using the indicator amino acid oxidation technique to study threonine requirements in horses fed

152 Pages·2017·1.72 MB·English

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Theses and Dissertations--Animal and Food
Animal and Food Sciences
Sciences
2015
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ChanHee Mok
University of Kentucky, [email protected]
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Mok, ChanHee, "USING THE INDICATOR AMINO ACID OXIDATION TECHNIQUE TO STUDY THREONINE
REQUIREMENTS IN HORSES FED DIFFERENT FEED COMPOSITIONS" (2015). Theses and Dissertations--
Animal and Food Sciences. 53.
https://uknowledge.uky.edu/animalsci_etds/53
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ChanHee Mok, Student
Dr. Kristine Urschel, Major Professor
Dr. David Harmon, Director of Graduate Studies
USING THE INDICATOR AMINO ACID OXIDATION TECHNIQUE
TO STUDY THREONINE REQUIREMENTS
IN HORSES FED DIFFERENT FEED COMPOSITIONS

________________________________

THESIS
________________________________

A thesis submitted in partial fulfillment of the
requirements for the degree of Master of Science
in the College of Agriculture, Food and Environment
at the University of Kentucky

By
ChanHee Mok
Lexington, Kentucky

Director: Dr. Kristine Urschel, Associate Professor of Animal Sciences
Lexington, Kentucky

2015
Copyright © ChanHee Mok 2015
ABSTRACT OF THESIS

USING THE INDICATOR AMINO ACID OXIDATION TECHNIQUE TO STUDY
THREONINE REQUIREMENTS IN HORSES FED DIFFERENT FEED COMPOSITIONS

Threonine has been reported to be the second limiting amino acid in typical equine diets,
but its actual requirement has not been determined in horses. The indicator amino acid oxidation
(IAAO) method has been successfully used for evaluating amino acid metabolism and
requirements in animals and humans. The objective of this research was to use the IAAO method
to estimate threonine requirements in mature horses fed two different feed compositions. In the
first study, 6 Thoroughbred mares (4 – 16 years old) received each of 6 levels of threonine intake
in a high concentrate diet, in a randomly determined order. The experimental diets consisted of
concentrate at 0.95% of body weight (BW), and chopped timothy hay at 0.95% of BW, and met
or exceeded NRC recommendations for all nutrients. Threonine intakes for the 6 treatments were
45, 56, 67, 79, 90 and 102 mg/kg BW/d and were generated by mixing the two experimental
concentrates, containing 2.9 and 14.8 g threonine/kg diet, in different ratios. In the second study,
horses received each of 6 levels of threonine intake, 41, 51, 61, 70, 80, and 89 mg/kg BW/d, in a
high forage diet, in a randomly determined order. The experimental diet was concentrate at 0.4%
of BW, and chopped timothy hay at 1.6% of BW. Study periods for each study were 7-d long and
on d 6, blood samples were collected before and 90 min after feeding to measure amino acid
concentrations using HPLC. On d 7, horses underwent IAAO procedures, which included a two
hour primed, constant intravenous infusion of [13C]sodium bicarbonate to measure total CO
2
production and a four hour primed, constant oral administration of [1-13C]phenylalanine to
estimate phenylalanine oxidation to CO . Blood and breath samples were collected to measure
2
blood [13C]phenylalanine, using GC-MS analysis, and breath 13CO enrichment, using an infrared
2
isotope analyzer. Experimental data were analyzed using a one-way ANOVA, including
orthogonal linear and quadratic contrasts, in the mixed procedures of SAS version 9.3, with
treatment as the fixed effect and horse nested in treatment as the random effect. Statistical
significance was declared at P<0.05. Phenylalanine oxidation rates were not affected by increased
threonine concentration in a high concentrate diet (P>0.05). Increasing threonine intake levels in
a high forage diet did not affect plasma phenylalanine oxidation by the ANOVA test (P>0.05) but
resulted in a linear decrease in phenylalanine oxidation (P=0.04) without a breakpoint by the
orthogonal linear contrast. Threonine requirements are still unknown in mature horses fed either
high concentrate or high forage diet. The present studies were the first attempt to evaluate
threonine requirements in horses by the indicator amino acid oxidation method.
Keywords: Equine, Maintenance, Requirement, Threonine
ChanHee Mok
September 29th, 2015
USING THE INDICATOR AMINO ACID OXIDATION TECHNIQUE TO STUDY
THREONINE REQUIREMENTS IN HORSES FED DIFFERENT FEED
COMPOSITIONS

By
ChanHee Mok

Dr. Kristine Urschel
Director of Thesis
Dr. David L. Harmon
Director of Graduate Studies
September 29th, 2015
ACKNOWLEDGEMENTS

First, I would like to extend my deepest gratitude to Dr. Urschel. She gave me a
chance to start equine research, which is currently scarce in Korea. Thanks to her, I have
learned a lot of knowledge and research skills that are needed in this filed.
Of course, I would like to thank Dr. BeobGyun Kim, who is a very influential
mentor and a great scientist, for stimulating me to pursue further education in animal
sciences and encouraging me to study abroad.
Also, I would like to acknowledge lots of helps and supports during my study
here at UK: Tammy, our lab technician, who helped me in any situations; the sweetest
fellow graduate students, who encouraged each other; the staffs of Maine Chance farm
and undergraduate students, who gave lots of help at the barn; the research horses, who
have been poked. I am sincerely grateful to Drs. Lawrence, Matthews, Lindemann, and
Adedokun for supporting me to go further steps.
Finally, a huge thank you goes out to my father and mother.

iii
Table of contents
Acknowledgments ......................................................................................................... iii
List of tables ................................................................................................................. vii
List of figures ................................................................................................................ ix
Chapter 1. Introduction ......................................................................................................1
Chapter 2. Literature review ..............................................................................................3
Crude protein and amino acid requirements in horses ....................................................3
Importance of providing appropriate amounts of dietary protein ...................................6
Consequences of overfeeding dietary protein ............................................................6
Low protein diets and supplementing nitrogen sources ...........................................11
Protein digestion in horses ............................................................................................13
Protein degradation ...................................................................................................13
Amino acid absorption .............................................................................................15
Amino acid bioavailability .......................................................................................16
Effect of feed composition on protein digestibility ..................................................19
Blood amino acid pool ..............................................................................................20
Limiting amino acids in equine diet ..............................................................................21
Threonine metabolism in animals .................................................................................23
Biological role of threonine ......................................................................................23
Threonine degradation ..............................................................................................25
Factors affecting threonine requirements ......................................................................26
Endogenous losses ....................................................................................................26
Dietary composition .................................................................................................27
Physiological state ....................................................................................................28
Methods in evaluating amino acids requirements .........................................................29
Calculations ..............................................................................................................29
Factorial approach ..............................................................................................29
Amino acids composition in the animal tissue ...................................................30
Dose-response feeding trials .....................................................................................32
Growth rate .........................................................................................................37
Nitrogen balance .................................................................................................37
iv
Blood amino acid concentration .........................................................................38
Blood urea concentration ....................................................................................39
Isotope methods ........................................................................................................40
Direct amino acid oxidation method ..................................................................42
Indirect amino acid oxidation method ................................................................43
Phenylalanine degradation ..................................................................................43
Conclusion ....................................................................................................................46
Chapter 3. Rationale and objectives of the reported study ................................................47
Chapter 4. Threonine requirements in horses fed a high concentrate diet .......................50
I. Introduction ...............................................................................................................50
II. Materials and methods .............................................................................................53
Animals, housing, and feeding .................................................................................53
Diets and ingredients ................................................................................................54
Study design and procedures ....................................................................................60
Sample collection .....................................................................................................61
Sample analyses ........................................................................................................62
Feed samples .......................................................................................................62
Pre-, post-feeding blood samples ........................................................................62
Breath and blood samples from isotope procedure ............................................63
Calculations ..............................................................................................................64
Statistical analyses ....................................................................................................66
III. Results .......................................................................................................................66
Plasma amino acids concentrations ..........................................................................69
Plasma urea nitrogen, glucose and insulin concentrations .......................................76
Whole-body phenylalanine kinetics .........................................................................78
IV. Discussion .................................................................................................................81
Chapter 5. Threonine requirements in horses fed a high forage diet ...............................89
I. Introduction ...............................................................................................................89
II. Materials and methods ...............................................................................................91
Experimental diets ....................................................................................................91
III. Results .....................................................................................................................95
v
Plasma amino acids concentrations ..........................................................................95
Plasma urea nitrogen, glucose and insulin concentrations .....................................103
Whole-body phenylalanine kinetics .......................................................................105
IV. Discussion .............................................................................................................108
Chapter 6. Overall summary and direction for future studies ........................................116
Literature cited ............................................................................................................125
Vita ..............................................................................................................................139

vi
List of tables
Table 2.1 Maintenance crude protein requirements in horses at various physiological
states ..................................................................................................................5
Table 2.2 Additional crude protein requirements in horses at various physiological
states ..................................................................................................................5
Table 2.3 Summary of ammonia emissions from the U.S. animal husbandry operations
in 2002 ...............................................................................................................8
Table 2.4 Estimated threonine requirement based on the amino acid composition of
equine muscle ..................................................................................................31
Table 2.5 Estimated threonine requirement based on the amino acid composition of
equine milk ......................................................................................................31
Table 2.6 Estimated indispensable amino acid in adult human using different methods
...........................................................................................................................36
Table 4.1 Composition of low threonine concentrate ...................................................56
Table 4.2 Composition of high threonine concentrate ..................................................57
Table 4.3 Nutrient composition of the feeds used for generating the treatments with 6
levels of threonine intake ................................................................................58
Table 4.4 Feed composition for generating 6 levels of dietary threonine in a high
concentrate diet ................................................................................................59
Table 4.5 Daily nutrient intake levels in the experimental diet containing 1:1 ratio of
forage to concentrate for each treatment .........................................................68
Table 4.6 Plasma amino acids concentrations in mature horses that were receiving
graded levels of dietary threonine in 1:1 ratio of forage to concentrate ..........72
Table 4.7 Plasma amino acids, urea nitrogen, glucose, and insulin concentrations pre-
feeding in mature horses that were receiving graded levels of dietary threonine
in 1:1 ratio of forage to concentrate ................................................................74
Table 4.8 Plasma amino acids, urea nitrogen, glucose, and insulin concentrations post-
feeding in mature horses that were receiving graded levels of dietary threonine
in 1:1 ratio of forage to concentrate ................................................................75
Table 4.9 Plasma metabolites concentrations in mature horses that were receiving
graded levels of dietary threonine in 1:1 ratio of forage to concentrate ..........77
Table 4.10 Whole-body phenylalanine kinetics in mature horses that were receiving
graded levels of dietary threonine in 1:1 ratio of forage to concentrate ..........80
Table 5.1 Feed composition for generating 6 levels of dietary threonine in a high forage
diet ...................................................................................................................93
Table 5.2 Daily nutrient intake levels in the experimental diet containing 4:1 ratio of
forage to concentrate for each treatment .........................................................94
Table 5.3 Plasma amino acids concentrations in mature horses that were receiving
graded levels of dietary threonine in 4:1 ratio of forage to concentrate ..........99
vii