The Effect of Dietary Amino Acid Balance on Nitrogen and Lysine Utilization in Lactating Sows

163 Pages·2016·1.69 MB·English

The Effect of Dietary Amino Acid Balance on
Nitrogen and Lysine Utilization in Lactating Sows

by
Lee-Anne Huber

A Thesis
presented to
The University of Guelph

In partial fulfilment of requirements
for the degree of
Doctor of Philosophy
in
Animal Biosciences

Guelph, Ontario, Canada

© Lee-Anne Huber, August, 2016
ABSTRACT

The Effect of Dietary Amino Acid Balance on Nitrogen and
Lysine Utilization in Lactating Sows

Lee-Anne Huber Advisor:
University of Guelph, 2016 Professor C.F.M. de Lange

Decreasing dietary crude protein (CP) concentration and increasing crystalline amino
acid (CAA) supplementation, in order to maintain constant daily intakes of Lys and other key
amino acids (AA), improves dietary AA balance. The effect of improved dietary AA balance on
lactation performance, nitrogen (N) and AA utilization efficiency, and AA fates must be
determined for precise lactating sow diet formulation and to meet specific production objectives.
Performance, N balance, mammary biopsy, and isotope tracer studies were conducted to
determine the effects of improving dietary AA balance on sow and litter lactation performance,
N and Lys utilization efficiencies for milk production, mRNA abundance of mammary AA (Lys)
transporters, and the partitioning of dietary AA between maternal and milk protein pools. Litter
growth rates and milk protein production increased with improved AA balance, at the expense of
maternal N retention, particularly in peak lactation. At identical Lys intakes, there was minimal
improvement in Lys utilization efficiency for milk production with improved dietary AA
balance, and there were no corresponding changes in the expression of mRNA for several Lys
transporters within the mammary gland. Whole-body protein turnover and tissue-specific
fractional rates of protein synthesis were not influenced by dietary AA balance. Feeding lactating
sows reduced CP diets with increased inclusion of CAA, to improve AA balance and to meet the
requirements of limiting AA, is a feasible way to improve the utilization of N and AA for milk
protein production and decrease N losses to the environment, without negatively impacting sow
and litter lactation performance. Amino acid and N requirements differ from those estimated by
the NRC (2012) model and among genotypes and parity of sows. The utilization efficiencies of
N and AA may change across a lactation period. All of these factors should be considered when
planning future research and formulating lactating sow diets.
Dedication
To Kees: my teacher, my leader, my inspiration.

iv
Acknowledgements
First and foremost, I would like to extend my deepest gratitude to Kees de Lange for his
inspiration and guidance throughout this research; it would have not been possible without him. I
would also like to thank the members of my committee John Cant and Brian McBride for their
insight on this thesis and for stepping up while our leader was hors de combat. Thanks also to
Nathalie Trottier for allowing me to conduct several studies under her supervision at Michigan
State University.
There is a long list of funding sponsors who made this research possible. Thank you to
the National Pork Board, the Michigan Animal Agriculture Initiative, Ontario Pork, Royal De
Heus, Heartland Lys Inc./Ajinomoto, the Ontario Ministry of Agriculture Food and Rural
Affairs, and the Natural Sciences and Engineering Research Council of Canada.
I would also like to thank a long list of graduate student colleagues for all of their
assistance in various aspects of these studies - milking sows, wrangling piglets, risking life and
limb during sow restraint, and for staying by my side during the 16-h sampling days and months
of tedious laboratory work: Jennifer De Vries and David Chamberlin from MSU, Uffe Krogh
from Aarhus University, and Emily Miller, Melissa Wiseman, Wilfredo Mansilla, Quincy Buis,
Heather Reinhardt, and especially Marko Rudar from the U of G. My summer students Jenna Lubitz
and Hannah Golightly, both of whom were stolen away by vet school, were also a tremendous help.
Doug Wey and Julia Zhu were quintessential in the development of the ear vein catheterization
technique and for keeping me sane during those stressful days. The staff at the Michigan State
University Swine Teaching and Research Center and Arkell Swine Research Station were
exceptionally valuable during these trials, especially Kevin Turner who went above and beyond
the call of duty on weekends and after-hours – and for giving me a place to live. The technical
support I received was phenomenal from Julie Moore, Dave Main, Jim Leisman, Nancy Raney
from MSU, as well as Drs. Robert Templeman and Margaret Quinton for statistical assistance,
Dr. Cathy Ernst for help with qPCR methodology and interpretation, Dr. Paul Luimes for
mentorship on the ear vein catheter technique, and Drs. Armen Carchoglyan and Dyanne Brewer
for assistance with the GC-MS analysis.
Last but not least, I would like to thank my parents, siblings, and friends for their
continuing support, as well as my husband, John Legault, for sticking by my side for the whole
journey – especially the 4 am sample collections – even though the only thing he likes about pigs
is their bacon.

v
List of abbreviations
4E-BP1 eIF4E-binding protein 1
4F2hc 4F2 cell-surface antigen heavy chain
AA Amino acid
ADFI Average daily feed intake
ADG Average daily gain
AUC Area under the Phe enrichment curve for casein
c
AUC Area under the Phe enrichment curve for plasma
p
ATB0,+ Neutral amino acid transporter B
B0,+AT b(0,+)-type amino acid transporter 1
BCAA Branched-chain amino acids
BW Body weight
B Phe released from whole-body protein breakdown
wb
C Candidate gene
C Threshold cycle
t
CAA Crystalline amino acid
CAT-1/2b High affinity cationic amino acid transporter 1/ 2b
CP Crude protein
DM Dry matter
E Phe enrichment in tissue protein-bound pool
b
E Phe enrichment in tissue free pool
f
E Predicted Phe enrichment at plateau in casein
maxc
E Predicted Phe enrichment at plateau in plasma
maxp
E Predicted Phe enrichment at time, t
p
EAA Essential amino acid
EDTA Ethylenediamine tetraacetic acid
eIF2 Eukaryotic initiation factor 2
eIF4E Eukaryotic initiation factor 4E
ER Endoplasmic reticulum
Exp Experiment
FSR Fractional synthesis rate
FSR Casein fractional synthesis rate
c
GC Gas chromatograph
GCN2 Non-depressible 2 protein kinase
GLUT Glucose transporter
HCP High crude protein
I Phe supplied by infusate
I
I Phe supplied by diet
F
IGF-1 Insulin-like growth factor 1
IGFBP Insulin-like growth factor binding protein
IM Intramuscularly
IRS1 Insulin receptor substrate 1
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ISR Integrated stress response
Jak/STAT Janus kinase/signal transducers and activators
k Rate constant
K Michaelis constant
m
KAA Amino acid utilization efficiency for milk protein production
L Linear
LCP Low crude protein
LEA Loin eye area
LGR Litter growth rate
MHCP Medium-high crude protein
MLCP Medium-low crude protein
MRPL39 Mitochondrial ribosomal protein L39
MS Mass spectrometer
mTORC1 Mammalian target of rapamycin complex 1
MUN Milk urea nitrogen
N Nitrogen
Na+/K+-ATPase Sodium/potassium adenosine triphosphate ion transporter
NEAA Non-essential amino acid
NEFA Non-esterified fatty acid
O Whole-body Phe oxidation
wb
P Probability
PDK1 3-phosphoinositide dependent protein kinase 1
PERK Protein kinase-like endoplasmic reticulum kinase
PI3K Phosphoinositide 3-kinase
PKB Protein kinase B
PPARα Peroxisome proliferator-activated receptor alpha
Q Quadratic
Q Whole-body Phe flux
wb
R Reference gene
RAG Ras-related GTP binding protein
rBAT Neutral and basic amino acid transport protein rBAT
Rheb Ras homolog enrichment in brain
RSP23 Ribosomal protein 23
S Whole-body use of Phe for protein synthesis
wb
SEM Standard error of the mean
SID Standardize ileal digestible
SUN Serum urea nitrogen
t Time
UCP Uncoupling protein
V Maximal transport velocity
max
VAPB Vesicle associated membrane protein-associated protein B/C
y+LAT1/2 y+L amino acid transporter 1/2

vii
List of tables
Table Page
Key amino acid transport systems in lactating mammary cells and their
1.1 physiological substrates……………………………………………………… 15

Ingredient composition and nutrient content of experimental diets (as-
3.1 fed)……………………………………………………………………………. 49

Sow and litter performance in experiment 1over a 22-d lactation period…….
3.2 51

Sow and litter growth performance in experiment 2 for sows fed high CP
3.3 (16.0 %) or reduced CP diets over a 21-d lactation period…………………... 52

Milk composition from sows fed high CP (16.0 %) or reduced CP diets
between d 3 and 7 of lactation (early lactation) and between d 14 and 18 of
3.4 53
lactation (peak lactation)…………………………………………………….

Nitrogen utilization in sows fed high CP (16.0%) or reduced CP diets
between d 3 and 7 of lactation (early lactation) and between d 14 and 18 of
3.5 54
lactation (peak lactation)……………………………………………………...

Serum urea nitrogen concentration in sows fed high CP (16.0 %) or reduced
3.6 CP diets during lactation……………………………………………………... 56

Ingredient composition and nutrient content of experimental diets used in
4.1 experiment 2 (as-fed)…………………………………………………………. 76

Assay information (TaqMan® Gene Expression Assays) for candidate and
4.2 reference genes……………………………………………………………….. 78

Amino acid utilization efficiency for milk protein production in sows fed
high CP (16.0 %) or reduced CP diets between d 3 and 7 of lactation (early
4.3 lactation) and between d 14 and 18 of lactation (peak 79
lactation)……………………………………………………………………….

Post-absorptive serum concentration of essential, non-essential and selected
4.4 metabolites for sows fed high CP (16.0%) or reduced CP diets……………… 81

Overall 21-d lactation performance for sows fed high or low CP diets in
4.5 experiment 2………………………………………………………………….. 82

5.1 Ingredient composition and nutrient content of experimental diets (as-fed)…. 107
Gilt and litter growth performance for gilts fed diets varying in protein
5.2 109
content and similar amounts of Lys over a 17-d lactation period…..................
Milk composition on d 16 of lactation for gilts fed diets varying in protein
5.3 110
content and similar amounts of Lys……………………………………..…….
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Nitrogen utilization between d 13 and 17 of lactation (peak lactation) for
5.4 111
gilts fed diets varying in protein content and similar amounts of Lys……..….
Fractional rate constants for plasma and milk casein Phe labelling, predicted
plateaux Phe enrichment of observed and adjusted plasma and casein ,
5.5 estimated time that 90 % of plateau was reached for both pools, and mean 112
isotopic enrichment of L- Phenylalanine in free pools on d 17 of lactation for
gilts fed diets varying in protein content and similar amounts of Lys………...
Aspects of whole-body Phe flux and liver weight on d 17 of lactation for
5.6 gilts fed diets varying in protein content and supplying similar amounts of 113
Lys……………………………………………………………………………..
Mean isotopic enrichment of L- Phenylalanine in free and bound pools and
fractional synthesis rate of protein in select tissues on d 17 of lactation for
5.7 114
gilts fed diets varying in protein content and supplying similar amounts of
Lys……………………………………………………………………………..
Brief summary of N utilization in parity 2+ sows and gilts in peak lactation;
6.1 127
means across dietary treatments……………………………………………….
Plasma concentrations of essential and non-essential AA on d 17 of lactation
A1 146
for lactating gilts fed high or low CP diets……………………………………

ix
List of figures
Figure Page
Simplified model demonstrating the major maternal protein pools that
contribute free AA for milk protein synthesis and how free AA are used
1.1 5
within mammary epithelial cells…………………………………………….

Visual representation of major transport systems in mammary epithelial
1.2 cells………………………………………………………………………….. 16

Post-absorptive serum concentration of Lys, Thr, His, Val, Phe, and Tyr for
sows fed high CP (16.0%) or reduced CP diets over a 21-day lactation
4.1 83
period………………………………………………………………………...

The mRNA abundance presented as fold change for genes encoding Lys
transporters in mammary tissue of lactating sows fed high CP (17.6 % CP)
4.2 85
diets relative to sows fed low CP (14.6 % CP) diets………………………..

The mRNA abundance presented as fold change for SLC6A14 mRNA
encoding the ATB+,0 transporter protein on d 14 relative to d 4 of lactation
(panel A) and SLC7A6 mRNA encoding the Y+LAT2 transporter protein
4.3 86
for sows fed high CP (17.6 % CP) diets relative to sows fed low CP (14.6
% CP) diets on d 4 and d 14 of lactation (panel B)………………………….

Temporal changes in the jugular adjusted plasma (Panel A) and casein-
5.1 bound (Panel B) isotopic enrichment of Phe on d 17 of lactation for gilts 115
fed HCP (16.0 % CP) and LCP diets (12.7 % CP)…………………………..
Schematic representing the flow of nitrogen into and out of the maternal
5.2 and milk protein pools, as well as, nitrogen intake from the diet and urinary 116
nitrogen losses for HCP-fed gilts on d 17 of lactation………………………
Schematic representing the flow of nitrogen into and out of the maternal
5.3 and milk protein pools, as well as, nitrogen intake from the diet and urinary 117
nitrogen losses for LCP-fed gilts on d 17 of lactation……………………….
Temporal changes in the jugular adjusted plasma isotopic enrichment of
A1 147
Phe on d 17 of lactation for individual gilts fed LCP diets (12.7 % CP)……
Temporal changes in the jugular adjusted plasma isotopic enrichment of
A2 149
Phe on d 17 of lactation for individual gilts fed HCP diets (16.0 % CP)……

x