
the animal’s requirement for P. Additionally, NRC (2001) adopted this approach and
estimated the true P maintenance requirement, based on German research (Spiekers et al.,
1993; Klosch et al., 1997).

Two German studies were found that reported inevitable fecal P, thus, the
apparent absorbed P maintenance requirement (Spiekers et al., 1993; Klosch et al., 1997).
Spiekers et al. (1993) determined the inevitable fecal P loss from two groups of ﬁve

multiparous lactating German Black Pied and German Red Pied dairy cows with similar

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BW (603 versus 597 kg) but different MY (20.8 versus 10.0 kg/d). The MYs were
different because average stage of lactation was different between groups (191 and 249
DIM) and allowed for two different daily DMI (16.9 versus 10.9 kg). Both treatment
groups were fed a similar diet at two different DMI rates based on milk production with
similar concentrations of P (0.22 versus 0.20%, dry basis). Phosphorus balance was
slightly negative for both groups indicating that the animals were fed close to their true P
requirement. They determined the inevitable fecal loss (thus, the fecal portion of the P
maintenance requirement) of dairy cows to be 1.2 g/kg of DMI.

Klosch et a1. (1997) determined the inevitable fecal loss from two groups of
growing bulls that weighed approximately 230 and 435 kg, respectively. Five calves of
each weight category were fed either low dietary P or high dietary P concentrations (0.91
or 1.08% P, respectively). Weight did not inﬂuence the amount of fecal P excretion and
it was determined that inevitable fecal P loss (thus, the fecal portion of the P maintenance
requirement) averaged 1.0 g/kg DMI. The value for growing bull calves is similar to the
inevitable fecal P reported by Spiekers et a1 (1993) for low producing cows.

Using the average inevitable P loss from Spiekers et a1. (1993) and assuming that
P from the diet was 80% available, the NRC (2001) calculated the inevitable fecal P
portion of the absorbed P maintenance requirement of lactating and non-lactating dairy
cows (1.0 g P/kg DMI per d). This was determined by the following equation: inevitable
fecal loss multiplied by P availability = absorbed P maintenance requirement or, 1.2 g
P/kg DMI x 0.80 = 0.96 g P/kg DMI per day. The NRC (2001) rounded this value to 1.0
g P/kg DMI. An additional 0.002 g P/kg BW was included to account for the additional

endogenous P excreted in urine that is associated with the P maintenance requirement.

11

 

31

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Br
Ste
(111:

191

Additionally, secretion of P in milk is the second major route of removal of P
from the body. The requirements for absorbed P for lactation is equal to daily milk yield
multiplied by the P concentration of milk (NRC, 2001). Milk P concentration was found
to range between 0.085 to 0.10% (Knowlton and Herbein, 2002; Wu et al., 2000;
Brintrup et al., 1993; Spiekers et al., 1993; Brodison et al., 1989; Flynn and Power,
1985). The NRC (2001) used milk P concentration of 0.09% P to estimate the P

requirement for lactation.

Inﬂuence of Dietary Phosphorus on Performance

Dry Matter Intake, Body Weight, and Body Condition Score. Dry matter intake
responses to varying dietary P have been reported in numerous studies. In 11 of these
studies, it was reported that dietary P concentration had no effect on DMI when lactating
cows were fed diets ranging from 0.30 to 0.69% P (Wu et al., 2002; Kohn et al., 2002;
Knowlton and Herbein, 2002; Wu et al., 2001; Wu et al., 2000; Wu and Satter, 2000;
Brintrup et al., 1993; De Boer et al., 1981; Forar et al., 1982; Carstairs et al., 1981;
Steevens et al., 1971). Only three studies were found that reported a reduction in DMI
due to dietary P concentration (V alk and Sebek, 1999; Call et al., 1987; Kincaid et al.,
1981). Valk and Sebek (1999) found that DMI was reduced when cows consumed diets
with 0.24 versus 0.28 or 0.34% P. Similarly, Call et a1. (1987) concluded that DMI I
declined when cows were fed 0.24 versus 0.32 or 0.42% P. Kincaid et a1. (1981) also
observed a DMI reduction when cows were fed diets containing 0.30 versus 0.54% P. As
previously mentioned, microbes have a P requirement in order to digest cellulose

(Burroughs et al., 1951). Inadequate P would result in less digested cellulose and

12

reduced passage rate, which could decrease DMI by increasing gut ﬁll. Therefore, it is
not surprising that MY also was reduced when compared with that of cows fed the higher
P diets in these three studies.

Varying dietary P concentration for lactating dairy cattle also inﬂuenced BW
change. Along with the DMI reduction mentioned previously, cows consuming 0.24
versus 0.28 to 0.42% P had greater BW change and weighed less (V alk and Sebek, 1999;
Call et al., 1987). However, dietary P concentration did not have an effect in cows fed
diets containing 0.31 to 0.69% P (Knowlton and Herbein, 2002; Wu et al., 2001; Wu et
al., 2000; Wu and Satter, 2000; Brintrup et al., 1993; De Boer et al., 1981). In general,
diets providing less P than the recommendation made by the NRC (2001) may decrease
BW as shown by Valk and Sebek (1999) and Call et a1. (1987) in diets of 0.24% P. Body
condition score also was evaluated in some studies. It was concluded that dietary P did
not affect BCS when cows were fed diets containing 0.31 to 0.49% P (Wu et al., 2000;
Wu and Satter, 2000; Brodison et al., 1989).

Lactation Performance. Several studies have addressed the effect of dietary P
concentration on milk production. Three short-term studies (< 5 mo) showed that feeding
diets to cows with 0.32 to 0.69% P had no effect on milk production when cows produced
14 to 48 kg/d (Wu etal., 2002; Kohn et al., 2002; Knowlton and Herbein, 2002;
Knowlton et al., 2001; De Boer et al., 1981). However, it should be noted that there was
a 3 kg/d non-signiﬁcant numerical decrease in milk production when Knowlton and
Herbein (2002) fed 0.67 versus 0.34 or 0.51% P (46 and 49 kg/d, respectively). This
suggested that extremely high dietary P relative to the P requirement might reduce milk

production.

13

 

(1'5

Long-term studies (10 mo to 3 yr) also addressed the effect of dietary P
concentration on milk production. Seven studies reported that feeding lactating cows
diets containing 0.30 to 0.60% P had no inﬂuence on MY (cows producing 25 to 40 kg
milk/d; Wu et al., 2002; Kohn et al., 2002; Wu et al., 2001; 2000; Wu and Satter, 2000;
Brintrup et al., 1993; F orar et al., 1982). However, Wu et a1. (2000) determined that
feeding cows a diet containing 0.31% P for a full lactation was marginally deﬁcient,
exhibited by a signiﬁcant decrease in milk yield during late lactation (> 200 DIM),
compared with cows receiving diets with 0.40 and 0.49% P (overall MY for the lactation
were not different among P treatment groups). This suggested that if lactating dairy cows
received the 0.31% P diet during a subsequent lactation, overall milk yield might be
reduced if a P-deﬁcient diet also was fed through the dry period. Additionally, Steevens
et a1. (1971) found that there was no difference in milk production (18 kg solids-corrected
milk/d) due to dietary P concentration from cows consuming diets containing 0.40 and
0.60% P.

Other studies showed that feeding low P diets reduced milk yield. Valk and
Sebek (1999) fed diets with 0.24 versus 0.28 or 0.34% P to cows for two lactations and
two dry periods. Dietary P concentration in the ﬁrst lactation did not affect milk yield
(overall average of 25 kg/d). However, during the second lactation, cows receiving the
0.24% P diet produced less milk than cows on the other two treatments (average MY of
38 versus 44 kg/d, respectively). Similarly, Call et a1. (1987) determined that feeding
diets containing 0.24% P to lactating cows reduced fat-corrected milk yield compared
with cows consuming 0.32 or 0.42% P (17 and 22 kg/d, respectively). In a similar

experiment, Kincaid et a1. (1981) showed that feeding cows 0.30 versus 0.54% P reduced

14

 

."e

CO

milk yield during the ﬁrst 10 months of lactation by about 3 kg/d (27 and 30 kg/d,
respectively). Two additional studies were reported in which cows consuming a diet
lower in P had higher MYs compared with cows fed the higher dietary P concentration.
In one Michigan study, cows consuming a diet of 0.40% P tended to produce more milk
for the ﬁrst 4 wk of lactation than cows consuming a diet of 0.50% P (23 and 21 kg/d,
respectively) (Carstairs et al., 1981). Likewise, when cows consumed diets with 0.35 or
0.44% P for 3 yr, P concentration inﬂuenced milk production in the ﬁrst year but not in
the last 2 yr (Brodison et al., 1989). British Friesian cattle were grazed during the
summer period on a grass-legume mixed pasture and fed grass silage plus concentrate
during the winter period. Cows were estimated to have dietary P concentrations of 0.35
and 0.44%. Cows consuming the 0.35% P diet produced more milk than the cows
consuming the 0.44% P diet during the ﬁrst year (16.8 and 15.8 kg/d). In years 2 and 3
cows produced similar amounts of milk (16.8 and 16.2 kg/d, respectively). Cumulative
effect of dietary P concentration for all three lactations was not reported.

Milk Composition. In addition to MY results, most of these same experiments
reported the inﬂuence of dietary P concentration on milk components. Dietary P
concentration ranging from 0.24 to 0.60% P had no effect on milk fat percentage or yield
(Wu et al., 2002; Wu etal., 2001; Knowlton and Herbein, 2002; Wu et al., 2000; Valk
and Sebek, 1999; Call et al., 1987; Carstairs et al., 1981; Steevens et al., 1971).
However, Brodison et a1. (1989) reported that milk fat percentage was lower (in the ﬁrst
year of a 3-yr study but not the subsequent 2-yr) when cows consumed a diet of 0.3 5% P

compared with cows that consumed a diet of 0.44% P (3.49 versus 3.6 % milk fat).

15

Additionally, Brintrup et a1. (1993) reported that milk fat percentage was decreased when
cows consumed diets with 0.39 versus 0.33% P (4.21 and 4.38%).

Milk lactose percentage, SNF% and SCC 1000 cell/ml milk were not affected by
dietary P concentrations ranging from 0.31 to 0.49% P (Wu et al., 2002; 2000; Knowlton
and Herbein, 2002; Wu and Satter, 2000). Steevens et a1. (1971) also reported that
SNF% was not affected by dietary P concentrations of 0.40 or 0.60% P. Knowlton et a1.
(2001) reported that milk lactose and SNF percentages and yields were not affected by
dietary P (0.34, 0.36, and 0.38%) concentration.

The effect of dietary P concentration on milk protein yield and percentage also
was studied. Neither milk protein concentration nor yield was affected by dietary P
concentrations ranging from 0.24 to 0.67% P (Wu et al., 2002; 2000; Knowlton and
Herbein, 2002; Knowlton et al., 2001; Valk and Sebek, 1999; Brintrup et al., 1993;
Brodison et al., 1989). In two studies opposite results were reported. Call et a1. (1987)
found that feeding 0.24 versus 0.32 or 0.42% P lowered milk protein concentration.
Additionally, Wu et a1. (2000) found that feeding 0.38 versus 0.48% P tended to reduce
milk protein percentage (3.05 and 3.16%. respectively) without affecting milk protein
yield.

De Boer et a1. (1981) found that dietary P concentration had no effect on milk fat
percentage, protein yield. or lactose percentage when cows consumed diets containing
0.34, 0.51, or 0.69% P. Milk protein percentage was affected by the 0.34% P diet
compared to the other two diets (3.15 versus 3.32%, respectively). However, caution
should be used in evaluating these results. Varying dietary Cu, Zn. and Mn in addition to

P confounded the interpretation of the effect of P. Thus, a deﬁnitive conclusion cannot

l6

be drawn because the effect of P cannot be separated from the potential effects of the
other minerals. Given the above results, published results show inconsistency in the

effect of dietary P concentration on milk composition and component yield.

Inﬂuence of Dietary Phosphorus on Fecal and Urinary Phosphorus Excretion and
Milk Phosphorus Secretion

Phosphorus excretion has become a widely discussed subject as it relates to
environmental pollution. Federal and state governments are reviewing current waste
regulations to determine if more stringent laws are required. In order to determine what
courses of action to take when new regulations are considered, it is important to
understand routes of excretion and the role that P intake has on the amount of P excreted.
Fecal excretion of P is the primary route (95 to 98%) in ruminants (NRC. 2001; Morse et
al., 1992b), with the remaining portion (2 to 5%) excreted in urine.

Several studies were reviewed that reported the effect of dietary P on fecal P
excretion. Lomba et a1. (1969), in a series of balance trials fed lactating and non-lactating
dairy cows diets varying in P concentration that resulted in P intakes of 39.8 to 75.4 and
7.9 to 54.1g/d, respectively. They concluded that P intakes in these ranges had no effect
on the amount of P excreted in feces or urine in lactating and non-lactating dairy cows.
However, several recent studies provided evidence that dietary P concentration does
inﬂuence the amount of P excreted in feces and urine. When cows were fed diets
containing 0.30 to 0.67% P, fecal P excretion increased with increasing dietary P
concentrations (Kohn et al., 2002; Knowlton and Herbein, 2002; Wu et al., 2002; 2001;

2000; Brintrup et a1, 1993', Morse et al., 1992b; Martz et a1, 1990). Similarly, reports

17

from studies measuring urine P excretion showed that as dietary P concentration
increased (0.30 to 0.67%), urine P excretion also increased (Knowlton and Herbein,
2002; Morse et al., 1992b).

Increased dietary P concentration almost always increases P intake. As a result,
generally more P is absorbed in the small intestine and enters the bloodstream. The
results of Wu et al. (2000) suggested that there might be a renal threshold that determines
when cows start spilling excess P into urine. Challa and Braithewaite (1988) concluded
that the renal threshold is determined by P concentration in blood serum. This
concentration was determined to be around 7.0 mg/dl for growing calves fed a P deﬁcient
diet (0.40% P/d) with an abomasal infusion of phosphorus to supply an additional 0, 3, 6,
or 9 g P/d. However, no study was found to support this renal threshold concentration for
lactating cows. The results of Wu et al. (2000) showed that the blood concentration
required to get the kidneys to increase P excretion was not obtained when cows were fed
diets consisting of 0.3 1, 0.40, or 0.49% P. They reported blood serum P concentrations
of 5.5, 6.2, and 6.1 mg/dl for the 0.31, 0.40, and 0.49% P treatments, respectively. Serum
P concentrations for lactating cows generally range between 4.0 to 6.0 mg/dl (Goff,
1998); Therefore, blood serum P concentrations of 7.0 mg/dl are rare in lactating cows,
suggesting that this concentration reported by Challa and Braithwaite (198 8) for calves is
too high to be used as a threshold concentration for mature lactating cows.

Additionally, apparent P digestibility (percent of intake) has been studied.
Apparent P digestibility [(P intake — (fecal P + urine P + milk P)) x 100%) describes how
well cattle utilize P. Increased P intake caused a decrease in apparent P digestibility

(Knowlton and Herbein, 2002). Apparent digestibility was 49, 34, and 33% when

18

lactating cows were fed 0.34, 0.51, and 0.67% P (Knowlton and Herbein, 2002).
However, Wu et a1 (2000) reported that apparent P digestibility was not affected by
dietary P concentration when cows were fed 0.31, 0.40, and 0.49% and averaged 47%
across the three treatments.

The inﬂuence of dietary P on milk P concentration also was measured in six
studies. No effect on milk P concentration was observed when cows were fed diets with
P concentrations ranging from 0.24 to 0.67% P (Knowlton and Herbein, 2002; Wu et al.,
2000; Brintrup et al., 1993; Brodison et al., 1989; Call et al., 1987; Forar et al., 1982).
Additionally, Morse et al. (1992b) determined that P retention affected milk P
concentration. They reported that P retention greater than 10 g/d increased milk P
secretion. This increase resulted in a shift of P loss in milk from 30 to 40% of total P
output. Morse et al. (1992b) reported that a decrease in MY with the same P intake

shifted more P excretion to feces.

Inﬂuence of Other Factors on Fecal Phosphorus Excretion

Diet Type. Diet has been shown to affect P excretion. Knowlton et al. (2001)
reported that diets fed to lactating cows supplemented with wheat bran decreased P. intake
compared with diets supplemented with soybean meal, soybean meal plus blood meal, or
soybean meal plus calcium phosphate. However, MY, milk composition, and apparent
digestibility of P was not affected. Decreased P intake resulted in lower fecal P that
which resulted in lower total P excretion compared with the other diets. Additionally,
Martz et al. (1990) found that cows consuming corn silage plus alfalfa hay-based diets

had increased fecal P excretion in addition to increased milk P percentage compared with

19

 

 

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cows consuming alfalfa hay-based diets. The increased fecal P excretion would be
expected due to increased DMI of the corn silage plus alfalfa hay diet compared with the
alfalfa hay diet (22.7 and 20.6 kg/d). Additionally, dietary P concentration was higher
(0.15 or 0.21% P for alfalfa plus corn silage versus alfalfa hay diets, respectively) when
corn silage was added. Thus, it is more likely that the increased P excretion is a function
of P intake and not type of diet in the study of Martz et al. (1990). The increase in milk P
concentration may be explained by a tendency of increased fat corrected milk production
from (35 and 32 kg/d) cows consuming the corn silage plus alfalfa hay-based diet.
Additionally, Wu et al. (2002) hypothesized that a high forage diet would increase fecal P
excretion because of increased salivary P in feces. They reported that high forage diets
increase salivation and therefore, increase the endogenous P loss. However, they
reported that feeding low forage (48%, DM basis) versus high forage (58%, DM basis)
did not inﬂuence fecal P excretion. Similarly, Klosch et al. (1997) fed high forage or low
forage diets (50 versus 25%, DM basis) to growing bull calves. They reported that there
was no inﬂuence on P excretion by diet type.

Dietary Energy, Protein, and Calcium Content. Braithwaite (1976), in a review
of P metabolism, reported that low energy diets and low protein diets decreased
absorption of P. Thus, fecal P excretion could be increased because a larger portion of
potentially absorbable P would be excreted. Additionally, Braithwaite (1976) reported
that low Ca diets might increase urine P excretion because of the additional P that enters
the blood stream as a result of accompanying Ca resorption from bone. Additionally, it

could be hypothesized that this could lead to an increase in fecal P because of increased P

20

 

 

 

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pool size in blood due to Ca resorption. The increased pool size could increase salivary P
recycling which can lead to increased fecal P excretion.

Body Weight and Age. The inﬂuence of BW and age on fecal excretion also has
been studied. Klosch et al. (1997) fed diets containing 0.1% P to growing bulls. They
concluded that BW (228 and 435kg) did not inﬂuence P excretion (1 g P/kg DMI). The
NRC (1989) reported that the efﬁciency of P absorption decreases with age. Absorption
efﬁciency decreases from 90% in calves to 50% in animals less than 1 yr of age. Thus,
there is potential for increased P excretion in older animals due to increased P supplied to
compensate for less efﬁcient absorption.

Phosphorus Source. Source of P inﬂuences P excretion to some extent. Different
sources of P have differing bioavailabilities (amount available to the animal for
absorption). Each source of phosphorus has an absorption coefﬁcient (AC), or
efﬁciency, with which it is absorbed. Forages generally have lower ACs than
concentrates although there is not an abundance of information available (NRC, 2001).
Absorption coefﬁcients for P in alfalfa hay and corn silage were 67 and 80 %,
respectively, for lactating cows consuming 22 kg/d of DM and producing 34 kg/d of
3.5% fat corrected milk (Martz et al. 1990). New research has provided additional.
insight into P availability from soybean meal, cottonseed, corn gluten feed, distillers
grain, and porcine meat and bone meal (Aguerre et al., 2002). They reported that the P
absorption coefﬁcients for these concentrates were 0.74, 0.81, 0.73, 0.83. and 0.64.
respectively. Signiﬁcance was not reported in this abstract.

There is much more information available about P mineral supplements than

forages and concentrates. The NRC (2001) compiled AC information from studies into

tabular values for reference. Some P sources commonly used by the dairy industry
included in the NRC (2001) tabular values are calcium phosphate, dicalcium phosphate.
mono- and dibasic ammonium phosphate, monobasic sodium phosphate, and phosphoric
acid. The ACs of these P sources range from 0.75 to 0.90%. An example follows to
provide a better understanding of how the AC affects P absorption. Rock phosphate has
an AC of 0.30 whereas ammonium phosphate has an AC of 0.80 (NRC, 2001). If 10g of
P in a diet were supplied by rock phosphate, 7g would be unavailable for absorption and
excreted in feces. In contrast, if 10g P in a diet were supplied by ammonium phosphate,
only 2g would be unavailable for absorption and excreted in feces. In order to meet the P
requirements of a lactating dairy cow using rock phosphate, 40% more P would have to
be supplied than if ammonium phosphate was used. The rock phosphate source would
result in more fecal P excretion compared with the ammonium phosphate source based on
their ACs.

Phytate Phosphorus. Phytate, a multi-phosphorylated inositol ring, also has been
studied for its signiﬁcance as a P source. Fifty to 75% of P in concentrates (grains.
vegetable, and protein sources) is present as phytate (Nelson et al., 1968). Phytate-P is
mostly or totally unavailable to nonruminants (Soares, 1995). Therefore, nonruminants
excrete most or all phytate-P they ingest. However, ruminants can utilize phytate-P
because microbes in the rumen have the enzyme phytase that hydrolyzes phytate-P to
inorganic P. Morse et al. (1992a) reported that 99% of phytate-P was hydrolyzed when
11 Holstein cows were fed 34g phytate-P/d. Similarly, Clark et a1. (1986) concluded that
98% of the phytate-P P hydrolyzed to inorganic P in 30 Holstein cows fed 38 or 43 g

phytate-P/d. Therefore. it can be concluded that phytate-P can be used to meet the P

requirements of dairy cows. Because phytate-P can be used to meet dairy cows’ P
requirements, less supplemental P needs to be fed. Therefore, fecal P excretion can be
reduced compared with the situation where phytate-P was not considered available to
cows. Because cows have ruminal phytase present, less supplemental P, if any, needs to
be fed and fecal P excretion potentially can be reduced.

Presence of Dietary Aluminum. The presence of dietary aluminum affects P
utilization and, therefore, P excretion. Aluminum forms insoluble salts with phosphate,
making it unavailable for absorption (Jones, 1938). Crowe et al. (1990) fed Holstein bull
calves 0 or 0.20% Al as aluminum chloride. They reported that apparent P absorption
was decreased (1 versus -3 g P/d for low and high Al dietary treatments, respectively),
thus, fecal P excretion was increased (3 versus 6 g PM for low and high Al dietary
treatments, respectively). Additionally, Rosa et al. (1982) reported that feeding lambs 0
or 1.4% A1 as aluminum chloride decreased P utilization determined from bone ash and P
content (36 versus 34.4 % ash and 16.8 and 16.5% P, ash basis for 0 and 1.4% A1,

respectively) absorption by binding phosphate.

Prediction of Phosphorus Excretion
Prediction of Fecal Phosphorous Excretion. There have been four papers that
reported an equation to predict P excretion. The ASAE (1980; 1996) published two
models (Model 1: P excretion (g/d) = BW (kg) x 0.0724 and Model 2: P excretion (g/d)
= BW (kg) x 0.094, respectively) developed from published and unpublished P balance

data to predict P excretion.

Morse et al. (1992b) developed a P excretion prediction equation (Model 3: P
excretion (g/d) = 14.67 + 0.6786 x intake P (g/d) + 0.00196 x [intake P (g/d)]2 — 0.317 x
MY (kg/d)) based on 93 P balances from 3-d total excreta collections. They used this
model to predict P excretion from cows consuming P ranging from 60 to 112 g/d.

Van Horn et al. (1994) expanded the dataset used to develop the equation above
to include a total of 93 individual P balances from lactating cows and 15 from non-
lactating cows. They developed a new model [Model 4: P excretion (g/d) = 9.6 + 0.472
x intake P (g/day) + 0.00196 x [intake P (g/d)] 2 + 0.323 x MY (kg/d)].

Additionally, Davidson and Beede (1999) deve10ped two additional models to
predict P excretion. The ﬁrst model [Model 5: P excretion (g/d) = -24.06 + 81.67 x .
dietary P (% of DM) + 0.07 x BW (kg) — 0.45 x milk P (g/day) was developed from
results published by Hibbs and Conrad (1983). The second model [Model 6: P excretion
(g/d) = P intake (g/d) — P excretion (g/d)] developed was based on a suggestion made by
Van Horn et al. (1994).

In a review of the literature, Davidson and Beede (1999) compiled a dataset of P
excretion including 11 treatment means from 85 intake-output balances. The dataset was
derived from ﬁve different experiments of lactating cows (Martz et al., 1990; Brintrup et
al., 1993; Spiekers et al., 1993; Wu et al., 1998; Rodriguez, 1998). This dataset was used
to evaluate the accuracy and precision with which P excretion was predicted using
Models 1 through 6.

For the following paragraphs measured P excretion pertains to experiments that
used total fecal collections to determine daily fecal P excretion and predicted P excretion

refers to P excretion as predicted from known values of variables in an equation

24

developed to estimate P excretion. Additionally, the full range of measured P excretion
was 13.6 to 62.7 g/d. The full range was then sub-divided into low and high measured P
excretion. Low measured P excretion was 13.6 to 22.2 g/d and high measured P
excretion was 40.4 to 62.7 g/d.

Accuracy (bias of prediction) of a model was determined by comparing the
average of [predicted — measured P excretion (g/d)] for each model to zero by t-test.
Accuracy is deﬁned as the closeness of predicted P to measured P excretion. In a model
that is not biased, predicted P — measured P excretion = 0. Only Models 5 and 6 were
unbiased (P < 0.01) in their prediction of actual P excretion.

Precision (over— or under-prediction) is the consistency of the model at predicted
P excretion at a given measured P excretion. This is determined by: (predicted -
measured) -:— measured P excretion (g/d) x 100%. Precision of each model differed.

Models 1 and 2 are used most often in developing dairy start-ups, manure
management systems, and whole farm nutrient management plans [better known as
Comprehensive Nutrient Management Plans (CNMP)] by the Natural Resource
Conservation Service (Davidson and Beede, 1999). These plans are intended to serve as
guidelines, to how much manure can be applied legally to a given amount of acreage
based on soil tests and the crop to be planted. According to the analyses doneby
Davidson and Beede (1999), the ASAE models (Models 1 and 2) were the least accurate
compared with measured P excretion. Model 1 greatly over-predicted P excretion
(155%) at low measured P excretion. However, it only slightly over-predicted (18%) at
high measured P excretion. Across all values of measured P excretion, Model 1 had a

mean over—prediction of 68%. The revised model (Model 2) was not an improvement

25

 

0C

CO

over Model 1. The major change in the standards was that the ASAE (1996) changed the
estimate of fecal DM excretion from 10 to 12 kg/d per 1000 kg BW and estimated P in
manure was changed from 0.70 to 0.78%, dry basis. At high and low measured P
excretion, Model 2 over-predicted (35 and 230%, respectively) P excretion. Across all
ranges of measured P excretion, model 2 over-predicted by an average of 106%.
Davidson and Beede (1999) suggested that P excretion. as a function of BW alone is not
appropriate and should not be used to predict P excretion.

Models 3 and 4 were improvements over Models 1 and 2, but still are relatively
inaccurate and imprecise. At low measured P excretion, models 3 and 4 over-predicted (81
and 93%, respectively) P excretion, and at high measured P they over-predicted (57 and
49%, respectively) P excretion. Both models had an average over-prediction of 65% over
the full range of measured P excretions.

Models 5 and 6 were the most accurate and precise models evaluated. At high
and low measured P excretion, Model 5 over-predicted P excretion (31 and 54%,
respectively). This model had an average over-prediction of 39% across the range of
measured P excretions.

Model 6, the simple relationship of intake P (g/d) minus milk P (g/d) was the most
accurate model according to Davidson and Beede (1999). The difference between
measured and predicted P excretion was within 18%. However, greater over-prediction
occurred at low measured P excretion compared with high measured P excretion when
comparing against an independent data set. This model was further evaluated using all
data available including an independent data set that included estimates in P excretion

from Hibbs and Conrad ( 1983) and Morse et al. (1992b). The ﬁnal data set included 71

26

treatment means. Davidson and Beede (1999) found that this model over-predicted P
excretion by 39% across the full range of measured values. The difference between
predicted P excretion and measured P excretion (bias) was 14.5% and was different from
zero. The relatively small bias may have been caused by experimental error in
determining P excretion or not properly accounting for P retained in bone and soft
tissues. This calculation, if indeed more accurate and precise, would be a simplistic
calculation to use for estimating P excretions in commercial dairy farms. Once intake P —
milk P was determined to be the most accurate and precise. Davidson and Beede (1999)
attempted to improve this model by regressing P excretion on intake P — milk P.
However, they did not evaluate this model for accuracy and precision in predicting P
excretion.

Since the review of Davidson and Beede (1999), Wu et al. (2000) deve10ped
another model [Model 7: P excretion (g/d) = 0.643X — 5.2, where X = P intake (g/d)] to
predict P excretion. This equation was developed from 26 individual cow means for P
intake and measured P excretion. Wu et al. (2000) reported that it explained 81% of the
variation in P excretion. However, this equation needs to be evaluated with an

independent data set before its practical application will be valid.

27

 

3C
10
We
Tr

101

CHAPTER 3

EVALUATING ESTIMATES OF THE PHOSPHORUS MAINTENANCE
REQUIREMENT OF LACTATIN G HOLSTEIN COWS WITH VARYING DRY

MATTER INTAKES

ABSTRACT

The objective was to evaluate estimates of the P maintenance requirement for
lactating dairy cows over a range of DMI rates. By deﬁnition, endogenous fecal P
excretion of an animal fed very near its true P requirement is the major part of the total P
maintenance requirement. Inevitable fecal P includes endogenous fecal P plus ’
unavailable dietary P excreted in feces and is expressed as g of fecal P/kg of DMI. Seven
lactating Holstein cows each were selected from high, medium, and low milk production
groups at the Michigan State University Dairy Teaching and Research Centei'. To
achieve a wider range in DMI, rations fed to the medium and low groups were restricted
to 75 and 50% of their pre-trial ad libitum intake. Treatments were based on DMI and
were 11.3, 15.3, and 25.1 kg/d, respectively for Treatment 1 (T1), Treatment 2 (T2), and
Treatment 3 (T3). All cows were fed the same low P diet (0.26% P, dry basis) so that
total P intake was low relative to requirement. Phosphorus balances were 1.5 g/d for all
treatments and not affected by DMI; nor, were they different from zero. Average daily
total inevitable fecal P excretion was 15.3, 18.2, and 26.3 g/cow for T1, T2, and T3,

respectively. Inevitable fecal P excretion (g kg DMI) was 1.36, 1.19, and 1.04 for T1.

28

 

1h.

E\

T2, and T3 and decreased linearly with increasing DMI. Therefore, an equation was
developed by regression to predict inevitable fecal P excretion across a range of DM1:
(g/d) = [0.85 i 0.070 (g/d)] x DMI (kg/d) + [5.30 :t 1.224 (g/d)]; (R2 = 0.90; P < 0.01).
This equation can be used to estimate the inevitable fecal P component of the total P
requirement for maintenance of lactating cows. Because P balances were not different
from zero, inevitable fecal P excretion was the fecal P portion of the total P maintenance

requirement. Therefore, the P maintenance requirement (g/kg DMI) was calculated.

INTRODUCTION

Accurately estimating the total dietary P requirement of dairy cattle is important
for optimal performance and to decrease excess manure P entering the environment from
dairy farms. The factorial method has been used to estimate P requirements (NRC, 2001;
ARC, 1980). The total dietary P requirement is the sum of the absorbed P requirements
for maintenance, milk production, growth, and pregnancy divided by the absorption
coefﬁcient. The P requirement for maintenance is deﬁned as the inevitable loss of P in
feces plus endogenous urine P when an animal is fed very near or just below its true P
requirement (NRC, 2001). The inevitable fecal component typically is by far the largest
part of the total P requirement for maintenance. The approach to estimate and the values
reported for the P requirement for maintenance vary (ARC, 1980; NRC, 1989; INRA,
1989; NRC, 2001). Previously, the daily P requirement for maintenance was expressed
as g/kg of BW (ARC, 1980; NRC, 1989; INRA, 1989). The ARC (1980) suggested that
the net P requirement for maintenance was 12 mg P/ kg BW per day. This was based on

experimental data of fecal P excretion extrapolated to zero P intake. Based on the ARC

29

(1980) approach, the NRC (1989) estimated the total dietary requirement of P for
maintenance as: g/d = [0.0143 x BW (kg)] + 0.50, where 0.50 was an overall estimate of
the absorption coefﬁcient for P from the diet. The Institut National de la Recherche
Agronomique (INRA; 1989) suggested that the P maintenance requirement of 12 mg P/
kg BW per (1 reported by the ARC (1980) was too low and suggested the P maintenance
requirement to be 23 to 32 mg P/ kg BW per (1 for cows producing 5 to 50 kg of milk/d.
Also, the AFRC (1991) noted that previous research (ARC, 1980) estimated P
maintenance requirements for growing and non-lactating animals in which BW and DMI
were correlated highly. The AFRC (1991) contended that this was not an adequate
representation of the maintenance requirement of P for a mature lactating cow. A mature
lactating cow likely has varying feed intake through the lactating and non-lactating cycle,
but BW may not change as much relative to the BW change of a growing animal. Also, a
lactating cow may have a greater maintenance demand for P due to the physiological
demands associated with milk production. Therefore, the AF RC (1991) estimated the P
maintenance requirement as: (g/d) = 0.693 x DMI (kg/d) -— 0.06; in this case the
requirement was equal to the daily inevitable fecal P excretion. The AFRC (1991) did
not include endogenous urine P in their approach of estimating the requirement. This
equation was developed using sheep, but used for lactating cattle because insufﬁcient
data from cattle were available.

Subsequently, Spiekers et al. (1993), using the AFRC (1991) approach,
determined the inevitable fecal P loss of high and low production groups each with ﬁve
lactating cows with similar BW but different DMI (17 vs. 11 kg/d) and MY (21 vs. 10

kg/d). The two groups were fed the same diet with 0.21% P. dry basis. Phosphorus

30

\

 

 

 

(3

IE

11

intakes (37 vs. 22 g/d) differed between groups. Inevitable fecal P loss, or the fecal
component of the total dietary maintenance requirement for P was estimated to be 1.20
and 1.22 g P/kg DMI for the high and low groups, respectively.

The NRC (2001) used the results of Spiekers et al. (1993) to set the fecal
component of the P requirement for maintenance (1.2 g/ kg of DMI for a diet with an
absorption coefﬁcient of 0.8 for P; or 1.0g of absorbed P/ kg of DMI) for lactating dairy
cows. urinary P excretion (0.002 g/d) is added to the fecal component to calculate the
total dietary P requirement for maintenance. The objective of this experiment was to
evaluate the estimates of the P maintenance requirement of lactating cows with a wider
range in DMI and MY to test and compare with other results in the literature and evaluate

the approach taken by the NRC (2001).

MATERIALS AND METHODS

Experimental Design, Cows, Experimental Treatments, and Diet

The Michigan State University All-University Committee on Animal Use and
Care approved (AUF# 11/00-151-00) methods for care and use of the cows. Twenty-one
lactating Holstein cows were selected from available animals at the Michigan State.
University Dairy Teaching and Research Center to provide seven cows for each treatment
group; the treatment groups initially were classiﬁed as early (55 i 6.5 DIM), mid (164 i
6.0 DIM), or late (253 i 6.0 DIM) lactation. Initially, treatments of different DIM were
chosen to theoretically provide different ad libitum intakes.

A diet was formulated to supply P at or very close to the cow’s true requirement

to be able to measure inevitable P excretion (NRC, 2001). The basal diet met or

31

exceeded requirements for all other nutrients (Table 1). The diet was formulated to
contain 0.24% P. The analyzed dietary P concentration was 0.26% P, dry basis. Once
treatments were assigned, all cows were fed this same basal diet for the duration of the
experiment (35 d). Cows were fed their diets once daily at approximately 1000 h.

The experiment lasted for 35 d and consisted of a 6-d evaluation period, a 24-d
diet adaptation period (d 7 through 30), and one 5-d collection period. Samples collected
included fecal grab samples, urine spot samples, milk samples, and feed ingredient
samples. The ﬁrst 6 d were used to determine if treatments, as deﬁned by DIM, would
meet the objective of having a wide range of ad libitum DMI

At the end of the 6-d evaluation period, it was determined that there was minimal
DMI difference among treatment groups [20.6, 20.7, and 21.8 i 0.86 kg/d for early, mid,
and late lactation groups, respectively (P = 0.53)]. Also during the pretrial period, MY
was affected by DIM [47.3, 29.1, and 25.3 i 1.23 kg/d for early, mid, and late lactation,
respectively (P < 0.01)]. It was desired to increase the range in DMI among the three
treatment groups. Therefore, the DMI needed to supply enough NEL to support the
average MY of each cow recorded during the 6-d pretrial period was calculated for the
mid and late lactation treatment groups. Once the DMI to supply adequate energy for
lactation was determined for the mid and late treatment groups, the average restriction by
treatment was approximately 75 % of ad libitum intake determined during the evaluation
period. This still did not produce the range in DMI desired because MY for the mid- and
late lactation groups were numerically similar. Therefore, cows in the late lactation
group were restricted an additional 25% of their 6-d evaluation period ad libitum DMI to

increase the range of DMI and MY. The treatments based on targeted DMI were:

32

treatment 1 (T1), average DMI of 11.3 kg/d; treatment 2 (T2) average DMI of 15.3 kg/d;
and, treatment 3 (T3) average DMI of 25.1 i 0.74 kg/d when the sample collection
started on d 30 (Table 2). Targeted DMI was accomplished by a stepwise reduction over
d 7 through 11 of the experiment; these daily amounts were offered and completely
consumed for the remainder of the experiment. Cows in the early lactation treatment
(highest milk yield) were allowed to continue ad libitum intake throughout the
experimental period to maximize ad libitum DMI. Therefore, DMI as the deﬁnition of
the treatments expressed as a percentage of the average daily ad libitum intake of each
individual cow during the evaluation period was: 50, 75 and 100%, for T1, T2, and T3,
respectively. Two cows were removed from T1. One cow died of causes unrelated to the
experiment during the sample collection period and a second cow was determined to be

an outlier (i3 standard deviations) in P balance. Their data were subsequently removed.

Experimental Procedures

Chromium oxide was used as an external marker to estimate daily fecal excretion.
An initial dose of 45 g chromium oxide was given orally via balling gun in two equal
amounts of 22.5 g at 0600 and 1800 h in lock ring gelatin capsules (Torpac Inc., Fairﬁeld,
NJ) on d 25 of the experiment. The initial dose of 45 g was given to help ensure that a
steady state concentration of Cr was reached prior to fecal sample collections. On d 26
through 35, cows were given chromium oxide orally in capsules via balling gun in two
equal doses of 7.5 g at 0600 and 1800 h, (Torpac Inc., F airﬁeld, NJ) so that a total of 15
g/d were supplied. Approximately 1 g of spelt hulls was added to the gelatin capsules to

provide buoyancy to the capsule in the rumen until the gelatin capsule dissolves (Oba and

33

Allen, 2000). Spelt hulls contained 0.22% P and contributed only 53 mg dietary P over
the entire ll-d dosing period; therefore, P supplied by spelt hulls was ignored in P-related
calculations of the data.

Approximately 500 g of feces (rectal grab samples) were collected into plastic
containers with snap-on lids (Sweetheart Cup Company, Inc., Chicago, IL) starting on d
31 at 0600 h. Fecal grab samples were collected every 15-h through 1500 h on d 35 to
minimize diurnal variation in chromium oxide and P excretion. A total of eight samples
were collected from each cow, representing every 3 h of a 24-h clock (Table 3). Fecal
samples were frozen at —4 °C for later processing and P, Cr, and DM analyses. Spot
samples of urine were taken via manual stimulation of the area around the vulva during
the same time frame as fecal samples. Approximately 100 ml of urine were collected into
150-ml graduated screw-top urine specimen containers and frozen. (—4 °C) for later
processing and P and speciﬁc gravity analyses. Urine samples were missed occasionally
because of failure to urinate after manual stimulation, but at least 6 of the 8 samples were
available from each cow for compositing and analyses.

Cows were milked twice-daily at 0400 and 1400 h. Two milk samples (25 ml
each) per cow were collected daily at each milking into sample vials containing a
potassium dichromate pellet during the 5-d collection period. One milk sample was for
analysis of milk fat % (MF), true protein % (MProt) lactose % (ML), SNF, and SCC
(Michigan DHIA, East Lansing). The second milk sample was frozen at —4 °C for later
analysis of P concentration.

Corn and alfalfa silages were sampled twice weekly on Monday and Thursday for

DM concentration determination via Koster tester (Seedburo Equipment Co, Chicago,

34

IL). As necessary, adjustments were made in the as-fed amounts of forages in the diet to
insure the same DM proportions of the dietary ingredients throughout the experiment.
Weekly alfalfa and corn silage samples also were taken and frozen (—4 °C) for later DM
concentration and chemical analysis. Alfalfa and corn silages were stored in upright
silos. To obtain samples, approximately 100 kg of forage were unloaded from the silo
and then grab samples were taken from different sections of the pile to form a 7-kg
sample. A sub-sample was then taken by the roll and quartering method. The sample
was rolled and quartered twice and the resulting l-kg sub-sample was stored in a sealed
plastic bag at —4 °C for later analysis of DM concentration and chemical composition.
Bagged feed ingredients (concentrates and beet pulp pellets) were sampled bi-weekly by
taking a grab sample from 10% of the bags. These samples were stored in sealed plastic
bags at —4 °C for later analysis of DM concentration and chemical composition. Bulk
ingredients (whole cottonseed and vitamin and trace mineral premix) were sampled
weekly by taking grab samples from separate places from the pile and stored in'sealed

plastic bags and —4 °C for later analysis.

Laboratory Procedures

Feed and Fecal Sample Analyses. After the completion of the sample collection
period, feed and fecal samples were thawed at room temperature. Samples were then
dried in a forced-air oven at 52 °C for 48 h for DM concentration determination. Once
DM concentration was determined samples were ground through 5 and 2mm screens in a
Thomas-Wiley Mill (Arthur Thomas Company. Philadelphia, PA). Feed samples were

composited by ingredient type by taking a 5 g aliquot from each sample and combining

35

them into one new sample and stored in a capped plastic container. The composite
sample represented the average composition of the ingredient used throughout the
experimental period. A sub-sample of the each composite was analyzed for chemical
composition (Dairy One Forage Lab, Ithaca, NY). Composite fecal samples (40 g each)
were made from the eight individual samples from each cow and stored in a capped
plastic container.

Phosphorus concentration in feed and feces was measured by colorimetric assay
adapted from AOAC (1990) method 965.17. All laboratory glassware was acid-washed
before use. Duplicate 1 g sub-samples of individual feed and fecal composites were
taken after thorough mixing and dry-ashed at 600 °C for 4 h. The resulting ash was
digested in 20 ml of 4N HCI plus 5 drops of concentrated H2804. Molybdovanadate
reagent was added to a l-ml aliquot of the digested sample to react with P for color
development. Color intensity increased with greater P concentrations. Duplicate
samples were pipetted into 96-well microtiter plates and P concentration was determined
by a spectrophotometer (SpectraMax 190, Molecular Devices, Sunnyvale, CA). Samples
were read at a wavelength of 400 nm and compared against the standard curve (0, 0.5.
1.0, 1.5, and 2.0 mg P/dl). The standard curve solutions contained the same volumes and
proportions of reagents and deionized, distilled water as the samples. The 0 mg P/ml
standard also was used as a blank to adjust the spectrophotometer for any background P
present in the reagents or water used in the digestion or assay processes. New sub-
samples from the composite were re-digested and assayed if the coefﬁcient of variation

(CV) between the duplicate samples was greater than 3%. If the P concentration of the

36

unknown sample was greater than the highest concentration of the standard curve, the
unknown was diluted 1—to-20 with deionized, distilled water and re-assayed.

Two known controls were included in each assay. One control was a composite
fecal sample (pooled sample) made of 8 g each from 10 individual cow fecal sample
composites. The pooled sample of constant P concentration was used in each run of the
digestion and assay processes in order to calculate overall intra- and inter-assay CV for
all plates read on the spectrophotometer. For all assays of fecal samples in the

experiment, the intra- and inter-assay CVs were 0.72 and 2.22%, respectively.

 

A second known control was a 1.0 mg/dl purchased certiﬁed standard P solution

311

(Fisher Scientiﬁc, Chicago, IL). This solution was assayed along with unknowns to .
evaluate the assay-to-assay variation of the spectrophotometer. The intra- and inter-assay
variations among all assays were 0.58 and 1.20%, respectively.

Chromium concentration in feces was analyzed by ﬂame atomic absorption
spectroscopy (SpectrAA 220, Varian, Inc., Walnut Creek, CA) using a method adapted
from Williams et al. (1962). Duplicate 1 g sub-samples were taken from fecal
composites after thorough mixing and dry-ashed at 600 °C for 4 h. Resulting ash was
then digested on a hot plate in a phosphoric acid, manganese sulfate, and potassium
bromate solution at 400 °C for approximately 10 min. Chromium concentration was
determined against the standard curve (1, 2, 3, 4, and 5 ug Cr/ml). The standard curve
solutions contained the same volume and proportions of reagents and deionized. distilled
water as the sample unknowns. A 0 pg Cr/ ml was used to adjust the atomic absorption
spectrometer for any background Cr present in reagents and water used in the digestion or

assay procedures. This solution contained the same reagents and volumes as the

37

 

nu

unknowns. Unknowns were redone beginning with sub-sampling for the dry-ashing step
if the CV between duplicates was greater than 5%.

Two known controls were included in each assay. A pooled fecal sample
containing Cr similar to the pooled fecal sample for P analysis was made to digest and
assay in duplicate along with each separate run of unknowns to obtain the intra- and
inter-assay CV. The intra- and inter-assay variations were 2.26 and 4.44%, respectively.

The second known control was a 3 mg Cr/ml solution made from purchased
certiﬁed chromium oxide powder (Fisher Scientiﬁc, Chicago, IL). This control was used
to evaluate the assay-to-assay variation of the atomic absorption spectrometer. The intra-
and inter-assay variations of the Cr control solution were 2.65 and 6.9%, respectively.

Urine Sample Analysis. After the conclusion of sample collection, urine samples
were thawed to room temperature and 10 ml were taken from each urine spot sample and
composited. Composite samples were acidiﬁed with 1 m1 concentrated HCl and ﬁltered
through cheesecloth to remove any large particulate matter. Once ﬁltered composite
urine samples were analyzed for P concentration. Also, speciﬁc gravity of each
composite was measured using an Adams® Midget Urinometer (Bristol-Myers Squib
Company, New York, NY). The speciﬁc gravity value was used to estimate daily urine
excretion , kg/d = 332.91 + 0.2885 x DMI, kg/d — 0.2175 x MY, kg/d — 0.083 x dietary
DM % + 0.524 x dietary CP, % DM - 326.5 x urine speciﬁc gravity + 0.2375 x
apparently absorbed water, kg/d (Holter and Urban, 1992).

Urine P concentration was determined by colorimetric assay adapted from F iske
and Subbarow (1925). Duplicate l-ml sub—samples were taken from urine composites

and 2 ml 20% TCA was added. The sample was then centrifuged at 4000 g for 15 min.

38

A 175-ul aliquot of the supernatant was used for determination of P concentration. A
molybdate plus arninonaptholsulfonic acid reagent was used for color development, with
color intensity increasing as P concentrations increased. The P concentration of urine
was determined at 660 run against the standard curve (0, 0.25, 0.5, 0.75, 1.0, and 1.25 mg
P/dl) by using a SpectraMax 190 spectrophotometer (Molecular Devices, Sunnyvale,
CA). The standard curve solutions contained the same volume and proportions of
reagents and deionized, distilled water as the samples. The 0 mg P/ml standard also was
used as a blank to adjust the spectrophotometer for any background P present in the
reagents or water. Urine samples were redone if the CV between duplicates was greater
than 10%. Additionally, if the P concentration of urine was greater than the highest
concentration of the standard curve, an aliquot of the original urine sample composite
was diluted l-to-6 with deionized, distilled water and re-assayed.

A pooled sample of urine as a known control was made by compositing S-ml
aliquots from six individual urine composites. Duplicate 1 ml pooled urine sample
aliquots were treated as unknowns in each separate assay in order to evaluate the
variation in the spectrophotometer from assay-to—assay and to calculate the intra- and
inter-assay variation. The intra- and inter-assay variations were 4.8 and 11.3%,
respectively.

Milk Sample Analysis. After the conclusion of the sample collection period milk
samples were thawed in a warm water bath (40 °C) and mixed thoroughly by inversion
and shaking. A single 10-g aliquot of whole raw milk was weighed into 150 ml beakers
for P analysis. Samples were dried overnight at 100 °C in a forced-air oven and DM

concentration of the samples was determined. Once dried. samples were dry-ashed and

39

 

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S011m.
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Statist

assayed according to the same protocol as the fecal samples described above except that
the standard curve for milk P concentration was 0, 0.5, 0.75, 1.0, and 1.25 mg P/dl. Once
milk P% (DM basis) was computed it was converted to a wet raw milk basis. Each milk
sample from each cow at each individual milking was analyzed for P concentration and
then a 5-d weighted average was computed for each cow based on milk yield at each
milking. A 5-d weighted average also was computed for MF, MProt, ML, SNF, and SCC
for each cow. Statistics reported are based on the 5-d weighted average for each cow for
these dependent variables.

Two separate milk sample pools as known controls were made to digest and assay
along with the unknowns. Milk pools were made from 2 L of milk obtained from an .
early lactation (10 DIM) cow. A high milk P pool was a 25-ml aliquot of whole raw
milk, and a low milk pool was made by diluting whole raw milk approximately 1-to-2.5
with water for a total volume of 25 ml. The milk pools were digested and analyzed in
duplicate along with the unknowns to estimate intra- and inter-assay variation. The intra-
and inter-assay variations were 1.73 and 6.39%, respectively for the high pool and 1.70
and 6.18%, respectively for the low pool.

A third known control was a solution containing 0.80 mg P/dl made from a.
purchased 1.0 mg P/dl certiﬁed standard (Fisher Scientiﬁc, Chicago, IL). This control
solution was assayed with the unknowns to provide an estimate of the assay-to-assay
variation in the spectrophotometer. The intra- and inter- assay variations of this control

were 0.56 and 1.02%. respectively.

Statistical Analysis

40

 

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Statistical inferences were made using SAS (1999). Prior to ANOVA, the mean,
standard deviation, minimum, and maximum values were calculated by the Means
procedure for all dependent variables to help validate the datasets. Next, an outlier
analysis was conducted using the calculated mean i 3 standard deviations of each
dependent variable. Based on this analysis it was determined that there was one outlier
that should be removed from the dataset. One cow in T1 was an outlier in P balance.

The Mixed Models procedure was used to determine that the residuals of each
dependent variable in the dataset were normally distributed. This was done using the
linear model Xijk = u + T, + Pj + T, x Pj + Ck (T, x Pj) + Eijk where:

Xijk is the expected response,

u is the overall mean of the response,

T, is the effect of treatment i,

Pj is the effect of parity j,

T, x Pj is the interaction between treatment i and parity j,

Ck is the random effect of cow k and is nested within treatment i and parity j, and;

Eijk is the experimental error.

There were two parity classiﬁcations for this experiment, S second parity and 2 third
parity. Distribution of residuals was determined by F-test to be normal if P > 0.001.

Dependent variables also were tested for heterogeneity (unequal variances). This
was done using the Mixed Procedure of SAS (1999). The absolute value of the residuals
was computed and used to determine heterogeneity. The linear model was Xik = u + T, +
+ C,- (Ti) + E“, where:

X“,- is the absolute value of the residual,

41

p. is the overall mean of the residuals,

T, is the effect of treatment i.

C, is the random effect of cow k and is nested within treatment I, and;

E“, is the experimental error.

Cow was random and nested within treatment. Variances were determined by F -test to
be heterogeneous (unequal) if P < 0.05.

For ANOVA, statistical signiﬁcance was declared if P < 0.05, and tendencies
were noted if P < 0.10. The least squares AN OVA was used to determine differences
among treatment means of each dependent variable by the mixed model procedure of
SAS (1999). The linear model used was Xik = u + T, + Ck (Ti) + Eik where:

Xik is the response of the dependent variable,

11 is the overall mean of the response,

T, is the effect of treatment i.

C,- is the random effect of cow k and is nested within treatment i. and;

E”, is the experimental error.

Parity and the treatment x parity interaction were included in the original model, but were
inconsequential and omitted in the ﬁnal model. Orthogonal contrasts (linear and
quadratic effect of treatment) were used to compare treatment meanresponses.

A major dependent variable of interest relative to the experimental objective was
fecal P excretion, g/d + DMI, kg/d. Regression analysis with daily fecal P excretion as
the dependent variable and DMI as the independent variable was done according to SAS

(1999). The ﬁt of the model was determined by the coefﬁcient of multiple determination

42

(R2) also using the regression procedure. A scatter plot of fecal P excretion, g/d (y-axis)

versus DMI, kg/d (x-axis) and the best-ﬁt regression line was graphed.

RESULTS AND DISCUSSION

Experimental Diet. Ingredient composition and analyzed chemical composition
of the basal diet is presented in Table 1. The diet was formulated to be as low as possible
in P concentration and therefore, contained 25% beet pulp pellets (without molasses),
comparable to corn silage and alfalfa silage in energy content, but with less P (0.08 vs.
0.21 and 0.20% P, DM basis for beet pulp pellets, corn silage, and alfalfa silage,
respectively). Cornstarch in effect, replaced a portion of corn grain and soybean meal
(44% CP) and supplied similar energy, but with less P (0.009%, dry basis vs. 0.30 and
0.71% P for corn grain and soybean meal, respectively). However, beet pulp pellets and
cornstarch did not contain as much protein (8.4 and 0.6% CP, DM basis, respectively,) as
alfalfa silage, corn silage, or corn grain (20.0, 8.5, or 9.1% CP, DM basis, respectively).
Therefore. urea and. biuret were added to meet the dietary crude protein requirement
without adding more P. The analyzed CP concentration of the diet was higher than-
formulated perhaps because of mixing error or a lower value for %CP used for urea or
biuret during formulation than what they actually were.

Performance Characteristics of Cows. General characteristics of the cows and
performance variables are present as treatment means with statistical signiﬁcance are in
Table 2. Differences among treatments in BW, BCS and DIM resulted from the initial

assignment of cows based on DIM and length of time cows had been lactating. Daily

43

DMI differences were as experimentally set and expressed as a percentage of body
weight and ranged from 1.8, 2.9 and 4.2% (P < 0.02; quadratic effect). It is important to
show differences in these variables as they were used as the criteria to deﬁne the
treatments and estimate the effects of DMI on inevitable fecal P excretion and that
component of P maintenance requirement.

Apparent DM digestibility averaged about 70% (Table 2) and did not differ
among experimental treatments (P > 0.10). The reduction in milk yield and energy-
corrected milk yield was caused by the combination of reduced DMI and increasing DIM.
Milk fat%, MProt, ML, SNF, and SCC were not affected by treatment (P > 0.05).

Characteristics of Feces, Urine, and Milk. Some characteristics of feces, urine, .
and milk are summarized in Table 3. Fecal DM% tended to decrease slightly (P =
0.0865; linear effect) with increasing DMI. Fecal DM excretion increased linearly (P <
0.01) as DMI increased (3598, 4610, and 7630 g/d) for T1, 2, and 3, respectively. This
response would be expected because apparent DM digestibility was similar among
treatments. This is similar to results reported by Spiekers et al. (1993), where two groups
of ﬁve cows in different stages of lactation had different DMI (17 vs 11 kg/d), and fecal
excretion increased as DMI increased. In the current experiment, there was an inverse
relationship between fecal P% (dry basis) and DMI, kg/d (0.43, 0.40 and 0.3 5% P, dry
basis; P < 0.0012; linear effect). Total daily fecal P excretion (15.3, 18.2. and 26.3 g/d
for T1, 2, and 3, respectively) increased linearly (P < 0.01) with increasing DMI; cows on
all treatments were fed the same diet (0.26% P, dry basis). These results differed slightly
from the results of Spiekers et al. (1993) who reported no difference in fecal P

concentration between treatments (0.48 vs.0.49% for high and low DMI, respectively).

44

However, they reported an increase in daily fecal P excretion due to increased DMI and
fecal DM excretion.

Predicted urine excretion was 11.4, 14.3, and 21.0 kg/d for T1, 2. and 3,
respectively, increasing linearly (P < 0.01) with increasing DMI. Urine P concentrations
on a wet basis, were 0.0024, 0.0005, and 0.0003% for T1, 2, and 3, respectively, but not
different (P = 0.50). Total daily urine P excretion was not different among treatments.
These results are similar to those of Wu et al. (2000) in which there was no difference in
urine P excretion of cows fed diets with 0.31 to 0.49% P. In other studies, with dietary P
from 0.30 to 0.67% P, urinary P excretion increased with increasing dietary P
concentration (Knowlton and Herbein, 2002; Morse et al., 1992b). Because urine P
concentration was low, total urinary P excretions across all treatments were only 1.8 and
1.7 g/d for Knowlton and Herbein (2002) and Morse et al. (1992b), respectively.

In the current experiment, milk P concentration was not different among
treatments and averaged about 0.081% (Table 3), which is similar to results reported by
Knowlton and Herbein (2002), Wu et al. (2000), and Brintrup et al. (1992). Milk P
concentration was not different between treatments when dietary P ranged from 0.31 to
0.67%. Average milk P% for these three studies was 0.087% and ranged from 0.085 to
0.091% P in these three studies. Due to differences in milk yield (Table 2), milk P
secretion increased linearly for T1 , 2, and 3, respectively (P < 0.01).

Phosphorus Excretion and Balance. Phosphorus balance [P intake, g/d — (fecal P
excretion + urinary P excretion + milk P secretion (g/d))] was important in identifying
whether or not the treatments as deﬁned would allow appropriate evaluation of inevitable

fecal P excretion and that component of the P maintenance requirement of lactating dairy

45

cows. Table 4 summarizes P intake, excretion, secretion, and balance by treatment. P
intake increased linearly (29.4, 39.8, and 65.2 g/d for T1, 2, and 3, respectively; P < 0.01)
as DMI increased. Additionally, total P excretion (fecal P + urinary P) increased linearly
(P < 0.01; 15.6, 18.3, and 26.4 g/d for T1, 2, and 3, respectively). Feces is the major
route of P excretion (NRC, 2001) and urinary P excretion constituted only between 0.04
and 1.6% of total P excretion across treatments (Table 4).

Maintenance Requirement for Phosphorus. Phosphorus balance was 1.5 g/d for
T1, 2, and 3 (Table 5). Phosphorus balances were not different from zero (P > 0.05)
providing support for the idea that cows were fed very close to their true requirements for
P. Because the cows consumed amounts of P that resulted in P balance very near zero,
the P excretion in feces and urine was considered inevitable P loss and the requirement
for maintenance was calculated from these data (NRC, 2001). Two assumptions are made
when determining the P maintenance requirement in this way. Firstly, because P is fed at
a low dietary concentration, the consumed P will be utilized with high efﬁciency. AFRC
(1991) and NRC (2001) assumed that the coefﬁcient of absorption was 0.8. That is, most
of the P consumed from the diet will be absorbed from the digestive tract. Secondly,
because most of the dietary P is absorbed. most of the P found in feces and all of that in
urine is of endogenous origin. Phosphorus of endogenous origin in feces means that
dietary P was absorbed across the digestive tract, secreted back into the rumen via saliva,
and subsequently excreted in feces. As mentioned previously, a small amount of P is
excreted through urine and is considered as part of the P maintenance requirement
because it is part of the absorbed P fraction. Inevitable fecal P excretion includes

endogenous fecal P plus the unavailable dietary P excreted in feces. The AFRC (1991)

46

acknowledged that urine P theoretically should be added to inevitable fecal P for
determining the maintenance requirement, but ignored it in their ﬁnal recommendation
due to the low amount of P generally excreted in urine by cattle. However, NRC (2001)
added an additional 0.002 g urine P/kg BW to their ﬁnal recommended maintenance
requirement.

In the current experiment, inevitable fecal P excretion/kg of DMI declined with
increasing DMI (1.36, 1.19, and 1.04 g of P/kg of DMI for T1, T2, and T3, respectivley;
linear effect of treatment, P < 0.01). Using a similar experimental approach, Spiekers et
al. (1993) found that inevitable fecal P excretion was 1.20 and 1.22 g /kg of DMI for
lactating cows consuming 17 and 11 kg DM/cow per day. Additionally, the NRC (2001)
reported that as much as 50% of the endogenous fecal P loss is associated with microbial
cells excreted in feces. Because T3 cows had the highest DMI, more microbes would be
present in their rumens. Therefore, these cows will have more endogenous fecal P
excretion associated with microbial cells.

Using a constant (e.g., 1.2 g of P/ kg of DMI; NRC, 2001) as an estimate of
inevitable fecal P component of the maintenance requirement does not allow for the
difference found as DMI changed in the current experiment. The estimate of NRC .
(2001) could be modiﬁed to account for decreasing inevitable fecal P (component of the
maintenance requirement)as DMI increased. To assess modiﬁcation of the NRC (2001)
approach for estimating the fecal fraction of the P maintenance requirement regression
analysis was done. Inevitable fecal P excretion (g/d) was regressed on DMI (kg/d)
(Figure 1) using all of the data collected in this experiment. The regression analysis

indicated a linear relationship between fecal (inevitable) P excretion and DM1 (P < 0.01).

47

The equation that described this relationship was: inevitable fecal P excretion, g/d =
[0.85 i 0.070 (g/d)]x DMI, kg/d + [5.30 i 1.224 (g/d)], where 0.68 is the slope of the
regression line and 5.3 is the y-intercept (Figure 1). This means that for every 1 kg
increase in DMI an additional 0.85 g P was excreted in feces and that if a cow could have
a DMI of 0 kg then she would still excrete 5.30 g P in her feces. This relationship
explained 90% of the variation associated with fecal P excretion and had a root mean
square error (RMSE) of 1.75 g/d. Additionally, the y-intercept was different from zero
and the slope was different from 1. There was more variation in inevitable fecal P
excretion at greater DMI (cows on T3 with ad libitum intake). Because cows were
allowed to consume ad libitum intake there was less control over their P intake relative to
the DMI of intake-restricted cows in T1 and T2. Therefore, the opportunity was greater
for T3 cows to consume more dietary P than their true requirement, increasing fecal P
excretion. When more dietary P is consumed than needed the additional P will be
excreted in feces and potentially urine (Knowlton and Herbein. 2002; Wu et al., 2001;
Wu et al., 2000; Brintrup et al, 1993; Morse et al., 1992b; Martz et a1, 1990). When DMI
was restricted (T1 and 2) there was a tighter relationship between fecal P excretion, g/d
and DMI, kg/d because there was less potential for variation in DMI and P intake in
excess of requirement for milk production plus maintenance. Therefore, feeding cows
closer to P needs resulted in a tighter relationship between inevitable fecal P excretion
and DMI and presumably allowed for a more accurate determination of the fecal
component of the P maintenance requirement in T1 and T2.

The total dietary P maintenance requirement for a 700 kg cow consuming 22 kg

DMI and producing 40 kg milk/d accounting for endogenous urinary P loss, would be

48

9.8, 20.0, 19.3, 15.2, and 23.4 g P/d for the ARC (1980), NRC (1989), INRA (1989),
AFRC (1991), and the NRC (2001), respectively. As a comparison, the P requirement for
maintenance of this same cow would be 24.1 g/d based on the model [inevitable fecal P
excretion (g/d) = 0.85 x DMI (kg) + 5.30 (g/d)] developed in the current experiment.
This example P maintenance requirement includes an additional 0.002 g P/kg BW to
account for the endogenous urinary P loss part of the total, maintenance requirement.

In the current research, cows with greater DMI utilized P more efﬁciently (Table
5) based on apparent P digestibility. Apparent P digestibility increased with increased
DMI (linear effect of treatment; P < 0.01). More efﬁcient use of ingested P may be
explained by MY. Cows with higher milk had increased demands for P relative to the .
other two experiments; therefore P would be more efﬁciently used to support the higher

MY.

CONCLUSIONS

The relationship of inevitable fecal P excretion (g/d) + DMI (kg/d), the approach
taken by the NRC (2001) to express the P maintenance requirement, could be evaluated
because P balance was not different from zero (P > 0.05) so inevitable fecal P excretion
was equal to that portion of the P requirement for maintenance of lactating cows. The
NRC (2001) set the P requirement for maintenance to be constant at 1.2 g P/kg DMI.
However, in the current research this relationship decreased linearly with increased DMI.
It is unknown whether or not these are statistically different from the NRC (2001)
recommendation, but due to low variation (0.040 g P/kg DMI) we were able to detect a

difference in inevitable fecal P related with DMI. Therefore, the approach taken by the

49

NRC (2001) was modiﬁed to account for increased inevitable P excretion with decreased
DMI. In this experiment, inevitable fecal P excretion (g P/kg DMI) decreased linearly
with DMI (Table 4). Therefore, regression analysis was used to determine the
relationship between inevitable P excretion and DMI. The relationship was: inevitable
fecal P excretion (g/d) = [0.85 i 0.070]x DMI (kg/d) + [5.30 i 1.244 (g/d)]; (RMSE =
2.75 g/d; R2 = 0.90; P < 0.01). However, this new approach needs to be evaluated by

further research to determine whether or not it is valid.

50

Table l. Formulated ingredient and analyzed chemical composition of the basal diet.

 

 

Item Percent of dietary DM

Ingredient
Alfalfa silage 1 1.4
Corn silage 19.3
Beet pulp pellets, dehydrated

without molasses 25.0

Whole cottonseed 1 1.0
Corn starch 10.9
Corn, dry ground 13.4
Soybean meal, 44% CP 3.9
Blood meal 1.3
Corn Oil 0.1
Limestone 0.8
Salt (NaCl) 0.4
Urea 0.7
Biuret' 0.8
Vitamin and mineral premix2 1.0

Chemical composition3
DM 58.9
CP 20.3
NEL‘, Meal/kg 1.69
ADF 21.7
NDF 39.0
Ash 3.
Ca 0.94
P 0.26
Mg 0.22
K 0.95
Na 0.39
Cl 0.36
S 0.19
Cu. mg/kg 20
Fe, mg/kg 346
Mn. mg/kg 82
Zn, mg/kg 83

 

lGraciously provided byMoorman's Manufactoring Compancy, Quincy IL.
2Trace mineral and vitamin base mix was 71.4% dry ground corn, 25.5%
vitamins and minerals. and 3.1% corn oil. The concentrations of the vitamins
and minerals were: 0.05% Ca, 0.28% P 0.60% Mg. 3.3% K, 1.4% S,

0.01% Na, 332 mg/kg Cu. 699 mg’kg Mn. 894 mg/kg Zn. 90 mg/kg Fe,

4 mg/kg Co, 7 ppm Se, 8 mg/kg 1. 24 1(1ng vitamin A, 5 KIU/kg

vitamin D, 0.09 KlU/kg vitamin E, dry basis.

3Nutrient composition was determined by Dairy One F oragc Lab

(Ithaca. NY) except for P and ash.

‘Energy value estimated based on computation per NRC (2001).

51

Table 2. Performance characteristics of cows as inﬂuenced by experimental treatments.1

 

 

 

 

Treatment2 P-value’
Item T1 12 T3 SEM Treatment Linear Quadratic
Number of cows 7 7 5 -- - - --
BW, kg 640 530 603 25.3 NS" NS <0.01
BCS 3.3 2.5 2.1 0.19 <0.01 <0.01 0.04
DIM 285 169 85 6.6 <0.01 <0.01 <0.01
DMI, kg/d 11.3 15.3 25.1 0.74 <0.01 <0.01 NS
DMI, %BW 1.8 2.9 4.2 0.13 <0.01 <0.01 <0.01
Apparent DM digestibility, % 68.1 70.0 69.7 0.91 NS <0.01 NS
MY, kg/d 15.7 23.9 47.0 1.56 <0.01 <0.01 NS
ECM", kg/d 15.7 23.5 50.6 1.87 <0.01 <0.01 NS
Milk fat. % 3.69 3.51 4.17 0.254 NS NS NS
Milk protein (true), % 2.94 2.88 3.04 0.062 NS NS NS
Milk lactose, % 4.69 4.65 4.72 0.064 NS NS NS
SNF, % 8.60 8.49 8.78 0.118 NS NS NS
SCC, 1000 cells/m1 271 286 191 74.3 NS NS NS

 

IValues are least squares means.

2Treatments; T1 = 50% ad libitum intake, T2 = 75% ad libitum intake; and, T3 = 100% ad libitum
intake.

3Signiﬁcant effects (P < 0.05) and tendencies (P < 0.10).

‘Ns = Not signiﬁcant (P > 0.10).

5ECM = Energy corrected milk yield (kg/d) = ([0.3246 x MY (lb/d)] + [12.86 x milk fat yield (lb/d)] +
[7.04 x milk protein yield (lb/d)]) + 2.2 (kg/lb) (Dairy Record Management Systems, 1999).

52

 

Tablt

 

BEE.
Paul
Fecal
Fecal

lnexit
Unne
Unne
Unne
Xhml
1Van
2Trea

intak-
351531
‘.\'S =

s .
Emm

Table 3. Characteristics of feces, urine, and milk as inﬂuenced by eiggerimental treatments.l

 

 

 

 

Treatment2 P-value3
Item T1 T2 T3 SEM Treatment Linear Quadratic
Fecal DM, % 18.3 17.6 16.6 0.58 Ns‘ 0.09 NS
Fecal DM excretion, g/d 3599 4610 7631 331.6 <0.01 <0.01 NS
Fecal P, % of DM 0.43 0.40 0.35 0.01 <0.01 <0.01 NS
Inevitable fecal P excretion, g/d 15.3 18.2 26.5 1.16 <0.01 <0.01 NS
Urine excretions, kg/d 11.4 14.3 20.2 0.93 <0.01 <0.01 NS
Urine P, % (wet basis) 0.0024 0.0005 0.0003 0.003 NS NS NS
Urine P excretion, g/d 0.26 0.07 0.06 0.14 NS NS NS
Milk P, % (wet basis) 0.080 0.084 0.079 0.003 NS NS NS
Milk P secretion, gd 12.3 20.1 37.3 1.23 <0.01 <0.01 NS

 

lValues are least squares means.

2Treatments; T1 = 50% ad libitum intake, T2 = 75% ad libitum intake; and, T3 = 100% ad libitum

intake.

3Signiﬁcant effects (P < 0.05) and tendencies (P < 0.10).

4NS = Not signiﬁcant (P > 0.10).

5Estimated from equation (Holter and Urban, 1992).

53

Table 4. Phosphorus excretion and balance as inﬂuenced by experimental treatments.1

 

 

 

 

Treatment2 P—value3
Item T1 T2 T3 SEM Treatment Linear Quadratic
P intake, g/d 29.4 39.8 65.2 1.93 <0.01 <0.01 Ns‘
Total P excretions, g/d 15.56 18.27 26.41 1.17 <0.01 <0.01 NS
Fecal P excretion, g/d 15.30 18.20 26.35 1.16 <0.01 <0.01 NS
Urine P excretion, g/d 0.26 0.07 0.06 0.14 NS NS . NS
Milk P secretion, g/d 12.3 20.1 37.3 1.23 <0.01 <0.01 NS
P balance“, g/d 1.5 1.5 1.5 1.51 NS NS NS
Inevitable fecal P 10557, g P/kg
DMI 1.36 1.19 1.04 0.040 0.0002 0.000] NS

 

1Values are least squares means.

2Treatments; T1 = 50% ad libitum intake, T2 = 75% ad libitum intake; and, T3 = 100% ad libitum
intake.

3Signiﬁcant effects (P < 0.05) and tendencies (P < 0.10).

‘NS = Not signiﬁcant (P > 0.10).

5Total P Excretion, g/d = the total maintenance requirement for P.

6P balance, g/d = P intake (g/d) - [total P excretion (g/d) + milk P secretion (g/d)]; P balance was not
different from zero (P > 0.05).

7Inevitable fecal P loss. g P/kg DMI = fecal P excretion (g/d) / DMI (kg/d), which is the major part
of the total maintenance requirement for P.

54

Tabel 5. Dietary P utilization as inﬂuenced by experimental treatments.’

 

 

 

 

Treatment2 P-value3

Item T1 T2 T3 SEM Treatment Linear Quadratic
Apparently absorbed p, g/d 14.1 21.6 33.9 1.15 <0.01 <0.01 NS"
Apparent P digestibility, % 47.0 54.1 59.7 1.54 <0.01 <0.01 NS
Total P excretion, % P intake 53.0 45.9 40.3 1.36 <0.01 <0.01 0.0401
Fecal P, % P intake 52.2 45.7 40.2 1.54 <0.01 <0.01 NS
Fecal P, % total P excretion 98.4 99.6 99.8 0.85 NS NS NS
Urine P, % P intake 0.79 0.16 0.09 0.412 NS NS NS
Urine P, % total P excretion 1.6 0.4 0.2 0.85 NS NS NS
Milk P, % P intake 41.9 50.7 57.6 3.38 0.02 <0.01 NS
Milk P, % total P output 43.4 52.3 58.8 2.33 0.0012 <0.01 NS

 

1Values are least squares means.

2Treatments; T1 = 50% ad libitum intake, T2 = 75% ad libitum intake; and, T3 = 100% ad

libitum intake.

3Signiﬁcant effects (P < 0.05) and tendencies (P < 0.10).

‘Ns = Not signiﬁcant (P > 0.10).

55

Endogenous fecal P excretion, g/d

 

 

 

 

l l l 4 l l
5 1O 15 20 25 30
DMI, kg/d

Figure 1. The relationship between inevitable fecal
P and daily DMI: inevitable fecal P (g/d) = [5.30 i 1.224
(g/d)] + [[0.85 i 0.70] x DMI (kg/d)] (RMSE = 2.75; y-
intercept different from zero; slope different than one; R2 =
0.90; P < 0.01).

 

 

BI

balar
datas
30-d

Were

MY the

Therefo

CHAPTER 4

INCREASING DIETARY PHOSPHORUS: UTILIZATION OF PHOSPHORUS

BY DAIRY CATTLE AT DIFFERENT POINTS OF THE LACTATION CYCLE

ABSTRACT

The objectives were to evaluate the effect of varying dietary P concentration on P
balance and utilization of lactating and non-lactating Holstein cattle and to construct a
dataset for future research to evaluate P excretion prediction mOdels. A series of seven
30-d experiments were conducted. Experiments were designed so that P balance data I
were collected at various points of the lactation cycle.

Experiments 1 and 2 were conducted with 21 pregnant nulliparous heifers at 57
and 28 i 1.0 (1 prior to parturition, respectively. Experiments 3 and 4 were conducted
with 21 pregnant non-lactating primi- and multiparous cows at 59 and 30 i 1.0 d prior to
parturition, respectively. Experimental treatments in Experiments 1 through 4 were diets
with 0.19 (low; LP), 0.28 (moderate; MP), and 0.35% P (high; HP), dry basis.
Experiments 5, 6, and 7 each consisted of 35 lactating primi-and multiparous cows. In
Experiment 5 cows were 97 i 10.0 DIM, in Experiment 6 cows were 183 i 6.2 DIM, and
in Experiment 7 cows were 294 i 1 1.6 DIM.

Experiments 5, 6, and 7 were designed to have different DIM so that DMI and
MY theoretically would be different, so that P intake, utilization and excretion would
differ for future modeling work. However. the DMI range desired did not occur.

Therefore, DMI was restricted in Experiment 7 to the NEL intake required to support MY

57

 

 

 

 

lneas
C ow:
inuﬂi
FIB:
dkns
Expel
ueret

equal

increa
Expen
eﬁecti
incons
00063
increag
then“;
Collecr

mOdels

(NRC.

measured during the ﬁrst 21 d of the experiment for any cow producing less than 25 kg/d.
Cows producing greater than 25 kg/d were allowed to consume ad libitum. Dry matter
intakes during the 5-d collection period for Experiments 5, 6, and 7 were 19.8, 19.3, and
17.8 :t 0.59 kg/d, respectively. Treatment groups for Experiments 5, 6, and 7 were fed
diets with 0.27 (T1), 0.36 (T2), 0.42 (T3), 0.46 (T4), and 0.52% P (T5), dry basis. In
Experiment 7 because of the DMI restriction of some cows, dietary P treatment and DMI
were confounded because the distribution of the cows with unrestricted DMI was not
equal across all treatments. Therefore, ANOVA were not conducted on this dataset.

In all experiments, fecal P concentration and fecal P excretion increased with
increased dietary P concentration and ranged from 0.43 to 2.21% P, respectively, for
Experiments 1 through 4 and 0.40 to 1.54, respectively, for Experiments 5 and 6. The
effect of dietary P concentration on urine P concentration and excretion was small but
inconsistent and averaged 0.0012% P and 0.82 g P/d for Experiments 1 through 4 and
0.0063% and 1.65 g PM for Experiments 5 and 6. In general. P balance decreased with
increased dietary P concentration in all experiments. Milk yield was not affected by
dietary P in Experiment 5 or 6 (P > 0.05). Three datasets were constructed from the data
collected in these experiments to be used in subsequent research to evaluate published

models for predicting P excretion.
INTRODUCTION

There are more known biological functions involving P than any other element

(NRC, 2001). Therefore, it is important to understand how dietary P is utilized and what

58

 

Fecal
total .
Durir
Milk .
et al..

Power

When <
Herbei
Martz.
fed die1
P excre
COncent
from 0.:
1993: B.
and Herl
ﬁddum

pregnant

Cattle 7]]
data to be .

happens to P consumed in excess of the P needed to sustain the body and production.
Fecal excretion is the main route of P removal from the body consisting of 95 to 98% of
total P excretion (NRC, 2001; Morse et al., 1992b) with the remaining 2 to 5% in urine.
During lactation, the second major route of P removal from the body is milk secretion.
Milk P concentration ranged between 0.085 to 0.10% (Knowlton and Herbein, 2002; Wu
et al., 2000; Brintrup et al., 1993; Spiekers et al., 1993; Brodison et al., 1989; Flynn and
Power, 1985).

Increasing dietary P concentration increased fecal P concentration and excretion
when cows were fed diets with 0.3010 0.67% P (Kohn et al., 2002; Knowlton and
Herbein, 2002; Wu et al., 2002; 2001; 2000; Brintrup et a1, 1993; Morse et al., 1992b; _
Martz et a1, 1990). Increasing dietary P also increased urine P excretion when cows were
fed diets with 0.30 to 0.67% P (Knowlton and Herbein, 2002; Morse et al., 1992b). Urine
P excretion ranged from 0.3 to 4.1 g/d in these three experiments. No effects on milk P
concentration was observed when cows were fed diets with P concentrations ranging
from 0.24 to 0.67% P (Knowlton and Herbein, 2002; Wu et al., 2000; Brintrup et al.,
1993; Brodison et al., 1989; Call et al., 1987; Forar et al., 1982). Additionally, Knowlton
and Herbein (2002), Wu et al. (2000), and Brintrup et al. (1993) reported that milk P
yield was not affected by dietary P fed at 0.31 to 0.67%. Phosphorus excretion data from
pregnant non-lactating data was not found.

The ﬁrst objective of this research was to characterize the effects of dietary P
concentration on P balance and routes of excretion from non-lactating and lactating dairy
cattle. The second objective was to assemble a dataset from all P balance and excretion

data to be used in subsequent research to evaluate models used to predict P excretion.

59

 

Expel

State 1
00). .1
\vere c

EXCICI

penod
CoHect
Smhpk
cons,

COllecp
Body v
COHecu
deterrn,
foreac}

Period.

mmem.

formula

MATERIALS AND METHODS

Experimental Design, Cows, and Diets

All procedures for care and sampling of animals were approved by the Michigan
State University All-University Committee on Animal Use and Care (AUF# 11/00-151-
00). A total of four experiments with non-lactating and three lactating Holstein animals
were conducted to address the effect of increasing dietary P concentration P balance, P
excretion. and performance.

Experimental Similarities. Each experiment consisted of a 30 (1 experimental ,
period. The ﬁrst 25 d were used for diet adaptation and the last 5 d were the sample
collection period. Samples collected included feed, fecal grab samples, and urine spot
samples for non-lactating animals, with milk samples collected in addition for lactating
cows. Lactating cows were milked twice-daily at 0400 and 1400 h. Milk samples were
collected for each milking during the 5-d collection period for a total of 10 milk samples.
Body weight and BCS were recorded 1 d prior to sample collection and 1 d after sample
collection to obtain an average BW and BCS for the collection period. Because BCS
determination is subjective, BCS was recorded by four separate technicians and averaged
for each cow. All results reported are from data collected during the 5-d collection
period.

Four experiments with non-lactating (nulliparous and multiparous) animals had
three dietary P treatments each with 7 cows/treatment. Dietary treatments were

formulated according to NRC (2001) recommendations for all nutrients except P (Table

60

 

1). A
treatm
formu
diet. 7
MP. a1
three c
dailya

that da

dietar}
accord
diet w;
represt
0.44, C
11101103
COncer
TCSpem
Oomph
reﬁgaj
detenn

exPErir

2] rlull

1). A basal diet was formulated to contain 0.16% P. This represented the low P dietary
treatment (LP). Moderate (0.28% P; MP) and high (0.40% P; HP) P diets were
formulated by replacing urea and cornstarch with monoarnmonium phosphate in the basal
diet. The analyzed dietary P concentrations were 0.19, 0.28, 0.3 5% P (dry basis) for LP,
MP, and HP, respectively (Table 1). Non-lactating animals were assigned to one of the
three dietary treatments in a completely randomized design and fed respective diets once-
daily at approximately 1000 h. Feed refusals were weighed every day prior to delivering ’
that days ration for feed intake determination.

Three experiments with lactating cows (primiparous and multiparous) had ﬁve
dietary P treatments each with 7 cows/treatment. Dietary treatments were formulated ,
according to NRC (2001) recommendations for all nutrients except P (Table 2). A basal
diet was formulated without supplemental P to contain 0.24% P. This treatment
represented Treatment 1 (T1). Treatments 2 through 5 were formulated to contain 0.34,
0.44, 0.54, and 0.64% P, dry basis. This was accomplished by substituting
monoammonium phosphate for urea and cornstarch in the basal diet. Analyzed dietary P
concentrations were 0.27 (T1), 0.36 (T2), 0.42 (T3), 0.46 (T4), and 0.52% (T5),
respectively. Lactating cows were assigned to one of the ﬁve dietary treatments in a
completely randomized design and fed once-daily at approximately 1000 h. Feed
refusals were weighed every day prior to delivering that days ration for feed intake
determination. All dependent variables either measured or estimated in the seven
experiments with lactating and non-lactating animals are deﬁned in Table 3.

Experimental Diﬂerences with Non-lactating Animals. Experiment 1 consisted of

21 nulliparous animals. Experiment 1 was started at an average of 57 i 1.0 (1 prior to

61

 

panunt
2c0nsi
continc
nudﬁpz
approx
lactatir
unﬁll'
Expen.

experil

parturition and continued until approximately 28 i 1.0 d prior to parturition. Experiment
2 consisted of 21 nulliparous animals and began at 38 i 1.0 d prior to parturition and
continued until 9 i 1.0 (1 prior to parturition. Experiment 3 consisted of 21 non-lactating
multiparous cows and began at 59 i 1.0 (1 prior to parturition and continued until
approximately 30 i 1.0 d prior to parturition. And, Experiment 4 consisted of 21 non-
lactating multiparous cows and began at 40 i 1.0 d prior to parturition and continued
until 11 i 1.0 (1 prior to parturition. However, one cow in each LP treatment of
Experiments 3 and 4 developed a displaced abomasum and was removed from the

experiment.

Experimental Diﬂerences with Lactating Cows. Experiment 5 consisted of 35
primiparous and multiparous cows with average DIM of 97 i 10.0 and average DMI of
21.8 i 2.47 kg/d for the ﬁrst 21 d of the experiment. Experiment 6 consisted of 35
primiparous and multiparous cows with average DIM of 183 i 6.2 and average DMI 21.6
i 2.16 kg/d for the ﬁrst 21 d of the experiment. However, two cows were removed from
the experiment. One cow in T1 contracted mastitis and data from one cow in T5 were
determined to be outliers (i 3 standard deviations) in fecal P excretion and P balance and
they were removed from the dataset used for statistical analyses. Experiment 7 consisted
of 35 primiparous and multiparous cows with average DIM 294 i l 1.6 and average DMI
23.0 .+_ 2.42 kg/d for the ﬁrst 21 d of the experiment. However. one cow was removed
from the study because she quit consuming feed for 48 hours during the 5-d collection
period.

Experiments 5, 6, and 7 followed each other sequentially, but each was conducted

with a separate set of cows. The purpose of designing the experiments based on DIM.

62

 

was 10
Howe\
lmsedt
cohect
todmr
expen
NRC (
contin

libitur

libitur
0fc0\
Collec
TESpe.
restrh
93cm
and d
HOW

DMI

Expe

“018C

was to get a wide range of ad libitum DMI and presmnably P utilization and excretion.
However, DMI (21.8, 21.6 and 23.0 kg/d, respectively) did not vary as much as expected
based on DIM among these three experiments. Therefore, 4 d prior to the sample
collection period during Experiment 7, DMI was restricted so that NEL intake was equal
to the energy needed to support measured MY of each cow during d 1 through 21 of the
experiment. Restrictions were made using the energy requirements recommended by the
NRC (2001). However, any cow producing greater than 25 kg milk/d was allowed to
continue consuming ad libitum intake. A total of 11 cows were allowed to consume ad
libitum.

Because cows producing greater than 25 kg milk/d were allowed to continue ad
libitum intake in Experiment 7, the overall DMI average still did not vary much from that
of cows in Experiments 5 and 6. When DMI was compared across treatments for the 5-d
collection period, least square means for DMI were 19.8, 19.3, and 17.8 i 0.59 kg/d,
respectively for Experiments 5, 6, and 7. Even though not all cows in Experiment 7 had
restricted feed intake, the restriction did reduce the overall DMI. Restriction of feed
offered for part (11 of 34) of the cows in Experiment 7, resulted in a confounding of DMI
and dietary P treatments. Therefore, ANOVA was not conducted for this experiment.
However, the highest priority objective was to gather some data of P excretion at lower

DMI for future modeling work.

Experimental Procedures

Experimental procedures were identical for all seven experiments except where

noted. Chromium oxide was used as an external marker to estimate daily fecal excretion.

63

 

An

NJ 1
can
On c
of 7.
Appr
to the
conta
d dosl

0f the

contai:
26 at C
10 min
Collecr.

fI’OZen

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Speciﬁc

SampleS

An initial dose of 45 g chromium oxide were given orally via balling gun in two equal
amounts of 22.5 g at 0600 and 1800 h in lock ring gelatin capsules (Torpac Inc., Fairﬁeld,
NJ) on d 20 of the experimental period. The initial dose of 45 g was given to help
ensure that a steady state Cr concentration was reached prior to fecal sample collections.
On (I 21 to 30 of the experiment, cows were given chromium oxide in two equal amounts
of 7.5 g at 0600 and 1800 h in lock ring gelatin capsules for a total of 15 g daily.
Approximately 1 g of spelt hulls was added to each gelatin capsule to provide buoyancy
to the capsule in the rumen until the capsule dissolved (Oba and Allen, 2001). Spelt hulls
contained 0.22% P, DM basis, which contributed only 53 mg dietary P over the entire 11-
d dosing period; therefore, P supplied by spelt hulls was ignored in P-related calculations
of the data.

Approximately 500 g of feces (grab samples) were collected into plastic
containers with snap-on lids (Sweetheart Cup Company, Inc., Chicago, IL) starting on d
26 at 0600 h. Fecal grab samples were collected at 15-h intervals through 1500 h on d 30
to minimize diurnal variation in Cr and P excretion. A total of eight fecal samples were
collected, with one sample representing every 3 h of a 24—h clock. Fecal samples were
frozen at —4 °C for later processing and P, Cr, and DM analyses.

Eight urine samples were taken via manual stimulation of the area around the
vulva during the same time fecal samples were collected. Approximately 100 ml of urine
were collected into 15 O-ml graduated urine specimen containers with screw-on lids (Life
Sciences Products, Inc., Denver. CO) and frozen (—4 °C) for later processing and P and
speciﬁc gravity analyses. Urine samples were occasionally missed, but at least 6 of the 8

samples were available for compositing and analyses.

64

 

140(
durir
pelle
lacto

samp

D.\1 c
IL). .
insurt
Week
Chem.
Silage
unloa.

Pile tc

Lactating cows in Experiments 5, 6, and 7 were milked twice-daily at 0400 and
1400 h. Two milk samples (25 ml each) per cow were collected daily at each milking
during the 5-d collection period into sample vials containing a potassium dichromate
pellet. One milk sample was for analysis of milk fat % (MF), true protein % (MProt)
lactose % (ML), SNF, and SCC (Michigan DHIA, East Lansing). The second milk
sample was frozen at (—4 °C) for later analysis of P concentration.

Corn and alfalfa silages were sampled twice weekly on Monday and Thursday for
DM concentration determination via Koster tester (Seedburo Equipment Co, Chicago,
IL). As necessary, adjustments were made in the as-fed amounts of forages in the diet to
insure the same DM proportions of dietary ingredients throughout the experiment.
Weekly alfalfa and corn silage samples were taken for later DM concentration and
chemical determination. Alfalfa silage was stored in bunker and upright silos and corn
silage was stored in bunker silos. For upright silos, approximately 100 kg of silage was
unloaded from the silo and then grab samples were taken from different sections of the
pile to form a 7-kg composite sample. An l-kg sub-sample was then taken by the roll
and quartering method. The sample was rolled and quartered twice and the resulting sub-
sarnple was stored in a sealed plastic bag (—4 °C) for later analysis of DM concentration
and chemical composition. For bunker silos, grab samples from the freshly exposed face
were taken from random spots across the entire face of the silo until a 7-kg composite
sample was obtained. The sample was then processed the same as previously mentioned.
Bagged feed ingredients (premix ingredients and beet pulp pellets) were sampled bi-
weekly by taking a 100-g grab sample from 10% of the bags. These samples were stored

in sealed plastic bags (—4 °C) for later analysis of DM concentration and chemical

65

 

 

compc
twere 5
they v1

and nu

Labor

period.
dﬁedil
DM co
m sc1
Feed sz
Salnple
reDrese
period

deterrm

cow, A
SamPle
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‘091 dai

composition. Bulk ingredients (whole cottonseed and vitamin and trace mineral mix)
were sampled weekly by taking 100-g grab samples from separate places from the pile:
they were stored in sealed plastic bags at —4 °C for later analysis of DM concentration

and nutrient composition.

Laboratory Procedures

Feed and Fecal Sample Analyses. After the completion of the sample collection
period, feed and fecal samples were thawed at room temperature. Samples were then
dried in a forced-air oven at 52 °C for 48 h for DM concentration determination. Once
DM concentration was determined for each sample, they were ground through 5 and 2 1
mm screens in a Thomas-Wiley Mill (Arthur Thomas Company, Philadelphia, PA).
Feed samples were composited by ingredient type by taking a 5 g aliquot from each
sample, which was then stored in a capped plastic container. The composite sample
represented the pooled composition of the ingredient used throughout the experimental
period. An approximate 5-g sub-sample of the each composite was used for
determination of chemical composition (Dairy One Forage Lab, Ithaca, NY).

Composite fecal samples were made from the eight individual samples from each
cow. Approximately 5 g from each individual sample were taken and combined into one
sample and stored in a capped plastic container. Each composite sample was the pooled
diurnal representation of total fecal excretion of the 5-d period for each cow represented
total daily excretion because feces were collected over a 5-d period to theoretically

reduce diurnal variation in chromium oxide and P excretion.

66

 

Pl
adapted f:
prior to e
were tale
digested
reagent '
molybd.
Color 11
(Inplice
by a Sp
Were n
1.0. 1.
Pmpo

uSed .
reage
lCOm
a853,:

gTEa

Phosphorus concentration in feed and feces was measured by colorimetric assay
adapted from AOAC (1990; method 965.17). All laboratory glassware was acid-washed
prior to each use. Duplicate 1 g sub-samples of individual feed and fecal composites
were taken after thorough mixing and dry-ashed at 600 °C for 4 h. The resulting ash was
digested in 20 ml of 4N HC1 and ﬁve drops of concentrated H2804. A molybdovanadate
reagent was added to a 1-ml aliquot of the digested sample for color development. The
molybdovanadate reagent reacted with P to form a phosphomolydenum yellow complex.
Color intensity increased with greater P concentration. Two aliquots from each digested
duplicate were pipetted into 96-well microtiter plates and P concentration was determined
by a spectrophotometer (SpectraMax 190, Molecular Devices, Sunnyvale, CA). Samples
were read at a wavelength of 400 nm and compared against the standard curve (0, 0.5,
1.0, 1.5, and 2.0 mg P/dl). The standard curve solutions contained the same volumes and
proportions of reagents and water as the unknowns. The 0 mg P/ml standard also was
used as a blank to adjust the spectrophotometer for any background P present in the
reagents or deionized, distilled water used in the digestion or assay processes. Samples
(composite feed and fecal samples) with unknown P concentrations were re-digested and
assayed if the coefﬁcient of variation (CV) between the original duplicate samples was
greater than 3%. The volume in the microtiter plate represented a 1-to-10 dilution of the
original sample. Therefore, if the P concentration of the unknown was greater than the
highest standard, the unknown was diluted 1-to-20 with deionized, distilled water and re-
assayed.

Two known controls were included in each assay. An assay was deﬁned as the P

determination of all the samples digested at the same time. Therefore, each assay may

67

 

have inc
pooled c
individt
and ass:
plates It
the intr;

respecti

solutiol
unknox

and int

Spectre
Proced

digest:

have included more than one microtiter plate of unknown samples. One control was a
pooled composite fecal sample made from 8 g of feces from the composites of 10
individual cows. The pooled sample was treated as an unknown throughout the digestion
and assay processes in order to calculate overall intra- and inter-assay variations for all
plates read by the spectrophotometer. For all assays of fecal samples in the experiment,
the intra- (within plate) and inter-assay (plate-to-plate) variations were 0.72 and 2.22%,
respectively.

The second known control was a 1.0 mg/dl purchased certiﬁed standard P
solution (Fisher Scientiﬁc, Chicago, IL). This solution was assayed along with the
unknowns to evaluate the assay-to-assay variation of the spectrophotometer. The intra-
and inter-assay variations across all assays were 0.58 and 1.2%, respectively.

Chromium concentration in feces was analyzed by ﬂame atomic absorption
spectroscopy (SpectrAA 220, Varian, Inc., Walnut Creek, CA) adapted from the
procedure of Williams et al. (1962). Duplicate l-g sub-samples were taken from fecal
composites after thorough mixing and ashed at 600 °C for 4 h. The resulting ash was
digested on a hot plate in a phosphoric acid, manganese sulfate, and potassium bromate
solution at 400 °C for approximately 10 min. Chromium concentrations were determined
by against the standard curve (1 , 2. 3, 4. and 5 mg Cr/ml). The standard curve solutions
contained the same volumes and proportions of reagents and deionized, distilled water as
the unknowns. A Stande with just reagents and deionized. distilled water and no added
Cr was used to adjust the atomic absorption spectrometer for any background Cr present
in reagents or deionized, distilled water used in the digestion or assay procedures.

Unknowns were redone if the CV between duplicates was greater than 5%. If samples

68

read greater than the highest concentration standard, the original sample was diluted 1-10-
12 and re-assayed.

Two known controls were included in each assay. A pooled fecal sample
containing Cr similar to the pooled fecal sample for P analysis was made to digest and
assay in duplicate along with each separate digestion of unknowns to obtain the intra- and
inter-assay variation. The intra- and inter-assay variation was 2.26 and 4.44%,
respectively.

The second known control of 3 mg Cr/ml solution made with the same reagents
and proportions as the unknowns from a purchased certiﬁed chromium oxide powder
(Fisher Scientiﬁc, Chicago, IL). This control was used to evaluate the assay-to-assay .
variation of the atomic absorption spectrometer. The intra- and inter-assay variations of
the Cr control solution were 2.65 and 6.90%, respectively.

Urine Prediction and Sample Analysis. After the conclusion of the experimental
period, urine spot samples were allowed to thaw and equilibrate to room temperature.
Ten ml of urine were taken from each urine spot sample and composited. Composited
samples were then acidiﬁed with concentrated HCl to dissolve any salts that may have
formed during storage and ﬁltered through four layers of cheesecloth to remove any large
particulate matter. Urine sample speciﬁc gravity was measured using an Adams®
Midget Urinometer (Bristol-Myers Squib Company, New York, NY). Once ﬁltered,
composite urine samples were analyzed for P concentration.

Urinary P concentration was determined by colorimetric assay adapted from the
procedure of Fiske and Subbarow (1925). Duplicate l-ml sub—samples were taken from

urine composites and treated with 20% TCA. The sample was then centrifuged at 4000 g

69

for 15 min. A l75-ul aliquot of the supernatant was then assayed for determination of P
concentration. A molybdate aminonaptholsulfonic acid reagent was used for color
development. The reagents formed a phosphomolybdenum blue complex with P. The
color development intensiﬁed with greater P concentrations. The P concentration of
urine was determined at 660 run against the standard curve (0, 0.25, 0.5, 0.75, 1.0, and
1.25 mg P/dl) by using a SpectraMax 190 spectrophotometer (Molecular Devices,
Sunnyvale, CA). The standard curve solutions contained the same volumes and
proportions of reagents and water as the unknowns. The 0 mg P/ml standard also was
used as a blank to adjust the spectrophotometer for any background P present in the
reagents or deionized, distilled water used in the assay. Urine samples were redone if the
CV between original duplicates was greater than 10%. The total volmne in the microtiter
plate represented a 1-to-3 dilution of the original sample. Additionally, if urine P
concentration was above the range of the standard curve, original samples were diluted 1-
to-6 and reread.

A pooled urine sample as a known control was made by compositing 5-ml
aliquots from six individual urine composites. Duplicate l-ml pooled urine sample
aliquots were treated as unknowns in each assay in order to evaluate the variation in the
spectrophotometer from assay-to-assay and to calculate the intra- and inter-assay
variation. The intra- and inter-assay variations were 4.77 and 11.3%, respectively.

Total urine excretion (kg/d) was predicted to use in the calculation of total urine P
excretion (g/d). Two equations were used to determine daily urine excretion, one for
non-lactating and one for lactating cows. The equation to predict urine excretion for non-

lactating cows was: (kg/d) = 212.1 + 0.8822 x DMI (kg/d) — 0.03452 x dietary DM (%)

70

+ 1.001 x dietary CP (% DM) — 216.4 x urine speciﬁc gravity (mg/ml) + 0.1414 x
apparently absorbed water (kg/d) (R2 = 0.92; P < 0.005: Holter and Urban, 1992), where
apparently absorbed water (kg/d; AA) = [feed water intake (kg/d; FWI) + drinking water

intake (kg/d; DWI)] — fecal water excretion (kg/d). Drinking water intake was predicted
by the equation: DWI (kg/d) = -10.34 + 0.2296 x dietary DM (%) + 2.212 x DMI (kg/d)
+ 0.03944 x dietary CP (% DM)2 (R2 = 0.64; P < 0.001; Holter and Urban, 1992). The
equation to predict urine excretion for lactating cows was: (kg/d) = 332.91 + 0.3885 x

DMI (kg/d) — 0.2175 x MY (kg/d) — 0.0830 x dietary DM (%) + 0.524 x dietary CP (%

 

DM) — 326.5 x urine speciﬁc gravity (mg/ml) + 0.2375 x AA (kg/d) (R2 = 0.70; P <
0.0001; Holter and Urban, 1992). Again, DWI was be predicted to determine AA and the
equation used was: DWI (kg/d) = —32.39 + 2.47 x DMI (kg/d) + 0.6007 x milk (kg/d) +
0.6205 x dietary DM (%) + 0.0911 x Julian Day -- 0.000257 x (Julian Day)2, where
Julian Day is the day of the year when January 1 = Julian Day 1 and December 31 =
Julian Day 365 (R2 = 0.69; P < 0.0001; Holter and Urban, 1992). Feed water intake and
fecal water for both types of cows were determined by: FWI (kg/d) = as-fed feed intake
(kg/d) — DMI (kg/d) and; fecal water (kg/d) = [fecal DM excretion (kg/d) + (fecal DM
(%) + 100)] — fecal DM excretion (kg/d). '
Milk Sample Analysis. After the conclusion of the sample collection period milk
samples were thawed in a warm water bath (40 °C) and mixed thoroughly by inversion.
A single 10-g aliquot of each whole raw milk sample was weighed for P analysis.
Samples were dried overnight in a 100 cC forced-air oven. Once dried. samples were
dry-ashed and assayed according to the same protocol as the fecal samples described

previously. The standard curve developed for determination of P concentration in milk

71

was 0, 0.5, 0.75, 1.0, and 1.25 mg P/dl. Once milk P% (DM basis) was determined it was
converted back to a whole raw milk basis. All milk samples taken (1 O/cow) were
analyzed separately for P concentration and then a 5-d weighted average P concentration
was computed for each cow. A 5-d weighted average also was computed for ME, MProt,
ML, SNF, and SCC for each cow. Values from statistical analyses are based on the 5-d
weighted average for each cow. [
Two separate milk sample pools as known controls were made to digest and assay

along with the unknowns. Milk pools were made from 2 L of milk obtained from an

 

early lactation (10 DIM) cow. A high milk P pool was made by pipetting a 25-ml aliquot
of the milk into a vial, and a low milk pool was made by adding water (approximately .
1.0-to-2.5) with water for a total volume of 25 ml. The pools were digested in duplicate
along with each digestion of unknowns.

By making milk pools in this manner a check of the assay was created.
Completing an analysis of the high and low known control duplicate samples allowed the
intra- and inter-assay variations to be calculated. The intra- and inter-assay variations
were 1.73 and 6.39%, respectively for the high pool and 1.70 and 6.18%, respectively for
the low pool. The overall intra- and inter-assay variations were 1.72 and 6.29%.

A third known control was a 0.80 mg P/dl solution made from a purchased 1.0 mg
P/dl‘certiﬁed standard (Fisher Scientiﬁc, Chicago, IL). This control solution was
assayed with the unknowns to provide an estimate of the assay-to-assay variation of the
spectrophotometer. The intra- and inter- assay variations were 0.56 and 1.02%,

respectively.

72

Statistical Analyses

Data from each experiment were analyzed seperately. Statistical inferences were
made using SAS (1999). Prior to conducting AN OVA for each dependent variable, the
arithmetic mean, standard deviation, minimum, and maximum values were calculated to
assist in verifying the data sets. Next, an outlier analysis was conducted using the
calculated mean i 3 SD for each dependent variable. Based on this analysis it was
determined that one cow on the T5 diet in Experiment 6 was an outlier in fecal P
excretion and P balance and all her data were removed from analysis.

The mixed models procedure was used to determine if the data for each dependent
variable in each dataset were normally distributed. This was done using the linear model
thk = p. + T, + PJ- + T, x Pj + Ck (T, x Pj ) + Eijk where:

Xijk is the expected response;

11 is the overall mean of the response;

T, is the effect of treatment i;

P,- is the effect of parity j;

T, x P,- is the interaction between treatment 1 and parity j;

C1, is the random effect of cow k and is nested within treatment i and parity j; and,

Eijk is the experimental error.

Within each experiment. cow was random and nested within treatment and parity. The
distribution of dependent variable residuals was determined by F—test to be normal if P >
0.001. No skewed distributions were detected in the dataset.

Data of dependent variables in each dataset also were tested for heterogeneity.

The absolute value of the residuals was computed and used to determine heterogeneity

73

using the Mixed procedure of SAS (1999). The linear model was X“, = p + T, + Ck (T,) +
E“, where:

X“, is the absolute value of the residual;

u is the overall mean of the residuals;

T, is the effect of treatment i;

Ck is the random effect of cow k and is nested within treatment i; and,

E,k is the experimental error.

Variances were determined by F-test to be heterogeneous (unequal) if P < 0.05.
Variances for each dependent variable were determined to be equal.

A difference among treatment means was signiﬁcant if P < 0.05. A difference.
with a probablility value > 0.05 but < 0.10, was denoted as a tendency. The least squares
method of ANOVA was used to determine the signiﬁcance of a dependent variable by the
mixed model procedure of SAS (1999). The linear model used for Experiments 1
through 7 was X“, = u + T, + Ck (T,) + E”, where:

X”,- is the response of the dependent variable;

11 is the overall mean of the response:

T, is the effect of treatment i;

Ck is the random effect of cow k and is nested within treatment i; and.

E, is the experimental error.

RESULTS AND DISCUSSION

Non-lactating Animals

74

Experiment 1. Performance descriptions for pregnant non-lactating heifers in
Experiment 1 are in Table 4. Body weight, BCS, DMI, and DMI (% of BW) were not
affected by treatment. Apparent DM digestibility decreased linearly with increasing
dietary P concentration (P = 0.03). The diet was exactly the same except P concentration
so the linear decrease was not expected and cannot be explained. Dietary P concentration
should not affect apparent DM digestibility in similar diets unless the concentration was
very low (NRC, 2001).

Phosphorus balance and characteristics of P excretion are found in Table 5.
Phosphorus intake linearly increased with increased dietary P concentration. Which was
expected because there were no effects of P concentration on DMI. Fecal DM% was not
different among treatments. However, Fecal DM excretion increased linearly (0.03) with
increased dietary P%. Fecal P concentration and excretion increased with increased
dietary P. Because apparent DM digestibility decreased with increased dietary P
concentration, fecal P % and excretion was should increase due to the undigested DM
excreted in feces. This is consistent to results reported by Valk et al. (2002). Fecal P
excretion increased when non-lactating cows were fed dietary P of 0.16, 0.19, and 0.26%.
In the current experiment fecal P excretion (g/kg DMI) increased linearly with increasing
dietary P concentration.

Total urine collections were not considered necessary due to the relatively small
amount of P excreted in urine. Estimated urine excretion was not affected by treatment.
However, urinary P% (0.0009, 0.0008, and 0.0017% for LP, MP, HP, respectively) was
linearly (P = 0.04) inﬂuenced by increased dietary P. Therefore, urinary P excretion

(0.1 1, 0.10, and 0.19 g/d) tended (P = 0.08) to increase with increasing dietary P.

75

Urinary P is generally regarded as minimal since urine is not a major route of excretion
and is usually assumed to be 2 to 5% of total P excretion (NRC, 2001). Total P excretion
[fecal P + urinary P (g/d)] increased linearly with increased dietary P concentration.
Urine excretion was estimated; therefore, estimated urine P may be inaccurate. However.
if the largest average urine excretion across Experiments 1 through 6 is doubled (22 kg/d
x 2 = 44; Table 11) and multiplied by the average urine P% (0.001%P) associated with
that treatment average, only 0.44 g/d would be excreted. Even if urine excretion were
under-estimated, the small amount of P in urine would not increase P excretion in urine
much.

Phosphorus balance for non-lactating animals equals P intake — total P excretion
(Table 5). Phosphorus balance decreased linearly with increased dietary P%.
Additionally, P balances were negative and not different from zero for LP and were
different from zero doe MP and HP. Salivary P can supply twice as much P to the
digestive tract than dietary P (NRC, 2001). Therefore, daily P excretion can be greater
than daily intake because P turnover rate can be decreased due to salivary recycling of P.

Dietary P concentration effects on fecal and urinary P excretion (% of P intake)
and fecal and urinary P excretion (% of total P excretion) are in Table 6. Total P
excretion (% of P intake) was linearly affected by treatment and greater than 100%.
Fecal P excretion (% of intake) increased linearly with dietary P concentration and again,
is probably due to salivary recycling of P absorbed in excess of P requirements. Urine P
excretion (% of P intake) decreased with increased dietary P (quadratic effect; P = 0.09)
and was a small percentage of P intake as expected. Fecal P excretion (% of total P

excretion) tended to increase (99.6, 99.8, and 99.8%, respectively; linear tendency; P =

76

0.09) with increased dietary P concentration. As expected, urine P excretion (% of total P
excretion) tended to decrease (0.40, 0.20, 0.18%, respectively; linear tendency; P = 0.09)
with increased dietary P% because feces tended to increase with increased dietary P.

This is contradictory to the NRC (2001) that suggested fecal P excretion is 95 to 98% and
urine is 2 to 5% of total P excretion.

Experiment 2. Performance descriptions for pregnant non-lactating heifers in
Experiment 2 are in Table 7. Body weight, BCS, DMI, and DMI (% of BW) were not
different among treatments. However, apparent DM digestibility tended to be affected
quadratically by dietary P concentration. Again, this was not expected and cannot be
explained. Dietary P concentration should not affect apparent DM digestibility in similar
diets unless the concentration was very low (NRC, 2001).

. Phosphorus balance and characteristics of P excretion are found in Table 8.
Dietary P concentrationlinearly affected P intake. Dietary P concentration did not affect
fecal DM% and excretion. Fecal P% increased with increased dietary P concentration;
therefore, fecal P excretion also increased with increased dietary P. Again this is
consistent with the results of Valk et al. (2002). Inevitable fecal P excretion (g/kg DMI)
was linearly decreased with increased dietary P.

Estimated urine excretion tended to be quadratically affected (P = 0.08) by
treatment. Urinary P% increased linearly (P = 0.03) with increasing dietary P.
Therefore, urinary P excretion (0.04, 0.10, and 0.14 g/d) increased linearly (P = 0.01)
with increasing dietary P%.

Phosphorus balance was —1.13, 1.38, and —7.66 g/d for LP, MP, and LP,

respectively and tended to be quadratically (P = 0.10) affected by treatment. Phosphorus

77

balance was not different from zero for LP and MP; P balance for HP was different than
zero indicating they were over-fed P.

Total P excretion (% of P intake), fecal P (% of P intake), and urine P (% of P
intake) were not different among treatments (Table 9). Fecal P excretion (% of total P
excretion) was about 99.7% among treatments and was not affected by dietary treatments.
Urinary P excretion (% of total P excretion) was about 0.35% and not affected by dietary
P concentration.

Experiment 3. Performance descriptions for pregnant non-lactating cows are
reported in Table 10. Body weight, BCS, and DMI (% of BW) were not different among
treatments. However, DMI was linearly different and decreased with increasing dietary
P. However, the DMI difference was more likely due to randomization of cows to
treatments because 30 d is a short period of time to cause a DMI different due to
treatment. Apparent DM digestibility was not different between treatments.

Phosphorus balance and characteristics of P excretion are found in Table 11.
Phosphorus intake was linearly inﬂuenced by dietary P. The HP treatment, despite the
lowest DMI, had the greatest P intake. Fecal DM% and excretion were not different
between treatments despite the difference in DMI mentioned previously. However, fecal
P% and excretion increased linearly with increased dietary P. Which again is similar to
results of Valk et al. (2002). Inevitable fecal P excretion (g/kg DMI) was linearly (P <
0.01) affected by increased dietary P.

Estimated urine excretion decreased linearly (P = 0.02) with increased dietary P
and cannot be explained by dietary P concentration. Urinary P% and excretion were not

affected by dietary treatments. Phosphorus balance was linearly affected by dietary P

78

concentration. Additionally, P balance was different from zero in HP, tended be different
for MP, and was not different for LP. The results from this experiment, like previously
mentioned experiments, indicate that the cows on all three treatments were overfed P
relative to their requirements because P balance was different from zero.

Total, fecal, and urine P excretion (% of P intake) were not different among
treatments. Fecal P and urine P excretion (% of total P excretion) also were not different
among treatments.

Experiment 4. Performance descriptions for pregnant non-lactating cows are
found in Table 13. Body weight tended to be quadratic (P = 0.10) that is, the cows on
MP tended to weigh more than the cows on the LP and HP treatments. This probably .
occurred during random assignment of cows to treatments. Body condition score, dry
matter intake, DMI (% of BW), and apparent DM digestibility did not differ between
treatments.

Results relative to P balance and excretion are in Table 14. Phosphorus intake
increased linearly with dietary P concentration. Fecal DM% and fecal DM excretion
were not different between treatments (P = 0.15 and 0.23, respectively). Fecal P% and
excretion increased linearly as dietary P concentration increased, and is similar the results
of Valk et al. (2002). Inevitable fecal P excretion (g/kg DMI) was linearly affected by
increased dietary P.

Estimated urine excretion decreased linearly as dietary P increased and cannot be
explained by dietary treatments. Dietary P concentration did not affect urine P%. And,
despite the linear decrease in urine excretion, urinary P excretion (0.08, 0.06, and 0.07

g/d) was not different among treatments. Phosphorus balance tended to decrease linearly

79

as dietary P was increased. Additionally, P balance was not different from zero for LP
and MP. P balance tended to be different from zero in HP (P = 0.07).

Phosphorus excretion results are reported in Table 15. Fecal P (% of P intake)
increased linearly with increased dietary P concentration and urine P (% of P intake)
decreased linearly with increased dietary P concentration. Total P excretion (% of P
intake) followed fecal P (% of P intake) and tended to increase linearly with dietary P
concentration. Fecal P excretion (% of total P excretion) tended to quadratically increase
with increased dietary P concentration; urinary P excretion (% of total P excretion)
tended to quadratically decrease with increased dietary P concentration.

Experiment 5. Performance descriptions for lactating cows (106 :1: 10.0 DIM) are
in Table 16. Body weight, BCS, DMI, DMI (% of BW), and apparent DM digestibility
were not different among treatments. Days in milk tended to be quadratic (P = 0.07)
between treatments. This occurred when cows were randomly assigned to treatments.
Despite the quadratic relationship of DIM, spread was not sufﬁcient for MY or energy
corrected milk (ECM) to be different among treatments. Milk fat concentration was
different between treatments (quartic effect; P = 0.05). This does not agree with other
studies that reported the effect of dietary P concentration on MF. Dietary P concentration
ranging from 0.24 to 0.67% P had no effect on milk fat percentage or yield (Wu et al.,
2002; Wu et al., 2001; Knowlton and Herbein, 2002; Wu et al., 2000; Valk and Sebek,
1999; Call et al., 1987; Carstairs et al., 1981; Steevens et al., 1971). Milk protein%
tended to be quadratically (P = 0.09) affected by dietary P concentration. Again, this is
different from previous research. When cows were fed dietary P concentrations ranging

from 0.24 to 0.67% P, MP was not affected (Wu et al., 2002; Knowlton and Herbein,

80

2002; Knowlton et al., 2001; Wu et al., 2000, Valk and Sebek, 1999; Brintrup et al.,
1993; Brodison et al., 1989).

Dietary P had a quartic (P = 0.03) effect on Milk lactose%. Again, this was
different than other studies. When dietary P ranging from 0.31 to 0.49% P was fed to
lactating cows, ML was not affected (Wu et al., 2002; Wu et al., 2001; Knowlton and
Herbein, 2002; Knowlton et al., 2001; Wu et al., 2000; Wu and Satter, 2000). Milk
solids-not-fat concentration was not affected by dietary treatments. This was similar to
other studies. Dietary P ranging from 0.31 to 0.60% P did not affect SNF% (Wu et al.,
2002; Wu et al., 2001; Knowlton and Herbein, 2002; Knowlton et al., 2001; Wu et al.,
2000; Wu and Satter, 2000; Steevens et al., 1971).

Results relative to P balance and excretion are reported in Table 17'. Phosphorus
intake increased with increased dietary P concentration. Fecal DM% and fecal DM
excretion were not different between treatments. Fecal P% and excretion increased with
increased dietary P concentration when cows were fed dietary P ranging from 0.24 to
0.67% P (Kohn et al., 2002; Knowlton and Herbein, 2002; Wu et al., 2002; 2001; 2000;
Valk et al., 2002; Brintrup et al, 1993; Morse et al., 1992b; Martz et al, 1990). The
results from this experiment support these observations. Fecal P concentration tended to
increase with increased dietary P concentration (quadratic effect; P = 0.09). This
difference led to a tendency for increase fecal P excretion (cubic effect; P = 0.09).
Inevitable fecal P excretion (g/kg DMI) tended to quadratically decrease with increased
dietary P concentration.

Estimated urine excretion was not different between treatments. A cubic

relationship tended to occur in urine P%. However, urine P excretion did not follow this

81

same trend and was not affected by dietary P concentration. This disagrees with previous
research that measured urine P excretion; as dietary P concentration increased (0.30 to
0.67%), urine P excretion also increased (Knowlton and Herbein, 2002; Morse et al.,
1992b). Total P excretion increases linearly as dietary P concentration was increased.
Which would be expected because the major component of total P excretion (fecal P
excretion) was linearly decreased with increased dietary P%.

Milk P concentration tended to be affected by dietary P concentration (quartic
effect; P = 0.10). This disagrees with other studies in which feeding dietary P from 0.24
to 0.67% did not affect milk P% (Knowlton and Herbein, 2002; Wu et al., 2000; Brintrup
et al., 1993; Brodison et al., 1989; Call et al., 1987; Forar et al., 1982). Milk P
concentration was found to range between 0.085 — 0.10% (Knowlton and Herbein, 2002;
Wu et al., 2000; Brintrup et al., 1993; Spiekers et al., 1993; Brodison et al., 1989; Flynn
and Power 1985). The quartic tendency may be partially explained by the quartic effects
reported for ME and MProt. Approximately 32% of P in milk is associated with milk fat
and protein (Walstra and Jenness, 1984). Therefore, increased milk fat or protein
concentrations potentially will increase milk P concentration. Treatments with higher
MF and MProt had higher milk P%, most notably T3 had 10% higher MF and 6% higher
milk P% (3.52 and 0.088%, respectively) than the average of the other four treatments
(3.16 and 0.083%, respectively). However, this slight difference in milk P% was not
enough to inﬂuence milk P yield (P =.0.30). Phosphorus balance (P balance (g/d) = P
intake (g/d) — [fecal P (g/d) + urinary P (g/d) + milk P (g/d)]) had a cubic relationship
with dietary P excretion. Phosphorus balance for T1 and 2 was not different from zero (1-

test; P = 0.20 and 0.38) and P balance for T3 through 5 was different from zero (P < 0.01

82

for T3 and P < 0.01 for T4 and 5). A P balance different from zero indicates that dietary
P was overfed.

Results relative to P excretion are presented in Table 18. Fecal P (% of P intake)
tended to decrease with increased dietary P. This means that fecal P decreased with
increased dietary P%. However, urine P (% of P intake) tended to increase cubically with
P intake. Since fecal P (% of P intake) decreased and urine P (% of intake) tended to
increase, some of the excess P was shifted to urine. As a result total P excretion (% of
intake) increased linearly with dietary P concentration. Dietary P concentration tended to
have a cubic affect on fecal and urine P excretion (% of total P excretion). Overall, these
values were within the ranges assumed by the NRC (2001). Fecal and urine P excretion
(% of total P excretion) averaged 96.7 and 3.3%, respectively. Milk P (% of P intake)
decreased linearly with increased dietary P concentration. This would be expected since
milk composition is relatively constant. Milk P % tended to increase (quartic effect; P =
0.10), but milk P secretion was not different among treatments (Table 17). Milk P (% of
total P output) tended to decrease with dietary P concentration (quartic effect; P = 0.09).

Experiment 6. Performance descriptions for lactating cows (183 i 6.2 DIM) are
in Table 19. Body weight, DIM. DMI. and DMI (% of BW) were not different among
treatments. Body condition score had a quartic (0.0248) relationship between treatments.
This was unexpected and occurred when cows were randomly assigned to treatments.
Apparent DM digestibility was different between treatments (quadratic effect; P = 0.04).
This was not expected because all cows were fed the same diet except for P
concentration; therefore, there should be no digestibility difference unless the dietary P

concentration was low (NRC. 2001).

83

Milk yield tended to be different and ECM had a cubic relationship with dietary P
concentration (quartic tendency and cubic effect; P = 0.08 and P =0.02, respectively)
among treatments. This MY and ECM difference may be partially explained by DMI.
Although DMI was not signiﬁcant, T2 cows had numerically less DMI than the other
treatments and subsequently less MY. Dry matter intake is directly related to MY (NRC,
2001). Milk fat concentration tended to be affected by dietary treatment (quadratic
effect; P = 0.07). This disagrees with other studies that reported MF as previously
mentioned (Wu et al., 2002; Wu et al., 2001; Knowlton and Herbein, 2002; Wu et al.,
2000; Valk and Sebek, 1999; Call et al., 1987; Carstairs et al., 1981; Steevens et al.,
1971). Milk protein,vlactose, and solids-not-fat concentrations were not affected by
dietary P concentration (P = 0.94, 0.57, and 0.87, respectively). This agrees with other
research mentioned previously where dietary P concentration did not inﬂuence these milk
components (Wu et al., 2002; Knowlton and Herbein, 2002; Knowlton et al., 2001; Wu et
al., 2000, Valk and Sebek, 1999; Brintrup et al., 1993; Brodison et al., 1989; Steevens et
aL,1971)

Results relative to P balance and excretion are in Table 20. Fecal DM
concentration was different between treatments (quartic effect; P < 0.01). This was
unexpected and cannot be explained by dietary concentration. However, this did not
translate to a difference in fecal DM concentration, which was not different between
treatments (P = 0.98). Phosphorus intake (56.0, 64.0, 80.1, 90.3, and 102.1 g/d,
respectively for T1, 2, 3, 4, and 5) was linearly (P < 0.01) affected by dietary P
concentration. Fecal P concentration and excretion increased with increased dietary P

concentration (quadratic effect; P = 0.04; and, linear effect; P < 0.01). Again, this is

84

similar to the results reported previously (Wu et al., 2002; 2000, Knowlton and Herbein.
2002; Valk et al., 2002).

Estimated urine excretion increased quadratically between treatments. Dietary P
concentration tended to increase urine P concentration linearly and did increase urine P
excretion linearly. Which is supported by results reported by Knowlton and Herbein
(2002) and Morse et al. (1992b) per previously mentioned.

Milk P concentration was not affected by dietary P concentration (P = 0.61). This
agrees with the other studies that reported no affect on milk P concentration mentioned
previously (Knowlton and Herbein, 2002; Wu et al., 2000; Brintrup et al., 1993; Brodison
et al., 1989; Call et al., 1987; Forar et al., 1982). Dietary P concentration tended to affect
milk P yield (cubic effect; P = 0.07). This may be partially explained by the quartic
difference between MYs. The treatment with the lowest MY (T2) had the lowest milk P
yield. Additionally, because T5 had numerically the lowest milk P concentration, less
milk P was secreted in milk.

Phosphorus balance decreased with increased dietary P (cubic affect; P = 0.04).
Additionally. P balance was not different from zero for T1, T2, and T3, but was different
from zero in T4 and T5. Again, P balance different from zero indicates that P was .
overfed relative to P requirements.

Results relative to P excretion are presented in Table 21. Fecal P (% of P intake)
increased linearly with increased dietary P concentration, and urine P (% of P intake)
tended to increase linearly with increased dietary P. Meaning that more of the intake P
was being excreted rather than utilized. Milk P (% of intake) and increased linearly with

increased P intake. Again, this would be expected because milk composition is relatively

85

constant. Milk P (% of total P output) tended to decrease quadratically. However, this
may be partially due to a tendency for decreased milk yield with higher dietary P
concentration. Dietary P concentration did not affect fecal or urine P excretion (% of
total P excretion) and was not in the range (average of 99.1 and 0.9%) of what the NRC
(2001) assumed fecal and urine P excretion (% of total P excretion) to be (2 to 5%).
Experiment 7. Due to the method of intake restriction mentioned previously,
treatment effects are confounded with DMI. Intake was restricted based on milk
production and cows with MY greater than 25 kg/d were not intake restricted. Therefore,
cows consuming ad libitum DMI were not evenly distributed across treatments. Eleven
of 34 cows were allowed to consume ad libitum intake, three in T1, ﬁve in T2, two in T3,
one in T4, and zero in T5. Therefore, ANOVA of dependent variables were not
conducted on this dataset. However, these data were very valuable for evaluation of

models to predict P excretion.

CONCLUSIONS

Throughout this series of experiments there are several dependent variables .that
were affected by dietary P concentration. In general, DMI and MY were not affected by
dietary P excretion. This is supported by other studies. Seven studies reported that
feeding lactating cows diets containing 0.3010 0.60% P had no inﬂuence on MY (cows
producing 25 to 40 kg milk/d; Wu et al., 2002; 2001; 2000; Kohn et al., 2002; Wu and
Satter, 2000; Brintrup et al., 1993; F orar et al., 1982). However, the duration for the

lactating experiments was only 30 d which probably was not long enough to pick up a

86

difference in DMI or MY. This was not a problem because the priority objective was to
collect P excretion data. Phosphorus excretion differences will occur more quickly
because DM excretion is correlated with DM1 and rumen turnover is approximately 48 h.

All experiments showed increased fecal P concentration and excretion when
dietary P concentration increased. This is supported by several studies where dietary P
concentration was increased from 0.30 to 0.67% (Kohn et al., 2002; Knowlton and
Herbein, 2002; Wu et al., 2002; 2001; 2000; Valk et al., 2002 Brintrup et al, 1993; Morse
et al., 1992b; Martz et al, 1990). In general, increased dietary P also increased urinary P
concentration and excretion. Similarly, reports from studies that reported urine P
excretion showed that as dietary P concentration increased (0.30 to 0.67%), urine P
excretion also increased (Knowlton and Herbein, 2002; Morse et al., 1992b). However,
Wu et a1. (2000) found that there was no difference in urine P excretion when cows were
fed diets with 0.31, 0.41, and 0.49% P. Urinary P excretion was small, which is typical
for ruminants. Urine P excretion in the current experiments generally represented less
than the 2 to 5% of total P excretion generally assumed when urine P excretion is
estimated (NRC, 2001).

Phosphorus balance is an important indicator of P status. For Experiments 1
through 4, P balance was negative and different from zero when animals consumed the
HP treatment diet. For Experiments 5 and 6, treatments 3, 4, and 5 had negative P
balances that were different from zero. Furthermore, Wu et al. (2000) suggested that

apparent P digestibility S 40% for lactating cows was indicative of overfeeding P. In

experiments 5 and 6, apparent P absorption was S 40% in all treatments except Tl

[0.27% P (DM basis)].

87

Data generated from these seven experiments construct an invaluable dataset that

will be used in subsequent research to evaluate models used to predict P excretion.

88

Table l. Formulated ingredient and analyzed chemical composition of
the basal diet for Experiments 1, 2, 3, and 4.

 

 

 

 

Treatmentl
LP MP HP
Item Percent of dietary DM
Ingredients
Alfalfa silage 17.5 17.5 17.5
Corn silage 27.0 27.0 27.0
Beet pulp pellets, dehydrated
without molasses 27 .0 27.0 27.0
Corn starch 9.1 9.1 9.1
Corn, ground 9.0 9.0 9.0
Mineral-vitamin mix2 3.3 3.3 3.3
SoyChlor16-7TM3 2.1 2.1 2.1
Rice hulls 1.8 1.3 0.9
Blood meal 1.1 1.1 1.1
BiuretTM “ 0.9 0.9 0.9
Urea 0.8 0.7 0.5
Ammonium chloride 0.4 0.4 0.4
Monoammonium phosphate 0 0.7 1.2
Chemical composition5
DM 58.1 58.1 58.1
CP 14.6 14.1 14.7
N13,“, Meal/kg 0.72 0.72 0.72
ADP 25.8 25.6 25.3
NDF 39.6 39.4 39.2
Ash 5.0 5.2 5.4
Ca 0.62 0.61 0.61
P 0.19 0.28 0.35
Mg 0.33 0.33 0.34
K 1.1 1.1 1.1
Na 0.1 0.1 0.10
CI 0.1 0.1 0.10
S 0.24 0.24 0.25
Cu, mg/kg 22.4 21.9 21.6
Fe, mg/kg 299 328 355
Mn, mg/kg 85.3 82.6 79.7

Zn, mglkg 44.68 41.6 42.0

 

Treatments: LP = 0.19, MP = 0.28, and HP = 0.35% dietary P.
2Composition (DM basis): 91.4% soybean hulls; 8% magnesium sulfate;
0.14% manganese sulfate; 0.11% zinc sulfate ; 0.06% copper sulfate;
0.06% Se; 0.001% cobalt sulfate; 0.001% ethylenediaminodihydroiodide
(EDDI) 488 KIU vitamin A/kg; 71 KIU vitamin D/kg; and, 2.5 KIU vitamin
E/kg.
3Graciously provided by West Central® Soy, Ralston, 1A.
4Graciously provided by Moorman's Manufactoring Compancy, Quincy, IL.
5Chemical composition was determined by Dairy One Forage Lab (Ithaca, NY)
except for P and ash.
6Energy value estimated based on computations per NRC (2001).

89

Table 2. Formulated ingredient and analyzed chemical composition of the basal diet for
Experiments 5. 6, and 7.

 

 

 

 

Treatmentl
T1 T2 T3 T4 T5

Item Percent of dietary DM

Ingredient
Alfalfa silage 11.4 1 1.4 11.4 11.4 1 1.4
Corn silage 19.3 19.3 19.3 19.3 19.3
Beet pulp pellets, dehydrated 25.0 25.0 25.0 25.0 25.0

without molasses

Whole cottonseed 11.0 1 1.0 11.0 1 1.0 1 1.0
Corn starch 10.9 10.6 10.3 10.0 9.7
Corn, dry ground 13.4 13.4 13.4 13.4 13.4
Soybean meal, 44% CP 3.9 3.9 3.9 3.9 3.9
Blood meal 1.3 1.3 1.3 1.3 1.3
Corn oil 0.1 0.1 0.1 0.1 0.1
Limestone 0.8 0.8 0.8 0.8 0.8
Salt (NaCl) 0.4 0.4 0.4 0.4 0.4
Urea 0.7 0.6 0.5 0.4 0.3
Monoammonium phosphate 0.0 0.4 0.8 1.2 1.6
BiutetTM 2 0.8 0.8 0.8 0.8 0.8
Vitamin and mineral premix3 1.0 1.0 1.0 1.0 1.0

Chemical composition‘
DM 59.7 59.7 59.8 59.8 59.8
CP 20.8 20.4 20.2 20.2 20.2
NEL’, Meal/kg 0.76 0.76 0.76 0.76 0.76
ADF 22.1 22.1 21.9 21.9 21.9
NDF 37.3 37.5 37.2 37.0 37.2
Ash 4.] 4.4 4.8 5.0 5.3
Ca 0.94 0.94 0.94 0.95 0.95
P 0.27 0.36 0.42 0.46 0.52
Mg 0.23 0.24 0.24 0.24 0.25
K 1.1 1.1 1.1 1.1 1.1
Na 0.39 0.39 0.39 0.39 0.40
Cl 0.36 0.36 0.36 0.36 0.36
S 0.21 0.21 0.22 0.22 0.23
Cu, mg/kg 19 18 18 18 19
Fe. mg/kg 325 364 404 446 534
Mn, mg/kg 80 82 83 84 87
Zn, mg/kg 82 82 82 82 83

 

’Treatments: T1 = 0.27, T2 = 0.36, T3 = 0.42, T4 = 0.46, and T5 = 0.52.

2Graciously provided by Moonnan's Manufacturing Company, Quincy IL

3Trace mineral and vitamin premix was 71.4% dry ground corn, 25.5% vitamins and

minerals, and 3.1% corn oil. The concentrations of the vitamins and minerals were: 0.05% Ca;
0.28% P; 0.60% Mg; 3.3% K; 1.4% S; 0.01% Na; 332 mg/kg Cu; 699 mg/kg Mn; 894

mg/kg Zn; 90 mg/kg Fe; 4 mg/kg Co; 7 mg/kg Se; 8 mg/kg 1; 24 KIU vitamin A/kg; 5 KIU
vitamin D/kg; and, 0.09 KIU vitamin E/kg, dry basis.

4Chemical composition was determined by Dairy One Forage Lab (Ithaca, NY) except for P and
ash.

5’Energy values estimated based on computation per NRC (2001).

90

Table 3. Description of all variables used to evaluate experimental objectives.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Abbreviation,
Variable units Comments Calculated or Measured (M)
Animal descriptor, used in
calculating P excretion from
Body weight BW.‘kg rediction models
Body condition
score BCS, 1 to 5 scale descriptor
Dry matter descriptor and treatment for
intake DMI. kg/d Maintenance Experiment
Dry matter
intake as %
percent of DMI, %BW = (DMI, kg/d + BW,
body weight DMI, %BW descriptor kg) x 100
Days in milk DIM descriptor M
AppDMdig, % = ((DMI, kg/d -
Apparent DM (FecDM, g/d + 1000))/DM1, kg/d)
digestibility % AppDMdi&% Diet evaluation x 100
descriptor, used to calculate milk P
concentration and used in
calculating P excretion in prediction
Milk yield MY, kg/d models
Milk fat’, % MF. % descriptor M
Milk protein’,
% MProt, % descriptor M
Milk lactose’,
% ML, % descriptor M
Milk solids-
not-fat', % SNF, % descriptor M
Somatic cell SCC, 1000
countl cells/ml descriptor M
Treatment for most experiments and
Dietary P, % of used in calculating P excretion from
dietary DM DietP, % rediction models M
Used in calculating P excretion P1, g/d = DMI, kg/d x (DietP,% +
P intake PI, g/d from prediction models 100)
FecDM, g/d = (Cr fed, g/d -i- Cr in
Fecal DM Used to calculate daily fecal P fecal sample, %) x 100 [Harris,
excretion FecDM, g/d excretion 1970]
Used to calculate daily fecal P
Fecal P. % FecP, % excretion M
Fecal P FecPE, g/d = FecDM, g/d x (FecP,
excretion FecPE. g/d Used to calculate daily P balance % + 100)
UEZ, kg/d = 332.91 + 0.2885 x
DMI - 0.2175 x MY 0083 x
dietary DM % + 0.524 x dietary
CP, % DM - 326.5 x SG + 0.2375
Urine x AppAbsP [Holter and Urban,
excretion UE, kg/d Used to calculate daily P excretion 1992]
Urine P, % UP. % Used to calculate daily P excretion M
UP. g/d = (UE, kg/d x (UP. % +
Urine P yield UPY. g/d Used to calculate daily P balance 100)) x 1000

 

91

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Milk P’, % MP, % Used to calculate daily milk P yield M
MPY, g/d = MY, kg/d x (MP, % +
Milk P yield MPY, yd Used to calculate dailﬂ balance 100) x 1000
3Pbal= P1, g/d (MPY, g/d +
P balance PbaL,g/d Evaluation of P status F ecPE g/d + UPY, ad
Fecal P
excretion, g/kg PMR, g P/kg Estimate of the truly absorbed P PMR, g P/kg DMI = P1, g/d +
DMI DMI maintenance requirement DMI, kgi
Total P
excretion PE, g/d Estimate of total daily P excretion PE, g/d = FecPE, g/d + UPY, g/d
Fecal P, % of
total P
excretion FecPexc, % P utilization estimate (FecP, g/d -:— PE. g/d) x 100
Urine P, % of
total P
excretion UrPexc, % P utilization estimate (UrPﬂd + PE, g/d_)x 100
Used to calculate daily fecal DM
Fecal Cr F ecCr, % excretion and AppDMdig% M
Fecal DM, % FecDM, % Used to calculate fecal water M
FecW, kg/d = (FecDM, g/d +
Fecal water FecW, kg/d Used to calculate AppAbsW (FecDM, % + 100)) - FecDM
Feed water F WI, g/d = As Fed Feed Intake,
intake FWI, kg/d Used to calculate total water intake kw - DMI, kg/d
“’DWI, kg/d = -32.39 + 2.47 x
DMI, kg/d + 0.6007 x MY, kg/d +
0.6205 x Dietary DM, % + 0.0911
Drinking water Used to calculate Total Water x Julian Day - 0.000257 x (’Julian
intake DWI, kg/d Intake Day)2 [Holter and Urban, 1992]
Total water Total amount of water consumed TWI, kg/d = F WI, kg/d + DWI,
intake TWI. kg/d er day kg/d
Apparently Used to calculate urine excretion, AppAbsW, kg/d = TWI, kg/d -
absorbed water AppAbsW, kgld 1kg/d FecW, kg/d
Urine speciﬁc Used to calculate urine excretion,
gavity SG, mg/ml [kg/d M

 

' Component weighted averages calculated as: (a. m. component, % x a. m. MY, kg + p. m. component, %

xpm. MY, kg)-:- (a.m. MYxp.m. MY, kg).
For dry cows, the following equation was used: UE kg/d= 212 1 + 0.8822 x DMI, kg- 0. 03452 x

dietary DM, % + 1 .001 x dietary CP, % DM— 216.4 * SG,mg/m1 + 0.1414 x AppAbsP, g/d [Holter and

Urban. 1992].

3 For dry cows. the following equation was used: Pbal, g/d = P1, g/d — (FecPE, g/d + UPY, g/d).
For dry cows, the following equation was used: DWI, kg/d —— -10. 34 + 0. 2296 x Dietary DM, % + 2.212
x DMI, kg/d + 0.03944 x (Dietary CP, % DM)2 [Holter and Urban, 1992].
5Julian Day 15 the day of the year when January 1 = Julian day l and December 31 = Julian day 365.

92

 

Table 4. Performance characteristics of nulliparous heifers (57 to 28 i 1.0 d prior to
parturition) as inﬂuenced by experimental treatments (Experiment 1).1

 

 

 

 

Treatment2 P-value3

Item LP MP HP SEM Treatment Linear Quadratic
n 7 7 7 -- -- -- --
BW, kg 693 700 669 18.5 NS" NS NS
BCS 3.6 3.7 3.7 0.09 NS NS NS
DMI, kg/d 12.3 12.9 12.7 0.78 NS NS NS
DMI, %BW 1.8 1.9 1.9 0.13 NS NS NS
Apparent DM

digestibility, % 70.6 66.1 61.9 2.60 0.08 0.03 NS

 

1Values are least squares means.

2Treatments: LP = 0.19%; MP = 0.28%; and, HP = 0.35% dietary P, dry basis.
3Signiﬁcant effects (P < 0.05) and tendencies (P < 0.10).

‘Ns = Not signiﬁcant (P > 0.10).

93

Table 5. Phosphorus balance and characteristics of feces and urine for nulliparous heifers (57 to 28 :

 

 

 

 

1.0 d
prior to parturition) as inﬂuenced by exLerimental treatments (Experiment 1).1

Treatment2 P-value’
Item LP MP HP SEM Treatment Linear Quadratic
P intake, g/d 23.3 36.1 44.5 2.41 <0.01 <0.01 NS"
Fecal DM, % 15.8 15.6 15.7 0.44 NS‘ NS NS
Fecal DM excretion, g/d 3551 4373 4823 397.4 0.10 0.03 NS
Fecal P, % of DM 0.97 1.42 2.21 0.117 <0.01 <0.01 0.10
Fecal P excretion, g/d 34.1 63.5 107.5 9.56 <0.01 <0.01 NS
g fecal P/kg DMI 2.9 4.8 8.4 0.59 <0.01 <0.01 NS
Urine excretions, kg/d 12.3 12.9 11.6 1.02 NS NS NS
Urine P, % 0.001 0.001 0.002 0.0003 0.04 0.04 NS
Urinary P excretion, g/d 0.1 1 0.10 0.19 0.030 0.09 0.08 NS
Total P excretions, g/d 34.2 63.6 107.7 9.57 <0.01 <0.01 NS
P balance’, g/d 40.91" -2750" -63.39 8.17 <0.01 <0.01 NS

 

1Values are least squares means.

2Treatments: LP = 0.19%; MP = 0.28%: and, HP = 0.35% dietary P, dry basis.
3Signiﬁcant effects (P < 0.05) and tendencies (P < 0.10).

4NS = Not signiﬁcant (P > 0.10).

5Estimated from urine excretion prediction model (Holter and Urban, 1992).

6Total P excretion (g/d) = fecal P + urinary P (g/d) excretion.
7P balance (g/d) = P intake (g/d) - total P excretion (g/d).

“Mean is not different from zero (P > 0.10).

9Mean is different from zero (P < 0.05).

94

Table 6. Phosphorus excretion by nulliparous heifers (57 to 28 i 1.0 11 prior to parturition) as
inﬂuenced by experimental treatments (Experiment 1).’

 

 

 

 

Treatmentz P-value3
Item LP MP HP SEM Treatment Linear Quadratic
Total P excretion, % of P intake 150.8 172.2 241.4 21.60 0.02 <0.01 NS
Fecal P, % of P intake 150.3 172.0 241.0 21.62 0.02 <0.01 NS
Fecal P, % of total P excretion 99.6 99.8 99.8 0.09 NS 0.09 NS
Urine P, % of P intake 0.46 0.29 0.43 0.074 NS 0.09 NS
Urine P, % of total P excretion 0.40 0.20 0.18 0.090 NS 0.09 NS

 

1Values are least squares means.

zTreatrnents: LP = 0.19%; MP = 0.28%; and, HP = 0.35% dietary P, dry basis.
1’Signiﬁcant effects (P < 0.05) and tendencies (P < 0.10).

‘NS = Not significant (P > 0.10).

95

Table 7. Performance characteristics of nulliparous heifers (38 to 9 i 1.0 d prior to
parturition) as inﬂuenced by experimental treatments (Experiment 2).1

 

 

 

 

Treatment2 P-value3
Item LP MP HP SEM Treatment Linear Quadratic
n 7 7 7 - -- - -
BW, kg 715 723 692 17.2 Ns‘ NS NS
BCS 3.7 3.8 3.8 0.10 NS NS NS
DMI, kg/d 8.0 10.0 9.2 0.94 NS NS NS
DMI, %BW 1.1 1.4 1.3 0.14 NS NS NS

Apparent DM digestibility, % 53.3 65.0 56.2 4.18 NS NS 0.07
lValues are least squares means.
2Treatments: LP = 0.19%; MP = 0.28%: and, HP = 0.35% dietary P, dry basis.
3Signiﬁcant effects (P < 0.05) and tendencies (P < 0.10).
‘NS = Not signiﬁcant (P > 0.10).

96

Table 8. Phosphorus balance and characteristics of feces and urine for nulliparous heifers
(38 to 9 i 1.0 d prior to parturition) as inﬂuenced by experimental treatments (Experiment 2).l

 

 

 

 

Treatment2 P-value3
Item LP MP HP SEM Treatment Linear Quadratic
P intake, g/d 15.3 27.9 32.1 2.76 <0.01 <0.01 NS
Fecal DM, % 16.5 16.9 16.3 0.52 NS‘ NS NS
Fecal DM excretion, g/d 3718 3357 3931 395.9 NS NS NS
Fecal P, % of DM 0.44 0.81 1.03 0.04 <0.01 <0.01 NS
Fecal P excretion, g/d 16.4 26.5 39.6 2.64 <0.01 <0.01 NS
g fecal P/kg DMI 2.1 2.8 4.4 0.31 <0.01 <0.01 NS
Urine excretions, kg/d 8.1 1 1 9.2 0.97 NS NS 0.08
Urine P, % 0.0006 0.0009 0.0015 0.0003 0.08 0.03 NS
Urinary P excretion, g/d 0.04 0.10 0.14 0.94 0.04 0.01 NS
Total P excretion“, g/d 16.4 26.6 39.8 2.64 <0.01 <0.01 NS
P balance7. g/d -1.1‘ 1.58 -7.59 2.88 0.10 NS 0.10

 

1Values are least squares means.

2Treatments: LP = 0.19%; MP = 0.28%; and, HP = 0.35% dietary P, dry basis.

3 Signiﬁcant effects (P < 0.05) and tendencies (P < 0.10).
‘NS = Not signiﬁcant (P > 0.10).

5Estimated from urine excretion prediction model (I-Iolter and Urban, 1992).

“Total P excretion (g/d) = fecal P + urinary P (g/d) excretion.

7P balance (g/d) = P intake (g/d) - total P excretion (g/d).
I’Mean is not different from zero (P > 0.10).
9Mean is different from zero (P < 0.05).

97

 

 

Table 9. Phosphorus excretion by nulliparous heifers (38 to 9 i 1.0 d prior to parturition) as
inﬂuenced by experimental treatments (Experiment 2).1

 

 

 

 

Treatment2 P-value’
Item LP MP HP SEM Treatment Linear Quadratic
Total P excretion, % of P intake 108.7 100.9 127.2 10.50 NS NS NS
Fecal P, % ofP intake 108.4 100.5 126.7 10.47 NS NS NS
Fecal P, % of total P excretion 99.7 99.6 99.7 0.10 NS NS NS
Urine P, % of P intake 0.28 0.40 0.46 0.12 NS NS NS
Urine P, % of total P excretion 0.27 0.44 0.34 0.10 NS NS NS

 

’Values are least squares means.

2Treatments: LP = 0.19%; MP = 0.28%; and, HP = 0.35% dietary P, dry basis.
3Signiﬁcant effects (P < 0.05) and tendencies (P < 0.10).

4NS = Not signiﬁcant (P > 0.10).

98

Table 10. Performance characteristics of non-lactating multiparous cows (59 to 30 i 1.0 d
prior to parturition) as influenced by experimental treatments (Experiment 3).1

 

 

 

 

Treatmentz P-value3

Item LP MP HP SEM Treatment Linear Quadratic
n 6 7 7 -- -- -- --
BW, kg 784 800 748 27.2 NS4 NS NS
BCS 3.8 3.9 3.7 0.11 NS NS NS
DMI, kg/d 20.4 19.6 17.0 1.06 0.09 0.04 NS
DMI, %BW 2.6 2.4 2.3 0.16 NS NS NS
Apparent DM

digestibility, % 66.8 69.8 65.6 1.82 NS NS NS

 

1Values are least squares means.

2Treatments: LP = 0.19%; MP = 0.28%; and, HP = 0.35% dietary P, dry basis.
3Signiﬁcant effects (P < 0.05) and tendencies (P < 0.10).

‘NS = Not signiﬁcant (P > 0.10).

99

Table 11. Phosphorus balance and characteristics of feces and urine for non-lactating multiparous (59 to
30 i 1.0 d prior to parturition) as inﬂuenced bﬂxperimental treatments (Experiment 3).'

 

 

 

 

Treatment2 P-value3
Item LP MP HP SEM Treatment Linear Quadratic
P intake, g/d 38.8 54.8 59.4 3.16 <0.01 <0.01 NS
Fecal DM, % 15.2 16.3 15.5 0.37 Ns‘ NS 0.07
Fecal DM excretion, g/d 6730 5868 5899 473.6 NS NS NS
Fecal P, % of DM 0.77 1.18 1.65 0.13 <0.01 <0.01 NS
Fecal P excretion, g/d 50.3 70.9 98.3 10.87 0.02 <0.01 NS
g fecal P/kg DMI 1.61 3.09 4.36 0.87 NS NS NS
Urine excretions, kg/d 22.0 20.6 17.2 1.24 0.04 0.02 NS
Urine P, % 0.001 0.005 0.001 0.003 NS NS NS
Urinary P excretion, g/d 0.17 1.29 0.14 0.30 NS NS NS
Total P excretion‘, g/d 50.52 72.13 98.40 1 1.07 0.03 <0.01 NS
P balance7, g/d -1 1.78 -173" -39.0"’ 8.54 0.03 0.05 NS

 

1Values are least squares means.

2Treatments: LP = 0.19%; MP = 0.28%; and, HP = 0.35% dietary P, dry basis.
3Significant effects (P < 0.05) and tendencies (P < 0.10).

‘Ns = Not signiﬁcant (P > 0.10).

sEstimated from urine excretion prediction model (Holter and Urban, 1992).
(”Total P excretion (g/d) = fecal P + urinary P (g/d) excretion.

7P balance (g/d) = P intake (yd) - total P excretion (g/d).

8Mean is not different from zero (P > 0.10).

9Mean is different from zero (P < 0.05).

100

Table 12. Phosphorus excretion by non-lactating multiparous cows (59 to 30 i 1.0 d prior to
parturition) as inﬂuenced by experimental treatments (Experiment 3).1

 

 

 

 

Treatment2 P-value3
Item LP MP HP SEM Treatment Linear Quadratic
Total P excretion, % ofP intake 130.8 127.5 162.6 13.63 NS NS NS
Fecal P, % ofP intake 130.4 125.5 162.4 12.84 NS NS NS
Fecal P, % of total P excretion 99.6 99.0 99.9 0.50 NS NS NS
Urine P, % of P intake 0.44 1.97 0.23 1.07 NS NS NS
Urine P, % oftotal P excretion 0.36 1.03 0.15 0.50 NS NS NS

 

1Values are least squares means.

2Treatments: LP = 0.19%; MP = 0.28%; and, HP = 0.35% dietary P, dry basis.
3Signiﬁcant effects (P < 0.05) and tendencies (P < 0.10).

4NS = Not significant (P > 0.10).

101

Table 13. Performance characteristics of non-lactating multiparous cows (40 to 11 i 1.0 d
prior to parturition) as inﬂuenced by experimental treatments (Experiment 4).I

 

 

 

 

Treatment2 P-value3
Item LP MP HP SEM Treatment Linear Quadratic
n 6 7 7 - -- - --
BW, kg 817 855 762 30.1 NS‘ NS 0.10
BCS 3.9 4.0 3.7 0.13 NS NS NS
DMI, kg/d 15.8 15.7 13.1 1.24 NS NS NS
DMI, %BW 1.9 1.8 1.7 0.17 NS NS NS
Apparent DM
digestibility, % 62.6 60.1 60.1 2.91 NS NS NS

 

1Values are least squares means.

2Treatments: LP = 0.19%; MP = 0.28%; and, HP = 0.35% dietary P, dry basis.
3Significant effects (P < 0.05) and tendencies (P < 0.10).

‘Ns = Not signiﬁcant (P > 0.10).

102

Table 14. Phosphorus balance and characteristics of feces and urine for non-lactating multiparous cows
(40 to 11 i 1.0 d prior to estimated parturition) as inﬂuence by experimental treatments (Experiment 4).1

 

 

 

 

Treatment2 P-value3
Item LP MP HP SEM Treatment Linear Quadratic
P intake, g/d 30.0 44.0 46.0 3.83 0.02 <0.01 NS
Fecal DM, % 15.4 16.2 16.1 0.29 NS4 NS NS
Fecal DM excretion, g/d 5947 6062 5086 428.0 NS NS NS
Fecal P, % of DM 0.43 0.76 1.08 0.04 <0.01 <0.01 NS
Fecal P excretion, g/d 25.62 46.31 54.04 3.72 <0.012 <0.01 NS
Urine excretions, kg/d 17.0 16.4 13.3 1.19 0.08 0.05 NS
Urine P, % 0.0008 0.0004 0.0005 0.0001 NS NS NS
Urinary P excretion, g/d 0.08 0.06 0.07 0.02 NS NS NS
Total P excretion“, g/d 26.65 46.37 54.11 3.94 <0.01 <0.01 NS
g fecal P/kg DMI 1.60 3.10 4.40 0.39 <0.01 <0.01 NS
P ba1ance7,g/d 4.4“ -24“ .828 2.52 NS 0.08 NS

 

1Values are least squares means.

2Treatments: LP = 0.19%; MP = 0.28%; and, HP = 0.35% dietary P, dry basis.
3Signiﬁcant effects (P < 0.05) and tendencies (P < 0.10).

‘NS = Not signiﬁcant (P > 0.10).

sEstimated from urine excretion prediction model (Holter and Urban, 1992).
6Total P excretion (g/d) = fecal P + urinary P (g/d) excretion.

7P balance, g/d = P intake (g/d) - total P excretion (g/d).

8Mean is not different from zero (P > 0.10).

103

Table 15. Phosphorus excretion by non-lactating multiparous cows (40 to 11 i 1.0 d prior to
parturition) as inﬂuenced by egerimental treatments (Experiment 4).1

 

 

 

 

Treatment2 P-value3
Item T1 T2 T3 SEM Treatment Linear Quadratic
Apparently absorbed P, g/d 4.38 -2.31 -8.09 2.915 Ns‘ 0.08 NS
Apparent P digestibility, % 14.7 -10.3 -24.8 13.20 NS 0.06 NS
P retained/P intake, g/d 0.2 -1.0 -0.2 0.130 NS 0.06 NS
P retained/apparently
absorbed P, g/d 1.0 1.0 1.0 0.03 NS NS NS
Total P excretion, % of P
intake 85.3 110.3 124.8 13.20 NS 0.06 NS
Fecal P, % ofP intake 84.7 110.2 124.6 12.38 NS 0.04 NS
Fecal P, % of total P
excretion 99.7 99.9 99.8 0.03 <0.01 <0.01 0.08
Urine P, % of P intake 0.3 0.2 0.2 0.03 0.05 0.03 NS
Urine P, % of total P
excretion 0.3 0.1 0.1 0.03 <0.01 <0.01 0.08

 

1Values are least squares means.

2Treatments: LP = 0.19%; MP = 0.28%; and, HP = 0.35% dietary P, dry basis.
3 Signiﬁcant effects (P < 0.05) and tendencies (P < 0.10).

‘Ns = Not signiﬁcant (P > 0.10).

104

Table 16. Performance characteristics of early lactation cows (106 i 10.0 DIM) as inﬂuenced by
experimental treatments (Experiment 5).1

 

 

 

Treatment2 P-value3
Item T1 T2 T3 T4 T5 SEM Treatment Linear Quadratic Cubic Quartic
n 7 7 7 7 7 -- -- - -- -- --
BW,kg 598 578 617 610 592 23.1 NS4 NS NS NS NS
BCS 2.7 2.7 2.9 2.6 2.7 0.19 NS NS NS NS NS
DIM 119 119 82 99 115 10.0 0.05 NS 0.073 0.09 NS
DMl,kg/d 18.9 19.3 19.3 20.6 19.0 0.85 NS NS NS NS NS
DMI,%BW 3.2 3.5 3.2 3.5 3.3 0.14 NS NS NS NS 0.08
Apparent DM
digestibility,% 67.0 69.6 68.3 68 68.2 1.77 NS ' NS NS NS NS
MY, kg/d 35.0 37.0 38.1 38.3 33.6 2.66 NS NS NS NS NS
ECM5,kg/d 32.0 33.7 37 34.9 31.5 2.60 NS NS NS NS NS
Milk fat,% 3.16 3.10 3.52 3.13 3.26 0.15 NS NS NS NS 0.05
Milk protein (true),
% 2.69 2.62 2.67 2.64 2.76 0.05 NS NS 0.10 NS NS
Milk lactose, % 4.83 4.7 4.83 4.68 4.74 0.05 NS NS NS NS 0.03 -
SNF,% 9.96 8.17 8.37 8.16 8.38 0.71 NS NS NS NS NS

SCC,IOOO cells/ml 73 134 127 81 155 51.9 NS NS NS NS NS

 

1Values are least squares means.

2Treatments: T1 = 0.26%; T2 = 0.35%; T3 = 0.41%; T4 = 0.45%; and, T5 = 0.51% dietary P, DM basis.
3Signiﬁcant effects (P < 0.05) and tendencies (P < 0.10).

4NS = Not significant (P > 0.10).

5ECM = Energy corrected milk yield (kg/d) = [[03246 x MY (lb/d)] + [12.86 x milk fat yield (1b/d)] +
[7.04 x milk protein yield (lb/d)]] -:- 2.2 (lb/kg) (Dairy Records Management Systems. 1999).

105

Table 17. Phosphorus balance and characteristics of feces, urine, and milk of early lactation cows (76 to
106 :1: 10.0 DIM) as inﬂuenced by experimental treatments (Experiment 5).1

 

 

 

 

Treatmentz P«-value3
Item T1 T2 T3 T4 T5 SEM Treatment Linear Quadratic Cubic Quartic
Pintake,g/d 49.4 70.3 80.5 94.3 98.0 4.40 <0.01 <0.01 NS NS NS
Fecal DM,% 16.8 16.3 17.2 16.3 16.8 0.49 NS‘ NS NS NS NS
Fecal DM
excretion, g/d 6010 6085 6247 6715 6148 512.7 NS NS NS NS NS
Fecal P,%of
DM 0.40 0.68 0.94 1.27 1.49 0.05 <0.01 <0.01 0.09 NS NS
FecalP
excretion, g/d 23.7 41.9 58.2 85.2 90.4 5.18 <0.01 <0.01 NS 0.09 NS
gfecalP/kg DMI 1.3 2.1 3.0 4.1 4.7 0.20 <0.01 <0.01 0.09 NS NS
Urine excretion“,
kg/d 16.0 17.9 16.4 17.0 16.0 1.02 NS NS NS NS NS
Urine P, °/o 0.003 0.016 0.007 0.010 0.024 0.006 NS 0.07 NS 0.08 NS
UrineP
excretion, g/d 0.52 2.93 1.27 1.72 3.85 1.14 NS NS NS NS NS
TotalP
excretion5,g/d 24.23 44.83 59.45 86.88 94.22 2.23 <0.01 <0.01 NS NS 0.10
Milk P,% 0.082 0.078 0.088 0.085 0.09 0.003 0.07 NS NS 0.05 0.10
Milkuield,g/d 28.5 28.9 33.8 32.6 28.5 2.22 NS NS NS NS NS
Pbaiance’,g/d -5.4” -34“ -12.89 -2519 -2479 3.83 <0.01 <0.01 NS 0.04 NS

 

'Values are least squares means.
2Treatments: T1 = 0.26%; T2 = 0.35%; T3 = 0.41%; T4 = 0.45%; and, T5 = 0.51% dietary P, DM basis.

3Signiﬁcant effects (P < 0.05) and tendencies (P < 0.10).
‘NS = Not signiﬁcant (P > 0.10).

5Total P excretion (g/d) = fecal P + urine P excretion (g/d).

6Estmatied from urine prediction model (Holter and Urban, 1992).

7P balance (g/d) = P intake (g/d) - [total P excretion (g/d) + milk P yield (g/d).

8P balance is not different from zero (P > 0.10).

9P balance is different from zero (P < 0.05).

106

Table 18. Phosphorus excretion by early lactation cows (76 to 106 3: 10.0 DIM) as inﬂuenced by
experimental treatments (Experiment 5).'

 

 

 

 

Treatment2 P-value’

Item T1 T2 T3 T4 T5 SEM Treatment Linear Quadratic Cubic Quartic
Total P excretion,

%ofPintake 51.2 63.5 73.7 91.8 96.7 4.47 <0.01 <0.01 NS NS NS
Fecal P,%of P

intake 50.1 59.3 72.0 90.2 92.9 4.58 <0.01 <0.01 NS 0.09 NS
Fecal P, % of total

Pexcretion 97.9 93.7 97.8 98.4 95.9 1.87 NS NS NS 0.05 NS
Urine P, % of P

intake 1.1 4.2 1.6 1.6 3.8 1.36 NS NS NS 0.06 NS
Urine P, % of total

Pexcretion 2.1 6.3 2.2 1.6 4.1 1.87 NS NS NS 0.05 NS

Milk P, % P intake 58.5 41.4 41.8 34.5 29.0 2.45 <0.01 <0.01 NS NS NS
Milk P, % total P
output 54.4 39.5 36.4 27.5 23.3 1.97 <0.01 <0.01 NS NS 0.09

 

|Values are least squares means.

2Treatments: T1 = 0.26%; T2 = 0.35%; T3 = 0.41%; T4 = 0.45%; and, T5 = 0.51% dietary P, DM basis.
JSigniﬁcant effects (P < 0.05) and tendencies (P < 0.10).

4NS = Not signiﬁcant (P > 0.10).

107

Table 19. Performance characteristics of mid lactation cows (162 to 192 i 6.2 DIM) as inﬂuenced by
experimental treatments (Experiment 6).1

 

 

 

 

Treatment2 P-value3
Item T1 T2 T3 T4 T5 SEM Treatment Linear Quadratic Cubic Quartic
n 6 7 7 7 6 -- - -- - - --
BW, kg 643 589 606 639 649 28.1 NS‘ NS NS NS NS
BCS 3.0 2.9 2.6 3.4 3.0 0.23 NS NS NS NS 0.02
DIM 191 187 198 189 194 6.2 NS NS NS NS NS
DMl,kg/d 20.7 17.8 19.1 19.6 19.6 1.01 NS NS NS NS NS
DMI,%BW 3.2 3.0 3.1 3.1 3.1 0.16 NS NS NS NS NS
Apparent DM
digestibility,% 71.1 67.5 69.2 69.2 70.3 1.04 NS NS 0.04 NS NS
MY,kg/d 31.8 27.0 31.8 29.4 30.4 1.58 NS NS NS NS 0.08
ECMs,kg/d 29.5 23.9 27.8 27.5 28.0 1.17 0.03 NS 0.03 0.02 NS
Milk fat,% 3.29 3.01 2.91 3.23 3.18 0.13 NS NS 0.07 NS NS
Milk protein
(true),% 2.68 2.64 2.65 2.72 2.66 0.07 NS NS NS NS NS
Milk lactose,% 4.81 4.8 4.80 4.73 4.63 0.08 NS NS NS NS NS
SNF,% 8.50 8.41 8.47 8.45 8.28 0.15 NS NS NS NS NS
SCC, 1000
cells/ml 254 390 238 63 101 141.2 NS NS NS NS NS

 

1Values are least squares means.

2Treatments: T1 = 0.26%; T2 = 0.35%; T3 = 0.41%; T4 = 0.45%; and, T5 = 0.51% dietary P, DM
basis.

3 Signiﬁcant effects (P < 0.05) and tendencies (P < 0.10).

‘NS = Not signiﬁcant (P > 0.10).

5ECM = Energy corrected milk yield (kg/d) = [[0.3246 x MY (lb/d)] + [12.86 x milk fat yield (lb/d)]
+ [7.04 x milk protein yield (lb/d)]] + 2.2 (lb/kg) (Dairy Records Management Systems, 1999).

108

Table 20. Phosphorus balance and characteristics of feces. urine, and milk of mid lactation cows (162 to
192 i 6.2) as inﬂuenced b1 experimental treatments (Eyeriment 6).l

 

 

 

 

Treatmentz P-valueJ
Item T1 T2 T3 T4 T5 SEM Treatment Linear Quadratic Cubic Quartic
Pintake,
g/d 56.0 64.1 80.1 90.3 102.1 3.99 <0.01 <0.01 NS NS NS
Fecal DM,
% 643 589 606 639 648 28.1 NS4 NS NS NS NS
Fecal DM
excretion,
g/d 5982 5765 5884 6046 5812 345.9 NS NS NS NS NS
Fecal P,%

ofDM 0.48 0.72 1.02 1.28 1.54 0.05 <0.01 <0.01 0.04 NS NS
FecalP

excretion,
g/d 28.3 41.1 59.9 77.0 90.0 4.04 NS NS NS NS NS

gfecal

P/ngMI 1.4 2.3 3.1 3.9 4.6 0.09 <0.01 <0.01 NS NS NS
Urine

excretions,

kg/d 19.8 15.9 18.0 18.2 20.4 1.28 NS NS 0.02 NS NS_

Urine P, % 0.0003 0.0026 0.0016 0.0021 0.0114 0.003 NS 0.06 NS NS NS
Urine P

excretion,
g/d 0.07 0.44 0.30 0.42 1.76 0.46 NS 0.04 NS NS NS
TotalP
excretion“,
g/d 28.3 41.6 60.2 77.4 91.7 3.87 <0.01 <0.01 0.10 NS NS

MilkP,°/o 0.084 0.083 0.082 0.085 0.079 0.003 NS NS NS NS NS
MilkP

yield,g/d 26.4 22.6 25.9 24.8 23.9 1.29 NS NS NS 0.07 NS
P

balance7.
g/d 1.28 0.1” 6.08 -119" -13.69 3.08 <0.01 <0.01 NS NS NS

 

lValues are least squares means.

2Treatments: T1 = 0.26%; T2 = 0.35%; T3 = 0.41%; T4 = 0.45%; and, T5 = 0.51% dietary P, DM'basis.
3Signiﬁcant effects (P < 0.05) and tendencies (P < 0.10).

4NS = Not signiﬁcant (P > 0.10).

5Estmatied from urine prediction model (Holter and Urban, 1992).

°Total P excretion (g/d) = fecal P + urine P excretion (g/d).

7P balance (g/d) = P intake (g/d) - [total P excretion (g/d) + milk P yield (g/d).

8P balance is not different from zero (P > 0.10).

('P balance is different from zero (P < 0.05).

109

Table 21. Phosphorus excretion by mid lactation cows (162 to 192 i 6.2 DIM) as inﬂuenced by
experimental treatments (Experiment 6).l

 

 

 

 

Treatment2 P-value3
Item T1 T2 T3 T4 T5 SEM Treatment Linear Quadratic Cubic Quartic
TotalP
excretion, % P
intake 51.1 65.8 74.9 86.1 89.7 3.51 <0.01 <0.01 NS NS NS
Fecal P,%P
intake 51.0 65.1 74.5 85.6 87.7 3.60 <0.01 <0.01 NS NS NS
Fecal P, % total
Pexcretion 99.8 99.0 99.5 99.4 97.9 1.61 NS NS NS NS NS
Urine P,%P
intake 0.1 0.6 0.4 0.4 1.9 0.51 NS 0.06 NS NS NS
Urine P, % total
Pexcretion 0.2 1.0 0.5 0.6 2.1 0.54 NS NS NS NS NS
Milk P,%P
intake 47.4 35.3 32.5 27.6 23.5 1.40 <0.01 <0.01 NS NS NS
Milk P,%totalP
output 48.5 35.0 30.4 24.3 20.9 1.36 <0.01 <0.01 0.05 NS NS

 

1Values are least squares means.

2Treatments: T1 = 0.26%; T2 = 0.35%; T3 = 0.41%; T4 = 0.45%; and, T5 = 0.51% dietary P, DM
basis.

3Signiﬁcant effects (P < 0.05) and tendencies (P < 0.10).

‘NS = Not signiﬁcant (P > 0.10).

110

CHAPTER 5

PREDICTING PHOSPHORUS EXCRETION OF DAIRY CATTLE FED A

RANGE OF DIETARY PHOSPHORUS CONCENTRATIONS

ABSTRACT

The objective of this research was to evaluate published models for accuracy
(bias) and precision in predicting measured P excretion using newly generated data. The
newly generated data were obtained from 20 primi- and multiparous lactating Holstein
cows (Chapter 3), 73 non-lactating nulli- and multiparous pregnant Holsein cattle and
120 lactating primi- and multiparous Holstein cows (Chapter 4). Three datasets for
evaluation were constructed from these data. The full dataset (F D) consisted of data of
all 193 animals, the lactating cow dataset (LD) consisted of the 120 lactating cows, and
the non-lactating animal dataset (ND) consisted of the 73 non-lactating, pregnant
animals.

Eight models were found in the literature to evaluate. The new independent data
set was used to evaluate these models. The total range in P excretion was 12.1 to 108.7
g/animal per d. Accuracy, bias, and precision were determined for each of the eight
models. Phosphorus excretion was most appropriately predicted by: (g/d) = intake P —
milk P (IPMP) model and (g/d) = 5.92 + 0.741 x [intake P — milk P (g/d)]. In contrast,

the least appropriate model was the ASAE (1996).

III

Furthermore, to improve the IPMP model, regression analysis was conducted
using each of the three datasets. Measured P excretion was regressed on the difference of
intake P — milk P to develop three models, one for each the F D, LD, and ND. The P
excretion model for the FD was: (g/d) = 1.19 i 0.059 x [intake P — milk P (g/d)] + [1.43
:t 2.670 (g/d)]. This relationship was linear (P < 0.01) and explained 68% of the variation
associated with intake P — milk P (g/d). Additionally, the y-intercept was different from
zero (P < 0.05), but the slope was not different from one (P > 0.05). The P excretion
model for the LD was: (g/d) = 1.22 i 0.055 x [intake P — milk P (g/d)] - [2.11 i 2.747
(g/d)]. This relationship was linear (P < 0.01) and explained 80% of the variation
associated with intake P - milk P (g/d). The y-intercept of this regression equation was
not different from zero (P > 0.05), but the slope was different from one (P < 0.05). The P
excretion prediction for the ND was: (g/d) = 1.26 i 0.160 x [intake P — milk P (g/d)] +
[2.05 i 6.148 (g/d)]. This relationship was linear (P < 0.01) and explained 46% of the
variation associated with intake P — milk P (g/d). The y-intercept was not different from
zero and the slope was not different from one (P > 0.05). These models need to be
evaluated for accuracy and precision in predicting measured P excretion using a separate

independent dataset.

INTRODUCTION

Better prediction of the amount of P excreted by the dairy herd would be useful
information to dairy producers and planners of Comprehensive Nutrient Management

Plans (CNMPs). In an evaluation of published P prediction models, Davidson and Beede

112

(1999) determined that the two models (ASAE 1980; 1996) used to develop manure
management systems and plan comprehensive nutrient management, were the most
inaccurate and imprecise for predicting P excretion compared with other published P
excretion prediction models (Morse et al., 1992b; Van Horn et al., 1994; Davidson and
Beede, 1999). These two models over-predicted P excretion by 68 and 106%,
respectively, compared with measured P excretion in an independent dataset. The ASAE
(1980; 1996) models predict P excretion as a linear function of BW, as BW increases P
excretion increases, only the slope of the linear ﬁt is different. Neither model likely is
acceptable for the development of CNMPs because the estimated P excretion is inﬂated
excessively (Davidson and Beede, 1999). Therefore, the acreage needed to apply manure
also is inﬂated.

Alternatively, Van Horn et al. (1994) suggested that the equation P excretion (g/d)
= P intake (g/d) — P output in milk (g/d) might be a better and simpler estimator.
Davidson and Beede (1999) determined that this equation was the most accurate and
precise model relative to the other published models (Morse et al., 1992b; Van Horn et
al., 1994; Davidson and Beede, 1999) they suggested. They determined that this model
over-predicted P excretion by 39% over the range of measured P excretions in an .
independent dataset developed from the literature. This model is interesting from its
practical standpoint. Phosphorus intake and milk P yield potentially can be more easily
and accurately estimated than BW or other variables found in some prediction models.

The overall hypothesis of this research was that the P excretion prediction
equation, P excretion (g/d) = 5.92 + [P intake (g/d) - milk P yield (g/d)] x 0.741

developed, but not evaluated by Beede and Davidson (1999) is the most accurate and

113

precise prediction equation currently available. The objective to test this hypothesis was
to evaluate eight published P excretion prediction models for accuracy and precision.

The objective was made possible by assembling a new set of data from P balance trials
(Chapter 4) in a series of experiments designed to collect P excretion data from lactating
and non-lactating Holstein cows and late gestation nulliparous Holstein heifers. This was
done to represent cows at various points of the lactation cycle with a wide range in DMI,

MY, and P intake.

MATERIALS AND METHODS

Phosphorus Excretion Datasets

Phosphorus intake, secretion, and excretion data were collected from balance
trials with 20 lactating cows (Chapter 3) plus 82 non-lactating and 102 other lactating
cows (Chapter 4) to evaluate eight P excretion prediction models published in the
scientiﬁc literature. Non-lactating cows were 59 to 9 d prepartum and were fed dietary P
of 0.19 to 0.35%. Lactating cows ranged from 76 to 303 DIM and were fed 0.26 to
0.52% dietary P. From these data (P balance and excretion information from 204 total
animals) a dataset was constructed containing the following variables: P excretion (g/d),
BW (kg), P intake (g/d), MY (kg/d), dietary P (% of dietary DM), and milk P yield (g/d).
These variables were included in at least one of the eight P excretion prediction models
(Tablel).

An outlier analysis was conducted with the entire dataset to identify any values

greater or less than i 2 standard deviations from the mean. Based on this analysis, data

114

of seven cows (ﬁve non-lactating and two lactating) were determined to be outliers (> i 2
SD) and were removed from the dataset. Additionally, four cows (4 non-lactating) had
missing data that did not allow calculation of total P excretion; therefore. these data also
were removed from the dataset. The full dataset (FD) consisted of P intake. milk P yield,
and excretion data from 193 cows (73 non-lactating and 120 lactating cows). Measured P
excretion ranged from 12.1 to 108.7 g/animal per d. Two other subsets of data were
constructed from these data. The lactating cow dataset (LD) consisted of the 120
lactating cows (measured P excretion ranged from 20.7 to 108.5 g/cow per (1), and the
non-lactating animal dataset (ND) consisted of the 73 non-lactating animals (measured P
excretion ranged from 12.1 to 108.7 g/animal per d). These datasets were used to
evaluate P excretion prediction models. The models evaluated were found in ASAE
(1980; 1996), Morse et al. (1992b), Van Horn et al. (1994), Davidson and Beede (1999),

and Wu et al. (2000) and are listed in Table 1.

Evaluation of Phosphorus Excretion Prediction Models

Accuracy. The accuracy of the eight different P excretion prediction models was
evaluated using the following equation: accuracy = predicted — measured P excretion for
each observation in PD, LD, and ND. This value was then determined to be different or
not different from zero by t-test. Accuracy is the closeness of predicted P excretion to
actual or measured P excretion.

Bias. Regression analysis was conducted on the relationship between predicted P
excretion and measured P excretion to determine bias. A model was biased if the y-

intercept of the regression line was different from zero (t-test) and the slope was different

115

from 1 (F -test). The y-intercept and slope were different from zero and one, respectively.
if P < 0.05. The Regression procedure of SAS (1999) was used to evaluate the
relationship between predicted P excretion and measured P excretion with following
statistical model: Xi = u + M; +Ei where,

X; = predicted P excretion;

u = mean measured P excretion;

Mi = the effect of measured P excretion on model i; and,

E; = the experimental error;
Bias means the predicted P excretion does not equal the measured P excretion.

Precision. The precision of prediction by models of P excretion was evaluated
using the root mean square error (RMSE) and the relative over-prediction of each model
in PD, LD, and ND. The relative over-prediction of each of the datasets was computed
by the following equation: relative over-prediction = [absolute value of (predicted —
measured) + measured] x 100% for each observation in its respective dataset and the
RMSE was generated as part regression analysis in the bias evaluation. Precision is how

consistent a model predicts P excretion at a given measured P excretion.

RESULTS AND DISCUSSION

The eight models evaluated using the newly collected datasets are listed in Table
1. All eight models were evaluated with each of the FD, LD, and ND datasets separately
to determine how well they predicted measured P excretion. The ASAE (1980; 1996)

models were developed from published and unpublished data about P excretion and

116

composition of dairy manure. The ASAE (1996) model was deve10ped from a maximum
of 86 cow-trial observations. However, the origins, amount, and types of cattle
represented in the development of this model could not be ascertained from the
publications. The Morse et al. (1992b) model was developed with 93 individual cow
balance datum from multiparous lactating Holstein cows. Van Horn et al. (1994)
expanded the Morse et al. (1992b) dataset with 15 observations of P excretion from non-
lactating, non-pregnant Jersey cows. Additionally, Van Horn et al. (1994) suggested that
P excretion might be more simply estimated as P intake — milk P yield. In a review and
evaluation of six P excretion models, Davidson and Beede (1991) developed the H & C
model with data from Hibbs and Conrad (1983), who conducted a series of experiments
from 1958 to 1967 with lactating Jersey cows. They also evaluated the intake P — milk P
model (IPMP) suggested by Van Horn et al. (1994). They then developed another model,
by linear regression analysis of the relationship between measured P excretion and intake
P — milk P using a database of 71 treatment means assembled from the scientiﬁc
literature. Wu et al. (2000) developed a prediction equation from 26 multiparous
lactating Holstein cows, which simply is a linear relationship of P excretion with P intake
with a slope of 0.643 and an intercept of —5.7. Because the ND contains only non- .
lactating cow data, the model P excretion = P intake — milk P was simply P excretion = P
intake. Therefore, the model P excretion = intake P was evaluated for accuracy, bias, and
precision for the PD and LD to determine if this simple model was more accurate and
precise and less biased than the eight published models. Plots of this relationship are not
shown for FD and LD, however, statistics are provided for comparison to the other eight

models.

117

Only one (Van Horn et al., 1994) of the eight models included non-lactating
animals; they represented only 15 of 108 P excretion data points. Therefore, it was
considered useful to determine whether or not any of the eight models could accurately
and precisely predict measured P excretion of non-lactating animals as well as lactating
cows. Again, accuracy is the closeness of predicted P excretion to actual or measured P
excretion. Whereas, precision provides an estimate of how consistently a model predicts
P excretion over a range of measured P excretion.

Evaluation of Accuracy with the Full Dataset. Plots of predicted P excretion
versus measured P excretion by model are presented in Figure l for visualization of
accuracy, bias, and precision. Relevant statistics are reported in Table 2. All milk
production-related variables in all models were zero when P excretion was predicted for
non-lactating animals. Of the eight models, accuracy (predicted — measured P excretion)
of only one model (ASAE, 1980) was not different than zero (P > 0.05). The accuracies
of all other models (ASAE, 1996; Morse et al., 1992b; Van Horn et al., 1994; H & C;
IPMP; Davidson and Beede, 1999; Wu et al., 2000) were statistically different from zero
(P < 0.05). To determine if P intake was an accurate predictor of P excretion, the model
P excretion = intake P was also evaluated for accuracy. Accuracy for this model was
5.59 g/d, which was different from zero (P < 0.01).

Evaluation of Bias with the Full Dataset. The regression equation associated with
the FD for all models and relevant statistics are reported in Table 3. The regression
equations are plotted in Figure 1 for comparison to unity (measured P excretion =
predicted P excretion). The slopes were different from one and the y-intercepts were

different from zero (P < 0.01) for each model. Therefore, all models were biased in their

118

prediction of measured P excretion. Again P excretion = intake P was also evaluated for
precision. This model was biased with a y-intercept of 24.2 g/d (different from zero) and
slope of 0.630 g/d (different from one).

Evaluation of Precision with the Full Dataset. Relevant statistics are reported in
Table 4. Precision was estimated by RMSE. The ASAE models (1980; 1996) and H & C
models were relatively precise with RMSE of 6.9, 9.0, and 9.2 g/d compared to the other
six models. The models of Morse et al. (I992b) and Van Horn et a1. (1994) were not as
precise as the previous three models with RMSE of 13.3 and 16.3 g/d, respectively.
Models of IPMP, Davidson and Beede (1999), and Wu et al. (2000) had RMSE of 10.2,
15.0, and 10.4 g/d, respectively. Lastly, precision for the simple model P excretion = ‘
intake P was 11.1 g/d.

When determining the most or least accurate, biased, or precise model over-all,
bias was the main factor for determination followed by accuracy then precision. Bias
was weighted more heavily because without the graphs of predicted versus measured
excretion, accuracy and precision can be misleading. But, with the slope and y-intercept
values present the model can be compared to unity (predicted — measured = 0) with a
slope of one and y-intercept of zero to determine how well the data points follow or do
not follow unity. Accuracy was addressed secondly because this relationship addressed
how close the predicted value was to the actual measured value. Precision was addressed
lastly because it was an estimate of consistency in prediction of measured P excretion and
represented the standard deviation of predicted versus measured P excretion around the

regression line.

119

When evaluating the eight published models with the FD, the most accurate and
precise model was IPMP. It was also to least biased (Table 3). Its accuracy was different
from zero and it was biased, but its precision of prediction (RMSE = 10.2) was closer to
measured P excretion relative to the other seven models across the full range of measured
P excretion. The relatively small accuracy (9.7 g/d) could be due to experimental error
in measured P excretion or not accounting for P retained in bone and soft tissues (Beede
and Davidson, 1999). The accuracy of the ASAE (1980) model was not different from
zero. From the plot of ASAE (1980) predicted versus measured P excretion (Figure 1) it
can be visualized why mean accuracy is not different from zero. It predicted P excretion
within the same range (about 35 to 61 g/d) across the entire range (about 10 to over 100
g/d) of actual measured P excretion; therefore, the overall average will be close to unity
(measured = predicted P excretion). The ASAE (1996) model was the least accurate and
precise model evaluated (accuracy different from zero, prediction was biased, and over-
prediction 82.9% with RMSE of 9.0 g/d), which is important to note because this model
likely is used to determine how many head of cattle can be on a farm with a given amount
of acreage. Additionally, the evaluation of the simple relationship P excretion = intake P
was not an improvement over the other eight published models.

Evaluation of A ccuracy with the Lactating Cow Dataset. Plots of predicted P
excretion versus measured P excretion by model are presented in Figure 2 for
visualization of accuracy and precision. Relevant statistics are reported in Table 5. All
models evaluated with the LD were different from zero (P < 0.05), and thus biased in
their prediction of P excretion. However, relative to the other models the IPMP model

had less variation associated with accuracy of the model (Table 5). This disagreed with

120

results reported by Davidson and Beede (1999), in which the H & C and IPMP models
were not different from zero when evaluated with an independent dataset. The simple
predictor P excretion = intake P was also evaluated for accuracy. Accuracy was 16.8 g/d
and different from zero.

Evaluation of Bias with the Lactating Cow Dataset. The regression equation
associated with the LD for all models and relevant statistics are reported in Table 6. The
regression equations are plotted in Figure 2 for comparison to unity. All models were
biased in their prediction of measured P excretion. The slopes were different than one
and the y-intercepts were different from zero for all eight models.

Evaluation of Precision with the Lactating Cow Dataset. Relevant statistics are
reported in Table 7. The ASAE (1980; 1996), Morse et al. (1992b), Van Horn et al.
(1994), and H & C, IPMP, Davidson and Beede (1999), and Wu et al. (2000) models had
similar mean precisions of 5.5, 7.2, 9.1, 9.1. 7.3, 8.9, 6.6, and 7.2 g/d, respectively. The
ASAE (1980) model was the most precise (RMSE = 5.5 g/d) model of the eight
evaluated. Precision of P excretion = P intake was 11.1.

The most accurate and precise and least biased model was the IPMP (Tables 5, 6,
and 7). This agreed with results of Davidson and Beede (1999) when models were.
evaluated with an independent dataset. In that model evaluation, the IPMP model was
relatively accurate (average predicted — measured P excretion = 14.5 g/d), although
different from zero (P < 0.01) with a relative over-prediction of 39% across measured P
excretion, when compared with other models (ASAE 1980; 1996; Morse et al., 1992b;
Van Horn et al., 1994; H & C) evaluated with their independent dataset. Additionally,

the model developed but not evaluated by Davidson and Beede (1999) by regression

121

analysis of the relationship between P excretion and intake P — milk P was not an
improvement over the IPMP model (Tables 5, 6, and 7). The model P excretion = intake
P was not an improvement over intake P — milk P.

Evaluation of Accuracy with the Non-lactating Animal Dataset. Plots of
predicted P excretion versus measured P excretion by model is presented in Figure 3 for
visualization of accuracy and precision. Relevant statistics are reported in Table 8. All
milk production-related variables in all models were zero when evaluating models with
ND. The ASAE (1980) and H & C models were not determined to be different from zero
(P > 0.05). The accuracy of all other models was different from zero.

Evaluation of Bias with the Non-lactating Animal Dataset. The regression
equation associated with the LD for all models and relevant statistics are reported in
Table 9. The regression equations are plotted in Figure 3 for comparison to unity. All
models were biased with slopes different from one and y-intercepts different from zero.

Evaluation of Precision with the Non-lactating Animal Dataset. Relevant
statistics are reported in Table 10. Precision for all models was relatively close and
ranged from 5.1 to 9.0 g/d. Van Horn et a1. (1994) was the most precise at 5.1 g/d and
IPMP was the least precise at 9.0 g/d.

The most accurate and precise model for the ND was IPMP. It was not as precise
as the other seven models, but mean accuracy (~12.8 g/d) coupled with precision
predicted P excretion closer to measured P excretion in this dataset. For non-lactating
animals, P intake was the best explanatory variable for measured P excretion compared
with the other variables [BW. MY, milk P yield, dietary P% (dry basis)]. There were no

models found in the literature for predicting P excretion speciﬁcally by non-lactating

animals so it was useful to determine whether any of these eight models was useful for
both lactating and non-lactating animals.

Further Evaluation of Phosphorus Intake Minus Milk Phosphorus Yield. As
previously mentioned IPMP over-predicted P excretion in each of the FD, LD, and ND.
To offer a potentially more explanatory model involving the intake P — milk P
relationship an equation to account for more variation in P excretion was developed for
each newly created dataset by regressing measured P excretion on measured intake P —
measured milk P (g/d). The P excretion model for the FD was: (g/d) = 1.19 i 0.059 x
[intake P — milk P (g/d)] + [1.43 i 2.670 (g/d)]. This relationship was linear (P < 0.01)
and explained 68% of the variation associated with intake P — milk P (g/d). Additionally,
the y-intercept was different from zero (P < 0.05), but the slope was not different from
one (P > 0.05). The P excretion model for the LD was: (g/d) = 1.22 :1: 0.055 x [intake P
— milk P (g/d)] - [2.11 i 2.747 (g/d)]. This relationship was linear (P < 0.01) and
explained 80% of the variation associated with intake P - milk P (g/d). The y-intercept
of this regression equation was not different from zero (P > 0.05), but the slope was
different from one (P < 0.05). The P excretion prediction for the ND was: (g/d) = 1.26 i
0.160 x [intake P — milk P (g/d)] + [2.05 i 6.148 (g/d)], where milk P = 0. This
relationship was linear (P < 0.01) and explained 46% of the variation associated with
intake P — milk P (g/d). The y-intercept was not different from zero and the slope was
not different from one (P > 0.05). A plot of measured P excretion plotted against intake
P — milk P is provided in Figure 4 along with a plot of the regression line associated with

each dataset. However, before use of any of these newly developed models, they must be

123

evaluated for accuracy, bias, and precision by a separate independent dataset currently

not available.

Conclusions

The eight models that predict P excretion from dairy cattle in Table 1 were
evaluated with three independent datasets compiled from 73 non-lactating and 120
lactating cows. The hypothesis tested in this research was that the P excretion model
developed, but not evaluated by Davidson and Beede (1999): P excretion (g/d) = 5.92 +
0.741 x [intake P (g/d) — milk P (g/d)], was the best predictor of P excretion for lactating
dairy cows. However, based on evaluation using the newly collected dataset (LD) it Was
not an improvement over the simple difference intake P - milk P.

Over all datasets, the IPMP was more accurate and precise relative to the other
seven models evaluated. The most practical equation for P excretion prediction for
whole-farm nutrient management would be one equation that could be used for all mature
cows. It appears that the IPMP would be a better predictor of P excretion than what is
currently used for planning of nutrient management. The ASAE (1996) model is an
equation likely used to predict P excretion in a given dairy operation. However, the
ASAE (1980; 1996) models were overall the most inaccurate and imprecise predictors of
P excretion relative to the other ﬁve. Planners of comprehensive nutrient management

should not use the ASAE (1980; 1996) models.

124

 

Table 1. Phosphorus excretion models evaluated using the independent datasets.

 

 

Model Prediction equationl

ASAE (1980) Y‘ = BW (kg) x 0.0724

ASAE (1996) Y = BW (kg) x 0.094

Morse et al. (1992b) Y = 14.67 + 0.6786 x intake P (g/d) + 0.00196 x [intake P
(g/d)]2 — 0.317 x MY (kg/d)

Van Horn et a1. (1994) Y = 9.6 + 0.472 x intake P (g/d) + 0.00126 x [intake P
(g/d)] 2 + 0.323 x MY (kg/d)

H & c modelz Y = -24.06 + 81.67 x diet P (% of dietary DM) + 0.07 x
BW (kg) — 0.45 x milk P (g/d)

Intake P — milk P Y = intake P (g/d) - milk P (g/d)

Davidson and Beede (1999) Y = 5.92 + 0.741 x [intake P (g/d) -— milk P (g/d)]

Wu et al. (2000) Y = [0.643 x P intake (g/d)] -— 5.2

 

1Y = P excretion (g/d).
2The H and C model was developed from data of Hibbs and Conrad (1983).

125

 

Table 2. Accuracy of prediction of measured P excretion (g/d) of

lactating and non-lactating dairy animals by various models using
the full dataset.

 

 

 

Accuracyl
Model Mean i SEM P-value2
ASAE (1980) —3.0 1.93 0.13
ASAE (1996) 11.6 1.97 <0.01
Morse et al. (1992b) 3.8 1.26 <0.01
Van Horn et al. (1994) -4.2 1.60 <0.01
H & c model3 -7.8 1.63 <0.01
Intake P - milk P -9.7 1.09 <0.01
Davidson and Beede (1999) -14.6 1.22 <0.01
Wu et al. (2000) -20.0 1.42 <0.01
Intake P 5.59 1.27 <0.01

 

rAccuracy = predicted - measured P excretion (g/d).

2Mean is different from zero if P < 0.05; t-test.

3Developed by Davidson and Beede (1999) from data of Hibbs and
Conrad (1983).

126

 

Table 3. Bias of prediction of measured P excretion (g/d) of non-lactating and
lactating dairy animals by various models using the full dataset.

 

 

Model y-intercept i SE P-valuel Slope 1: SE P-value2
ASAE (1980) 47.9 1.09 <0.01 0.013 0.019 <0.01
ASAE (1996) 62.1 1.43 <0.01 0.018 0.025 <0.01
Morse et al. (1992b) 26.1 2.11 <0.01 0.566 0.037 <0.01
Van Horn et al. (1994) 25.6 2.59 <0.01 0.422 0.045 <0.01
H & c model3 32.8 1.47 <0.01 0.212 0.025 <0.01
Intake P - milk P 12.2 1.62 <0.01 0.574 0.028 <0.01
Davidson and Beede (1999) 15.0 1.20 <0.01 0.426 0.021 <0.01
Wu et al. (2000) 10.4 1.93 <0.01 0.411 0.033 <0.01
Intake P 24.2 3.00 <0.01 0.639 0.052 <0.01

 

Iy-intercept (g/d) is different from zero if P < 0.05; t-test.

2Slope (g/d) is different from one if P < 0.05; F-test.
3Developed (Davidson and Beede, 1999) from data of Hibbs and Conrad (1983).

127

 

Table 4. Precision of various models to predict P excretion (g/d) by lactating
and non-lactating dairy animals using the full dataset.

 

Mean‘ i RMSE2 95% Conﬁdence Interval3

 

ASAE (1980) 48.6 6.9 41.7 to 55.5
ASAE (1996) 63.1 9.0 54.1 to 72.1
Morse et al. ( 1992b) 55.3 13.3 42.0 to 68.6
Van Horn et al. (1994) 47.3 16.3 34.0 to 63.6
H & c model‘ 43.8 9.3 34.5 to 53.1
Intake P - milk P 41.8 10.2 31.6 to 52.0
Davidson and Beede (1999) 36.9 7.6 29.3 to 44.5
Wu et al. (2000) 31.5 12.2 19.3 to 43.7
P intake 57.1 11.1 46.0 to 68.2

 

lMean = average predicted P excretion (g/d).

2Precision: RMSE = root mean square error of the regression of the
relationship between predicted P excretion and measured P excretion (g/d).
395% conﬁdence interval = mean i 2 RMSE (g/d).

4Developed (Davidson and Beede, 1999) from data of Hibbs and Conrad
(1983)

128

Table 5. Accuracy of prediction of P excretion (g/d) of lactating dairy cows
by various models using the lactating cow dataset.

 

 

 

Accuracyl
Model Mean i SEM P-value2
ASAE (1980) -8.1 2.43 <0.01
ASAE (1996) 5.5 2.43 0.03
Morse et al. (1992b) 10.2 1.20 <0.01
Van Horn et al. (1994) 5.6 1.58 <0.01
H & C models -13.6 1.88 <0.01
Intake P - milk P -7.8 1.18 <0.01
Davidson and Beede (1999) -13.7 1.41 <0.01
Wu et al. (2000) —l3.5 1.52 <0.01
Intake P 16.8 127 <0.01

 

lAccuracy = predicted - measured P excretion (g/d).
2Mean is different from zero if P < 0.05; t-test.

3Developed (Davidson and Beede, 1999) from data of Hibbs and Conrad
(1983)

129

 

Table 6. Bias of prediction of measured P excretion (g/d) of lactating dairy animals by
various models using the lactating cow dataset.

 

 

Model y-intercept i SE P-valuel S10pe i SE P-value2
ASAE (1980) 43.2 1.12 <0.01 0.040 0.019 <0.01
ASAE (1996) 56.1 1.45 <0.01 0.052 0.024 <0.01
Morse et al. (1992b) 29.1 1.84 <0.01 0.646 0.031 <0.01
Van Horn et al. (1994) 34.7 1.84 <0.01 0.454 0.031 <0.01
H & C model3 24.4 1.49 <0.01 0.287 0.025 <0.01
Intake P - milk P 10.6 1.81 <0.01 0.656 0.030 <0.01
Davidson and Beede (1999) 13.8 1.34 <0.01 0.486 0.022 <0.01
Wu et al. (2000) 16.3 1.45 <0.01 0.443 0.024 <0.01
Intake P 33.4 2.25 <0.01 0.689 0.037 <0.01

 

ly-intercept (g/d) is different from zero if P < 0.05; t-test.

2Slope (g/d) is different from one if P < 0.05; F-test.

3 Developed (Davidson and Beede, 1999) from data of Hibbs and Conrad (1983).

I30

 

Table 7. Precision of various models to predict P excretion (g/d) by lactating
dairy cows using the lactating cow dataset.

 

 

 

Model Meanl i RMSE2 95% Conﬁdence Interval3
ASAE (1980) 45.4 5.5 39.9 to 50.9
ASAE (1996) 58.9 7.2 51.7 to 66.1
Morse et al. (1992b) 63.6 9.1 54.5 to 72.7
Van Horn et al. (1994) 59.0 9.1 49.9 to 68.1
H & C model4 39.8 7.3 32.5 to 47.1 '
Intake P - milk P 45.6 8.9 36.7 to 54.5
Davidson and Beede (1999) 39.7 6.6 33.1 to 46.3
Wu et al. (2000) 39.9 7.2 32.7 to 47.1
Intake P 70.2 11.1 59.1 to 81.3
1Mean = average predicted P excretion (g/d). '

 

2Precision: RMSE = root mean square error of the regression of the
relationship between predicted P excretion and measured P excretion (g/d).

395% conﬁdence interval = mean i 2 RMSE (g/d).

4Developed (Davidson and Beede, 1999) from data of Hibbs and Conrad
(1983)

131

Table 8. Accuracy of prediction of P excretion (g/d) of non-
lactating dairy animals by various models using the non-lactating

 

 

 

animal dataset.

Accuracyl
Model Mean 1r SEM P-valne2
ASAE (1980) 5.5 2.96 0.07
ASAE (1996) 21.5 3.00 <0.01
Morse et al. (1992b) -6.8 2.18 <0.01
Van Horn et al. (1994) -20.2 2.36 <0.01
H & C model3 1.9 2.66 0.47
Intake P - milk P -12.8 2.09 <0.01
Davidson and Beede
(1999) -16.1 2.23 <0.01
Wu et a1. (2000) -30.7 2.30 <0.01

 

IAccuracy = predicted — measured P excretion (g/d).

2Mean is different from zero if P < 0.05, t-test.
3 Developed (Davidson and Beede, 1999) from data of Hibbs and

Conrad (1983).

132

Table 9. Bias of prediction of measured P excretion (g/d) of non-lactating
dairy animals by various models using the non-lactating animal dataset.

 

 

Model tintercept i SE P-valuel Slope :t SE P-value2
ASAE (1980) 53.6 1.43 <0.01 0.006 0.026 <0.01
ASAE (1996) 69.5 1.85 <0.01 0.007 0.034 <0.01
Morse et al. (1992b) 26.7 1.93 <0.01 0.309 0.035 <0.01
Van Horn et al. (1994) 17.9 1.32 <0.01 0.212 0.024 <0.01
H & C model3 44.3 1.71 <0.01 0.123 0.031 <0.01
Intake P - milk P 17.4 2.33 <0.01 0.377 0.043 <0.01
Davidson and Beede (1999) 18.8 1.73 <0.01 0.279 0.032 <0.01
Wu et al. (2000) 6.0 1.50 <0.01 0.242 0.028 <0.01

 

Iy-intercept (g/d) is different from zero: if P < 0.05; t-test.

2Slope (g/d) is different from one if P < 0.05; F-test.
3Developed (Davidson and Beede, 1999) from data of Hibbs and Conrad (1983).

133

 

Table 10. Precision of various models to predict P excretion (g/d) by
non-lactating animals using the non-lactating dataset.

 

 

Model Meanl i RMSE2 95% Conﬁdence Interval3
ASAE (1980) 53.8 5.5 48.3 to 59.3
ASAE (1996) 69.9 7.2 62.7 to 77.1
Morse et al. (1992b) 41.6 7.5 34.1 to 49.1
Van Horn et al. (1994) 28.2 5.1 23.1 to 33.3
H & C model4 50.3 6.6 43.7 to 56.9
Intake P - milk P 35.6 9.0 26.6 to 44.6
Davidson and Beede (1999) 32.3 6.7 25.6 to 39.0
Wu et al. (2000) 17.7 5.8 11.9 to 23.5

 

IMean = average predicted P excretion (g/d).

2Precision: RMSE = root mean square error of the regression of the
relationship between predicted P excretion and measured P excretion (g/d).

395% conﬁdence interval = mean i 2 RMSE (g/d).

4Developed (Davidson and Beede, 1999) from data of Hibbs and Conrad
(1983)

 

Predicted P Excretion, g/d

ASAE (1980) ASAE (1996)

100 ~ -

80 _. - . Q

60 _ . L- -
40 -— . —

20 1.. .—
0 1 1 1 1 1 1 l l l 1
Morse et a1. (1992) . Van Horn et al. (1994)

100 — . _ ..
80 — t — . O 4
60 t. . . .. .. t—

.0
40 L O . O. _ O. O.
r O ’
20 _ f .
0 l l l l l l l I l l
H&C lntakeP-MilkP
100 — — .
80 — — S 1
o ’0’
60 - -
4 ._ - .
O C . O . .
20 ; O
O l l I l l l l l l 1
Davidson and Beede (1999) Wu et al. (2000)
100 '- l—
80 - -
O
60 _ .3“ .5}
O
40 - -
'° as?"
20 - -
, . a ..
0 l l 1 1 1 l . L l 1

 

 

 

 

 

 

 

 

o 20 40 60 80100 0 20 40 60 80100
Measured P Excretion, g/d

Figure 1. Predicted P excretion (0) plotted against measured P

excretion in the full dataset for visualization of the precision of each
model. The solid diagonal line is unity where predicted P excretion
equals measured P excretion. The dashed line is the regression line
of the relationship between predicted and measured P excretion.

 

 

ASAE 1996
I00 r ASAE(1980) _ ( )
0 h i-
8 . .
60 '- . :_ '-
4" F O o ”
20 P -
0 1 1 1 1 1 l 1 1 1 1
O
[00 FMorse et a1. (1992) }_VanHorn Ct al. (1994)
/. O. A,
80 — . - ‘
O O
6., _ o ’ _ O
"O O O
E) 40 O . .
.S r F
a _. 1—
2 20
U
x 0 1 l 1 L l l l l 1 1
DJ
0..
"U [00 .. H & C
8
,2 80 —
"o
E 60 r . ..
40 e 0
O
f
20 1-
0 l l l l l
1 00 Davidson and Beede (1999)
80 — ..
. a O
A
a - A _ e
O
40 - L-
O .0
20 ; P‘
0 1 1 1 l 1 l 1 1 1 1
0 20 40 60 80 100 0 20 40 60 80 100
Measured P Excretion, g/d

Figure 2. Predicted P excretion (o) plotted against measured P excretion in the
lactating cow dataset for visualization of the precision of each model. The solid
diagonal line is unity where predicted P excretion equals measured P excretion.
The dashed line is the regression line of the relationship between predicted and

 

 

 

 

 

 

 

 

 

measured P excretion.

136

 

Predicted P Excretion (g/d)

 

'00 _ ASAE (I980) ASAE (1996)

 

 

80 - _ .

60 e . _ 7..
_ O

40 — _.

20 - _

0 1 1 1 1 1 L I J 1 J

'00 Morse et al. (1992) LVanHom et al. (1994)

80 r- __

60 ” O .. IO' _

40 P :.O*.’ O. ._ OO...O"1
’ i

20 - . .

0 l I I I L I j l l l
tak - Mil
looeH‘g‘C Pln eP kp
80 - _
O

60 _ W“ ’ o 00 w
'" O

40 I . . . _ ::o*0" o.

20 - P’

0 1 l I J 1 L 1 1 1 1

 

'00 f‘ Davidson and Beede (199 _Wu et al. (2000)

 

 

 

 

80 — ..
60r- l—
40 - -—

‘OO’O.’
20 -- I’ " ’
0 l l l l l I .

 

0 20 40 60 80 100 0 20 40 60 80 100

Measured P Excretion (g/d)

Figure 3. Predicted P excretion (0) plotted against measured P excretion in
the non-lactating animal dataset for visualization of the precision of each
model. The diagonal line is unity where predicted P excretion equals measured
P excretion. The dashed line is the regression line of the relationship between
predicted and measured P excretion.

137

 

P excretion ('g/d)

100
80
60
40

20

 

 

a . .
+- 0. o
e
a o:
O
_ . C
o
O o.
— o
I L l l l

 

 

 

 

0 20 40 60 80 100 0 20 40 60 80 100

Intake P - milk P (g/d)

 

100 - o’

P excretion (g/d)

 

 

l l
0 20 40 60 80 100

Intake P (g/d) - milk P (g/d)

 

 

Figure 4. Phosphorus prediction models developed using linear regression of P
excretion on intake P -— milk P. (a) The model developed using the full dataset was: P
excretion (g/d) = [1.19 i 0.059 (g/d)] x [intake P — milk P (g/d)] + [1.43 i 2.670
(g/d)], (y-intercept not different from 0; slope different from 1: RMSE = 14.9 (g/d):
R2 = 0.68; P < 0.01). (b) P excretion (g/d) = [1.22 a 0.055 (g/d) x [intake P — milk P
(g/d)]. (y-intercept not different from 0: slope different from 1; RMSE = 12.1 (g/d);
R2 = 0.80: P < 0.01). (c) P excretion (g/d) = [1.26 a 0.160 (g/d)] x [intake P — milk P
(g/d)] + [2.05 i 6.148 (g/d)]. (y-intercept not different from 0: slope not different
from 1: RMSE = 18.1 (g/d): R2 = 0.46: P < 0.01).

 

Chapter 6

CONCLUSIONS AND IMPLICATIONS

Better deﬁned P requirements for dairy cattle are critical to understanding how to
feed lactating cows to reduce P excretion. The current total net P maintenance
requirement is 1.0 g P/kg DMI (the fecal component) plus an additional 0.002 g P/kg BW
to account for endogenous urine P excretion that also is part of the maintenance
requirement (NRC, 2001). This estimate was computed using an absorption coefﬁcient
of 0.8 of dietary P and inevitable fecal P excretion (major part of the total P maintenance
requirement) of 1.2 g P/kg DMI determined by Spiekers et al. (1993). However, based
on the current experiment with higher producing cows, inevitable fecal P excretion (g/kg
DMI) was 1.36, 1.19, and 1.04 and decreased with increase DMI (linear effect; P < 0.01).
An equation was developed by regression to predict inevitable fecal P of a cow fed P
close to the true requirement: (g/d) = 0.85 i- 0.070x DMI, kg/d + [5.30 :t 1.224 (g/d)] (R2
= 0.90; P < 0.01). This equation accounts for decreased inevitable fecal P excretion (g/kg
DMI) with increased DMI and allows an estimate of the P maintenance requirement to be
calculated that potentially is closer to true P maintenance requirement.

It is not known whether or not the fecal portions of the maintenance requirement
determined by this experiment are statistically different from the results reported by
Spiekers et al. (1993); we suspect that they probably are not. However, this relationship
may become even more important as milk production per cow increases. Dry matter

intake must increase to support increased production (NRC, 2001) or efﬁciency of

139

 

converting nutrients to milk must increase. As average milk production increases,
average DMI theoretically will increase. Because DMI affects the fecal portion of the
total maintenance requirement, it may become lower (on a basis of g P/kg DMI) than
observed in this study with cows consuming DMI > 25.1 kg/d but still consuming P close
to their true P maintenance requirement.

According to Figure 1 (Chapter 3), when DMI was restricted (T1 and 2) there was
a tighter relationship between fecal P excretion (g/d) and DMI (kg/d) because there was
less potential for variation in DMI and P intake in excess of requirement for milk
production plus maintenance. In future research, to help control this variation, perhaps
DMI in all groups should be restricted to the NEL intake needed to support higher milk
yield. Also, type of diet may inﬂuence inevitable fecal P excretion. It would be
beneﬁcial to see how different forages or different ratios of forages-to-concentrate
inﬂuence the relationship between inevitable fecal P excretion and DMI. However, the
basal diet used here was quite different than that of Spiekers et al. (1993). Developing
diets that are very different in nutrient composition may be a challenge because low P
diets are difﬁcult to formulate with typical ingredients.

Seven experiments were conducted using non-lactating and lactating cows at
different points in the lactation cycle to determine the effects of varying dietary P
concentration on P balance and excretion. Dietary P concentration affected P balance and
utilization of non-lactating and lactating dairy cows. Overall, fecal P excretion increasing
linearly with increased dietary P concentration. In general, urine P excretion increased

with increasing dietary P concentration; however, the effect on urine P excretion was

140

quite small and not consistent. Urine P% was low and total daily excretion generally
resulted in less than 1 g P/d excreted in urine.

Phosphorus balance decreased with increasing dietary P concentrations; generally
P balance was less than zero and became more negative with increased dietary P
concentration. This was largely driven by increased fecal P excretion. In general, P
balances of cows in the higher P dietary treatments (MP and HP for non-lactating
animals; T3, T4, and T5 for lactating cows) were different from zero. This suggested that
P supplied by the higher P diets was in excess of the P requirement. In general milk P%,
MF, ML, MProt, and SNF were not affected by dietary P concentration. The priority
objective of these seven experiments was to collect P balance data for use to evaluate
models to predict P excretion.

Having a more accurate and precise model to predict P excretion by cows at
different points in the lactation would be very useful important to the dairy industry.
Currently, the ASAE (1996) equation is available to develop nutrient management plans
for start-up and existing dairies. Beede and Davidson (1999) reported that this model
was the least accurate and precise model in their evaluation with an independent dataset.
The current evaluation with a separate new independent dataset agreed with Beede and
Davidson (1999). The ASAE (1996) was biased (slope different from one; y-intercept
different from zero) and accuracy was 12.8 g/d and different from zero. Precision was
7.8 g/d. However, it over-predicted measured P excretion by 85.3%. The range of
measured P excretion was 12.1 to 108.7 g P/animal per d. This model was not adequate
to be used for its current purpose. Inﬂated predicted P excretion also will unnecessarily

inﬂate the calculations of land needed to apply manure to cropland.

141

Beede and Davidson (1999) also determined that the simple diﬁerence of intake P
minus milk P suggested by VanHorn et al. (1999) was the most accurate and precise
model evaluated using their independent dataset. Once this was determined they
developed a model by regression: P excretion (g/d) = 5.92 + 0.741 x [P intake (g/d) —
milk P (g/d)]), using this relationship to hopefully explain more of the variation
associated with P excretion. The objective of the last chapter was to determine if this
new model was an improvement over intake P — milk P. The results of the current
experiment concluded that this model was not as accurate or precise as intake P — milk P.
We evaluated all models (Table 1; Chapter 5) reported by Davidson and Beede (1999)
plus one more (Wu et al., 2000) published after their review. Intake P — milk P was the
most accurate and precise predictor of P excretion for all three datasets developed using
the three datasets mentioned previously. When evaluating the intake P - milk P model
using all three datasets, P excretion was most accurately and precisely predicted for LD
and least for ND (Tables 4, 5, 6, and 7; Chapter 5). Additionally, the accuracy and
precision of this model was reduced when non-lactating animals were added to the
lactating dataset (FD). It was concluded that intake P — milk P, instead of the ASAE
(1996) model should be used to predict P excretion for development of nutrient
management plans. For non—lactating cows the model to predict P excretion was: (g/d) =
P intake (g/d). This relationship was more accurate and precise than the ASAE (1996)
for non-lactating cows so should be used instead of the ASAE (1996) model until a better
model for non-lactating cows is developed.

Additional model evaluation or development research would be beneﬁcial. Intake

P — milk P appears to be useful at predicting P excretion for lactating cows, but may not

142

 

be for non-lactating animals. This model was biased (slope different from one; y-
intercept different from zero) with mean accuracy of —12.8 g/d, which was different from
zero. Precision was 9.0 g/d and intake P — milk P over-predicted P excretion by 26.6%.
Additional model development can be completed from the datasets generated. Prediction
models can be developed separately for non-lactating (ND) and lactating (LD) cows or
for both together (FD) to determine whether or not a more accurate and precise model,

relative to intake P — milk P, can be developed.

143

 

 

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148

APPENDIX A

Digestion Procedure for Feed, Feces, and Milk Phosphorus

Reagents:

Hydrochloric Acid, 4N: Add 667ml ultra pure water to a 1L media bottle. Gradually add
333ml concentrated hydrochloric acid to the water while stirring. Cover with paraﬁlm
and cap. BE SURE TO ADD ACID TO WATER!!!

Concentrated Nitric Acid

Materials:

150ml beakers

100ml volumetric ﬂasks with stoppers

125ml erlenmeyer ﬂasks with #5 rubber stoppers
Watch glasses

Whatman 41 ashless ﬁlter paper (160 mm circles)
Funnels

Pasteur Pipette and bulb

Dispenser pipette capable of dispensing 20ml
Wash bottle ﬁlled with dd H20

Data sheet

Ashing Procedure:

1. Place labeled 150ml beakers in 100°C oven.

2. Let remain in oven for at least 1 hr to evaporate all water attached to beaker.

3. After 1 hr, hot weigh each beaker, one at a time, until all beakers have been
weighed. Place weight (in g) in the “Beaker Wt.” column of weigh sheet. Be
sure to tare scale in between each beaker.

4. Weigh out fecal sample. Place beaker on scale and tare. Weigh out ~1g of .fecal
or feed samples into beakers(Note: for milk digestion, add 10 g milk sample).
Record the sample weight accurately onto “Sample Wt.” column of weigh sheet.
Don’t forget to place the cow id into the appropriate spot on the weigh sheet.

5. Place in 100°C oven for DM determination for 8 hr or overnight.

6. Hot weigh samples, one at a time, until all beakers have been weighed. Record
weights accurately onto “Dry Wt.” column of weigh sheet.

7. Ash at 600°C for 4 hr.

8. Cool in mufﬂe furnace for 1 hr or until temperature in the furnace is <150°C.
Taking glassware out too soon results in breakage.

9. Return beakers to 100°C oven and let ﬁnish cooling for 20 min.

10. Record weights accurately onto “Ashed Wt.” coltunn of weigh sheet.

149

Digestion Procedure (Be sure to wear goggles, lab coat, and nitrile gloves
throughout digestion procedure):

I.

SQMPP’N

10.
11.

12.
13.
14.

15.
16.

Preheat hotplates located in rear ventilation hood. Set hot plate #1 and 2 to
400°C. Set hot plate #3 to 5.

Add 20ml hydrochloric acid, 4N.

Add 5 drops concentrated nitric acid with pasteur pipette.

Swirl to mix.

Place 4 beakers on each hotplate and cover with watch glasses.

Bring to boil and let boil for 7 minutes.

Set beaker off hotplate using beaker tongs, keeping watch glass in place. Do not
remove from hood until cool. Vapors may be harmful if inhaled.

Cool for 2 min and rinse watch glass with dd H20 into corresponding beaker.
Watch glass may be reused at this time.

Let cool an additional 5 min.

Pour beaker contents into labeled 100 ml volumetric ﬂask.

Rinse beaker with 5 — 10 ml dd H20 using wash bottle, 3x, pouring contents into
corresponding ﬂask each time.

Dilute ﬂasks to the mark with dd H20.

Stopper ﬂask and invert 10x to mix.

Filter solution, using Whatman 41 ﬁlter paper, into labeled 125 ml erlenmeyer
ﬂask.

Stopper Flask.

Sample is ready for analysis.

150

 

APPENDIX B

Procedure for Feed, Fecal, and Milk P Assay

Reagents:

Molybdovanadate Reagent: Dissolve 40g Ammonium Molybdate o 4 H20 in 400 ml of
hot dd H20 and cool. Dissolve 2g Ammonium meta-Vanadate in 250 ml hot dd H2O,
cool, and add 450ml Perchloric Acid. Gradually add molybdate solution to vanadate
solution with stirring, and dilute to 2L with dd H2O.

Phosphorus Standard Solution: a) STOCK SOLUTION - (2 mg P/ml) Dissolve 8.788 g
KH2PO4 in dd H2O water and dilute to 1L. b) WORKING SOLUTION - (0.1 mg P/ml)
Dilute 50ml of stock solution to IL with dd H2O.

Materials

16mm X 100 mm test tubes

10ml volumetric ﬂasks with stoppers

P 1000 pipette and tips

P 200 pipette and tips

Repeat pipette

Dispenser pipette capable of dispensing 7 ml water
Wash bottle ﬁlled with dd H2O

96-well plate

Spectrophotometer capable of reading at 400nm

Procedure (Feed and Fecal P):

1. Standard Curve (0.0, 0. 5, 1.0, 1.5, and 2.0 mg P/ml): From the 0.1 mg P/ml
Working Solution pipette the following amounts to create the standard curve:

 

 

Std. ConcentLation ml 0.1 mg P/ml to add
0.0 no addition
0.5 500111
1.0 1000111
1.5 1500111
2.0 2000 ul

Note: Actual concentrations in the final 10ml volume will be the [standard]/10.

2. Unknowns: Mix unknowns by inversion 10x

3. Pipette 1ml of each unknown into a test tube

4. Add 2ml of Molybdovanadate Reagent to each tube, including standards, using
the repeat pipette

5. Dilute Standards to mark with dd H2O using wash bottle and mix by inversion

6. Add 7 ml dd H2O to test tubes and mix by inversion

7. Let standards and unknown stand for 10 minutes

8. Transfer 2001.11 of standards and unknowns, in duplicate, to a 96-well plate (make
sure to ﬁll out “Data Sheet” to be used as a map for the plate

9. Read plate at 400nm on spectrophotometer

151

10. Save ﬁle and print a copy
Procedure (Milk P):

1. Standard Curve (0.0, 0.5, 0. 75, 1.0, and 1.25 mg P/ml): From the 0.1 mg P/ml
Working Solution pipette the following amounts to create the standard curve:

 

 

Std. Concentration ml 0.1 mLP/ml to add
0.0 no addition
0.5 500111
0.75 750111
1.0 1000111
1.25 12501.11

Note: Actual concentrations in the ﬁnal 10ml volume will be the standard
concentration + 10.

2. The rest of the procedure is the same as above.
Calculations:

(mg of P in aliquot ) x (dilution factor ) x 100 I
a) % P in ash = mg of ash in sample

b)%P in sample = (%Pinash)x(%ash in sample)x 100

Aliquots Dilution Factor
0.5 mL from 10 mL = 20
1.0mLfrom10mL = 10

Waste Container Label
0.4 % -Ammonium Molybdate
0.02 % -Ammonium meta-Vanadate
2.5 %- Perchloric Acid ( 70 %)
97 % - Water

REFERENCE: Assoc. Ofﬁcial Analytical Chemists (1990). Method 965.17.

152

APPENDIX C

Digestion for Chromium Analysis

Reagents:

1. Phosphoric acid-manganese sulfate solution:
30ml of 10% w/v, Mn8040 H2O + 970ml of 85% phosphoric acid.
2. 4.5%, w/v, potassium bromate solution.
3. 4000 ppm Ca solution: 11.09 g CaCl2/1iter dd H20.
4. Chromium atomic absorption standard solution (Aldrich Chemical Co., Inc.)

Standards:
1, 2, 3, 4, 5 ppm Cr

Materials

50ml beakers

Watch glasses

100ml volumetric ﬂasks with glass stoppers
Repeat pipette with three different tips

Wash bottle ﬁlled with dd H20

Whatman 41 ashless ﬁlter paper (160 mm circles)
Funnels

Digestion Procedure:
1. Weigh lg samples into 50 ml glass beakers
2. Place samples into 100°C oven overnight for DM determination
3. Ash at 600°C for 1.5 hours
4. Cool until furnace temperature is <150 (usually about 1 hour). Taking
beakers out too soon may cause breakage
Add 3ml of phosphoric acid-manganese sulfate solution; swirl to mix
6. Add 4 ml of 4.5%, w/v, potassium bromate solution; swirl to mix
7. Cover with a watch glass and digest on a previously heated hot plate (set between

2 and 3) until effervescence ceases and purple color appears (usually 5-7 min)
8. Cool for 5 min

9. Pour into labeled 100 ml volumetric ﬂask

10. Rinse, with wash bottle. 3 times with 5 — 10 ml of dd H20 and pour into 100 ml
vol. ﬂask

11. Add 12.5ml of a calcium chloride solution containing 4000 ppm of calcium

12. Dilute to mark with dd H20 with wash bottle, and invert 10X to mix thoroughly

£11

153

13. Filter sample with Let stand overnight to allow suspended material to settle or
ﬁlter
14. Dilute digested sample 1:6 and read on an atomic absorption spectrophotometer

154

APPENDIX D

Urine Speciﬁc Gravity Analysis

. Filter composited urine sample through four layers of cheesecloth into another

clean, dry specimen container.
Rinse original specimen container with water and acetone, let dry and pour
ﬁltered sample back into original container.
Place ~25 ml ﬁltered urine sample into urinometer cylinder (1” clearance from
top of cylinder).
Measure urine temperature (°F) and record on data sheet.
Grasp the hydrometer at its top and slowly insert into cylinder. AVOID
WETTING FLOAT STEM ABOVE LIQUID LINE. Excessive wetting of the
stern will cause the ﬂoat to sink below the true test reading.
Impart a slight spin to the ﬂoat as it is released to release any air bubbles that may
have been attached to the hydrometer.
Read the scale of the urinometer at the lowest portion of the meniscus of the urine
at eye level. Be sure to keep ﬂoat away from sides of cylinder while reading.
NOTE: If it is necessary to read the top of the meniscus, as in the case of opaque
specimens, add 0.002 to the speciﬁc gravity reading to correct for viewing error.
Apply temperature corrections, as necessary and record on data sheet. Note: the
urinometer was calibrated at 60°F, therefore, the SG must be corrected if the
temperature of liquid is not 60°F.

a. Turing - 60°F = ADS)

b. Ansl/5.4°F = An32

c. An52*0.001 = AnS3

d. Corrected SG = Measured SG + Ans;

9. Pour urine sample back into original container.

10.

11.

Rinse cylinder and hydrometer with water and then acetone and let dry between
each sample.
The urine sample is now ready for P analysis.

155

 

APPENDIX E

Procedure for Urine Phosphorus Assay
Materials Needed:

96-well plate

P 200 pipet and tips

P 1000 pipet and tips

Repeatable pipet and tip capable of pipetting 2 ml
12 x 75 mm test tubes and rack

Sharpie

Spectrophotometer

Urine Samples

Reagents Needed:
Trichloroacetic Acid, 20% (TCA)
Molybdate Reagent
Aminonaptholsulfonic acid

Ultra pure water (UPW)

TCA, 20%; Add 200 g trichloracetic acid in a 1 l volumetric ﬂask and ﬁll to the mark w/
UPW. Label a 1 1 glass container; pour solution into container, cover w/ paraﬁlm, and
close lid tightly for storage.

Sulfuric Acid, 10N; Add 250 m1 concentrated sulfuric acid to 750 ml UPW. Mix. Cool.
Label a 1 1 container; pour solution into container, cover w/ paraﬁlm, and close lid tightly
for storage in refrigerator. Don’t forget to add ACID TO WATER!

Molybdate Reagent; Dissolve 25 g ammonium molybdate in 200 ml UPW. Place 300
m1 of Sulfuric Acid, 10N into a 1 I volumetric ﬂask. Add molybdate solution and mix.
Fill to the mark w/ UPW. Label a 1 1 glass container; pour solution into container, cover
w/ paraﬁlm, and close lid tightly for storage. Discard if blanks show a blue color.

Aminonaptholsulfonic acid reagent; This reagent can be purchased as 1-amino-2-

napthol-4-sulfonic acid solution (for Phosphate) from Fisher Scientiﬁc. Check expiration
date prior to use.

156

Assay Procedure:

t—n

.U‘

9°>’.°‘

10.
ll.

12.
13.
14.

. Obtain thawed, room temperature urine sample.

Using a Sharpie, number a test tube for each sample to be analyzed. Each sample
should be done in duplicate.
Shake urine composite vigorously for 10 s and pipette 1 ml urine sample into
labeled test tube.
Repeat Step 4 until all samples to be analyzed have been completed.
Add 2 ml TCA, 20% (1:3 dilution) to each test tube containing 1 ml of urine
sample.
Mix by inversion and let stand for 5 min.
Spin sample at 4000 rpm in a centrifuge for 15 min.
While samples are spinning prepare plate.
Standard Curve:
Pipette 175 111 of the following standard P concentrations into rows A and
H of a 96-well plate:
0 mg/dl (TCA), A and H1;
0.25 mg/dl, A and H2;
0.50 mg/dl, A and H3;
0.75 mg/dl, A and H4;
1.0 mg/dl, A and H5;
1.25 mg/dl, A and H6.
Pipette 175 111 of supernatant, being careful not to disturb pellet, into a well of a
96-well plate (do in duplicate, 175 111 each).
Add 35 111 Molybdate reagent to all wells.
Add 14 111 Aminonaptholsulfonic acid reagent to all wells, agitate the plate
slightly to mix
Add 125 111 UPW, agitate the plate slightly to mix, and let sit for 5 min.
Read standards and unknowns at 660 nm with a spectrophotometer.
Repeat duplicates if the CV is greater than 10%.

157

 

 

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