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This is to certify that the

dissertation entitled

Amino Acid Trans-Membrane Transport, Net Uptake, and
Intracellular Kinetics in the Porcine Mammary Gland
During Lactation.

presented by

Xinfu Guan

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Animal Science

 

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Major professor

 

Date [0 " a 7'00

 

MS U is an Affirmative Action/Equal Opportunity Institution 0-12771

 

LlBRARY
Michigan State

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at--- _. _

AMINo ACID TRANS-MEMBRANE TRANSPORT, NET UPTAKE, AND
INTRACELLULAR KINETICS IN THE PORCINE MAMMARY GLAND
DURING LACTATION
By

XINFU GUAN

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY

Department of Animal Science

2000

ABSTRACT

AMINO ACID TRANS-MEMBRANE TRANSPORT, NET UPTAKE, AND
INTRACELLULAR KINETICS IN THE PORCINE MAMMARY GLAND
DURING LACTATION

By

Xinfu Guan

The lactating porcine mammary gland (MG) has a large demand for indispensable amino
acids (IAA) to meet a high rate of milk protein synthesis. Mammary trans-membrane
transport and net uptake of IAA can be rate-limiting for the synthesis of milk protein.
Endogenous contributions of IAA mobilized from body protein, their dietary needs for
milk synthesis by the MG, and ideal dietary IAA pattern of the lactating sow have not
been quantified. In the first study, 16 lactating sows were fed one of four isocaloric diets
varying in protein concentration (7.8, 13.0, 18.2, and 23.5 %). The sows were fitted with
catheters in the carotid artery and the main mammary vein. On d 10, 14, 18, and 22 of
lactation, arterial and venous blood samples were simultaneously obtained every 30 min
over 6 h. Milk yield on d 11 and 21 of lactation was estimated by the D20 dilution
method. Results were summarized as follows. (1) Dietary protein concentration affected
mammary IAA uptake pattern, but did not affect the IAA profile in milk. Uptake of
plasma Arg and the branched-chain AA by the MG exceeded their output in milk. (2)
Endogenous IAA contributions, defined by mammary uptake when intake was
extrapolated to zero, were 10.5, 2.1, 5.6, 6.4, and 4.8 g/kg body weight loss, respectively,

for Lys, Met, Phe, Thr, and Val. (3) Dietary needs of truly digestible IAA for milk

synthesis, defined by their maximal mammary uptake, were 28.8, 10.0, 20.2, 6.9, 12.1,
13.9, 4.5, and 22.4 g/kg litter weight gain, respectively, for Arg, His, Lys, Met, Phe, Thr,
Trp, and Val. In the second study, 9 lactating sows were fed ad libitum one of three diets
varying in Lys and Val concentrations (4.93, 9.87; 9.71,lO.15; and 9.76, 13.37 g/kg diet
as-fed, respectively, in the Lys-deficient diet (LD), the positive control diet (PC), and the
Val-excess diet (VE)). The jugular vein, the anterior main mammary vein, and the
carotid artery were cannulated. A mixture of the labeled IAA ([2-‘5N]-L-LySoHCl, [S-
methyl-2H3]-L-Met, and [1-13C]-L-Val) was infused continuously via the jugular vein for
20.5 h on d 18 of lactation; meanwhile, milk, carotid arterial, and mammary venous blood
samples were obtained. Milk yield, milk protein content, and litter weight gain were
decreased in the LD, but were not affected in the VE. In the LD, inward trans-membrane
transport (Fmg,a) and outward trans-membrane transport (Fv,mg) across the MG were
decreased for Lys, and tended to be decreased for Met, resulting in decreased net uptake
of plasma Lys and Met by the MG. In the VE, Fmg,a for Val was decreased numerically
and Fv,mg for Val was completely blocked, resulting in numerically increased net uptake
of plasma Val by the MG. However, Fv,mg for Lys was numerically increased to a
greater extent than F mg,a for Lys, resulting in a decreased net uptake of plasma Lys by
the MG. Protein synthesis and breakdown were decreased in the MG in the LD, resulting
in decreased net balance of mammary protein. Protein breakdown was decreased to a
greater extent compared to protein synthesis in the VE. In conclusion, endogenous IAA
contributions and dietary IAA needs for milk synthesis can be defined by their mammary
uptake. Moreover, trans-membrane transport, net uptake, and the intracellular kinetics of

IAA in the MG are tightly regulated by dietary AA regime.

To my wife, daughter, and family

for their love, support, and trust throughout my life

iv

ACKNOWLEDGMENTS

I would like to express my great appreciation to my advisor Dr. Nathalie Trottier for her
guidance, challenge, and support. Without her continuous encouragement and trust, I
could not have accomplished this work. I would like to thank my committee members:
Dr. David Beede, Dr. Michael Orth, Dr. Dale Romsos, Dr. Dale Rozeboom, and Dr.
Allen Tucker for their guidance and support. Special thanks also goes to Dr. Brian
Bequette for his great input and collaboration on amino acid kinetics, Dr. Pao Ku for his
great technical support, Dr. Kent Ames for his excellent surgical skills, Dr. Robert
Templeman for his statistical advice, and Dr. Karen Chou, Dr. James Pettigrew, Barb
Sweeney, Dr. Feng Yang, Dr. Mel Yokoyarna, and Dr. Adroaldo Zanella for their
fi'iendship and encouragement. I would like to thank my fellow graduate students Lara
Andersen, Emily Otto, Juliana Perez-Laspiur, and Janet Snow for their assistance. I
would like to thank Alan Snedegar and his team at Michigan State University Swine
Teaching and Research Farm for their help and the Department of Animal Science Lab
support staff, especially Robert Burnett, Jane Link, Ron Southwick, and Jackie Ying for
their support. In addition, I would like to express my sincere recognition to Michigan
State Agricultural Experiment Station, Minnesota Soybean Research Promotion Council,
Rowett Research Institute, and Scottish Agricultural Office for providing research
funding. Finally, I am indebted greatly to my wife Zhuo Yang, my lovely daughter Yue

Guan, and my parents for their love, encouragement, and support.

TABLE OF CONTENTS

List of Tables ....................................................................................... viii
List of Figures ........................................................................................ xi
List of Abbreviations ............................................................................. xiii
Introduction .......................................................................................... 1

Chapter 1. Effects of Dietary Protein Concentration on the Amino Acid Profile in

Milk and the Amino Acid Uptake Pattern by the Lactating Porcine Mammary

Gland

Abstract .................................................................................. 5
Introduction ............................................................................. 7
Materials and Methods .............................................................. 10
Results .................................................................................. 16
Discussion .............................................................................. 21

Implications ........................................................................... 29
Literature Cited ...................................................................... 30

Chapter 2. Quantitation of Indispensable Amino Acids Mobilized from Body

Protein Reserves of Extra-mammary Tissues in the Lactating Sow

Abstract ................................................................................. 60
Introduction ........................................................................... 62
Materials and Methods .............................................................. 64

vi

Results ................................................................................... 70
Discussion ............................................................................... 74

Literature Cited ...................................................................... 80

Chapter 3. Uptake of Plasma Amino Acids by the Lactating Porcine Mammary

Glands: A Novel Approach to Define Their Needs for Milk Synthesis

Abstract ................................................................................. 94
Introduction ........................................................................... 97
Materials and Methods ............................................................ 101
Results ................................................................................. 106
Discussion ............................................................................ 109
Literature Cited ..................................................................... 120

Chapter 4. Effects of Dietary Lysine Deficiency or Valine Excess on Trans-
membrane Transport of Indispensable Amino Acids and Intracellular Protein

Synthesis and Breakdown in the Lactating Porcine Mammary Gland

Abstract ............................................................................... 149
Introduction .......................................................................... 151
Materials and Methods ............................................................ 156
Results ................................................................................. 169
Discussion ............................................................................. 177
Literature Cited ..................................................................... 184
Chapter 5. Summary and Conclusions ....................................................... 218

vii

LIST OF TABLES

TABLE 1-1. Composition of diets for lactating sows (%, as-fed basis) ................... 36
TABLE 1-2. Effects of dietary protein concentration on lactation performance. . .....38

TABLE 1-3. Effects of dietary protein concentration and stage of lactation on milk
composition (%, Meal/kg) ....................................................... 40

TABLE 1-4. Effects of dietary protein concentration and stage of lactation on the
concentrations (mmol/L) of indispensable amino acids in defatted porcine
milk ................................................................................. 41

TABLE 1-5. Effects of dietary protein concentration and stage of lactation on output
(g/d) of indispensable amino acids in porcine milk ........................... 42

TABLE 1-6. Effects of dietary protein concentration on the amino acid profile of
defatted porcine milk ............................................................. 43

TABLE 1-7. Effects of stage of lactation on the amino acid profile of defatted porcine
milk .................................................................................. 44

TABLE 1-8. Effects of dietary protein concentration and stage of lactation on arterial
concentrations (umol/L) of plasma indispensable amino acids in lactating
sows ................................................................................. 45

TABLE 1-9. Effects of dietary protein concentration and stage of lactation on mammary
arterio-venous (umol/L) differences of plasma indispensable amino acids
in lactating sows ................................................................... 47

TABLE 1-10. Effects of dietary protein concentration and stage of lactation on
mammary extraction rates (%) of plasma indispensable amino acids in
lactating sows ...................................................................... 49

TABLE l-ll. Effects of dietary protein concentration on the mammary uptake patterns
for plasma indispensable amino acids. ......................................... 51

TABLE 1-12. Effects of stage of lactation on the mammary uptake patterns for plasma
indispensable amino acids ........................................................ 53

TABLE 1-13. Comparison of the ratios (wt/wt) of other indispensable amino acids to
lysine in porcine milk ............................................................. 55

TABLE 1-14. Comparison of mammary uptake ratios (wt/wt) of other indispensable
amino acids to lysine in the lactating sow ..................................... 56

TABLE 2-1. Composition of diets for lactating sows (g/kg diet, as-fed basis). . . . . . . . ....84

viii

TABLE 2-2. Initial characteristics of sows and litters on d l of lactation .................. 85
TABLE 3-3. Sow and litter performance over a 21-d lactation period ..................... 86
TABLE 2-4. Estimation of mammary plasma flow rate by the Fick principle ............ 87

TABLE 3-5. Regressions of log mammary A-V differences of plasma IAA against daily
intakes of IAA over a 21-d lactation period .................................... 88

TABLE 2-6. Contributions of endogenous IAA predicted by the mammary A-V
differences of plasma IAA model ............................................... 89

TABLE 2-7. Contributions of endogenous IAA estimated by the body composition
change approach .................................................................... 90

_ TABLE 2-8. Contributions of endogenous IAA derived by the factorial approach ...... 91

TABLE 2-9. Comparisons of the contributions of endogenous IAA estimated by three
methods ............................................................................. 92

TABLE 3-1. Regression of log mammary A-V differences of plasma IAA against daily
intakes of dietary total IAA over a 21-d lactation period .................. 144

TABLE 3-2. Dietary needs of TDIAA for milk production predicted at the maximal
mammary uptake of plasma IAA ............................................... 145

TABLE 3-3. Daily intake of dietary total IAA (g/d) predicted at the maximal log
mammary A-V difference of plasma IAA over a 21-d lactation
period .............................................................................. 146

TABLE 34. Dietary needs of TDIAA for milk production derived by the factorial
approach .......................................................................... 147

TABLE 3-5. Ideal ratios of other IAA to lysine for maintenance, milk synthesis, and
endogenous contribution ........................................................ 148

TABLE 4-1. Distinguishing characteristics of cationic amino acid transport
systems ........................... 195

TABLE 4-2. Distinguishing characteristics of neutral amino acid transport systems...l96
TABLE 4-3. Composition of experimental diets (as-fed basis) ........................... 197

TABLE 44. Effects of dietary amino acid regime on productive performance of the
lactating sow over a 21-d lactation period ................................... 198

TABLE 4-5. Effects of dietary amino acid regime on arterial concentrations of plasma
amino acids and urea in the lactating sow .................................... 199

ix

TABLE 4-6. Effects of dietary amino acid regime on mammary extraction rates of
plasma amino acids and urea in the lactating sow ........................... 200

TABLE 4-7. Effects of dietary amino acid regime on kinetics parameters of plasma
lysine across the lactating porcine mammary gland ........................ 201

TABLE 4-8. Effects of dietary amino acid regime on kinetics parameters of plasma
methionine across the lactating porcine mammary gland .................. 202

TABLE 4-9. Effects of dietary amino acid regime on kinetics parameters of plasma
valine across the lactating porcine mammary gland ........................ 203

TABLE 4-10. Effects of dietary amino acid regime on fractional ratios of plasma lysine
kinetics across the lactating porcine mammary gland ..................... 204

TABLE 4—11. Effects of dietary amino acid regime on fractional ratios of plasma
methionine kinetics across the lactating porcine mammary gland ....... 205

TABLE 4-12. Effects of dietary amino acid regime on fractional ratios of plasma valine
kinetics across the lactating porcine mammary gland ..................... 206

TABLE 4-13. Effects of dietary amino acid regime on mammary protein synthesis and
breakdown based upon protein equivalent methionine fluxes ............ 207

APPENDIX A. Glossary ........................................................................ 192

LIST OF FIGURES

FIGURE 1-1. Effects of dietary protein concentration on the mammary uptake
proportions of individual dispensable amino acids to total individual
dispensable amino acids ......................................................... 57

FIGURE 1-2. Comparison of the amino acid profile in porcine milk and the amino acid
uptake pattern by the lactating porcine mammary gland .................... 58

FIGURE 1-3. Mammary arterio—venous difference of plasma lysine or valine affected by
their intake ..................................................................... 59

FIGURE 2-1. Relationship between the log mammary A-V difference of plasma lysine
and daily intake of dietary lysine over a 21-d lactation period. . . . . . . . ..93

FIGURE 3-1. Comparison between uptake of plasma IAA by the porcine mammary
glands and their output in milk over a 21-d lactation ..................... 124

FIGURE 3-2a. The relation between log mammary A-V differences of plasma Arg (Y)
and daily intakes of dietary total Arg (X) over a 21-d lactation period... 126

FIGURE 3-2b. The relation between log marmnary A-V differences of plasma His (Y)
and daily intakes of dietary total His (X) over a 21-d lactation period...128

FIGURE 3-2c. The relation between log mammary A-V differences of plasma Lys (Y)
and daily intakes of dietary total Lys (X) over a 21-d lactation period. 130

FIGURE 3-2d. The relation between log mammary A-V differences of plasma Met (Y)
and daily intakes of dietary total Met (X) over a 21-d lactation period.
...................................................................................... 132

FIGURE 3-2e. The relation between log mammary A-V differences of plasma Phe (Y)
and daily intakes of dietary total Phe (X) over a 21-d lactation period..134

FIGURE 3-2f. The relation between log mammary A-V differences of plasma Thr (Y)
and daily intakes of dietary total Thr (X) over a 21-d lactation period. 136

FIGURE 3-2g. The relation between log mammary A-V differences of plasma Trp (Y)
and daily intakes of dietary total Trp (X) over a 21-d lactation period. 138

FIGURE 3-2h. The relation between log mammary A-V differences of plasma Val (Y)
and daily intakes of dietary total Val (X) over a 21-d lactation period... 140

FIGURE 3-3. A 3-D response surface of dietary truly digestible lysine requirements (Y)
for lactating sows fitted by the multiple regression model: Y = 0.83 +

xi

20.20 litter weight gain — 7.28 body weight loss (P < 0.0001, R2 = 0.64).
...................................................................................... 142

FIGURE 3-4. Dietary truly digestible lysine requirements of lactating sows derived from
the maximal mammary uptake model and the NRC model (1998) at
different litter weight gain and body weight loss over a 21-d lactation
period ............................................................................. 143

FIGURE 4-la. Enrichments of plasma free- and milk casein-bound lysine in the sows
fed the lysine-deficient diet .................................................... 208

FIGURE 4-lb. Enrichments of plasma free- and milk casein-bound lysine in the sows
fed the positive control diet ................................................... 209

FIGURE 4-lc. Enrichments of plasma free- and milk casein-bound lysine in the sows
fed the valine-excess diet ...................................................... 210

FIGURE 4-ld. Enrichments of plasma free- and milk casein-bound methionine in the
sows fed the lysine-deficient diet ............................................. 211

FIGURE 4-1e. Enrichments of plasma free- and milk casein-bound methionine in the
sows fed the positive control diet ............................................. 212

FIGURE 4-1f. Enrichments of plasma free- and milk casein-bound methionine in the
sows fed the valine-excess diet ................................................ 213

FIGURE 4-1g. Enrichments of plasma free- and milk casein-bound valine in the sows
fed the lysine-deficient diet .................................................... 214

FIGURE 4-1h. Enrichments of plasma free- and milk casein-bound valine in the sows
fed the positive control diet .................................................... 215

FIGURE 4-1i. Enrichments of plasma free- and milk casein-bound valine in the sows fed
the valine-excess diet ...................................................... 216

FIGURE 4-2. A three-compartmental model of amino acid kinetics across the lactating
porcine mammary gland ....................................................... 217

xii

LIST OF ABBREVIATIONS

Arginine ............................................................................................. Arg
Hstidine .............................................................................................. His
Isoleucine ............................................................................................ Ile
Leucine .............................................................................................. Leu
, Lysine ................................................................................................ Lys
Methionine .......................................................................................... Met
Phenylalanine ....................................................................................... Phe
Threonine ............................................................................................ Thr
Tryptophan ......................................................................................... Trp
Tyrosine ............................................................................................. Tyr
Valine ................................................................................................ Val
Nitrogen ............................................................................................... N
Crude protein ......................................................................................... CP
Indispensable amino acids ........................................................................ IAA
Branched-chain amino acids ................................................................... BCAA
Mammary gland .................................................................................... MG

xiii

Arterio-venous ..................................................................................... A-V

Nitrogen ............................................................................................... N
Low protein diet ..................................................................................... LP
Appropriate protein diet ............................................................................ AP
Median protein diet ................................................................................. MP
. High protein diet .................................................................................... HP
Truly digestible indispensable amino acids ................................................ TDIAA
Contributions of endogenous indispensable amino acids ................................... IAAc
Mammary aIterio-venous difference model ............................................... MAVD
Body composition change approach ......................................................... BCCA
Lysine-deficient diet. ............................................................................... LD
Positive-control diet ................................................................................ PC
Valine-excess diet ................................................................................... VE

xiv

INTRODUCTION

The porcine mammary gland has a large demand for indispensable amino acids (IAA) to
meet a high rate of milk protein synthesis. For example, a sow nursing a litter of 10 or
more piglets has daily output of protein and lysine in milk of 550 and 42 g/d,
respectively. The lactating sow must be provided with adequate amounts of IAA in
optimal proportions to maximize productive performance and utilization efficiency of
dietary protein. The total dietary requirement of an IAA defined by the factorial
approach is comprised of dietary needs for three components: maintenance need, milk
synthesis, and body protein gain (or loss). However, none of these individual component
needs have been defined experimentally. The total dietary requirement of an IAA
estimated by the empircal method is affected by the endogenous contributions of this
IAA mobilized from body protein. Since both dietary and endogenous IAA can be used
to meet the high rate of milk protein synthesis, low intake of an IAA with high
endogenous contribution may maintain a similar level of milk yield as high intake of this
IAA with low (or zero) endogenous contribution. Dietary needs of IAA other than lysine
have been derived by multiplying dietary lysine needs and ideal dietary IAA patterns.
The IAA profile in milk is considered an ideal dietary IAA pattern for milk synthesis
(ARC 1981, NRC 1998). However, this may be inappropriate for arginine and branched-
chain AA because their uptake by the mammary gland exceeds their output in milk
(Trottier et al. 1997). Mammary IAA uptake pattern has been proposed as a reference for
an ideal dietary IAA pattern for milk synthesis by the mammary gland (Trottier and Guan

2000). The uptake of lysine by mammary tissue is inhibited by cationic and branched-

chain AA in vitro (Hurley et a1. 2000, Sherman et a1. 1994), suggesting that intake of
protein or dietary AA may regulate the uptake of IAA by the mammary gland in vivo.
Moreover, the synthesis of milk protein may be restricted by the intracellular free IAA
availability, which is mainly controlled by their mammary net uptake (Mepham 1982).
Net uptake of an individual IAA is determined by its inward and outward trans-
membrane transport across the mammary epithelium. There is substantial protein
turnover in the lactating goat mammary gland (Oddy et al. 1988). Obviously, increased
V protein synthesis and/or decreased protein breakdown would result in an increase in net
balance of protein. Protein turnover in the porcine mammary gland has not been
investigated. Therefore, the objectives in this study were to: (l) evaluate the effects of
dietary protein concentration on the AA profile in milk and mammary uptake pattern of
plasma IAA; (2) quantify the contributions of endogenous IAA mobilized from body
protein in lactating sows; (3) estimate dietary needs of truly digestible IAA by the
mammary gland for milk production in the lactating sow; and (4) evaluate if dietary
lysine deficiency or valine excess affect trans-membrane transport of IAA (lysine,
methionine, and valine) and intracellular protein synthesis and breakdown in the lactating

porcine mammary gland.

The following chapters of this dissertation describe two studies that address these
objectives. In the first study, lactating sows were provided ad libitum access to one of
four diets varying in dietary protein concentration to establish a regression of mammary

IAA arterio-venous difference and intake of dietary IAA. The results are summarized in

Chapters 1, 2, and 3. In the second study, lactating sows were provided ad libitum access
to one of three diets varying only in Lys and Val (i.e., Lysine-deficient, positive control,
and valine-excess). The results are summarized in Chapter 4. In Chapter 1, the effects of
dietary protein concentration on the IAA profile in milk and the mammary IAA uptake
pattern are described. In Chapter 2, the endogenous IAA contributions from body protein
loss are quantified using a mammary IAA arterio-venous model, and are validated based
on both nitrogen balance and the AA composition of body protein, and a factorial
, approach. In Chapter 3, dietary needs of truly digestible IAA for milk synthesis by the
mammary gland are determined using their maximal mammary IAA uptake, and further
validated by a factorial approach. In Chapter 4, inward and outward trans-membrane
transport, net uptake, and intracellular kinetics of IAA (lysine, methionine, and valine)
across the mammary epithelium are defined using a three-compartmental model. Chapter

5 contains the summary and conclusions of this dissertation.

LITERATURE CITED

Bequette, B. J., Hanigan, M. D., Calder, A. G., Reynolds, C. K., Lobley, G. E. &
MacRae, J. C. (2000) Amino acid exchange by the mammary gland of lactating
goats when histidine limits milk production. J. Dairy Sci. 83: 765-775.

Biolo, G., Fleming, R. Y., Maggi, S. P. & Wolfe, R. R. (1995) Trans-membrane
transport and intracellular kinetics of amino acids in human skeletal muscle. Am.
J. Physiol. 268: E75-84.

Hurley, W. L., Wang, H., Bryson, J. M. & Sherman, D. B. (2000) Lysine uptake by
mammary gland tissue from lactating sows. J Anim Sci 78: 391-395.

3

Mepham, T. B. (1982) Amino acid utilization by lactating mammary gland. J. Dairy Sci.
65: 287-298.

NRC. (1998). Nutrient requirements of swine. 10th ed, National Academy Press,
Washington, DC.

Oddy, V. H., Lindsay, D. B. & Fleet, I. R. (1988) Protein synthesis and degradation in
the mammary gland of lactating goats. J. Dairy Res. 55: 143-154.

Sherman, D. B., McNeillie, S. A., Jamieson, E. A. & Calvert, D. T. (1994) Lysine
transport in lactating rat mammary tissue: evidence for an interaction between
cationic and neutral amino acids. Acta Physiol. Scand. 151: 461-466.

Trottier, N. L. & Guan, X. F. (2000) Research paradigms behind amino acid
requirements of the lactating sow: Theory and future application. J. Anim. Sci.
78(Suppl. 3): 48-58.

Trottier, N. L., Shipley, C. F. & Easter, R. A. (1997) Plasma amino acid uptake by the
mammary gland of the lactating sow. J. Anim. Sci. 75: 1266-1278.

CHAPTER 1

Effects of Dietary Protein Concentration on the Amino Acid Profile in Milk and the

Amino Acid Uptake Pattern by the Lactating Porcine Mammary Gland

ABSTRACT Objectives of this study were to evaluate the effects of dietary protein
concentration on the amino acid (AA) profile in milk and on mammary AA uptake
pattern to define an ideal pattern of indispensable amino acids (IAA) for milk synthesis
by the lactating sow. Sixteen Landrace x Yorkshire lactating sows were provided ad
libitum access to one of four isocaloric diets varying in protein concentration (7.8, 13.0,
18.2, and 23.5 % CP) with a similar pattern of IAA. The sows were fitted with catheters
in the carotid artery and the main mammary vein on d 4 of lactation. On d 10, 14, 18, and
22 of lactation, arterial and venous blood samples were obtained simultaneously every 30
min over 6 h. Milk yield on d 11 and 21 of lactation was estimated by the D20 dilution
method. Sows fed the diet containing 7.8% CP (the low protein diet, LP) had decreased
loin eye area, milk yield, and litter weight gain over a 21-d lactation period. Dietary
protein concentration and stage of lactation did not affect milk composition except
protein content. The content of true protein in milk decreased in the sows fed the LP and
on d 18 of lactation, which decreased concentrations of IAA in defatted milk. The AA
profile in milk, expressed either as the proportion of individual IAA to true protein or to
Lys in milk, was not affected by dietary protein concentration or stage of lactation.
Arterial concentrations and mammary arterio—venous (A-V) differences of plasma IAA
increased linearly with increasing dietary protein concentration and increasing day of

lactation (P < .05). Mammary A-V differences and extraction rates of plasma IAA

tended (P < 0.10) to be greatest in sows fed the diet containing 18.2% CF and d 18 of
lactation. Mammary uptake pattern, defined either as the proportions of individual IAA
to the total IAA or to Lys taken up by the mammary gland, was affected by dietary
protein concentration (P < .05). Mammary uptake proportions of branched-chain AA
(Ile, Leu, and Val) increased with increasing dietary protein concentration, while those of
Lys, Met, Phe, and Thr decreased. The mammary uptake pattern was not altered across
stage of lactation. In conclusion, mammary uptake ratios for Arg and the branched-chain
V AA were greater than their ratios in milk. The mammary uptake pattern of plasma IAA
under appropriate intake of protein would represent an ideal pattern of IAA for milk
synthesis by the mammary gland because they are presumably required for incorporation
into milk protein and other metabolic pathways. The ideal dietary AA pattern for milk
synthesis based only on the AA profile in milk seems inappropriate, especially for Arg

and the branched-chain AA.

KEY WORDS: amino acids omilk omammary glands ouptake olactation osows

INTRODUCTION

Swine must be provided with adequate amounts of indispensable amino acids (IAA) in
optimal proportions for maximal growth, reproduction, and lactation. The concept of an
ideal protein has been employed to establish dietary requirements for IAA and to balance
dietary IAA in diets for swine (ARC 1981, NRC 1988, NRC 1998). An ideal protein,
i.e., the ideal pattern of dietary IAA, is defined as the optimal balance among dietary IAA
and the relative proportion of IAA-nitrogen (N) to dispensable amino acid (AA)-N,
which corresponds to dietary requirements of IAA by the animal (ARC 1981, NRC
1998). Four sets of ideal patterns of IAA for maintenance, protein accretion, milk
synthesis, and body tissue, respectively, have been recommended by the NRC (1998) to
constitute the entire ideal pattern of dietary IAA for swine for particular productive
purposes. The ideal pattern of IAA for milk synthesis by the lactating sow has been

derived from the AA profile in milk (NRC 1998), but this approach may be inappropriate.

The AA profile in milk is presumably the ideal pattern of dietary IAA required for milk
synthesis based on the fact that dietary IAA absorbed by the lactating sow are mainly
used for milk synthesis (ARC 1981). The AA in porcine milk is comprised of two forms:
protein-bound AA (predominant) and free AA (negligible) (Wu and Knabe 1994a). The
protein in porcine milk is quantified by multiplying 6.38 and the total N % in milk
(Elliott et al. 1971, King et al. 1993a). Approximately 15.0% of the total N in porcine
milk is attributed to non-protein N (Klobasa et al. 1987). Thus, the AA profile in milk,
expressed as the percentage of total N % x 6.38 in milk, may not accurately represent the

AA composition of true proteins in milk.

The AA profile in milk depends mainly on the AA composition of milk proteins. Casein
and whey proteins are the predominant milk proteins. The ratio of casein to whey
proteins in rat milk is positively related to dietary protein intake (Ronayne-De-Ferrer and
Sambucetti 1993). The proportion of casein-N to the total N in porcine milk increases
approximately 10% as dietary Ile concentration increases; in contrast, the proportion of
whey protein-N and non-protein N decreases (Richert et al. 1997). The proportion of
casein-N to the total N in milk from sows fed Lys—adequate diet appears to be higher than
that from sows fed Lys-deficient diet (53.3% vs. 49.0% on d 18 of lactation) (Guan et al.
unpublished data). However, whether the ratio of casein to whey proteins in porcine milk
is affected by dietary protein intake is not known, though the concentration of the total N
in milk is decreased in sows fed a diet low in protein content (King et al. 1993a, Kusina
et al. 1999a). The ratio of whey protein-N to casein-N in porcine milk decreases from .40
to 3121.00, respectively, on d 7 and d 21 of lactation (Brent et al. 1973). Changes in the ,
ratio of caseins to whey proteins in milk may affect the AA profile in milk given the
differences in the AA composition between caseins and whey proteins (Swaisgood 1995).
Therefore, the AA profile in milk may be affected by dietary protein intake and the stage

of lactation.

Plasma free AA are taken up by the mammary gland through specific AA transport
systems situated on the basolateral membranes of the mammary epithelium (Sherman et
al. 1997). Mammary uptake of plasma free AA can be quantified by the technique of

their arterio-venous (A-V) differences across the mammary glands of the lactating sow

(Trottier et al. 1995). The amount of branched-chain amino acids (Ile, Leu, and Val,
BCAA) and Arg taken up by porcine mammary glands exceeds their output in milk by
approximately 25 to 30% (Nielsen et al. 2000, Trottier et al. 1997). The IAA taken up in
excess of their output in milk may be required for metabolism and protein synthesis in the
mammary gland. The rate of mammary uptake for plasma Arg, the precursor for de novo
synthesis of nitric oxide, may be involved in regulation of mammary blood flow (Lacasse
et al. 1996, Wu and Morris 1998). In addition to incorporation into milk protein, the
BCAA taken up by the mammary gland can provide a-amino N and carbon for
dispensable AA synthesis and provide energy for synthesis of other compounds (e.g.,
lactose and fatty acids) in the gland (Wohlt et al. 1977). Therefore, the ideal pattern of
IAA required for milk synthesis in the lactating sow, based only on the AA profile in

milk, may be inappropriate.

Nutritional regulation of plasma free AA uptake by the mammary gland is poorly
understood. In the lactating porcine mammary tissue, Lys uptake is inhibited in vitro by
cationic AA and neutral AA as shown in the lactating rat mammary tissue in vitro
(Calvert and Sherman 1996, Hurley et al. 2000, Sherman et al. 1994). Methionine uptake
by the mammary glands from the starved lactating mice is increased in vitro compared to
fed lactating mice (Verma and Kansal 1995). Taken together, we hypothesize that the
mammary uptake pattern of plasma IAA, defined as the proportion of an individual IAA
to the total IAA taken up by the mammary glands, is affected by dietary protein
concentration. Though the mammary AA uptake pattern has been proposed as a

reference for an ideal dietary AA pattern for milk synthesis (Trottier and Guan 2000), it is

not known if intake of dietary protein regulates the uptake pattern. It is imperative to use
the best dietary AA pattern for milk synthesis to derive dietary needs and balance dietary
AA so that the lactating sow can reach milk synthesis potential and utilize dietary protein
efficiently. The objectives of this study were to evaluate effects of dietary protein
concentration on the AA profile in milk and the mammary uptake pattern of plasma IAA

to derive an ideal pattern of IAA for milk synthesis by the lactating sow.

MATERIALS AND METHODS
Michigan State University All-University Committee on Animal Use and Care approved

all procedures in this study.

Experimental Design and Dietary Treatments. Sixteen Landrace x Yorkshire lactating
sows (parity 2 or 3) were allocated to dietary treatments according to a randomized block
design. Each block consisted of four sows. Each sow in one block was provided ad
libitum access to one of four diets. Diets contained different protein (CP) concentrations
(7.8, 13.0, 18.2, and 23.5% as-fed basis) and were balanced to contain a similar pattern of
indispensable amino acids (IAA) recommended by the NRC (1988). All diets were

isocaloric with a ME of 14.3 MJ/kg. The composition of diets is given in TABLE 1-1.

Animals. Litters were adjusted to 11 piglets per sow by cross-fostering within 48 h after
birth. Sows were housed individually in farrowing crates in a mechanically ventilated,
thermally controlled room (21 oC). Sows were provided free access to water. Rations

were offered in a stair step manner during the first 3 d post-surgery and sows were fed to

Ina-fl __ . _ "I,” "

appetite thereafter. Sow feed intake was recorded daily. Sow body weight, backfat
depth, and loin eye area were recorded after farrowing (d 1) and at weaning (d 21).
Backfat depth and loin eye area were scanned 5 cm lateral from the spine and centered
over the 10th rib (Ultrasound Scanner 200 Vet with ASP-l8 linear probe, Pie Medical
Equipment B.V., Maastricht, the Netherlands). Piglets were weighed individually

weekly.

_ Cannulation. For the catheter, microbore Tygon® tubing (1.0 mm i.d, 1.8 mm o.d.,
Norton Performance Plastics Co., Akron, OH) was used and the lumen was coated with
triododecyhnethylammonium chloride - heparin complex (7% (wt/wt), Polysciences, Inc.,
Warrington, PA). The anterior main mammary vein and the carotid artery were
cannulated on d 4 d: l of lactation following the surgical procedure described by Trottier
et al. (1995). Antibiotic (Naxcel®, Pharmacia and Upjohn Co., Kalamazoo, MI) and anti-
inflammatory (Banamine®, Schering-Plough Animal Health Corp., Kenilmorth, NJ) were
administered i.v. for 3 (1 following surgery. The catheters were flushed once daily with

heparinized saline (201U heparin/ml).

Milk Yield Estimation. Milk intake by piglets (i.e., milk yield) was estimated on d 11
and 21 by the D20 dilution method of Pettigrew et al. (1987) as modified by Pluske et al.
(1997) using piglet serum. In brief, piglets were fasted for l h, weighed, and injected i.m.
with a dose (1 mg/kg BW) of D20 (Cambridge Isotope Laboratories, Andover, MA). At
1 h after the dose, piglets were bled from the jugular vein using vacutainers containing

SST® gel and clot-activator (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ).

11

At 24 h after the dose, the piglets were fasted for l h, weighed, and bled as before.
During this 24-h period, piglets were not provided access to water. Blood samples were
centrifuged at 1500 x g for 15 min at 4 °C, and then serum samples were separated and
stored at -20 °C until assayed for D20. Serum D20 concentration was determined by an
infrared spectrophotometer (The Foxboro Co., East Bridgewater, MA) using a fixed-filter
length of 4 pm. Milk intake was computed on the basis of the D20 dilution principle

(Pettigrew et al. 1987).

Blood and Milk Sampling Protocol. Blood samples from sows were collected on d 9 i
1, 13 i 1, 17 d: 1, and 21 fl: I (referred to as d 10, 14, 18, and 22 of lactation,
respectively). Sows were fed 1 h prior to blood sampling and provided ad libitum access
to feed and water as usual. Matched sets of carotid arterial and main mammary venous
blood samples (10 ml each) were collected into sterile syringes from 0800 every 30 min
over 6 h. Thus, a total of 13 blood samples were collected from each sow per sampling
day. Blood was transferred into EDTA-coated tubes and centrifuged at 1500 x g for 15
min at 4 °C. Plasma was removed and stored at -20 °C. On the same day as blood
sampling, milk was collected and pooled from all functional teats on each sow following
an i.v. dose of oxytocin (10 IU). Approximately 20 m1 pooled milk was transferred into a
vial containing a preserver (Broad Spectrum Microtales‘m, D & F Control System, Inc.,
San Ramon, CA) and stored at 4 °C until assayed for milk composition. Another portion
of pooled milk was stored at -20 °C until assayed for nitrogen. The remainder of pooled
milk was defatted by centrifugation at 1500 x g for 15 min at 4 °C and stored at -20 °C

until assayed for amino acids (AA).

12

Laboratory measurements.

(1) Milk Composition and Amino Acid Analysis. Milk lipid, lactose, and protein
contents were assayed using a mid-infrared spectroscope (Multispec M, Berwind
Instrument Ltd, York, England, U.K.)(AOAC 1990). Frozen milk samples were thawed
at 37 °C and mixed by a vortex. Milk nitrogen was determined using a LECO Nitrogen
Analyzer FP-2000 (LECO Corp., St. Joseph, MI) with EDTA (Sigma Chemical Co., St.
Louis, MO) as a calibration standard. The energy content of milk was estimated by
multiplying the percentage of milk protein, lipid, and lactose by 5.70, 9.40, and 4.15 kcal,
respectively (Jenness 1974). Amino acid concentrations in the defatted milk were
quantitated by reverse-phase, high-performance liquid chromatography using
phenylisothiocyanate precolumn derivatization (Pico0Tag®, Waters Corp., Milford, MA).
In brief, 200 uL of defatted milk was hydrolyzed with 6 mol/L HCl for 24 h at 110 °C.
Amino acids in the hydrolysate were derivatized with phenylisothiocyanate and separated
by a Pico-Tag® column (3.9 mm x 150 mm) and detected at 254 nm by Waterstm 486
Tenable Absorbance Detector (Waters Corp., Milford, MA). Norleucine as an internal
standard was added into defatted milk samples before hydrolysis. Amino acid standard H
(Pierce, Rockford, IL) was used as a calibration standard. The method was validated

with certified AA standard (NIST, Gaithersburg, MD).
(2) Plasma Amino Acid Analysis. Frozen plasma samples were thawed at 4 °C overnight

and mixed using a vortex. Plasma samples were pooled from the 13 samples taken from

each sow on each sampling day. Glucosaminic acid was added to the pooled samples as

13

an internal standard. Amino acid concentrations were determined using a Beckman 6300
Amino Acid Analyzer following the method described by Lee and Slocum (1987). In
brief, plasma samples were deproteinized by 35% sulfosalicyclic acid precipitation.
Amino acids in the supernatant were separated by a Beckman cation-ion exchange
column charged in lithium citrate buffer. The eluted AA were measured

spectrophotometrically following postcolumn derivatization with ninhydrin.

_ (3) Dietary Nitrogen and Amino Acid Analysis. Feed samples were finely ground using
a Cyclotec® 1093 Sample Mill (Foss Tecator, Sweden). Feed nitrogen was analyzed by
the micro-Kjeldahl method (AOAC 1990). Hydrolysis of feed samples and determination
of AA in the hydrolysate were performed as described in (I). The content of Trp in the

diets was calculated using its content in feed ingredients (NRC 1988).

Calculations.

(1) Milk Output of Indispensable Amino Acids. Milk output of IAA (g/d) = milk yield
(L/d) x the concentrations (umol/L) of IAA in defatted milk x (l — the content of lipid in
whole milk) x molecular weight (g/mol) x 106. The concentrations of IAA in defatted
milk were corrected by the content of lipid in whole milk to obtain the concentrations of
IAA in whole milk. To quantify milk output of IAA, milk yield on d 11 was multiplied
with an average concentration of IAA in whole milk from d 10 and 14 of lactation, and

milk yield on d 21 with that from d 18 and 22 of lactation.

l4

(2) Mammary Extraction Rates of Plasma Indispensable Amino Acids. Mammary
extraction rate (%) = 100 x mammary A-V difference of plasma IAA / arterial

concentration of the plasma IAA.

(3) Mammary Uptake Pattern of Plasma Indispensable Amino Acids. Mammary uptake
pattern of plasma IAA was defined either as the proportion (%, mol/mol) of individual
IAA to total IAA taken up by the glands or the ratios (wt/wt) of other IAA to Lys taken
up by the glands. Note that mammary plasma flow rate as a variable was cancelled out in
the following calculations:

Mammary uptake proportions (%, mol/mol) = 100 x mammary arterio-venous (A-V)
difference (umol/L) of an individual IAA x mammary plasma flow rate (L/d) /
(mammary A-V difference (umol/L) of the total IAA x mammary plasma flow
rate (L/d)); i.e.,

Mammary uptake proportions (%, mol/mol) = 100 x mammary A-V difference
(umol/L) of an individual IAA / mammary A-V difference (umol/L) of the total
IAA. (Eq- 1)

Mammary uptake ratios (wt/wt) = mammary A-V difference (umol/L) of IAA x
mammary plasma flow rate (L/d) x its molecular weight (pg/umol) / ((marnmary
A-V difference (umol/L) of plasma Lys x mammary plasma flow rate (L/d) x Lys
molecular weight (pg/umol»; thus,

Mammary uptake ratios (wt/wt) = mammary A-V difference (umol/L) of IAA x its

molecular weight (ug/umol) / (mammary A-V difference (umol/L) of plasma Lys

x Lys molecular weight (ug/umol». (Eq. 2)

15

Statistical Analyses. Data were analyzed by the Mixed Procedure with a repeated
statement of SAS/STAT (Version 6.12, SAS Institute Inc., Cary, NC). The first-order
autoregressive was assumed in the covariance structure. Individual sow was considered
the experimental unit (the random effect) and day of lactation as the repeated effect. The
model for milk and plasma variables included block, dietary treatment, day of lactation,
and all two-way interactions with day of lactation in a repeated statement. The
interaction between dietary treatment and day of lactation was not significant (P > .10).
Therefore, only main effects (dietary treatment or day of lactation) are presented. The
model for performance variables included block and dietary treatment; sow body weight,
backfat depth, and loin eye area on d l of lactation were used as covariates, respectively,
to adjust their changes over a 21-d lactation. Least-squares means are presented because

of missing values in the data. Differences are considered significant at P < .05 or .01.

RESULTS

Sow and Litter Performance

Sows fed the 13% CP diet (the appropriate protein diet, AP) had higher feed intake over a
21-d lactation period than sows fed other diets (TABLE 1-2). Intake of protein was
lower and higher, respectively, for sows fed the 7.8% CP diet (the low protein diet, LP)
and the 23.5% CP (the high protein diet, HP) compared with that of sows fed the AP and
the 18.2% CP diet (the median protein diet, MP). Dietary treatments did not affect sow
body weight change and backfat depth change over the 21-d lactation period. However,

sows fed the LP lost two-fold loin eye area as sows fed other diets. Daily litter weight

16

gain over the 21-d lactation period was 1.88, 2.43, 2.07, and 2.23 kg/d, respectively, for
sows fed the LP, AP, MP, and HP. Sows fed the LP had the lowest litter growth rate and
milk yield on d 21, though sows fed other diets were not different in litter growth rate and

milk yield.

Milk Composition and Output

Dietary treatments and stage of lactation did not affect milk composition except protein
concentration (TABLE 1-3). Milk protein concentration increased linearly with
increasing dietary protein concentrations (P < .01). In contrast, the content of milk
lactose tended to decrease with increased dietary protein concentrations (P < .10). The
content of milk protein decreased approximately 10% in sows fed the LP and AP
compared to that in sows fed the MP and HP. Effect of stage of lactation on milk protein
concentration was quadratic (P < .05). It decreased from d 10 to 18 and then increased
by d 22 of lactation. The content of nitrogen in milk changed as the content of milk
protein did (data not shown). The concentrations of indispensable amino acids (IAA) in
defatted milk reflected the content of milk protein (TABLE 1-4). The concentrations of
IAA (except His and Met) in defatted milk increased linearly with increasing dietary
protein concentrations (P < .05). Effect of stage of lactation on concentrations was
quadratic (P < .05) for His, Thr, and Val, and tended to be quadratic for other IAA (P <

.10).

Dietary treatments affected output of protein in milk (P < .05) and tended to affect output

of dry matter in milk (P < .10), but did not affect output of lipid, lactose, or energy in

17

 

 

 

milk (data not shown). Sows fed the LP had the lowest output of macro-nutrients and
energy due to their lowest milk yield. Milk yield and output of macro-nutrients and

energy were not different between (71 11 and 21 oflactation (11.67 i .62 vs 11.41 i .62,
2.18 i .11 vs 2.09 i .11, .55 i .03 vs .55 i .03, and .66 i .04 vs .63 i .04 kg/d,
respectively, for milk yield, dry matter, protein, and lactose, and 13.19 i .69 vs 12.11 i
.67 Meal/d for energy), though output of lipid in milk tended to be higher on d 11 than
that on d 21 oflactation (.78 i .04 vs .68 i .04 kg/d, P < .10). Output ofindividual IAA
in milk increased linearly with increasing dietary protein concentrations (P < .05)
i (TABLE 1-5). The output of individual IAA in milk decreased approximately 25 to 30%
from sows fed the LP compared to that fed the AP. As reflected in the output of protein
in milk, output of individual IAA in milk was not different between d 11 and 21 of

lactation.

The Amino Acid Profile in Milk

The AA profile in milk was expressed as the proportion of individual IAA to milk protein
or the ratios of other IAA to Lys in milk. Dietary treatments did not affect the AA profile
in milk (TABLE 1-6). There were slight alterations in the ratios for Ile, Leu, and Met
among dietary treatments. Average ratios of other IAA to Lys in milk across dietary
treatments were .79, .45, .65, 1.15, .34, .60, .66, and 8421.00, respectively, for Arg, His,
Ile, Leu, Met, Phe, Thr, and Val. Stage of lactation did not affect the AA profile in milk
either (TABLE 1-7) except for His. The proportion of Lys and the ratio of Leu to Lys

were slightly altered across stage of lactation. Average ratios of other IAA to Lys in milk

18

across stage of lactation were .78, .45, .65, 1.15, .34, .59, .66, and 8321.00, respectively,

for Arg, His, Ile, Leu, Met, Phe, Thr, and Val.

Arterial Concentrations, Mammary Arterio-venous Differences, and Mammary
Extraction Rates of Plasma Indispensable Amino Acids

Arterial concentrations of plasma individual IAA (except His and Ile) and total IAA
increased linearly with increasing dietary protein concentration (P < .05) (TABLE 1-8).
. Arterial concentrations for the majority of plasma individual IAA (including Arg, Ile,

Leu, Lys, Phe, and Val) and total IAA increased as lactation progressed (P < .05).

Mammary A-V differences of plasma individual IAA and total IAA were affected
linearly by dietary treatments (P < .05) (TABLE 1-9). Mammary A-V differences of
plasma IAA other than branched-chain AA (Ile, Leu, and Val) tended to be greatest in
sows fed the MP (P < .10). Mammary A-V differences of plasma individual IAA and

total IAA tended to increase as lactation progressed (P < .10).

Mammary extraction rates of plasma individual IAA (except Arg, Lys, and Trp) were
affected by dietary treatments (P < .05) (TABLE 1-10). Mammary extraction rates for
the majority of plasma individual IAA (including Arg, His, Ile, Met, Phe, Thr, Trp, and
Val) tended to be the greatest in sows fed the MP (P < .01). In contrast, they decreased in
sows fed the HP compared to those in sows fed the MP (P < .05). Mammary extraction

rates of total IAA tended to be affected by dietary treatments (P < .10). Mammary

19

extraction rates of plasma IAA (except Ile, Trp, and Val) and total IAA increased on d 18

compared to those on d 14 of lactation.

Mammary Uptake Patterns of Plasma Indispensable Amino Acids
Mammary uptake pattern of plasma IAA was defined as the proportion of individual IAA
to total IAA taken up by the mammary glands or the ratio of other IAA to Lys taken up
by the glands. The proportions for Lys, Met, Phe, and Thr decreased with increasing
dietary protein concentration (FIGURE 1-1). In contrast, the proportions for the
branched-chain AA increased with increasing dietary protein concentration. The
proportions for Arg, His, and Trp were not affected significantly by dietary protein
concentration. Thus, the ratios of other IAA to Lys taken up by the glands for Arg, Met,
and the branched-chain AA increased with increasing dietary protein concentration (P <

.05) (TABLE 1-11).

The proportions of individual IAA to total IAA were not altered across stage of lactation
(TABLE 1-12). Those proportions were averaged across stage of lactation at 12.15,
3.60, 11.80, 19.33, 13.50, 4.30, 7.30, 9.93, 1.43, and 16.63 % (mol/mol), respectively, for
Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val. Based on the average proportions,
the ratios (wt/wt) of other IAA to Lys were 1.07, .28, .78, 1.28, 1.00, .33, .61, .60, .15,

and .99, respectively, for Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val.

20

Discussion

Lactation Performance
Sows fed LP had lower milk yield and lower litter growth rate. This is in agreement with
the results from other studies (Elliott et a1. , King et al. 1993b, Kusina et al. 1999b,
Sauber et al. 1998). Dietary protein concentration did not affect sow body weight loss
during lactation in the present study, which is in agreement with the results of King et al.
(1993). However, body weight loss during lactation increased in sows fed diets deficient

in CP and (or) Lys in other studies (Dourmad et al. 1998, Johnston et al. 1993, Jones and
Stahly 1999, Kusina et al. 1999b, Stahly 1992). The inconsistent effect of dietary protein
concentration on body weight loss may be confounded by intake of dietary energy during
lactation. In the present study, dietary protein concentration did not affect sow backfat
depth change during lactation as reported in other studies (Dourmad et al. 1998, Johnston
et al. 1993, Jones and Stahly 1999, Revell et al. 1998b, Sauber et al. 1998). In contrast,
sows fed the LP lost more than twice as much loin eye area compared with sows fed diets
higher in CF, indicating that sows mobilized body protein reserves to support milk

production when fed diets deficient in CP (Dourmad et al. 1998, Jones and Stahly 1999).

Milk Composition and Output

Dietary protein concentration did not affect milk composition except protein
concentration in the present study. Sows fed the LP and AP had low content of true
protein in milk and thus lower concentrations of IAA in milk, indicating that 7.8 and
13.0% of dietary protein concentrations were not adequate for milk synthesis. The

content of true protein in milk ranged from 4.5 to 5.0% in the present study, which agrees

21

with values (from 4.0 to 5.0%) of true protein in milk from sows fed diets containing 7.9
and 19.0% CF (Revell et al. 1998a). The content of true protein in milk may be lower by
15% than the content of CP (i.e., 6.38 x N %) in milk because non—protein N contributes
approximately 15% of the total N (Klobasa et al. 1987). The content of the CP in milk
decreases when sows fed diets low in CF (King et al. 1993b, Kusina et al. 1999b, Sauber
et al. 1998). Stage of lactation influenced milk protein concentration, but did not affect
the concentrations of dry matter, lipid, lactose, or energy in the present study. Similarly,
no changes in milk composition or the content of N-containing fractions in milk (such as
casein and whey) were found between d 7 and 21 of lactation (Brent et a1. 1973,

Dounnad et al. 1998, Klobasa et al. 1987, Pluske et al. 1998).

Output of macro-nutrients and IAA in milk decreased in sows fed the LP because of
lower milk yield and (or) the content of true protein and IAA in milk in this study.
Because there was neither difference in milk yield between (1 11 and 21 of lactation as
reported in other studies (King et al. 1993b, Pluske et al. 1998) nor difference in the
average content of macro-nutrients and IAA in milk between early lactation (d 10 to 14)

and late lactation (d 18 to 22), there was no difference in their output in milk.

The Amino Acid Profile in Milk
Dietary protein concentration did not affect the AA profile in milk in the present study.
Elliott et al. (1971) and King et al. (1993) found the concentrations of individual IAA in

milk decreased when sows were fed diets low in CF. This evidence suggests that output

of individual proteins in milk may be affected proportionally by intake of dietary protein;

22

and supports the notion that modification of the amount of milk protein, rather than the
AA profile of milk protein, may be an evolutionary mechanism used to meet the unique
AA needs by mammalian neonates (Davis et al., 1994). The AA profile in milk was not
altered between (I 10 to 22 of lactation. Similarly, Elliott et al. (1971) found that the AA
profile in milk was not altered between d 5 to 21 of lactation. Moreover, the AA profiles
in milk are consistent throughout all independent studies (TABLE 1-13). The
proportions of Lys were based on the true protein (in the present study), on the total
individual AA (in which Trp was not included) (Davis et al. 1994), or on the N x 6.38
(Dourmad et al. 1998, Dourmad et al. 1991, Elliott et al. 1971, King et al. 1993a). The
average proportion of Lys to true protein (7.83%) in the present study was slightly lower
than that in the study of Davis et al. (1994), but was higher than those in the other studies
(TABLE 1-13). In addition, the average concentration of Lys (including free and
protein-bound forms) in defatted milk was 26.5 mmol/L in the present study, which is
within the range of 24 to 30 mmol/L of protein-bound Lys in defatted porcine milk

reported by Wu and Knabe (1994b).

Dietary pattern of individual IAA required for milk synthesis is presumably dominated by
the AA composition of proteins secreted into milk (ARC 1981). Thus, the AA profile in
milk has been used to derive the ideal pattern of IAA required for milk synthesis in the

lactating sow by the NRC (1998).

Mammary Arterio-venous Difference of Plasma Indispensable Amino Acids

23

The mammary gland during lactation requires a large amount of AA for metabolism and
protein synthesis. Amino acids are extracted from blood by specific AA transport
systems situated in the blood-facing aspect of the mammary epithelial cells, as reflected
in mammary A-V differences of blood AA (Sherman et al. 1997). The transport of
certain AA by the mammary epithelial cells could be rate limiting for the synthesis of
milk protein (Mepham 1982). Milk yield is positively related to mammary A-V
difference of plasma IAA in the lactating cow and sheep when mammary blood flow
' rates are constant (Fleet and Mepharn 1985, Guinard et al. 1993, Madsen et a1. 1999).
Thus, mammary A-V difference of plasma IAA may be considered as a physiological
response criterion to determine the IAA needs for milk synthesis by the lactating porcine

mammary gland (Trottier and Guan 2000).

Mammary A-V differences of plasma IAA in the present study are within ranges reported
in the recent studies (Nielsen et a1. 2000, Trottier et al. 1997). The mammary A-V
differences increased in a quadratic manner with increasing dietary protein concentration
and with advancement of lactation in the present study. Similarly, mammary A-V
differences of plasma IAA increase in the lactating cow with duodenal infusion of casein
or whey protein (Guinard and Rulquin 1994a). Mammary A-V difference of plasma Lys
decreased approximately 25% in sows fed the HP compared to that in sows fed the MP,
which is possibly due to a decreased mammary extraction rate of plasma Lys. The
relationship between mammary A-V difference and arterial concentration of plasma Lys
is curvilinear in the lactating sow (Trottier 1997) and in the lactating cow infused

duodenally with Lys (Guinard and Rulquin 1994b). Furthermore, Lys uptake by the

24

lactating rat and porcine mammary tissue is inhibited in vitro by physiological
concentrations of cationic AA and high concentrations of neutral AA because these AA
inhibit Lys influx into the lactating rat mammary tissue and stimulate Lys efflux from the
tissue (Calvert and Sherman 1996, Hurley et al. 2000, Sherman et al. 1994). Thus, Lys
uptake by the porcine mammary gland may be inhibited in vivo by physiological

concentrations of arterial cationic AA and neutral AA.

Mammary A-V differences and extraction rates of plasma IAA increased by d 18 of
lactation, implying that mammary uptake may be up-regulated to meet increased IAA
needs for milk synthesis by the mammary glands. Similarly, in lactating sheep mammary
A-V differences of plasma IAA are regulated by stage of lactation (further corresponding
to milk yield) (Fleet and Mepham 1985). This regulation may be through actions of
lactogenic hormones (e. g. prolactin) on the AA transport systems in the mammary gland
(Sharma and Kansal 1999, Shanna and Kansal 2000, Sherman et al. 1997, Vina et al.

1981).

Mammary Uptake Pattern of Plasma Indispensable Amino Acids

An important finding in the present study was that the proportions of the branched—chain
AA to the total IAA taken by the mammary gland were increased for the lactating sows
with high intakes of protein; in contrast, the proportions of Lys, Met, Phe, Thr, and Trp to
the total IAA taken by the mammary gland maintained at high levels for the lactating
sows at low intake of protein (FIGURE 1-1). This may have important physiological

implications. On one hand, Lys, Met, Phe, Thr, and Trp might become the limiting AA

25

for the lactating sow at low intake of protein, leading to up-regulation of their uptake
(indicated by their increased proportions to the total IAA taken by the mammary gland)
to meet their needs for milk synthesis. On the other hand, the branched-chain AA (e.g.,
Val) might become the limiting AA for the lactating sow at high intakes of protein,
leading to up-regulation of their uptake (indicated by their increased proportions to the
total IAA taken by the mammary gland), which might be driven by their increased
intracellular removal (e. g., oxidation). Activities of branched-chain aminotransferase and
branched-chain a-keto acid dehydrogenase, the first two enzymes in the branched-chain
AA catabolic pathway, increase dramatically in rat mammary glands during lactation
(DeSantiago et al. 1998). Moreover, a high intake of dietary protein up-regulates the
expression of branched-chain aminotransferase and activity of branched-chain a-keto
acid dehydrogenase in rat (Torres et al. 1998). This up-regulation might also occur in the
lactating mammary gland to increase its capacity to dispose the branched-chain AA at a
high intake of protein. Though physiological consequences of their excess uptake by the
mammary gland are not known at this moment, we speculate that the BCAA may have
two aspects of roles: (1) providing substrates and energy for milk synthesis and (2)
participating in regulations of protein turnover, by decreasing protein breakdown

(MacLean et al. 1994) (Mitch and Clark 1984, Nair et al. 1992).

Mammary uptake patterns of individual IAA, defined as the ratios of other IAA to Lys
taken up by the lactating porcine mammary gland, are relatively constant across five
independent studies (TABLE 1-14), though the ratios were regulated by dietary protein

concentration in the present study. These patterns might be required by the mammary

26

glands for metabolism and protein synthesis. Thus, this set of average proportions for
individual IAA to total IAA may represent an ideal pattern of plasma IAA taken up by
the mammary glands. In comparison to the AA profiles in milk (TABLE 1-13), the
ratios of Arg and the branched-chain AA to Lys are higher in the mammary uptake
pattern (FIGURE 1-2). Arginine and the branched-chain AA are taken up by the
lactating porcine mammary gland in excess of their output into milk (Nielsen et al. 2000,
Trottier et al. 1997). Similar results have been found in other lactating species, such as
dairy cattle, goats, and sheep (Bequette et a1. 1997, Fleet and Mepham 1985, Guinard and

Rulquin 1994a).

Besides incorporation into milk protein, the IAA taken up by the mammary gland may
play an important role in other metabolic pathways: (1) They can provide or-amino N and
carbon for synthesis of dispensable AA (Wohlt et al. 1977). (2) They can meet energy
needs for high rates of protein turnover due to substantial synthesis and degradation of
protein within the lactating goat mammary gland (Oddy et al. 1988). (3) They can be
used for synthesis of structural proteins and enzymes to maintain mammary cell integrity
and function (Bequette et al. 1997), as partly accreted in the lactating porcine mammary
gland (Kim et al. 1999). (4) They can be oxidized to yield energy for the synthesis of
milk non-protein compounds (e.g., fatty acids and lactose) (Davis and Mepham 1976,
Roets et al. 1979). (5) They can be used as precursors for the synthesis of important
metabolites. For example, Arg is the precursor for de novo synthesis of nitric oxide that
is involved in local regulation of mammary blood flow (Lacasse et a1. 1996, Vina and

Williamson 1981, Wu and Morris 1998, Wu et al. 1999). These IAA taken up in excess

27

are presumably needed to meet these metabolic needs (other than milk protein synthesis)
by the mammary glands. Thus, we hypothesize that the mammary uptake pattern of
plasma IAA may be considered as an ideal pattern for milk production required by the

lactating porcine mammary gland.

One application of the mammary uptake pattern is to define an ideal ratio of Val to Lys
for milk synthesis in the lactating sow. Mammary A-V differences of plasma Val and
Lys increased in a quadratic manner with increasing intake of protein during lactation
(FIGURE 1-3). Note that the mammary A-V difference of plasma Val was greater than
that of plasma Lys when their intake was more than 28 g/d, i.e., the mammary uptake
ratio of plasma Val to Lys became larger than 1:1 at high intakes of dietary AA during
lactation. This finding provides a physiological explanation for the beneficial effect of
increasing dietary ratio of Val to Lys (up to 1:1) on litter growth rates in high producing
lactating sows (Moser et al. 2000, Richert et al. 1997, Richert et al. 1996). This finding
also supports the concept that Val is the co-limiting AA to Lys in corn soybean meal diets
for the lactating sow, and even becomes the first limiting AA at high intakes of dietary
AA (NRC 1998). Therefore, the ideal ratio of Val to Lys required for milk synthesis is
approximately 1:1, which is higher than the value of .85 to 1 recommended by NRC

(1998)

28

Implications

The ideal pattern for milk synthesis by the lactating sow should be defined by the
mammary uptake pattern of plasma indispensable amino acids, which represents the
physiological needs of indispensable amino acids for metabolism and protein synthesis
by the mammary gland. However, the mammary amino acid uptake patterns are
regulated by intake of protein, indicating that the ideal dietary amino acid pattern for milk
synthesis is dynamic. Specifically, dietary ratios of the branched-chain amino acids in
high concentrations of dietary protein should be increased to meet their increased uptake
by the mammary gland. Ideal ratios for milk synthesis defined by the mammary uptake
pattern are greater for arginine and the branched-chain amino acids than those based only
on the amino acid profile in milk. For example, an ideal ratio of valine to lysine for milk
synthesis is approximately at 1:1, which is higher than the value of .85:1 recommended
by the current NRC (1998). The ideal pattern of indispensable amino acids for milk
synthesis established in this study, together with ideal patterns for maintenance, protein
accretion, and (or) body protein loss, can be used to constitute the entire ideal pattern of

dietary truly digestible indispensable amino acids for the lactating sow.

29

LITERATURE CITED

AOAC. (1990). Official methods of analysis of the association of official analytical
chemists. 15 ed. (Helrich, K., Ed), 2. 2 vols, AOAC, Inc., Arlington.

ARC. (1981). The nutrient requirements of pigs. Commonwealth Agricultural Bureaux,
London.

Bequette, B. J., Backwell, F. R., Calder, A. G., Metcalf, J. A., Beever, D. E., MacRae, J.
C. & Lobley, G. E. (1997) Application of a U-13C—labeled amino acid tracer in
lactating dairy goats for simultaneous measurements of the flux of amino acids in
plasma and the partition of amino acids to the mammary gland. J. Dairy Sci. 80:
2842-2853.

Brent, B. E., Miller, E. R., Ullrey, D. E. & Kemp, K. E. (1973) Postpartum changes in
nitrogenous constituents of sow milk. J. of Anim. Sci. 36: 73-77.

Calvert, D. T. & Sherman, D. B. (1996) Evidence for an interaction between cationic
and neutral amino acids at the blood-facing aspect of the lactating rat mammary
epithelium. J. Dairy Res. 63: 25-33.

Davis, S. R. & Mepham, T. B. (1976) Metabolism of L-(U-14C)valine, L-(U-
14C)leucine, L-(U-14C)histidine and L-(U-14C) phenylalanine by the isolated
perfused lactating guinea-pig mammary gland. Biochem. J. 156: 553-560.

Davis, T. A., Nguyen, H. V., Garcia Bravo, R., Fiorotto, M. L., Jackson, E. M. & Reeds,
P. J. (1994) Amino acid composition of the milk of some mammalian species
changes with stage of lactation. Br. J. Nutr. 72: 845-853.

DeSantiago, S., Torres, N., Suryawarr, A., Tovar, A. R. & Hutson, S. M. (1998)
Regulation of branched-chain amino acid metabolism in the lactating rat. J. Nutr.
128: 1165-1171.

Dourmad, J. Y., Noblet, J. & Etienne, M. (1998) Effect of protein and lysine supply on
performance, nitrogen balance, and body composition changes of sows during
lactation. J. Anim. Sci. 76: 542-550.

Dourmad, J .-Y., Etienne, M. & Noblet, J. (1991) A contribution to the study of amino
acid requirement for lactation in sows. J. Rech. Porcine en France 23: 61-68.

Elliott, R. F., Vander Noot, G. W., Gilbreath, R. L. & Fisher, H. (1971) Effect of dietary
protien level on composition changes in sow colostrum and milk. J. Anim. Sci.

32: 1128-1137.

Fleet, I. R. & Mepham, T. B. (1985) Mammary uptake of amino acids and glucose
throughout lactation in friesland sheep. J. Dairy Res. 52: 229-237.

30

Guinard, J. & Rulquin, H. (1994a) Effect of graded levels of duodenal infusions of
casein on mammary uptake in lactating cows: 2. Individual amino acids. J. Dairy
Sci. 77: 3304-3315.

Guinard, J. & Rulquin, H. (1994b) Effects of graded amounts of duodenal infusions of
lysine on the mammary uptake of major milk precursors in dairy cows. J. Dairy
Sci. 77: 3565-3576.

Guinard, J., Rulquin, H. & Verite, R. (1993) Effect of graded levels of duodenal
infusions of casein on mammary uptake in lactating cows: 1.Major nutrients. J.
Dairy Sci. 77: 2221-2231.

Hurley, W. L., Wang, H., Bryson, J. M. & Sherman, D. B. (2000) Lysine uptake by
mammary gland tissue from lactating sows. J. Anim. Sci. 78: 391-395.

. Hurley, W. L., Wang, H., Bryson, J. M. & Sherman, D. B. (2000 Feb) Lysine uptake by
mammary gland tissue from lactating sows. J. Anim. Sci. 78: 391-395.

Jenness, R. (1974). The composition of milk. In Lactation: A comprehensive treatise
(Larson, B. L., ed), pp. 164. Iowa State University Press, Ames.

Johnston, L. J ., Pettigrew, J. E. & Rust, J. W. (1993) Response of matemal-line sows to
dietary protein concentration during lactation. J. Anim. Sci. 71: 2151-2156.

Jones, D. B. & Stahly, T. S. (1999) Impact of amino acid nutrition during lactation on
body nutrient mobilization and milk nutrient output in primiparous sows. J. Anim.
Sci. 77: 1513-1522.

Kim, S. W., Hurley, W. L., Han, I. K. & Easter, R. A. (1999) Changes in tissue
composition associated with mammary gland growth during lactation in sows. J.
Anim. Sci. 77: 2510-2516.

King, R. H., Rayner, C. J. & Kerr, M. (1993a) A note on the amino acid composition of
sow's milk. Anim. Prod. 57: 500-502.

King, R. H., Toner, M. S., Dove, H., Atwood, C. S. & Brown, W. G. (1993b) The
response of first-litter sows to dietary protein level during lactation. J. Anim. Sci.
71: 2457-2463.

Klobasa, F., Werhahn, E. & Butler, J. E. (1987) Composition of sow milk during
lactation. J. Anim. Sci. 64: 1458-1466.

Kusina, J ., Pettigrew, J. E., Sower, A. F., White, M. E., Crooker, B. A. & Hathaway, M.
R. (1999a) Effect of protein intake during gestation and lactation on the
lactational performance of primiparous sows. J. Anim. Sci. 77: 931-941.

31

Kusina, J ., Pettigrew, J. E., Sower, A. F., White, M. E., Crooker, B. A. & Hathaway, M.
R. (1999b) Effect of protein intake during gestation and lactation on the
lactational performance of primiparous sows. J. Anim. Sci. 77: 931-941.

Lacasse, P., Farr, V. C., Davis, S. R. & Prosser, C. G. (1996) Local secretion of nitric
oxide and the control of mammary blood flow. J. Dairy Sci. 79: 1369-1374.

Lee, P. & Slocum, R. (1987) Expanded physiological fluid analysis. In System
6300/7300 Application Note (No. A6300-AN-006). Beckman Instruments.

MacLean, D. A., Graham, T. E. & Saltin, B. (1994) Branched-chain amino acids
augment ammonia metabolism while attenuating protein breakdown during
exercise. Am. J. Physiol. 267: E1010-1022.

Madsen, T. G., Misciattelli, L. & Nielsen, M. O. (1999). Effect of amino acid versus
propionic acid absorption on the supply and uptake of amino acids and glucose by
the mammary gland of dairy cows (Abs.). The VIIIth international symposium on
protein metabolism and nutrition (Eds. Lobley, G. E., White, A. & MacRae, J. C. ),
Aberdeen, United Kingdom.

Mepham, T. B. (1982) Amino acid utilization by lactating mammary gland. J. Dairy Sci.
65: 287-298.

Mitch, W. E. & Clark, A. S. (1984) Specificity of the effects of leucine and its
metabolites on protein degradation in skeletal muscle. Biochem. J. 222: 579-5 86.

Moser, S. A., Tokach, M. D., Dritz, S. S., Goodband, R. D., Nelssen, J. L. &
Loughmiller, J. A. (2000) The effects of branched-chain amino acids on sow and
litter performance. J. Anim. Sci. 78: 658-667.

Nair, K. S., Schwartz, R. G. & Welle, S. (1992) Leucine as a regulator of whole body
and skeletal muscle protein metabolism in humans. Am. J. Physiol. 263: E928-
934.

Nielsen, T. T., Trottier, N. L., Stein, H. H., Bellaver, C. & Easter, R. A. (2000) The
effect of litter size and day of lactation on amino acid uptake by the porcine
mammary gland. J. Anim. Sci.

NRC. (1988). Nutrient requirements of swine. 9th ed, National Academy Press,
Washington, D. C.

NRC. (1998). Nutrient Requirements of Swine. 10th ed, National Academy Press,
Washington, DC.

Oddy, V. H., Lindsay, D. B. & Fleet, I. R. (1988) Protein synthesis and degradation in
the mammary gland of lactating goats. J. Dairy Res. 55: 143-154.

32

Pettigrew, J. E., Cornelius, S. G., Moser, R. L. & Sower, A. F. (1987) A refinement and
evaluation of the isotope dilution method for estimating milk intake by piglets.
Livest. Prod. Sci. 16: 163-174.

Pluske, J. R., Fenton, T. W., Lorschy, M. L., Pettigrew, J. E., Sower, A. F. & Aheme, F.
X. (1997) A modification to the isotope dilution technique for estimating milk
intake ofpigs using pig serum. J. Anim. Sci. 75: 1279-1283.

Pluske, J. R., Williams, I. H., Zak, L. J., Clowes, E. J., Cegielski, A. C. & Aheme, F. X.
(1998) Feeding lactation primiparous sows to establish three divergent metabolic
states: 111. Milk production and pig growth. J. Anim. Sci. 76: 1165-1171.

Revell, D. K., Williams, I. H., Mullan, B. P., Ranford, J. L. & Smits, R. J. (1998a) Body
composition at farrowing and nutrition during lactation affect the performance of
primiparous sows: 11. Milk composition, milk yield, and pig growth. J. Anim.
Sci. 76: 1738-1743. ‘

Revell, D. K., Williams, I. H., Mullan, B. P., Ranford, J. L. & Smits, R. J. (1998b) Body
composition at farrowing and nutrition during lactation affect the performance of
primiparous sows: 1. Voluntary feed intake, weight loss, and plasma metabolites.
J. Anim. Sci. 76: 1729-1737.

Richert, B. T., Goodband, R. D., Tokach, M. D. & Nelssen, J. L. (1997) Increasing
valine, isoleucine, and total branched-chain amino acids for lactating sows. J.
Anim. Sci. 75: 2117-2128.

Richert, B. T., Tokach, M. D., Goodband, R. D., Nelssen, J. L., Pettigrew, J. E., Walker,
R. D. & Johnston, L. J. (1996) Valine requirement of the high—producing
lactating sow. J. Anim. Sci. 74: 1307-1313.

Roets, E., Massart Leen, A. M., Verbeke, R. & Peeters, G. (1979) Metabolism of [U-
14C; 2,3-3H]-L-valine by the isolated perfused goat udder. J. Dairy Res. 46: 47-
57.

Ronayne-De-Ferrer, P. A. & Sambucetti, M. E. ( 1993) Casein to whey protein ratio in
rat and human milk: effects of maternal protein intake. J. Dairy Sci. 76: 1645-
1653.

Sauber, T. E., Stahly, T. S., Williams, N. H. & Ewan, R. C. (1998) Effect of lean growth
genotype and dietary amino acid regimen on the lactational performance of sows.
J. Anim. Sci. 76: 1098-1111.

Sharma, K. & Kansal, V. K. (1999) Characteristics of transport systems of l-alanine in
mouse mammary gland and their regulation by lactogenic hormones: evidence for
two broad spectrum systems. J. Dairy Res. 66: 385-398.

33

Sharma, R. & Kansal, V. K. (2000) Heterogeneity of cationic amino acid transport
systems in mouse mammary gland and their regulation by lactogenic hormones. J.
Dairy Res. 67: 21-30.

Sherman, D. B., McNeillie, S. A., Jamieson, E. A. & Calvert, D. T. (1994) Lysine
transport in lactating rat mammary tissue: evidence for an interaction between
cationic and neutral amino acids. Acta Physiol. Scand. 151: 461-466.

Sherman, D. B., Millar, I. D. & Calvert, D. T. (1997) Mammary-tissue amino acid
transport systems. Proc. Nutr. Soc. 56: 177-191.

Stahly, T. S., G. L. Cromwell, H. J. Monegue. (1992) Milk yield responses of sows
nursing large litters to dietary amino acid levels. J. Anim. Sci. 70: 238.

Swaisgood, H. (1995). Protein and amino acid composition of bovine milk. In Handbook
of Milk Composition (Jensen, R. G., ed), pp. 464-472. Academic Press, San
Diego.

Torres, N., Lopez, G., De Santiago, S., Hutson, S. M. & Tovar, A. R. (1998) Dietary
protein level regulates expression of the mitochondrial branched-chain
aminotransferase in rats. J. Nutr. 128: 1368-1375.

Trottier, N. L. (1997) Nutritional control of amino acid supply to the mammary gland
during lactation in the pig. Proc. Nutr. Soc. 56: 581-591.

Trottier, N. L. & Guan, X. F. (2000) Research paradigms behind amino acid
requirements of the lactating sow: Theory and firture application. J. Anim. Sci.
78(Suppl. 3): 48-58.

Trottier, N. L., Shipley, C. F. & Easter, R. A. (1995) A technique for the venous
cannulation of the mammary gland in the lactating sow. J. Anim. Sci. 73: 1390-
1395.

Trottier, N. L., Shipley, C. F. & Easter, R. A. (1997) Plasma amino acid uptake by the
mammary gland of the lactating sow. J. Anim. Sci. 75: 1266-1278.

Verrna, N. & Kansal, V. K. (1995) Characterization and starvation induced regulation of
Methionine uptake sites in mouse mammary gland. Indian J. Exp. Biol. 33: 516-
520.

Vina, J ., Puertes, I. R., Saez, G. T. & Vina, J. R. (1981) Role of prolactin in amino acid
uptake by the lactating mammary gland of the rat. FEBS Lett. 126: 250-252.

Vina, J. R. & Williamson, D. H. (1981) Effects of lactation on L-leucine metabolism in
the rat. Studies in vivo and in vitro. Biochem. J. 194: 941-947.

34

Wohlt, J. E., Clark, J. H., Derrig, R. G. & Davis, C. L. (1977) Valine, leucine, and
isoleucine metabolism by lactating bovine mammary tissue. J. Dairy Sci. 60:
1875-1882.

Wu, G. & Knabe, D. A. (19943) Free and Protein-Bound Amino Acids in Sow's
Colostrum and Milk. J. Nutr. 124: 415-424.

Wu, G. & Knabe, D. A. (1994b) Free and protein-bound amino acids in sow's colostrum
and milk. J. Nutr. 124: 415-424.

Wu, G. & Morris, S. M. (1998) Arginine metabolism: nitric oxide and beyond.
Biochem. J. 336: 1-17.

Wu, G. Y., Flynn, N. E., Flynn, S. P., Jolly, C. A. & Davis, P. K. (1999) Dietary protein
or arginine deficiency impairs constitutive and inducible nitric oxide synthesis by
young rats. J. Nutr. 129: 1347-1354.

35

TABLE 1-1 Composition of diets for lactating sows (%, as-fed basis)

 

Dietary protein, %

 

 

Item 7.8 13.0 18.2 23.5
Corn 15.43 25.72 36.00 46.37
Soybean meal (44% CP) 14.75 24.58 34.41 44.32
Corn starch 42.53 28.39 14.25 0
Sugar 14.18 9.46 4.75 0
Tallow 5.00 5.00 5.00 5.00
Solka floc 3.02 2.02 1.02 0
Dicalcium phosphate 3.14 2.64 2.14 1.63
Calcium carbonate .51 .70 .89 1.08
Sah .25 .25 .25 .25
Trace mineral premixa .23 .23 .23 .23
Vitamin premixb .90 .90 .90 .90
DL-Met .027 .044 .061 .079
L-Thr .011 .019 .025 .033
L-Val .038 .064 .090 .1 15
ME, MJ/kg° 14.3 14.3 14.3 14.3
Calciumc .90 .90 .90 .90
Phosphorusc .72 .72 .72 .72
CP" 8.2 13.2 18.2 23.0
Argd .60 .93 1.28 1.60
Hisd .30 .45 .58 .72
Ired .37 .57 .78 1.00
Leud .64 1.01 1.36 1.73
Lysd .41 .67 .90 1.19
Metd .18 .27 .34 .42
Phed .42 .66 .89 1.13
Thrd 32 .53 66 93
Trpc 11 .18 25 33
Val .46 .71 .96 1.21

 

36

TABLE 1-1 Continued

 

21Provided the following amounts of trace minerals in milligrams per kilogram of diet: copper, 5; iodine,

0.075; iron, 50; manganese, 5; selenium, 0.15; and zinc, 50.

t,Provided the following amounts of vitamins in milligrams per kilogram of diet: retinyl acetate, 8.3;

cholecalciferol, 0.0138; “-tocoperol, 44.1; menadione, 4.5; vitamin Biz, 0.033; riboflavin, 4.5; d-pantothenic
acid, 17.6; niacin, 26.4; thiamin, 1.1; pyridoxine, 1.0; choline, 385.0; folic acid, 1.65; and d-biotin, 0.22.

cCalculated values (NRC, 1988).

dAnalyzed values.

37

TABLE 1-2 Effects of dietary protein concentration on lactation performance”

 

Dietary protein, %

 

 

Item SEM
7.8 13.0 18.2 23.5
No. of sows 4 4 4 4 -
Intake, kg/d
Feed“ 4.62y 5.75" 4.67y 4.62y .20
Protein° .366Z .750y .845y 1.070" .037
Sow’s body weight, kg
d 1 209.5 216.1 220.0 230.3 8.4
Change -25.8 -254 -16.8 -26.6 4.6
Backfat depth, mm
d 1 22.5 21.1 24.4 20.8 2.6
Change 46"" -.2y -6.8" -5.8" 1.4
Loin eye area, cm2
(1 1 46.1 47.2 44.8 ' 51.2 2.4
Changeb -8.46" -3.72Y .126 370’ 1.44
Litter size
Pigs, d 1 11 11 11 11 -
Pigs, d 21 10.3xy 11.0x 9.5y 11.0" 0.4
Litter weight, kg
d 1 19.1 19.3 19.3 18.5 .8
Gain 37.5y 48.7" 41 .4”y 44.5xy 2.9
Milk yield, kg/d
d 11 10.03 13.07 10.57 12.99 1.25
d 21 9.18y 13.21" 10.82"y 12.43"y 1.25

 

38

TABLE 1-2 Continued

 

zilData are least-squares means :1: standard error.
b’ cEffect of dietary treatments (P < .05 and .01, respectively).

x’ y’ zLeast-squares means within a row lacking a common superscript letter differ (1D < .05).

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41

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42

TABLE 1-6 Effects of dietary protein concentration on the amino acid profile of

 

 

 

 

 

defatted porcine milk”
Dietary protein, %
IAA
7.8 13.0 18.2 23.5
No.f 16 14 14 13
g/ 100 g milk protein
Arg 6122.15 6102.18 5952.18 6.13:.23
His 3.592.09 3.682.16 3.382.11 3.451.13
Ile 4.95 i .06 5.15 :l: .07 4.99 i .07 5.18 :t .09
Leu 8.802.10 8902.10 8.782.10 8.982.13
Lys 7,762.12 7.832.15 7.872.15 7.852.19
Met 2.67 i: .06 2.67 :t .07 2.56 i .06 2.76 i .06
Phe 4.61 d: .05 4.63 :l: .06 4.61 :t .06 4.68 i .08
Thr 5272.10 5162.12 4992.12 5152.15
Val 6.50 i .07 6.49 :t .09 6.34 a: .09 6.54 i .11
Ratios of IAA to Lys, wt/wt

Arg .79 :t .02 .80 :1: .03 .76 i .03 .79 :1: .03
His .46 i .01 .47 i .03 .43 i .02 .44 i .02
Ile .642 .01y .66:t.01" .642 .01y .662 0]"
Leu 1.14:1:.01"y 1.172.02" 1.12zt.01y 1.152.02"y
Lys 1.00 1.00 1.00 1.00

Met 34:1: .01"y 36:1: .01" .33 d: .01y 341.01"y
Phe .60 :1: .01 .61 :1: .01 .59 :1: .01 .60 :1: .01
Thr .68 :t .01 .66 i .02 .63 i .01 .66 :1: .02
Val .84 i .01 .85 :1: .02 .81 i .01 .84 :t .02

 

aData are least-squares means 2 standard error.
fNumber of milk samples analyzed.
x‘ yLeast-squares means within a row lacking a common superscript letter differ (P < .05).

43

TABLE 1-7 Effects of stage of lactation on the amino acid profile of defatted porcine

 

 

 

 

 

milk”
I A A Day of lactation
10 14 18 22
No.f 15 13 13 16
g/l 00 g milk protein
Arg 6.10-1.17 6.13:t.19 5.82i.19 6.15:t.17
Hisd 3.563;.10x 3.62:t.13" 3.24:2.13y 3.69:: .12"
Ile 5.02 i .06 5.10 :1: .07 4.99 d: .07 5.05 d: .06
Leu 8.82:1:.10 8.93zt.11 8.80:t.11 8.86i.10
Lys 7.76 :t .12"y 7.74 i .13"y 7.57 :t .13y 7.93 d: .12x
Met 2.69 i .05 2.67 i .07 2.69 i .07 2.64 i .06
Phe 4.59 :t .05 4.65 i .06 4.57 i .06 4.66 :t .05
Thr 5.17:t.10 5.091.11 4.94i.11 5.21 $.10
Val 6.45 :t .08 6.49 :t .09 6.34 i .09 6.48 :1: .08
Ratios of IAA to Lys, wt/wt

Arg .79 i .02 .79 i .03 .77 :1: .03 .77 i .02
His .46 i .01 .46 d: .02 .42 i .02 .46 i .02
Ile .65 i .01 .66 i .01 .66 d: .01 .64 :l: .01
Leu 1.14i.01"y 1.16i.01" 1.16::01" 1.12:1:.01y
Lys 1.00 1.00 1.00 1.00

Met .35 :1: .01 .34 :t .01 .35 :t .01 .33 :t .01
Phe .59 :t .01 .60 :1: .01 .61 i .01 .59 :1: .01
Thr .67 i .01 .66 i .01 .65 i .01 .66 i .01
Val .83 :t .01 .84 i .01 .84 :t .01 .82 i .01

 

aData are least-squares means i standard error.

dEffect of stage of lactation (P < .05).
Number of milk samples analyzed.
x’ yLeast-squares means within a row lacking a common superscript letter differ (P < .05).

 

 

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49

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50

TABLE 1-11 Effects of dietary protein concentration on the mammary uptake patterns

for plasma indispensable amino acids“

 

 

 

 

 

Dietary protein, %
IAA
7.8 13.0 18.2 23.5
No.f 12 9 9 12
Proportions of individual IAA to total IAA, % (mol/mol)
Argb 11.7 :1: .4y 11.4 :5 .6y 14.3 :1:.6x 11.3 :1: .4y
Hn 185A“ 2.31.7y 4922* 3.32.4xy
15° 10.753y 1258.5x 10.855y 1322.3x
Leub 18.9 2: .8y 20.2 i 1.4"y 16.1 :h 1.4y 22.0 i .8"
Lys° 15.1 i .4" 13.8 .1: .7y 12.9 i .7yz 12.3 2t .42
Met 4.5 :t: .1" 4.3 i .2"y 4.2 i .2xy 4.2 i: .2y
Phe° 8.1 i .2" 7.7 i .4xy 6.6 :t .4y 6.8 :t .22y
Thr° 11.1 i .3" 8.9 a: .6yz 10.6 :1: .6” 9.2 :t .3y
Tm L4i3 L0i6 2356 L153
Val 14.5 i .9 18.0:t 1.6 17.45 1.6 16.6i .9
Ratios of IAA to Lys, wt/wt

Argb .94 i .052 1.00 :h .08y 1.31 :t: .08" 1.09 :1: .05y
His .29 :l: .03"y .20 :1: .06y .39 i .06" .28 :1: .03"y
116c .66 :i: .022 .81 :l: .03y .76 :1: .03y .98 a: .02x
Leu° 1.13 :t: .042 1.31 :1; .06y 1.14 :5: .06yz 1.63 i: .04x
Met” .31 i .01y .32 21:01"y .33 2t .01" .35 :1. .01K
Phe .61 :t: .02 .63 :i: .03 .58 :t .03 .62 :1: .02
Thr .60 i .02"y .54 a. .03y .67 a: .03" .61 :1: .02"y
Trp 445.04 .10i.06 235.06 .11 2.04
Val" .81 3. .07y 1.06 a .12xy 1.07 :t .12"y 1.09 :h .07"

 

51

TABLE 1-11 Continued

 

aData are least-squares means 1 standard error.
I" cEffect of dietary treatments (P < .05 and .01, respectively).
fNumber of plasma samples analyzed.

x, , z . . . .
y Least-squares means w1th1n a row lackmg a common superscnpt letter.

52

TABLE 1-12 Effects of stage of lactation on the mammary uptake patterns for plasma

indispensable amino acids”

Stage of lactation, d

 

 

 

 

IAA
10 14 18 22
No.f 12 10 10 10
Proportions of individual IAA to total IAA, % (mol/mol)
Arg 11.3 at .4y 12.2 i .5"y 12.3 i .5"y 12.8 i .5"
His 4.0 :h .4 3.2 d: .5 3.9 i .5 3.3 :t .5
Ile 11.7:t.3 12.3i.3 11.9:t.3 ll.3i.3
Leu 19.6 :t .8 20.2 i 1.0 18.4 21:10 19.1:t1.0
Lys 14.3 :1: .4" 12.8 5 .5y 13.0 :t .5y 13.9 :1: .5xy
Met 4.4 :1: .1 4.4 :l: .2 4.2 fl: .2 4.2 :1: .2
Phe 7.6 i .2 7.2 5: .3 7.1 :t .3 7.3 i .3
Thr 10.1 i .3 9.5 at .4 9.8 i .4 10.3 i .4
Trp l.3i.3 1.45.4 l.6i.4 1.45.4
Val 15.759 16.7il.2 17.8512 16.3i 1.2
Ratios of IAA to Lys, wt/wt
Arg .96i.05y 1.14506" 1.13i.06" 1.115;.06y
His .30 :t .03 .27 a: .04 .33 i- .04 .27 :1: .04
Ilee .75 i .02y .88 :1: .02" .83 i: .02" .75 :1: .02 y
Lend 1.25 t .04y 1.44 i .04" 1.28 :t .04y 1.25 :t .04y
Metd .32 :1: .01y .35 :t .01" .33 i .01"y 311.01’
Phe .60 :t .02 .64 :1: .02 .62 d: .02 .59 i .02
Thr .58 :1: .02 .61 :1: .02 .62 :1: .02 .61 a: .02
Trp .14i.04 .14i.04 .1721:.O4 .14:l:.04
Val .91 :h .07y 1.04 :t .09"y 1.12 d: .09" .97 i .09"y

 

53

TABLE 1-12 Continued

 

aData are least- squares means i standard error.
d’ eEffect of stage of lactation (P < .05 and .01. respectively).
fNumber of plasma samples analyzed.

x’ yLeast- squares means within a row lacking a common superscript letter differ (P < .05).

54

TABLE 1-13

Comparison of the ratios (wt/111) of other indispensable amino acids to lysine in porcine

milk

 

IAA Davis Elliott King Dourmad Dourmad Duee and This Average

et al. et al. et al. et al. et a1. Jung studyf
(1994)a (1971)b (1993)“ (1991)d (1998)6 (1973)

 

Arg .56 .68 .65 .74 .74 .65 .79 .69
His .30 .45 .40 .53 .53 .35 .45 .43
Ile .51 .51 .59 .58 .58 .55 .65 .57
Leu 1.13 1.16 1.14 1.18 1.18 1.15 1.15 1.16
Lys 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
Met .28 .23 .28 .27 .27 .27 .34 .28
Phe .54 .56 .55 .57 .57 .54 .60 .56
Thr .47 .61 .59 .59 .59 .55 .66 .58
Trp - .17 .19 .15 .15 - - .17
Val .58 .63 .77 .69 .69 .80 .84 .71

Lys, g/ 100 g milk protein

 

7.90 7.59 6.95 7.39 7.39 7.75 7.83 7.54

 

3Derived from TABLE 2, 3, and 4 in the paper.
bDerived from TABLE 4 in the paper.
cDerived from TABLE 3 in the paper.
dDerived from TABLE 3 in the paper.
eDerived from TABLE 5 in the paper.
{Derived from TABLE 1-7 in this paper.

55

TABLE 1-14
Comparison of mammary uptake ratios (wt/wt) of indispensable amino acids to lvsine in the

lactating sow

 

 

IAA Linzell et Spincer et Nielsen et Trottier et This Ave
al. (1969)a a1. (1969)b al. (2000)° al. (1997)d studye

No.r 5 7 10 40 42 -
Arg 1.14 1.07 1.17 1.20 1.09 1.13
His .58 .56 .30 .37 .29 .42
Ile .72 1.09 .85 .80 .80 .85
Leu 1.17 1.77 1.58 1.43 1.31 1.45
Lys 1.00 1.00 1.00 1.00 1.00 1.00
Met .20 .32 .27 .29 .33 .28
Phe .61 .70 .61 .67 .61 .64
Thr .52 .68 .64 .67 .61 .62
Trp - - - .35 .15 .25
Val 1.07 1.44 .88 1.00 1.01 1.08

 

aDerived from TABLE 6 in the paper.

bDerived from TABLE 2 in the paper.

cDerived from TABLE 3 in the paper.

dDerived from TABLE 3 and 4 in the paper.
cDerived from TABLE 1-12 in this paper.

rNumber of plasma samples analyzed.

56

25-

 

 

 

 

 

 

 

A * - 7.8V: CP diet
42 23.5 /o CP d1et
h
g 20 J
V
6
e\
a 4-
: 1‘
O 5f a
'a l 5 d 4'; If":
* hf. -.
.. , 4
2' if * ‘1?
t. it
a. L113 '33
.52 '41-. 5.11 g
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E .i ’41
a li' at"; i
2 343 :4
i E”: <'."- 7..
‘ ' El" fill 933 3‘1
Arg His Ile Leu Lys Thr Trp Val

 

FIGURE 1-1 Effects of dietary protein concentration on the mammary uptake
proportions of individual dispensable amino acids to total individual dispensable amino
acids. The mammary uptake proportions (%, mol/mol) = 100 x mammary A-V
difference of individual IAA / mammary A-V difference of the total IAA (See
Materials and Methods). Least-squares means with ' differ (P < .05) between diets.

57

2.00 -
_ The AA profile in milk

.. The AA uptake pattern

 

1.80 .

1.60 -

1.40 -

1.20 -

1.00 -

0.80 -

 

 

0.60 ..

 

 

 

0.40 -

Ratio (wt/wt) of other IAA to Lys

0.20 -

 

 

 

 

 

 

0.00 -

 

FIGURE 1-2 Comparison of the amino acid profile in porcine milk and the amino acid
uptake pattern by the lactating porcine mammary gland. The AA profile in milk was
the average ratios (wt/wt) of other IAA to Lys in porcine milk (TABLE 1-13). The AA
uptake pattern was the average ratios (wt/wt) of other IAA to Lystaken up by the

lactating porcine mammary gland (TABLE 1-14).

58

4.5 q

 

 

 

 

—— Lys
. Val

23
= 4.0 u
0
a“:
E O
‘6
>.
<

3.5 -
t:
a
E
E
a
E
g4 3.0 T
.4

2'5 I I I v I I i I I I i I I

Intake of dietary Lys or Val, g/d

FIGURE 1-3 Mammary arterio-venous difference of plasma lysine or valine affected
by their intake. The relationship between log mammary A-V difference of plasma Lys
or Val (Y) and daily intake of dietary total Lys or Val (X) over a 21—d lactation period
was best fitted by a quadratic regression model. The solid curve (for Lys) was best
fitted by Y = 2.4104 + .07902 X - .001059 X2 (P = .036; R2 = .49) and the dot curve
(for Val) by Y = 1.7441 + .1120x - .001308x2 (P = .0066, R2 = .63). The line with an

arrow distinguishes the mammary uptake ratio of plasma Val to Lys less (the left side)

and greater (the right side) than 1:1, which corresponds to low and high intake of

dietary Lys or Val, respectively.

59

CHAPTER 2

Quantitation of Indispensable Amino Acids Mobilized from Body Protein Reserves

of Extra-mammary Tissues in the Lactating Sow

ABSTRACT Sows mobilize body protein reserves to support milk synthesis
when intake of protein is not adequate, which complicates estimates of dietary
requirements for indispensable amino acids (IAA). We hypothesize that
contributions of IAA mobilized from body protein reserves of extra-mammary
tissues to the mammary glands can be predicted by a regression model of
mammary arterio-venous (A-V) differences of plasma IAA (MAVD). To
quantify the contributions of endogenous IAA to the mammary gland for milk
synthesis, 16 Landrace x Yorkshire lactating sows were fed isocaloric (14.3 M]
ME/kg) diets varying in protein concentrations (78, 130, 182, and 235 g/kg).
Sows were fitted with carotid arterial and main mammary venous catheters on d 4
of lactation. Beginning at 0800 h on d 10, 14, 18, and 22 of lactation, matched
sets of arterial and venous blood samples were obtained every 30 min over 6 h.
Milk yield was estimated by the D20 dilution method. Mammary plasma flow
rate was estimated at 6440 L/d by the Fick principle using lysine as an internal
indicator. The relationship between log mammary A-V differences of plasma
IAA (Y) and daily intakes of IAA (X) was best fitted by a polynomial regression
model: Y = [30 + [51X + [32X2 (P < 0.05). The reverse log intercept ([30) was
defined as the mammary A-V difference of plasma IAA when intake of IAA was

extrapolated to zero. The contribution of each IAA was defined as the sum of its

60

need for maintenance and the amount of mammary uptake of this IAA when
intake of this IAA was extrapolated to zero. The contributions of IAA to the
mammary gland for milk synthesis predicted by the MAVD were 10.5, 2.1, 5.6,
6.4, and 4.8 g/kg body wt loss, respectively, for lysine, methionine,
phenylalanine, threonine, and valine. These predicted values were validated by
two other approaches. Firstly, the contributions of IAA were quantified by the
algebraic product of body protein loss and the amino acid concentration in body
protein. Secondly, the contributions of IAA were estimated by a factorial
approach with assumed utilization efficiencies of dietary digestible IAA for milk
synthesis. Estimates of the contributions based on body protein loss and the
factorial approach were very close to the values obtained by the MAVD. We
concluded that in the lactating sow, the contributions of IAA mobilized from body

protein to the mammary gland for milk synthesis can be predicted by the MAVD.

KEY WORDS: 0 body protein mobilization o arterio-venous difference 0 mammary

glands osows o lactation

61

INTRODUCTION

Sows lose body weight to support high rates of milk production when their intakes of
protein, indispensable amino acids (IAA), and/or energy are not adequate (Clowes et al.
1998, Dourmad et a1. 1998, Jones and Stahly 1999, King and Williams 1984, Mullan and
Williams 1990, Revell et a1. 1998, Williams and Smits 1991). Sow body weight loss
varies widely from 0 to 1.35 kg/d during lactation (Jones and Stahly 1999). The protein
proportion of body weight loss ranges from 10 to 16% and the lipid proportion of body
weight loss from 37 to 50% (Jones and Stahly 1999, Mullan and Williams 1990,
Whittemore and Yang 1989, Williams and Smits 1991). The chemical composition of
body weight loss during lactation is diet-dependent. Lactating sows lose a large amount
of muscle tissue (the major body protein reserve) when fed diets deficient in protein or
lysine, and a massive amount of adipose tissue (the major body lipid reserve) when fed

diets deficient in energy (Brendemuhl et al. 1989, Dourmad et al. 1998).

Maternal body weight loss may be driven by a homeorhetic mechanism that involves
coordinated changes in the metabolism of other body tissues to support high rate of milk
synthesis in the mammary glands (Bauman 1999). Thus, sows fed diets low in protein
may maintain milk protein production by mobilizing body protein reserves. Inadequate
intake of IAA decreases rates of protein synthesis and increases rates of protein
degradation in muscle tissue, leading to mobilization of the major body protein reserve in
lactating goats, rats, and pigs (Champredon et al. 1990, Clowes et al. 1998, Jones and

Stahly 1999, Pine et al. 1994). Mobilization of body protein reserves in sows may occur

62

throughout the entire period of lactation (Jones and Stahly 1999). This mobilization
complicates estimates of dietary requirements for 1AA based on the milk production

response (Pettigrew 1993).

Dietary requirements for IAA by the lactating sow can be estimated by a factorial
approach, i.e., by integrating individual needs of IAA for the various physiological
components including body maintenance, milk production, and body protein accretion (or
loss) (NRC 1998, Pettigrew 1993). The contribution of endogenous lysine from body
protein loss is considered the sum of maintenance need and the intercept in the linear
regression of lysine intake against litter weight gain (NRC 1998, Pettigrew 1993).
Contributions of endogenous IAA other than lysine from body protein loss are derived by
multiplying the contribution of endogenous lysine and ideal ratios of other IAA to lysine
in body tissue (NRC 1998, Pettigrew 1993). However, the contributions of endogenous
IAA need to be defined experimentally to adjust dietary requirements for IAA at different

rates of body protein loss.

We have developed a regression model for mammary arterio-venous (A-V) differences of
plasma IAA to predict the contributions of endogenous IAA mobilized from extra-
marnmary tissues in the lactating sow. Being driven by the homeorhetic mechanism,
endogenous IAA mobilized from body protein reserves will be partitioned to the
mammary gland for milk synthesis. Nutrients are used first for maintenance and second
for milk production in sows (Pomar et al. 1991). Thus, we hypothesize that the

contributions of IAA from body protein reserves in the lactating sow can be predicted as

63

the sum of maintenance needs for IAA and mammary uptakes of plasma IAA when
dietary intakes of IAA are extrapolated to zero. Our objective in this study was to
quantify the contributions of IAA mobilized from body protein reserves in lactating sows
so that these estimates could be used in a factorial approach to define dietary

requirements for IAA.

MATERIALS AND METHODS

All procedures in this study were approved by the All-University Committee on Animal

Use and Care of Michigan State University.

Experimental design. Sixteen Landrace x Yorkshire multiparous lactating sows (parity 2
or 3) were allocated to treatments according to a randomized block design. Each block
consisted of four sows. Each sow in one block was provided ad libitum access to one of
four diets. Diets contained different crude protein concentrations (78, 130, 182, and 235
g/kg) and were balanced to contain an ideal pattern of IAA that met recommendations for
lactating sows (NRC 1988). Diets were formulated by diluting the highest concentration
of crude protein (235 g/kg diet) with starch and sugar. All diets were isocaloric with a
metabolizable energy (MB) of 14.3 MJ/kg. The composition of the diets is given in

TABLE 2-1.

Animals. Litters with 11 piglets per sow were cross-fostered within 48 h after birth. Sow
and litter characteristics at the stat of the study are given in TABLE 2-2. Sows were
individually housed in farrowing crates equipped with metal wire floors in a
mechanically ventilated, thermally controlled room (21 oC). The sows were fed twice
daily to appetite and provided free access to water. Sow food intake was recorded daily.
Sow body weight, backfat depth, and loin eye area were recorded after farrowing (d l)
and at weaning (d 21). Backfat depth and loin eye area were scanned (Ultrasound
Scanner 200 Vet with ASP-18 linear probe, Pie Medical Equipment B.V., Maastricht, the
Netherlands) 5 cm lateral from the spine and centered over the 10th rib. Piglets were

individually weighed weekly.

Cannulation. For the catheter, microbore Tygon® tubing (1.0 mm id, 1.8 mm o.d.,
Norton Performance Plastics Co., Akron, OH) was used and the lumen was coated with
triododecylmethylammonium chloride - heparin complex (7% w/w, Polysciences, Inc.,
Warrington, PA). The anterior main mammary vein and the carotid artery were
cannulated in sows on d 4 :t 1 of lactation following the surgical procedure described by
Trottier et al. (1995). Antibiotic Naxcel® (Pharmacia and Upjohn Co., Kalamazoo, MI)
and anti-inflammatory Banamine® (Schering-Plough Animal Health Corp., Kenilmorth,
NJ) were administered i.v. for three days following surgery. The catheters were flushed
daily with heparinized saline (20 IU heparin/ml). Food was offered in a stair step manner

during the first three days post surgery, and was available free access thereafier.

65

Milk intake estimation. Milk intake by piglets was estimated on d 11 and 21 by the D20
dilution method of Pettigrew et al. (1987) as modified by Pluske et a1. (1997) using piglet
serum. In brief, piglets were fasted for l h, weighed, and injected i.m. with a dose (1
mg/kg BW) of D20 (Cambridge Isotope Laboratories, Andover, MA). At 1 h afier the
dose, piglets were bled from the jugular vein using vacutainers containing SST® gel and
clot-activator (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ). At 24 h after
the dose, the piglets were fasted for 1 h, weighed, and bled as before. During this 24-h
period, piglets were not provided access to water. Blood samples were centrifuged at
1500 x g for 15 min at 4 °C, and then serum samples were separated and stored at -20 0C
until assayed for D20. Serum D20 concentration was determined by an infrared
spectrophotometer (The Foxboro Co., East Bridgewater, MA) using a fixed-filter length
of 4 pm. Milk intake was calculated on the basis of the D20 dilution principle (Pettigrew

et al. 1987).

Mammary plasma flow estimation. Mammary plasma flow rate was calculated by the
Fick principle of Davis et al. (1978) as modified by Trottier et al. (1997) using lysine as
an internal indicator. Given that the amount of lysine output in milk contributed from
vascular peptides is negligible (Bequette et al. 1999), mammary uptake of plasma lysine
can be based on the Fick principle as follows:
Mammary lysine uptake = Plasma lysine A-V difference x plasma flow rate

= Lysine output in milk + lysine's other metabolic fates (1)

Among other metabolic fates, there are two major pathways: lysine oxidation and lysine

accretion in the mammary tissue. In the guinea pig mammary tissue, the amount of

66

lysine oxidation is negligible (Mepham 1982). Because lysine is the first limiting amino
acid in a com-soybean meal diet for lactating sows (NRC 1998) and dietary metabolic
energy intake was adequate in the present study, it might be minimally oxidized in the
lactating mammary glands. In addition, accretion of lysine in lactating porcine mammary
glands was calculated to be around 1 g/d from the study of Kim et al. (1999), which is
negligible compared to lysine output in milk (41 g/d) in the present study. Therefore,
Equation (1) is simplified as follows:

Mammary lysine uptake = Plasma lysine A-V difference x plasma flow rate

= Lysine output in milk (2)

Thus, mammary plasma flow rate was estimated by dividing lysine output in milk by
plasma lysine A-V difference:

Mammary plasma flow rate (L/d) = lysine concentration in milk (mmol/L) x milk yield

(L/d) / (lysine concentration in carotid artery - lysine concentration in the main

mammary vein) (mmol/L).

Blood and milk sampling protocol. Blood samples were collected on d 9 :t 1, 13 a: l, 17
:1: l, and 21 d: I (referred to as d 10, 14, 18, and 22, respectively). On sampling days,
sows were provided ad libitum access to food and water as usual. The sows were fed one
h prior to blood sampling. Matched sets of carotid arterial and main mammary venous
blood samples (10 ml each) were collected into sterile syringes every 30 min over a 6-h
period beginning at 0800 h. Blood was transferred into EDTA-coated tubes and
centrifuged at 1500 x g for 15 min at 4 °C. Plasma was removed and stored at -20 °C.

0n the same day as blood sampling, milk was collected and pooled from all functional

67

teats on each sow following an iv administration of oxytocin (10 IU). Approximately
20 ml pooled milk was transferred into a vial containing a preserver (Broad Spectrum
Microtales‘m, D & F Control System, Inc., San Ramon, CA) and stored at 4 °C until
assayed for milk composition. Another portion of pooled milk was stored at -20 °C until
assayed for nitrogen. The remainder of pooled milk was defatted by centrifugation at

1500 x g for 15 min at 4 OC and stored at -20 0C until assayed for amino acids (AA).

Laboratory measurements

(1) Milk composition and amino acid analysis. Milk fat, lactose, and protein contents
were assayed using a mid-infrared spectroscope (Multispec M, Berwind Instrument Ltd.,
York, England, UK.) (AOAC 1990). Frozen milk samples were thawed at 37 °C and
mixed by vortex. Milk nitrogen was determined using LECO Nitrogen Analyzer FP-
2000 (LECO Corp., St. Joseph, MI) and EDTA (Sigma Chemical Co., St. Louis, MO) as
a calibration standard. Amino acid concentrations in the defatted milk were analyzed
using reversed-phase high-perfonnance liquid chromatography by a precolumn
derivatization with phenylisothiocyanate (Pierce, Rockford, IL) following the Pico-Tag®
method (Cohen et al. 1989). In brief, 200 11L of defatted milk was hydrolyzed using 6
mol/L HCl for 24 h at 110 °C. Amino acids in the hydrolysate were derivatized with
phenylisothiocyanate and separated by a Pico-Tag® column (3.9 mm x 150 mm, Waters
Corp., Milford, MA) using Alliancetm Waters 2690 Separations Module and detected by
Waterstm 486 Tenable Absorbance Detector (Waters Corp., Milford, MA). Norleucine
(Sigma, St. Louis, MO) as an internal standard was added into defatted milk samples

before hydrolysis. Amino acid standard H (Pierce, Rockford, IL) was used as a

68

calibration standard. The method was validated with certified amino acid standard

(NIST, Gaithersburg, MD).

(2) Plasma amino acid analysis. Frozen plasma samples were thawed at 4 °C overnight
and mixed by vortex. Plasma samples were pooled from the 13 samples taken from each
sow on each sampling day. Glucosaminic acid was added into the pooled samples as an
internal standard. Amino acid concentrations were determined using a Beckman 6300
Amino Acid Analyzer following the method described by Lee and Slocum (1987). In
brief, plasma samples were deproteinized by 35% sulfosalicyclic acid precipitation.
Amino acids in the supernatant were separated by a Beckman cation-ion exchange
column charged in lithium citrate buffer. The eluted amino acids (AA) were measured

spectrophotometrically following postcolumn derivatization with ninhydrin.

(3) Dietary nitrogen and amino acid analysis. Food samples were finely ground using a
Cyclotec® 1093 Sample Mill (Foss Tecator, Sweden). Food nitrogen was analyzed by the
micro-Kjeldahl method (AOAC, 1990). Hydrolysis of food samples and determination of

AA in hydrolysate were performed as described in (1).

Statistical analyses. The linear regression models were computed by the REG procedure
of SAS/STAT (Version 6.12, SAS Institute Inc., Cary, NC). Mammary A-V differences
of plasma 1AA were log-transformed based on their residual error distribution. The
relationship between log mammary A-V differences of plasma IAA and intakes of dietary

IAA followed a linear and quadratic pattern (P < 0.05), except for isoleucine and leucine.

69

The best fitting regression model was Y = 130 + [31X + B2X2, with Y being log mammary
A-V differences of plasma IAA (the predicted variable), X being daily intake of dietary
IAA (the predictor variable), and BO, 0., and B2 being the parameter estimates.
Confidence intervals were estimated using the reflection method by the Bootstrapping
procedure (Neter et al., 1996). A minimum of 750 bootstrapping samples was set up to
compute the confidence intervals. Analysis of variance for milk yield was cam'ed out by

the Mixed procedure with a repeated statement of the SAS/STAT.

RESULTS

Average sow and litter performance over a 21-d lactation is shown in TABLE 2-3.
Dietary ME intake met the level of 70.3 MJ/d recommended by the NRC (1998),
although intakes of protein and lysine varied across individual lactating sows. Changes
in sow body weight, backfat depth, and loin eye area were within ranges (PigCHAMP
1998). Average litter weight gain across diets met the expected level of 40 kg over a 21-
d lactation. Average daily milk yield did not change significantly (P > 0.05) between d
11 and 21 of lactation (11.67 i 0.62 and 11.41 i 0.62 kg/d, respectively). Therefore,
sows in the present study performed at levels comparable to the current US. national

average performance for lactating sows (PigCHAMP 1998).

Plasma flow rate across the lactating porcine mammary glands was estimated by the F ick
principle (TABLE 2-4). Lysine was chosen as an internal indicator for evaluating

mammary plasma flow rate based on the following reasons: (1) Lysine is the first limiting

70

amino acid in a corn-soybean meal diet for lactating sows (NRC 1998), implying that its
oxidation is minimal when dietary ME intake is adequate; and (2) Kim et al. (1999) have
shown that lysine accretion in lactating porcine mammary glands is only about 1 g/d,
which only represents approximately 2.5 % of the total lysine output in milk in the
present study. Mammary plasma flow rates estimated using phenylalanine plus tyrosine
as an internal indicator were not different from those using lysine as an internal indicator
(data not shown). The average volume ratio of mammary plasma flow to milk yield was

560:1 over a 21-d lactation period.

Firstly, we have developed a regression model for mammary A-V differences of plasma
IAA to predict the contributions of IAA (IAAC) mobilized from body protein reserves in
extra-mammary tissues to the mammary glands in lactating sows. TABLE 2-5 shows the
relationship between log mammary A-V differences of plasma IAA and daily intakes of
IAA by the sows over a 21-d period of lactation. The data were best fitted by a
polynomial regression model: Y = [30 + 01X + B2X2, where Y was log mammary A-V
differences of plasma IAA (the response variable); X was daily intake of IAA (the
predictor variable); and Bo, [31, and [322 were parameter estimates. Probability values were
significant (P < 0.05) for the model, intercept, and both linear and quadratic terms for
arginine, lysine, phenylalanine, threonine, and valine. Except for intercept, P—values
were significant (P < 0.05) for model, the linear terms, and quadratic terms for histidine,
methionine, and tryptophan. Although P—values were very significant (P < 0.01) for the
model, they were not significant for the quadratic term for isoleucine or leucine. We

focused on lysine, methionine, phenylalanine, threonine, and valine in the present study.

71

It should be pointed out that our model in the present study did not differentiate two
endogenous sources: de novo synthesis and protein breakdown. Because arginine can be
synthesized in body (NRC 1998), its endogenous contribution mobilized from body
protein can not be defined. In addition, either estimates of intercepts for histidine and
tryptophan or estimates of the quadratic term for isoleucine and leucine were not different
from zero (P > 0.05). Thus, their endogenous contributions can not be defined from the

models in the present study.

The relationship between log mammary A-V differences of plasma lysine and daily
intakes of lysine is shown in FIGURE 2-1. The reverse log intercept (Bo) is defined as
the mammary A-V difference of plasma lysine when lysine intake is extrapolated to zero.
Mammary uptakes of plasma IAA, defined by multiplying mammary A-V differences of
plasma IAA and mammary plasma flow rate, were derived from the contributions of
endogenous IAA mobilized from body protein reserves in extra-mammary tissues when
dietary intakes of IAA were extrapolated to zero. A small proportion of endogenous IAA
would be used first for obligatory metabolism. Predominant proportion of endogenous
IAA would be taken up second by the mammary glands for milk synthesis. Thus, IAAc is
defined as the sum of these two parts. The amount of IAAc used for obligatory
metabolism was considered as the maintenance need (NRC 1998). The amount of IAA
used for milk synthesis was predicted by the regressions (TABLE 2-6). We named this

technique the regression model for the mammary A-V differences of plasma IAA

(MAVD).

72

Secondly, the contributions of endogenous IAA in lactating sows was quantified as the
algebraic product of body protein loss during lactation and the respective amino acid
concentration in body protein (TABLE 2-7). First, nitrogen balance in the lactating sows
was predicted from the regression equation: Nitrogen balance (g/d) = -15.8 + 1.22 dietary
lysine intake (g/d) - 0.63 nitrogen output in milk (g/d) (Dourmad et al. 1998). Negative
nitrogen balance of the whole animal was defined as the amount of nitrogen loss from
body protein reserves. Second, amino acid concentrations in body protein were derived
from ARC (1981) and NRC (1998). Lysine concentration was estimated at 7 g/ 100 g
body protein (ARC 1981). Methionine, phenylalanine, threonine, and valine

concentrations were calculated on the basis of the amino acid profile in body protein

(NRC 1998).

Thirdly, the contributions of endogenous IAA were estimated by a factorial approach
with assumed utilization efficiencies of digestible IAA for milk synthesis (TABLE 2-8).
The utilization efficiency of digestible lysine for milk synthesis was assumed to be 100%.
This assumption was based on the fact that lysine was the first limiting amino acid and
dietary ME intake was adequate in this study. The ratio of valine output in milk (34.9
g/d) to valine uptake by the mammary glands (41.2 g/d) was approximately 85%, which
represented an approximate utilization efficiency of digestible valine for milk synthesis
by the mammary gland. The dietary ratio of digestible phenylalanine to tyrosine (1.44:1)
was higher by 50% compared to their ratio in milk (0.96:1). Phenylalanine might be
oxidized considerably and/or be converted to tyrosine in the body in the present study.

Thus, the utilization efficiency of digestible phenylalanine for milk synthesis was

73

arbitrarily assumed at 65%. Methionine and threonine might, to some extent, be oxidized
and/or converted to cysteine and glycine, respectively. Their utilization efficiencies for

milk synthesis were arbitrarily assumed to be 95%.

DISCUSSION

To quantify mammary uptake of plasma 1AA, we estimated mammary A-V differences of
plasma IAA and mammary plasma flow rate. Linzell et al. (1969) first developed a
mammary A-V difference method to estimate mammary uptake of plasma nutrients in
lactating sows. Trottier et al. (1995) further improved this method in lactating sows by
introducing a cannulation of the anterior main mammary vein to obtain representative
blood samples of the total mammary venous effluent. Mammary plasma flow has been
estimated based on the F ick principle using internal indicators such as lysine, methionine,
and phenylalanine plus tyrosine (Davis et al. 1978, Trottier et al. 1997). Mammary
plasma flow rates estimated using phenylalanine plus tyrosine as an internal indicator
were not significantly different from those using lysine as an intemal indicator in the
present study. Lysine would be minimally oxidized or negligibly accreted in the
mammary glands because it is the first liming amino acid for the lactating sow (Trottier et
al. 1997). Therefore, mammary plasma flow rate reported herein was calculated using
lysine as the internal indicator. An estimate of mammary plasma flow rate was 6440 L/d,
equivalent to a volume ratio of plasma flow to milk yield of 560:]. This ratio is in
agreement with that of 540:1 in sows (Trottier et al. 1997) and is comparable to the

volume ratio of blood flow to milk yield of 500:1 in cows and goats (Linzell 1974).

74

We have developed a regression model for mammary A-V differences of plasma IAA to
predict the contributions of endogenous IAA (IAAc) mobilized from the body protein
reserves of extra-mammary tissues to the lactating porcine mammary glands. When
intake of protein is not adequate, sows mobilize body protein reserves to support milk
production (Mullan and Williams 1990). Mobilization of body protein reserves may be
driven by the homeorhetic mechanism that involves coordinated changes in the
metabolism of other body tissues to support milk synthesis (Collier 1999). Sows alter
secretion of metabolic hormones to favor this mobilization to support milk synthesis
(Quesnel et al. 1998). This mobilization is mainly associated with a decreased rate of
protein synthesis and/or an increased rate of protein degradation in the muscle tissue
(Baracos et al. 1991, Clowes et a1. 1998, Jones and Stahly 1999, Pine et al. 1994).
Through this mobilization, IAA from extra-mammary tissues is partitioned to the
mammary glands for milk synthesis. Nutrients are used first for maintenance and second
for milk production in sows (Pomar et a1. 1991). Therefore, we hypothesize that IAAc
mobilized from body protein reserves can be predicted as the sum of the amount of the
IAA captured for obligatory metabolism (or maintenance need) and the amount of the
IAA taken up by the mammary glands when dietary intake of the IAA is extrapolated to

zero .

To validate the MAVD, we quantified the contributions of endogenous IAA by

multiplying body protein loss and the concentrations of IAA in body protein. This

approach is referred as the body composition change approach (BCCA). In the present

75

study, body protein loss of the sows was estimated to be 177 g/d or 150 g/kg BW loss
using nitrogen balance assuming that negative nitrogen balance of the whole animal
represented body protein loss. This estimate of body protein loss is in agreement with
previous studies (Jones and Stahly 1999, Mullan and Williams 1990, Whittemore and
Yang 1989). Body protein loss can be calculated by multiplying body weight loss and
the protein proportion of the body weight loss. The protein proportion of body weight
loss is relatively constant compared to the lipid proportion in lactating sows (Whittemore
and Yang 1989). The protein proportion of the body weight loss for Large White x
Landrace crossbred sows ranges from 122 to 160 g/kg BW loss (Mullan and Williams
1990, Whittemore and Yang 1989). The NRC (1988) assumed the protein proportion to
be 130 g/kg BW loss; however, the current NRC (1998) used a value of 94 g/kg BW loss
from the study of Beyer et al. (1993a,b). Lysine concentration in body protein was
assumed at 70 g/kg body protein in the present study. This assumption was based on
estimates of 65 (NRC 1998), 70 (ARC 1981), 70.5 (Kyriazakis and Emmans 1993), and
73 g/kg body protein (Mahan 1998). Moreover, the amino acid composition of the whole
body protein was assumed to represent that of the body protein mobilized during
lactation. It should be pointed out that protein turnover was not taken into an account for
the BCCA. In fact, oxidation rates of the branched-chain, aromatic, and sulfur AA
liberated from tissue proteolysis might be increased to maintain their low tissue
concentrations, while oxidation rates of lysine and threonine liberated from tissue
proteolysis might not be further increased due to their larger pool sizes (Millward 1998).
In addition, lactation significantly increases the rate of oxidation for branched-chain AA

(DeSantiago et al. 1998), which may decrease the availability of endogenous valine from

76

extra-mammary tissues to the mammary glands. Therefore, the amount of lysine and
threonine mobilized from body protein reserves defined by the BCCA could be close to
the estimates of those contributions predicted by the MAVD, while the amount of
methionine, phenylalanine, and valine defined by the BCCA are slightly higher than

those contributions predicted by the MAVD (TABLE 2-9).

Furthermore, the MAVD was validated to some extent by the factorial approach using
assumed utilization efficiencies of digestible IAA for milk synthesis. The utilization
efficiency of digestible lysine for milk synthesis was assumed at 100% in the lactating
sows in the present study. Branched-chain AA are taken up by the mammary glands in
excess of their output in milk protein (Davis and Mepham 1976, Trottier et al. 1997,
Wohlt et al. 1977). Approximately 30% of valine molecules taken up by goat mammary
glands were oxidized to C02 and 70% were incorporated into casein (Roets et al. 1979).
The ratio of valine output in milk to valine uptake by the mammary glands was about
85% in the present study. This ratio might be defined as the utilization efficiency of
digestible valine for milk synthesis by the mammary glands assuming that the amount of
milk valine contributed from vascular peptides is negligible. The hydroxylation of
phenylalanine to tyrosine in the whole body increased from 10 to 18% with the infusion
of phenylalanine in lactating goats (Bequette et al. 1999). Dietary ratio of digestible
phenylalanine to tyrosine was higher by 50% compared to their ratio in milk in the
present study, implying that hydroxylation of phenylalanine to tyrosine might be
considerable. Approximately 3% of threonine molecules taken up by the lactating goat

mammary gland were metabolized (Verbeke et al. 1972). To what degree oxidation of

77

methionine or its conversion to cysteine occurs in the mammary gland is not known.
Because of the absence of data, utilization efficiencies of digestible methionine,
phenylalanine, and threonine for milk synthesis were assumed to be 95, 65, and 95%,
respectively. The contributions of endogenous lysine, methionine, phenylalanine, and
threonine derived by the factorial approach are quite comparable to those predicted by the
MAVD (TABLE 2-9). The contribution of endogenous valine derived by the factorial

approach is slightly higher compared to that predicted by the MAVD.

The contribution of endogenous lysine, average of 10.4 g/kg BW loss for the three
methods in the present study, is in agreement with the study of (Jones and Stahly 1999)
where lactating sows fed diets low in protein lost 10.3 g/kg BW loss. However, it is
much higher than the value of 6.2 g/kg BW loss assumed by the current NRC (1998). As
discussed above, the current NRC (1998) might underestimate the contribution of
endogenous lysine by underestimating the protein proportion of the body weight loss and

lysine concentration in body protein for modern genotypes of lactating sows.

In summary, the contributions of IAA mobilized from the body protein reserves to the
mammary glands for milk synthesis can be predicted by the MAVD, which is validated to
some extent by the BCCA and the factorial approach. The MAVD predicted the amount
of IAA from body protein loss to be available to the mammary glands. The BCCA
estimated the maximal amount of IAA from body protein loss because it did not take into
an account for protein turnover and rates of oxidation for individual IAA. The factorial

approach could derive the contributions of IAA if utilization efficiencies of digestible

78

IAA for milk synthesis were defined. The contribution of endogenous lysine (10.4 g/kg
BW loss) estimated in the present study is much higher than the value of 6.2 g/kg BW

loss assumed by the current NRC (1998).

79

LITERATURE CITED

ARC. (1981). The nutrient requirements of pigs. Commonwealth Agricultural Bureaux,
London.

Baracos, V. E., Brun Bellut, J. & Marie, M. (1991) Tissue protein synthesis in lactating
and dry goats. Br. J. Nutr. 66: 451-465.

Bauman, D. E. (1999) Bovine somatotropin and lactation: from basic science to
commercial application. Domestic Anim. Endocrinol. 17: 101-116.

Bequette, B. J ., Backwell, F. R., Kyle, C. E., Calder, A. G., Buchan, V., Crompton, L. A.,
France, J. & MaCrae, J. C. (1999) Vascular sources of phenylalanine, tyrosine,
lysine, and methionine for casein synthesis in lactating goats. J. Dairy Sci. 82:
362-377.

Beyer, M., Jentsch, W., Hoffrnann, L. & Schiemann, R. (1993a) [The energy and
nitrogen metabolism of pregnant and lactating sows and suckling piglets. 2.
Chemical composition and energy content of the animal bodies of pregnant, non-
pregnant and lactating sows]. Arch. Tieremahr. 44: 317-338.

Beyer, M., Jentsch, W., Hoffrnann, L. & Schiemann, R. (1993b) [Energy and nitrogen
metabolism of pregnant and lactating sows and suckling piglets. 3. Chemical
composition and energy content of the animal body fractions as well as portions
of the animal body fractions of empty body of nonpregnant, pregnant and
lactating sows]. Arch. Tieremahr. 45: 13-34.

Brendemuhl, J. H., Lewis, A. J. & Peo Jr., E. R. (1989) Influence of energy and protein
intake during lactation on body composition of primiparous sows. J. Anim. Sci.
67: 1478-1488.

Champredon, C., Debras, E., Mirand, P. P. & Amal, M. (1990) Methionine flux and
tissue protein synthesis in lactating and dry goats. J. Nutr. 120: 1006-1015.

Clowes, E. J ., Williams, I. H., Baracos, V. E., Pluske, J. R., Cegielski, A. C., Zak, L. J. &
Aheme, F. X. (1998) Feeding lactating primiparous sows to establish three
divergent metabolic states: 11. Effect on nitrogen partitioning and skeletal muscle
composition. J. Anim. Sci. 76: 1 154-1 164.

Cohen, S. A., Meys, M. & Tarvin, T. L. (1989) The Pico.Tag Method: A manual of
advanced techniques for amino acid analysis. Millipore Corp.

Collier, R. J. (1999). Lactation, Nonhuman. In Encyclopedia of Reproduction (Knobil, E.
& Neill, J. D., eds.), Vol. 4, pp. 973-978. 2 vols. Academic Press, New York.

Davis, S. R., Bickerstaffe, R. & Hart, D. S. (1978) Amino acid uptake by the mammary
gland of the lactating ewe. Aust. J. Biol. Sci. 31: 123-132.

80

Davis, S. R. & Mepham, T. B. (1976) Metabolism of L-(U-14C)valine, L-(U-
14C)leucine, L-(U-14C)histidine and L-(U-14C) phenylalanine by the isolated
perfused lactating guinea-pig mammary gland. Biochem. J. 156: 553-560.

DeSantiago, S., Torres, N., Suryawan, A., Tovar, A. R. & Hutson, S. M. (1998)
Regulation of branched-chain amino acid metabolism in the lactating rat. J. Nutr.
128:1165—1171.

Dourmad, J. Y., Noblet, J. & Etienne, M. (1998) Effect of protein and lysine supply on
performance, nitrogen balance, and body composition changes of sows during
lactation. J. Anim. Sci. 76: 542-550.

Jones, D. B. & Stahly, T. S. (1999) Impact of amino acid nutrition during lactation on
body nutrient mobilization and milk nutrient output in primiparous sows. J. Anim.
Sci. 77: 1513-1522.

Kim, S. W., Hurley, W. L., Han, I. K. & Easter, R. A. (1999) Changes in tissue
composition associated with mammary gland growth during lactation in sows. J.
Anim. Sci. 77: 2510-2516.

King, R. H. & Williams, I. H. (1984) The effect of nutrition on the reproductive
performance of first-litter sows: 2. Protein and energy intakes during lactation.
Anim. Prod. 38: 249-256.

Koketsu, Y. (1998) PigCHAMP 1998 Summary Report.

Kyriazakis, I. & Emmans, G. C. (1993) Whole body amino acid composition of the
growing pig. J. Sci. Food. Agric. 62: 29-33.

Lee, P. & Slocum, R. (1987) Expanded physiological fluid analysis. System 6300/7300
Application Note. No. A6300-AN-006. Beckman Instruments.

Linzell, J. L. (1974). Mammary blood flow and methods of identifying and measuring
precursors of milk. In Lactation: A comprehensive treatise (Larson, B. L. &
Smith, V. R., eds.), Vol. 1, pp. 143-225. 1 of 4 vols. Academic Press, New York.

Linzell, J. L., Mepham, T. B., Annison, E. F. & West, C. E. (1969) Mammary
metabolism in lactating sows: arteriovenous differences of milk precursors and
the mammary metabolism of [14C]glucose and [14C]acetate. J. Nutr. 23: 319-
332.

Mahan, D. C. (1998) Relationship of Gestation Protein and Feed Intake Level over a
Five-Parity Period Using a High-Producing Sow Genotype. J. Anim. Sci. 76: 533-
541.

Mepham, T. B. (1982) Amino acid utilization by lactating mammary gland. J. Dairy Sci.
65: 287-298.

81

Millward, D. J. (1998) Metabolic demands for amino acids and the human dietary
requirement: Millward and Rivers (1988) revisited. J. Nutr. 128: 25638-25768.

Mullan, B. P. & Williams, I. H. (1990) The chemical composition of sows during their
first lactation. Anim. Prod. 51: 375-387.

NRC. (1998). Nutrient requirements of swine. lOth ed., National Academy Press,
Washington, DC.

Pettigrew, J. E. ( 1993) Amino acid nutrition of gestating and lactating sows. Biokyowa
Technical Review-5: 2-1 8.

Pettigrew, J. E., Cornelius, S. G., Moser, R. L. & Sower, A. F. (1987) A refinement and
evaluation of the isotope dilution method for estimating milk intake by piglets.
Livest. Prod. Sci. 16: 163-174.

Pine, A. P., Jessop, N. S. & Allan, G. F. (1994) Maternal protein reserves and their
influence on lactational performance in rats 4. Tissue protein synthesis and
turnover associated with mobilization of maternal protein. Br. J. Nutr. 72: 831-
844.

Pluske, J. R., Fenton, T. W., Lorschy, M. L., Pettigrew, J. E., Sower, A. F. & Aheme, F.
X. (1997) A modification to the isotope dilution technique for estimating milk
intake of pigs using pig serum. J. Anim. Sci. 75: 1279-1283.

Pomar, C., Harris, D. L. & Minvielle, F. (1991) Computer simulation model of swine
production systems: II. Modeling body composition and weight of female pigs,
fetal development, milk production, and growth of suckling pigs. J. Anim. Sci. 69:
1489-1502.

Quesnel, H., Pasquier, A., Mounier, A. M., Louveau, I. & Prunier, A. (1998) Influence
of feed restriction in primiparous lactating sows on body condition and metabolic
parameters. Reprod. Nutr. Dev. 38: 261-274.

Revell, D. K., Williams, I. H., Mullan, B. P., Ranford, J. L. & Smits, R. J. (1998) Body
composition at farrowing and nutrition during lactation affect the performance of
primiparous sows: 1. Voluntary feed intake, weight loss, andplasma metabolites.
J. Anim. Sci. 76: 1729-1737.

Roets, E., Massart Leen, A. M., Verbeke, R. & Peeters, G. (1979) Metabolism of [U-
14C; 2,3-3H]-L-valine by the isolated perfused goat udder. J. Dairy Res. 46: 47-
57.

Trottier, N. L., Shipley, C. F. & Easter, R. A. (1995) A technique for the venous
cannulation of the mammary gland in the lactating sow. J. Anim. Sci. 73: 1390-

1395.

82

Trottier, N. L., Shipley, C. F. & Easter, R. A. (1997) Plasma amino acid uptake by the
mammary gland ofthe lactating sow. J. Anim. Sci. 75: 1266-1278.

Verbeke, R., Roets, E., Massart-Leen, A.-M. 8.: Peeters, G. (1972) Metabolism of [U-
l4C]—L-threonine and [U-l4C]-L—phenylalanine by the isolated perfused udder. J.
Dairy Res. 39: 239-249.

Whittemore, C. T. & Yang, H. (1989) Physical and chemical composition of the body of
breeding sows with differing body subcutaneous fat depth at parturition, differing
nutrition during lactation and differing litter size. Anim. Prod. 48: 203-212.

Williams, I. H. & Smits, R. J. (1991). Body protein losses can be minimized during
lactation. In Manipulating pig production (Batterham, E. 8., ed.), Vol. III, pp. 73.
Australian Pig Science Association, Werribee, Australia.

Wohlt, J. E., Clark, J. H., Derrig, R. G. & Davis, C. L. (1977) Valine, leucine, and
isoleucine metabolism by lactating bovine mammary tissue. J. Dairy Sci. 60:
1875-1882.

83

TABLE 2-1 Composition of diets for lactating sows (g/kg diet, as-fed basis)

 

 

 

Item Dietary protein
235 182 130 78
Ingredient
Corn 463.7 360.0 257.2 154.3
Soybean meal (44% CP) 443.2 344.1 245.8 147.5
Corn starch 0 142.5 283.9 425.3
Sugar 0 47.5 94.6 141.8
Tallow 50.0 50.0 50.0 50.0
Solka floc 0 10.2 20.2 30.2
Dicalcium phosphate 16.3 21.4 26.4 31.4
Calcium carbonate 10.8 8.9 7.0 5.1
Salt 2.5 2.5 2.5 2.5
Trace mineral premix' 2.3 2.3 2.3 2.3
Vitamin premix2 9.0 9.0 9.0 9.0
DL-Met 0.79 0.61 0.44 0.27
L-Thr 0.33 0.25 0.19 0.11
L-Val 1.15 0.90 0.64 0.38
Calculated nutrient content
ME, MJ/kg 14.3 14.3 14.3 14.3
Calcium 9.0 9.0 9.0 9.0
Phosphorus 7.2 7.2 7.2 7.2
Analyzed nutrient content
Crude protein 230 182 132 82
Arg 16.0 12.8 9.3 6.0
His 7.2 5.8 4.5 3.0
'Ile 10.0 7.8 5.7 3.7
Leu 17.3 13.6 10.1 6.4
Lys 11.9 9.0 6.7 4.1
Met 4.2 3.4 2.7 1.8
Phe 11.3 8.9 6.6 4.2
Thr 9.3 6.6 5.3 3.2
Trp3 3.3 2.5 1.8 1.1
Val 12.1 9.6 7.1 4.6

 

1 Provided the following amounts of trace minerals in milligrams per kilogram of diet: copper,
5; iodine, 0.075; iron, 50; manganese, 5; selenium, 0.15; and zinc, 50.

2 Provided the following amounts of vitamins in milligrams per kilogram of diet: retinyl
acetate, 8.3; cholecalciferol, 0.0138; a-tocoperol, 44.1; menadione, 4.5; vitamin B”, 0.033; riboflavin,
4.5; d-pantothenic acid, 17.6; niacin, 26.4; thiamin, 1.1; pyridoxine, 1.0; choline, 385.0; folic acid,
1.65; and d-biotin, 0.22.

3 Calculated value (NRC, 1988).

84

TABLE 2-2

Initial characteristics of sows and litters on d I of lactation

 

 

Item n Mean SD
Sow body wt, kg 16 219.0 25.5
Sow backfat depth, mm 16 22.2 6.0
Sow loin eye area, cm2 16 47.3 5.7
Litter size 16 l l 0
Litter wt , kg 16 18.6 2.3
Piglet body wt, kg 16' 1.69 0.21

 

I Piglets in the same litter are considered an experimental unit.

85

TABLE 2-3

Sow and litter performance over a 21 -d lactation period

 

 

Item n Mean SD
Sow feed intake, kg/d 16 4.92 0.96
Lysine intake, g/d 16 38.5 14.5
Protein intake, g/d 16 758.0 292.6
Metabolic energy intake, MJ/d 16 70.3 13.7
Average sow body wt during 16 206.25 22.51
lactation, kg
Sow body wt change, kg/d 16 -1.18 0.56
Sow backfat depth change, mm/d 16 -0.22 0.18
Sow loin eye area change, mmz/d 16 -21.4 19.7
Litter size at weaning, No. 16 10.44 1.03
Litter wt gain, kg/21 d 16 43.00 6.72
Daily litter wt gain, kg/d 16 2.15 0.34
Piglet body wt gain, kg 16' 4.15 0.69
Milk yield2 , kg/d 30 11.48 2.19

 

l Piglets in the same litter are considered an experimental unit.

2 Average of daily milk intakes estimated on d 11 and 21 of lactation.

86

TABLE 2-4

Estimation of mammary plasma flow rate by the F ick principle

 

l

 

Item n Mean
Plasma lysine arteriovenous differencez, umol/L 15 43.40
Lysine concentration in skim milkz, mmol/L 15 26.00
Lipid concentration in milkz, % 15 6.35
Milk yield, L/d 15 1 1.48
Plasma flow rate3, L/d 6440.7

 

1 Individual lactating sow is considered an experimental unit.

2 Averaged four repeated measurements on d 10, 14, 18, and 22 of lactation of individual sows.

3 Plasma flow rate (L/d) = Lysine output in milk x 103 / plasma lysine arteriovenous difference;

where Lysine output in milk = Lysine concentration in skim milk x (1 - lipid concentration in milk) x milk

yield.

87

TABLE 2-5
Regressions of log mammary A- V diflerences of plasma 1AA against daily intakes of IAA

over a 21 -d lactation period]

 

 

 

 

Amino Parameter estimate R2 P value

acid _0 -1 _2 Model _0 -1 _2
Lys 2.4104 0.0790 -0.001 1 0.49 0.036 0.000 0.015 0.020
Met 0.7216 0.2710 -0.0091 0.60 0.015 0.254 0.022 0.039
Phe 1.6599 0.0859 -0.0012 0.54 0.020 0.003 0.012 0.018
Thr 1.7894 0.1284 -0.0022 0.55 0.027 0.007 0.012 0.018
Val 1.7441 0.1120 -0.0013 0.63 0.007 0.017 0.012 0.026

 

l A polynomial regression model is described by: I = [30 + [SIX + B2X2. With Bo, Bi, and B2 being

parameter estimates, 3 and X are log mammary arteriovenous differences of plasma 1AA (the predicted

variable) and daily intakes of dietary 1AA (the predictor variable), respectively.

88

TABLE 2-6 Contributions of endogenous IAA predicted by the mammary A- V differences of
plasma IAA model]

 

Amino Intercept A-V differenceo2 Plasma Molecular Uptakeo3 OM4 IAAc

 

acid umol/L flow rate wt g/mol g/d g/d g/d
L/d

Lys 2.4104 11.14 6440.7 146.19 10.49 1.96 12.45

Met 0.7216 2.06 6440.7 149.21 1.98 0.55 2.53

Phe 1.6599 5.26 6440.7 165.19 5.60 0.98 6.58

Thr 1.7894 5.99 6440.7 119.12 4.60 2.96 7.56

Val 1.7441 5.72 6440.7 117.15 4.32 1.31 5.63

 

1 The contributions of endogenous IAA (IAAC) are defined as the sum of the amount of the IAA captured

for obligatory metabolism and the amount of the IAA taken up (uptake) by the mammary glands in lactating

sows when daily intake of dietary IAA is extrapolated to zero (See Text).

2 Mammary A-V differenceo of plasma IAA = the reverse log intercept.

3 Mammary uptakeo of plasma IAA (g/d) = Mammary A-V differenceo of plasma 1AA (umol/L) x
mammary plasma flow rate (L/d) x molecular wt of IAA (g/mol) x 106.

4 Obligatory metabolism (OM) is considered as maintenance requirement (NRC 1998). Daily

maintenance requirement of the lactating sows (gld) = Metabolic BW (54.36, kg BWO'”) x maintenance

requirement (10'3 x mg/kg BW0'75od").

89

TABLE 2-7 Contributions of endogenous IAA estimated by the body composition change

 

 

approach
Item Mean Equation
Dietary lysine intake, g/d 38.48 = Average daily feed intake (kg/d) x dietary

lysine concentration (g/kg diet)‘

Milk nitrogen output, g/d 94.36 = Milk nitrogen concentration (8.227 g/L) x
milk yield (11.47 L/d)'

Nitrogen balance, g/d -28.30 = -15.8 + 1.22 dietary lysine intake (g/d) - 0.63
nitrogen output in milk (g/d)2

Body protein loss, g/d 176.90 = Nitrogen balance (g/d) x 6.25

Amino acid from body protein loss, g/d

Lys 12.38 = Body protein loss (g/d) x lysine concentration
in body tissue (g/ 100 g protein)

= Body protein loss x 7.0 %3

Met 3.36 = Body protein loss x 1.9 %4
Phe 7.43 = Body protein loss x 4.2 %4
Thr 7.25 = Body protein loss x 4.1 %4
Val 8.49 = Body protein loss x 4.8 %‘

 

1Obtained from sows in the present study (11 = 16).
2 This regression equation was established by Dourmad et al. (1998).
3 Lysine concentration in body tissue was estimated at 7.0 g/ 100 g protein (ARC 1981).

4 Methionine, phenylalanine, threonine, and valine concentrations in body tissue were calculated to be

1.9, 4.2, 4.1, and 4.8 g / 100 g protein, respectively, from the amino acid profile in body tissue (NRC 1998).

90

TABLE 2-8

Contributions of endogenous IAA derived by the factorial approach

 

Amino Intake of Mainten- Accretion Output in Efficiency IAAc5

 

acid TDIAA', ance needz, 3 milk, 4 g/d
g/d g/d g/d g/d

Lys 33.43 1.96 1.11 42.35 1.00 11.99

Met 13.36 0.55 0.29 14.20 0.95 2.44

Phe 33.66 0.98 0.65 25.00 0.65 6.78

Thr 25.24 2.96 0.63 27.85 0.95 7.70

Val 35.55 1.31 0.84 34.90 0.85 7.81

 

] Based on true ileal digestible AA calculated from regression equations (NRC 1998).

2 Calculated from NRC (1998) by multiplying metabolic body weight (54.36, kg body wto”) and
maintenance requirement (g/kg body wt0‘75-d").
3 Calculated from data of Kim et al. (1999) by multiplying daily protein accretion (14.81 g/d) and

amino acid composition (7.50, 1.99, 4.40, 4.26, and 5.70 g/100 g protein, respectively, for lysine,

methionine, phenylalanine, threonine, and valine) in lactating porcine mammary glands.
4 Assumed utilization efficiencies of truly digestible IAA for milk synthesis and accretion (See
Text).
5 Contributions of endogenous IAA were derived from the following equation:
Dietary intake of truly digestible IAA (g/d) = maintenance requirement for truly digestible 1AA

(g/d) + [accretion of [AA in lactating mammary glands (g/d) + output of IAA in milk (g/d)]/ utilization

efficiency of truly digestible 1AA for milk synthesis — the contribution of endogenous IAA (g/d)

91

TABLE 2-9

Comparisons of the contributions of endogenous IAA estimated by three methods]

 

MAVD BCCA Factorial approach Mean4

 

 

 

AA
8/d2 84483 g/d g/kg3 g/d 8/k83 g/kg

 

Lys 12.45 10.55 12.38 10.49 11.99 10.16 10.40

Met 2.53 2.14 3.36 2.85 2.44 2.07 2.35
Phe 6.58 5.58 7.43 6.30 6.78 5.75 5.88
Thr 7.56 6.41 7.25 6.15 7.70 6.52 6.36
Val 5.63 4.77 8.49 7.20 7.81 6.62 6.20

 

' Based on true ileal digestible IAA.
2 Adjusted daily contribution of endogenous IAA (g/d) = maintenance requirement for IAA
(g/d) + Mammary uptakeo of plasma IAA (g/d).

3 Daily contribution of endogenous 1AA (g/d) divided by daily body wt loss (1.18, kg/d).
4 Average of estimates by the mammary A-V differences of plasma IAA model, the body

composition change approach, and the factorial approach.

92

Log Lys A-V difference

 

- ' Y = 2.4104 + 0.07902 x - 0.001059 x2
(P = 0.036; R2 = 0.49)

 

Reverse log intercept = lysine A-V differenceo

 

 

I I I I I l I

10 20 30 40 50 60 70
Dietary Lys intake, g/d

FIGURE 2-1 Relationship between the log mammary A-V difference of plasma

lysine and daily intake of dietary lysine over a 21-d lactation period. The solid curve

was best fitted by a polynomial regression model: Y = 2.4104 + 0.07902 X - 0.001059

X2 (P = 0.036; R2 = 0.49). Extrapolation of the curve (dotted line) was made on the

basis of this model. Each dot represents an individual sow. The reverse log intercept

is defined as the plasma lysine A-V difference() when dietary lysine intake is

extrapolated to zero.

93

CHAPTER 3

Uptake of Plasma Amino Acids by the Lactating Porcine Mammary Glands: A

Novel Approach to Define Amino Acid Needs for Milk Synthesis

ABSTRACT We hypothesize that the maximal uptake of plasma indispensable
amino acids (IAA) by porcine mammary glands can be defined as dietary needs of
truly digestible IAA for milk synthesis in the lactating sow. To determine dietary
needs of truly digestible IAA (TDIAA) for milk synthesis, 16 Landrace x
Yorkshire lactating sows were used in a randomized block design. Sows were fed
isocaloric (14.3 MJ ME/kg) diets varying in protein concentrations (78, 130, 182,
and 235 g/kg) with the same pattern of dietary IAA. Sows were fitted with
catheters in the carotid artery and the main mammary vein on d 4 of lactation. 0n
(1 10, 14, 18, and 22 of lactation, arterial and venous blood samples were
simultaneously obtained every 30 min over 6 h. Dietary needs of TDIAA for
milk synthesis were determined by a regression model of the mammary
arteriovenous (A-V) differences of plasma IAA (Y) against daily intakes of
dietary IAA (X) over a 21-d lactation period. The relationship between Y and X
was best fitted by a quadratic regression: Y = 00 + [31X + [32X2 (P < 0.05). The
vertex (the maximal Y, X) predicted from this regression was employed in two
aspects. (1) The maximal mammary uptake of plasma IAA was quantified by
multiplying the maximal mammary A-V difference of plasma IAA (= the reverse

log maximal Y) and mammary plasma flow rate. The maximal mammary uptake

94

was considered as dietary needs of TDIAA for milk synthesis by the mammary
glands. Dietary needs of TDIAA for milk synthesis predicted by the maximal
mammary uptake were 28.78, 10.04, 20.15, 6.92, 12.10, 13.91, 4.49, and 22.40
g/kg litter wt gain, respectively, for arginine, histidine, lysine, methionine,
phenylalanine, threonine, tryptophan, and valine. (2) Corresponding to the
maximal Y, dietary intake of TDIAA (derived from X,) would stand for the total
dietary requirement of TDIAA by the lactating sow, which is equal to the sum of
maintenance need and milk synthesis need after adjustment by the contribution of
endogenous IAA from body protein loss. Estimates of dietary needs of TDIAA
for milk synthesis derived by a backward factorial approach were 29.01, 11.68,
20.18, 7.57, 18.40, 13.95, 4.00, and 20.86 g/kg litter wt gain, respectively, for
arginine, histidine, lysine, methionine, phenylalanine, threonine, tryptophan, and
valine. Furthermore, dietary need of truly digestible lysine for milk synthesis
defined by the maximal mammary uptake was validated by a multiple regression
of the total dietary requirements of truly digestible lysine of lactating sows against
litter weight gain and maternal body weight loss (data from the literature). We
concluded that the maximal mammary uptakes of plasma IAA could be used to
determine simultaneously dietary needs of TDIAA for milk synthesis by porcine
mammary glands. Dietary need of truly digestible lysine for milk synthesis was
estimated at 20.2 g/kg litter wt gain at zero maternal body weight loss. Dietary
needs of TDIAA other than lysine for milk synthesis, specifically for arginine and

valine, were higher than those recommended by NRC (1998).

95

KEY WORDS: 0 amino acid requirement 0 mammary arterio-venous difference

0 mammary uptake 0 sow o lactation

96

INTRODUCTION

The lactating sow must be provided with adequate amounts of indispensable amino acids
(IAA) in optimal proportions for maximal productive performance and utilization
efficiency of dietary protein. However, this has been hindered considerably by the
paucity of data on standards for optimal proportions of dietary IAA required by the
lactating sow. Dietary requirements of IAA by the lactating sow have been estimated
using an empirical method and a factorial approach. The empirical method involves
exploring response curves of physiological and/or productive criteria by the animal under
graded dietary intakes or concentrations of the test amino acid. This method only
estimates the total dietary requirement for one amino acid at the specified conditions. It
is difficult to extrapolate the total dietary requirement determined by this method to other
conditions such as faster or slower litter growth rate. The factorial approach derives the
total dietary requirements of the test amino acid by summing up individual needs of the
various physiological components (e.g., body maintenance, milk production, and body
protein accretion in the lactating sow). The factorial approach has been used to predict
dietary requirements of IAA for the lactating sow at various conditions (NRC 1998,
Pettigrew 1993). However, application of the factorial approach is limited due to lack of
experimental estimates of individual needs of dietary IAA for the specific components

and is also limited due to lack of utilization efficiency of dietary IAA for milk synthesis

(Trottier and Guan 2000).

97

0f dietary requirements for IAA, lysine has been the most intensively investigated
because it is the first limiting amino acid for the lactating sow fed a com-soybean meal
diet. Estimates of dietary lysine requirements of lactating sows, based on the empirical
method, vary widely from 25 to 55 g/d (Boomgaardt et al. 1972, Coma et al. 1996, Lewis
and Speer 1973, Johnston et a1. 1993, Touchette et al. 1998a, Wilkinson et al. 1982, Yang
et al. 2000a). These variations in the estimates might be attributed to changes in maternal
body weight, differences in lactational performance, and/or diversity in response criteria
selected in individual studies (Guan et al. 2000a, Pettigrew 1993, Trottier and Guan
2000). When dietary intake of protein and/or lysine is inadequate during lactation, sows
mobilize body proteins to support milk production (Dourmad et al. 1998, Jones and
Stahly 1999, King et al. 1993, Touchette et al. 1998). The contribution of endogenous
lysine mobilized from the maternal body protein will compensate for part of the total

dietary lysine requirement of the lactating sow (Guan et al. 2000a, King et al. 1993).

Dietary lysine requirement of the lactating sow can also be predicted using the factorial
approach (NRC 1998, Pettigrew 1993). The total dietary lysine requirements (depending
on litter grth rate and maternal body weight gain or loss) can be estimated by the sum
of individual needs of dietary lysine for body maintenance, milk production, and body
protein accretion (or adjusted for the contribution from body protein loss). This study
will focus on the major component in the factorial approach for predicting dietary IAA
requirements of the lactating sow, i.e., dietary IAA need for milk production by the
mammary glands. The NRC (1998) and Pettigrew (1993) derived dietary lysine need for

milk production from simple linear regression of the total dietary lysine requirements

98

against litter weight gain. Pettigrew (1993) defined the slope in the linear regression as
dietary lysine need for milk production. In contrast, the NRC (1998) used the slope
minus the intercept to predict dietary lysine need for milk production. However, dietary

lysine need for milk production of the lactating sow has not been defined experimentally.

Dietary requirements of IAA other than lysine have not been extensively estimated in the
lactating sow. The NRC (1998) derived dietary requirements for IAA other than lysine
by multiplying dietary lysine needs and the ratios of other IAA to lysine in ideal amino
acid patterns for maintenance, milk synthesis, and body protein accretion (or body
tissue), respectively. The amino acid profile in milk is assumed an ideal amino acid
pattern for milk synthesis (NRC 1998, Pettigrew 1993). However, the mammary AA
uptake pattern (defined as the relative proportions of other IAA to lysine taken up by the
mammary glands) is not identical to the amino acid profile in porcine milk (Nielsen et al.
2000, Trottier et al. 1997), in which lactating sows were fed standard com-soybean meal
diet and attended by different litter size, respectively. The amount of branched-chain
amino acids (isoleucine, leucine, and valine) and arginine taken up by porcine mammary
glands exceeds their output in milk by approximately 25 to 30% (Nielsen et al. 2000,
Trottier et al. 1997). Similar results have been found in lactating dairy cattle, sheep, and
goats (Bequette et al. 1997, Davis et al. 1978, Fleet and Mepham 1985, Guinard and
Rulquin 1994, Wohlt et al. 1977). In addition to direct incorporation into milk protein,
branched-chain amino acids can provide carbon and a-amino nitrogen for dispensable
amino acid synthesis and provide energy for synthesis of other compounds (e.g. lactose

and fatty acids) in lactating bovine mammary tissue and guinea-pig mammary gland

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(Davis and Mepham 1976, Wohlt et al. 1977). The optimal dietary ratio (approximately
1:1) of valine to lysine for the lactating sow, based on litter weight gain, is higher than
the value (0.85:1) recommended by the NRC (1998)(Moser et al. 2000, Richert et al.
1997a, Richert et al. 1997c, Rousselow and Speer 1980). Arginine, the precursor for de
novo synthesis of nitric oxide (the endothelium-derived relaxing factor), may be involved
in regulation of mammary blood flow (Lacasse et al. 1996, Wu and Morris 1998).
Therefore, estimates of dietary needs of IAA for milk synthesis based only on the amino
acid profile in milk are appropriate in the absence of other data, but they are the estimates

of minimal needs.

Milk production is positively related to marmnary uptake of plasma lysine in sows
(Trottier 1997), which may be extended to mammary uptake of other limiting IAA for
milk synthesis. Furthermore, milk production is directly related to mammary arterio-
venous (A-V) differences of plasma IAA in dairy cows duodenally infused with casein or
whey proteins where mammary blood flow rates are constant (Guinard et a1. 1994,
Madsen et al. 1999). There is a quadratic relation between mammary A-V differences of
plasma lysine and arterial concentrations of plasma lysine in lactating sows and cows
(Guinard 1994, Trottier 1997), implying that mammary uptake of plasma lysine will peak
at certain concentrations of arterial lysine. However, the relationship between mammary
A-V differences of plasma IAA and daily intakes of dietary IAA has not been explored in
the lactating sow. Maximal mammary uptake of plasma 1AA may provide the
intracellular IAA required for milk synthesis potential in the lactating sow. Thus, we

hypothesize that the maximal mammary uptake of plasma 1AA can be defined as the

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mammary need for IAA, and further considered as dietary need of truly digestible IAA
for milk synthesis by the mammary glands in the lactating sow. Dietary needs of truly
digestible IAA for milk synthesis defined by the maximal mammary uptake of plasma
IAA include the amount of direct incorporation into milk and the metabolic need by the
mammary gland. Moreover, a set of dietary needs of truly digestible IAA for milk
production could be estimated concurrently by maximal mammary uptakes. The
objective of this study was to quantify dietary needs of truly digestible IAA by the
mammary glands for milk production in the lactating sow at zero change in body protein.
This information can be used to constitute an ideal dietary IAA pattern for milk synthesis

and predict total dietary IAA requirements of the lactating sow.

MATERIALS AND METHODS

The All-University Committee on Animal Use and Care of Michigan State University

approved all procedures in the study.

Experimental design and dietary treatments. Sixteen Landrace x Yorkshire multiparous
lactating sows (parity 2 or 3) were allocated to treatments according to a randomized
block design. Each block consisted of four sows. Each sow in one block was provided
ad libitum access to one of four diets. Diets contained different crude protein
concentrations (78, 130, 182, and 235 g/kg) and were balanced to contain a similar

pattern of IAA that met recommendations for lactating sows (NRC 1988). Diets were

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formulated by diluting the highest level of crude protein (235 g/kg diet) with starch and

sugar. All diets were isocaloric with a metabolizable energy (MB) of 14.3 MJ/kg.

Animals. Litters with 11 piglets per sow were cross-fostered within 48 h after birth.
SOWs were individually housed in farrowing crates in a mechanically ventilated,
thermally controlled room (21 0C). Sows were provided free access to water. Food was
offered in a stair step manner during the first three days post surgery and to appetite
thereafter. Sow food intake was recorded daily. Sow body weight was recorded after

farrowing (d 1) and at weaning (d 21). Piglets were individually weighed weekly.

Cannulation. For catheter, microbore Tygon® tubing (1.0 mm id, 1.8 mm o.d., Norton
Performance Plastics Co., Akron, OH) was used and the lumen was coated with
triododecylmethylammonium chloride - heparin complex (7% w/w, Polysciences, Inc.,
Warrington, PA). The anterior main mammary vein and the carotid artery were
cannulated on d 4 i 1 of lactation following the surgical procedure described by Trottier
et al. (1995). Antibiotic Naxcel® (Pharmacia and Upjohn Co., Kalamazoo, MI) and anti-
inflarnmatory Banarnine® (Schering-Plough Animal Health Corp., Kenilmorth, NJ) were
administered i.v. for three days following surgery. The catheters were flushed once daily

with heparinized saline (20 IU heparin/ml).

Blood sampling protocol. Blood samples were collected on d 9 i 1, 13 i 1, 17 i 1, and
21 :t: I (referred to as d 10, 14, 18, and 22, respectively). Sows were fed 1 h prior to

blood sampling and provided ad libitum access to food and free access to water as usual.

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Carotid arterial and main mammary venous blood samples (10 ml each) were
simultaneously collected into sterile syringes from 0800 every 30 min over 6 h. Thus, a
total of 13 blood samples were collected from each sow per sampling day. Blood was
transferred into EDTA-coated tubes and centrifuged at 1500 x g for 15 min at 4 0C.

Plasma was removed and stored at -20 °C.

Laboratory measurements

(1) Plasma amino acid analysis. Frozen plasma samples were thawed at 4 °C overnight
and mixed using a vortex. Plasma samples were pooled from the 13 samples taken from
each sow on each sampling day. Glucosaminic acid as an internal standard was added to
the pooled samples. Amino acid concentrations were determined using a Beckman 6300
Amino Acid Analyzer following the method described by Lee and Slocum (1987). In
brief, plasma samples were deproteinized by 35% sulfosalicyclic acid precipitation.
Amino acids in the supernatant were separated by a Beckman cation-ion exchange
column charged in lithium citrate buffer. The eluted amino acids were measured

spectrophotometrically following postcolumn derivatization with ninhydrin.

(2) Dietary nitrogen and amino acid analysis. Food samples were finely ground using a
Cyclotec® 1093 Sample Mill (Foss Tecator, Sweden). Concentrations of amino acids in
food were analyzed by HPLC following the Pico-Tag® method (Cohen et al. 1989). In
brief, food samples were hydrolyzed using 6 mol/L HCl for 24 h at 110 OC. Norleucine
as an internal standard was added into food samples before hydrolysis. Amino acids in

the hydrolysate were derivatized with phenylisothiocyanate and separated by a Pico-Tag®

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column (3.9 mm x 150 mm, Waters Corp., Milford, MA) using Alliance‘m Waters 2690
Separations Module and detected by Waterstm 486 Tenable Absorbance Detector (Waters
Corp., Milford, MA). Amino acid standard H (Pierce, Rockford, IL) was used as a
calibration standard. The method was validated with certified amino acid standard
(NIST, Gaithersburg, MD). Tryptophan concentrations in the diets were calculated using

the value of tryptophan content in food ingredients (NRC 1988).

Calculations
(1) Milk output of IAA.

Milk output of IAA (g/d) = Milk yield (L/d) x Concentrations (umol/L) of IAA in
defatted milk x (l - Content of fat in whole milk) x (1 - Proportion of non-mammary
synthesized proteins in milk) x molecular weight (g/mol) x 10'6. Milk yield was
measured on d 11 and 21 of lactation by the D20 dilution method (Guan et al. 2000a).
Concentrations of IAA in defatted milk (collected on d 10, 14, 18, and 22 of lactation)
were analyzed by the Pico.Tag® Method and the content of fat in whole milk by the mid-
infrared spectroscopic method (AOAC 1990). Concentrations of IAA in defatted milk
were corrected by the content of fat in whole milk to obtain the concentrations of IAA in
whole milk. They were firrther corrected by the proportion of non-mammary synthesized
proteins (such as albumin and immunoglobins) in milk, which was calculated to be 7.25
% of the total milk proteins over a 21-d lactation according to the studies (Bourne and
Curtis 1973, Klobasa and Butler 1987, Klobasa et al. 1987). To quantify milk output of

IAA, milk yield on d 11 and 21 was multiplied by the average concentration of IAA in

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whole milk (adjusted by non-mammary synthesized proteins in milk) from d 10 and 14,

and from d 18 and 22 of lactation, respectively.

(2) Mammary uptake of plasma IAA.

Mammary uptake of plasma IAA (g/d) = Mammary A-V difference (umol/L) of plasma
1AA x Mammary plasma flow rate (L/d) x molecular weight (g/mol) x 10'6. Mammary
plasma flow rate was estimated by the Fick principle using lysine as an internal indicator
(Guan et al. 2000a). To quantify mammary uptake of plasma IAA, mammary plasma
flow rate (on d 11 and 21) was multiplied by the average mammary A-V difference of

plasma IAA from d 10 and 14, and from d 18 and 22 of lactation, respectively.

(3) The vertex of the quadratic regression.
The relationship of log mammary A-V differences of plasma IAA (Y) and daily
intakes of dietary IAA (X) was best fitted by a quadratic regression: Y = [30 + [31X + [32X2 (132
< 0). The vertex (the maximal Y, X.) of this regression was predicted at dY/dX = 0, i.e., X, =

431/202, thus the maximal Y = Bo - 512/4132.

( 4) Dietary needs of T DIAA for milk production derived by the factorial approach.

Based on the factorial approach, the total dietary requirement of TDIAA (considered as
daily intake of TDIAA at the maximal Y) = maintenance need of TDIAA + dietary need of
TDIAA for milk production — the contribution of endogenous IAA (expressed as an
equivalent of TDIAA). Thus, dietary need of TDIAA for milk production = daily intake of

TDIAA at the maximal Y + the contribution of endogenous IAA - maintenance need of

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TDIAA. Daily intake of TDIAA at the maximal Y was converted from daily intake (X;) of
IAA at the maximal Y based on the regressions of TDIAA against total IAA (NRC 1998).

The contribution of endogenous IAA was estimated by (Guan et al. 2000a). Maintenance

need of TDIAA was calculated by the NRC (1998).

Statistical analyses. The linear regression models were computed with the REG
procedure of SAS/STAT (Version 6.12, SAS Institute Inc., Cary, NC). The relationship
between log mammary A-V differences of plasma IAA and daily intakes of dietary IAA
followed a linear and quadratic pattern (P < 0.05), except for isoleucine and leucine. The
best fitting regression model was Y = 130 + [51X + B2X2, with Y being the predicted log
mammary A-V differences of plasma IAA (the predicted variable), X being daily intake
of dietary IAA (the predictor variable), and BO, [31, and [32 being the parameter estimates.
Confidence intervals were estimated using the reflection method by the Bootstrapping
procedure (Neter et al., 1996). A minimum of 750 bootstrapping samples was set up for
computing the confidence intervals. Comparison between mammary uptake of plasma

IAA and their output in milk was analyzed with the TTEST procedure of SAS/STAT.

RESULTS

Mammary uptake vs. milk output. Mammary uptakes of plasma lysine, methionine,
phenylalanine, threonine, and tryptophan were not different from their output in milk
over a 21-d lactation (FIGURE 3-1); mammary uptake of plasma histidine was lower by

18% than its output in milk; and mammary uptakes of plasma arginine, isoleucine,

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leucine, and valine were higher by approximately 20 to 50% than their output in milk.
This study focused on arginine, histidine, lysine, methionine, phenylalanine, threonine,
tryptophan, and valine because they might become limiting IAA for milk production in

the lactating sow fed a com-soybean meal diet.

Mammary needs of IAA for milk production predicted by the maximal mammary
uptake of plasma IAA. The mammary need for IAA includes both incorporation of IAA
into milk and metabolic need of IAA by the mammary glands (e.g., oxidation, accretion,
and conversion to other substrates such as dispensable amino acids, lactose, and fatty
acids). The mammary need of IAA was defined by the maximal uptake of plasma IAA
across the porcine mammary glands. Relations between log mammary A-V differences
of plasma IAA (Y) and daily intakes of dietary IAA (X) over a 21-d lactation period were
best fitted by quadratic regressions (TABLE 3-1). Quadratic coefficients in the
regressions were negative (P < 0.05), indicating that Y can be maximized in the vertex.
The vertex of the quadratic regression for arginine, histidine, lysine, methionine,
phenylalanine, threonine, tryptophan, and valine is represented, respectively, in FIGURE
3-2a to 4-2h. Maximal mammary uptakes of plasma IAA were predicted by multiplying
maximal A-V differences of plasma IAA (the reverse log maximal Y) and mammary
plasma flow rate of 6440 L/d estimated by the F ick principle using lysine as an internal
indicator (TABLE 3-2) (Guan et al. 2000a). The sows had an average litter weight gain
of 2.15 kg/d and an average milk yield of 11.48 kg/d over a 21-d lactation period in this
study. Thus, the maximal mammary uptake was standardized for milk production need

by dividing the maximal mammary uptake with the litter weight gain (TABLE 3-2). The

107

maximal mammary uptakes were defined as mammary needs of IAA, and were further
considered as dietary needs of truly digestible (absorbed) IAA for milk production
(TABLE 3-2). This was based on the following assumptions. (1) While the
contributions of endogenous IAA from body protein loss are zero, the IAA taken up by
the mammary glands are entirely supplied by post-hepatic available IAA of dietary
origin. (2) Dietary intake of truly digestible (absorbed) IAA by the lactating sow meets
two parts: body maintenance need (including the loss of absorbed IAA metabolized on
the first pass by the splanchnic bed) and the mammary need of post-hepatic available
IAA (of dietary origin) for milk production. Ratios relative to lysine for the maximal
mammary uptake were at 1.43, 0.50, 0.34, 0.60, 0.69, 0.22, and 1.11:1, respectively, for
arginine, histidine, methionine, phenylalanine, threonine, tryptophan, and valine for the

lactating sows (TABLE 3-5).

Mammary needs of IAA for milk production derived from the factorial approach.
Corresponding to the maximal A-V differences of plasma IAA (i.e, the reverse log
maximal Y), daily intakes of dietary total IAA (Xi) were estimated from regressions in
TABLE 3-1. Means of intakes of dietary IAA with 95% confidence intervals were
computed by the Bootstrapping reflection method (TABLE 3—3). These intakes of
dietary IAA, afier subtracting maintenance need, were considered as dietary needs of
IAA for milk production at given contributions of endogenous IAA. We named this
approach as the backward factorial approach. Based on the backward factorial approach,
dietary intake of TDIAA at the maximal Y = maintenance need of dietary TDIAA + milk

production need of dietary TDIAA — the contribution of endogenous IAA (expressed as

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an equivalent of dietary TDIAA). Thus, dietary needs of TDIAA for milk production
were derived from this equation (TABLE 3-4). Ratios of other TDIAA to lysine required
for milk production (determined by the backward factorial approach) were 1.44, 0.58,
0.38, 0.91, 0.69, 0.20, and 1.03, respectively, for arginine, histidine, methionine,

phenylalanine, threonine, tryptophan, and valine for the lactating sows.

DISCUSSION

Mammary uptake of plasma branched-chain amino acids and arginine exceeded their
output in milk by approximately 20 to 50%. Thus, it might be inappropriate to estimate
dietary needs of TDIAA other than lysine for milk production based only on the amino
acid profile in milk. A novel approach to determine dietary needs of TDIAA for milk
production was developed from the relationship between log mammary A-V differences
of plasma IAA and daily intakes of IAA in lactating sows. Dietary needs of TDIAA for
milk production were determined by maximal mammary uptakes of plasma IAA and
firrther evaluated using the backward factorial approach. Dietary need of truly digestible
lysine for milk production was estimated at 20.15 and 20.18 g/kg litter wt gain,
respectively, by these two methods. Dietary needs of TDIAA other than lysine for milk
production in the present study were higher than the values recommended by the NRC

(1998), especially for arginine and valine.

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The ratios of mammary uptake to output in milk are approximately at 1:1 for lysine,
methionine, phenylalanine, threonine, and tryptophan, while the ratio for arginine and the
branched-chain AA exceeds 1:1 (see FIGURE 3-1), suggesting that mammary uptake of
the plasma free form of these IAA may be sufficient for milk production in lactating
sows. However, the ratio of mammary uptake to output for histidine was less than 1:1,
possibly indicating that the mammary gland takes up peptides from plasma to meet this
deficiency for milk synthesis. In the lactating goat, however, the ratio of mammary
uptake to output in milk is less than 1:1 for phenylalanine (Backwell et al. 1996). It is
suggested that this shortfall could be met by mammary uptake of the AA derived from
plasma peptides. Vascular peptides are hydrolyzed by membrane—bound peptidases at the
basolateral surface of the mammary epithelium, which is followed by uptake of the
released free AA (Backwell et al. 1994, Backwell et al. 1996, Sherman et al. 1998,
Sherman et al. 1999). The present study was the first to provide direct comparison
between uptake of plasma free AA and output of AA in milk after corrected for the
contribution of protein-bound AA to their output from non-mammary synthesized
proteins (e.g., albumin and immunoglobins). In a previous study, this contribution was
not taken into account in the comparison (Trottier et a1. 1997), leading to no negative
differences between mammary uptake of plasma IAA and their output in milk in the
lactating sow (Trottier et al. 1997). In fact, any comparison between the mammary IAA
uptake and their output is complicated by multiple sources of entrance and outlet. On the
entrance end, there are at least (1) uptake of free AA fi'om plasma and blood cells (if
nay), (2) direct transport of immune proteins and peptides (if any), and (3) intracellular

contribution from protein breakdown and de novo synthesis (if any). On the outlet end,

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there are (1) incorporation of peptide-bound and free AA into milk, (2) accretion as
synthesized constitutive proteins in the mammary gland, and (3) metabolized loss
(including oxidation and conversions to other compounds, if nay, in the mammary gland).
In addition to these complications, output of IAA might be not responsive to intake of
dietary protein due to lactational homeorhesis (Bauman 1999). For example, sows lose
body weight to support high rate of milk production when their intakes of protein,
indispensable amino acids (IAA), and/or energy are inadequate (Clowes et al. 1998,
Dourmad et al. 1998, Jones and Stahly 1999, King and Williams 1984, Mullan and
Williams 1990, Revell et al. 1998, Williams and Smits 1991). Therefore, output of IAA
in milk might not be the “gold standar ” response to intake of dietary AA. In contrast,
mammary uptake of plasma IAA is highly responsive to intake of dietary IAA and might
be considered a new response criterion for estimating dietary IAA requirements of the

lactating sow (Guan et al. 2000b, Trottier and Guan 2000).

The maximal mammary uptakes of plasma IAA were defined as mammary need of IAA
for milk production, and further considered as dietary needs of TDIAA for milk
production by the mammary glands in this study. Without any contributions of
endogenous IAA, dietary intake of TDIAA would meet both maintenance need and
mammary need for milk production. Thus, mammary need of IAA for milk production is
equivalent to dietary need of TDIAA for milk production. Dietary needs of TDIAA for
milk production predicted by the maximal mammary uptakes are supported by estimates
derived from the backward factorial approach except for phenylalanine (see TABLE 3-2

and 4-4). Maximal mammary uptakes can quantitatively encompass both direct

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incorporation into milk and metabolic needs occurring in mammary glands. The
metabolic needs includes oxidation, accretion, de novo synthesis of dispensable AA, and
conversions to other substrates, if any, in mammary glands (Mepham 1982, Trottier

1997).

The total requirement of dietary lysine for the lactating sow has been estimated
empirically based on productive performance (e.g. milk yield, litter weight gain, and
body weight loss) and nutritional status of body nitrogen (e.g. plasma amino acid profile,
plasma urea nitrogen, and nitrogen balance) (Trottier and Guan 2000). Considerable
variation in empirical estimates may be attributed to differences in lactation performance,
high variability in selected response criteria, and changes in body protein reserves
(Pettigrew 1993, Trottier and Guan 2000). The total requirements of dietary lysine are
strongly related to lactation performance, such as litter weight gain (Dourmad et al. 1998,
Pettigrew 1993, Wilkinson et a1. 1982). The total requirements of dietary lysine can be
met by both exogenous intake of dietary lysine and the contribution of endogenous lysine
from the mobilization of liable protein reserves. Sows mobilize body protein reserves to
maintain milk production when intake of lysine is not adequate during lactation
(Touchette et al. 1998). This contribution of endogenous lysine makes it complicated to
empirically estimate the total requirements of dietary lysine for lactating sows (Guan et

al. 2000a, King et al. 1993, Pettigrew 1993).

To use these estimates of the total dietary lysine requirements of lactating sows, we

developed a response surface model to fit empirical estimates reported in the literature.

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The response surface of the total requirements of dietary truly digestible lysine were
regressed against litter weight gain and body weight loss during lactation, which
corresponds to milk production and the contribution of endogenous lysine, respectively.
The total requirements (Y) of dietary truly digestible lysine for lactating sows can be
predicted by the response surface model: Y (g/d) = 0.83 + 20.20 litter weight gain (kg/d)
— 7.28 body weight loss (kg/d) (P < 0.0001, R2 = 0.64). The partial coefficient of litter
weight gain (20.20 g/kg, P < 0.0001) can be considered as dietary need of truly digestible
lysine for milk production at zero body weight loss, and agrees closely with an estimate
of 20.15 g/kg predicted by the maximal mammary uptake in this study. Dietary need of
apparent digestible lysine for milk production was predicted at 22 g/kg litter wt gain
minus 6.39 g (NRC, 1998). As discussed in Chapter 2, the NRC (1998) may have
underestimated the contribution of endogenous lysine and consequently had to correct
dietary need of apparent digestible lysine for milk production by subtracting the intercept
(6.39) from the slope in the simple linear regression. The 3-D response surface is shown
in FIGURE 3-3. It clearly demonstrates that (1) the total requirement of dietary truly
digestible lysine increases with increasing milk production, i.e., with increasing litter
weight gain; (2) the total requirement decreases with increasing contribution of
endogenous lysine from body protein reserves, i.e., with increasing maternal body weight
loss; and (3) the total requirement (considered as the daily intake of lysine at the maximal
A-V difference of plasma lysine) in this study is reliable because it fits well on the 3-D
response surface. Moreover, the total requirements of truly digestible lysine were
predicted at various conditions based on dietary need of truly digestible lysine for milk

production (20.15 g/kg litter wt gain, in the present study), the contribution of

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endogenous lysine (10.40 g/kg BW loss, Guan et al. 2000), and maintenance need (NRC
1998). The predicted estimates of the total requirements were compared with those
derived by the NRC (1998) in FIGURE 3-4. It can be seen that two sets of predicted
values are very close at zero body weight loss. However, this does not necessarily reflect
the identity in estimates used for individual components between the two models.
Especially, the differences (in the total dietary lysine requirement between the two
models) increase with increased body weight loss, possibly indicating that the value for
the endogenous lysine contribution is underestimated in the current NRC (NRC 1998). In
the firture, the NRC should use estimates of dietary lysine needs for individual
components by the lactating sow, which have been generated experimentally to replace

totally model-derived values to improve the current model.

Dietary ratio of truly digestible valine to lysine required for milk production was
estimated at 1.1:1 based on their maximal mammary uptakes, which is higher than the
value of 0.85:1 for milk synthesis recommended by the NRC (1998). The NRC (1998)
may underestimate dietary need of truly digestible valine for milk production when based
mainly on the ratio of valine to lysine in milk though it had been adjusted up from 0.73 to
0.85:1 (of valine to lysine) according to the study of Richert et al. (1996). The amount of
valine taken up by the mammary glands is much higher than its output in milk (Linzell et
al. 1969, Roets et al. 1979, Trottier et al. 1997). The branched-chain AA can be
catabolized in vitro by bovine, guinea-pig, and porcine mammary tissues (Davis and
Mepham 1976, Richert, 1998, Wohlt, 1977). The capacity of rat mammary gland to

catabolize the branched-chain AA is increased dramatically during lactation (DeSantiago

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et al. 1998). Approximately 30% of valine taken up by the mammary gland was oxidized
to CO2 and 70% was incorporated in casein in isolated goat mammary gland in vitro
(Roets et al. 1979), though a small proportion of the branched-chain AA taken up by
porcine mammary tissue is oxidized as an energy source (Richert et al. 1998). Moreover,
they may provide carbon and a-amino nitrogen for synthesis of dispensable AA in bovine
mammary tissue (Wohlt et al. 1977). The ratio for the total requirement of dietary valine
to lysine in the lactating sow was estimated to be 0.91:1 and 1.17:1 based on plasma AA
profile plus nitrogen balance and litter weight gain plus milk production, respectively
(Rousselow and Speer 1980). Effects of high dietary valine on lactational performance
are still controversial. Litter weight gain increased with increased dietary ratio of valine
to lysine from 0.89:1 to 1.33:1 (Moser et al. 2000) or from 0.80:1 to 1.20:1 for the
lactating sow nursing a large size of more than 10 piglets per sow (Richert et al. 1997a,
Richert et al. 1997b). However, increasing dietary ratio of valine to lysine (up to 1.20:1)
did not improve lactational performance of the lactating sow nursing a large size of
piglets of more than 12 piglets per sow (Carter et al. 2000). Differences in these feeding
trials might be attributed to uncontrolled treatments (e.g., balance of dietary AA). Note
that estimates in the present study represent dietary needs of IAA for milk synthesis by
the mammary gland for the lactating sow at zero change in body protein; in contrast, all
the estimates in the literature are for the total dietary requirements of IAA for the
lactating sow at their given conditions. Ideal dietary ratios of other IAA to lysine for the
lactating sow should be expressed on a truly digestible basis and be constituted using
three sets of ideal ratios for maintenance, milk production, and body protein accretion or

endogenous contribution (TABLE 5).

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Dietary ratio of threonine to lysine required for milk production was estimated at 0.69:1
in this study, which is higher than the value (0.58:1) recommended by the NRC (1998).
Approximately 3% of threonine molecules taken up by perfused goat mammary gland
was oxidized to C02 and converted to dispensable AA (Verbeke et al. 1972). The ratio
for the total dietary requirement of threonine to lysine for the lactating sow was estimated
at 0.82:1 and 0.70:1 when based on maximal milk yield plus litter weight gain and on
minimal plasma urea nitrogen plus urinary urea nitrogen output, respectively (Lewis and
Speer 1975). This ratio was further evaluated at 0.66:1 and 0.68:1 based on maximal
litter weight gain and minimal plasma urea N, respectively (Cooper et al. 2000). Dietary
need of truly digestible threonine for milk production was estimated at 30 g/d or 13.9
g/kg litter wt gain at zero body weight loss in the present study, which is slightly higher
than the estimates of Cooper et al. (2000). The total requirement of dietary truly
digestible threonine for lactating sows was estimated at 30 g/d or 11.8 g/kg litter wt gain
when based on maximal litter weight gain and minimal maternal body weight loss

(Cooper et al. 2000).

Dietary ratio of truly digestible tryptophan to lysine required for milk production was
estimated at 0.22:1 in this study, which is slightly higher than the value (0.18:1) for milk
synthesis recommended by the NRC (1998) and much higher than an estimate of 0.14:1
for the lactating sow (Lewis and Speer 1974). Lactating sows had higher voluntary feed
intake, lower plasma urea nitrogen, and less body weight loss when fed a higher dietary

ratio of tryptophan to lysine (0.23:1 vs 0.16:1)(Libal et a1. 1997). Increased intake of

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dietary tryptophan improved nitrogen utilization by the lactating sows although it did not
influence litter weight gain (Libal et al. 1997), implying that the dietary ratio of 0.16:]

might be inadequate in terms of nitrogen balance.

Dietary ratio of truly digestible methionine to lysine required for milk production was
estimated at 0.34:1 in this study, which is higher than the value of 0.26:1 recommended
by NRC (1998). Dietary need of truly digestible methionine for milk production was
estimated at 14.9 and 16.3 g/d, respectively, based on the maximal mammary uptake and
the factorial approach, which is in agreement with the study of Schneider et al. (1992).
Daily intake of dietary methionine plus cysteine (33.2 g/d) was required by lactating sows
based on minimal body weight loss (Schneider et al. 1992). Thus, approximately 17 g/d
of the total requirement of dietary methionine might be required by the lactating sows

assuming that 50% of S-containing AA could be met by dietary cysteine.

Dietary ratio of truly digestible phenylalanine to lysine required for milk production was
estimated at 0.60:1 based on the maximal mammary uptake, which is higher than the
value of 0.55:1 for milk synthesis recommended by the NRC (1998). However, it was in
agreement with the result of Lellis and Speer (1985). Dietary ratio of phenylalanine to
lysine required for lactating sows was calculated at 0.63:1 based on milk yield and
plasma amino acid profile and on the assumption that 50% of the total dietary
requirement of aromatic AA is met by tyrosine (Lellis and Speer 1985, 1987, NRC 1998).
Dietary need of truly digestible phenylalanine for milk production derived from the

backward factorial approach is higher than that predicted by the maximal mammary

117

uptake (39.6 vs 26.0 g/d). Dietary ratio of truly digestible phenylalanine to tyrosine was
higher by 50% compared to their ratio in milk in the present study. The hydroxylation of
phenylalanine to tyrosine in the whole body increases with increased available
phenylalanine in lactating goats (Bequette et al. 1999). The hydroxylation may have
occurred in the present study, resulting in a decreased utilization efficiency of dietary
truly digestible phenylalanine for milk production. Thus, dietary need of truly digestible
phenylalanine for milk synthesis might be overestimated by the backward factorial

approach.

The present study was the first to estimate dietary needs of arginine and histidine, for milk
synthesis by the mammary gland for the lactating sow. We found that dietary needs of
truly digestible arginine for milk synthesis is much higher than the value recommended
by the current NRC (1998). This is due to the fact that the mammary gland during
lactation takes up arginine in excess of its output in milk (Trottier et a1. 1997). As
discussed previously, arginine may play a very important role involved in local regulation
of mammary blood flow (Lacasse et a1. 1996). Thus, this additional amount of dietary
arginine for this metabolism need should be considered in estimation of its dietary need
for milk synthesis. Dietary needs of truly digestible histidine for milk synthesis in the

present study is also higher than the value recommended by the current NRC (1998).
In summary, dietary needs of TDIAA for milk production can be determined by their

maximal mammary uptake, and their estimates were further validated by the backward

factorial approach. Dietary need of truly digestible lysine for milk synthesis by the

118

mammary gland was estimated at 20.15 and 20.18 g/kg litter wt gain, respectively, by
these two methods, which was further supported by the multiple regression analysis of
empirical estimates of the total requirements of dietary lysine for lactating sows in the
literature. Dietary ratios of truly digestible arginine, histidine, methionine,
phenylalanine, threonine, tryptophan, and valine to lysine required for milk synthesis
were higher than values recommended by the current NRC (1998). Therefore, it is
inappropriate to derive dietary needs of TDIAA for milk synthesis based only on the AA
profile in milk, especially for arginine and the branched-chain AA (e. g. valine). Dietary
needs of TDIAA defined by their maximal mammary uptake include the amount of direct
incorporation into milk and the additional metabolic needs (if any) by the mammary

gland as well.

119

LITERATURE CITED

AOAC. (1990). Official methods of analysis of the association of official analytical
chemists. 15th ed. (Helrich, K., Ed.), 2. 2 vols, AOAC, Inc., Arlington.

Bauman, D. E. (1999) Bovine somatotropin and lactation: from basic science to
commercial application. Domest. Anim. Endocrinol. 17: 101-116.

Bequette, B. J ., Backwell, F. R., Kyle, C. E., Calder, A. G., Buchan, V., Crompton, L. A.,
France, J. & MaCrae, J. C. (1999) Vascular sources of phenylalanine, tyrosine,
lysine, and methionine for casein synthesis in lactating goats. J. Dairy Sci .82:
362-377.

Boume, F. J. & Curtis, J. (1973) The transfer of immunoglobins IgG, IgA and IgM from
serum to colostrum and milk in the sow. Immunology 24: 157-162.

Carter, S. D., Hill, G. M., Mahan, D. C., Nelssen, J. L., Richert, B. T. & Shurson, G. C.
(2000) Effects of dietary valine concentration on lactational performance of sows
nursing large litters. J. Anim. Sci. 78: 2879-2884.

Clowes, E. J ., Williams, I. H., Baracos, V. E., Pluske, J. R., Cegielski, A. C., Zak, L. J. &
Aheme, F. X. (1998) Feeding lactating primiparous sows to establish three
divergent metabolic states: 11. Effect on nitrogen partitioning and skeletal muscle
composition. J. Anim. Sci. 76: 1 154-1164.

Cohen, S. A., Meys, M. & Tarvin, T. L. (1989) The Pico.Tag Method: A manual of
advanced techniques for amino acid analysis. Millipore Corp.

Davis, S. R. & Mepham, T. B. (1976) Metabolism of L-(U-14C)valine, L-(U-
14C)leucine, L-(U-14C)histidine and L—(U-14C) phenylalanine by the isolated
perfirsed lactating guinea-pig mammary gland. Biochem. J. 156: 553-560.

DeSantiago, S., Torres, N., Suryawan, A., Tovar, A. R. & Hutson, S. M. (1998)
Regulation of branched-chain amino acid metabolism in the lactating rat. J. Nutr.
128: 1165-1171.

Dourmad, J. Y., Noblet, J. & Etienne, M. (1998) Effect of protein and lysine supply on
performance, nitrogen balance, and body composition changes of sows during
lactation. J. Anim. Sci. 76: 542-550.

Guan, X. F., Ku, P. K. & Trottier, N. L. (2000a) Quantitation of indispensable amino
acids mobilized from body protein reserves of extra-mammary tissues in the
lactating sow. J. Nutr.

Guan, X. F., Pettigrew, J. E., Ku, P. K. & Trottier, N. L. (2000b) Effects of dietary
protein concentration on the amino acid profile in milk and the amino acid uptake
pattern by the lactating porcine mammary gland. J. Anim. Sci.

120

Guinard, J. a. H. R. (1994) Effects of graded amounts of duodenal infusions of lysine on
the mammary uptake of major milk precursors in dairy cows. J. Dairy Sci. 77:
3565—3576.

Jones, D. B. & Stahly, T. S. ( 1999) Impact of amino acid nutrition during lactation on
body nutrient mobilization and milk nutrient output in primiparous sows. J. Anim.
Sci. 77: 1513-1522.

King, R. H., Toner, M. S., Dove, H., Atwood, C. S. & Brown, W. G. (1993) The
response of first-litter sows to dietary protein level during lactation. J. Anim. Sci.
71: 2457-2463.

King, R. H. & Williams, 1. H. (1984) The effect of nutrition on the reproductive
performance of first-litter sows: 2. Protein and energy intakes during lactation.
Anim. Prod. 38: 249-256.

Klobasa, F. & Butler, J. E. (1987) Absolute and relative concentrations of
immunoglobulins G, M, and A, and albumin in the lacteal secretion of sows of
different lactation numbers. Am. J. Vet. Res. 48: 176-182.

Klobasa, F., Werhahn, E. & Butler, J. E. (1987) Composition of sow milk during
lactation. J. Anim. Sci. 64: 1458-1466.

Lacasse, P., Farr, V. C., Davis, S. R. & Prosser, C. G. ( 1996) Local secretion of nitric
oxide and the control of mammary blood flow. J. Dairy Sci. 79: 1369-1374.

Lee, P. & Slocum, R. (1987) Expanded physiological fluid analysis. System 6300/7300
Application Note. No. A6300-AN-006. Beckman Instruments.

Lellis, W. A. & Speer, V. C. (1985) Aromatic amino acid requirement of the lactating
sow. J. Anim. Sci. 61: 1448-1453.

Lewis, A. J. & Speer, V. C. (1974) Tryptophan requirement of the lactating sow. J.
Anim. Sci. 38: 778-784.

Libal, G. W., Uttecht, D. J. & Hamilton, C. R. (1997) Tryptophan needs of lactating
sows fed diets supplements with crystalline lysine. J. Anim. Sci. 75: 417-422.

Moser, S. A., Tokach, M. D., Dritz, S. S., Goodband, R. D., Nelssen, J. L. &
Loughmiller, J. A. (2000) The effects of branched-chain amino acids on sow and
litter performance. J. Anim. Sci. 78: 658-667.

Mullan, B. P. & Williams, I. H. (1990) The chemical composition of sows during their
first lactation. Anim. Prod. 51: 375-387.

Nielsen, T. T., Trottier, N. L., Stein, H. H., Bellaver, C. & Easter, R. A. (2000) The
effect of litter size and day of lactation on amino acid uptake by the porcine
mammary gland. J. Anim. Sci.

121

NRC. (1998). Nutrient requirements of swine. lOth ed., National Academy Press,
Washington, DC.

Pettigrew, J. E. (1993) Amino acid nutrition of gestating and lactating sows. Biokyowa
Technical Review 5: 1-17.

Revell, D. K., Williams, I. H., Mullan, B. P., Ranford, J. L. & Smits, R. J. (1998) Body
composition at farrowing and nutrition during lactation affect the performance of
primiparous sows: 1. Voluntary feed intake, weight loss, and plasma metabolites.
J. Anim. Sci. 76: 1729-1737.

Richert, B. T., Goodband, R. D., Tokach, M. D. & Nelssen, J. L. (1997a) Increasing
valine, isoleucine, and total branched-chain amino acids for lactating sows. J.
Anim. Sci. 75: 2117-2128.

Richert, B. T., Tokach, M. D., Goodband, R. D., Nelssen, J. L., Campbell, R. G. &
Kershaw, S. (1997b) The effect of dietary lysine and valine fed during lactation
on sow and litter performance. J. Anim. Sci. 75: 1853-1860.

Richert, B. T., Tokach, M. D., Goodband, R. D., Nelssen, J. L., Campbell, R. G. &
Kershaw, S. (1997c) The effect of dietary lysine and valine fed during lactation
on sow and litter performance. J. Anim. Sci. 75: 1853-1860.

Richert, B. T., Tokach, M. D., Goodband, R. D., Nelssen, J. L., Pettigrew, J. E., Walker,
R. D. & Johnston, L. J. (1996) Valine requirement of the high-producing
lactating sow. J. Anim. Sci. 74: 1307-1313.

Roets, E., Massart Leen, A. M., Verbeke, R. & Peeters, G. (1979) Metabolism of [U-
14C; 2,3-3H]-L-valine by the isolated perfused goat udder. J. Dairy Res. 46: 47-
57.

Rousselow, D. L. & Speer, V. C. (1980) Valine requirement of the lactating sow. J.
Anim. Sci. 50: 472-478.

Touchette, K. J., Allee, G. L., Newcomb, M. D. & Boyd, R. D. (1998) The lysine
requirement of lactating primiparous sows. J. Anim. Sci. 76: 1091-1097.

Trottier, N. L. (1997) Nutritional control of amino acid supply to the mammary gland
during lactation in the pig. Proc. Nutr. Soc. 56: 581-591.

Trottier, N. L. & Guan, X. F. (2000) Research paradigms behind amino acid
requirements of the lactating sow: Theory and future application. J. Anim. Sci.
78(Suppl. 3): 48-58.

Trottier, N. L., Shipley, C. F. & Easter, R. A. (1995) A technique for the venous
cannulation of the mammary gland in the lactating sow. J. Anim. Sci. 73: 1390-
1395.

122

Trottier, N. L., Shipley, C. F. & Easter, R. A. (1997) Plasma amino acid uptake by the
mammary gland ofthe lactating sow. J. Anim. Sci. 75: 1266-1278.

Verbeke, R., Roets, E., Massart-Leen, A.-M. & Peeters, G. (1972) Metabolism of [U-
14C ]-L-threonine and [U-14C]-L-phenylalanine by the isolated perfused udder. J.
Dairy Res. 39: 239-249.

Wilkinson, R., Cole, D. J. A. & Lewis, D. (1982) Amino acid nutrition of the lactating
sow: the requirement for dietary lysine. Anim. Prod. 35: 15-23.

Williams, 1. H. & Smits, R. J. (1991). Body protein losses can be minimized during
lactation. In Manipulating pig production (Batterham, E. 3., ed.), Vol. III, pp. 73.
Aust. Pig Sci. Assoc, Werribee, Australia.

Wohlt, J. E., Clark, J. H., Derrig, R. G. & Davis, C. L. (1977) Valine, leucine, and
isoleucine metabolism by lactating bovine mammary tissue. J. Dairy Sci. 60:
1875-1882.

Wu, G. & Morris, S. M. (1998) Arginine metabolism: nitric oxide and beyond.
Biochem. J. 336: 1-17.

123

- Mammary uptake of plasma IAA

 

60 q Milk output of IAA
+
50 -
**
:44. *
40 . 4';
. 4;; .9
74. Si
at 3° ' :.:.- 4:
.; ' 54.1
g 11
g :14 ii
'2 1' '3 33'.
f4 l: :9" E (4'1:
:71 "3. ' - 1-
‘0 4 i: it 4’ at '4 .-
a; 8’4 ii 4 ii 44 t
3, {i st. 4 44 .
' *3: 44‘ a "43 ti
fi if is .13 '5 § 93

   

Arg His Ile Len Lys Met Phe Thr Trp Val

FIGURE 3-1 Comparison between uptake of plasma IAA by the porcine mammary
glands and their output in milk over a 21-d lactation. Mammary uptake of plasma IAA
was quantified by multiplying mammary plasma flow rate and mammary A-V differences
of plasma IAA. Milk output of IAA was estimated by multiplying milk yield and the
concentrations of IAA in milk. Calculations in detail are shown in MATERIALS AND
METHODS. Values (Mean i SD, n = 26) between uptake and output with * differ (P <

0.05) and with + tend to differ (P < 0.10), respectively.

124

FIGURE 3-2a The relation between log mammary A-V differences of plasma
Arg (Y) and daily intakes of dietary total Arg (X) over a 21-d lactation period.
The curve was best fitted by a quadratic regression model: Y = 1.2767 + 0.0992X
- 0.0009X2 (P = 0.006, R2 = 0.64). Each dot represents an individual sow. The
vertex (the maximal Y, X,) derived from this regression had two implications: (1)
The maximal mammary uptake of plasma Arg could be quantified by multiplying
the maximal mammary A-V difference of plasma Arg (i.e, the reverse log
maximal Y) and mammary plasma flow rate. The maximal mammary uptake of
plasma Arg was defined as mammary need for Arg, and further considered as
dietary need of truly digestible Arg for milk production by the mammary glands.
(2) Daily intake of dietary total Arg (Xi) would correspond to the entire
requirement of dietary total Arg by the lactating sow, i.e., the sum of maintenance
need and dietary need for milk production adjusted by the maternal body weight
loss of this study. Thus, dietary need of truly digestible Arg for milk production
could be derived by the factorial approach. A Bootstrapping method was
employed to compute the confidence intervals (95% CI) for daily intake of
dietary total Arg (Neter et al. 1996).

125

 

Log mammary Arg A-V difference

 

 

v = 1.2767 + 0.0992X - 0.0009x2
(P = 0.006, R2 = 0.64)
O

    

 

 

    

I Daily dietary Arg intake
' xi = 53.35 i 3.50; 95% CI: 48.91

* ~ 63.04 (gld)

 

 

O
4 ‘ Predicted maximal log Arg A-V difference
re
3 -
2 .
10 20

30 40 50 60 70 80
Daily dietary Arg intake, g/d

126

FIGURE 3-2b The relation between log mammary A-V differences of plasma
His (Y) and daily intakes of dietary total His (X) over a 21-d lactation period.
The curve was best fitted by a quadratic regression model: Y = -1.4929 +
0.4054X - 0.0090X2 (P = 0.022, R2 = 0.53). Each dot represents an individual
sow. The vertex (the maximal Y, X.) derived from this regression had two
implications: (1) The maximal mammary uptake of plasma His could be
quantified by multiplying the maximal mammary A-V difference of plasma His
(i.e, the reverse log maximal Y) and mammary plasma flow rate. The maximal
mammary uptake of plasma His was defined as mammary need for His, and
further considered as dietary need of truly digestible His for milk production by
the mammary glands. (2) Daily intake of dietary total His (X,) would correspond
to the entire requirement of dietary total His by the lactating sow, i.e., the sum of
maintenance need and dietary need for milk production adjusted by the maternal
body weight loss of this study. Thus, dietary need of truly digestible His for milk
production could be derived by the factorial approach. A Bootstrapping method
was employed to compute the confidence intervals (95% CI) for daily intake of

dietary total His (Neter et al. 1996).

127

 

 

 

 

 

 

Log mammary His A-V difference

 

Predicted maximal log His A-V difference

 

  
  
  

Y = 4.4929 + 0.4054X - 0.0090x2
(:4 = 0.022, n2 = 0.53)

Daily dietary His intake 0

* Xi = 22.57 i 0.93; 95% CI: 20.85 ~ 24.51 (g/d)

 

I I I I I

10 15 20 25 30 35

Daily dietary His intake, g/d

128

FIGURE 3-2c The relation between log mammary A-V differences of plasma
Lys (Y) and daily intakes of dietary total Lys (X) over a 21-d lactation period.
The curve was best fitted by a quadratic regression model: Y = 2.4104 + 0.0790X
- 0.0011X2 (P = 0.036, R2 = 0.49). Each dot represents an individual sow. The
vertex (the maximal Y, X) derived from this regression had two implications: (1)
The maximal mammary uptake of plasma Lys could be quantified by multiplying
the maximal mammary A-V difference of plasma Lys (i.e, the reverse log
maximal Y) and mammary plasma flow rate. The maximal mammary uptake of
plasma Lys was defined as mammary need for Lys, and further considered as
dietary need of truly digestible Lys for milk production by the mammary glands.
(2) Daily intake of dietary total Lys (Xi) would correspond to the entire
requirement of dietary total Lys by the lactating sow, i.e., the sum of maintenance
need and dietary need for milk production adjusted by the maternal body weight
loss of this study. Thus, dietary need of truly digestible Lys for milk production
could be derived by the factorial approach. A Bootstrapping method was
employed to compute the confidence intervals (95% CI) for daily intake of
dietary total Lys (Neter et al. 1996).

129

 

 

4.

Predicted maximal log Lys A-V difference

 

1‘

Log mammary Lys A-V difference

 

 
 
 
 

Y = 2.4104 + 0.0790x - 0.001 1x1
(P = 0.036, R2 = 0.49)

Daily dietary Lys intake

' Xi = 38.08 i 4.65; 95% CI: 33.48 ~ 47.53 (g/d)

I I I I I I I

 

10

20 30 40 50 60 70
Daily dietary Lys intake, g/d

130

FIGURE 3-2d The relation between log mammary A-V differences of plasma
Met (Y) and daily intakes of dietary total Met (X) over a 21-d lactation period.
The curve was best fitted by a quadratic regression model: Y = 0.7216 +
0.02710x - 0.0091x2 (P = 0.015, R2 = 0.60). Each dot represents an individual
sow. The vertex (the maximal Y, X.) derived from this regression had two
implications: (1) The maximal mammary uptake of plasma Met could be
quantified by multiplying the maximal mammary A-V difference of plasma Met
(i.e, the reverse log maximal Y) and mammary plasma flow rate. The maximal
mammary uptake of plasma Met was defined as mammary need for Met, and
further considered as dietary need of truly digestible Met for milk production by
the mammary glands. (2) Daily intake of dietary total Met (X,-) would correspond
to the entire requirement of dietary total Met by the lactating sow, i.e., the sum of
maintenance need and dietary need for milk production adjusted by the maternal
body weight loss of this study. Thus, dietary need of truly digestible Met for milk
production could be derived by the factorial approach. A Bootstrapping method
was employed to compute the confidence intervals (95% CI) for daily intake of

dietary total Met (Neter et a1. 1996).

131

 

 

 

 

 

Y = 0.7216 + 0.2710x - 0.0091x2

  

 

o
g (P = 0.015, 1?.2 = 0.60)
0 o
a... 3 a
g o
:5 Predicted maximal log Met A-V difference
2
E
2
t:
a
E
E
a
E 2 -
go Daily dietary Met intake
"1 X, = 15.49 i- 2.42; 95% CI: 13.68
~ 21.13 (g/d)
6 10 12 14 16 18 20 22

Daily dietary Met intake, g/d

132

FIGURE 3-2e The relation between log mammary A-V differences of plasma
Phe (Y) and daily intakes of dietary total Phe (X) over a 21-d lactation period.
The curve was best fitted by a quadratic regression model: Y = 1.6599 + 0.0859X
- 0.0012X2 (P = 0.020, R2 = 0.54). Each dot represents an individual sow. The
vertex (the maximal Y, X.) derived from this regression had two implications: (1)
The maximal mammary uptake of plasma Phe could be quantified by multiplying
the maximal mammary A-V difference of plasma Phe (i.e, the reverse log
maximal Y) and mammary plasma flow rate. The maximal mammary uptake of
plasma Phe was defined as mammary need for Phe, and further considered as
dietary need of truly digestible Phe for milk production by the mammary glands.
(2) Daily intake of dietary total Phe (X.) would correspond to the entire
requirement of dietary total Phe by the lactating sow, i.e., the sum of maintenance
need and dietary need for milk production adjusted by the maternal body weight
loss of this study. Thus, dietary need of truly digestible Phe for milk production
could be derived by the factorial approach. A Bootstrapping method was
employed to compute the confidence intervals (95% CI) for daily intake of
dietary total Phe (Neter et al. 1996).

133

 

 

_ _- m—

Log mammary Phe A-V difference

 

10

Predicted maximal log Phe A-V difference

 

 

  
 

Y = 1.6599 + 0.0859X - 0.0012x2
(P = 0.020, R2 = 0.54)

Daily dietary Phe intake
X. = 37.59 3: 3.52; 95% CI: 33.86 ~ 48.58 (g/d)

I I ' I I I

20 30 40 50 60 70
Daily dietary Phe intake, g/d

134

FIGURE 3-2f The relation between log mammary A-V differences of plasma
Thr (Y) and daily intakes of dietary total Thr (X) over a 21-d lactation period.
The curve was best fitted by a quadratic regression model: Y = 0.7894 + 0.1284X
- 0.0022X2 (P = 0.027, R2 = 0.55). Each dot represents an individual sow. The
vertex (the maximal Y, X.) derived from this regression had two implications: (1)
The maximal mammary uptake of plasma Thr could be quantified by multiplying
the maximal mammary A-V difference of plasma Thr (i.e, the reverse log
maximal Y) and mammary plasma flow rate. The maximal mammary uptake of
plasma Thr was defined as mammary need for Thr, and further considered as
dietary need of truly digestible Thr for milk production by the mammary glands.
(2) Daily intake of dietary total Thr (X.) would correspond to the entire
requirement of dietary total Thr by the lactating sow, i.e., the sum of maintenance
need and dietary need for milk production adjusted by the maternal body weight
loss of this study. Thus, dietary need of truly digestible Thr for milk production
could be derived by the factorial approach. A Bootstrapping method was
employed to compute the confidence intervals (95% CI) for daily intake of
dietary total Thr (N eter et al. 1996).

135

 

 

Log mammary Thr A-V difference

   

Predicted maximal log Thr A-V differ ce
Y = 1.7894 + 0.1284X - 0.0022xz

 

 

   

(P = 0.027, R2 = 0.55)

. Daily dietary Thr intake
l xi = 29.88 i 2.17; 95% C]: 26.74 ~ 34.65 (g/d)

 

10

I T I I I I I

15 20 25 30 35 40 45 50
Daily dietary Thr intake, g/d

136

FIGURE 3-2g The relation between log mammary A-V differences of plasma
Trp (Y) and daily intakes of dietary total Trp (X) over a 21-d lactation period.
The curve was best fitted by a quadratic regression model: Y = -0.8l73 +
0.6476X - 0.0373X2 (P = 0.007, R2 = 0.63). Each dot represents an individual
sow. The vertex (the maximal Y, X.) derived from this regression had two
implications: (1) The maximal mammary uptake of plasma Trp could be
quantified by multiplying the maximal mammary A-V difference of plasma Trp
(i.e, the reverse log maximal Y) and mammary plasma flow rate. The maximal
mammary uptake of plasma Trp was defined as mammary need for Trp, and
fiirther considered as dietary need of truly digestible Trp for milk production by
the mammary glands. (2) Daily intake of dietary total Trp (X.) would correspond
to the entire requirement of dietary total Trp by the lactating sow, i.e., the sum of
maintenance need and dietary need for milk production adjusted by the maternal
body weight loss of this study. Thus, dietary need of truly digestible Trp for milk
production could be derived by the factorial approach. A Bootstrapping method
was employed to compute the confidence intervals (95% CI) for daily intake of

dietary total Trp (Neter et al. 1996).

137

 

 

 

 

 

2 Y =- 0.8173 + 0.6476X - 0.0373x2
g (P = 0.007, R2 = 0.63)
E 2 Predicted maximal log Trp A-V difference
‘6
>.
<
E-
I-
t:
a
g 1 .
a
E
on
c
A 0
Daily dietary Trp intake
X. = 8.65 i 0.34; 95% CI: 7.93 ~ 9.26 (g/d)
0 I I I I T I
2 4 6 8 10 12 14

Daily dietary Trp intake, g/d

138

FIGURE 3-2h The relation between log mammary A-V differences of plasma
Val (Y) and daily intakes of dietary total Val (X) over a 21-d lactation period.
The curve was best fitted by a quadratic regression model: Y = 1.7441 + 0.1120X
- 0.001308X2 (P = 0.0066, R2 = 0.63). Each dot represents an individual sow.
The vertex (the maximal Y, X.) derived from this regression had two implications:
(1) The maximal mammary uptake of plasma Val could be quantified by
multiplying the maximal mammary A-V difference of plasma Val (i.e, the reverse
log maximal Y) and mammary plasma flow rate. The maximal mammary uptake
of plasma Val was defined as mammary need for Val, and further considered as
dietary need of truly digestible Val for milk production by the mammary glands.
(2) Daily intake of dietary total Val (X.) would correspond to the entire
requirement of dietary total Val by the lactating sow, i.e., the sum of maintenance
need and dietary need for milk production adjusted by the maternal body weight
loss of this study. Thus, dietary need of truly digestible Val for milk production
could be derived by the factorial approach. A Bootstrapping method was

employed to compute the confidence intervals (95% CI) for daily intake of
dietary total Val (Neter et al. 1996).

139

 

Log nranrrnary Val A—V difference

.,040_'

...s.~'

.....

Log mammary Val A-V difference

 

 

10

   
 

Y = 1.7441 + 0.1120x - 0.001308x2
(P = 0.0066, n2 = 0.63)

Daily dietary Val intake
l X; = 44.3 :t 6.3; 95% CI: 39.1 ~ 60.3 (g/d)

 

I I I I I I

20 30 40 50 60 70
Daily dietary Val intake, g/d

140

FIGURE 3-3 A 3-D response surface of dietary truly digestible lysine requirements (Y)
for lactating sows fitted by the multiple regression model: Y = 0.83 + 20.20 litter weight
gain — 7.28 body weight loss (P < 0.0001, R2 = 0.64). Each dot represents individual
estimate of dietary truly digestible lysine requirement in the literature (Boomgaardt et al.
1972, Chen et al. 1978, Coma et al. 1996, Dourmad et al. 1998, Guan et al. 1998,
Johnston et al. 1993, 1999, King and Dunkin 1986, King et al. 1993, Knabe et al. 1996,
Kusina et al. 1999, Lewis and Speer 1973, Richert et al. 1997, Sauber et al. 1998,
Touchette et al. 1998a, b, Wilkinson et al. 1982, Yang et al. 2000). Two estimates for
high and low lean growth genotypes (Sauber et al. 1998) and three estimates for parity 1,
2, and 3 (Yang et al. 2000) are shown, respectively. Dietary lysine requirements are
expressed on truly digestible basis as cited from the literature or following conversion

from total basis (NRC 1998).

141

 

 

l 2' r Cur or _', 4
43w.!mu.i~m~r.iir~t§~¢' ~ ~ M.,
.EEEEEEnauig ~

 

 

 

 

Eu Jae—=23?! ~59: 935896 2:?

0 Estimated dietary true digestible lysine requirements in literature
142

E Predicted 3-D surface of dietary true digestible lysine requirements
I Estimated dietary true digestible lysine requirement in this study

 

 

Dietary digestible lysine requirements, g/d

 

25 I j I I I
1.6 1.8 2.0 2.2 2.4 2.6

Litter weight gain, kg/d

FIGURE 3-4 Dietary truly digestible lysine requirements of lactating sows
derived from the maximal mammary uptake model and the NRC model (1998)
at different litter weight gains and body weight losses over a 21-d lactation
period. Lines with solid symbols were fitted by the maximal mammary uptake
model. Lines with empty symbols were fitted by the NRC model. Lines with
circles, triangles, and squares represent 0, 0.5, and 1.0 kg/d of body weight

losses, respectively.

143

2.8

TABLE 3-1
Regression of log mammary A- V differences of plasma IAA against daily intakes of

dietary total IAA over a 21 -d lactation period]

 

AA Parameter estimate R2 P value

 

 

-0 —1 _2 Model _0 -1 _2

 

Arg 1.2767 0.0992 -0.0009 0.64 0.006 0.055 0.004 0.007

His -1.4629 0.4054 «0.0090 0.53 0.022 0.257 0.007 0.007

Lys 2.4104 0.0790 -0.0011 0.49 0.036 0.000 0.015 0.020

Met 0.7216 0.2710 -0.0091 0.60 0.015 0.254 0.022 0.039

Phe 1.6599 0.0859 -0.0012 0.54 0.020 0.003 0.012 0.018

Thr 1.7894 0.1284 -0.0022 0.55 0.027 0.007 0.012 0.018

Trp -0.8173 0.6476 -0.0373 0.63 0.007 0.217 0.002 0.002

Val 1.7441 0.1120 -0.0013 0.63 0.007 0.017 0.012 0.026

 

I A polynomial regression model was best fitted by: 3 = Bo + [3.x + B2X2. Where 130, 15., and

B2 are parameter estimates, 3 and X are the log mammary A-V differences of plasma IAA (the

predicted variable) and daily intakes of dietary total 1AA (the predictor variable), respectively.

144

Dietary needs of T DIAA for milk production predicted at the maximal mammary

TABLE 3-2

 

 

 

uptake of plasma 1AA
Amino acid Max. '1" Max. A-V difference2 Max. mammary uptake3 Milk production need4
umol/L mmol/d g/d g/kg LWG

Arg 4.01 55.16 355.22 61.88 28.78
His 3.07 21.59 139.06 21.58 10.04
Lys 3.829 46.0 296.3 43.31 20.15
Met 2.739 15.5 99.7 14.87 6.92

Phe 3.197 24.5 157.5 26.02 12.10
Thr 3.663 39.0 251.0 29.90 13.91
Trp 1.994 7.3 47.3 9.66 4.49

Val 4.156 63.8 411.1 48.17 22.40

 

I When X. = -B./2[32, the maximal Y = [30 - 13.2/4B2, where [30, 13., and B2 are the intercept, the linear

coefficient, and the quadratic coefficient in the regression, respectively (See TABLE 3-1).

2 Maximal mammary A-V difference of plasma IAA (umol/L) = the reverse log maximal Y.

3 Maximal mammary uptake of plasma IAA (mmol/d) = the maximal mammary A-V difference of

plasma IAA (umol/L) x mammary plasma flow rate (6440 L/d) x 10‘3(Guan et al. 2000). Maximal

mammary uptake in mass (g/d) = maximal mammary uptake (mmol/d) x molecular weight of the

respective amino acid (g/mol) x 103.

4 Milk production need is obtained by the maximal mammary uptake (g/d) adjusted for litter

weight gain (LWG) of 2.15 kg/d over a 21-d lactation period.

145

TABLE 3-3
Daily intake of dietary total IAA (g/d) predicted at the maximal log mammary A-V

difference of plasma IAA over a 21-d lactation period]

 

 

Amino acid Mean 5 so 95% CI2
Arg 53.35 a 3.50 48.91 ~ 63.05
His 22.58 a 0.93 20.85 ~ 24.51
Lys 38.08 a 4.65 33.48 ~ 47.53
Met 15.494242 13.68~21.13
Phe 37.59 a 3.52 33.86 ~ 48.58
Thr 29.88 a 2.17 26.74 ~ 34.65
Trp3 8.65 i- 0.34 7.93 ~ 9.26
Val 44.29 a 6.30 39.05 ~ 60.27

 

] Daily intakes of dietary total IAA (X, g/d) were predicted at the maximal log mammary A-V
difference of plasma IAA (Y) based on the regressions in TABLE 3-1. When dY/dX = 0, i.e., X = -
[3.432, Y reaches its maximal value since [32 < 0.

2 Confidence intervals (95% CI) for daily intakes of dietary total IAA were computed

using the Bootstrapping reflection method with Bootstrapping sample size of 750.

146

TABLE 3-4

Dietary needs of T DIAA for milk production derived by the factorial approach

 

 

 

Amino acid Maintenance Contribution of Intake of TDIAA 3 Milk production need4
need' endogenous IAA 2 g/d
g/d g/d g/d 14,/kg LWG
Arg 0 10.92 49.48 62.37 29.01
His 0.63 4.68 20.21 25.10 11.68
Lys 1.96 10.40 33.07 43.38 20.18
Met 0.55 2.35 14.04 16.27 7.57
Phe 0.98 5.88 33.60 39.55 18.40
Thr 2.96 6.36 25.45 30.00 13.95
Trp 0.51 1.24 7.65 8.60 4.00
Val 1.31 6.20 38.84 44.84 20.86

 

1 Based on average body weight of 206 kg over a 21-d lactation period (NRC 1998).

2 Estimated by Guan et al. (2000) except for endogenous Arg, His, and Trp contributions which

were calculated by multiplying endogenous lysine contribution and the ratio of Arg, His, and Trp to
Lys in body tissue, respectively (NRC 1998).

3 Dietary intakes of TDIAA were converted from dietary intakes of total IAA (in TABLE 3-3)
based on the regressions of dietary TDIAA against dietary total IAA (NRC 1998).

4 Dietary needs of TDIAA for milk production were derived by the factorial approach: daily

intake of TDIAA = maintenance need + milk production need - the contribution of endogenous IAA.
Lactating sows had an average body weight loss of 1.18 kg/d and litter weight gain of 2.15 kg/d over a

21-d lactation period. Milk production needs were adjusted for litter weight gain.

147

TABLE 3-5
Ideal ratios of other IAA to lysine for maintenance, milk synthesis, and

endogenous contribution

 

 

 

 

 

Amino acid Maintenance Endogenous contribution Milk synthesis

NRC Guan et al. NRC Maximal NRC

(1998) (2000) (1998) uptake (1998)
Lys 1.00 1.00 1.00 1.00 1.00
Arg 0 -' 1.05 1.43 0.66
His 0.32 - 0.45 0.50 0.40
Met 0.28 0.23 0.27 0.34 0.26
Phe 0.50 0.56 0.60 0.60 0.55
Thr 1.51 0.61 0.58 0.69 0.58
Trp 0.26 - 0.10 0.22 0.18
Val 0.67 0.60 0.69 1.11 0.85

I not estimated.

148

CHAPTER 4

Effects of Dietary Lysine Deficiency or Valine Excess on Trans-membrane
Transport of Indispensable Amino Acids and Intracellular Protein Synthesis and

Breakdown in the Lactating Porcine Mammary Gland

ABSTRACT The objectives in the study were to evaluate if dietary lysine deficiency
or valine excess affect trans-membrane transport of indispensable amino acids
(lysine, methionine, and valine) and intracellular protein synthesis and breakdown in
the porcine mammary gland during lactation. From (I 1 of lactation, nine Landrace x
Yorkshire lactating sows were provided ad libitum access to one of three diets which
varied only in lysine and valine contents (g/kg diet): lysine-deficient (LD; 4.93 vs
9.87), positive control (PC; 9.71 vs 10.15), and valine-excess (VE; 9.76 vs 13.37).
Dietary methionine content was kept constant (3.26 g/kg diet). Permanent catheters
were inserted into a jugular vein, a carotid artery, and the anterior main mammary
vein on d 9 i l of lactation. On (1 18 of lactation, a solution containing [2-‘5N]-L-
lysine-HCl, [S-methyl-2H3]-L-methionine, and [1-13C]-L-valine was infused
continuously into the jugular vein for 20.5 h. Matched sets of carotid arterial and
mammary venous blood samples were obtained at 3-h intervals from 0 to 12 h, and at
1-h intervals from 14 to 20 h. An individual anterior gland was milked-out
completely by hand at 2-h intervals from 12.5 to 20.5 h for measurement of milk
protein content and casein-bound amino acid enrichment. Pre-planned comparisons

were made between the PC diet versus LD and VE diets. Milk yield, milk protein

149

content, and litter weight gain were reduced (P < 0.05) by LD whereas the effects of
VE were not significant. The whole body plasma flux of lysine was lower (P <0.01)
due to LD, and the whole body plasma flux of valine was higher (P < 0.05) due to
VE. The LD diet decreased (P < 0.05) mammary net uptake of lysine and methionine
as a result of a decrease (P < 0.05) in inward (Fmg,a) and outward (Fv,mg) trans-
membrane transport rate for lysine and a decrease (P < 0.10) in Fmg,a for methionine.
Although F mg,a and Fv,mg for valine were decreased in the VB, net uptake of valine
was not affected. The VE diet reduced the net uptake of lysine. Estimated rates of
mammary protein synthesis and breakdown were decreased on the LD diet, which
decreased net protein synthesis (secretion plus constitutive protein) by the gland.
Protein breakdown was decreased to a greater extent than protein synthesis in the VE,
resulting in comparable net protein synthesis compared with the PC. Model-derived
estimates of mammary net protein synthesis were not different from the direct
measurements of mammary synthesized milk protein output and net accretion. The
proportion of protein breakdown to protein synthesis was decreased in the VE, but
was not affected in the LD. The partition of valine within the mammary gland toward
protein synthesis (66%) compared to oxidation (34%) was not affected by dietary AA
regime. In conclusion, trans-membrane transport of lysine, methionine, and valine

and the intracellular protein turnover in the porcine mammary gland were regulated

by dietary AA regime.

KEY WORDS: o uptake 0 protein metabolism - mammary gland o pig 0 lactation o

lysine deficiency - valine excess

150

INTRODUCTION
The lactating mammary gland has a large demand for amino acids (AA) to meet the high
rate of milk protein synthesis. Dietary needs of indispensable AA for milk synthesis by
the mammary gland account for more than 95% of the total dietary needs of the lactating
sow under zero change of body protein (Guan et al. 2000c, NRC 1998). The transport of
certain AA by the mammary epithelial cells could be rate-limiting for the synthesis of
milk protein (Gomez Angelats et a1. 1995, Mepham 1982). The transport of AA from
blood into the mammary gland is mediated by AA transport systems across the
basolateral membranes of the mammary epithelium (Sherman and Peaker 2000). Most
mammalian cells have both ubiquitous AA transport systems (e.g., A, ASC, L, and y+)
and tissue-specific AA transport systems (e.g., bO’+ and B°'+) (TABLE 4-1 and 5-2)
(Deves and Boyd 1998, Palacin et al. 1998). Each AA transport system is distinct but
exhibits overlapping substrate specificity (Kilberg et al. 1993). This elaborate
arrangement of AA transport systems on the plasma membranes permits fine regulation
of intracellular availability, cellular influx and efflux, and interorgan flux of AA

(Christensen 1990).

Amino acid transport systems in the basolateral membrane are up-regulated by lactogenic
hormones (cortisol and prolactin) at the onset of lactation to increase uptake of AA by the
mammary gland for high rate of milk protein synthesis (Rillema et al. 1992, Sharma and
Kansal 1999, Sharma and Kansal 2000). Furthermore, the transport of AA across plasma
membranes is influenced by mass effects (such as substrate saturation, analogue

competition, trans-stimulation, and trans-inhibition) (Christensen 1982). The endogenous

151

B0’+ transporter of Xenopus oocytes is adaptively regulated (i.e., derepression and
repression with deprivation and supplementation of AA) (Taylor et al. 1996). The
transport of lysine across the intestinal brush border membrane (epithelium) adapts to
intake of dietary protein (Wolffram and Scharrer 1984), suggesting that the uptake of

lysine by the marmnary epithelium may be regulated by intake of dietary lysine.

The transport of cationic AA (arginine, lysine, and ornithine) across the mammary tissue
is carried out at least by two AA transport systems: one is specific for the cationic AA
(system y+) and the other is shared with the cationic and neutral AA (system b0‘+, B°’+, or
y+L). These AA transport systems are situated in the basolateral membrane of the
lactating (bovine, mouse, and rat) mammary epithelium (Baumrucker 1984, Calvert and
Sherman 1996, Sharma and Kansal 2000). Thus, lysine uptake by the lactating mammary
gland can be inhibited in vitro by high concentrations of neutral AA (e.g., leucine)
(Hurley et al. 2000, Sherman et al. 1994, Sherman et al. 1997). A Na+-dependent cationic
and neutral AA transporter (hATB°’+) has been cloned from the human mammary gland.
When expressed in Xenopus oocytes, hATBO’+ transports both cationic and neutral AA;
and its kinetics characteristics are consistent with system BO’+ (Sloan and Mager 1999).
The evidence suggests that uptake of plasma lysine by the mammary gland in vivo may

be reduced by neutral AA.
The transport of branched-chain AA (isoleucine, leucine, and valine, BCAA) is mediated

at least by system L (situated in the basolateral membrane of the mammary epithelium) as

demonstrated in lactating bovine, guinea-pig, mouse, porcine, and rat mammary tissue

152

(Jackson et al. 2000, Kansal and Kansal 1996, Sherman et a1. 1997, Sherman and Peaker
2000). The transport system for valine in porcine mammary gland has a K... of 640 uM
(Jackson et al. 2000), implying that the transport of valine by the mammary gland might
be saturated under physiological concentrations of plasma valine (~ 600 uM). The
uptake of valine by the lactating porcine mammary gland is inhibited in vitro by high
concentrations of lysine, suggesting an interaction in the transport between the cationic

AA and BCAA (Jackson et al. 2000).

The uptake of methionine by the mammary gland is mediated by multiple AA transport
systems A, ASC, and L, having a K... at ~ 460 uM for mouse mammary tissue
(Baumrucker 1985, Sherman and Peaker 2000). Moreover, the transport of L-methionine
across the apical membrane of the chicken jejunum is carried out by four systems (b°‘+,
y+m, L, and B°) (Soriano Garcia et al. 1998). Thus, transport of methionine across the
plasma membranes may be regulated by the cationic and neutral AA because they share
AA transport systems (e.g., ASC, L, Bo, and B°’+) (Soriano Garcia et al. 1998,
Utsunomiya Tate et al. 1996). Dietary excess of L-methionine (4 g/kg diet) down-
regulates uptake of L-methionine across the apical membrane of the brush-border
membrane to reduce the risk of toxicity of L-methionine (Soriano Garcia et al. 1999). In
addition, methionine can be dearninated to a-keto-y-methiolbutyric acid, which is further
decarboxylated by branched-chain or-keto acid dehydrogenase (Dixon and Benevenga
1980, Mitchell and Benevenga 1978). Oxidation of BCAA may be up-regulated by high

intake of protein or BCAA through increased expression of branched-chain

aminotransferase and/or increased activity of branched-chain or-keto acid dehydrogenase

153

(Langer et al. 2000, Torres et al. 1998). In this connection, dietary valine may affect
metabolism of methionine as suggested by Langer et al. (2000). Effects of dietary AA
regime on trans-membrane transport of individual indispensable AA and their
intracellular kinetics are not well understood in the lactating porcine mammary gland in

vivo.

There is substantial protein synthesis and breakdown in the lactating goat mammary
gland (Oddy et a1. 1988). Milk protein synthesis accounts for 30 to 80% of the total
synthesis in the mammary gland (Bequette et al. 1998). This high rate of protein turnover
would help to maintain mammary metabolism and functions by sustaining structural and
regulatory proteins (Bequette et al. 1998). Mammary protein mass is determined by net
balance between protein synthesis and protein breakdown. Increased protein synthesis
and/or decreased protein breakdown would increase net balance of mammary protein,
thus leading to an increase in output of protein in milk. However, dietary effects on
protein synthesis and breakdown in the lactating porcine mammary gland have not been

studied.

Dietary ratio of indispensable AA is important to maximize milk synthesis and efficiency
in the lactating sow. The lactating porcine mammary gland takes up branched-chain AA
in excess of their output in milk (Guan et al. 2000b, Nielsen et al. 2000, Trottier et al.
1997), questioning whether dietary ratio of valine to lysine required for milk synthesis
may be underestimated when only based on their ratio in milk. The data are equivocal

with regards to the optimal ratio of valine to lysine required for the lactating sow. For

154

example, increasing dietary ratio of valine to lysine (up to 1.20:1) increased milk yield
and litter grth rate (Moser et al. 2000, Richert et al. 1997, Richert et al. 1996,
Rousselow and Speer 1980); where in another study increasing the ratio to 1.22:1 did not
improve lactational performance (Carter et al. 2000). Thus, an optimal dietary ratio of

valine to lysine for the lactating sow remains to be determined.

Taken together, supplementation of synthetic AA may cause imbalance in their uptake at
the tissue level, resulting in a decrease in their utilization. We hypothesize that dietary
AA regime affects net uptake of individual AA by the mammary gland through fine
regulation of unidirectional trans-membrane transport of individual AA, and further
affects kinetics of the mammary intracellular free AA. The objectives of the present
study were to evaluate effects of dietary lysine deficiency or valine excess on trans-
membrane transport of indispensable AA (lysine, methionine, and valine) and on
intracellular protein synthesis, breakdown, and net protein synthesis in the lactating
mammary gland. A kinetic model previously developed for the forearm of humans
(Biolo et al. 1995), and recently adapted to the goat mammary gland (Bequette et al.
2000) was used to monitor in vivo changes in inward and outward fluxes of AA by the
porcine mammary gland. The present study was the first in vivo to measure the rates of
inward and outward trans-membrane transport of AA under their deficiency and excess;
to estimate protein synthesis, protein breakdown, and oxidation of AA; and to describe
quantitatively AA transport by the porcine mammary gland during lactation. This

information provides insight into AA interactions on net uptake across the mammary

155

 

gland that may further influence the intracellular protein metabolism; and defines the

optimal ratio of valine to lysine for milk synthesis by the mammary gland.

MATERIALS AND METHODS

Michigan State University All-University Committee on Animal Use and Care approved

all procedures in this study.

Animals and diets. Nine Landrace x Yorkshire lactating sows (parity 2, an average body
wt of 212.6 i 12.0 kg on d 1 of lactation) were allocated to dietary AA regimes according
to a randomized block design. Each block consisted of three sows. Each sow in one
block was provided ad libitum access to one of three dietary AA regimes from d 1 to 21
of lactation. The three dietary AA regimes contained the same concentrations of
metabolizable energy, calcium, available phosphorus, and indispensable AA other than
lysine and valine. Dietary ratio of indispensable to dispensable AA-N (1.00:1.00)
remained constant. The composition of the diets is given in TABLE 4-3. The lysine-
deficient diet contained 50% of lysine content in the positive control and valine-excess
diets. Dietary ratios of valine to lysine were at 1.05:1.00 and 1.37:1.00, respectively, for
the positive control and valine-excess diets. Litters were cross-fostered within 48 h after

birth to ensure 12 piglets per sow. Sows were housed in individual farrowing crates in a

156

thermally controlled room (21 0C). The sows were fed twice daily to appetite and
provided free access to water. Sow food intake was recorded daily. Milk yield on d 21
was estimated by the weigh-suckle-weigh method (Pettigrew et al. 1985). Piglets were

individually weighed on d 1 and 21.

Cannulation. Catheters were constructed from microbore tubing (Tygon®, ID 1.0 mm,
OD 1.8 mm, Norton Performance Plastics Corp., Akron, OH) and the lumen coated with
triododecylmethylammonium chloride - heparin complex (Polysciences Inc., Warrington,
PA). A jugular vein, the anterior main mammary vein, and A carotid artery were
cannulated in sows on d 9 i 1 of lactation as described by Trottier et al. (1995).
Halothane (1.5%) was administered as the anesthetic agent via the nasal/tracheal tube
during all surgeries. Antibiotic (Naxcel®, Pharrnacia and Upjohn Co., Kalamazoo, MI)
and anti-inflammatory medicine (Banamine®, Schering-Plough Animal Health Corp.,
Kenilmorth, NJ) were administered i.v. for the first three days post surgery. The
catheters were flushed daily with heparinized sterile saline (20 TU heparin/ml). Food was
offered in a stair step manner during the first three days post surgery, and was available

free access thereafier.

Protocol. Sows were provided ad libitum access to food and water as usual on d 18 of
lactation when a mixture of stable isotope labeled AA was infused via the jugular vein for
measurement of AA transport and metabolism by the mammary gland. Prior to the i.v.
infusion, a blood and milk sample were collected to obtain a measurement of 13 C, 2H, or

15N natural abundance (background). In a previous study, the enrichment of plasma free

157

leucine and casein-bound leucine was found to reach a plateau at 12 h of a non-primed,
continuous isotope infusion in the lactating sow (Hoffman et al. 1997). So the infusion
herein was extended to 20.5 h to allow repeated measurements over the period of plateau.
The infusion rates of labeled AA were at 24.2, 5.2, and 17.0 umol/min, respectively, for
[2-‘5N]-L-1ysineoHC1, [S-methyl-2H3]-L-methionine, and [1-13C]-L-valine. All isotopes
were purchased from Mass Trace (Wobum, MA) and 99 atoms of '3 C, 2H, or 15N. All
solutions were prepared in sterile saline (0.9% wt/vol) and cold sterilized through an in-
line filter (Nalgene® with surfactant-free cellulose acetate membrane of 0.20 pm pore
size, Nalge Nunc Int. Corp., Rochester, NY). The infusion flow rate was controlled with
a peristaltic pump (Minipuls 3, Gilson Medical Electronics, Middleton, WI). Matched
sets of carotid arterial and mammary venous blood samples were withdrawn
simultaneously into sterile syringes at 3-h intervals from 0 to 12 h of infusion and at l-h
intervals from 14 to 20 h of infusion. Blood was transferred into Li-heparin-coated tubes
and centrifuged at 1500 x g for 15 min at 4 oC. Plasma was removed and stored at -20
0C. After an i.v. dose of oxytocin (10 11.1), a single anterior gland (the 3rd on sow’s right
lateral side) was milked-out by hand at 2-h intervals from 12.5 to 20.5 of infusion;
otherwise, this gland was suckled by its piglet. Milk samples were defatted by

centrifugation at 1500 x g for 15 min at 4 °C and stored at -20 °C.

Analytical techniques
(1) Concentrations of nitrogen in food and milk. Food samples were finely ground
using a sample mill (Cyclotec® 1093, Foss Tecator, Sweden). Concentrations of total N

in food, defatted milk, and casein were determined in a nitrogen analyzer (FP-2000,

158

LECO Corp., St. Joseph, MI) using EDTA (Sigma, St. Louis, MO) as a calibration
standard. The concentration of total N in defatted milk was converted to true protein in
milk as follows:

True protein in milk (%) = Total N in defatted milk (%) x (100 - the concentration of

lipid in milk) x 6.38 x (1 - 0.15) x 1042

Where the concentration of total N in defatted milk was corrected by the concentration of
lipid in milk to obtain the concentration of total N in milk. The concentration of lipid in
milk was assayed using a mid-infrared spectroscope (Multispec M, Berwind Instrument
Ltd., York, England, UK). The concentration of crude protein (total N x 6.38) in milk
was corrected by the proportion of non-protein N to obtain the concentration of true
protein in milk. The proportion of non-protein N was assumed to be 15% of the total N

in porcine milk (Bourne and Curtis 1973, Klobasa and Butler 1987, Klobasa et al. 1987).

(2) Concentrations of amino acids in food and milk. Concentrations of AA in food and
milk were analyzed by reverse-phase HPLC with pre-column phenylisothiocyanate
derivatization (Pico-Tag®, Waters Corp., Milford, MA). In brief, food or milk samples
were hydrolyzed in 6 M HCl at 110 °C for 24 h. Amino acids in the hydrolysates were
derivatized with phenylisothiocyanate (Pierce, Rockford, IL) and separated on a
Pico-Tag® column (3.9 mm x 150 mm) and detected at 254 nm on a tenable absorbance
detector (Waterstm 486, Waters Corp., Milford, MA). Norleucine (Sigma, St. Louis, MO)
as an internal standard was added into food samples before hydrolysis. Amino acid
standard H (Pierce, Rockford, IL) was used as a calibration standard. The method was

validated with certified AA standard (N 1ST, Gaithersburg, MD).

159

(3) Enrichment of casein-bound and plasma free amino acids. Casein in defatted milk
was precipitated at room temperature by adjusting the pH to 4.60 with 1.0 M HCl, and
centrifuged at 1500 x g for 15 min at 4 °C. Precipitated casein pellets were washed twice
with distilled water, solubilized at pH 7.0, freeze-dried, and stored at -20 °C. Casein was
hydrolyzed in 6 M HCl (containing 0.05% (wt/vol) dithiothreitol) at 110 0C for 18 h. The
hydrolysate was desalted with cation-ion exchange resin (AG®, 50W—X8, H+-form, Bio-
Rad Laboratories, Hercules, CA) and eluted with 2 M NH40H. Frozen plasma were
thawed at 4 °C, deproteinized with 5-sulfosalicylic acid by centrifugation at 1500 x g for
15 min at 4 °C, and desalted as above. Isolated AA from casein and plasma were freeze-
dried and derivatized with methyl-t-butyldimethylsilyl trifluoroacetamide to form their
MTBMS-derivatives. Isotopic enrichments were determined by electron-impact on gas
chromatography-mass spectrometry (Trio-1, VG Masslab., Manchester, UK) as described
by Bequette et al. (1994). Because of a small (~ 3%) contamination of the labeled L-
lysine with D-lysine, chiral separation was necessary (Lobley et al. 1996). All
enrichments were expressed as molar percent excess (MPE) with respect to the pre-

infusion natural abundance of the casein-bound or free AA.

(4) Concentrations of plasma amino acids. The curves of plasma AA enrichments over
the isotope infusion time showed that their plateaus occurred by approximately 12 h post-
infusion. Thus, plasma samples from 15 to 20 h of infirsion from each sow were pooled
for plasma AA assay. Glucosaminic acid as an internal standard was added into the

pooled samples. The concentrations of plasma AA were determined using a Beckman

160

6300 Amino Acid Analyzer. In brief, proteins in plasma were precipitated by 5-
sulfosalicylic acid. Amino acids in the deproteinized supernatant were separated by a
Beckman cation-ion exchange column charged in Li citrate buffer. The eluted AA were

measured spectrophotometrically following post-column derivatization with ninhydrin.

Mammary plasma flow estimation. Mammary plasma flow rate was calculated by the
Fick principle based on the assumption that output of phenylalanine and tyrosine (FY) in
milk equates to their mammary uptake (Cant et al. 1993). Thus,
Mammary uptake of plasma FY = mammary A-V difference in concentrations of
plasma FY x mammary plasma flow
= Output of FY in milk + the amount of FY metabolized in the mammary gland
(1)
There might be some factors involved in the balance between marmnary uptake of plasma
FY and their output in milk. (1) The amount of FY metabolized in the mammary gland is
negligible. As demonstrated in the guinea pig mammary tissue, the activity of
phenylalanine hydroxylation is insignificant (Davis and Mepham 1976). Phenylalanine is
not oxidized in the lactating goat mammary gland (Verbeke et al. 1972). In addition, the
amount of FY accretion in the lactating porcine mammary gland is approximately 1 g/d
(Kim et al. 1999), which is negligible compared to their output in milk (50 g/d) in the
present study. (2) Approximately 5% of the total proteins in milk is derived from non-
marnmary synthesized proteins (Bourne and Curtis 1973, Klobasa and Butler 1987,
Klobasa et al. 1987). (3) The contribution of vascular peptide-bound FY to mammary

synthesized proteins might be negligible because 96% of casein-bound phenylalanine

161

originates from plasma phenylalanine (Verbeke et al. 1972). (4) Losses of FY from the
mammary gland via the lymph are negligible as is output of free FY in milk (Linzell
1974). The pr0portion of free FY is approximately 0.5% of their peptide-bound residues
in porcine milk (Wu and Knabe 1994). Therefore, Eq.1 was simplified as follows:
Mammary uptake of plasma FY = mammary A-V difference in concentrations of
plasma FY x mammary plasma flow
= Output ofFY in milk x (1 — 0.05), i.e.,
Mammary plasma flow rate (L/h) = Concentrations of FY in milk (mmol/L) x milk

yield (L/h) x (l — 0.05) x 103 / mammary A-V difference of plasma FY (pmol/L)

(2)

Calculations.
The whole body irreversible loss rate (wa, See the Appendix) of plasma individual AA
was calculated from average arterial enrichment over the last 5 h of the isotope infusion
period when the enrichment reached a quasi-isotopic plateau (FIGURE 4-1a to 11):
Iin=waan+Ian, i.e.,
wa = (Bi/Ea - 1) x I (3)
Where Ba and Bi are the enrichments of plasma free AA in the carotid artery and AA in

the mixture of labeled AA, respectively, and where I is the rate of infusion (see Protocol).

The unidirectional flux (gross) of plasma free AA from mammary artery to the mammary
gland (F mg) was calculated from labeled AA (tracer) and then converted to its tracee:

Fmg (mmol/h) = (Ca x Ea - Cv x Ev) x MPF x 103/Ea (4)

162

Where Ca and Cv are the concentrations (umol/L) of plasma AA in the carotid artery and
the main mammary vein, respectively. The concentrations of plasma AA in the carotid
artery were presumably the same as in the mammary artery. Ev is an average enrichment
of plasma free AA in the main mammary vein (over the last 5 h of the infusion). MPF is

the plasma flow rate (L/h) across the mammary gland estimated by the Fick principle.

(I) Trans-membrane transport of free amino acids in the mammary gland. Trans-
membrane transport and intracellular kinetics of free AA in the lactating porcine
mammary gland were derived from a three-compartmental model (FIGURE 4-2)
originally based on the model of Biolo et al. (1995) for measurements of AA trans-
membrane transport across human leg, but modified for the lactating goat mammary
gland (Bequette et al. 2000). The model assumes that AA enter and leave the mammary
gland via the mammary artery (Fa,o) and the main mammary vein (Fo,v), respectively.
Fmg,a and Fv,mg refer to the net movements of free AA from mammary artery to
mammary intracellular free AA compartment and from this compartment to mammary
vein, i.e., inward and outward trans-membrane transport, respectively. Fv,a refers to
direct flow of AA from artery to vein which does not enter intracellular compartment.
Fmg,o refers to the rate of mammary intracellular free AA appearance from endogenous
sources (i.e., release from protein breakdown (PB) and de novo synthesis (DS), if any).
Fo,mg refers to the rate of mammary intracellular free AA disappearance (i.e., the rate of
utilization of the intracellular free AA for protein synthesis (PS), oxidation (OX), and
other metabolic fates (OM), if any). Fa,o and Fo,v were calculated as follows:

Fa,o (mmth) = Ca x MPF x 10'3 (5)

163

Fo,v (mmol/h) = Cv x MPF x 10'3 (6)
Net mass uptake (NB) of AA (including labeled and unlabeled AA) across the mammary
gland was determined by the difference between Fa,o and Fo,v:

NB (mmth) = Fa,o - Fo,v, i.e.,

NB (mmol/h) = (Ca - Cv) x MPF x 10'3 (7)
Inward (Fmg,a) and outward (Fv,mg) transport rates can be calculated if the intracellular
enrichment of AA is known. This measure is usually obtained by biopsy, for example,
the muscle (Biolo et al. 1995), but this is often difficult to obtain, especially for the
mammary gland where considerable vascularisation lends itself to excessive bleeding. As
an alternative, Bequette et al. (2000) used the enrichment of casein-bound AA in milk,
which was assumed to represent the enrichment of the immediate precursor pool within
the mammary gland at the site of milk protein synthesis. Thus, the enrichment of casein
was also assumed to reflect that of the mammary intracellular free pool at steady state.
The average enrichment of casein-bound AA in milk over the last 4 h (16.5 to 20.5 h) of
infusion was used in calculations. (Fig. 1a to 1i). The net mass balance and tracer
balance of AA across the mammary gland were calculated as follows:

(Ca - Cv) x MPF x 10'3 = Fmg,a - Fv,mg (8)

(Ca x Ea - Cv x Ev) x MPF x10'3 = Fmg,a x Ea - Fv,mg x Ec (9)
Where Ec was the enrichment of casein-bound AA in milk at steady state. The net mass
balance and tracer balance across the mammary gland were estimated using the
concentrations and the enrichments of plasma AA because of the limited equilibration of
unlabeled and labeled AA between plasma and red blood cells and between the latter and

the mammary gland. Thus, Fmg,a and Fv,mg could be solved from Eq. 5, 6, 8, and 9:

164

Fmg,a (mmol/h) ={[(Ec - Ev)/(Ea - Ev)] x Cv + Ca}x MPF x 10'3 (10)

Fv,mg (mmol/h) ={[(Ec - Ev)/(Ea - Ev)] x Cv + Cv}x MPF x 10'3 (11)
At steady state, F a,o = Fmg,a + Fv,a; and Fo,v = Fv,a + Fv,mg. Thus, F v,a was
calculated as follows:

Fv,a = Fa,o - Fmg,a (12)
Or

Fv,a = Fo,v - Fv,mg (l3)

(2) Kinetics of intracellular free amino acids in the mammary gland. Total rate of
mammary intracellular free AA appearance (Ra) was calculated by the intracellular tracer
dilution approach. The only source of tracer appearing in the mammary intracellular free
AA compartment was transported inward fi'om plasma. Thus, any dilution of tracer AA
in casein is assumed to derive from the intracellular free AA from endogenous sources
(Fmg,o) (e.g., protein breakdown and de novo synthesis, if any). Therefore,

Ra x Ec = Fmg,a x Ea, i.e.,

Ra (mmol/h) = (Fmg,a x Ea) / Ec (14)
Where Ra is the sum of inward trans-membrane transport (Fmg,a) and the rate of
intracellular free AA appearance from the endogenous sources (F mg,o):

Ra = Fmg,a + Fmg,o
Thus,

Fmg,o (mmth) = Fmg,a x (Ea/Ec - 1) (15)
At steady state, the total fluxes into the mammary intracellular free AA compartment are
equal to the total fluxes out of this compartment, i.e.,

Fmg,a + Fmg,o = Fv,mg + Fo,mg

165

Thus,

Fo,mg (mmol/h) = (Fmg,a - Fv,mg) + Fmg,o (16)
Or,

Fo,mg (mmol/h) = NB + Fmg,o (17)
The rate of intracellular free AA disappearance (F o,mg) could be also directly calculated
as the tracer balance divided by the precursor enrichment (Ec):

Fo,mg (mmol/h) = (Ca x Ea - Cv x Ev) x MPF x 10'3 / Ec (18)
(3) Protein synthesis and breakdown in the mammary gland. Fmg,o and Fo,mg may
reflect rates of different metabolic pathways for each AA. Fmg,o refers to the rate of
intracellular free AA endogenous appearance from protein breakdown and de novo
synthesis within the mammary gland. Thus, Fmg,o represents the rate of intracellular
protein breakdown because indispensable AA (e.g., lysine, methionine, and valine) can
not be synthesized de novo in the mammary gland. Fo,mg refers to the rate of
intracellular free AA utilized for protein synthesis, oxidation, and other metabolic
pathways, if any, in the mammary gland. Fo,mg for the intracellular methionine may
represent the rate of protein synthesis because their oxidation and utilization for other
metabolic pathways might be negligible as demonstrated in the guinea-pig mammary
gland (Peters et al. 1979). In contrast, Fo,mg for intracellular valine could be subdivided
into two fractional fluxes: Fo,mg(p) for intracellular valine utilized for protein synthesis
and Fo,mg(o) for the intracellular valine utilized for oxidation and other metabolic
pathways, if any. Therefore,

PB (g protein/d) = Fmg,o (mmol/h) x AA mol wt (mg/mmol) x 24 (h/d) x 10'1 / AA %

of protein (1 9)

166

Where AA % of protein in the mammary tissue was averaged at 7.52, 2.05, and 5.53,
respectively for lysine, methionine, and valine based on the AA composition in
constitutive proteins in the mammary gland (Kim et al. 1999) and that in milk proteins
(Guan et al. 2000b).
PS"... (g protein/d) = Fo,mg (mmol/h) x methionine mol wt (mg/mmol) x 24 (h/d) x
10" / methionine % of protein . (20)
Where PS"... refers to the rate of protein synthesis defined by Fo,mg for the intracellular
methionine. On the basis of the equivalent protein mass, the rate of protein synthesis
defined by Fo,mg(p) for the intracellular valine would be the same as those defined by
Fo,mg for the intracellular methionine, i.e.,
PSval = PSmet
Where PSvat refers to the rate of protein synthesis for intracellular valine. Thus, Fo,mg(p)
for intracellular valine utilized for protein synthesis was calculated as follows:
Fo,mg(p) x valine mol wt/valine % of protein = Fo,mg (of methionine) x methionine
mol wt/methionine % of protein; i.e.,
Fo,mg(p) (mmol/h) = Fo,mg (of methionine, mmol/h) x [(methionine mol
wt/methionine % of protein)/( valine mol wt/valine % of protein)] (21)
Or,
F o,mg(p) (mmol/h) = PSval (g/d) x valine % of protein x 10/ [24 (h/d) x valine mol wt
(mg/11101)] (22)
Thus, Fo,mg for the intracellular valine = Fo,mg(p) + Fo,mg(o), i.e.,

Fo,mg(o) (mmol/h) = F o,mg - F o,mg(p) i (23)

167

Where Fo,mg(o) may represent the rate of the intracellular valine utilized for oxidation

and other metabolic pathways.

Net production of protein in the mammary gland was derived from the compartmental
model, i.e., the difference between protein synthesis and breakdown (Eq. 19 and 20). The
values for net production of protein derived from the model could be validated by
estimates of accretion of mammary tissue proteins and output of mammary synthesized
proteins in milk. Average accretion of mammary tissue proteins was 14.81 g/d over a 21-
d lactation period in the lactating sow (Kim et al. 1999). Output of mammary
synthesized proteins in milk was calculated as follows:
Output of mammary synthesized proteins in milk (g/d) = Milk yield (kg/d) x 1.15 x
the concentration of true protein in milk (%) x (1 - 0.05) x 10 (24)
Where an estimate of milk yield was adjusted by 1.15 because the weigh-suckle-weigh
method underestimates milk consumption compared to D20 dilution method (Guan et al.
2000a, Pettigrew et a1. 1987, Pettigrew et al. 1985). The contribution of non-mammary
synthesized proteins was assumed at 5.0% of true protein in milk (Bourne and Curtis

1973, Klobasa and Butler 1987, Klobasa et al. 1987).

Statistical Analyses. Data were analyzed by the Mixed Procedure (SAS/STAT Version
6.12, SAS Institute, Cary, NC). The model for the concentration of total N in defatted
milk included block, dietary treatment, and sampling time, and all two-way interactions
with sampling time in a repeated statement. The model for other variables included block

and dietary treatment. Based on the residual distribution, data for Fa,o, Fv,a, Fmg,a,

168

Fv,mg, F o,v, and NB were log-transformed prior to their ANOVA. Their least-squares
means were then converted to actual values. Least-squares means are presented because
of unbalanced data in blocks. Differences between the lysine-deficient diet and positive
control diet or between the positive control diet and valine-excess diet are considered

significant at P < 0.05 or 0.01 and tended to be significant at P < 0.10.

RESULTS

Productive performance and mammary plasma flow rate. Productive performance over
a 21-d lactation period was affected by the dietary amino acid regime (TABLE 4-4).
Sows fed the lysine-deficient diet had lower litter weight gain and milk yield than sows
fed the positive control diet (P < 0.05 and 0.01, respectively). Total nitrogen content in
defatted milk and protein content in milk were decreased (P < 0.05) in the lysine-
deficient diet compared to those fed the positive control diet. The proportion of casein-N
to total N in milk was numerically lower in the lysine-deficient diet than that in the
positive control diet. No differences were observed in the productive performance
between the valine-excess diet and the positive control diet. The dietary amino acid
regime did not affect plasma flow rate across the mammary gland (214.4 :1: 26.0, 236.8 :t
26.0, and 265.3 :t 26.0 L/h, respectively, for the lysine-deficient diet, the positive control

diet, and the valine-excess diet).

Arterial concentrations of plasma amino acids and urea and their extraction rates by

the mammary gland. Plasma amino acid and urea profiles are shown in TABLE 4-5.

169

Concentration of plasma lysine in the carotid artery was decreased (P < 0.01) in the sows
fed the lysine-deficient diet compared to that in the sows fed the positive control diet. In
contrast, plasma threonine concentration tended to be increased in the lysine-deficient
diet (P < 0.10). The concentration of plasma urea was increased (P < 0.10) in the lysine-
deficient diet, possibly indicating that deamination from indispensable amino acids other
than lysine was increased. Taken together, the plasma amino acid and urea profiles in the
lysine-deficient diet reflected a deficiency of lysine in the body. The concentration of
plasma valine in the artery was increased considerably (P < 0.01) in the sows fed the
valine-excess diet, indicating the excess of valine in the body. Concentrations of plasma

indispensable amino acids other than valine were not altered in the valine-excess diet.

Mammary extraction rates (i.e., the ratios of mammary arteriovenous differences to
arterial concentration) of plasma amino acids were affected by the dietary amino acid
regime (TABLE 4-6). The extraction rate of plasma lysine was increased (P < 0.05) in
the lysine-deficient diet compared to that in the positive control diet. Moreover, the
extraction rates of certain indispensable amino acids (e. g., methionine and leucine) were
decreased (P < 0.05), possibly due to their increased arterial concentrations in the lysine-
deficient diet. In contrast, extraction rates of plasma cationic amino acids (e.g., arginine)
and certain neutral arrrino acids (e.g., leucine, threonine and tryptophan) dended to be

decreased (P < 0.05) in the valine-excess diet.

Kinetics of plasma lysine across the mammary gland. Kinetics of trans-membrane

transport for lysine across the mammary gland was affected by the dietary amino acid

170

regime (TABLE 4-7). The whole body irreversible loss rate (F wb) and the unidirectional
flux from the mammary artery to the mammary gland (Fmg) of lysine were decreased (P
< 0.01) in the lysine-deficient diet compared to those in the positive control diet. The
flux of plasma lysine entrance into the gland from the mammary artery (Fa,o) and the
flux of lysine exit from the gland to the mammary vein (F o,v) were decreased (P < 0.01
and 0.05, respectively) in the lysine-deficient diet compared to those in the positive
control diet. Inward trans-membrane transport (F mg,a) of free lysine from the mammary
artery to mammary intracellular free lysine compartment and outward trans-membrane
transport (Fv,mg) from this compartment to the mammary vein were decreased (P <
0.05), resulting in decreased net uptake (NB) of plasma lysine by the mammary gland.
The rate of mammary intracellular free lysine appearance from endogenous sources
(Fmg,o) was decreased in the lysine-deficient diet, indicating a decreased protein
breakdown in the mammary gland. Because of decreased Fmg,a and Fmg,o for lysine,
the total rate of mammary intracellular free lysine appearance (Ra) was decreased by
approximately 85% in the lysine-deficient diet. Decreased Ra indicated decreased
availability of the mammary intracellular free lysine for protein synthesis and other
metabolisms, if any, as indicated by decreased rate of the intracellular free lysine

disappearance (Fo,mg) (P < 0.01) in the lysine-deficient diet.

Though wa for lysine was not different between the valine-excess diet and the positive
control diet, Fmg for lysine was decreased (P < 0.01) in the valine-excess diet. There
were no differences in Fa,o and Fo,v for lysine between the valine-excess diet and the

positive control diet. Both Fmg,a and Fv,mg for lysine were increased at approximately

171

35% and 80%, respectively, in the valine-excess diet compared to those in the positive
control diet, resulting in decreased NB for lysine in the valine-excess diet. The rate of
mammary intracellular free lysine disappearance (Fo,mg) was decreased (P < 0.01) in the

valine-excess diet.

Fractional proportion of the whole body irreversible loss of plasma lysine partitioned to
the mammary gland (Fmg/wa) was decreased in the lysine-deficient diet and the valine-
excess diet compared to that in the positive control diet (P < 0.10 and 0.01, respectively)
(TABLE 4-10). The ratio of net uptake by the mammary gland to total entrance of free
lysine from the mammary artery (NB/Fa,o), being identical to mammary extraction rate,
was increased by 120% in the lysine-deficient diet compared to that in the positive
control diet. The dietary amino acid regime did not affect the fractional proportion
(Fmg,a/Fa,o) of inward trans-membrane transport to the total entrance of free lysine from
the mammary artery, the fractional proportion (Fv,a/Fa,o) of direct flow (from artery to
vein) to the total entrance of free lysine from the mammary artery, or the volume of
arterial plasma completely cleared of lysine via trans-membrane transport per unit time
(Fmg,a/Ca). In the lysine-deficient diet, the contribution of inward trans-membrane
transport to Ra (Fmg,a/Ra) was decreased (P < 0.05); coincidentally, the contribution
from protein breakdown (the only endogenous source in the mammary gland) to Ra
(Fmg,o/Ra) was increased (P < 0.05). In contrast, the contribution of inward trans-
membrane transport was increased (P < 0.05) and the contribution from protein
breakdown was decreased (P < 0.05) in the valine-excess diet. The ratio of the flux of

the intracellular free lysine utilized for protein synthesis to the total rate of the

172

intracellular free lysine appearance (i.e., availability) (Fo,mg/Ra) was increased in the
lysine—deficient diet, indicating an increased efficiency of the intracellular lysine for
protein synthesis. In contrast, the ratio of Fo,mg to Ra was decreased (P < 0.10) in the

valine-excess diet.

Kinetics of plasma methionine across the mammary gland. Although concentrations of
dietary and arterial plasma methionine were not different among the dietary AA regimes,
fluxes of methionine were affected (TABLE 4-8). The whole body irreversible loss rate
(wa) and the unidirectional flux to the mammary gland (Fmg) were decreased in the
lysine-deficient diet. In the lysine-deficient diet, inward trans-membrane transport of free
methionine (F mg,a) was decreased by 43% (P < 0.05); outward trans-membrane transport
(Fo,mg) was decreased by 48%, resulting in decreased net uptake (NB) of plasma
methionine by the mammary gland (P < 0.05). The rate of mammary intracellular free
methionine appearance (Fmg,o) from endogenous sources was decreased (P < 0.05) in
the lysine-deficient diet, indicating a decreased protein breakdown in the mammary
gland. The total rate of the mammary intracellular free methionine appearance (Ra) was
decreased (P < 0.10) due to decreased Fmg,a and Fmg,o in the lysine-deficient diet. The
decreased Ra indicated a decrease in the intracellular methionine availability to protein
synthesis and other metabolism (if any) in the mammary gland, as indicated by decreased
rate of the intracellular free methionine disappearance (F o,mg) in the lysine-deficient diet
(P < 0.05). There were no differences between the lysine-deficient diet and the positive
control diet in the flux of methionine entrance into the mammary gland (Fa,o), the direct

flow fi'om the artery to the vein (F v,a), or the flux of methionine exit from the mammary

173

gland to the vein (Fo,v). Except for Fv,a and Fmg,o, the other fluxes were not different
between the valine-excess diet and the positive control diet. Note that decreased Fmg,o

implied that mammary protein breakdown was decreased in the valine-excess diet.

The proportion of the whole body irreversible loss of plasma methionine partitioned to
the mammary gland (Fmg/wa) was decreased in the lysine-deficient diet (P < 0.05)
compared to that in the positive control diet (TABLE 4-11), possibly due to decreased
extraction rate (NB/Fa,o). The dietary amino acid regime did not affect the fractional
proportion (Fmg,a/Fa,o) of inward trans-membrane transport to the total entrance of free
methionine from the mammary artery, the fractional proportion (Fv,a/Fa,o) of direct flow
(from the artery to the vein) to the total entrance of free methionine from the mammary
artery, or the ratio of the flux of the intracellular free methionine for utilization to the
total rate of the intracellular free methionine appearance (availability) (Fo,mg/Ra). More
than 50% of the total entrance of free methionine from the mammary artery did not enter
the intracellular free methionine compartment, but directly flowed from the artery to the
vein, resulting in low extraction efficiency of plasma methionine by the mammary gland.
The major proportion (above 70%) of the total appearance of the intracellular free
methionine was contributed from inward trans-membrane transport and the minor
proportion (below 30%) from endogenous sources (i.e., protein breakdown). In the
valine-excess diet, the contribution of inward trans-membrane transport was increased (P

< 0.05) and that from mammary protein breakdown was decreased (P < 0.05).

Kinetics of plasma valine across the mammary gland. Effects of the dietary AA regime

174

on kinetics of plasma valine across the mammary gland are represented in TABLE 4-9
and 5-12. No differences in the whole body irreversible loss rate (wa) or the
unidirectional flux to the mammary gland (Fmg) were found between the lysine-deficient
diet and the positive control diet. The flux of valine entrance into the mammary gland
(Fa,o), the direct flow from the artery to the vein (Fv,a), and the flux of valine exit from
the mammary gland to the vein were decreased (P < 0.05) in the lysine-deficient diet.
There were no differences in inward trans-membrane transport (F mg,a) or outward trans-
membrane transport (Fv,mg) between the lysine-deficient diet and the positive control
diet. Though the rate of the intracellular free valine disappearance (Fo,mg) was not
different between the lysine-deficient diet and the positive control diet, the fractional flux
of Fo,mg utilized for protein synthesis (Fo,mg(p)) was decreased (P < 0.05) by 40% in
the lysine-deficient diet; in contrast, the fractional flux of Fo,mg utilized for oxidation
and other metabolism (Fo,mg(o)) was numerically increased by 55% in the lysine-

deficient diet.

In the valine-excess diet, the whole body irreversible loss (wa), the flux of valine
entrance into the mammary gland (Fa,o), the flux of mammary free valine exit to the
mammary vein (Fo,v), and the direct flow from the artery to the vein (Fv,a) were
increased dramatically (P < 0.05 or 0.01). However, inward tranS-membrane transport
(Fmg,a) was numerically decreased by 35%; and outward trans-membrane transport
(Fv,mg) was completely blocked in the valine-excess diet. Therefore, net uptake of
plasma valine was slightly increased in the valine-excess diet. The rate of the

intracellular valine appearance from endogenous sources (i.e., protein breakdown)

175

(Fmg,o) was decreased (P < 0.05) by 56.8% in the valine-excess diet, indicating a
decreased protein breakdown in the mammary gland. The total rate of appearance of the
intracellular valine was decreased by approximately 40% due to decreased Fmg,a and
Fmg,o. There were no differences in the rate of the intracellular valine disappearance
(Fo,mg), the fractional flux of the intracellular valine utilized for protein synthesis
(Fo,mg(p)), or the fractional flux of the intracellular valine utilized for oxidation and

other metabolism (F o,mg(o)) between the valine-excess diet and the positive control diet.

In the valine-excess diet, the proportion of inward trans-membrane transport to the total
entrance of arterial plasma valine (Fmg,a/Fa,o) was decreased (P < 0.10) due to
decreased Fmg,a and increased F a,o; accordingly, the proportion of direct flow (from the
artery to the vein) to the total entrance of arterial plasma valine (Fv,a/Fa,o) was increased
(P < 0.10); thus, mammary extraction rate of plasma valine (NB/Fa,o) was decreased by
50%. The proportion of the whole body irreversible loss of plasma valine partitioned to
the mammary gland (Fmg/wa) was decreased due to increased F wb in the valine-excess
diet. The dietary amino acid regime did not affect the fractional proportion (Fmg,a/Ra)
of inward trans-membrane transport to the total appearance of the intracellular valine, the
fractional proportion (Fmg,o/Ra) of the flux of the intracellular valine appearance from
endogenous sources (i.e., protein breakdown) to the total appearance of the intracellular
valine, the proportion of Fo,mg utilized for protein synthesis (Fo,mg(p)/Fo,mg), or the
proportion of Fo,mg utilized for oxidation and/or other metabolic pathways
(F o,mg(o)/F o,mg). Averaged the ratio of Fo,mg(p) to Fo,mg and the ratio of Fo,mg(o) to

Fo,mg were at 0.66 and 0.34, respectively. The volume of arterial plasma completely

176

cleared of valine through the trans-membrane transport per unit of time was decreased
numerically in the valine-excess diet, possibly due to increased size of the extracellular

free valine compartment.

Protein synthesis and breakdown in the mammary gland. Protein turnover in the
mammary gland was derived from the compartmental model based on methionine fluxes
(TABLE 4-13). In the lysine-deficient diet, protein synthesis and breakdown were
decreased (P < 0.05), resulting in decreased net balance of protein in the mammary gland.
In the valine-excess diet, protein breakdown was decreased (P < 0.05). Moreover, the
ratio of protein breakdown to protein synthesis was decreased by approximately 25% in
the valine-excess diet compared to that in the positive control diet. Net production of
protein, estimated by the sum of output of protein in milk and accretion of protein in the

mammary gland, was very comparable to the model-derived values.

DISCUSSION
A three-compartmental model has been developed to quantify the integrated trans-
membrane transport of an individual AA across human skeletal muscle (Biolo et al. 1992,
Biolo et al. 1995); and has been modified to define the unidirectional influx and efflux of
an individual AA across the lactating goat mammary gland by Bequette et a1. (2000)
using enrichment of casein-bound AA in milk instead of mammary intracellular free AA.

In the present study, we demonstrated effects of dietary AA regime on the trans-

177

membrane transport kinetics and intracellular metabolism of indispensable AA (lysine,

methionine, and valine) in the lactating porcine mammary gland.

Milk yield and protein output. Milk yield and milk protein output were significantly
decreased in sows fed a lysine-deficient diet as shown in the study of Wilkinson
(Wilkinson et al. 1982). However, milk yield was not improved in the valine-excess diet.
An optimal ratio of valine to lysine for milk synthesis by the lactating sow was estimated
approximately at 1:1 at zero loss of maternal body protein (Guan et al. 2000c). Thus,
dietary ratio of valine to lysine (1.05:1) in the positive control diet may be the most
optimal for the lactating sow. These results of the present study were supported by a
recent feeding trial, in which dietary ratio of valine to lysine at 1.03:1.00 yields the
highest litter growth rate, and increased dietary ratio (up to 1222100) does not improve

litter growth rate (Carter et a1. 2000).

T mus-membrane transport and net uptake. Net uptake of indispensable AA by the
mammary gland is determined by the difference between inward (Fmg,a) and outward
trans-membrane transport (Fv,mg). Degrees of asymmetry as to inward versus outward
trans-membrane transport are inherent characteristics of AA transport systems of the
plasma membranes to favor accumulation or release of the intracellular AA (Christensen
1990, Sherman and Peaker 2000). Trans-membrane transport and net uptake of plasma
lysine was decreased in the lysine-deficient diet due to a very low concentration of

arterial plasma lysine (~ 30 uM), which was below the K... value (~ 100 pM) for systems

178

y+ and B0‘+. Transport of L-lysine across the brush-border membrane of the chicken
jejunum is regulated by dietary lysine concentration: systems y+ and b0'+ are up-regulated
by increasing dietary lysine concentration (from 4.8 to 6.8 g/kg diet)(Torras-Llort et al.
1998). Thus, AA transport systems for lysine in the basolateral membranes of the

mammary epithelium might be down-regulated by dietary lysine deficiency.

Dietary valine excess decreased net uptake of plasma lysine, which resulted from
increased F v,mg for lysine to a greater extent than Fmg,a for lysine. Uptake of lysine by
the mammary tissue is inhibited in vitro by high concentrations of neutral AA (e.g.,
leucine) (Baumrucker 1984, Hurley et al. 2000, Sherman et al. 1994, Sherman et al.
1997), by stimulating efflux of lysine from rat mammary tissue (Sherman et a1. 1997). In
addition, dietary supplementation of valine inhibits uptake of lysine by the brush-border
membrane of mouse jejunum (Stein et al. 1987). Recently, a Na+-dependent AA
transporter (hATB°‘+) cloned fi'om human mammary gland has expressed the same
kinetics properties of system B°’+ (Sloan and Mager 1999). This AA transport system has
high affinity for both lysine and valine (K... ~ 110 to 140 11M), trans-stimulated, and
adaptively regulated. Note that arterial plasma concentrations of lysine and valine were,
respectively, approximately at 250 and 650 11M in the lactating sow fed the positive
control diet. In this context, uptake of lysine by the mammary gland might be inhibited
by physiological (or high) concentrations of plasma valine, through stimulating outward
trans-membrane transport of lysine. Decreased net uptake of plasma lysine by the

mammary gland in the valine-excess diet was unlikely attributed to mammary plasma

179

flow rate because it was not decreased. Trans-membrane transport of methionine was not
affected in the valine-excess diet possibly due to its multiple AA transport systems in the

basolateral membranes.

Inward trans-membrane transport of valine was decreased numerically and its outward
trans-membrane transport was completely blocked in the valine-excess diet, resulting in a
numerically increased net uptake of plasma valine by the mammary gland. The transport
system for valine in the lactating porcine mammary gland has a K... of 640 11M in vitro
(Jackson et a1. 2000), indicating that AA transport system for valine may be saturated in
vivo. Transport of BCAA into muscle is regulated in vivo mainly by their concentrations
in the extracellular fluid and by intracellular metabolic removal (e. g., through catabolism
and protein synthesis) (Tovar et al. 1991). Outward trans-membrane transport for valine
was blocked possibly due to a very high concentration of free valine in the extracellular
fluid. The mammary venous plasma valine concentration (supposed to be lower than that
in the extracellular fluid) was as high as 1150 uM in the lactating sow fed the valine-
excess diet. Transport of the BCAA into the soleus muscle (presumably through system
L) is reduced by their high concentrations in vitro (Tovar et al. 1991). This down-
regulation of trans-membrane transport for valine would protect it from the risk of
toxicity of the intracellular valine excess since the BCAA may be toxic at high

concentrations in the tissue (Millward 1998).

Protein synthesis and breakdown. The present study demonstrated significant protein

180

synthesis and breakdown in the lactating porcine mammary gland. The proportion of
protein breakdown to protein synthesis ranged from 0.31 to 0.41 in the present study,
which supports an estimate of 0.33 in the goat mammary gland (Bequette et al. 1998).
With the lysine-deficient diet, protein synthesis was inhibited to slightly greater extent
than protein breakdown, resulting in decreased net balance of mammary protein.
Decreased protein synthesis in the lysine-deficient diet was indicated by decreased fluxes
of disappearance of the intracellular free lysine and methionine, the fractional flux of
disappearance of the intracellular free valine (F o,mg(p)). Protein synthesis in the lysine-
deficient diet might be restrained by decreased intracellular availability of lysine and

methionine, indicated by their decreased Ra.

We found that protein breakdown in the mammary gland was decreased to a greater
extent than protein synthesis in the valine-excess diet, resulting in a comparable net
balance of mammary protein with the positive control diet. Mammary protein breakdown
was significantly inhibited by dietary valine excess, indicating by fluxes (Fmg,o) of
appearance for the intracellular lysine, methionine, and valine from intracellular
endogenous sources. Moreover, the proportion of protein breakdown to protein synthesis
was decreased by 25% in the valine-excess diet. This inhibitory effect on mammary
protein breakdown by dietary valine excess, is possibly through its intracellular
metabolism (e. g., oxidation) or its extracellular regulatory sites on the basolateral
membranes of the mammary epithelial cells. Branched-chain amino acids (e.g., leucine)

inhibit muscle protein breakdown, presumably by their increased intracellular

181

accumulation or their keto-acids (e.g.,or-ketoisocaproate and isovalerate) (MacLean et al.
1994, Mitch and Clark 1984, Nair et al. 1992). We did not measure the mammary
intracellular concentrations of BCAA and their keto-acids. Though the total flux of
appearance of the intracellular free valine (Ra) was not increased in the valine-excess
diet, it can not be ruled out that the intracellular valine was oxidized rapidly in the
mammary gland. Oxidation of BCAA in the rat mammary gland is increased
significantly during lactation (DeSantiago et al. 1998). Their oxidation may be up-
regulated by high intake of protein or BCAA through increased expression of branched-
chain aminotransferase and/or increased activity of branched-chain or-keto acid

dehydrogenase (Langer et al. 2000, Torres et al. 1998).

However, the inhibitory effect of high valine on mammary protein breakdown was more
likely to occur on the plasma membranes since arterial plasma valine concentration
almost doubled without significant increase in the intracellular valine appearance in the
valine-excess diet. High concentrations of extracellular leucine and or-ketoisocaproate
inhibit protein breakdown in vitro, possibly through interactions with their regulatory
sites on the plasma membrane in the liver and heart where the activity of branched-chain

aminotransferase is minimal (Chua 1994, Miotto et al. 1992, Venerando et al. 1994).

Metabolism other than protein synthesis. In the present study, the proportion of the
mammary intracellular free valine utilized for metabolisms other than protein synthesis
was not affected by the dietary AA regime and averaged at 0.34, indicating that valine

may participate considerately in metabolic pathways (e.g., oxidation) other than protein

182

synthesis in the mammary gland. As discussed above, the catabolism of BCAA in the rat
mammary gland increases significantly during lactation due to increased expression of
mitochondrial branched-chain aminotransferase and increased activity of branched-chain
or-keto acid dehydrogenase (DeSantiago et al. 1998). The amount of the BCAA taken up
by the lactating porcine mammary gland exceeds their output in milk by approximately
25 to 30% (Nielsen et al. 2000, Trottier et al. 1997) (Guan et al. 2000b). Similar results
have been found in other lactating species, such as dairy cattle, sheep, and goat (Bequette
et al. 1997, Davis et al. 1978, Davis and Mepham 1976, Fleet and Mepham 1985,
Guinard 1994, Roets et al. 1979, Vina and Williamson 1981, Wohlt et al. 1977). This
positive difference between uptake and output of valine actually reflects its fractional flux
utilized for metabolism other than protein synthesis (e.g., oxidation). For example,
BCAA can be catabolized to yield carbon and or-amino nitrogen for synthesis of
dispensable AA and energy for milk synthesis by the mammary gland (Wohlt et al.

1977)

In summary, trans-membrane transport of indispensable AA (lysine, methionine, and
valine) across the basolateral membrane of the mammary epithelium is finely and
individually regulated to control their net uptake by dietary AA regime. Inward and
outward trans-membrane transport of certain indispensable AA (e. g., lysine) may be the
rate-limiting for milk synthesis (as shown in the lysine-deficient diet). Mammary protein
synthesis and breakdown are associated with net uptake of plasma individual AA, which
determines net balance of mammary protein. Dietary valine excess inhibits protein

breakdown in the mammary gland, possibly through its intracellular metabolism (e.g.,

183

oxidation) and/or its interactions with extracellular regulatory sites on the basolateral
membrane. In practice, dietary ratio of valine to lysine higher than 1.05:1 does not show

any improvement in milk yield, output of protein in milk, or litter grth rate.

LITERATURE CITED

Baumrucker, C. R. ( 1984) Cationic amino acid transport by bovine mammary tissue. J.
Dairy Sci. 67: 2500-2506.

Baumrucker, C. R. (1985) Symposium: Nutrient uptake across the mammary gland:
Amino acid transport systems in bovine mammary tissue. J. Dairy Sci. 68: 2436-
2451.

Bequette, B. J., Backwell, F. R., Dhanoa, M. S., Walker, A., Calder, A. G., Wray Cahen,
D., Metcalf, J. A., Sutton, J. D., Beever, D. E. & Lobley, G. E. (1994) Kinetics
of blood free and milk casein-amino acid labeling in the dairy goat at two stages
of lactation [see comments]. Br. J. Nutr. 72: 211-220.

Bequette, B. J., Backwell, F. R., Calder, A. G., Metcalf, J. A., Beever, D. E., MacRae, J.
C. & Lobley, G. E. (1997) Application of a U-13C-labeled amino acid tracer in
lactating dairy goats for simultaneous measurements of the flux of amino acids in

plasma and the partition of amino acids to the mammary gland. J. Dairy Sci. 80:
2842-2853.

Bequette, B. J., Backwell, F. R. & Crompton, L. A. (1998) Current concepts of amino
acid and protein metabolism in the mammary gland of the lactating ruminant. J.
Dairy Sci. 81: 2540-2559.

Bequette, B. J., Hanigan, M. D., Calder, A. G., Reynolds, C. K., Lobley, G. E. &
MacRae, J. C. (2000) Arrrino acid exchange by the mammary gland of lactating
goats when histidine limits milk production. J. Dairy Sci. 83: 765-775.

Biolo, G., Chinkes, D., Zhang, X. J. & Wolfe, R. R. (1992) Harry M. Vars Research
Award. A new model to determine in vivo the relationship between amino acid
trans-membrane transport and protein kinetics in muscle. JPEN J. Parenter
Enteral. Nutr. 16: 305-315.

Biolo, G., Fleming, R. Y., Maggi, S. P. & Wolfe, R. R. (1995) Trans-membrane
transport and intracellular kinetics of amino acids in human skeletal muscle. Am.

184

J. Physiol. 268: E75-84.

Boume, F. J. & Curtis, J. (1973) The transfer of immunoglobins IgG, IgA and IgM from
serum to colostrum and milk in the sow. Immunology 24: 157-162.

Calvert, D. T. & Sherman, D. B. (1996) Evidence for an interaction between cationic

and neutral amino acids at the blood-facing aspect of the lactating rat mammary
epithelium. J. Dairy Res. 63: 25-33.

Cant, J. P., DePeters, E. J. & Baldwin, R. L. (1993) Mammary amino acid utilization in
dairy cows fed fat and its relationship to milk protein depression. J. Dairy Sci. 76:
762-774.

Carter, S. D., Hill, G. M., Mahan, D. C., Nelssen, J. L., Richert, B. T. & Shurson, G. C.
(2000) Effects of dietary valine concentration on lactational performance of sows
nursing large litters. J. Anim. Sci. 78: 2879-2884.

Chillaron, J ., Estevez, R., Mora, C., Wagner, C. A., Suessbrich, H., Lang, F., Gelpi, J. L.,
Testar, X., Busch, A. E., Zorzano, A. & Palacin, M. ( 1996) Obligatory amino
acid exchange via systems bo,+-like and y+L-like. A tertiary active transport

mechanism for renal reabsorption of cystine and dibasic amino acids. J. Biol.
Chem. 271: 17761-17770.

Christensen, H. N. (1982) lnterorgan amino acid nutrition. Physiol. Rev. 62: 1193-1233.

Christensen, H. N. (1990) Role of amino acid transport and countertransport in nutrition
and metabolism. Physiol. Rev. 70: 43-77.

Chua, B. H. (1994) Specificity of leucine effect on protein degradation in perfused rat
heart. J. Mol. Cell Cardiol. 26: 743-751.

Davis, S. R., Bickerstaffe, R. & Hart, D. S. (1978) Amino acid uptake by the mammary
gland of the lactating ewe. Aust . J. Biol. Sci. 31: 123-132.

Davis, 8. R. & Mepham, T. B. (1976) Metabolism of L-(U-14C)va1ine, L-(U-
14C)leucine, L-(U-14C)histidine and L-(U-14C) phenylalanine by the isolated
perfused lactating guinea-pig mammary gland. Biochem. J. 156: 553-560.

DeSantiago, S., Torres, N., Suryawan, A., Tovar, A. R. & Hutson, S. M. (1998)
Regulation of branched-chain amino acid metabolism in the lactating rat. J. Nutr.
128: 1165-1171.

Deves, R. & Boyd, C. A. (1998) Transporters for cationic amino acids in animal cells:
discovery, structure, and function. Physiol. Rev. 78: 487-545.

Dixon, J. L. & Benevenga, N. J. (1980) The decarboxylation of alpha-keto-gamma-
methiolbutyrate in rat liver mitochondria. Biochem. Biophys. Res. Commun. 97:

185

93 9-946.

Fleet, I. R. & Mepham, T. B. (1985) Mammary uptake of amino acids and glucose
throughout lactation in friesland sheep. J. Dairy Res. 52: 229-237.

F ukasawa, Y., Segawa, H., Kim, J. Y., Chairoungdua, A., Kim, D. K., Matsuo, H., Cha,
S. H., Endou, H. & Kanai, Y. (2000) Identification and characterization of a
Na(+)-independent neutral amino acid transporter that associates with the 4F2

heavy chain and exhibits substrate selectivity for small neutral D- and L-amino
acids. J. Biol. Chem. 275: 9690-9698.

Gomez Angelats, M., Ruiz Montasell, B., Felipe, A., Marin, J. J ., Casado, F. J. & Pastor
Anglada, M. (1995) Effect of protein malnutrition on neutral amino acid
transport by rat hepatocytes during development. Am. J. Physiol. 268: E368-374.

Guan, X. F., Ku, P. K. & Trottier, N. L. (2000a) Quantitation of indispensable amino
acids mobilized from body protein reserves of extra-mammary tissues in the
lactating sow. J. Nutr.

Guan, X. F ., Pettigrew, J. E., Ku, P. K. & Trottier, N. L. (2000b) Effects of dietary
protein concentration on the amino acid profile in milk and the amino acid uptake
pattern by the lactating porcine mammary gland. J. Anim. Sci.

Guan, X. F., Pettigrew, J. E., Ku, P. K. & Trottier, N. L. (2000c) Uptake of plasma
amino acids by the lactating porcine mammary gland: A novel approach to define
their needs for milk synthesis. J. Nutr.

Guinard, J. a. H. R. (1994) Effects of graded amounts of duodenal infusions of lysine on
the mammary uptake of major milk precursors in dairy cows. J. Dairy Sci. 77:
3565-3576.

Hoffman, L., Trottier, N. L., Bequette, B. J., Nielsen, T. T. & Easter, R. A. (1997)
Leucine kinetics and incorporation into milk casein in the lactating sow. J. Anim.
Sci. 75(Suppl. 1): 77(Abs.).

Hurley, W. L., Wang, H., Bryson, J. M. & Sherman, D. B. (2000 Feb) Lysine uptake by
mammary gland tissue from lactating sows. J. Anim. Sci. 78: 391-395.

Jackson, S. C., Bryson, J. M., Wang, H. & Hurley, W. L. (2000) Cellular uptake of
valine by lactating porcine mammary tissue. J. Anim. Sci. 78: 2927-2932.

Kamath, S. G., Furesz, T. C., Way, B. A. & Smith, C. H. (1999) Identification of three
cationic amino acid transporters in placental trophoblast: cloning, expression, and
characterization of hCAT-l. J. Membr. Biol. 171: 55-62.

Kanai, Y., Segawa, H., Miyamoto, K., Uchino, H., Takeda, E. & Endou, H. (1998)
Expression cloning and characterization of a transporter for large neutral amino
186

acids activated by the heavy chain of 4F2 antigen (CD98). J. Biol. Chem. 273:
23629-23632.

Kansal, R. & Kansal, V. K. (1996) Lack of functional correlation between gamma-
glutamyl transpeptidase and amino acid transport in the lactating mouse
mammary gland. Indian J. Exp. Biol. 34: 267-269.

Kekuda, R., Prasad, P. D., Fei, Y. J., Torres Zamorano, V., Sinha, S., Yang Feng, T. L.,
Leibach, F. H. & Ganapathy, V. (1996) Cloning of the sodium-dependent,

broad-scope, neutral amino acid transporter Bo from a human placental
choriocarcinoma cell line. J. Biol. Chem. 271: 18657-18661.

Kekuda, R., Torres Zamorano, V., Fei, Y. J., Prasad, P. D., Li, H. W., Mader, L. D.,
Leibach, F. H. & Ganapathy, V. (1997) Molecular and functional
characterization of intestinal Na(+)-dependent neutral amino acid transporter BO.
Am. J. Physiol. 272: G1463-1472.

Kilberg, M. S., Stevens, B. R. & Novak, D. A. (1993) Recent advances in mammalian
amino acid transport. Annu. Rev. Nutr. 13: 137-165.

Kim, S. W., Hurley, W. L., Han, I. K. & Easter, R. A. (1999) Changes in tissue
composition associated with mammary gland growth during lactation in sows. J.
Anim. Sci. 77: 2510-2516.

Klobasa, F. & Butler, J. E. (1987) Absolute and relative concentrations of
immunoglobulins G, M, and A, and albumin in the lacteal secretion of sows of
different lactation numbers. Am. J. Vet. Res. 48: 176-182.

Klobasa, F., Werhahn, E. & Butler, J. E. (1987) Composition of sow milk during
lactation. J. Anim. Sci. 64: 1458-1466.

Langer, S., Scislowski, P. W., Brown, D. S., Dewey, P. & Fuller, M. F. (2000)
Interactions among the branched-chain amino acids and their effects on
methionine utilization in growing pigs: effects on plasma amino- and keto-acid

concentrations and branched-chain keto-acid dehydrogenase activity. Br. J. Nutr.
83: 49-58.

Linzell, J. L. (1974). Mammary blood flow and methods of identifying and measuring
precursors of milk. In Lactation: A comprehensive treatise (Larson, B. L. &
Smith, V. R., eds.), Vol. 1, pp. 143-225. 1 of 4 vols. Academic Press, New York.

Lobley, G. E., Connell, A., Revell, D. K., Bequette, B. J., Brown, D. S. & Calder, A. G.
(1996) Splanchnic-bed transfers of amino acids in sheep blood and plasma, as
monitored through use of a multiple U-13C-labelled amino acid mixture
[published erratum appears in Br J Nutr 1996 Aug;76(2):319]. Br. J. Nutr. 75:
217-235.

187

MacLean, D. A., Graham, T. E. & Saltin, B. (1994) Branched-chain amino acids
augment ammonia metabolism while attenuating protein breakdown during
exercise. Am. J. Physiol. 267: E1010-1022.

Mepham, T. B. (1982) Amino acid utilization by lactating mammary gland. J. Dairy.
Sci. 65: 287-298.

Millward, D. J. (1998) Metabolic demands for amino acids and the human dietary
requirement: Millward and Rivers (1988) revisited. J. Nutr. 128: 2563S-2576S.

Miotto, G., Venerando, R., Khurana, K. K., Siliprandi, N. & Mortimore, G. E. (1992)
Control of hepatic proteolysis by leucine and isovaleryl-L-carnitine through a
common locus. Evidence for a possible mechanism of recognition at the plasma
membrane. J. Biol. Chem. 267: 22066-22072.

Mitch, W. E. & Clark, A. S. (1984) Specificity of the effects of leucine and its
metabolites on protein degradation in skeletal muscle. Biochem. J. 222: 579-586.

Mitchell, A. D. & Benevenga, N. J. (1978) The role of transamination in methionine
oxidation in the rat. J. Nutr. 108: 67-78.

Moser, S. A., Tokach, M. D., Dritz, S. S., Goodband, R. D., Nelssen, J. L. &
Loughmiller, J. A. (2000) The effects of branched-chain amino acids on sow and
litter performance. J. Anim. Sci. 78: 658-667.

Nair, K. S., Schwartz, R. G. & Welle, S. (1992) Leucine as a regulator of whole body
and skeletal muscle protein metabolism in humans. Am. J. Physiol. 263: E928-
934.

Nielsen, T. T., Trottier, N. L., Stein, H. H., Bellaver, C. & Easter, R. A. (2000) The
effect of litter size and day of lactation on amino acid uptake by the porcine
mammary gland. J. Anim. Sci.

NRC. (1998). Nutrient requirements of swine. 10th ed. National Academy Press,
Washington, DC.

Oddy, V. H., Lindsay, D. B. & Fleet, I. R. (1988) Protein synthesis and degradation in
the mammary gland of lactating goats. J. Dairy Res. 55: 143-154.

Palacin, M., Estevez, R., Bertran, J. & Zorzano, A. (1998) Molecular biology of
mammalian plasma membrane amino acid transporters. Physiol. Rev. 78: 969-
1054.

Peters, A. R., Alexandrov, S. & Mepham, T. B. ( 1979) Effects of increased supply of
essential amino acids on the metabolism of the isolated perfused guinea-pig
mammary gland. J. Dairy Res. 46: 59-67.

188

Pettigrew, J. E., Cornelius, S. G., Moser, R. L. & Sower, A. F. (1987) A refinement and
evaluation of the isotope dilution method for estimating milk intake by piglets.
Livest. Prod. Sci. 16: 163—174.

Pettigrew, J. E., Sower, A. F., Cornelius, S. G. & Moser, R. L. (1985) A comparison of
isotope dilution and weigh-suckle-weigh methods for estimating milk intake by
pigs. Can. J. Anim. Sci. 65: 989-992.

Pineda, M., Fernandez, E., Torrents, D., Estevez, R., Lopez, 0, Camps, M., Lloberas, J .,
Zorzano, A. & Palacin, M. (1999) Identification of a membrane protein, LAT-2,
that Co-expresses with 4F2 heavy chain, an L-type amino acid transport activity
with broad specificity for small and large zwitterionic amino acids. J. Biol. Chem.
274: 19738-19744.

Richert, B. T., Goodband, R. D., Tokach, M. D. & Nelssen, J. L. (1997) Increasing
valine, isoleucine, and total branched-chain amino acids for lactating sows. J.
Anim. Sci. 75: 2117-2128.

Richert, B. T., Tokach, M. D., Goodband, R. D., Nelssen, J. L., Pettigrew, J. E., Walker,
R. D. & Johnston, L. J. (1996) Valine requirement of the high-producing
lactating sow. J. Anim. Sci. 74: 1307-1313.

Rillema, J. A., Golden, K. & Jenkins, M. A. (1992) Effect of prolactin on alpha-
aminoisobutyric acid uptake in mouse mammary gland explants. Am. J Physiol.
262: E402-405.

Roets, E., Massart Leen, A. M., Verbeke, R. & Peeters, G. (1979) Metabolism of [U-
14C; 2,3-3H]—L—valine by the isolated perfused goat udder. J. Dairy Res. 46: 47-
57.

Rousselow, D. L. & Speer, V. C. (1980) Valine requirement of the lactating sow. J.
Anim. Sci. 50: 472-478.

Segawa, H., Fukasawa, Y., Miyarnoto, K., Takeda, E., Endou, H. & Kanai, Y. (1999)
Identification and functional characterization of a Na+-independent neutral amino
acid transporter with broad substrate selectivity. J. Biol. Chem. 274: 19745-
19751.

Sharma, K. & Kansal, V. K. (1999) Characteristics of transport systems of l-alanine in
mouse mammary gland and their regulation by lactogenic hormones: evidence for
two broad spectrum systems. J. Dairy Res. 66: 385-398.

Shanna, R. & Kansal, V. K. (2000) Heterogeneity of cationic amino acid transport
systems in mouse mammary gland and their regulation by lactogenic hormones. J.
Dairy Res. 67: 21-30.

189

Sherman, D. B., McNeillie, S. A., Jamieson, E. A. & Calvert, D. T. (1994) Lysine
transport in lactating rat mammary tissue: evidence for an interaction between
cationic and neutral amino acids. Acta Physiol. Scand. 151: 461-466.

Sherman, D. B., Millar, I. D. & Calvert, D. T. (1997) Mammary-tissue amino acid
transport systems. Proc. Nutr. Soc. 56: 177-191.

Sherman, D. B. & Peaker, M. (2000) Transport of milk constituents by the mammary
gland. Physiol. Rev. 80: 925-951.

Sloan, J. L. & Mager, S. (1999) Cloning and functional expression of a human Na(+)
and Cl(-)-dependent neutral and cationic amino acid transporter B(0+). J. Biol.
Chem. 274: 23740-23745.

Soriano Garcia, J. F., Torras Llort, M., Ferrer, R. & Moreto, M. (1998) Multiple
pathways for L-methionine transport in brush-border membrane vesicles from
chicken jejunum. J. Physiol. Lond. 509: 527-539.

Soriano Garcia, J. F., Torras Llort, M., Moreto, M. & Ferrer, R. (1999) Regulation of L-
methionine and L-lysine uptake in chicken jejunal brush-border membrane by
dietary methionine. Am. J Physiol. 277: R1654-1661.

Stein, E. D., Chang, S. D. & Diamond, J. M. (1987) Comparison of different dietary
amino acids as inducers of intestinal amino acid transport. Am. J. Physiol. 252:
G626-635.

Taylor, P. M., Kaur, S., Mackenzie, B. & Peter, G. J. (1996) Amino-acid-dependent
modulation of amino acid transport in Xenopus laevis oocytes. J. Exp. Biol. 199:
923-931.

Torras-Llort, M., Soriano-Garcia, J. F., Ferrer, R. & Moreto, M. (1998) Effect of a
lysine-enriched diet on L-lysine transport by the brush-border membrane of the
chicken jejunum. Am. J. Physiol. 274: R69-R75.

Torrents, D., Estevez, R., Pineda, M., Fernandez, E., Lloberas, J ., Shi, Y. B., Zorzano, A.
& Palacin, M. (1998) Identification and characterization of a membrane protein
(y+L amino acid transporter-1) that associates with 4F 2hc to encode the amino

acid transport activity y+L. A candidate gene for lysinuric protein intolerance. J.
Biol. Chem. 273: 32437-32445.

Torres, N., Lopez, G., De Santiago, S., Hutson, S. M. & Tovar, A. R. (1998) Dietary
protein level regulates expression of the mitochondrial branched-chain
aminotransferase in rats. J. Nutr. 128: 1368-1375.

Tovar, A. R., Tews, J. K., Torres, N. & Harper, A. E. (1991) Neutral amino acid
transport into rat skeletal muscle: competition, adaptive regulation, and effects of

190

insulin. Metabolism 40: 410-419.

Trottier, N. L., Shipley, C. F. & Easter, R. A. (1995) A technique for the venous
cannulation of the mammary gland in the lactating sow. J. Anim. Sci. 73: 1390-
1395.

Trottier, N. L., Shipley, C. F. & Easter, R. A. (1997) Plasma amino acid uptake by the
mammary gland of the lactating sow. J. Anim. Sci. 75: 1266-1278.

Utsunomiya Tate, N., Endou, H. & Kanai, Y. (1996) Cloning and firnctional
characterization of a system ASC-like Na+-dependent neutral amino acid
transporter. J. Biol. Chem. 271: 14883-14890.

Venerando, R., Miotto, G., Kadowaki, M., Siliprandi, N. & Mortimore, G. E. (1994)
Multiphasic control of proteolysis by leucine and alanine in the isolated rat
hepatocyte. Am. J. Physiol. 266: C455-46l.

Verbeke, R., Roets, E., Massart-Leen, A.-M. & Peeters, G. (1972) Metabolism of [U-
l4C]-L-threonine and [U-14C]-L-phenylalanine by the isolated perfused udder. J.
Dairy Res. 39: 239-249.

Vina, J. R. & Williamson, D. H. (1981) Effects of lactation on L-leucine metabolism in
the rat. Studies in vivo and in vitro. Biochem. J. 194: 941-947.

Wilkinson, R., Cole, D. J. A. & Lewis, D. (1982) Amino acid nutrition of the lactating
sow: the requirement for dietary lysine. Anim. Prod. 35: 15-23.

Wohlt, J. E., Clark, J. H., Derrig, R. G. & Davis, C. L. (1977) Valine, leucine, and
isoleucine metabolism by lactating bovine mammary tissue. J. Dairy Sci. 60:
1875-1882.

Wolffram, S. & Scharrer, E. (1984) Effect of feeding a high protein diet on amino acid
uptake into rat intestinal brush border membrane vesicles. Pflugers Arch 400: 34-
39.

Wu, G. & Knabe, D. A. (1994) Free and protein-bound amino acids in sow's colostrum
and milk. J. Nutr. 124: 415-424.

191

Appendix A Glossary

 

 

Term Unit Definition

The whole body irreversible loss rate of plasma individual
wa mmol/h

AA

The unidirectional flux of plasma free AA from mammary
F mg mmol/h

artery to the mammary gland

Rate of free AA entrance into the mammary gland from
Fa,o mmol/h . . . _

systemic crrculatron vra mammary artery

Direct flow of free AA from mammary artery to mammary
Fv,a mmol/h . . . . .

vern wrthout entering intracellular flurd

Rate of free AA exit from the mammary gland to systemic
Fo,v mmol/h . _ . _

crrculatron vra mammary vem

Net mass balance of plasma free AA across the mammary
NB mmol/h

gland

Inward trans-membrane transport of free AA from
Fmg,a mmol/h mammary artery to mammary intracellular free AA

compartment

Outward trans-membrane transport of free AA from
Fv,mg mmol/h mammary intracellular free AA compartment to mammary

vein

Rate of mammary intracellular free AA appearance from
Fmg,o mmol/h endogenous sources (i.e., from protein breakdown and de

novo synthesis, if any)

 

192

 

Fo,mg

Fo,mg(p)

F o,mg(o)

Ra

Fm g/F wb

Fmg,a/Fa,o

Fv,a/Fa,o

NB/Fa,o

mmol/h

mmol/h

mmol/h

mmol/h

Rate of mammary intracellular free AA disappearance (or
utilization) (i.e., for protein synthesis, oxidation, and other

metabolic fates, if any)

Fractional rate of mammary intracellular valine

disappearance utilized for protein synthesis

Fractional rate of mammary intracellular valine
disappearance utilized for oxidation and other metabolic

pathways

Total rate of mammary intracellular free AA appearance,
i.e., the sum of inward trans-membrane transport (Fmg,a)

and intracellular appearance from endogenous sources

(F 1448.0)

Fractional proportion of wa partitioned to the mammary

gland

Fractional proportion of inward trans-membrane transport to
total entrance of free AA fiom mammary artery, indicating
the ability of mammary AA transport systems to take up
circulating AA

Fractional proportion of direct flow (from artery to vein) to
total entrance of fi'ee AA from mammary artery, indicating
inability or regulation of mammary AA transport systems to

take up circulating AA

Extraction rate of net mass balance to total entrance of free

AA from mammary artery

 

193

 

Fmg,a/Ra

F m g,o/Ra

Fo,mg/Ra

Fo,mg(p)/Fo,mg

F o,mg(o)/F o,mg

Fmg,a/Ca L/min

Fractional contribution of inward trans-membrane transport

to total rate of mammary intracellular free AA appearance

Fractional contribution from protein breakdown to total rate

of mammary intracellular free AA appearance

Ratio of the flux for protein synthesis to total flux of
mammary intracellular free AA appearance (availability),
an indicator of the utilization efficiency for protein

synthesis

Ratio of fractional flux for protein synthesis to total flux of

mammary intracellular free AA disappearance

Ratio of fractional flux for oxidation and other metabolic
rates to total flux of mammary intracellular free AA

disappearance

The volume of arterial plasma completely cleared of the AA
via trans-membrane transport per unit of time, indicating the

ability of any particular AA to be transported

 

194

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TABLE 4-3 Composition of experimental diets (as-fed basis)

 

Dietary amino acid regime

 

Item

 

Lysine Positive Valine
deficient control excess
Ingredient, gfl<g diet
Corn 679.5 679.5 679.5
Soybean meal 125.0 125.0 125.0
Tallow 50.0 50.0 50.0
Solk floca 25.0 25.0 25.0
Calcium phosphate 22.0 22.0 22.0
Calcium carbonate 10.0 10.0 10.0
Salt 2.5 2.5 2.5
Mineral and vitamin premixb 1 1.5 11.5 11.5
Amino acid mixturec 14.8 14.8 14.8
Lysine-HCl 0 8.0 8.0
Valine 4.9 4.9 9.3
Glutamic acid 24.1 36.8 42.4
Corn starch 30.7 10.0 0
Analyzed contents of nitrogen and indispensable amino acids, g/kg diet
Nx6.25 138.1 151.1 158.8
Histidine 4.90 4.94 4.79
Arginine 7.08 7.15 6.83
Threonine 5.17 5.28 5.13
Valine 9.87 10.15 13.37
Methionine 3.15 3 .26 3.39
Isoleucine 6.54 6.56 6.52
Leucine 12.90 12.91 12.51
Phenylalanine 8.00 7.87 7.73
Lysine 4.93 9.71 9.76
Tryptophand 2.10 2.10 2.10

 

aThe content of cellulose was 98.5%.

bProvided the following amounts of trace minerals and vitamins in milligrams per kilogram of
diet: copper, 5; iodine, 0.075; iron, 50; manganese, 5; selenium, 0.15; zinc, 50; retinyl acetate, 8.3;
cholecalciferol, 0.0138; a-tocoperol, 44.1; menadione, 4.5; vitamin 8.2, 0.033; riboflavin, 4.5; d-
pantothenic acid, 17.6; niacin, 26.4; thiamin, 1.1; pyridoxine, 1.0; choline, 385.0; folic acid, 1.65;
and d-biotin, 0.22.

cProvided the following amounts of indispensable amino acids in grams per kilogram of diet:
histidine-HCl, 1.9; threonine, 3.2; DL-methionine, 1.4; isoleucine, 2.0; leucine, 1.6; phenylalanine,
2.5, tyrosine, 1.3, and tryptophan, 0.9.

dCalculated value (NRC, 1998).

197

TABLE 4-4 Effects of dietary amino acid regime on productive performance of the
lactating sow over a 21—d lactation period

 

Dietary amino acid regime

 

 

Item Lysine Positive Valine SEM
deficient control excess
Feed intake, kg/d 5.34 5.71 5.83 0.56
Litter size on d 21, n 11.0 11.8 12.0 0.60
Litter growth rate, kg/d 1.69. 2.37 2.50 0.11
Milk yield, kg/d 6.26" 9.20 9.06 0.16
N in defatted milk, % 0.95‘ 1.01 0.99 0.01
Protein in milk, % 4.88. 5.11 5.03 0.04
Casein-N, % of total N in milk 49.30 53.28 52.69 2.30

 

Values (least-squares means) with * and *"' differ respectively at P < 0.05 and 0.01 between the lysine-

deflcient diet and the positive control.

198

TABLE 4-5 Effects of dietary amino acid regime on arterial concentrations of plasma
amino acids and urea in the lactating sow

 

Dietary amino acid regime

 

 

Amino acid SEM
Lysine deficient Positive control Valine excess

Taurine 75.51‘ 19.89 20.51 9.04
Urea 551419" 279498 4329.01 816.71
Aspartic acid 34.94 4751 48.59 6.84
Threonine 470.70+ 22500 224.63 67.48
Serine 111.05 88°88 72.85 7.63
Asparagine 57.58 61'” 67.11 13.45
Glutamic acid 368.08 41 1‘96 398.73 77.45
Glutamine 512.73 559.21 450.32 37.68
Proline 266.28 256.11 250.98 32.75
Glycine 774.64 939.87 785.19 93.02
Alanine 466.62 685.86 666.15 55.19
Citrulline 103.86 86.79 86.62 17.11
Valine 529.96 664.85 1236.68” 60.88
Cystine 606* 2.20 4.23 0.91
Methionine 57.15 60.83 51.57 7.38
Isoleucine 109.80 87.38 98.70 11.02
Leucine 192.25 123.52 148.46 24.14
Tyrosine 155.88 88.73 68.31 36.94
Phenylalanine 101.56 61.12 64.27 14.77
Tryptophan 51.69 3 5.01 34.46 7.83
Omithine 21.37 30.82 28.33 5.61
Lysine 32.67” 253.98 203.94 27.05
Histidine 108.98 113.21 101.08 9.07
Arginine 102.18 113.84 125.77 15.58

 

Values (least-squares means, mol/L) with T , ’, and "”" differ at P < 0.10, 0.05, and 0.01, respectively,

between the lysine-deficient diet and the positive control or between the valine-excess diet and the positive

control.

199

TABLE 4-6 Effects of dietary amino acid regime on mammary extraction rates of plasma
amino acids and urea in the lactating sow

 

Dietary amino acid regime

 

 

Amino acid SEM
Lysine deficient Positive control Valine excess
Taurine 12.59 -223 -1040 9.47
Urea 5.93 2.98 -9.91 5.03
Aspartic acid -2.25 2.57 -10.72 4.40
Threonine 10.60 17.64 459+ 3.05
Serine 26.18‘ 46.70 38.18 4.37
Asparagine 26.05 25.43 26.29 1 1.90
Glutamic acid 16.78 15.27 6.27 7.54
Glutamine 12.52 13.26 5.72 5.30
Proline 12.92 13.74 9.00 2.49
Glycine 9.69 5.94 -2.56 5.62
Alanine 10.71 9.73 -O.83 5.04
Citrulline 14.82 -4.52 -7.71 9.57
Valine 15.30 14.30 6.90 3.39
Cystine 19.53‘ 41.25 19.11‘ 2.90
Methionine 18.65‘ 24.45 24.65 0.45
Isoleucine 28.85 38.28 27.63 4.55
Leucine 28.46‘ 49.64 35.37“ 4.34
Tyrosine 15.76 25.34 20.59 3.32
Phenylalanine 20.75 36.50 30.21 5.17
Tryptophan 6.71 2.40 1.46 7.36
Ornithine 32.06 30.22 15.14’r 3.54
Lysine 51.15‘ 22.75 15.35 4.53
Histidine 12.00 17.12 082* 3.63
Arginine 34.79 28.04 14.02‘ 2.52

 

Values (least-squares means, %) with i and * differ respectively at P < 0.10 and 0.05 between the lysine-

deficient diet and the positive control or between the valine-excess diet and the positive connol.

200

TABLE 4-7

Effects of dietary amino acid regime on kinetics parameters of plasma lysine across the

lactating porcine mammary gland

 

Dietary amino acid regime

Item . . . . . SEM
Lys1ne defiment Posmve control Valine excess

 

 

‘

M 14.89‘ 28.58 29.67 0.48
Fmg 7.71" 15.07 9.58“ 0.46
Fa,o 11.22" 55.41 52.16 4.38
Fv,a 2.59 9.13 0.07 7.01
Fmg,a 965* 38.47 52.64 7.83
Fv,mg 4.18”“ 24.87 44.58 7.98
Fo,v 5.57‘ 42.19 44.02 3.80
Fmg,o 2.57 5.98 3.29 1.07
Fo,mg 9.55** 17.43 10.35** 0.60
Ra 732* 48.17 58.23 8.41
NB 6.47" 10.91 7.10‘ 0.77

 

Values (least-squares means, mmol/h) with T , ‘, and ‘”" differ at P < 0.10, 0.05, and 0.01, respectively,
between the lysine-deficient diet and the positive control or between the valine-excess diet and the positive

control.

201

TABLE 4-8

Effects of dietary amino acid regime on kinetics parameters of plasma methionine across

the lactating porcine mammary gland

 

Dietary amino acid regime
Item SEM
Lysine deficient Positive control Valine excess

 

 

wa 6.06”r 6.93 6.86 0.23
Fmg 2.42‘ 4.05 3.71 0.37
Fa,o 11.43 13.77 13.37 0.84
Fv,a 7.83 7.55 695* 0.15
Fmg,a 3.421 6.00 6.54 0.94
Fv,mg 1.43 2.73 3.35 0.68
Fo,v 9.20 10.35 10.08 0.59
Fmg,o 1.32‘ 2.29 1.39‘ 0.15
Fo,mg 3.34‘ 5.58 4.51 0.41
Ra 4.74“ 8.34 7.94 1.08
NB 1.96‘ 3.28 3.11 0.26

 

Values (least-squares means, mmth) with T and * differ respectively at P < 0.10 and 0.05 between the

lysine-deficient diet and the positive control or between the valine-excess diet and the positive control.

202

TABLE 4-9

Effects of dietary amino acid regime on kinetics parameters of plasma valine across the

lactating porcine mammary gland

 

Dietary amino acid regime
Item SEM
Lysine deficient Positive control Valine excess

 

 

M 29.97 34.81 43.17’ 1.85
Fmg 20.11 23.08 24.02 4.65
Fa,o 112.57” 150.91 333.99” 4.80
Fv,a 8361* 111.15 313.68" 8.79
Fmg,a 28.26 36.21 23.47 8.44
Fv,mg 11.82 15.95 0.00* 4.21
Fo,v 95.50‘ 129.35 310.66" 6.35
Fmg,o 5.79 6.72 2.90" 0.67
Fo,mg 24.02 27.25 27.12 4.99
Fo,mg(p) 11.47‘ 19.17 15.49 1.41
Fo,mg(o) 12.55 8.08 11.63 4.97
Ra 36.36 44.78 26.34 9.09
NB 15.30 20.19 23.99 4.35

 

Values (least-squares means, mmth) with T , *, and *"' differ at P < 0.10, 0.05, and 0.01, respectively,
between the lysine-deficient diet and the positive control or between the valine-excess diet and the positive

control.

203

TABLE 4-10

Effects of dietary amino acid regime on fractional ratios of plasma lysine kinetics across

the lactating porcine mammary gland

 

Dietary amino acid regime
Item SEM
Lysine deficient Positive control Valine excess

 

 

Fmg/wa 048* 0.54 0.33" 0.02
Fmg,a/Fa,o 0.86 0.74 1.00 0.09
Fv,a/Fa,o 0.14 0.26 0.00 0.09
NB/Fa,o 0.51* 0.23 0.15 0.05
Fmg,a/Ra 080* 0.87 093* 0.01
Fmg,a/Ra 0.20* 0.13 0.07* 0.01
Fo,mg/Ra 067* 0.42 020* 0.05
Fmg,a/Ca, L/min 7.42 7.52 10.94 1.39

 

Values (least-squares means) with f , *, and *"' differ at P < 0.10, 0.05, and 0.01, respectively, between

the lysine-deficient diet and the positive control or between the valine-excess diet and the positive control.

204

TABLE 4-11

Effects of dietary amino acid regime on fractional ratios of plasma methionine kinetics

across the lactating porcine mammary gland

 

Dietary amino acid regime
Item SEM
Lysine deficient Positive control Valine excess

 

 

Fmg/wa 041* 0.60 0.55 0.06
Fmg,a/Fa,o 0.32 0.45 0.49 0.04
Fv,a/Fa,o 0.68 0.55 0.51 0.04
NB/Fa,o 0.19“ 0.24 0.25 0.00
Fmg,a/Ra 0.73 0.72 0.82‘ 0.02
Fmg,o/Ra 0.27 0.28 0.18‘ 0.02
Fo,mg/Ra 0.69 0.67 0.57 0.04
Fmg,a/Ca, L/min 2.97 4.60 5.40 0.73

 

Values (least-squares means) with f , *, and ** differ at P < 0.10, 0.05, and 0.01, respectively, between

the lysine-deficient diet and the positive control or between the valine-excess diet and the positive control.

205

TABLE 4-12

Effects of dietary amino acid regime on fractional ratios of plasma valine kinetics across

the lactating porcine mammary gland

 

Dietary amino acid regime
Item SEM
Lysine deficient Positive control Valine excess

 

 

Fmg/wa 0.63 0.69 0.55 0.13
Fmg,a/Fa,o 0.26 0.26 006* 0.06
Fv,a/Fa,o 0.75 0.74 094* 0.06
NB/Fa,0 0.15 0.14 0.07 0.03
Fmg,a/Ra 0.84 0.85 0.90 0.02
Fmg,o/Ra 0.16 0.15 0.10 0.02
Fo,mg(p)/Ra 0.33 0.46 0.63 0.12
Fo,mg(p)/Fo,mg 0.58 0.73 0.62 0.18
Fo,mg(o)/Fo,mg 0.42 0.27 0.38 0.18
Fmg,a/Ca, L/min 2.27 2.52 074* 0.52

 

Values (least-squares means) with i differ at P < 0.10 between the valine-excess diet and the positive

control.

206

TABLE 4-13

Effects of dietary amino acid regime on mammary protein synthesis and breakdown based

upon protein equivalent methionine fluxes

 

Dietary amino acid regime
Item SEM
Lysine deficient Positive control Valine excess

 

 

Protein synthesis 583.3‘ 975.1 787.1 71.6
(PS), 8/da

Protein breakdown 229.5’ 399.6 243.1 ‘ 25.5
(PB). g/d"

PB/PS 0.39 0.41 0.31” 0.01
Net balance of 353.7‘ 575.5 544.0 46.2
protein, g/dC

Net production of 355.0" 529.0 514.2 10.1
protein, g/dd

 

Values (least-squares means) with "' and ‘”“ differ at P < 0.05 and 0.01, respectively, between the lysine-

deflcient diet and the positive control or between the valine-excess diet and the positive control.

aProtein synthesis is reflected by rate of disappearance of mammary intracellular methionine (Fo,mg,

see Figure 2).

b . . . . .
Proteln breakdown is reflected by rate of appearance of mammary intracellular meth1on1ne from

endogenous source (Fmg,o, see Figure 2).

cNet balance of protein = protein synthesis - protein breakdown.

dEstimated from the sum of output of protein in milk and accretion (14.81 g/d) of protein in the lactating

porcine mammary gland (Kim et a1. 1999).

207

 

Lysine enrichment, MPE
(It

 

 

I I I 1 I I I j I

I
12 13 14 15 16 l7 18 19 20 21 22

Time post-infusion, h

FIGURE 4-1a Enrichment of plasma free- and milk casein-bound lysine in the sows fed
the lysine-deficient diet. Lines with closed circles, open circles, and closed squares
represent the enrichments of plasma lysine in the carotid artery, plasma lysine in the

mammary vein, and casein-bound lysine in milk, respectively.

208

Lysine enrichment, MPE
U!

 

 

 

I I T I I I I I 1 I

12 13 14 15 l6 17 18 19 20 21 22

Time post-infusion, h

FIGURE 4-1b Enrichment of plasma free- and milk casein-bound lysine in the sows fed
the positive control diet. Lines with closed circles, open circles, and closed squares
represent the enrichments of plasma lysine in the carotid artery, plasma lysine in the

mammary vein, and casein-bound lysine in milk, respectively.

209

 

Lysine enrichment, MPE
UI

 

 

I I I I f I I I I I

12 13 14 15 16 17 18 19 20 21 22

Time post-infusion, h

FIGURE 4-1c Enrichment of plasma free- and milk casein-bound lysine in the sows fed
the valine-excess diet. Lines with closed circles, open circles, and closed squares
represent the enrichments of plasma lysine in the carotid artery, plasma lysine in the

mammary vein, and casein-bound lysine in milk, respectively.

210

 

 

Methionine enrichment, MPE
A

 

I I T 1 I r I I I I

12 l3 14 15 16 17 18 19 20 21 22

 

Time post-infusion, h

FIGURE 4-1d Enrichment of plasma free- and milk casein—bound methionine in the
sows fed the lysine-deficient diet. Lines with closed circles, open circles, and closed
squares represent the enrichments of plasma methionine in the carotid artery, plasma

methionine in the mammary vein, and casein-bound methionine in milk, respectively.

211

 

 

 

 

 

 

Methionine enrichment, MPE
&

 

 

I I I I I I I I I

I
12 13 14 15 l6 17 18 19 20 21 22

Time post-infusion, h

FIGURE 4-1e Enrichment of plasma free- and milk casein-bound methionine in the
sows fed the positive control diet. Lines with closed circles, open circles, and closed
squares represent the enrichments of plasma methionine in the carotid artery, plasma

methionine in the mammary vein, and casein-bound methionine in milk, respectively.

212

 

 

 

Methionine enrichment, MPE
A

 

 

I I I j 1 I I T I

I
12 13 14 15 16 1‘7 18 19 20 21 22

Time post-infusion, h

FIGURE 4-1f Enrichment of plasma free- and milk casein-bound methionine in the
sows fed the valine-excess diet. Lines with closed circles, open circles, and closed
squares represent the enrichments of plasma methionine in the carotid artery, plasma

methionine in the mammary vein, and casein-bound methionine in milk, respectively.

213

 

 

Valine enrichment, MPE
w

 

 

2 i

1 .

o I I I I I I I I I I
12 13 14 15 16 17 18 19 20 21 22

Time post-infusion, h

FIGURE 4-1g Enrichment of plasma free- and milk casein-bound valine in the sows fed
the lysine-deficient diet. Lines with closed circles, open circles, and closed squares
represent the enrichments of plasma valine in the carotid artery, plasma valine in the

mammary vein, and casein-bound valine in milk, respectively.

214

 

Valine enrichment, MPE
b)

 

 

I I I I j I I I I I

12 13 14 15 16 17 18 19 20 21 22

Time post-infusion, h

FIGURE 4-1h Enrichment of plasma free- and milk casein-bound valine in the sows fed
the positive control diet. Lines with closed circles, open circles, and closed squares
represent the enrichments of plasma valine in the carotid artery, plasma valine in the

mammary vein, and casein-bound valine in milk, respectively.

215

 

 

Valine enrichment, MPE
w

 

 

I I I I I I I I I I

12 13 14 15 16 17 18 19 20 21 22

Time post-infusion, h

FIGURE 4-1i Enrichment of plasma free- and milk casein-bound valine in the sows fed
the valine-excess diet. Lines with closed circles, open circles, and closed squares
represent the enrichments of plasma valine in the carotid artery, plasma valine in the

mammary vein, and casein-bound valine in milk, respectively.

216

Fa,o

PS. OX, OM
PB. DS

 

Fo,v

FIGURE 4‘2 A three-compartmental model of amino acid kinetics across the lactating porcine mammary
gland. Free amino acid compartments in artery (A), main mammary vein (V), and mammary tissue (MG)
are connected by arrows indicating unidirectional fluxes of free amino acids between each compartment.
Amino acids enter the mammary gland via the mammary artery (Fa,o) and leave the mammary gland via
the main mammary vein (Fo,v). Other fluxes are designated as follows: Fv,a, direct flow of amino acids
from artery to vein without entering intracellular fluid (by the arterial shunt); Fmg,a and Fv,mg, inward and
outward trans-membrane transport of amino acids from artery to the mammary tissue and from the
mammary tissue to vein, respectively; Fo,mg, the rate of intracellular amino acid appearance from
endogenous sources (i.e., release from protein breakdown (PB) and de novo synthesis (DS), if any); and
Fo,mg, the rate of the intracellular amino acids disappearance (i.e., the rate of utilization of intracellular

amino acids for protein synthesis (PS), oxidation (OX), and other metabolic fates (OM), if any).

217

CHAPTER 5

Summary and Conclusions

This dissertation presents data that provide new insight into amino acid nutrition of the
porcine mammary gland during lactation. The present study demonstrated that the
lactating porcine mammary gland has a large demand for IAA to meet high rate of milk
protein synthesis. The lactating sow adapts its physiological needs to the nutritional
environment at least on three levels: lactational homeorhesis of the whole body, fine
regulation of unidirectional trans-membrane transport across the mammary epithelium,

and intracellular control of protein turnover in the mammary gland.

The AA profile in milk is not affected by intake of dietary protein, which supports the
notion that modification of the amount of milk protein, rather than the AA profile of milk
protein, is the evolutionary mechanism used to meet the unique AA needs of mammalian
neonates (Davis et al. 1994a, Davis et al. 1994b). Intake of dietary protein regulates the
mammary AA uptake pattern. Interestingly, mammary uptake proportions of the BCAA
to the total IAA are up-regulated with increasing intake of dietary protein, implying an
increased capacity for the mammary gland to metabolize these AA. This is associated
with unique characteristics of intracellular BCAA metabolisms as follows. (1) The
BCAA can provide a-amino nitrogen and carbon for the synthesis of dispensable AA
(Wohlt et al. 1977); yield energy from their oxidation for high rate of protein turnover
and for the synthesis of milk non-protein compounds (e.g., fatty acids and lactose) (Davis

and Mepham 1976, Oddy et al. 1988, Roets et al. 1979, Vina and Williamson 1981);

218

and/or participate in regulation of protein breakdown or in interorgan partitioning of the
BCAA (e.g., possibly providing branched-chain a-keto acids for hepatic oxidation)
(DeSantiago et al. 1998, MacLean et al. 1994, Mitch and Clark 1984, Nair et al. 1992).
(2) The catabolism of the BCAA can be up-regulated to remove their excess (if any) after
high intake of dietary protein through increased expression of branched-chain
aminotransferase and increased activity of branched-chain OL-keto acid dehydrogenase
(Langer et al. 2000, Torres et al. 1998). In contrast, mammary uptake proportions of the
limiting AA (e.g., Lys, Met, Thr, Phe, and Trp) to the total IAA maintained at a relatively
high level under low intake of protein to meet high rate of milk synthesis. Therefore,
mammary AA uptake pattern is adaptively regulated by intake of dietary protein to

maintain synthesis of milk protein.

Lactational homeorhesis was described in the lactating animal to explain metabolism of
the extra-mammary tissues and partitioning of nutrients to the mammary gland to support
high rate of milk synthesis (Bauman 1999, Collier 1999). The lactating sow loses body
protein and/or lipid to support a high rate of milk production when intake of protein,
IAA, and/or energy are inadequate (Clowes et al. 1998, Jones and Stahly 1999, Revell et
al. 1998). Thus, we hypothesize that when intake of dietary IAA is extrapolated to zero,
the amount of mammary IAA uptake originates from the mobilization of body protein
(i.e., endogenous IAA contributions). Endogenous IAA contributions defined by the
mammary uptake are supported by two indirect estimations: (1) the algebraic product of
body protein loss (defined from nitrogen balance) times the AA composition of body

protein, and (2) the backward factorial approach-derived values. These estimates of

219

 

 

endogenous IAA contributions can be used to adjust the total dietary IAA requirements

of the lactating sow at different rates of body weight loss.

The factorial approach has been widely used to predict nutrient requirements of domestic
animals. The total dietary IAA requirement of the lactating sow encompasses individual
components (e.g., maintenance, milk synthesis, and body protein accretion or loss, if
any). The present study is the first to estimate dietary IAA needs for milk synthesis by
the porcine mammary gland. The estimates are higher than the values recommended by
the current NRC (1998), especially for Arg and Val. In the present study, dietary needs
of truly digestible IAA for milk synthesis were determined by the maximal mammary
uptake. This novel approach has remarkable advantages as follows. ( 1) Estimates
defined by the maximal mammary uptake are independent from any change in body
protein, in contrast to estimates of the total dietary IAA requirements determined by
conventional approaches. (2) Estimates defined by the maximal uptake account for
physiological needs for incorporation into milk protein and metabolism other than protein
synthesis (e.g., oxidation) in the mammary gland. In contrast, estimates based only on
output of protein in milk might be underestimated, especially for Arg and the BCAA. (3)
This novel approach can estimate dietary needs of multiple IAA concurrently; in contrast,
the conventional approaches can determine dietary requirement of a single IAA at one

time.

Finally, the present study demonstrated that trans-membrane transport, net uptake, and
intracellular kinetics of IAA in the porcine mammary gland during lactation are finely

regulated by dietary AA regime. Trans-membrane transport and net uptake of an

220

individual IAA (e.g., Lys) can be rate limiting for the synthesis of milk protein as
demonstrated in vitro (Mepham 1982). Moreover, kinetics properties of the intracellular
free IAA are coordinated with their cellular transport and intracellular metabolism. For
example, net uptake of lysine is inhibited by excess Val, which would result in a decrease
in protein synthesis due to decreased availability of the intracellular free Lys. However,
intracellular protein breakdown is decreased to a greater degree compared to protein
synthesis, which would result in a comparable net balance of protein in the mammary

gland.

In conclusion, the present study demonstrated that intake of dietary AA regulates trans-
membrane transport, net uptake, and intracellular kinetics of IAA in the porcine
mammary gland during lactation to meet high rate of milk protein synthesis. Moreover,
trans-membrane transport and net uptake of an IAA (e.g., Lys) is rate limiting for milk
synthesis in vivo. Endogenous IAA contributions (mobilized from body protein to the
mammary gland for milk synthesis) and dietary IAA needs for milk synthesis by the
mammary gland can be estimated by their mammary uptake. Endogenous contributions
are 10.40, 2.35, 5.88, 6.36, and 6.20 g/kg body weight loss, respectively, for Lys, Met,
Phe, Thr, and Val. Dietary needs of truly digestible indispensable AA for milk synthesis
by the mammary gland (at zero change in body protein) are 28.78, 10.04, 20.15, 6.92,
12.10, 13.91, 4.49, and 22.40 g/kg litter weight gain, respectively, for Arg, His, Lys, Met,
Phe, Thr, Trp, and Val. While the ideal dietary IAA pattern for milk synthesis based only

on the IAA profile of milk protein provides appropriate estimates in absence of

221

experimental data, it is underestimating for Arg and the BCAA. An optimal dietary ratio
of Val to Lys for milk synthesis is approximately at 1:1, which is higher than the ratio of
0.85:1 recommended by the current NRC (1998); however, an increased ratio of dietary

Val to Lys above 1:1 will not improve lactational performance.

LITERATURE CITED

Bauman, D. E. (1999) Bovine somatotropin and lactation: from basic science to
commercial application. Domest. Anim. Endocrinol. 17: 101-116.

Clowes, E. J ., Williams, I. H., Baracos, V. E., Pluske, J. R., Cegielski, A. C., Zak, L. J. &
Aheme, F. X. (1998) Feeding lactating primiparous sows to establish three
divergent metabolic states: II. Effect on nitrogen partitioning and skeletal muscle
composition. J. Anim. Sci. 76: 1154-1164.

Collier, R. J. (1999). Lactation, Nonhuman. In Encyclopedia of Reproduction (Knobil, E.
& Neill, J. D., eds), Vol. 4, pp. 973-978. 2 vols. Academic Press, New York.

Davis, S. R. & Mepham, T. B. (1976) Metabolism of L-(U-14C)valine, L-(U-
14C)leucine, L-(U-14C)histidine and L-(U-14C) phenylalanine by the isolated
perfused lactating guinea-pig mammary gland. Biochem. J. 156: 553-560.

Davis, T. A., Nguyen, H. V., Garcia Bravo, R., Fiorotto, M. L., Jackson, E. M., Lewis, D.
S., Lee, D. R. & Reeds, P. J. (1994a) Amino acid composition of human milk is
not unique. J. Nutr. 124: 1126-1132.

Davis, T. A., Nguyen, H. V., Garcia Bravo, R., Fiorotto, M. L., Jackson, E. M. & Reeds,
P. J. (1994b) Amino acid composition of the milk of some mammalian species
changes with stage of lactation. Br. J. Nutr. 72: 845-853.

DeSantiago, S., Torres, N., Suryawan, A., Tovar, A. R. & Hutson, S. M. (1998)
Regulation of branched-chain amino acid metabolism in the lactating rat. J. Nutr.
128:1165-1171.

Jones, D. B. & Stahly, T. S. (1999) Impact of amino acid nutrition during lactation on
body nutrient mobilization and milk nutrient output in primiparous sows. J. Anim.

222

T . Egon-'11

 

 

Sci. 77: 1513-1522.

Langer, S., Scislowski, P. W., Brown, D. S., Dewey, P. & Fuller, M. F. (2000)
Interactions among the branched-chain amino acids and their effects on
methionine utilization in growing pigs: effects on plasma amino- and keto-acid
concentrations and branched-chain keto-acid dehydrogenase activity. Br. J. Nutr.
83: 49-58.

MacLean, D. A., Graham, T. E. & Saltin, B. (1994) Branched-chain amino acids
augment ammonia metabolism while attenuating protein breakdown during
exercise. Am. J. Physiol. 267: E1010-1022.

Mepham, T. B. (1982) Amino acid utilization by lactating mammary gland. J. Dairy Sci.
65: 287-298.

Mitch, W. E. & Clark, A. S. (1984) Specificity of the effects of leucine and its
metabolites on protein degradation in skeletal muscle. Biochem. J. 222: 579-586.

Nair, K. S., Schwartz, R. G. & Welle, S. (1992) Leucine as a regulator of whole body
and skeletal muscle protein metabolism in humans. Am. J. Physiol. 263: E928-
934.

Oddy, V. H., Lindsay, D. B. & Fleet, I. R. (1988) Protein synthesis and degradation in
the mammary gland of lactating goats. J. Dairy Res. 55: 143-154.

Revell, D. K., Williams, I. H., Mullan, B. P., Ranford, J. L. & Smits, R. J. (1998) Body
composition at farrowing and nutrition during lactation affect the performance of
primiparous sows: 1. Voluntary feed intake, weight loss, and plasma metabolites.
J. Anim. Sci. 76: 1729-1737.

Roets, E., Massart Leen, A. M., Verbeke, R. & Peeters, G. (1979) Metabolism of [U-
14C; 2,3-3H]-L-valine by the isolated perfused goat udder. J. Dairy Res. 46: 47-
57.

Torres, N., Lopez, G., De Santiago, S., Hutson, S. M. & Tovar, A. R. (1998) Dietary
protein level regulates expression of the mitochondrial branched-chain
aminotransferase in rats. J. Nutr. 128: 1368-1375.

Vina, J. R. & Williamson, D. H. (1981) Effects of lactation on L-leucine metabolism in
the rat. Studies in vivo and in vitro. Biochem. J. 194: 941-947.

Wohlt, J. E., Clark, J. H., Derrig, R. G. & Davis, C. L. (1977) Valine, leucine, and
isoleucine metabolism by lactating bovine marmnary tissue. J. Dairy Sci. 60:
1875-1882.

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