Investigation of Methionine and Lysine Derivatives as a Source of Rumen-Protected Amino Acids for Lactating Dairy Cows

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Investigation of Methionine and Lysine Derivatives as a Source of Investigation of Methionine and Lysine Derivatives as a Source of

Rumen-Protected Amino Acids for Lactating Dairy Cows Rumen-Protected Amino Acids for Lactating Dairy Cows

Mark Avila Fagundes

Utah State University

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INVESTIGATION OF METHIONINE AND LYSINE DERIVATIVES AS A SOURCE

OF RUMEN-PROTECTED AMINO ACIDS FOR LACTATING DAIRY COWS

Mark Avila Fagundes

A dissertation submitted in partial fulfillment

of requirements for the degree

DOCTOR OF PHILOSOPHY

Animal, Dairy, and Veterinary Sciences

Approved:

______________________ ____________________

Jeffery O. Hall, D.V.M., Ph.D. Jong-Su Eun, Ph.D.

Major Professor Committee Member

______________________ ____________________

Kerry A. Rood, D.V.M., M.P.H., M.S. Allen J. Young, Ph.D.

Committee Member Committee Member

______________________ ____________________

Juan J. Villalba, Ph.D. Dirk K. Vanderwall, D.V.M., Ph.D.

Committee Member Department Head

_______________________________________

D. Richard Cutler, Ph.D.

Interim Vice Provost of Graduate Studies

UTAH STATE UNIVERSITY

Logan, Utah

2021

iii

ABSTRACT

Investigation of Methionine and Lysine Derivatives as a Source of Rumen-Protected

Amino Acids for Lactating Dairy Cows

Mark Avila Fagundes, Doctor of Philosophy

Utah State University, 2021

Major Professor: Dr. Jeffery O. Hall

Department: Animal, Dairy, and Veterinary Sciences

The N-acetyl amino acids, such as N-acetyl-L-methionine (NALM), ƐN-acetyl-L-

lysine, and Nα, ε-acetyl-L-Lysine are amino acid (AA) derivatives and developmental

forms of rumen-protected (RP) AAs for dairy cows (CJ CheilJedang). The AA derivates

are produced via N-acetylation of the L-Met α-amino, L-Lys α-amino or ε-amino group,

respectively. Three independent studies were conducted to investigate the use of Met and

Lys acetylation derivatives as sources of RPAAs for lactating dairy cows. The initial

study investigated production responses and ruminal fermentation characteristics of

lactating dairy cows when supplemented with NALM as a source of RPMet in

metabolizable protein (MP) -deficient or MP-adequate diets. Supplementation of NALM

increased milk fat concentration and yield and 3.5% fat-corrected milk (FCM) yield and

tended to increase energy-corrected milk (ECM) yield regardless of MP difference.

Dietary treatments had similar effects on ruminal fermentation characteristics and

microbial protein yield. Results from the first study suggest that NALM exerted minor

influence on ruminal metabolism, but increased milk fat concentration, resulting in

increases in milk fat yield and feed efficiency.

The subsequent study investigated lactational performance of dairy cows when

supplemented with N-acetyl-L-Lysine (NALL), N, -acetyl-L-Lysine (diNALL), or

AjiPro



-L (AP) as a source of RPLys to a control diet. Feeding the diNALL and AP diets

decreased dry matter intake. Stage of lactation may have altered DMI in cows fed the

diNALL and AP diets because energy requirements decrease during mid-lactation and

control of feed intake is regulated by hepatic oxidation. Milk yield was not altered by

RPLys supplementation. Findings from the study indicate that supplementing N, -

acetyl-L-Lysine in mid to late lactation dairy cows decreases DMI and maintained milk

yield leading to improved milk production efficiency (milk yield/DMI).

The final study evaluated the bioavailability (BA) and lactational performance of

lactating dairy cows supplemented with NALM at two different supplementation rates.

Overall, production parameters were not influenced by NALM supplementation.

Bioavailability, as assessed by plasma appearance of Met was higher for the 30g NALM

dose compared with 60g NALM and control, suggesting that optimal supplementation

rate for NALM is 30 g/cow/d. Residue of NALM was not detected in plasma, milk, liver

or muscle samples. Overall, findings suggest that NALM is deacetylated before reaching

central circulation, increases plasma Met concentrations, therefore supplying lactating

dairy cows with a BA source of RPMet.

(208 pages)

PUBLIC ABSTRACT

Investigation of Methionine and Lysine Derivatives as a Source of Rumen-Protected

Amino Acids for Lactating Dairy Cows

Mark Avila Fagundes

Cows have a protein requirement for growth, maintenance, and lactation. In order to

meet those protein requirements, dairy farmers can supplement or feed cattle with

specific amino acids, the building blocks that make-up protein. However, in order for the

amino acid product to be effective it must avoid degradation in the rumen and be

delivered in the small intestine for absorption. Lysine and methionine have traditionally

been recognized as the most limiting amino acids for lactating dairy cows. Therefore,

nutrition companies have focused on finding ways to encapsulate or protect lysine and

methionine from rumen microbes. The N-acetyl-L-methionine and N-acetyl-L-lysine are

amino acid derivatives and developmental forms of amino acids for dairy cows. Three

separate nutrition studies were conducted to investigate the use of lysine and methionine

acetylated derivatives as sources of amino acids for lactating dairy cows. The initial study

investigated milk production responses of lactating dairy cows when supplemented with

N-acetyl-L-methionine. Supplementation of N-acetyl-L-methionine increased milk fat

concentration and yield and 3.5% fat-corrected milk yield. Results from the first study

suggest that N-acetyl-L-methionine did not impact rumen metabolism, but increased milk

fat concentration by providing methyl donors to the mammary gland, resulting in

increases in milk fat yield. The second study investigated lactational performance of

dairy cows when supplemented with N-acetyl-L-lysine, N, -acetyl-L-lysine, or

AjiPro



-L as a source of rumen-protected lysine. Feeding the N, -acetyl-L-lysine and

AjiPro



-L diets decreased dry matter intake. Milk yield was not altered by rumen-

protected lysine supplementation. Findings from the study suggest that supplementing

N, -acetyl-L-lysine to dairy cows decreases dry matter intake, but maintained milk

yield leading to improved milk production efficiency. The final study evaluated the

bioavailability and lactational performance of dairy cows supplemented with N-acetyl-L-

methionine. Overall, milk production parameters were not affected by N-acetyl-L-

methionine supplementation. Bioavailability was higher for diets with N-acetyl-L-

methionine compared with the control diet. Residue of N-acetyl-L-methionine was not

detected in plasma, milk, liver, or muscle samples. Overall, findings suggest that N-

acetyl-L-methionine is deacetylated and absorbed as methionine into the bloodstream of

cows, supplying lactating dairy cows with a bioavailable source of rumen-protected

methionine.

vii

ACKNOWLEDGMENTS

First, I would like to thank all the employees of the Caine Dairy Research Center,

veterinary staff of the USU Clinical Veterinary Services and students in the ruminant

nutrition lab for aiding in sample collections, maintaining animal health, and feeding of

experimental animals. A special thanks is given to both the USU School of Veterinary

Medicine and CJ Bio for financial support. Upmost gratitude is expressed to all

committee members for accepting the task to direct me through graduate school at USU. I

appreciate the time given by Dr. Rood, Young, and Villalba attending committee

meetings and mentoring me in your areas of expertise. Dr. Hall, I am grateful for your

willingness to take me on as your graduate student, partway through my program, which I

know has not always been the most convenient with your busy schedule. To this day your

mineral course is one of the best I have taken in my academic career and I have enjoyed

our discussions of veterinary medicine. Dr. Eun, I am especially appreciative that you

accepted me as your graduate student. Over the last few years, you have not only

provided mentorship, but you have become a great friend. Although, you are over 5,000

miles away that doesn’t prevent you from having an active role in my graduate program.

This willingness speaks to your passion, thoughtfulness, and leadership in advising

students to help them succeed. Lastly, I would like to give a special thanks to my wife,

friends, and family members who have supported me throughout this process.

Mark Avila Fagundes

viii

CONTENTS

Page

ABSTRACT……………………………………………………………………………...iii

PUBLIC ABSTRACT………………………………………………………………….....v

ACKNOWLEDGMENTS………………………………………………………….…....vii

LIST OF TABLES………………………………………………………………………..xi

LIST OF FIGURES……………………………………………………………………..xiii

LIST OF ABBREVIATIONS………………………………….………………………..xiv

INTRODUCTION……………………………………………………………………..….1

REVIEW OF LITERATURE…………………………………………………………......3

AA…………….………………………………………………………………..….3

AA Nutrition in Dairy Cows………………….……………….……..…....3

Structural Configuration and Biological Functions of Met and Lys………7

RPMET and RPLYS..…………………………………………………………......9

RPMet Supplements………………………………………………….…...9

RPLys Supplements………………..…………………………………….14

Effects on Intake………………………………………………..………..17

Effects on Milk Yield and Composition………...…………........…….....20

Effects on Immunity………………………...……………………….…..26

INTESTINAL ABSORPTION OF AA…...……………………………………..30

MET METABOLISM……………………………………...…...…………….....32

MAMMARY GLAND AA METABOLISM………………….....……………...34

Lys….……………………..……………………………...……………...34

Met……………………………………………………………………….35

MAMMALIAN TARGET OF RAPAMYCIN (mTOR) PATHWAY……….….36

BA…………………..……………………………………………………………37

Current Approaches and Limitations………………………...………......37

RPMet……………………………………………………………………39

RPLys………………...…………………………………………………..41

CONCLUSIONS………………………..............……………………………….41

REFERENCES…………………………………………………………………..43

CHAPTER 1. INFLUENCE OF SUPPLEMENTING A METHIONINE

DERIVATIVE, N-ACETYL-L-METHIONINE IN DAIRY DIETS ON

PRODUCTION AND RUMINAL FERMENTATION BY LACTATING

COWS DURING EARLY TO MID-LACTATION……..………………………65

ABSTRACT……………………………………………………………………...65

INTRODUCTION……………………………………………………………….66

MATERIALS AND METHODS…………………………………………...…....68

RESULTS AND DISCUSSION……..…………………………….………….....75

CONCLUSIONS…………………………………………………………….…..83

ACKNOWLEDGMENTS…………………………...…………………………..84

REFERENCES…………….……………………………………...……………..84

CHAPTER 2. EFFECTS OF SUPPLEMENTING THREE DIFFERENT

FORMS OF RUMEN-PROTECTED LYSINE ON LACTATIONAL

PERFORMANCE OF MID TO LATE LACTATION DAIRY COWS...……..104

ABSTRACT……………………………………………………………………104

INTRODUCTION……………………………………………..……………….105

MATERIALS AND METHODS…………………………………………….…107

RESULTS AND DISCUSSION…………………………..……………………113

CONCLUSIONS….…………………………………………………………....121

ACKNOWLEDGMENTS…………………………………….…………...…...122

REFERENCES…………………………………...……..…………………...…122

CHAPTER 3. BIOAVAILABILITY AND LACTATIONAL

PERFORMANCE OF DAIRY COWS SUPPLEMENTED

WITH N-ACETYL-L-METHIONINE AT DIFFERENT DOSES………...…..140

ABSTRACT…………………………………………………………………….140

INTRODUCTION……………………………………………..……………….141

MATERIALS AND METHODS…………………………………………….…144

RESULTS AND DISCUSSION…………………………..……………………151

CONCLUSIONS….…………………………………………………………....161

ACKNOWLEDGMENTS…………………………………….…………...…...162

REFERENCES…………………………………...……..……………………...163

CONCLUSIONS……………………………………………………………………….182

REFERENCES…………………………………...……..……………………...186

CURRICULUM VITAE……………………………………………………………….188

LIST OF TABLES

Table Page

1-1. Ingredient and chemical composition of the experimental diets without

or with N-acetyl-L-Met (NALM) at 2 different levels of MP fed to

lactating Holstein dairy cows (n = 4)…………………………….………….…..94

1-2. Intake, milk production, feed efficiency, and net energy utilization of

lactating Holstein dairy cows supplemented without or with

N-acetyl-L-Met (NALM) at 2 different levels of MP……………………………97

1-3. Plasma AA concentrations (µmol/L) of lactating Holstein dairy cows

supplemented without or with N-acetyl-L-Met (NALM) at 2 different

levels of MP …………..........................................................................................99

1-4. Ruminal fermentation characteristics of lactating Holstein dairy cows

supplemented without or with N-acetyl-L-Met (NALM) at 2 different

levels of MP………………………………………………..…………………...101

2-1. Ingredient and chemical composition of the experimental diets

supplemented with rumen-protected lysine fed to lactating Holstein dairy

cows (n = 40)………...........................................................................................129

2-2. Dry matter intake, productive performance, body weight (BW) change,

and net energy utilization of mid to late lactation Holstein dairy cows

supplemented with rumen-protected lysine…..………………………………...132

2-3. Plasma AA concentrations (µmol/L) of mid to late lactation Holstein dairy

cows supplemented with rumen-protected lysine…………...………………….135

2-4. Utilization of N by mid to late lactation Holstein dairy cows supplemented

with rumen-protected lysine……………………………………………………137

2-5. Ruminal fermentation characteristics of mid to late lactation Holstein dairy

cows supplemented with rumen-protected lysine………………………...….....139

3-1. Ingredients and chemical composition of the basal diet……………………..…170

3-2. Energy and protein supply and AA balance of the basal diet supplemented

with N-acetyl-L-Met (NALM) at 2 different doses…………………...……..…172

xii

3-3. Intake, milk production, and feed efficiency of lactating Holstein dairy

cows supplemented with N-acetyl-L-Met (NALM) at 2 different doses…….....174

3-4. Plasma AA concentrations (µmol/L) of lactating Holstein dairy cows

supplemented with N-acetyl-L-Met (NALM) at 2 different doses……..……....176

3-5. Determination of bioavailability and kinetic parameters in lactating Holstein

dairy cows supplemented with N-acetyl-L-Met (NALM) at 2 different

doses…………………………………………………………………………….178

3-6. Residue potential in lactating Holstein dairy cows supplemented with

N-acetyl-L Met (NALM) at 2 different doses………………………………….180

xiii

LIST OF FIGURES

Figure Page

1. Biological functions of Met and hepatic Met metabolism. BHMT =

Betaine-homocysteine methyltransferase; CBS = cystathionine-β-synthase;

CSE = cystathionase; DMG = dimethylglycine; GSH = glutathione;

GNMT = glycine N-methyltransferase; HCY = homocysteine; MAT1A =

Met adenosyltransferase 1A; MTR = Met reductase; MT =

methyltransferase; 5-MTHF = 5-methyltetrahydrofolate; SAH = S-

adenosylhomocysteine; SAHH = SAH hydrolase; SAM = S-adenosyl-

Met and THF = tetrahydrofolate. Adapted from Zhang and White, (2017)……..64

1-1. Proposed mechanism of N-acetyl-L-Met (NALM) on its potential effects

on milk fat and milk production. SAM = S-adenosyl-Met; VLDL = very

low-density lipoprotein (Nelson and Cox, 2013)………………………….…....103

3-1. Plasma Met concentration (µM) versus time plot for lactating Holstein

dairy cows abomasally infused with N-acetyl-L-Met (NALM) at 2

different supplementation doses……………………….……………………….181

xiv

LIST OF ABBREVIATIONS

AA = amino acid

ADF = acid detergent fiber

AP = AjiPro



-L

AUC = area under the curve

BA = bioavailability

BCAA = branched chain amino acids

BW = body weight

CL = control

CNCPS = cornell net carbohydrate and protein system

CP = crude protein

DIM = days in milk

diNALL = Nα, Ɛ-acetyl-L-lysine

dLys = digestible lysine

DM = dry matter

dMet = digestible methionine

DMI = dry matter intake

EAA = essential amino acid

ECM = energy corrected milk

εNALL = ƐN-acetyl-L-lysine

FCM = fat corrected milk

His = histidine

HMB = DL-2-hydroxy-4-(methylthio)-butanoic acid

HMTBa = DL-2-hydroxy-4-(methylthio)-butanoic acid

HMBi = isopropyl ester of 2-hydroxy-4-(methylthio)-butanoic acid

Ile = isoleucine

KMB = 2-keto-4-(methylthio) butanoic acid

Leu = leucine

LFI = liver functionality index

LPS = lipopolysaccharide

Lys = lysine

MCP = microbial crude protein

ME = metabolizable energy

Met = methionine

MHA = methionine hydroxy analogue

MP = metabolizable protein

MPA = metabolizable protein adequate

MPD = metabolizable protein deficient

mTOR = mammalian target of rapamycin

MUN = milk urea nitrogen

N = nitrogen

NALL = N-acetyl-L-lysine

NALM = N-acetyl-L-methionine

xvi

NAM = N-acetyl-DL-methionine

NDF = neutral detergent fiber

NEAA = non-essential amino acids

NEFA = non-esterified fatty acid

= net energy for lactation

NFC = non fiber carbohydrates

-N = ammonia nitrogen

NRC = national research council

OM = organic matter

PD = purine derivatives

PMNL = polymorphonuclear leukocytes

RDP = rumen degradable protein

RP = rumen-protected

RPAA = rumen-protected amino acid

RPLys = rumen-protected lysine

RPMet = rumen-protected methionine

RUP = rumen undegradable protein

SAM = S-adenosyl-methionine

TMR = total mixed rations

Val = valine

VFA = volatile fatty acids

VLDL = very low-density lipoproteins

INTRODUCTION

Lysine (Lys) and methionine (Met) have traditionally been recognized as the most limiting

amino acids (AA) for lactating dairy cows fed corn-based diets (Schwab et al., 1992; NRC,

2001). Protein nutrition of dairy cows in recent years has shifted from the use of dietary crude

protein (CP) towards meeting the ammonia and AA needs for ruminal fermentation in order to

maximize microbial protein (MCP) synthesis (Schwab and Broderick, 2017). Another nutritional

shift seen recently is the balancing of individual AA in metabolizable protein (MP) as a method

to deliver bioavailable AAs to the small intestine. However, in order for an AA product to reach

the small intestine for absorption, it must be protected from rumen microbial degradation.

Protection of free AAs from ruminal degradation dates to the 1960s (Schwab and Broderick,

2017) and numerous rumen-protected AA (RPAA) products, have been developed using a

variety of technologies. In terms of Met, encapsulation of a Met molecule with coating materials,

such as carbohydrates or polymer allows RPAA products to avoid ruminal degradation (Schwab

and Ordway, 2003). Altering the structural configuration of AAs to avoid microbial degradation

is another method to escape rumen degradation. Methionine analogues utilize a hydroxyl group

to protect them from ruminal degradation and are converted to useable Met by enzymatic

reactions (Schwab and Ordway, 2003). Rumen-protected Lys (RPLys) products are often coated

by a series of lipid or fatty acid calcium-based salts (Ji et al., 2016). Encapsulated rumen-

protected Met (RPMet) and Met analogues can impact milk yield, but their effects tend to be

more prominent in altering milk fat and protein composition (Patton, 2010; Zanton et al., 2014).

Encapsulated RPLys supplementation has minimal impact on production parameters, unless

RPLys is supplemented with a RPMet product (Robinson, 2010). Lactational responses to both

supplemental RPMet and RPLys in the literature are inconsistent and require further research.

Inconsistent findings may be related to AA protection methods, varying supplementation rates,

or metabolizable protein concentrations in the diets.

The N-acetyl-L-Met (NALM), ƐN-acetyl-L-Lys (εNALL), and Nα, ε-acetyl-L-Lys

(diNALL) are AA derivatives and developmental forms of RPAAs for lactating dairy cows.

Based on prior research, it is thought that the acetyl group acts as a barrier to block the

hydrolysis of the AA N-terminal (Wallace, 1992). However, to our knowledge there is limited

production and bioavailability data available on the supplementation of acetylated AA

derivatives to lactating dairy cows (Amaro et al., 2019; Digenis et al., 1974; Liang et al., 2019;

Windschitl and Stern 1988).

Therefore, a series of lactation studies were performed to investigate the use of acetylated

Met and Lys derivatives as a source of RPAAs for lactating dairy cows. Three independent

studies were conducted, and it was hypothesized that; for study 1) increased Met from NALM

would increase milk protein synthesis and milk yield, but the degree of the response would be

dependent upon the MP supply in the diets; for study 2) increased post-ruminal Lys from

developmental NALL products would increase milk protein synthesis and milk yield compared

with the control diet and for study 3) Met from abomasal infusion of NALM would increase

plasma Met concentration, and the bioavailability of NALM would be greater for each dose

compared with the control leading to a dose response, while lactational performance would be

similar across all treatments due to the short study length.

REVIEW OF LITERATURE

Methionine and Lys are recognized as the 2 most limiting AAs for milk protein synthesis and

milk production in lactating dairy cows. Individual or combined supplementation of RPAAs in

ruminant diets is not a new concept, but in recent years has become more popular due to the

beneficial effects of RPLys and RPMet on lactational performance (Robinson, 2010; Zanton et

al., 2014). Supplementation of RPMet can improve milk fat (Overton et al., 1998; Samuelson et

al., 2001) and protein yield (Rulquin and Delaby, 1997; Patton, 2010), while RPLys tends to

exert its beneficial effects on milk protein yields (Robinson, 2010). As the dairy industry

continues to find solutions to reduce environmental impact, nitrogen (N) efficiency can be

improved by reducing protein concentrations in ruminant diets with strategic supplementation of

RPAAs. For these reasons, feeding RPAAs has gained popularity in the dairy industry. This

literature review will cover the common RPLys and RPMet products available for use in

ruminants, as well as their effects on feed intake, milk and milk component production,

immunity, metabolism, intestinal absorption of AA, mammary gland AA metabolism, and

bioavailability. Main focus will be placed on encapsulated RPAAs, Met analogues, and AA

derivatives, specifically NALM and N-acetyl-L-Lys (NALL).

AA Nutrition in Dairy Cows

In recent decades, significant progress has been made in understanding the AA nutrition of

dairy cows. Ruminal protein metabolism and the discovery of bacterial protein synthesis have

been comprehensively researched (Wegner et al., 1940). Dairy cows can utilize nonprotein N

(e.g., urea) and free ammonia from ruminal AA degradation for MCP synthesis. Ruminal MCP is

the major form of MP supplied to (50-80%) and absorbed from the small intestine of dairy cows

(Storm and Ørskov, 1983). Microbial protein is considered a high-quality protein for dairy cows

because of its high digestibility and favorable AA composition (Schwab and Broderick, 2017).

However, MCP synthesis requires large amounts of energy with the efficiency of energetic

transformations for protein synthesis from AA being less than 75% (Wu, 2013). Therefore, much

attention has been placed on the development of RPAAs to escape rumen degradation and supply

high-quality, systemically available AAs in ruminant diets.

Historically, AA dietary formulations for dairy cows have been based on the single-limiting

AA theory (Mitchell and Block, 1946), which was adapted from the law of the minimum

proposed by von Lieberg (1863; Liu et al., 2017). Recently, the single-limiting AA theory has

been refuted (Appuhamy et al., 2012; Liu et al., 2017). A revised conceptual framework with the

NRC (2001) model that represents the individual and additive effects of all 10 essential AA

(EAA) is believed to improve model accuracy compared with the single-limiting AA model in

ruminants (Liu et al., 2017). Thus, when a ration is formulated based on the single-limiting AA

theory, the additive effects of EAA for lactational AA requirements are ignored, and production

responses may be altered. Balancing rations for EAA requirements of dairy cows and for

multiple-limiting AAs may help to reduce overfeeding of protein, improve post-ruminal AA

absorptive efficiency, reduce N excretion, and improve lactational performance (Liu et al.,

2017).

Of the 20 primary AAs that occur in proteins, 10 are classified as being ‘‘essential’’ (or

indispensable) (NRC, 2001). Essential AAs cannot be synthesized by animal tissues or if they

can (arginine and histidine), not at rates sufficient to meet requirements at early stages of growth

or for high levels of production (NRC, 2001). Lysine and Met have been identified as the 2 most-

limiting EAAs for lactating dairy cows, with current evidence suggesting that histidine (His)

may be limiting in grass silage-based diets (Vanhatalo et al., 1999; Giallongo et al., 2016). Since

His concentrations are lower in ruminal MCP compared with milk protein and common

feedstuffs (NRC, 2001), His may be a limiting EAA for lactating cows fed MP-deficient diets.

This is of most significance when MCP provides or encompasses most of the MP supplied to

ruminants (Patton et al., 2014; Giallongo et al., 2016).

Branched chain amino acids (BCAA) are used by the mammary gland for cellular, milk

protein synthesis and comprise 50% of the EAA in milk proteins (Mackle et al., 1999). It has

been suggested that BCAAs may be as limiting as Met and Lys in high producing dairy cows

(Appuhamy et al., 2011), because of obligate use of BCAAs (isoleucine (Ile), leucine (Leu), and

valine (Val)) by intestinal tissue as energy sources. A meta-analysis was performed (Lean et al.,

2018) to predict the effects of metabolizable AAs on dairy cattle performance. The researchers

found a positive association of Cornell Net Carbohydrate and Protein System estimated

metabolizable Leu with milk protein and milk yield responses, suggesting that Leu should be

given greater consideration as a potential co-limiting AA (Lean et al., 2018). However, the

effects of BCAA supplementation on dairy cow production is varying and further evidence is

needed to determine if BCAAs should be considered as limiting AAs in dairy diets for

incorporation into a multiple-limiting AA model.

Methionine and Lys are the most researched AAs in regards to dairy cattle nutrition. They are

considered the most limiting EAA for the synthesis of milk and milk protein in the high-

producing dairy cow (Schwab et al., 1992; Rulquin et al., 1993). Methionine is one of 4 sulfur-

containing AAs and along with cysteine is proteogenic (Brosnan and Bronson, 2006). In

ruminants, Met is not only used for protein synthesis, but serves multiple biological roles (Figure

1). Milk protein synthesis by lactating dairy cattle can be altered if Met is in the shortest dietary

supply of available dietary AAs requirements based on the single-limiting AA theory (Mitchell

and Block, 1946). Methionine functions as a methyl donor and precursor to antioxidant and

lipotropic compounds including carnitine, choline, creatine, cysteine, glutathione,

metallothionine, taurine and S-adenosyl methionine (SAM) (Lehninger, 1977; Seymour, 2016;

Figure 1).

Previously, research has focused on the role of Met as a co-limiting AA for milk protein and

fat synthesis, and maintaining metabolic balance (Polan et al., 1991; Seymour, 2016). Recently,

the metabolic functions of Met such as its role in supporting liver function, oxidative balance,

and immunity have been assessed (Osorio et al., 2013). Methionine deficiency in non-ruminants

produces fatty liver disease, a reduction in cellular antioxidants, reduced methyl donors, and

generalized hepatic inflammation and fibrosis (Schugar and Crawford, 2012). Unlike the

variability of Met’s biological functions, the predominant biological function of Lys is protein

synthesis (milk protein, growth, pregnancy, and maintenance) (NRC, 2001). It is evident that

Met and Lys are essential for milk and milk component production and serve many biological

roles. However, the chemical form of AA fed to dairy cattle can alter the absorption, utilization

efficiency, and subsequent production response to AA supplementation.

Structural Configuration and Biological Functions of Met and Lys

Methionine and all protein AAs, except glycine, can have both D- and L-isomers. L-AAs are

physiological isomers in organisms and account for greater than 99.98% of the total AAs in the

body (Wu, 2013). In dairy cows, the D-isomer is not bioavailable (Lapierre et al., 2012). The D-

isomer must be converted to the L-isomer before it can be incorporated into mammalian proteins.

Supplementation of the L-isomer of Met can be utilized directly for protein synthesis without

conversion to any other isoforms. Since chemical synthesis of AAs results in a racemic mixture

of the D- and L-enantiomers of Met, the bioavailability (BA) of the D- isomer of Met in dairy

cows depends on the rate of transformation into the L-isomer (Lapierre et al., 2012).

For ruminants, BA data of D-Met are limited and somewhat unclear. In small ruminants,

lower utilization of D-Met than L-Met has been described (Doyle, 1981), while the utilization of

D-Met and L-Met appear to be equally effective for supporting wool growth (Doyle, 1981; Reis

et al., 1989). In growing steers, D- and L-Met produced similar increases in N retention

(Campbell et al., 1996), although this tended to be lower with DL-Met infusion compared with

an equimolar dose of L-Met (Titgemeyer and Merchen, 1990). In both bovine studies, plasma

total Met concentrations (D plus L) were greater when either D- or DL-Met was provided

compared with similar supplementation of the L-enantiomer (Lapierre et al., 2012). Lobley et al.,

(2001) found that feeding RPMet with the 2 enantiomers (D- and L-) to dairy cows results in

accumulation of plasma D-Met, which could be an indication of poorer systemic utilization of D-

Met. From this research, it seems that both enantiomers increase D-Met plasma concentration,

but is D-Met utilized by the dairy cow?

To date, the research by Lapierre et al., (2012) is the most thorough to determine the BA of

D-Met in dairy cows. The researchers sought to quantify the conversion of D-Met to the L-

isomer, proportion of D-Met plasma removal, and L-Met conversion, and determine if the

conversion of D- to L-Met occurs in the mammary gland of dairy cows (Lapierre et al., 2012).

The findings from this study indicate that the D-enantiomer of Met, constituting half of the Met

in rumen-protected products, is transformed to the L-enantiomer in dairy cows. The whole body

rate of disappearance of D-Met is, however, much slower than that of L-Met, with the half-life of

D-Met being 6 to 7 times longer than that of L-Met (Lapierre et al., 2012). The non-utilization of

D-Met by the mammary gland and a lower hepatic extraction of D-Met compared with L-Met

would contribute to the slower rate of disappearance (Lapierre et al., 2012). The longer half-life

of D-Met could be useful as it offers the opportunity to delay the clearance of the absorbed Met

or act as a reservoir for L-Met synthesis (Lapierre et al., 2012).

The diversity of AAs is influenced by structural configuration (e.g., side chains), which

determines a specific AAs chemical property, such as reactivity, solubility, stability, and taste

(Wu, 2013). The R-group of Met contains a methyl group covalently bonded to a sulfur atom.

Methionine is among the most hydrophobic of the AAs and its electrophilic qualities are

attributed to the terminal methyl group and sulfur atom (Brosnan et al., 2007). This means that

most of the Met residues in globular proteins are found in the interior hydrophobic core, in

membrane-spanning protein domains, and Met is often found to interact with the lipid bilayer

(Brosnan and Brosnan, 2006). Methionine must bind to initiator tRNA in order for the pre-

initiation complex to form during the beginning of protein synthesis in eukaryotes (N-formyl-

Met in bacteria) and it is believed that the hydrophobic nature of Met is essential for protein

synthesis (Drabkin et al., 1998).

Lysine is a basic AA characterized by the presence of an amino group at the end of a 4-

carbon aliphatic side chain and bears a positive charge at physiological pH (Tom and Bos,

2007). This structure makes Lys a reactive component in different chemical reactions including

carbonyl-amine interactions. As a consequence, a segment of Lys can be degraded, and another

part can become unavailable in different food systems because of its involvement in different

interactions with AA side chains or with other components including carbohydrates or lipids

(Tom and Bos, 2007). As a result, its ε-amino group side chain easily forms covalent bonds

with other AAs and reducing sugars known as the Maillard reaction (Ordway and Aines, 2010).

Even when Lys is incorporated into peptides or proteins, its ε-amino group remains reactive and

can still form covalent bonds (Nursten, 2005). Although all AA, peptides, and proteins can

participate in the Maillard reaction, Lys is the most common and prevalent AA that participates

(Ordway and Aines, 2010).

RPMet and RPLys

RPMet Supplements

Protection of free AAs from ruminal degradation dates back to the 1960s when researchers

began to understand the importance of balancing AA in order to meet dairy cows AA

requirements (Schwab and Broderick, 2017). The first RPMet supplement was developed by

Delmar Chemicals (LaSalle, QC, Canada). This product consisted of a core of 20% DL-Met,

colloidal kaolin, and tristearin (Schwab and Broderick, 2017). The encapsulated DL-Met product

when fed to lactating cows at 5, 15 and 45 g/d had no effect on milk production or milk

component yields (Broderick et al., 1970). However, plasma Met concentrations were increased

at the higher supplementation rates (Broderick et al., 1970). Abomasal infusion of the

encapsulated DL-Met product to lactating cows increased both milk protein yield and

concentration. Plasma AA concentrations were altered when casein plus the encapsulated DL-

Met were abomasally infused. These results suggest that the Delmar product was a successfully

developed commercial RPMet product (Broderick et al., 1970); however, it was later found to

have poor intestinal digestibility (Grass and Unangst, 1972). These findings highlighted the

reality that developing encapsulated products with both high rumen protection and intestinal

release was not an easy task.

Since this initial RPMet research, many approaches have been investigated to increase

ruminal protection of Met products. In the 1980s and 1990s, Met analogues and derivatives were

screened to find alternatives to encapsulation. Methionine analogues and derivatives provide the

precursors for enzymatic conversion to Met. However, the screening was unsuccessful, and

attention was again focused on improving encapsulation technologies (Schwab and Broderick,

2017). Today RPAA products are marketed as being either; physically encapsulated or protected

AA, AA analogues, or AA derivatives. The most popular RPMet products currently fed to dairy

cows are physically protected and Met analogues. A brief review will be given on the 2 popular

physically protected RPMet products, and special attention will be given to Met analogues in this

review as they are structurally and functionally similar to Met derivatives.

Physical protection approaches have been utilized other than lipid coating to protect Met with

a surface coated carbohydrate (Mepron M85

, Evonik Inc., Kennesaw, GA). The Mepron

pellets consist of an 85% DL-Met core and are coated with several thin layers of ethylcellulose

and stearic acid. Rumen enzymatic digestion of ethyl cellulose is negligible; therefore,

degradation of the products occurs through physical action and abrasion. This results in minimal

rumen degradation and slow release of DL-Met in the small intestine (Schwab and Ordway,

2003). Smartamine

M (Adisseo Inc., Alpharetta, GA) is a lipid/pH-sensitive polymer coated

product. The core contains 75% DL-Met and is covered with ethylcellulose, stearic acid, and a

small droplet of 2-vinylpyridine-co-styrene. This copolymer arrangement allows the product to

escape ruminal degradation and become solubilized at the low abomasal pH of 2, thus allowing

free DL-Met to be released in the abomasum (Schwab and Ordway, 2003).

The Met analogues currently used in the dairy industry are Met hydroxy analogue (MHA or

HMTBa) (ALIMET

, Novus International Inc., St. Louis, MO), calcium bis MHA (MFP

Novus International Inc.), the isopropyl ester of 2-hydroxy-4-methylthiobutanoic acid (HMBi)

(MetaSmart

Liquid, Adisseo Inc.) and butanoic acid, 2-hydroxy-4-(methylthio)-1-methylethyl

ester (MetaSmart

Dry, Adisseo Inc.). The ALIMET

is a liquid 2-hydroxy-4-

methylthiobutanoic acid (HMB) product with the substitution of the α-amino group of Met with

a hydroxyl group. The manufacturers’ data sheet does not state a minimum L-Met or DL-Met

hydroxy analogue percentage (Novus International Inc. 2015). The minimum Met activity as a

percent of DM in beef and dairy is 100%. The minimum activity is 88% in all other species. The

product has a chemical formula of C

S. The ALIMET

is a light brown to brown liquid

with a characteristic sulfur odor. The pH of the product is less than 1. Boiling point is observed

at 121°C and the flash point using Pensky-Martens Closed Cup is greater than 116°C. The vapor

pressure is 16 mm Hg at 25°C. The material density is heavier than water, but is completely

soluble. Shelf life is a minimum of 2 years when stored as directed (Novus International Inc.

2015).

The MFP

is the dry-pelleted HMB calcium with substitution of the α-amino group of Met

with a hydroxyl group (Novus International Inc. 2015). The product has a minimum DL-Met

hydroxy analogue calcium content of 95%. The minimum Met activity is 84%. The product has a

chemical formula of C

S . ½ Ca. The MFP

is a light tan to tan granular powder with a

cherry odor/flavor and a pH of 11 at 5% solution. The MFP

has a solubility of 74 g/kg at 25°C,

and the material density is 800-850 kg/m

(loose). The MFP

is not oxidative and has dust

explosion class ST1 (weak explosion). The shelf life is a minimum of 5 years when stored as

directed (Novus International Inc. 2015).

The MetaSmart

Liquid (Adisseo Inc. 2018) is a liquid HMBi (95%) product with the

substitution of the α-amino group of Met with an isopropyl ester group. The product has a

chemical formula of C

S. It has a colorless to brown liquid appearance and a pH of 3.6. It

has a melting/freezing point of -35.2°/-30.4°C. The product degrades prior to boiling. The flash

point using Pensky-Martens Closed Cup is 115°C. The vapor pressure (Pa) is 0.6% at 25°C. The

specific gravity is 1.07 at 20°C and has a solubility in water of 25.1 g/L at 30°C (Adisseo Inc.

2018).

The MetaSmart

Dry is a dry HMB product with the substitution of the α-amino group of

Met with an ester group (Adisseo Inc. 2018). The product has a chemical formula of C

It is free-flowing, cream to beige-colored powder, consisting of butanoic acid, 2-hydroxy-4-

(methylthio)-1-methylethyl ester (50-75% by weight) adsorbed onto a silicon dioxide carrier (25-

50% by weight). The product has a flash point of > 93°C. The specific gravity is 0.75 g/cm

(loose), 0.80 g/cm

(packed), and the product is insoluble in water (Adisseo Inc. 2018).

The first Met analogue extensively studied was MHA (DL-α-hydroxy-γ-mercaptobutyrate) or

more appropriately called HMB (Schwab and Ordway, 2003). The HMB is more resistant to

ruminal degradation than free Met and its production is less expensive than that of DL-Met

protected supplements such as Smartamine M (Wilson et al., 2008). Estimated bypass rates of

HMB range from 99 (Jones et al., 1988) to 6% (Noftsger et al., 2005). Supplementation of HMB

has a moderate effect on increasing plasma Met concentrations in dairy cows (Koenig et al.,

1999; Koenig et al., 2002). In order for the body to utilize HMB as useable Met, HMB must be

converted to L-Met in a two-step process. In the first step, HMB is enzymatically converted to 2-

keto-4-(methylthio) butanoic acid (KMB). In the second step, transamination of KMB yields

useable body Met (L-Met) (Dibner and Knight, 1984).

Several esters of HMB have been developed in an attempt to increase its rumen protection.

Comparing microbial degradation of several HMB esters, HMBi was estimated to have 60%

protection, but methyl, ethyl, butyl, and cyclohexyl esters were more readily hydrolyzed to HMB

in the rumen (Robert et al., 2001). The ability of HMBi to avoid hydrolysis suggests that

isopropyl esters present a steric hindrance of ruminal bacterial esterases. In order for HMBi to be

converted to useable body Met, HMBi must be hydrolyzed to HMB and isopropanol. Then,

HMB is converted to KMB and L-Met (Dibner and Knight, 1984).

Methionine derivatives are the least studied form of RPMet. A Met derivative is a free Met

molecule with a chemical blocking group added to the α-amino group or a Met molecule in

which the acyl group has been modified (Schwab and Ordway, 2003). Methionine derivatives

add a chemical tail to the α-amino group, while Met analogues change the structural

configuration of the α-amino group. There are 2 enzymes capable of converting AA derivatives

to their corresponding AAs, to be utilized as body AAs. The enzyme aminoacylase I is present in

multiple mammalian organs (small intestine, liver, and kidney) (Giardina et al., 1997). The

bovine liver contains the enzyme, α-N-acylamino acid hydrolase which is capable of hydrolyzing

α-N-acylated AAs to yield acetate and the corresponding AAs (Met or Lys) (Gade and Brown,

1981).

Digenis et al. (1974) screened 31 nitrogenous compounds with possible Met activity. The

compounds were screened in vitro with rumen microbes and compounds not supporting cellulose

digestion were included in studies to confirm resistance to rumen microbial degradation. N-

acetyl-DL-Met (NAM) and DL-homocysteine thiolactone hydrochloride were the most stable

toward microbial degradation (Digenis et al., 1974).

Windschitl and Stern (1988) reported that Met flowing out of continuous cultures increased

with NAM supplementation without a change in bacterial Met flow, which was attributed to

greater escape (67%) of the NAM from bacterial degradation. Daabees et al., (1984)

supplemented NALM to young pigs and revealed that plasma Met concentration increased, but

no NALM was present in either portal or vena caval plasma at any sampling time, thus indicating

NALM deacetylation to Met in the intestinal lumen or mucosal cells with release of free Met to

the plasma. Additionally, Amos et al. (1974) abomasally infused sheep with NAM and found

that plasma Met concentration increased 1 h post infusion compared with the control treatment.

Based on the prior data, it seems that NAM can bypass the rumen and that both NAM and

NALM can increase plasma Met concentrations. However, research for NALM has not reported

the BA of NALM fed to lactating dairy cows. These in vitro and in vivo findings revealed the

potential use of Met derivatives as a RPMet source to lactating dairy cows; however, it was not

until the past few years that interest in these products has grown.

RPLys Supplements

Extensive research has focused on RPMet products. However, there are limited publications

on RPLys products. This is primarily due to difficulties with development and production of

rumen stable and efficacious RPLys products. As previously discussed, the Lys side chain is

highly reactive and can react with encapsulation materials, such as polymer. Encapsulated Lys

has a lack of resistance to TMR mixing and ruminant mastication (Swanepoel et al., 2010).

Product stability of RPLys in wet TMR has been overlooked in recent years. A study conducted

by Ji et al. (2016) assessed whether mechanical mixing of TMR or TMR with low or high

moisture content alters Lys release from 6 RPLys products. The authors concluded that

mechanical feed mixing, dietary moisture content, and exposure of RPLys to other feed

ingredients in the TMR over time may damage the protective coating and reduce efficacy of

RPLys products (Ji et al., 2016). Although differences in Lys release among RPLys products

were found, inferences about RPLys product BA is cautioned, as altered protection of Lys does

not determine free Lys delivery to the plasma and systemic availability to the cow (Block and

Jenkins, 1994; Ji et al., 2016).

Inconsistent post-ruminal polymer degradation and high production costs have also limited

the development of RPLys products (Swanepoel et al., 2010). However, there has been success

since the early 1990s in developing rumen stable AA matrixes and a number of RPLys products

have become available for commercial use (Robinson, 2009b). Available commercial RPLys

products use encapsulation, matrix technology, or a combination of both to protect a Lys core

from ruminal degradation (Ji et al., 2016). These RPLys products are often coated by a series of

lipid or fatty acid calcium-based salts (Ji et al., 2016). Wu and Papas (1997) found that RPLys

coating protection efficacy in the rumen depends on coating composition, pellet surface

smoothness, inner core pellet strength, pellet solubility, and pellet size (Wu and Papas, 1997).

Because the majority of RPLys products are lipid-coated, questions have arisen about the

rumen environment (i.e., water content and acidity) and stability of lipid coated RPLys products.

Acid can catalyze the hydrolysis of triglycerides to fatty acids and glycerol (Bender et al., 1961;

Carey, 2003), and acid-catalyzed hydrolysis occurs in environments with high water

concentrations, such as in the rumen (Bender et al., 1961; Carey, 2003). Thus, Reiners et al.

(2018) sought to determine the effects of acidity and silage source on Lys retained by 2 RPLys

products and to estimate ruminal degradation of Lys retained by RPLys after mechanical mixing

with alfalfa or corn silage with different amounts of acidity (pH). The authors concluded that

physical and chemical dietary characteristics can affect Lys provided to ruminants from RPLys

products (Reiners et al., 2018). They state that Lys concentration was reduced from the two

RPLys products when mixed in silage-based diets before cows consume the diet (Reiners et al.,

2018). Because chemical and physical dietary factors along with on-farm feeding practices

(mixing times) can impact RPLys efficacy, additional research is needed to find ways to

minimize Lys loss. The most popular RPLys products currently fed to dairy cows are physically

protected. A brief review will be given on 2 popular physically protected lipid RPLys products.

AjiPro-L

(Ajinomoto Co. Inc., Tokyo, Japan) is a small white oval bead composed of L-Lys

monohydrochloride (50% minimum) core, vegetable oil (49%) and lecithin (1%). The

hydrogenated vegetable oil matrix in AjiPro-L resists physical damage while its hydrophobic

layer prevents the microbial breakdown of Lys (Reiners et al., 2018). USA Lysine

(Kemin

Industries, Des Moines, IA) is a beige to light brown particle composed of a L-Lys

monohydrochloride (65% minimum) core. It is manufactured by extrusion of L-Lys-HCl and

lipid (vegetable oil) into small particles and subsequently the Lys lipid particles are coated with

multiple layers of a lipid matrix (Reiners et al., 2018).

Lysine derivatives, like Met derivatives are the least studied form of RPLys. A Lys derivative

is a free Lys molecule that has a chemical blocking group added to either the α-amino or ε-amino

group. The focus of this dissertation is on AA derivatives, specifically NALM and NALL. For

now, the review will focus on production responses of lactating dairy cows to encapsulated AA

supplementation, Met analogue supplementation, and briefly cover NALM supplementation. To

our knowledge, NALL supplementation to lactating dairy cows has not been described in the

literature and therefore only encapsulated RPLys production responses will be discussed.

Effects on Intake

Dry matter intake (DMI) is the amount of feed an animal consumes that does not include

moisture content. Dry matter intake is significant in nutrition, because it determines the amount

of nutrients available to an animal for health, production, and other physiologic processes (NRC,

2001). Actual or estimated DMI is important for the formulation of diets to prevent underfeeding

or overfeeding of nutrients and to promote efficient nutrient use (NRC, 2001). A variety of

factors can influence DMI including forage quality, TMR nutrient balance, feeding delivery,

feeding method, TMR palatability, ration dry matter (DM), environmental conditions, housing

facilities, and managerial practices.

Amino acids, especially His have been shown to increase DMI in lactating dairy cows (Lee et

al., 2012b; Giallongo et al., 2016). The exact mechanism of increased DMI is unknown, but it is

speculated that His and other EAA regulate DMI via a direct effect in the anterior prepyriform

cortex of the brain (Rudell et al., 2011). Methionine on the other hand if overfed can have a

negative impact on DMI (Benevenga, 1974). A meta-analysis conducted by Zanton et al. (2014)

reported that DMI response was affected by supplemental Met source (Smartamine, Mepron,

HMTBa, and DL-Met). Cows fed Smartamine had higher DMI, while cows fed Mepron had

lower DMI compared to control cows. Supplementation of HMBTa and DL-Met did not affect

DMI compared to control cows (Zanton et al., 2014).

Responses to supplementation of RPMet and HMBi on DMI in the literature have been

inconsistent. Kung and Rode (1996), Leonardi et al. (2003), and Chen et al. (2011) found that

RPMet supplementation did not improve DMI. Limited studies have shown an increase in DMI

with RPMet supplementation (Broderick et al., 2009; Zanton et al., 2014), whereas Ordway et al.

(2009) and Zanton et al. (2014) reported decreases in DMI. Supplementation of HMBi may

increase DMI (Osorio et al., 2014) because of the potential stimulatory effect of HMBi on

cellulolytic bacteria and the subsequent increase in ruminal passage rate and DMI (Lee et al.,

2015).

Lee et al. (2012b) assessed supplementation of RPMet products in MP-deficient diets. The

authors reported that MP-deficient diets below 15% of the NRC requirements decreased DMI.

However, the authors observed that a treatment supplemented with a combination of 3 RPAA

consisted of Lys (100 g/cow), Met (30 g/cow), and His (50 g/cow) increased DMI (Lee et al.,

2012b). In contrast, Lee et al. (2015) found that supplementation with RPLys and RPMet in MP-

deficient diets (−256 to −305 MP g/d) did not affect DMI. Recent research suggests that

transition cows may benefit from balancing for limiting EAA (Zhou et al., 2016). Supplementing

Lys adequate diets with RPMet resulted in increases in DMI (Osorio et al., 2013; Zhou et al.,

2016). Improved DMI during the transition period leads to better postpartum performance in

dairy cows supplemented with RPMet (Zhou et al., 2016).

Responses to supplementation of NALM on feed intake is scarce, but a recent study by Liang

et al. (2019) supplemented mid-lactating cows with 0, 15, 30, or 60 g/d of NALM. Dry matter

intake was not affected by NALM supplementation. This finding is in agreement with Amaro et

al. (2019), in which dosage rates of 0, 15, 30, and 45 g/d of NALM were fed to 60 multiparous

Holstein dairy cows in early lactation (27 ± 4.3 DIM) and the dose did not affect DMI. However,

a study by Gresenti (2017) supplemented mid to late lactating cows with 0, 15, 30, or 60 g/d of

NALM. Increasing NALM supplementation linearly increased DMI, which the research group

speculated may potentially be related to a linear increase in rumen MCP yield (Gresenti, 2017).

However, the potential stimulatory effects of NALM supplementation on rumen microbiota

needs to be further investigated. Contrasting results from Met supplementation may be due to

differences in length of feeding of RPMet, varying Met supplementation amounts or rates (based

on % DM), Met products, stage of lactation, or a combination of factors (Osorio et al., 2013).

Research of RPLys and its effect on DMI is limited. However, RPLys supplementation does

not appear to affect DMI of lactating dairy cows (Paz et al., 2014). These findings of RPLys and

no effect of DMI are in agreement with Blauwiekel et al. (1997) and Robinson et al. (2011).

Swanepoel et al. (2010) found that supplementation of RPLys did not influence DMI in mid-

lactation cows (77 ± 3.2 DIM). However, the late lactation group (262 ± 6.70 DIM) had a

tendency toward lower DMI with addition of RPLys (Swanepoel et al., 2010). Recently, Girma

et al. (2019) investigated providing transition cows with an increased level of energy and

supplementing RPLys to improve both pre- and post-partal DMI. Supplementation of the TMR

with RPLys improved DMI in postpartum cows and RPLys supplementation of the close-up diet

increased DMI of cows compared to control animals (Girma et al., 2019). The authors contribute

the findings of increased DMI to the endogenous synthesis of carnitine from RPLys

supplementation. The authors state that supplementing RPLys is an efficient strategy to increase

the supply of Lys that serves as the carbon backbone for carnitine synthesis (Vaz and Wanders,

2002; Robinson et al., 2006). Carnitine is the methylated form of Lys (Shug et al., 1982) that

buffers acetyl residues from lipid mobilization, reduces ketone body formation, and plasma β-

hydroxybutyrate concentrations (Jacobs, 2002). Carnitine is important for oxidation of long-

chain fatty acids, regulation of ketosis, support of the immune system, and enhancement of the

antioxidant system (Citil et al., 2009; Pirestani et al., 2011). As previously mentioned, DMI is

controlled by many factors, so further research needs to be completed to fully understand RPAA

(RPLys and RPMet) supplementation and DMI regulation.

Effects on Milk Yield and Composition

The adoption of the multiple component pricing system by the Federal Milk Marketing

Administration (2000) changed the method by which producers are compensated for their milk.

The system adjusted the pricing structure to pay producers based on the amounts of milk fat,

protein and solids in milk. Milk was previously priced based on total fluid milk volume.

Economically, milk protein and fat are the most important milk components to dairy farmers. As

the value of milk protein continues to increase, there is greater interest among dairy farmers to

find alternative feeding strategies such as RPAA supplementation to increase milk protein yields

(Vyas and Erdman, 2009). Not only are productive responses important (i.e., increasing milk fat

and protein yield), reducing environmental pollution by improving dietary N conversion into

milk protein is of concern in the dairy industry (Lapierre et al., 2005). As mentioned previously,

Liu et al. (2017) suggest the incorporation of a multiple-limiting AA concept to improve milk

and milk protein yield model predictions, thus allowing modification of diets to increase post-

absorptive N efficiency and reduced N excretion by dairy cows.

Encapsulated RPMet products, especially Smartamine and Mepron have been extensively

studied in the literature (Patton., 2010). The evidence for one product being more effective than

the other is inconclusive, as milk yield responses to supplemental RPMet products is variable.

Patton (2010) reported increased milk production for both RPMet products (0.43 kg/d for

Mepron and 0.14 kg/d for Smartamine) based on a meta-analysis from 36 studies with 17 studies

evaluating Mepron and 18 evaluating Smartamine. Zanton et al. (2014) evaluated the lactational

performance of dairy cows to supplemental Met sources. The authors assessed HMTBa provided

as either a liquid or Ca salt form (17 papers with 34 control diets and 46 treatment comparisons),

Mepron (18 papers with 35 control diets and 42 treatment comparisons), and Smartamine (20

papers with 30 control diets and 39 treatment comparisons). Overall, milk yield did not respond

to any form of Met supplementation; however, it tended to increase for HMTBa and Mepron

supplementation (Zanton et al., 2014).

Although there is variability in milk yield responses to supplemental Met products, strategic

supplementation of RPAAs to low CP diets is becoming more popular. The observed production

responses to supplementation of RPMet in cows fed low CP diets (i.e., MP-deficient diets) have

been inconsistent and marginal (Arriola Apelo et al., 2014a; Sinclair et al., 2014). Milk yield was

not improved when a low CP diet (15.0%) was fed alone or in combination with RPMet

supplementation (Arriola Apelo et al., 2014b). However, further research with MP-deficient diets

(ranging 5–10% below MP requirement) have sustained milk yield (Lee et al., 2015; Giallongo et

al., 2016). Peri-partal RPMet supplementation has been shown to improve post-partal

performance. Osorio et al. (2013) and Zhou et al. (2016) found that cows supplemented with a

RPMet source increased milk yield in the subsequent lactation.

A Met analogue, HMTBa, is converted to Met in the body and used as a supplemental Met

source in dairy production. Results of published studies assessing the effects of supplementing

Met sources, including HMTBa, on performance variables are inconsistent as well. A recent

meta-analysis was performed to summarize the accumulated results of HMTBa supplementation

on animal performance (Feng et al., 2018). Supplementation of HMTBa had no effect on milk

yield, which was in agreement with the meta-analysis done by Zanton et al. (2014). Noftsger et

al. (2005) reported no difference in milk production when cows were supplemented with HMBi

at 0.13% DM. However, St-Pierre and Sylvester (2005) showed an increase in milk yield (2.9

kg/d) to cows supplemented with HMBi at 0.15% DM. Lee et al. (2015b) found that

supplementing HMB (the unprotected form), numerically decreased milk production when %

DM inclusion rate of dietary HMB increased. Alternatively, HMB supplementation increased

milk production in a quadratic manner (Piepenbrink et al., 2004).

Supplementation of NALM to mid lactation dairy cows improved milk yield in a quadratic

manner, with 30 g/d of NALM seeming to be the optimal supplementation rate for milk yield

under the experimental conditions (Liang et al., 2019). Amaro et al. (2019) found that milk yield

of early lactation Holstein dairy cows tended to be greater (P = 0.07) with intermediate

concentrations (15 and 30 g/d) of NALM compared to control animals. The authors noted that

NALM supplementation at 30 g/d resulted in the highest milk yield (Amaro et al., 2019), which

is in agreement with Liang et al. (2019). Encapsulated RPMet, Met analogues, and Met

derivatives can impact milk yield, but their effects tend to be more prominent in altering milk

composition (milk fat and protein).

Supplementing RPLys to pre-calving cows during the close-up period (-21 to 0 d) at a rate of

40 g/cow/d had no effect on post-partum milk yield (Girma et al., 2019). These results agree with

Swanepoel et al. (2010) who observed that supplementing dairy cows with RPLys (41 g/cow/d)

did not affect milk yield in early lactation. Paz and Kononoff (2014) did not observe a difference

in milk yield when RPLys was supplemented to diets with altering inclusion rates of low-fat

distillers dry grains. Cows supplemented with RPLys (37 g/cow/d) after peak lactation showed

no improvement in milk yield (Bernard et al., 2014). In contrast, Robinson et al. (2011) observed

a 2 kg increase in milk yield when RPLys was supplemented to deliver 15 to 21 g of MP Lys.

Historically, HMB/HMBTa and HMBi supplementation tends to exert their effects on milk

fat production in lactating dairy cows (Zanton et al., 2014). Supplementation of HMTBa had no

effect on milk yield, fat percent, protein percent, or protein yield, but increased milk fat yield

(Feng et al., 2018). Zanton et al. (2014) reported HMTBa supplementation increased milk fat by

5.38 g/g of MP Met from HMTBa, whereas Feng et al. (2018) reported HMTBa supplementation

increased milk fat by 5 g/g of MP Met. A meta-analysis by Zanton et al. (2014) concluded

HMTBa supplementation increases milk fat yield by 45 g/d and milk protein yield by 13 g/d. St-

Pierre and Sylvester (2005) observed a 166 g/d increase of milk fat yield with HMBi

supplementation.

The beneficial effects of HMTBa supplementation on milk fat have been investigated (Polan

et al., 1970; Wang et al., 2010). Patton et al. (1970) reported that in vitro HMTBa

supplementation can stimulate polar lipid formation and the authors suggest that the polar lipid

class may increase with rumen protozoal growth. Increased ruminal biohydrogenation

intermediates of the alternative pathway (e.g., trans-10 C18:1) exert antilipogenic effects in the

mammary gland of ruminants which can lead to milk fat depression (Shingfield and Griinari,

2007). Baldin et al. (2018) witnessed that HMTBa supplemented to either high-producing (44.1

± 4.50 kg/d) or low-producing (31.4 ± 4.30 kg/d milk yield) lactating dairy cows at 0.1% of diet

DM maintained lower concentrations of the biohydrogenation intermediates, trans-10 C18:1 in

milk samples. This finding suggests that HMTBa may stabilize ruminal biohydrogenation and

help prevent the shift to the alternative biohydrogenation pathway (Baldin et al., 2018).

Therefore, it is speculated that supplemental HMTBa exert their effects on milk fat production

by shifting ruminal biohydrogenation intermediates.

Physically encapsulated RPMet products and Met derivatives generally do not increase milk

fat concentrations, because, unlike Met analogues, they lack a ruminal mode of action. However,

encapsulated forms of RPMet have been shown to increase milk fat yield. Chen et al. (2011)

found trends (P = 0.08) for increased milk fat concentration and yield when lactating dairy cows

were supplemented with approximately 15 g of RPMet (Smartamine M) at 0.06% on a DM basis.

Recently, Toledo et al. (2017) evaluated the effects of daily top-dressing a TMR with 21.2 g of

RPMet from 30 ± 3.0 to 126 ± 3.0 DIM on productive performance in lactating dairy cows. The

group found that top-dressing RPMet to multiparous cows increased milk fat percentages (3.45

vs. 3.14%), although no effects were seen in primiparous cows. Based on limited data in the

literature, NALM supplementation to either mid or early lactating dairy cows has failed to

exhibit beneficial effects on milk fat concentrations (Amaro et al., 2019; Liang et al., 2019).

Lactational performance of dairy cows in response to feeding RPLys has been researched

over the past 30 yr, but sufficient data are lacking for beneficial effects of single AA

supplementation of Lys in dairy rations. Supplementing combinations of Met and Lys is a much

more common practice in the dairy industry. Swanepoel et al. (2010) reported reduced milk fat

synthesis to feeding a RPLys product to high-producing dairy cows. Třináctý et al. (2009) fed a

RPLys tablet to high-producing Holstein dairy cows (33.5 kg/d); milk protein concentration

increased, while milk fat concentration and fat yield declined. However, according to Robinson

(2010) the results described above for Lys supplementation on milk fat are inconsistent with the

literature of post-ruminal Lys supplementation. A review of literature by Robinson (2010)

reported that RPLys supplementation generally resulted in minimal negative DMI and

production responses, possibly due to an AA imbalance or poor AA delivery to the intestinal

absorptive site. Productive benefits of RPLys supplementation is disappointing and overall

lactational performance effects can only be judged as negligible (Robinson, 2010).

Less convincing evidence exists for the effect of L-Lys fed to lactating cows, but when L-Lys

was deleted from a mixture of abomasal infused AAs, milk and milk protein production was

reduced in the Lys-deficient infusion (Weeks et al., 2006). The exact relationship of L-Lys to AA

deficiencies has yet to be established (Patton et al., 2014). However, increased L-Lys supply

appears important in early lactation when microbial synthesis is reduced because of lower DMI,

to help drive milk and milk protein production (Robinson et al., 2011).

Although RPLys has minimal effects on production performance, increases in milk protein

yield are one of the most likely observed responses due to supplementation with RPMet (Zanton

et al., 2014). As previously described, Zanton et al. (2014) evaluated the lactational performance

in dairy cows receiving supplemental Met sources (HMTBa, Mepron, and Smartamine) or post-

ruminal infusion of DL-Met. Milk protein yield was increased due to supplementation from all

Met sources or from infusion of DL-Met and protein concentration was greater for all

supplements or infusion of DL-Met, except for cows supplemented with HMTBa. Average milk

protein yield responses ranged from 13 g/d for cows supplemented with HMTBa to 35 g/d for

Mepron (Zanton et al., 2014). These results are in agreement with the meta-analysis by Patton

(2010) who noted similar milk protein production responses when cows were supplemented with

RPMet (Mepron and Smartamine). Osorio et al. (2013) observed an increase in protein yield

post-partal for cows supplemented with Smartamine and MetaSmart (1.24 and 1.23 kg/d) versus

control cows (1.11 kg/d) during the transition period (-21 to 30 DIM). Supplementing RPMet can

also result in an increase in milk protein percentage (Osorio et al., 2013), but one study

(Benefield et al., 2009) reported a negative effect on milk protein concentration. Milk protein

concentration has been used to indicate the effectiveness of a Met source, leading to changes in

milk protein synthesis (Schwab et al., 2001). This is based on the idea that if milk yield is

unchanged, an increase in milk protein concentration will allow for an inference to be made on

milk protein yield and therefore efficacy of Met supplementation (i.e., bioavailability) (Zanton et

al., 2014). The take-home message is that if Met can escape ruminal degradation and be made

available post-ruminally, whether by supplementing RPMet products or Met analogues, milk

protein can be increased. Methionine, if not utilized by the cow for milk and milk component

production, can also potentially play a pivotal role in both cow and calf immunity.

Effects on Immunity

It is well-known that Lys and Met are the most limiting EAA for milk production, but these

EAAs also play a crucial role in immune function. Methionine and its derivate metabolites (e.g.,

glutathione, taurine, polyamines) are immunonutrients in nonruminants (humans), which aid and

improve immune functionality and activity (Vailati-Riboni et al., 2017). Recently, Vailati-Riboni

et al. (2017) supplemented dairy cows with RPMet at 0.08% DM (8 to 12 g/d DL-Met) during

the transition period (-21 to 30 DIM) to determine periparturient immune response using an ex

vivo whole blood approach and the inflammatory response of the blood immune cells were tested

with a LPS challenge (Vailati-Riboni et al., 2017). Overall, Met supplemented cows had a lower

post-partum IL-1ß (pg/µL) concentration compared to the control cows. The Met treated cows

had a greater phagocytosis capacity of neutrophils and oxidative burst activity by both

monocytes and neutrophils. The authors suggest that the above results indicate that Met

supplementation improved immune functional activity, while dampening the innate immune

response to a LPS challenge (Vailati-Riboni et al., 2017). They also mention that Met probably

acts on the oxidative status of the animal or indirectly through an immunologically active

metabolite (e.g., taurine). Taurine is the most plentiful AA in the cytosol of neutrophils that can

trap hypochlorous acid to form taurine monochloramine (Vailati-Riboni et al., 2017). Greater

oxidative burst in neutrophils could be attributed to higher taurine monochloramine

concentrations, leading to peri-partal immune cell hyper-responsiveness. Therefore, Met may act

as an immunomodulator of the immune response in transition cows when cows undergo a period

of immunosuppression (Vailati-Riboni et al., 2017).

Zhou et al., (2017) investigated differences in liver functionality indexes (LFI) in peri-partal

dairy cows fed RPMet or choline. The LFI is a composite index that is used to determine changes

in plasma concentrations of blood biomarkers (albumin, cholesterol, and bilirubin) (Bertoni and

Trevisi, et al., 2013). The researchers retrospectively assigned cows to low (LFI < 0) and high

(LFI > 0) groups. Animals with a high LFI had greater liver functionality and are considered to

be at a lower risk for developing health/metabolic disorders during the transition period. The

liver regulates the metabolic activity aimed at mobilizing and directing nutrient flow to the

mammary gland and the gastrointestinal tract (Seymour, 2016). Therefore, any reduction in liver

function can impair lactational performance, reproductive efficiency, and immune status

(Seymour, 2016). A larger number of Met-supplemented cows were in the high LFI group. The

high LFI cows had an overall greater liver functionality and decreased state of oxidative stress,

indicating that the high LFI cows had a better immunometabolic status during the transition

period (Zhou et al., 2017).

In dairy cows, Met can also have a large impact on cow health through a variety of reasons

other than immunomodulation or liver functionality (Figure 1). Methionine serves as a source of

cysteine for glutathione synthesis via homocysteine produced in the Met cycle (Martinov et al.,

2009). Liang et al. (2019) supplemented RPMet to transition cows (-28 d to 30 d) at a rate of

0.10% DM to determine if RPMet alters glutathione metabolism in peri-partal Holstein cows.

Adipose tissue biopsies were collected on d −10, 10, and 30, relative to parturition, from 7 cows

in each group to measure various proteins and genes related to glutathione metabolism. Overall,

increased mRNA abundance of 4 key glutathione metabolism-related genes and upregulation of

4 protein abundance components related to glutathione metabolism were found (Liang et al.,

2019). The authors state that RPMet supplementation may alter glutathione metabolism in

adipose tissue (Liang et al., 2019). Altered glutathione metabolism in adipose tissue may

contribute to reduced oxidative stress and inflammation during the transition period. However,

further research is needed to assess the impacts of adipose tissue glutathione metabolism on

immunity of dairy cattle.

Methionine plays a role in the liver as a lipotropic agent (Martinov et al., 2009). Methionine

is needed for synthesis of SAM, which is a key methyl donor necessary for the packaging of very

low-density lipoproteins (VLDL) for exportation out of the liver (Osorio et al., 2014).

Triglycerides, if not exported from the liver, can accumulate causing fatty liver disease in

ruminants. Sun et al. (2016) observed increased plasma non-esterified fatty acids (NEFA) and

plasma VLDL concentrations in post-partum transition cows supplemented with RPMet. This

finding suggests that Met may affect hepatic lipid metabolism and exportation of VLDLs from

the liver (Sun et al., 2016).

Transition cows benefit from Met supplementation through lipotropic agent activity,

antioxidant substrate formation, immunomodulation and immune functionality. In addition,

maternal nutritional status may also benefit neonatal innate immune function (Jacometo et al.,

2016). Abdelmegeid et al. (2017) performed an in vitro study with neonatal Holstein calf

polymorphonuclear leukocytes (PMNL). Supplementation with either Met, choline, or taurine

altered the inflammatory response and affected oxidative stress, suggesting that Met and other

nutrients may be needed for an adequate immune response in calves (Abdelmegeid et al., 2017).

Jacometo et al. (2018) supplemented RPMet at 0.08% DM during the close-up period to

multiparous Holstein cows. Calves born to RPMet supplemented cows showed positive changes

in PMNL molecular pathways compared to control calves. Although the data are inconclusive,

maternal RPMet supplementation may play a role in neonatal calf immunity, but further research

is needed to identify the link between maternal AA supply and calf immunity.

Methionine’s role in ruminant immunity has been investigated more extensively in the

literature compared with that of Lys. However, Lys deficiency can impair both antibody

responses and cell-mediated immune responses in monogastric animals (Chen et al., 2003; Wu,

2013). Lysine deficiency limits animal immunity-related protein synthesis which includes

antibodies and cytokines that are necessary for normal immune function (Liao et al., 2015). Han

et al. (2018) reported that Lys deficiency in piglets impaired inflammatory responses in the liver,

kidney, and spleen by mediating serum antibody concentrations (i.e., IgG and IgM) and

inflammatory cytokines. Additionally, toll-like receptors, which are needed to active the

inflammatory response were altered by Lys restriction in piglets (Han et al., 2018). It is apparent

that Lys is necessary for immune function in monogastrics (i.e., humans, swine, and fowl), but

data are lacking for the potential beneficial effects of Lys in ruminant immunology.

Intestinal Absorption of AA

The absorption of AAs through the gastrointestinal tract of animals is an important biological

function because growth, lactation, reproduction, and maintenance requirements depend on the

adequate supply of AAs for protein synthesis. Amino acids that reach the small intestine can be

incorporated into proteins, converted into other AA for biosynthetic processes, oxidized to CO

or transported through enterocytes into the mesenteric portal vein (Stoll and Burrin, 2006).

Protein reaching the small intestine of the ruminant can come from a variety of sources:

including rumen undegradable protein (RUP), MCP, and endogenous proteins. Ruminants use

similar intestinal and pancreatic proteases as monogastric animals for protein breakdown.

Absorption rates of AAs differ by anatomical location of the small intestine (i.e., duodenum,

jejunum, and ileum), enterocyte maturity, and the site of AA absorption varies by species. In

monogastrics, the jejunum is believed to hold the greatest capacity for AA absorption (Webb,

1990). In ruminants, the majority of AA absorption increases with distance from the pylorus and

the majority of absorption occurs in the jejunum and ileum (William, 1969; Phillips et al., 1976).

Amino acid absorption site and rate can also be influenced by individual AA. Williams

(1969) observed that sheep absorbed the majority of their AAs in the ileum; however, the order

of absorption varied with anatomical location, suggesting that AA affinities for transport systems

vary among intestinal sites. Phillips et al. (1976) reported that the largest amount of threonine

and valine absorption occurs in the ileum and Met absorption was similar between the jejunum

and ileum in sheep. Webb (1990) reported that total Met and Lys uptake was higher by ileal

brush border membrane vesicles than by jejunal brush border membrane vesicles. Webb (1990)

also reported that in ileal and jejunal tissue, Met transporters had lower affinities and higher

capacities for Met than the Lys transporter. This suggests that the mode of transportation and

mechanism of AA transport through the enterocytes can differ according to the individual AA

being absorbed.

Amino acid transport is affected by size, charge, and AA structural configuration (i.e., R-

group side chains). Spencer et al. (1962) suggested that all transport carriers require the presence

of an amino/imino group and a carboxyl group. Hydrophilic small weight neutral amino acids are

taken up less readily than the larger weight hydrophobic amino acids and basic amino acid

transport is intermediate (Webb, 1990). The various carriers are located on the basolateral

membrane, brush border, or on both sides of the small intestine (Webb, 1990). To date many AA

transport systems have been identified in the small intestine. However, the exact number is

unknown, but the classification of amino acid transport systems is based on substrate preference

and is determined by kinetic and inhibition analysis measurements (Christensen, 1984).

The major mechanisms for AA transport from the intestinal lumen to the enterocytes occur

through simple diffusion, facilitated diffusion, or active transport (Wilson and Webb, 1990).

Active transport requires energy and is believed to be Na

-dependent, while facilitated diffusion

mechanisms are Na

-independent systems. The relative significance of each route is highly

dependent on the concentration of substrate present. The Na

-dependent systems require

metabolic energy to transport AAs against a concentration gradient. The Na

gradient which has

high extracellular Na

and low intracellular Na

concentrations is regulated by Na

-ATPase

located in the basolateral membrane of the enterocyte (Webb, 1990). The Na

-ATPase pump

co-transports Na

and an AA into the cell and K

out of the cell (Webb and Matthews, 1994).

The Na

-independent systems do not require metabolic energy to move along a concentration

gradient. Simple diffusion is the predominant form of transport, but when substrate

concentrations are low, facilitated diffusion and active transport will account for more transport

(Webb, 1990).

Met Metabolism

Metabolism of Met is divided into 3 pathways: transmethylation, remethylation, and

transsulfuration. Transmethylation is a sequence of reactions present in Met metabolism and is

universally present in cells. Transmethylation (i.e., Met metabolism) begins when Met is

converted to SAM by Met adenosyltransferase (Brosnan and Brosnan, 2006). Numerous

methylases comprise the user group for SAM that donates its methyl group to an acceptor to

produce S-adenosylhomocysteine (Brosnan and Brosnan, 2006). The S-adenosylhomocysteine is

hydrolyzed to adenosine and homocysteine in a reaction catalyzed by S-adenosylhomocysteinase

(Martinov et al., 2009).

Remethylation occurs when homocysteine is methylated back to Met by Met synthase, which

is ubiquitous in the body (Brosnan and Brosnan, 2006). Betaine homocysteine methyltransferase

which is present in the liver and kidneys of some species can also methylate homocysteine to

Met. Methionine synthase uses 5-methyl-tetrahydrofolate as a methyl donor, while betaine

homocysteine methyltransferase uses betaine, from the diet or betaine produced during choline

oxidation (Stead et al., 2006). Betaine homocysteine methyltransferase and Met synthase solely

affect remethylation. The combined steps of transmethylation and remethylation makeup what is

called the Met cycle and these steps occur in most cells of the body (Brosnan and Brosnan,

2006). Methionine catabolism does not occur during transmethylation or remethylation.

In the transsulfuration pathway, homocysteine is converted to cysteine by cystathionine β-

synthase and cystathionine γ-lyase. The transsulfuration pathway exists in most tissues except

muscle (including heart) and endothelium (Martinov et al., 2009). The conversion of Met to

cysteine is an irreversible process. Methionine metabolism plays a vital role in folate

assimilation. Methionine synthase converts 5-methyl-tetrahydrofolate to tetrahydrofolate,

making it free to support DNA synthesis and various biological functions (Brosnan and Brosnan,

2006). The transsulfuration pathway is responsible for the majority of Met catabolism in the

body. The transsulfuration is also vital for the production of glutathione and taurine through its

synthesis of cysteine. Glutathione synthesis is often limiting based on cellular cysteine

concentrations and the transsulfuration pathway provides half of the cellular cysteine

requirements (Mosharov et al., 2000).

It is important to remember that Met metabolism is dependent upon the availability of

coenzymes and enzymes. For animals to properly metabolize Met, dietary requirements for

vitamins and minerals must be met. Dietary intake of B vitamins, specifically B2, B9, and B12

are necessary for proper Met metabolism in the body. Methionine synthase uses the derivative of

vitamin B9 (folic acid), using 5-methyl-tetrahydrofolate as a methyl donor. 5-methyl-

tetrahydrofolate donates a methyl group to homocysteine to regenerate Met and the enzyme Met

synthase catalyzes this step and utilizes vitamin B12 (cobalamin) as a cofactor (Locasale, 2013).

Vitamin B2 (riboflavin) is a cofactor for 5-methyl-tetrahydrofolate synthesis. In monogastrics,

Met metabolism can be altered if the diet is limiting in B-vitamins. Because rumen microbes can

synthesize B vitamins, ruminants are usually able to meet B vitamin requirements once rumen

microbes reach the small intestine (NRC, 2001).

Mammary Gland AA Metabolism

Knowledge of mammary gland AA metabolism is just as important as body or hepatic AA

metabolism, as the mammary gland is responsible for milk protein synthesis and can impact

lactational performance. Historically, AA metabolism is classified by the balance between

arteriovenous uptake and milk casein-AA secretion (Manjarin et al., 2014), while catabolism is

represented by excessive AA uptake which is not available for milk protein synthesis. Complex

intracellular interactions exist within mammary tissue. Therefore, AA metabolism in the

mammary gland may give insight on mechanisms responsible for improved AA utilization in

lactating dairy cows. Thus, if molecular mechanisms can be targeted by AA supplementation,

then lactational responses may be regulated at the cellular level and add to the complexity of

lactation.

Lys

Lysine has one of the highest fractional extraction rates by the mammary gland in bovids,

ovids, and suids (Trottier et al., 1997; Mabjeesh et al., 2000; Manjarin et al., 2014). The primary

metabolic fate of Lys in the mammary gland is for milk protein synthesis. Mabjeesh et al. (2000)

described that the uptake of Lys in the mammary gland of dairy cows and goats is greater than

milk Lys output. Hurley et al. (2000) reported that a small proportion of Lys is oxidized by

mammary tissue. Lapierre et al. (2009) assessed the responses to mammary and splanchnic

metabolism to altered Lys supply in dairy cows. Six catheterized dairy cows received a control

diet with low CP (15.0% DM) plus an abomasal infusion of AAs (560 g/d) without or with Lys

(at 50.3 g/d) (Lapierre et al., 2009). The researchers found that limiting Lys did not alter the

uptake of Lys by the mammary gland. The mammary uptake to milk output ratio of Lys

decreased from 1.37 to 1.12 without Lys in the infusion, but Lys was still in excess of mammary

supply (Lapierre et al., 2009). The excessive uptake of Lys points toward Lys serving roles, other

than milk protein synthesis. The swine NRC (2012) estimates the rate of post-absorptive Lys

utilization into milk protein to be 67%, leaving 33% of Lys to be utilized for other processes,

including oxidative losses (Manjarin et al., 2014). According to Manjarin et al., (2014), Lys must

be involved in other roles, such as utilization of Lys into nonessential AA synthesis and the

utilization rate of 67% is a gross underestimation for the amount of Lys used for milk protein

synthesis.

Met

Methionine extraction by the mammary gland, unlike Lys, closely matches the amounts

required for milk protein synthesis (Bequette et al. 1998). Methionine, as previously stated has

many biological functions (Figure 1) and Met can be utilized to synthesize phospholipids,

carnitine, creatine, and polyamines (Wu, 2013a). To date there is a clear deficit in the literature

on the understanding of Met’s role in mammary tissue except for its incorporation into milk

proteins (Manjarin et al. 2014). According to Manjarin et al. (2014), none of the catabolic

pathways of Met known to occur in other tissues and organs has been studied nor identified in

mammary tissue. The swine NRC (2012) used a coefficient of utilization efficiency for Met of

66%. The utilization efficiency for Met of 66% is most likely underestimated based on the idea

that mammary extraction of Met matches the amount required for milk protein output in ovids

and suids (Bequette et al. 1998; Manjarin et al., 2014). Guan et al. (2004) estimated the

efficiency of Met incorporation into porcine milk protein as being closer to 95%. Lysine and Met

catabolic pathways in mammary tissues need to be further investigated in cattle to get a better

understanding of the function these EAAs play in the mammary gland and on lactation.

Mammalian Target of Rapamycin (mTOR) Pathway

The mammalian target of rapamycin (mTOR) pathway regulates cell cycle progression, cell

growth, and protein synthesis through its ability to integrate nutrient and growth factor signals to

increase protein translation (Manjarin et al., 2014). The effect of individual AA supplementation

on translational protein synthesis has become an important research focus in recent years. The

mTOR pathway in the mammary gland has been fully described, but the molecular mechanisms

that AA influence for mTOR activation to increase protein synthesis in monogastrics and

ruminants is unclear (Manjarin et al., 2014). It is believed that AA availability regulates the

mTOR signaling pathway, by controlling translational initiation rates and by suppressing the

inhibitory activity of eIF4E binding protein 1 (Arriola Apelo et al., 2014a). Amino acids may

also increase elongation rates by stimulating eukaryotic elongation factor 2 and enhance

ribosomal activity through activation of ribosomal protein S6 (Arriola Apelo et al., 2014a).

Eukaryotic elongation factor 2 has been thought to be a limiting factor for milk protein synthesis

(Christophersen et al., 2002).

Recently, Ma et al. (2019) sought to evaluate the effect of supplementing RPMet around

parturition on the quantity and phosphorylation of mTOR related signaling proteins in post-partal

bovine mammary tissue. The researchers found lower phosphorylation of mTOR complex 1 and

were unable to find the exact mechanisms responsible for increased protein synthesis due to

RPMet supplementation (Ma et al., 2019). However, they attributed the increased milk protein

percentage post-partal to increased DMI which may increase AA flux into the mammary gland.

Increased protein synthesis could also be attributed to increases in AKT serine/threonine kinase 1

phosphorylation status and a cascade of intracellular events, leading to upregulation of AA and

glucose transporters (Ma et al., 2019). It is evident that the exact molecular mechanisms that AA

influence for mTOR activation is unclear and further research needs to be conducted. The

findings from such studies could lead to a better understanding of why production responses to

supplementation of RPAAs are inconsistent and how molecular mechanisms could potentially

influence milk protein responses in lactating dairy cows.

Current Approaches and Limitations

Bioavailability of AA in dairy cows has been assessed in vivo by a few techniques which

include the production response approach (Schwab et al., 2001; Fleming et al., 2019), the area-

under-the-curve method (Graulet et al., 2005; Fleming et al., 2019), the plasma free AA dose-

response technique (Rulquin and Kowalczyk, 2003; Borucki Castro et al., 2008; Hanigan et al.,

2009), and the revised plasma free Lys dose-response technique (Whitehouse et al., 2017). The

most recent technique described in the literature is the stable isotope-based approach which is

used for the assessment of intestinally absorbed AAs from individual feed ingredients (Estes et

al., 2018). Although each approach has been described in the literature, caution should be taken

when reviewing each BA method, because each approach has limitations as is discussed below.

The major limitation for the production response approach is being able to ensure an AA

deficiency, for all treatment animals on the trial, over the entire range of the treatment dosages

(Whitehouse et al., 2017). The complexity of AA interactions in the mammary gland,

specifically with the mTOR pathway, also limits the utility of the production response approach.

If animals respond to AA supplementation with a positive linear response in milk protein

concentration or yield, this could either be due the AA exerting its effect directly in the

mammary gland, through activation of a molecular pathway, or nutrient interactions to stimulate

milk protein synthesis. The AA deficiency is necessary in order to obtain a linear response to AA

supplementation in either milk protein concentration (%) or milk protein yield (kg/d)

(Whitehouse et al., 2017).

For the area-under-the-curve technique, animals receive a single pulse dose of the RPAA in

high quantities not normally encountered by rumen microbes, which may limit or alter the

ruminal RPAA degradation, thus affecting the amount of AA that reaches the small intestine for

absorption (Whitehouse et al., 2017). The limitation for the plasma dose-response technique was

that all trials that used this technique, prior to the revised Lys technique, failed to assign dietary

treatments simultaneously within the same Latin square, thus ignoring potential animal variation

(Whitehouse et al., 2017).

A stable isotope non-invasive method for measuring BA of AA has been studied in ruminants

(Borucki Castro et al., 2008; Estes et al., 2017). There are 2 isotope dilution techniques that

either utilize a bolus or constant rate infusion of the AA isotope of interest (Estes, 2016). The

isotopic measurements are used to estimate the total AA flux from the labeled AA infused

dosage and subsequent isotopic enrichment of the samples selected to represent the compartment

(i.e., pool) of interest (Estes, 2016). The bolus approach uses a single isotope dose delivered into

the compartment of interest (Estes, 2016). The constant infusion approach uses a similar

principal, but the infusion is continued until enrichment in the compartment of interest reaches a

steady state to calculate a rate of total entry into the compartment (Estes, 2016).

Borucki Castro et al. (2008) assessed the efficacy of a whole-body Lys net flux method to

determine BA of Lys in dairy cows fed 3 different forms of soybean meal. The whole-body net

flux method (i.e., isotopic method) was tested against the plasma dose-response technique and

duodenal flow/intestinal digestion method. Borucki Castro et al. (2008) found that the plasma

response method recovered 97% of the omasally infused Lys and the isotope method recovered

100% of the omasally infused Lys, suggesting that there was no loss of the infused dose. The

researchers concluded that the isotope method is sensitive and accurate in determining Lys

availability in dairy cattle, which could be beneficial in eliminating the use of cannulated animals

for BA studies (Borucki Castro et al., 2008). However, the plasma response curve yielded similar

results to the isotopic method and further research is needed to assess the validity of an isotope

approach.

RPMet

For RPMet products, the BA is generally calculated based on a positive or negative dietary

control. Most often, Smartamine M is the positive control of reference value with a known BA of

80%. However, the equation to derive BA of AA will be dependent on the method used to

measure BA (e.g., production response approach, the area-under-the-curve method, the plasma

free AA dose-response technique, or the stable isotope-based approach). Before a reference BA

value for Smartamine M was established, Berthiaume et al. (2000) stated that RPMet BA should

be based on a product’s AA concentration, ruminal stability, and intestinal digestibility. Koenig

and Rode (2001) defined BA of RPMet as a combination of effective ruminal degradability and

intestinal disappearance with the calculation being equal to: (100 – effective ruminal

degradability) × intestinal digestibility. Koenig and Rode (2001) estimated the BA of Mepron to

be a 23.6% based on the above calculation. Recently, Abdi-Benemar (2016) reported a BA of

61% for Mepron, which is closer to that of Smartamine M.

Bioavailability of Smartamine M was first estimated to be 80% from nylon bag studies and

determination of its ruminal stability exceeding 90% at 24 h and intestinal release values close to

90% (Schwab and Ordway, 2003). Robert and Williams (1997), estimated that the BA of

Smartamine M falls somewhere between 75 to 97%, with an average BA value of 88%. Graulet

et al. (2005) determined the BA of Smartamine M to be 74%, while Schwab (1995) found the

BA to be 80%.

The BA of HMBi is between 40 and 58%, based on blood kinetics (Robert et al., 2001;

Robert et al., 2002) and milk true protein indices (Schwab et al., 2001). Bioavailability

determined by blood Met concentration was 53 and 74% for HMBi and Smartamine M (Graulet

et al., 2005). The measurements from Graulet et al. (2005) indicate BA of HMBi to be 71% of

that from Smartamine M, whereas another study indicated that BA of HMB was 50% of that of

Smartamine M (Robert et al., 2001).

RPLys

Limited data is available in the literature on the BA of RPLys products. However, the most

studied RPLys product is AjiPro-L and multiple generations of AjiPro-L have been developed

(i.e., generations 1-3). Recently, Whitehouse et al. (2017) estimated the BA of AjiPro-L 2G and

AjiPro-L 3G using the plasma free Lys dose-response method. The slope for the AjiPro-L 3G

was numerically greater than that for the AjiPro-L 2G, which resulted in a 12% improvement in

the BA (Whitehouse et al., 2017). Tucker et al. (2015) compared the BA of 3 commercially

available RPLys products using the plasma free Lys dose-response method (MetaboLys; H.J.

Baker & Bro., Inc., Westport, CT and Lysine USA; Kemin Industries Inc., Des Moines, IA

against the second generation AjiPro-L; Ajinomoto Heartland, Inc.). The BA of MetaboLys and

Lysine USA were estimated as 18.3 and 38.2% of the second generation AjiPro-L BA,

respectively (Tucker et al., 2015).

CONCLUSIONS

Methionine and Lys are recognized as the 2 most limiting AA for milk protein synthesis and

milk production in dairy cows. With this in mind, considerable advancements have been made in

both protein and AA nutrition of dairy cows. These include the development of RPAA sources

with varying encapsulation coatings, the development of Met analogues, and the development of

cost-effective Met and Lys derivatives. As prices of feed protein continue to rise and as

environmental issues remain a concern, producers and nutritionists will need to evaluate the

potential of feeding lower CP diets with strategic AA supplementation. Incorporation of a

multiple limiting AA concept in an animal requirement model will likely add to the complexity

of feeding dairy cattle. Although not necessary for animal requirement models, molecular and

cellular mechanisms (i.e., mTOR pathway) can identify sources of variation in response to AA

supplementation of dairy cows and potential discrepancies in BA data. A sound approach to

determine BA of AA in lactating dairy cattle may not be practical, but the prospect of an in vivo

stable isotope-based method could help with precision feeding of cattle.

There is an overall insufficiency of information on Met and Lys derivatives in lactating

cattle. The dissertation emphasis will be on Met (NALM) and Lys (NALL) acetylated derivatives

supplemented to lactating dairy cows. Their impacts on lactational performance (NALM and

NALL) and BA of NALM in lactation cows will be assessed. It was hypothesized that increased

Met and Lys supply from the AA derivatives being fed (NALM and NALL) would improve

lactational performance by increasing milk yield; however, the degree of response would depend

upon the AA derivative supplemental dose in the dietary treatments. It was anticipated that

NALM will act as an effective source for delivering BA Met equivalents to the small intestine to

be absorbed, transported into the bloodstream, and utilized by the animal for various biological

functions.

REFERENCES

Abdelmegeid, M. K., M. Vailati-Riboni, A. Alharthi, F. Batistel, and J. J. Loor. 2017.

Supplemental methionine, choline, or taurine alter in vitro gene network expression of

polymorphonuclear leukocytes from neonatal Holstein calves. J. Dairy Sci. 100:3155–3165.

https://doi.org/10.3168/jds.2016 -12025.

Adbi-Benemar, H., M. Dehghan-Banadaky, K. Rezayazdi, and Y. Abdollahi. 2016. The

comparison of the amount of methionine supply by different rumen-protected methionine

sources. J. Agr. Sci. Tech. 18:1773-1780.

Adisseo Inc. 2018. Safety data sheet MetaSmart Dry. https://adisseo.biz/products/

metasmart/msds.

Adisseo Inc. 2018. Safety data sheet MetaSmart Liquid. https://adisseo.biz/products/

metasmart/msds.

Amaro, F. X., K. G. Arriola, Y. Jiang, D. Kim, A. P. Cervantes, V. P. Silva, M. C. N. Agarussi,

J. T. Silva, A. T. Adesogan, L. F. Ferraretto, C. R. Staples, J.-S. Eun, J. S. Park, J. O. Moon,

and D. Vyas. 2019. Lactational performance of dairy cows in response to supplementing N-

acetyl-L-methionine as a source of rumen protected methionine. J. Dairy Sci. 101, Suppl. 2

(Abstract 485).

Amos, H. E., C. O. Little, G. A. Digenis, G. T. Schelling, R. E. Tucker, and G. E. Mitchell. 1974.

Methionine, DL-Homocysteine thiolactone and N-Acetyl-L-Methionine for ruminants. J.

Ani. Sci. 39(3):612-617.

Appuhamy, J. A. D. R. N., J. R. Knapp, O. Becvar, J. Escobar, and M. D. Hanigan. 2011. Effects

of jugular-infused lysine, methionine, and branched-chain amino acids on milk protein

synthesis in high-producing dairy cows. J. Dairy Sci. 94:1952-1960.

Appuhamy, J. A. D. R. N., N. A. Knoebel, W. A. D. Nayananjalie, J. Escobar, and M. D.

Hanigan. 2012. Isoleucine and leucine independently regulate mTOR signaling and protein

synthesis in MAC-T cells and bovine mammary tissue slices. J. Nutr. 142:484–491.

Arriola Apelo, S. I., A. L. Bell, K. Estes, J. Ropelewski, M. J. de Veth, and M. D. Hanigan.

2014a. Effects of reduced dietary protein and supplemental rumen-protected essential amino

acids on the nitrogen efficiency of dairy cows. J. Dairy Sci. 97:5688–5699.

Arriola Apelo, S. I., J. R. Knapp, and M. D. Hanigan. 2014b. Invited review: Current

representation and future trends of predicting amino acid utilization in the lactating dairy

cow. J. Dairy Sci. 97:4000–4017.

Baldin, M., G. I. Zanton, and K. J. Harvatine. 2018. Effect of 2-hydroxy-4-(methylthio)butanoate

(HMTBa) on risk of biohydrogenation-induced milk fat depression. J. Dairy Sci. 101:376-

385.

Bender, M. L., H. Matsui, R. J. Thomas, and S. W. Tobey. 1961. The concurrent alkaline

hydrolysis and isotopic oxygen exchange of several alkyl benzoates and lactones. J. Am.

Chem. Soc. 83:4193–4196.

Benefield, B. C., R. A. Patton, M. J. Stevenson, and T. R. Overton. 2009. Evaluation of rumen-

protected methionine sources and period length on performance of lactating dairy cows

within Latin squares. J. Dairy Sci. 92:4448–4455.

Benevenga, N. J. 1974. Toxicities of methionine and other amino acids. J. Agric. Food Chem.

22:2-9.

Bequette, B. J., F. R. C. Backwell , and L. A. Crompton. 1998. Current concepts of amino acid

and protein metabolism by the lactating ruminant mammary gland. J Dairy Sci 8:2540–2559.

Bernard, J., P. Chandler, C. Sniffen, and W. Chalupa. 2014. Response of cows to rumen-

protected lysine after peak lactation. Prof. Anim. Sci. 30:407–412.

Berthiaume, R., H. Lapierre, M. Stevenson, N. Cote, and B. W. McBride. 2000. Comparison of

the in situ and in vivo intestinal disappearance of ruminally protected methionine. J. Dairy

Sci. 83:2049-2056.

Bertoni, G., and E. Trevisi. 2013. Use of the liver activity index and other metabolic variables in

the assessment of metabolic health in dairy herds. Vet. Clin. North Am. Food Anim. Pract.

29:413–431.

Blauwiekel, R., S. Xu, J. H. Harrison, K. A. Loney, R. E. Riley, and M. C. Calhoun. 1997. Effect

of whole cottonseed, gossypol, and ruminally protected lysine supplementation on milk yield

and composition. J. Dairy Sci. 80:1358–1365.

Block, S. M., and T. C. Jenkins. 1994. The use of prilled fat to coat and protect amino acids from

ruminal degradation. J. Sci. Food Agric. 65:441–447.

Borucki Castro, S. I., H. Lapierre, L. E. Phillip, P. W. Jardan, and R. Berthiaume. 2008. Towards

non-invasive methods to determine the effect of treatment of soya-bean on lysine availability

in dairy cows. Animal 2:224–234.

Broderick, G. A., M. J. Stevenson, and R. A. Patton. 2009. Effect of dietary protein

concentration and degradability on response to rumen-protected methionine in lactating dairy

cows. J. Dairy Sci. 92:2719–2728.

Broderick, G. A., T. Kowalczyk, and L. D. Satter. 1970. Milk production response to

supplementation with encapsulated methionine per os or casein per abomasum. J. Dairy Sci.

53:1714–1721.

Brosnan, J.T. and M.E. Brosnan. 2006. The sulfur-containing amino acids: an overview. J. Nutr.

136:1636S-1640S.

Brosnan, J.T., M.E. Brosnan, R.F.P. Bertolo, J.A. Brunton. 2007. Methionine: A metabolically

unique amino acid. Livestock Science 112:2-7.

Campbell, C. G., E. C. Titgemeyer, and G. St-Jean. 1996. Efficiency of D- vs L-Methionine

utilization by growing steers. J. Anim. Sci. 74:2482–2487.

Carey, F. A. 2003. Carboxylic acid derivatives: Nucleophilic acyl substitution. Pages 830–885 in

Organic Chemistry. 5th ed. S. R. Oberbroeckling, ed. McGraw-Hill, New York, NY.

Chen, C., J. E. Sander and N. M. Dale. 2003.The effect of dietary lysine deficiency on the

immune response to Newcastle disease vaccination in chickens. Avian Dis. 47:1346–1351.

doi: 10.1637/7008.

Chen, Z. H., G. A. Broderick, N. D. Luchini, B. K. Sloan, and E. Devillard. 2011. Effect of

feeding different sources of rumen-protected methionine on milk production and N-

utilization in lactating dairy cows. J. Dairy Sci. 94:1978–1988.

Christensen. H. N. 1984. Organic ion transport during the seven decades: The amino acids.

Biochim. Biophys. Acta 7792%.

Christophersen, C. T., J. Karlsen, M. O. Nielsen, and B. Riis. 2002. Eukaryotic elongation factor-

2 (EEF-2) activity in bovine mammary tissue in relation to milk protein synthesis. J. Dairy

Res. 69:205–212.

Citil, M., M. Karapehlivan, H. M. Erdogan, R. Yucayurt, E. Atakisi, and O. Atakisi. 2009. Effect

of orally administered L-carnitine on selected biochemical indicators of lactating Tuj-ewes.

Small Rumin. Res. 81:174–177.

Daabees, T. T., D. W. Andersen, W. L. Zike, L. J. Filer Jr., and L. D. Stegink. 1984. Portal and

vena caval methionine concentrations in young pigs administered L-methionine, N-acetyl-L

methionine and N-acetyl-D-methionine. J. Nutr. 114:1541–1547.

Dibner, J.J. and C.D. Knight. 1984. Conversion of 2-hyrdoxy-4-(methylthio)butanoic acid to L-

methionine in the Chick: A stereospecific pathway. J.N. 144:1716-1723.

Digenis, G. A., H. E. Amos, K. Yang, G. E. Mitchell, C. O. Little, J. V. Swintosky, R. C. Parish,

G. T. Schelling, E. M. Dietz, and R. E. Tucker. 1974. Methionine substitutes nutrition I:

stability of nitrogenous compounds related to methionine during in vitro incubation with

rumen microorganisms. Journal of Pharmaceutical Sciences. 67(5):745-750.

Doyle, P. T. 1981. Sulfur and methionine metabolism in sheep. V. Utilization of methionine

isomers. Aust. J. Biol. Sci. 34:47–59.

Drabkin, H. J. and U. L. RajBhandary. 1998. Initiation of protein synthesis with codons other

than AUG and amino acids other than methionine. Mol. Cell. Biol. 15, 5140-5147.

Estes, K. A. Assessing intestinal absorption of amino acids utilizing an isotope based approach.

Masters Theses. http://hdl.handle.net/10919/84352.

Estes, K. A., R. R. White, P. S. Yoder, T. Pilonero, H. Schramm, H. Lapierre, and M. D.

Hanigan. 2018. An in vivo stable isotope-based approach for assessment of absorbed amino

acids from individual feed ingredients within complete diets. J. Dairy Sci. 101:7040-7060.

Feng, X., R. R. White, H. A. Tucker, and M. D. Hanigan. 2018. Meta-analysis of 2-hydroxy-4-

methylthio-butanoic acid supplementation on ruminal fermentation, milk production, and

nutrient digestibility. J. Dairy Sci. 101:7182-7189.

Fleming, A. J., K. A. Estes, H. Choi, B. A. Barton, C. A. Zimmerman, and M. D. Hanigan. 2019.

Assessing bioavailability of ruminally protected methionine and lysine prototypes. J. Dairy

Sci. 102:1-11.

Gade, W., and J. L. Brown. 1981. Purification, characterization and possible function of alpha-

N-acylamino acid hydrolase from bovine liver. Biochim. Biophys. Acta. 662:86–93.

Giallongo, F., M. T. Harper, J. Oh, J. C. Lopes, H. Lapierre, R. A. Patton, C. Parys, I. Shinzato,

and A. N. Hristov. 2016. Effects of rumen-protected methionine, lysine, and histidine on

lactation performance of dairy cows. J. Dairy Sci. 99:4437–4452.

Giardina, T., A. Biagini, F. Dalle Ore, E. Ferre, M. Reynier, and A. Puigserver. 1997. The hog

intestinal mucosa acylase I: subcellular localization, isolation, kinetic studies and biological

function. Biochimie. 79:265–273.

Girma, D. D., L. Ma, F. Wang, Q. R. Jiang, T. R. Callaway, J. K. Drackley, and D.P. Bu. 2019.

Effects of close-up dietary energy level and supplementing rumen-protected lysine on energy

metabolites and milk production in transition cows. 2019. J. Dairy Sci. 102.7059-7072.

Grass, G. M., and R. R. Unangst. 1972. Glycerol tristearate and higher fatty acid mixtures for

improving digestive absorption. Smith Kline French Lab, assignee. U.S. patent 3,655,864.

Graulet, B., C. Richard and J.C. Robert. 2005. Methionine availability in plasma of dairy cows

supplemented with methionine hydroxyl analog isopropyl ester. J. Dairy Sci. 88:3640-3649.

Gresenti, T. G. 2017. Lactational performance and energy partitioning of dairy cows

supplemented with N-acetyl-L-methionine during mid to late lactation. All Graduate Theses

and Dissertations. 6906. https://digitalcommons.usu.edu/etd/6906.

Guan, X., J. E. Pettigrew, P. K. Ku, K. N. Ames, B. J. Bequette, N. L. Trottier. 2004. Dietary

protein concentration affects plasma arteriovenous difference of amino acids across the

porcine mammary gland. J Anim Sci 82:2953–2963.

Han, H., J. Yin, B. Wang, X. Huang, J. Yao, J. Zheng, W. Fan, T. Li, and Y. Yin. 2018. Effects

of dietary lysine restriction on inflammatory responses in piglets. Scientific Reports 8,

Article number: 2451. DOI:10.1038/s41598-018-20689-3.

Hanigan, M. D., C. Vanderhoof, S. Garbade, O. Becvar, C. A. Umberger, and M. J. de Veth.

2009. In vivo determination of lysine bioavailability of rumen protected lysine in lactating

dairy cows. J. Dairy Sci. 92(E-Suppl. 1):290. (Abstr.)

Jacobs, S. 2002. Practical experiences with L-carnitine. Lohmann Inf. Int. 26:21–24.

Jacometo, C. B., A. S. Alharthi, Z. Zhou, D. Luchini, and J. J. Loor. 2018. Maternal supply of

methionine during late pregnancy is associated with changes in immune function and

abundance of microRNA and mRNA in Holstein calf polymorphonuclear leukocytes. J.

Dairy Sci. 101:8146-8158. https://doi.org/10.3168/jds.2018-14428.

Jacometo, C. B., Z. Zhou, D. Luchini, E. Trevisi, M. N. Correa, and J. J. Loor. 2016. Maternal

rumen-protected methionine supplementation and its effect on blood and liver biomarkers of

energy metabolism, inflammation, and oxidative stress in neonatal Holstein calves. J. Dairy

Sci. 99:6753–6763. https:// doi .org/ 10 .3168/jds .2016 -11018.

Ji, P., H. A. Tucker, R. E. Clark, M. Miura, and C. S. Ballard. 2016. Short communication:

Effect of on-farm feeding practices on rumen protected lysine products. J. Dairy Sci.

99:1242–1246.

Jones, B.A., O.E. Mohamed, R.W Prange, and L.D. Satter. 1988. Degradation of methionine

hydroxyl analog in the rumen of lactating dairy cows. J. Dairy Sci. 71:525-529.

Koenig, K. M. and L. M. Rode. 2001. Ruminal degradability, intestinal disappearance, and

plasma methionine response of rumen-protected methionine in dairy cows. J. Dairy Sci.

84:1480-1487.

Koenig, K.M., L.M. Rode, C.D. Knight, and P.R. McCullough. 1999. Ruminal escape,

gastrointestinal absorption, and response of serum methionine to supplementation of liquid

methionine hydroxyl analog in dairy cows. J. Dairy Sci. 82:355-361.

Koenig, K.M., L.M. Rode, C.D. Knight, and M. Vàzquez-Añon. 2002. Rumen degradation and

availability of various amounts of liquid methionine hydroxyl analog in lactating dairy cows.

J. Dairy Sci. 85:930-938.

Kung, L., Jr., and L. M. Rode. 1996. Amino acid metabolism in ruminants. Anim. Feed Sci.

Technol. 59:167–172.

Lapierre, H., G. Holtrop, A. G. Calder, J. Renaud, and G. E. Lobley. 2012. Is d-methionine

bioavailable to the dairy cow? J. Dairy Sci. 95:353–362.

Lapierre H, L. Doepel, E. Milne, and G. E. Lobley. 2009. Responses in mammary and splanchnic

metabolism to altered lysine supply in dairy cows. Animal 3:360–371.

Lapierre, H., R. Berthiaume, G. Raggio, M. C. Thivierge, L. Doepel, D. Pacheco, P. Dubreuil,

and G. E. Lobley. 2005. The route of absorbed nitrogen into milk protein. J. Anim. Sci.

80:11-22.

Lean, I. J., M. B. de Ondarza, C. J. Sniffen, J. E. P. Santos, and K. E. Griswold. 2018. Meta-

analysis to predict the effects of metabolizable amino acids on dairy cattle performance. J.

Dairy Sci. 101:340–364.

Lee, C., A. N. Hristov, T. W. Cassidy, K. S. Heyler, H. Lapierre, G. A. Varga, M. J. de Veth, R.

A. Patton, and C. Parys. 2012b. Rumen protected lysine, methionine, and histidine increase

milk protein yield in dairy cows fed a metabolizable protein-deficient diet. J. Dairy Sci.

95:6042–6056.

Lee, C., F. Giallongo, A. N. Hristov, H. Lapierre, T. W. Cassidy, K. S. Heyler, G. A. Varga, and

C. Parys. 2015. Effect of dietary protein level and rumen-protected amino acid

supplementation on amino acid utilization for milk protein in lactating dairy cows. J. Dairy

Sci. 98:1885–1902.

Lehninger, A.L. 1977. Biochemistry. 2nd Ed. Worth Publishers Inc., New York.

Leonardi, C., M. Stevenson, and L. E. Armentano. 2003. Effect of two levels of crude protein

and methionine supplementation on performance of dairy cows. J. Dairy Sci. 86:4033–4042.

Liang, Y., F. Batistel, C. Parys, and J. J. Loor. 2019. Glutathione metabolism and nuclear factor

erythroid 2-like 2 (NFE2L2)-related proteins in adipose tissue are altered by supply of ethyl-

cellulose rumen-protected methionine in peripartal Holstein cows. J. Dairy Sci. 102:5530-

5541.

Liang, S. L., Z. H. Wei, J. J. Wu, X. L. Dong, J. X. Liu, and D. M. Wang. 2019. Effect of N-

acetyl-L-methionine supplementation on lactation performance and plasma variables in mid-

lactating dairy cows. J. Dairy. Sci. 102:5182-5190.

Liao, S. F., Wang, T., & Regmi, N. 2015. Lysine nutrition in swine and the related monogastric

animals: muscle protein biosynthesis and beyond. SpringerPlus, 4, 147. doi:10.1186/s40064-

015-0927-5.

Liu, G. M., M. D. Hanigan, X. Y. Lin, K. Zhao, F. G. Jiang, R. R. White, Y. Wang, Z. Y. Hu,

and Z. H. Wang. 2017. Methionine, leucine, isoleucine, or threonine effects on mammary cell

signaling and pup growth in lactating mice. J. Dairy Sci. 100:4038–4050.

Lobley, G. E., H. Lapierre, J. Dibner, D. S. Parker, and M. Vazquez-Anon. 2001. HMB

metabolism in ruminants. Pages 15–23 in Proc. Southwest Nutrition & Management Conf.

College of Agriculture, University of Arizona, Tucson.

Locasale, J. W. Serine, glycine and one-carbon units: cancer metabolism in full circle. Nat. Rev.

Cancer. 2013 Aug;13(8):572-83.

Ma, Y. F., F. Batistel, T. L. Xu, L. Q. Han, R. Bucktrout, Y. Liang, D. N. Coleman, C. Parys,

and J. J. Loor. 2019. Phosphorylation of AKT serine/threonine kinase and abundance of milk

protein synthesis gene networks in mammary tissue in response to supply of methionine in

periparturient Holstein cows. J. Dairy Sci. 102:1-11.

Mabjeesh, S. J., C. E. Kyle, J. C. MacRae, and B. J. Bequette. 2000. Lysine metabolism by the

mammary gland of goats at two stages of lactation. J Dairy Sci 83:996–1003

Mackle, T. R., D. A. Dwyer, and D. E. Bauman. 1999. Effects of branched-chain amino acids

and sodium caseinate on milk protein concentration and yield from dairy cows. J. Dairy Sci.

82:161–171.

Manjarin, R., B. J. Bequette, G. Wu, and N. L. Trottier. 2014. Linking our understanding of

mammary gland metabolism to amino acid nutrition. Amino Acids. 46:2447-2462.

Martinov, M. V., V. M. Vitvitsky, R. Banerjee, and F. I. Ataullakhanov. 2009. The logic of the

hepatic methionine metabolic cycle. Biochim. Biophys. Acta 1804:89–96.

Mosharov E, Crawford MR, Banerjee R. 2000. The quantitatively important relationship

between homocysteine metabolism and glutathione synthesis by the transulfuration pathway

and its regulation by redox changes. Biochemistry. 39:13005–11.

Mitchell, H. H., and R. J. Block. 1946. Some relationships between the amino acid contents of

proteins and their nutritive values for the rat. J. Biol. Chem. 163:599–620.

Noftsger, S., N.R. St-Pierre, and J.T. Sylvester. 2005. Determination of rumen degradability and

ruminal effects of three sources of methionine in lactating dairy cows. J. Dairy Sci. 88:223-

237.

Novus International Inc. 2015. GPS safety summary Alimet

https://novusint.my.salesforce.com/sfc/p/#E0000000Xz5h/a/44000000MEPp/rSkGZ5ojfXn_.

clwu_cDyzaeuVaaR5qa.MpBsBEgvm0.

Novus International Inc. 2015. GPS safety summary MFP

https://na98.salesforce.com/sfc/p/#E0000000Xz5h/a/E00000008n5E/DyWOSIzYmJbL6VcG

9u8MseXdqsLWWK8rFMEilkRLEhk.

NRC. 2001. Nutrient Requirements of Dairy Cattle. 7th ed. Natl. Acad. Press, Washington, DC.

Nursten, H. E. 2005. Nutritional Aspects. Pages 101-108 In: The Maillard Reaction: Chemistry,

Biochemistry, and Implications.

Ordway, R., and G. Aines. 2010. Feeding lysine: A nutritionist and dairy producer’s perspective.

Industry Presentation. The high plains dairy conference.

Ordway, R. S., S. E. Boucher, N. L. Whitehouse, C. G. Schwab, and B. K. Sloan. 2009. Effects

of providing two forms of supplemental methionine to periparturient Holstein dairy cows on

feed intake and lactational performance. J. Dairy Sci. 92:5154–5166.

Osorio, J. S., E. Trevisi, P. Ji, J. K. Drackley, D. Luchini, G. Bertoni, and J. J. Loor. 2014.

Biomarkers of inflammation, metabolism, and oxidative stress in blood, liver, and milk

reveal a better immunometabolic status in peripartal cows supplemented with Smartamine M

or MetaSmart. J. Dairy Sci. 97:7437–7450.

Osorio, J. S., P. Ji, J.K. Drackley, N.D. Luchini, and J.J. Loor. 2013. Supplemental Smartamine

M or MetaSmart during the transition period benefits postpartal cow performance and blood

neutrophil function. J. Dairy Sci. 96:6248.

Overton, T. R., L. S. Emmert, and J. H. Clark. 1998. Effects of source of carbohydrate and

protein and rumen-protected methionine on performance of cows. J. Dairy Sci. 81:221–228.

Patton, R. A. 2010. Effect of rumen-protected methionine on feed intake, milk production, true

milk protein concentration, and true milk protein yield, and the factors that influence these

effects: A meta-analysis. J. Dairy Sci. 93:2105–2118.

Patton, R. A., R. McCarthy, and L. Griel. 1970. Observations on rumen fluid, blood serum, and

milk lipids of cows fed methionine hydroxy analog 1, 2. J. Dairy Sci. 53:776–780.

Patton, R. A., A. N. Hristov, and H. Lapierre. 2014. Protein feeding and balancing for amino acid

in lactating dairy cattle. Pages 599–621 in Veterinary Clinics of North America. R. J. Van

Saun and R. A. Smith, ed. Elsevier, Philadelphia, PA.

Paz, H. A., and P. J. Kononoff. 2014. Lactation responses and amino acid utilization of dairy

cows fed low-fat distillers dried grains with solubles with or without rumen-protected lysine

supplementation. J. Dairy Sci. 97:6519-6530.

Phillips, W. A., K. E. Webb, Jr. and J. P. Fontenot. 1976. In vitro absorption of amino acids by

the small intestine of sheep. J. Anim. Sci. 42:201.

Piepenbrink, M.S., A.L. Marr, M.R. Waldron, W.R. Butler, T.R. Overton, M. Vasquez-Anon,

and M.D. Holt. 2004. Feeding 2-hydroxy-4-(methylthio)-butanoic acid to periparturient dairy

cows improves milk production but not hepatic metabolism. J. Dairy Sci. 87:1071-1084.

Pirestani, A., M. Aghakhani, S. Tabatabaei, G. Ghalamkari, and F. Baharlo. 2011. Effects of

dietary L-carnitine and choline chloride compound on reproduction indices and udder

immune system in Holstein dairy cattle. Pages 59–61 in 2011 International Conference on

Life Science and Technology. IPCBEE vol. 3. IACSIT Press, Singapore.

Polan, C.E., K.A. Cummins, C.J. Sniffen, T.V. Muscato, J.L. Vicini, B.A. Crooker, J.H. Clark,

D.G. Johnson, D.E. Otterby, and B. Guillaume. 1991. Responses of dairy cows to

supplemental rumen-protected forms of methionine and lysine. J. Dairy Sci. 74 (9):2997-

3013.

Polan, C. E., P. T. Chandler, and C. N. Miller. 1970. Methionine hydroxy analog: Varying levels

for lactating cows. J. Dairy Sci. 53:607–610.

Reis, P. J., D. A. Tunks, and L. F. Sharry. 1989. Incorporation of abomasal and intravenous

doses of [35S] cysteine and [35S] methionine into wool. J. Agric. Sci. 112:313–319.

Reiners, J. N., J. E. Held, C. L. Wright, and D. W. Brake. 2017. Short communication: Lysine

retained among 2 lipid-coated lysine products after exposure to alfalfa or corn silage with

different amounts of acidity. J. Dairy. Sci. 101:5159-5165.

Robert, J. C. and P. Williams. 1997. Influence of forage type on the intestinal availability of

methionine from a rumen protected form. J. Dairy Sci. 80:(Suppl.1):248. (Abstr.)

Robert, J.-C, C. Richard, and B. Bouza. 2001. Influence of monomer and dimer forms of

isopropyl ester of HMB on the supply of metabolizable methionine to the blood of ruminants.

J. Dairy Sci. 84:(Suppl.1):281.

Robert, J.-C, T. d’Alfonso, G. Etave, E. Depres, and B. Bouza. 2002. Quantifying the

metabolizable methionine contribution of a liquid or powder presentation of 2-hydroxy-4-

(methylthio) butanoic acid isopropyl ester (HMBi). J. Dairy Sci. 85:(Suppl.1):71.

Robinson, P., E. DePeters, I. Shinzato, and H. Sato. 2006. Rumen lysine escape, rumen

fermentation, and productivity of early lactation dairy cows fed free lysine. Anim. Feed Sci.

Technol. 128:31–41.

Robinson, P. H. 2009b. Can manipulating ration metabolizable protein amino acid levels really

be expected to effect environmental impacts of cows? Proceedings of the Southwest

Nutrition Conference, Arizona State University, Tucson, AZ, USA pp. 34-46.

Robinson, P. H. 2010. Impacts of manipulating ration metabolizable lysine and methionine levels

on the performance of lactating dairy cows: A systemic review of the literature. Livestock

Science. 127:115-126.

Robinson, P. H., N. Swanepoel, and E. Evans. 2010. Effects of feeding a ruminally protected

lysine product, with or without isoleucine, valine and histidine, to lactating dairy cows on

their productive performance and plasma amino acid profiles. Anim. Feed Sci. Technol.

161:75-84.

Robinson, P. H., N. Swanepoel, I. Shinzato, and S. O. Juchem. 2011. Productive responses of

lactating dairy cattle to supplementing high levels of ruminally protected lysine using a

rumen protection technology. Anim. Feed Sci. Technol. 168:30–41.

Rudell, J. B., A. J. Rechs, T. J. Kelman, C. M. Ross-Inta, S. Hao, and D.W. Gietzen. 2011. The

anterior piriform cortex is sufficient for detecting depletion of an indispensable amino acid,

showing independent cortical sensory function. J. Neurosci. 31:1583–1590.

Rulquin, H., and J. Kowalczyk. 2003. Development of a method for measuring lysine and

methionine bioavailability in rumen-protected products for cattle. J. Anim. Feed Sci. 12:465–

474.

Rulquin, H., and L. Delaby. 1997. Effects of the energy balance of dairy cows on lactational

responses to rumen-protected methionine. J. Dairy Sci. 80:2513–2522.

Rulquin, H., P. M. Pisulewski, R. Ve´rite´, and J. Guinard. 1993. Milk production and

composition as a function of postruminal lysine and methionine supply: a nutrient response

approach. Livest. Prod. Sci., 37:69–90.

Samuelson, D. J., S. K. Denise, R. Roffler, R. L. Ax, D. V. Armstrong, and D. F. Romagnolo.

2001. Response of Holstein and Brown Swiss cows fed alfalfa hay-based diets to

supplemental methionine at two stages of lactation. J. Dairy Sci. 84:917–928.

Schugar, R.C., and P.A. Crawford. 2012. Low-carbohydrate ketogenic diets, glucose

homeostasis and nonalcoholic fatty liver disease. Curr. Opin. Clin. Nutr. Metab. Care. 15(4):

374-380.

Schwab, C. G. and G. A. Broderick. 2017. A 100-year review: protein and amino acid nutrition

in dairy cows. J. Dairy Sci. 100:10094-10112.

Schwab C. G. and R. S. Ordway. 2003. Methionine supplementation options. In: Proc Four-State

Applied Nutrition Conference, Paper 2, Schwab.

Schwab, C. G., C. K. Bozak, N. L. Whitehouse, and M. M. A. Mesbah. 1992. Amino acid

limitation and flow to the duodenum at four stages of lactation. I. Sequence of lysine and

methionine limitation. J. Dairy Sci. 75:3486–3502.

Schwab, C.G., N.L. Whitehouse, A.M. McLaughlin, R.K. Kadariya, N.R. St-Pierre, B.K. Sloan,

R.M. Gill, and J.-C. Robert. 2001. Use of milk protein concentration to estimate the

methionine bioavailability of two forms of 2-hydroxy-4-(methylthio) butanoic acid (HMB)

for lactating cows. J. Dairy Sci. 84:(Suppl.1):35

Seymour, W. M. 2016. Role of methionine and methionine precursors in transition cow nutrition

with emphasis on liver function. Proc. Florida Ruminant Nutrition Symposium.

Shingfield, K., and J. M. Griinari. 2007. Role of biohydrogenation intermediates in milk fat

depression. European Journal of Lipid Science and Technology.

Shingfield, K., and R. Wallace. 2014. Synthesis of conjugated linoleicacid in ruminants and

humans. Pages 1–65 in Conjugated Linoleic Acids and Conjugated Vegetable Oils. B. Sels

and A. Philippaerts, ed. Royal Society of Chemistry, London, UK.

Shug, A. L., M. J. Schmidt, G. T. Golden, and R. G. Fariello. 1982. The distribution and role of

carnitine in the mammalian brain. Life Sci. 31:2869–2874.

Sinclair, K. D., P. C. Garnsworthy, G. E. Mann, and L. A. Sinclair. 2014. Reducing dietary

protein in dairy cow diets: Implications for nitrogen utilization, milk production, welfare and

fertility. Animal 8:262–274.

Spencer, R. P., T. M. Bow and M. A. Markulis. 1962. Amino group requirement for in vitro

intestinal transport of amino acids. Am. J. Physiol. 20:22-71.

St-Pierre, N. R., and J. T. Sylvester. 2005. Effects of 2-hydroxy4- (methylthio) butanoic acid

(HMB) and its isopropyl ester on milk production and composition by Holstein cows. J.

Dairy Sci. 88:2487–2497.

Stead L.M., M. E. Brosnan, J. T. Brosnan, D. E. Vance, and R. L. Jacobs. 2006. Is it time to re-

evaluate methyl balance in humans? Am J Clin Nutr. 83:5–10.

Stoll, B. and D. G. Burrin. 2006. Measuring splanchnic amino acid metabolism in vivo using

stable isotopic tracers. J. Anim. Sci. 84:E60-E72.

Storm, E., and E.R. Ørskov. 1983. The nutritive value of rumen microorganisms in ruminants. 1.

Large-scale isolation and chemical composition of rumen mircroorganisms. Br. J. Nutr.

50:463-470.

Sun, F., Y. Cao, C. Cai, S. Li, C. Yu, and J. Yao. 2016. Regulation of nutritional metabolism in

transition dairy cows: Energy homeostasis and health in response to post-ruminal choline and

methionine. PLoS One 11:e0160659.

Swanepoel, N., P. H. Robinson, and L. Erasmus. 2010. Amino acid needs of lactating dairy

cows: impact of feeding lysine in a ruminally protected form on productivity of lactating

dairy cows. Anim. Feed Sci. Technol. 157:79–94.

Titgemeyer, E. C., and N. R. Merchen. 1990. Sulfur-containing amino acid requirement of

rapidly growing steers. J. Anim. Sci. 68:2075–2083.

Toledo, M. Z., G. M. Baez, A. Garcia-Guerra, N. E. Lobos, J. N. Guenther, E. Trevisol, D.

Luchini, R. D. Shaver, and M. C. Wiltbank. 2017. Effect of feeding rumen-protected

methionine on productive and reproductive performance of dairy cows. PLOS ONE 12(12):

e0189117. https://doi.org/10.1371/journal.pone.0189117

Tom, D. and C. Bos. 2007. Lysine requirement through the human life cycle. The Journal of

Nutrition Volume 137. Issue 6. Pages 1642S–1645S, https://doi.org/10.1093/jn/137.6.1642S

Třináctý, J., L. Křížová, M. Richter, V. Černý, and J. Říha. 2009. Effect of rumen-protected

methionine, lysine or both on milk production and plasma amino acids of high-yielding dairy

cows. Czech Journal of Animal Science. 54. 10.17221/1730-CJAS.

Trottier, N. L., C. F. Shipley, and R. A. Easter. 1997. Plasma amino acid uptake by the mammary

gland of the lactating sow. J Anim Sci 75:1266–1278.

Tucker, H. A., M. Miura, I. Shinzato, and C. S. Ballard. Comparative bioavailability of lysine in

three commercial rumen-protected lysine products using the in vivo plasma lysine response

method. ADSA/ASAS Joint Animal Meeting. (Abstract W351).

Vailati-Riboni, M., Z. Zhou, C. B. Jacometo, A. Minuti, E. Trevisi, D. N. Luchini, and J. J. Loor.

2017. Supplementation with rumen-protected methionine or choline during the transition

period influences whole-blood immune response in periparturient dairy cows. J. Dairy Sci.

100:3958-3968.

Vanhatalo A, Huhtanen P, Toivonen V, et al. 1999. Response of dairy cows fed grass silage diets

to abomasal infusions of histidine alone or in combination with lysine and methionine. J

Dairy Sci. 82:2674–2685.

Vaz, F. M., and R. J. Wanders. 2002. Carnitine biosynthesis in mammals. Biochem. J. 361:417–

429

Vyas D. and R. A. Erdman. 2009. Meta-analysis of milk protein yield responses to lysine and

methionine supplementation. J. Dairy Sci. 92:5011-5018.

Wallace, R. J. 1992. Acetylation of peptides inhibits their degradation by rumen micro-

organisms. British J. Nutr. 68:365-372.

Wang, C., H. Y. Liu, Y. M. Wang, Z. Q. Yang, J. X. Liu, Y. M. Wu, T. Yan, and H. W. Ye.

2010. Effects of dietary supplementation of methionine and lysine on milk production and

nitrogen utilization in dairy cows. J. Dairy Sci. 93:3661–3670.

Webb, K. E. Jr. 1990. Intestinal absorption of protein hydrolysis products: A review. J. Anim.

Sci. 68:3011-3022.

Webb, K. E. Jr. and J. C. Matthews. 1994. “Absorption of amino acids and peptides.” Principles

of Protein Nutrition of Ruminants, edited by J. Malcolm Asplund, CRC Press, 127-146.

Weeks T.L., P. H. Luimes, and J. P. Cant. 2006. Responses to amino acid imbalances and

deficiencies in lactating dairy cows. J. Dairy Sci. 89:2177–87.

Wegner, M. I., A. N. Booth, G. Bohstedt, and E. B. Hart. 1940. The “in vitro” conversion of

inorganic nitrogen to protein by microorganisms from the cow’s rumen. J. Dairy Sci.

23:1123–1129.

Whitehouse, N. L., A. Brito, C. Schwab, I. Shinzato, and M. Muira. 2017. Bioavailability of

AjiPro-L 2G and AjiPro-L 3G using the plasma free lysine dose-response technique. J. Dairy

Sci. 101: Suppl. 2 (Abstract M257).

Whitehouse, N. L., C. G. Schwab, and A. F. Brito. 2017. The plasma free amino acid dose-

response technique: A proposed methodology for determining lysine relative bioavailability

of rumen-protected lysine supplements. J. Dairy Sci. 100:9585-9601.

Williams, V. J. 1969. The relative rates of absorption of amino acids from the small intestine of

the sheep. Comp. Biochem. Physiol. 29:865.

Wilson, J. W. and K. E. Webb Jr. 1990. Lysine and methionine transport by bovine jejunal and

ileal brush border membrane vesicles. J. Anim. Sci. 68:504-514.

Wilson, K.R., C.S. Abney, J.T. Vasconcelos, M. Vàzquez-Añon, J.P. McMeniman, and M.L.

Galyean. 2008. Effects of 2-hydroxy-4-(methylthio)-butanoic acid on performance and

carcass characteristics of finishing beef cattle and on fermentation in continuous culture. J.

Anim. Sci. 86:1951-1962.

Windschitl, P. M., and M. D. Stern. 1988. Influence of methionine derivatives on effluent flow

of methionine from continuous culture of ruminal bacteria. J. Anim. Sci. 66:2937–2947.

Wu, G. 2013. Functional amino acids in nutrition and health. Amino acids 45, 407–411,

https://doi.org/10.1007/s00726-013-1500-6.

Wu, G. 2013a Amino Acids: Biochemistry and Nutrition. CRC Press, Boca Raton

Wu, S. H., and A. Papas. 1997. Rumen-stable delivery systems. Adv. Drug Deliv. Rev. 28:323–

334.

Zanton, G. I., G. R. Bowman, M. Vázquez-Añon, and L. M. Rode. 2014. Meta-analysis of

lactation performance in dairy cows receiving supplemental dietary methionine sources or

postruminal infusion of methionine. J. Dairy Sci. 97:7085–7101.

Zhang., Q., and H. M. White. 2019. Short communication: The effect of increasing

concentrations of different methionine forms and 2-hydroxy-4-(methylthio)-butanoic acid on

genes controlling methionine metabolism in primary bovine neonatal hepatocytes. J. Dairy

Sci. 102:1-5.

Zhou, Z., E. Trevisi, D. N. Luchini, and J. J. Loor. 2017. Differences in liver functionality

indexes in peripartal dairy cows fed rumen-protected methionine or choline are associated

with performance, oxidative stress status, and plasma amino acid profiles. J. Dairy Sci.

100:6720-6732.

Zhou, Z., M. Vailati-Riboni, E. Trevisi, J. K. Drackley, D. N. Luchini, and J. J. Loor. 2016.

Better postpartal performance in dairy cows supplemented with rumen-protected methionine

compared with choline during the peripartal period. J. Dairy Sci. 99:8716–8732.

Figure 1. Biological functions of Met and hepatic Met metabolism. BHMT = Betaine-

homocysteine methyltransferase; CBS = cystathionine-β-synthase; CSE = cystathionase; DMG =

dimethylglycine; GSH = glutathione; GNMT = glycine N-methyltransferase; HCY =

homocysteine; MAT1A = Met adenosyltransferase 1A; MTR = Met reductase; MT =

methyltransferase; 5-MTHF = 5-methyltetrahydrofolate; SAH = S-adenosylhomocysteine;

SAHH = SAH hydrolase; SAM = S-adenosyl-Met and THF = tetrahydrofolate. Adapted from

Zhang and White, (2017).

CHAPTER 1

Influence of supplementing a methionine derivative, N-acetyl-L-methionine in dairy diets

on production and ruminal fermentation by lactating cows during early to mid-lactation

-Journal of Dairy Science, 2018, Vol. 101, Issue 8, p7082-7094

ABSTRACT

The present study investigated production responses and ruminal fermentation characteristics

of lactating dairy cows when supplemented with N-acetyl-L-Met (NALM) as a source of rumen-

protected Met in metabolizable protein (MP) -deficient (MPD) or MP-adequate diet (MPA).

Eight lactating dairy cows (53  10.4 d-in-milk, on average) were blocked by parity and days-in-

milk, and the experiment was performed in a replicated 4 × 4 Latin square design. Within each

square, cows were randomly assigned to a sequence of 4 diets during each of the four 21-d

periods (14 d of treatment adaptation and 7 d of data collection and sampling). A 2 × 2 factorial

arrangement was used; MPD or MPA was combined without or with NALM: MPD without

NALM, MPD with NALM (MPD+NALM), MPA without NALM, and MPA with NALM

(MPA+NALM). A NALM product was supplemented in the MPD+NALM and the

MPA+NALM at 30 g/cow/d. Supplementation of NALM did not affect dry matter intake (DMI)

and milk yield regardless of MP concentration. In addition, supplementing NALM resulted in a

similar milk true protein concentration and yield. In contrast, NALM supplementation increased

milk fat concentration and yield and 3.5% fat-corrected milk (FCM) yield and tended to increase

energy-corrected milk (ECM) yield regardless of MP difference. Additionally, trends were

observed for increased 3.5% FCM yield/DMI and ECM yield/DMI, and the positive effects were

greater under the MPA than the MPD diet, resulting in trends toward interactions between MP

and NALM. Dietary treatments had similar effects on ruminal fermentation characteristics and

microbial protein yield. Plasma concentration of Met increased under the MPD but not the MPA

diet, leading to an MP × NALM interaction. Overall results in the current study suggest that

NALM exerted minor influence on ruminal metabolism, but increased milk fat concentration,

resulting in increases in milk fat yield and feed efficiency. Yet, potential effects of NALM on

intermediary metabolism between the gastrointestinal tract, the liver, and the mammary gland

need to be explored to understand utilization efficiency for production of dairy cows.

Key Words: N-acetyl-L-methionine, feed efficiency, lactational performance, milk fat

concentration

INTRODUCTION

Dietary sources of N in lactation diets are inefficiently utilized by dairy cows, as they excrete

substantially more N in manure over milk, which increases milk production costs and

environmental N excretion (Noftsger et al., 2005). Optimizing the balance of total AA in MP has

been used as the most promising means to maximize lactational performance with minimal

dietary CP supply (Lee et al., 2012a). Studies have suggested that Lys and Met are the most

limiting AA, especially in high producing dairy cows fed alfalfa and corn silage-based

commercial diets typically used in the United States, and optimal concentrations of these limiting

AA in MP are required to increase milk protein synthesis (Schwab et al., 1976). Thus, balancing

for these AA is of practical significance as an effective approach to enhance milk protein

production. With an increasing economic value of milk protein, there has been strong interest by

dairy producers and industries in nutritional modifications such as supplementation of rumen-

protected AA that will increase milk protein yield (Arriola Apelo et al., 2014a).

Considerable research has been conducted over the past 3 decades to develop technologies to

protect AA from ruminal degradation, and consequently ruminally protected Met (RPMet)

products in different chemical forms have been developed and extensively used to alleviate Met

deficiencies in lactating dairy cows (Overton et al., 1998; Patton, 2010). However, attempts to

increase milk protein yield by increasing the supply of Met by addition of RPMet have given

mixed results mainly due to complex inter-organ metabolisms in the rumen, the liver, and the

mammary gland (Bequette et al., 2003) and substantial variations on DMI and CP concentration

in feeds (Sinclair et al., 2014; Zanton et al., 2014).

The N-acetyl-L-Met (NALM) is a Met derivative produced via protection of L-Met α-amino

group with an N-acetyl group. The cost of production for N-acetyl DL-Met by addition of an α-

amino N-acetyl group to DL-Met is economically infeasible, but a cost-effective method for

NALM production was recently developed (CJ CheilJedang, Seoul, South Korea) by addition of

an α-amino N-acetyl group to L-Met. The NALM has been shown to be bioavailable and capable

of replacing dietary Met in animals and humans according to Rotruck and Boggs (1975). In

addition, NALM was readily incorporated into tissue proteins and supported growth of rats

(Rotruck and Boggs, 1975). The NALM has also been used in foods as an approved food

additive (Baxter et al., 2002). Windschitl and Stern (1988) reported that Met flowing out of

continuous cultures increased with N-acetyl DL-Met administration without a change in bacterial

Met flow, which was attributed to greater escape (67%) of the N-acetyl DL-Met from bacterial

degradation. In the literature, however, there is no research reported on the effects of

supplementing NALM in lactation diet on production and ruminal fermentation in dairy cows.

We conducted the present study to investigate lactational performance and nutrient utilization

by lactating dairy cows supplemented without or with NALM in MP-deficient (MPD) or MP-

adequate diet (MPA), according to NRC (2001). We hypothesized that increased Met supply

from NALM would increase milk protein synthesis and milk yield, but the degree of the response

would depend upon the MP supply in the diets.

MATERIALS AND METHODS

The dairy cows used in the present study were cared for according to the Live Animal Use in

Research Guidelines of the Institutional Animal Care and Use Committee at Utah State

University. The study was conducted at the Caine Dairy Research Center (Wellsville, UT), Utah

State University from May to July, 2016.

Cows, Experimental Design, and Diets

Eight multiparous lactating Holstein cows were used during this trial. Cows began the

experiment averaging 53 ± 10.4 DIM, producing 39.2 ± 5.13 kg/d of milk yield, consuming 24.2

± 3.51 kg/d of DMI and an average BW of 696 ± 66.2 and 721 ± 57.3 kg at the beginning and the

end of the experiment, respectively. The experiment was performed in a replicated 4 × 4 Latin

square design. Within each square, cows were randomly assigned to a sequence of 4 diets during

each of the four 21-d periods (14 d of treatment adaptation and 7 d of data collection and

sampling). Although primary production parameters were measured in the present study, caution

needs to interpret the data due to the short-term observation. Within each square, cows were

randomly assigned to a sequence of 4 dietary treatments with a 2 × 2 factorial arrangement: MPD

without NALM diet (MPD−NALM); MPD with NALM diet (MPD+NALM); MPA without

NALM diet (MPA−NALM), and MPA with NALM (MPA+NALM; Table 1-1).

The developmental NALM product from CJ CheilJedang was used as a supplemental source

of RPMet in this study. The NALM product was in powder form, and contained Met

concentration of 78.0%, with 99.5% purity. The NALM product was added at a rate of 0.13%

DM, and the rate was determined for cows to consume 30 g/cow/d of Met in order to supply 15.6

g of digestible Met to the small intestine according to manufacturer’s recommendation. This rate

was also based on the requirements of digestible Met for cows fed the MPD diet. The

recommendation of required digestible Lys and Met were assumed as 6.6 and 2.2% of the MP

requirements, respectively (NRC, 2001). The NALM product was top-dressed onto

corresponding experimental diets.

The chemical composition of alfalfa hay was 21.6, 38.9, and 27.9% DM for CP, NDF, and

ADF, respectively, whereas corn silage contained 5.32, 41.5, and 23.9% DM for CP, NDF, and

ADF, respectively. The diets had a forage-to-concentrate ratio of 61:39 (DM basis) on average

and were typical of high-producing dairy cows in the Intermountain West (i.e., Utah, Idaho,

Wyoming, Montana, and parts of Arizona and Nevada) with 44% of the forage coming from

good-quality alfalfa hay. Diets were formulated based on the NRC (2001) recommendations to

provide sufficient NE

, MP, vitamins, and minerals with an estimated intake of 25.0 kg/d to

produce 40 kg/d of milk with 3.5% fat and 3.0% true protein.

Cows were housed individually in tie stalls fitted with rubber mattresses covered with straw,

allowing free access to water. Cows were individually fed twice daily for ad libitum intake at a

level of 110% expected daily intake with 70% of allotted feed fed at 0600 h and 30% fed at 1500

h. Feed offered and refused was recorded daily, and samples were taken during the sampling

week to determine DMI. Cows were milked twice daily at 0400 and 1600 h, and milk production

was recorded throughout the entire experiment.

Energy Partitioning Calculations

Energy partitioning was determined during treatment periods using data of milk yield, milk

composition, and BW of experimental animals. Cows were weighed for 2 consecutive d after the

a.m. milking and before the a.m. feeding at the beginning (d 1 and 2) and end (d 20 and 21) of

each period. These weights were used to calculate the mean BW of cows for each experimental

period. Energy used for maintenance was calculated as BW

0.75

× 0.08 (NRC, 2001). Energy of

BW change was assumed to be 5.114 Mcal/kg of gain or 4.924 Mcal/kg of loss (NRC, 2001).

Milk energy was calculated as (0.0929 × milk fat concentration) + (0.0563 × milk true protein

concentration) + (0.0395 × milk lactose concentration) (NRC, 2001). Estimated NE

value was

calculated by total net energy utilization (maintenance, BW gain, and milk) divided by DMI

(Neal et al., 2014).

Feed and Milk Samplings and Analyses

Samples of alfalfa hay and corn silage were taken weekly to determine DM, and diets were

adjusted accordingly for change in DM concentration. Samples were composited by month,

ground to pass a 1-mm screen (standard model 4; Arthur H. Thomas Co., Swedesboro, NJ), and

stored for chemical analysis. Samples of TMR and orts were collected from individual cows on d

15 to d 21, composited, dried at 60°C for 48 h, and ground as previously described. The DM

concentrations of samples were used to calculate intakes of DM and nutrients.

Analytical DM concentration of samples was determined by oven drying overnight at 105°C,

and OM was determined by ashing at 550°C for 5 h (AOAC, 2000; method 942.05).

Concentration of CP was determined using an automated N combustion analyzer (Elementar,

Analysensysteme GmbH, Hanau, Germany; AOAC, 2000; method 968.06). Concentrations of

NDF and ADF were sequentially determined using a fiber analyzer (200/220, ANKOM

Technology, Macedon, NY) according to the methodology supplied by the company, which is

based on the methods described by Van Soest et al. (1991). Sodium sulfite was used in the

procedure for NDF determination and pre-treated with heat-stable amylase (Type XI-A from

Bacillus subtilis; Sigma-Aldrich Corporation, St. Louis, MO). Ether extract was measured using

a fat analyzer (XT20, ANKOM Technology; AOAC, 2000; method 2003.05). In addition,

samples of TMR were analyzed for starch by Dairyland Laboratories, Inc. (Arcadia, WI)

according to Knudsen (1997).

Milk was sampled for 2 consecutive days (d 15 and 16) during the a.m. and p.m. milkings

each period. Individual milk samples were analyzed by the Rocky Mountain DHIA Laboratory

(Providence, UT) for fat, true protein, lactose, and MUN. Milk composition was expressed on

weighted milk yield of a.m. and p.m. samples. Milk fat and protein yields were calculated by

multiplying milk yield from the respective day by fat and true protein concentration of the milk

from an individual cow.

Blood and Urine Samplings and Analyses

Blood samples were collected immediately before the morning feeding as well as at 1000 and

1400 h into 10-mL plasma vacuum tubes from the coccygeal artery or vein on d 19 and 20 in

each period. Blood samples were centrifuged at 2,300 × g for 20 min, and plasma was collected

and stored at ‒40°C for subsequent AA analysis. The plasma samples were prepared for AA

analysis using the EZ:faast GC-FID Free (Physiological) Amino Acid Analysis Kit (Phenomenex

Inc., Torrance, CA). Concentrations of plasma free AA were determined in accordance with the

user manual provided with the kit. Extraction of free AA from the plasma consisted of a

combination of solid-phase extraction, derivatization, and liquid/liquid extraction. The organic

phase containing the AA in question were analyzed using a GC (model 5890, Hewlett-Packard

Lab) with a capillary column (30 m × 0.32-mm i.d., 1-μm phase thickness, Zebron ZB-FAAP;

Phenomenex Inc., Torrance, CA) and flame-ionization detection.

On d 15 to 17, spot urine samples were collected from each cow at 0600 and 1800 h for a

total of 6 samples per cow (Holt et al., 2013). Using 4 M HCl urine samples were acidified to pH

< 4.0 and composited by cow per period. Samples were frozen and stored at –40°C. At a later

date, the samples were thawed in preparation for analysis and diluted with 39 parts diluent to 1

part urine. The diluent utilized consisted of 0.202% sodium 1-heptanesulfonate and 0.086%

ammonium dihydrogen phosphate (NH

). The solution was brought to a pH of 2.1 using 4

M HCl. Utilization of the ratio of the urinary purine derivatives (PD) to creatinine is an accepted

way to estimate the microbial protein (MCP) flow to the duodenum (Shingfield and Offer,

1998). The PD and creatinine were analyzed using an HPLC instrument (Waters Corp., Milford,

MA) according to the procedures set forth by Shingfield and Offer (1999). In order to estimate

urine volume, creatinine was used as a marker (Valadares et al., 1999), and an average creatinine

output of 28 mg/kg of BW as estimated by Whittet (2004) was assumed. Similar creatinine

outputs have been reported (25 to 30 mg/kg of BW daily) (McCarthy et al., 1983; Jones et al.,

1990). In order to estimate the relative differences in MCP production, the ratio of urinary PD

(allantoin and uric acid) to creatinine was used (Shingfield and Offer, 1998), and the supply of

MCP was estimated based on estimates of urinary excretion of PD according to the method of

Chen et al. (1990) and Janicek et al. (2008).

Ruminal Fermentation Characteristics

Ruminal fluid samples were obtained using a Geishauser probe at 0, 3, and 6 h after the

morning feeding on d 18 and 19 in each period. The fluid was collected with a solid, tube-like

probe with rows of small holes on the end (Geishauser, 1993). The first 100 mL of ruminal fluid

was discharged to avoid contamination from saliva, and then 10 mL was collected for analysis.

The pH of the ruminal fluid was measured within 5 min of collecting the samples using a

portable pH meter (Oakton pH 6; Oakton Instruments, Vernon Hills, IL). Five milliliters of the

ruminal fluid were mixed with 1 mL of 1% sulfuric acid and stored frozen (−40°C) for ammonia-

N (NH

-N) analysis. Concentration of NH

-N in the ruminal contents was determined as

described by Rhine et al. (1998), using a plate reader (MRXe; Dynex Technologies Inc.,

Chantilly, VA). Another 5 mL of the ruminal fluid was collected and mixed with 1 mL of 25%

metaphosphoric acid, and then stored at −40°C for VFA content determination. Ruminal VFA

were separated and quantified using a GLC (model 6890 series II; Hewlett-Packard Co.,

Avondale, PA) with a capillary column (30 m × 0.32-mm i.d., 1-μm phase thickness, Zebron ZB-

FAAP; Phenomenex Inc., Torrance, CA) and flame-ionization detection. The oven temperature

was held at 170°C for 4 min, increased to 185°C at a rate of 5°C/min, then increased by 3°C/min

to 220°C, and held at this temperature for 1 min. The injector and the detector temperatures were

225 and 250°C, respectively, and the carrier gas was helium (Eun and Beauchemin, 2007).

Statistical Analysis

All data were analyzed as a 4 × 4 replicated Latin square with a factorial arrangement of

treatments using the Proc Mixed procedure of SAS (version 9.4; SAS Institute Inc., Cary, NC).

Data for intake, milk production, and energy partitioning were analyzed with a model that

accounted for effects of square, MP difference (MPD vs. MPA), supplementation of NALM

(−NALM vs. +NALM), day nested within period, and the interaction between MP, NALM, and

day as fixed effects and cow within square and period within square as random effects with day

included as a repeated measure. Data for ruminal pH, VFA, and NH

-N were analyzed using the

model described above except that time after feeding was included as the repeated option. For

each variable analyzed, 3 covariance structures (compound symmetry, autoregressive order 1,

and unstructured covariance) were evaluated. The covariance structure that resulted in the lowest

values for the Akaike information criterion and the Schwartz Bayesian criterion was used (Littell

et al., 1998). Kenward-Roger's option was used to calculate the denominator degrees of freedom.

Residual errors were used to test main effects and interactions. Differences were considered

significant at P ≤ 0.05, and trends were discussed at 0.05 < P ≤ 0.10.

RESULTS AND DISCUSSION

To the best of our knowledge, this is the first time that the effects of supplementing NALM

in lactation dairy diets have been described. The lack of data in the literature regarding NALM

led us to indirectly compare our results with data reported for HMBi as well as traditional

polymer-protected Met (RPMet) products.

A continuous culture study estimated ruminal undegradability of N-acetyl-DL-Met at 67%

(Windschitl and Stern, 1988). Monogastric data involving young pigs supplemented with NALM

revealed that no NALM was present in either portal or vena caval plasma at any sampling time

(Daabees et al., 1984), which indicates hydrolysis of NALM to Met in the intestinal lumen and

mucosal cells. The research reported in this paper represents initial attempts to investigate

lactational performance and ruminal fermentation profiles of dairy cows fed with NALM as an

alternative RPMet source.

Characteristics of Diet Composition and Treatment

Ingredients and chemical composition of experimental diets are presented in Table 1-1. The

MPA diet provided adequate MP, whereas the MPD diet was 6% MP-deficient (-181 to -190

g/d). The MPD diet was formulated to contain less MP compared to the MPA diet by reducing

the amount of dietary concentrations of canola and soybean meal in the diets. The 6% MP

deficiency was chosen for the MPD based on the result from Giallongo et al. (2016) who fed an

MP-deficient diet (5-10%) supplemented with RPMet. All diets supplied NE

in excess of

requirement (NRC, 2001). The MPA diet met or exceeded the MP, RDP, and RUP requirements,

while the MPD diet was deficient for MP, RDP, and RUP as expected.

The NRC (2001) model suggests that Lys and Met concentrations in MP for optimal milk

protein production are 7.2 and 2.4%, respectively. These concentrations are difficult to achieve

without feeding very high dietary concentrations of protein. Therefore, it is recommended that

the first step in balancing diets for Lys and Met is to maintain a Lys:Met in MP of 3.0 with more

practical concentrations of Lys and Met in MP being 6.6 and 2.2%, respectively (Schwab et al.,

2005). In this study, supplementing NALM (assuming 67% rumen undegradability) substantially

improved Met status, as indicated by the reduction of the predicted (NRC, 2001) digestible Lys-

to-digestible Met ratio in MP from 3.5 to 2.7 (Table 1-1). The digestible Lys-to-digestible Met

ratio for maximum milk and milk protein yield have traditionally been reported as a 3.00:1

(NRC, 2001, Whitehouse et al., 2009), but optimal ratios as low as 2.69:1 were reported by Van

Amburgh et al. (2015) when using the CNCPS model after calculating the optimum efficiency of

utilization of Lys and Met using data derived by Lapierre et al. (2007) and from a meta-analysis

of 40 published papers (Doepel et al., 2004).

Feed Intake, Productive Performance, and Net Energy Utilization

Intake of DM did not differ among dietary treatments, averaging 25.3 kg/d (Table 1-2). The

MPD and the MPA diet with supplementation of NALM increased CP intake with a greater

increase under the MPA diet, resulting in an MP × NALM interaction. Responses to

supplementation of polymer-coated RPMet and HMBi on feed intake and productive

performance in the literature have been quite variable. Kung and Rode (1996) found that rumen-

protected AA supplementation did not improve DMI, which corresponds to the findings of other

studies performed by Leonardi et al. (2003) and Chen et al. (2011). A limited number of studies

have shown an increase in DMI with RPMet supplementation (Broderick et al., 2009; Zanton et

al., 2014), while Ordway et al. (2009) and Zanton et al. (2014) reported decreases in DMI.

Supplementation of HMBi has also increased DMI (Osorio et al., 2014) because of the potential

stimulatory effect of HMBi on cellulolytic bacteria and the subsequent increase in passage rate

and DMI (Lee et al., 2015). On the other hand, Lee et al. (2012b) assessed supplementation of

RPMet products to MP-deficient diets, as was explored in the current study and reported that

MP-deficient diets below 15% of the NRC requirements decreased DMI. However, the authors

observed that a treatment supplemented with a combination of 3 rumen-protected AA consisted

of Lys (100 g/cow), Met (30 g/cow), and His (50 g/cow) increased DMI (Lee et al., 2012b). An

additional study by the same group (Lee et al., 2015) found that supplementation with rumen-

protected Lys and RPMet in MP-deficient diets (-256 to -305 MP g/d) did not affect DMI. In the

current study, stage of lactation may have interfered with a potential effect of NALM on DMI

under the MPA diet, because peak DMI typically tends to lag behind peak milk in early to mid-

lactating cows that consume feed in order to meet energy needs.

Feeding the MPA diet increased milk yield by 2.2 kg/d relative to the MPD diet, resulting in

more efficient feed utilization for milk production (1.64 vs. 1.57; Table 1-2). Milk yield was not

affected by NALM supplementation, while yields of 3.5% FCM and ECM increased or tended to

increase (P = 0.06) with NALM addition. Noftsger et al. (2005) reported no difference in milk

production when cows were supplemented with HMBi at 0.13% DM. However, St-Pierre and

Sylvester (2005) showed an increase in milk yield (2.9 kg/d) to cows supplemented with HMBi

at 0.15% DM. The observed production responses to supplementation of RPMet in cows fed

low-protein diets (i.e., MP-deficient diets) have been, in most cases, minimal and/or inconsistent

(Arriola Apelo et al., 2014a; Sinclair et al., 2014). No negative effect on milk yield was detected

by feeding a low CP diet (15%) alone or in combination with RPMet supplementation (Arriola

Apelo et al., 2014b). In the current study, a 6% MP-deficient diet decreased milk yield, while

previous research with MP-deficient diets (5-10% below MP requirement) have maintained milk

yield (Lee et al., 2015; Giallongo et al., 2016). In the present study, there was no improvement in

milk yield in response to NALM supplementation in both of the MPD and the MPA diet, thus

indicating that NALM was not utilized for milk production. Alternatively, NALM

supplementation increased yields of 3.5% FCM and ECM, suggesting a potential role on fatty

acid synthesis and delivery to the mammary gland possibly through a methyl donation

mechanism in the liver, which will be discussed later in this paper.

There was a tendency toward MP × NALM interaction on milk fat concentration (P = 0.10),

because NALM supplementation increased milk fat concentration under the MPA, but not the

MPD diet (Table 1-2). However, milk true protein concentration was similar across the diets.

Ordway et al. (2009) showed that HMBi did not increase milk fat concentration, but increased

milk protein concentration in comparison to the control diet. In contrast, milk fat concentration

increased with HMBi supplementation (Dalbach et al., 2011; Osorio et al., 2013). Supplementing

RPMet typically results in an increase in milk protein percentage (Rulquin and Delaby, 1997;

Samuelson et al., 2001; Lara et al., 2006), but one study (Benefield et al., 2009) reported a

negative impact on milk protein concentration. It has been well established that Met acts as a

methyl donor needed in the synthesis of milk fat (Osorio et al., 2016; Sun et al., 2016). The

primary methyl carrier during bovine hepatic methyl metabolism is S-adenosyl-Met (SAM). It

works to donate methyl groups for the generation of phosphatidylethanolamine, acting as

phospholipids for cell membranes in the packaging of very low-density lipoprotein (VLDL;

Purohit et al., 2007). Once packaged, the VLDL consist of triglycerides, apolipoproteins B and

E, phospholipids, and cholesterol, and consequently Met plays a vital role in both the

development of apolipoproteins and phospholipids. The VLDL are then exported from the liver

to the mammary gland for milk fat synthesis (Figure 1-1). Thus, as a potential mode of action,

NALM after its digestion and absorption may have been involved in hepatic Met metabolism

pertaining to fatty acid trafficking, leading to the increased milk fat production in early to mid-

lactation observed in the current study.

It has been reported that aminoacylase I is present in multiple mammalian organs (i.e., small

intestine, liver, and kidney; Giardina et al., 1997). The bovine liver contains the enzyme, α-N-

acylamino acid hydrolase which is capable of hydrolyzing α-N-acylated AA to yield acetate and

the corresponding AA (Gade and Brown, 1981). However, it is unclear how cows utilize the

NALM into conversion of milk fat, once NALM was hydrolyzed by aminoacylase I and

absorbed in the small intestine as acetate and Met (Baxter et al., 2002) as was illustrated in the

Figure 1-1.

Feeding the MPA diet increased milk fat yield compared with the MPD diet, and

supplementing NALM further increased milk fat yield regardless of MP level (Table 1-2).

Although feeding the MPA diet increased milk protein yield compared with the MPD diet,

NALM supplementation did not influence milk protein yield. Noftsger et al. (2005) and St-Pierre

and Sylvester (2005) demonstrated an increase in milk protein yield with HMBi

supplementation. Supplementing HMBi and polymer-coated RPMet typically has more impact

on milk protein vs. milk fat yield (Lara et al., 2006; Rulquin et al., 2006; Ordway et al., 2009).

This is most likely due to Met being the first-limiting essential AA in lactation dairy diets, and

consequently supplementation of HMBi or polymer-coated RPMet is first partitioned to improve

milk protein yield in dairy cows. Contradictory effects have been reported (Phipps et al., 2008

and Ordway et al., 2009) for HMBi on increased milk fat yield. Patton (2010) performed a meta-

analysis on 35 studies evaluating production performance in lactating dairy cows supplemented

with various RPMet products to dairy diets and found increased production of true milk protein

both as a percentage (0.07%) and yield (27 g/d). However, NALM supplementation did not

increase milk protein yield, and thus NALM may have been used as a source of Met in methyl

donation toward VLDL production as previously discussed. Further research is needed to

determine the actual contribution of Met in NALM for VLDL formation and its impacts on milk

production parameters.

For the diets supplemented with NALM, the increased milk fat concentration and no change

in DMI resulted in a tendency toward MP × NALM interaction on 3.5% FCM yield/DMI and

ECM yield/DMI (P = 0.06; Table 1-2). A 12-wk study performed by Chen et al. (2011) showed

that supplementing HMBi to lactating cows averaging 143 DIM increased ECM yield/DMI. Feed

efficiency is a reasonable indicator evaluated on dairy farms, as it helps measure the relative

ability of cows to convert feed nutrients into milk or milk components. The increased feed

efficiencies observed in the current study suggest that NALM supplementation would be

economically favorable in early to mid-lactating cows, but only when fed a diet adequate in MP

concentration. However, caution should be exercised to extrapolate these benefits of NALM in

the current study due to the short length of data collection and, therefore, further investigation is

needed to confirm our data with a relatively long period of experiment.

No changes were seen in either BW gain or net energy utilization with supplementation of

NALM in the diets (Table 1-2). However, feeding the MPA diet increased milk yield relative to

the MPD diet, leading to an increased net energy utilization for milk. Research on RPMet

supplementation on BW gain in ruminants is scarce, but some studies have reported increases in

BW gain upon RPMet supplementation (Deetz et al., 1985; Oke et al., 1986). Methionine is a

limiting AA in protein synthesis for growing cattle (Richardson et al., 1978), and therefore an

increase in available Met can support protein deposition in the body (Loest et al., 2002). The role

of Met in the formation of phospholipids through conversion of Met to SAM will also support fat

deposition in growing cattle (Obeid et al., 2009). In addition, acetate, a component of NALM, is

also recognized for its role as a lipogenic precursor (Bauman and Griinari, 2001). No effect on

BW gain due to NALM supplementation suggests that energy was partitioned for milk fat

production rather than BW gain in early to mid-lactation in the present study.

Plasma AA Profiles

Among EAA, feeding the MPA diet increased plasma concentrations of Ile and Leu

compared with the MPD diet, but supplementing NALM decreased those AA only under the

MPA diet, resulting in an interaction between MP and NALM (Table 1-3). Supplementing

NALM decreased plasma concentration of Val under the MPA but not the MPD diet, leading to a

MP × NALM interaction. In contrast, Noftsger et al. (2005) did not find any difference in Val

concentration when cows were supplemented with HMB, HMBi, or DL-Met. Similarly, St-Pierre

et al. (2005) supplemented a control diet with HMB, HMBi, or a combination of HMB and

HMBi, and the dietary treatments had no effect on free plasma amino acids, including Val.

Plasma concentration of Met increased under the MPD but not MPA, leading to an MP × NALM

interaction. The no effect of NALM supplementation on plasma Met concentration in the MPA

diet observed in this study was unexpected, and its mechanism is difficult to explain. The liver

plays a central role in Met metabolism (Finkelstein, 1990); approximately 50% of Met

metabolism and up to 85% of all methylation reactions occur in the mammalian liver (Mato et

al., 2002). Hepatic gene regulation serves as an important regulatory point for controlling Met

metabolism (Finkelstein, 2003; Mato et al., 2008). However, effects of RPMet supplementation

on genes for Met metabolism in bovine hepatocytes in vivo has not been clearly understood in

lactating dairy cattle. Zhang et al. (2016) demonstrated decreased mRNA expression for genes

that catalyze regeneration of Met and showed the decreased need for cellular regeneration of Met

with increasing Met concentrations from 2-hydroxy-4-(methylthio)-butanoic acid (HMB) and

DL-Met on primary bovine hepatocytes. The authors described that increased Met supply from

HMB and DL-Met in primary bovine hepatocytes was not directed at increased glucose

synthesis, probably due to other metabolic priorities for Met (Zhang et al., 2016). Likewise, in

the current study it is speculated that increased Met supply from NALM supplementation in the

MPA diet may have been prioritized in the bovine hepatocyte to the primary methyl donor SAM,

allowing SAM to be used for increased VLDL synthesis and exportation to the mammary gland

for milk fat synthesis (Figure 1-1).

Ruminal Fermentation Characteristics

Ruminal pH decreased in the MPD relative to the MPA diet (6.24 vs. 6.37; Table 1-4), but

NALM supplementation did not influence ruminal pH. The mean ruminal pH from all dietary

treatments were at least above 6.2, so even the effect of MP difference on overall microbial

physiology may have been minor. Concentration of VFA and their compositions were in general

not affected by MP level as well as NALM supplementation. Similarly, Noftsger et al. (2005)

and Lee et al. (2015) reported no effects of RPMet supplementation on ruminal pH and VFA

production. Concentration of NH

-N also did not differ across the dietary treatments, while MCP

yield increased due to feeding the MPA diet compared with the MPD diet, but NALM did not

affect MCP yield regardless of MP level (Table 1-4). Supplementation of RPMet traditionally

has not had an impact on microbial production, with the exception of Met analogues such as

HMBi because of its partial ruminal degradation and potential stimulatory effects on MCP

synthesis (Robert et al., 2001). Similarly, Lee et al. (2015) reported increased MCP yield with

polymer-coated RPMet supplementation. Although NALM is partially degraded in the rumen

(approximately 33%, Figure 1-1), the degraded fraction of the NALM would not contribute to

MCP synthesis under the experimental condition of our study. Further research is needed to

understand the mode of action of NALM on ruminal metabolism, particularly how the

aminoacylase I enzyme acts on NALM.

CONCLUSIONS

The current study investigated the production responses and ruminal fermentation profiles of

early to mid-lactating dairy cows supplemented without or with a developmental NALM product

in the MPD or the MPA diet. The NALM supplementation did not affect DMI, milk yield, and

milk protein yield or alter ruminal fermentation characteristics in either the MPD or the MPA

diet, suggesting that early to mid-lactating dairy cows tested in this study may have other

metabolic priorities for Met. However, supplementation of NALM regardless of MP difference

increased both 3.5% FCM and ECM yields coupled with increased milk fat concentration which

was more impactful in the MPA diet, leading to better feed efficiencies with the MPA diet. Thus,

supplementing NALM, particularly in the MPA diet, in early to mid-lactation can be a good

approach to formulate an optimal dairy feeding program for improving feed efficiency. Yet, the

potential effects of NALM on intermediary metabolism between the gastrointestinal tract, the

liver, and the mammary gland needs to be explored to understand the metabolic impacts of

NALM on lactational and physiological parameters of dairy cows. Overall, the results in the

current study suggest that NALM exerted minor influence on ruminal metabolism, but increased

milk fat concentration possibly due to accelerated fatty acid delivery to the mammary gland,

resulting in sizable increases in milk fat yield and feed efficiency.

ACKNOWLEDGMENTS

This study was supported by CJ CheilJedang (Seoul, South Korea). The authors thank S.

Sharp at Utah State University (Logan) for technical assistance and the staff of the Caine Dairy

Research Center (Wellsville, UT) for their conscientious care of the experimental cows.

REFERENCES

AOAC. 2000. Official Methods of Analysis. Vol. 1 and 2. 17th ed. AOAC Int., Gaithersburg,

MD.

Arriola Apelo, S. I., A. L. Bell, K. Estes, J. Ropelewski, M. J. de Veth, and M. D. Hanigan.

2014a. Effects of reduced dietary protein and supplemental rumen-protected essential amino

acids on the nitrogen efficiency of dairy cows. J. Dairy Sci. 97:5688–5699.

Arriola Apelo, S. I., J. R. Knapp, and M. D. Hanigan. 2014b. Invited review: Current

representation and future trends of predicting amino acid utilization in the lactating dairy

cow. J. Dairy Sci. 97:4000–4017.

Bauman, D. E., and J. M. Griinari. 2001. Regulation and nutritional manipulation of milk fat:

low-fat milk syndrome. Livest. Prod. Sci. 70:15–29.

Baxter, J., Y. Kim, and M. Snowden. 2002. Hydrolysis of N-acetyl-L-glutamine by acylase I. J.

Food Sci. 66:1428–1433.

Benefield, B. C., R. A. Patton, M. J. Stevenson, and T. R. Overton. 2009. Evaluation of rumen-

protected methionine sources and period length on performance of lactating dairy cows

within Latin squares. J. Dairy Sci. 92:4448–4455.

Bequette, B. J., M. D. Hanigan, and H. Lapierre. 2003. Mammary uptake and metabolism of

amino acids by lactating ruminants. Pages 347–365 in Amino Acids in Animal Nutrition. J.

P. F. D’Mello, ed. CAB International, Wallingford, Oxon, U.K.

Broderick, G. A., M. J. Stevenson, and R. A. Patton. 2009. Effect of dietary protein

concentration and degradability on response to rumen-protected methionine in lactating dairy

cows. J. Dairy Sci. 92:2719–2728.

Chen, X. B., E. R. Ørskov, and F. D. D. Hovell. 1990. Excretion of purine derivatives by

ruminants: endogenous excretion, differences between cattle and sheep. Br. J. Nutr. 63:121–

129.

Chen, Z. H., G. A. Broderick, N. D. Luchini, B. K. Sloan, and E. Devillard. 2011. Effect of

feeding different sources of rumen-protected methionine on milk production and N-

utilization in lactating dairy cows. J. Dairy Sci. 94:1978–1988.

Daabees, T. T., D. W. Andersen, W. L. Zike, L. J. Filer, Jr., and L. D. Stegink. 1984. Portal and

vena caval methionine concentrations in young pigs administered L-methionine, N-acetyl-L-

methionine and N-acetyl-D-methionine. J. Nutr. 114:1541–1547.

Dalbach, K. F., M. Larsen, B. L. M. Raun, and N. B. Kristensen. 2011. Effects of

supplementation with 2-hydroxy-4-(methylthio)-butanoic acid isopropyl ester on splanchnic

amino acid metabolism and essential amino acid mobilization in postpartum transition

Holstein cows. J. Dairy Sci. 94:3919–3927.

Deetz, L. E., A. M. Papas, and C. H. Benton. 1985. Performance of finishing steers fed rumen-

protected methionine and/or lysine. J. Anim. Sci. 61(Suppl. 1):486 (Abstr.).

Doepel, L., D. Pacheco, J. J. Kennelly, M. D. Hanigan, I. F. Lopez, and H. Lapierre. 2004. Milk

protein synthesis as a function of amino acid supply. J. Dairy Sci. 87:1279–1297.

Eun, J. S., and K. A. Beauchemin. 2007. Enhancing in vitro degradation of alfalfa hay and corn

silage using feed enzymes. J. Dairy Sci. 90:2839–2851.

Finkelstein, J. D. 1990. Methionine metabolism in mammals. J. Nutr. Biochem. 1:228–237.

Finkelstein, J. D. 2003. Methionine metabolism in liver diseases. Am. J. Clin. Nutr. 77:1094–

1095.

Gade, W., and J. L. Brown. 1981. Purification, characterization and possible function of alpha-

N-acylamino acid hydrolase from bovine liver. Biochim. Biophys. Acta. 662:86–93.

Giallongo, F., M. T. Harper, J. Oh, J. C. Lopes, H. Lapierre, R. A. Patton, C. Parys, I. Shinzato,

and A. N. Hristov. 2016. Effects of rumen-protected methionine, lysine, and histidine on

lactation performance of dairy cows. J. Dairy Sci. 99:4437–4452.

Geishauser, T. 1993. An instrument for the collection and transfer of ruminal fluid and for the

administration of water soluble drugs in adult cattle. Bovine Pract. 27:38–42.

Giardina, T., A. Biagini, F. Dalle Ore, E. Ferre, M. Reynier, and A. Puigserver. 1997. The hog

intestinal mucosa acylase I: subcellular localization, isolation, kinetic studies and biological

function. Biochimie. 79:265–273.

Holt, M. S., K. Neal, J. S. Eun, A. J. Young, J. O. Hall, and K. E. Nestor. 2013. Corn silage

hybrid type and quality of alfalfa hay affect dietary nitrogen utilization by early lactating

dairy cows. J. Dairy Sci. 96:6564–6576.

Janicek, B. N., P. J. Kononoff, A. M. Gehman, and P. H. Doane. 2008. The effect of feeding

dried distillers grains plus solubles on milk production and excretion of urinary purine

derivatives. J. Dairy Sci. 91:3544–3553.

Jones, S. J., D. L. Starkey, C. R. Calkins, and J. D. Crouse. 1990. Myofibrillar protein turnover

in feed-restricted and realimented beef cattle. J. Anim. Sci. 68:2707–2715.

Knudsen, K. E. B. 1997. Carbohydrate and lignin contents of plant materials used in animal

feeding. Anim. Feed Sci. Technol. 67:319–338.

Kung Jr., L., and L. M. Rode. 1996. Amino acid metabolism in ruminants. Anim. Feed Sci.

Technol. 59:167–172.

Lapierre, H., G. E. Lobley, D. R. Quellet, L. Doepel, and D. A. Pacheco. 2007. Amino acid

requirements for lactating dairy cows: Reconciling predictive models and biology. Pages

39–60 in Proc. Cornell Nutr. Conf., Dept. Anim. Sci., Cornell Univ., Ithaca, NY.

Lara A., Mendoza G.D., Landois L., Barcena R., Sánchez-Torres M.T., Rojo R., Ayala J., and

Vega S. 2006. Milk production in Holstein cows supplemented with different levels of

ruminally protected methionine. Livest. Sci. 105:105–108.

Lee, C., A. N. Hristov, K. S. Heyler, T. W. Cassidy, H. Lapierre, G. A. Varga, and C. Parys.

2012a. Effects of metabolizable protein supply and amino acid supplementation on nitrogen

utilization, milk production, and ammonia emissions from manure in dairy cows. J. Dairy

Sci. 95:5253–5268.

Lee, C., A. N. Hristov, T. W. Cassidy, K. S. Heyler, H. Lapierre, G. A. Varga, M. J. de Veth, R.

A. Patton, and C. Parys. 2012b. Rumen-protected lysine, methionine, and histidine increase

milk protein yield in dairy cows fed a metabolizable protein-deficient diet. J. Dairy Sci.

95:6042–6056.

Lee, C., F. Giallongo., A.N. Hristov., H. Lapierre., T. W. Cassidy., K. S. Heyler., G. A. Varga.,

and C. Parys. 2015. Effect of dietary protein level and rumen-protected amino acid

supplementation on amino acid utilization for milk protein in lactating dairy cows. J. Dairy

Sci. 98:1885–1902.

Leonardi, C., M. Stevenson, and L. E. Armentano. 2003. Effect of two levels of crude protein

and methionine supplementation on performance of dairy cows. J. Dairy Sci. 86:4033–4042.

Littell, R. C., P. R. Henry, and C. B. Ammerman. 1998. Statistical analysis of repeated measures

data using SAS procedures. J. Anim. Sci. 76:1216–1231.

Loest, C. A., E. C. Titgemeyer, G. St-Jean, D. C. Van Metre, and J. S. Smith. 2002. Methionine

as a methyl group donor in growing cattle. J. Anim. Sci. 80:2197–2206.

Mato, J. M., F. J. Corrales, S. C. Lu, and M. A. Avila. 2002. S-Adenosylmethionine: A control

switch that regulates liver function. FASEB J. 16:15–26.

Mato, J. M., M. L. Martínez-Chantar, and S. C. Lu. 2008. Methionine metabolism and liver

disease. Annu. Rev. Nutr. 28:273–293.

McCarthy, F. D., W. C. Bergen, and D. R. Hawkins. 1983. Muscle protein turnover in cattle of

differing genetic backgrounds as measured by urinary N’-methylhistidine excretion. J. Nutr.

113:2455–2463.

Neal, K., J.-S. Eun, A. J. Young, and J. O. Hall. 2014. Feeding protein supplements in alfalfa

hay-based lactation diets improves nutrient utilization, lactational performance, and feed

efficiency of dairy cows. J. Dairy Sci. 97:7716–7728.

Nelson, D. L., and M. M. Cox. 2013. Lehninger Principles of Biochemistry. 6th ed. W. H.

Freeman and Company, New York, NY.

Noftsger, S., N. R. St-Pierre, and J. T. Sylvester. 2005. Determination of rumen degradability

and ruminal effects of three sources of methionine in lactating cows. J. Dairy Sci. 88:223–

237.

National Research Council. 2001. Nutrient Requirements of Dairy Cattle. 7th rev. ed. Natl. Acad.

Press. Washington, DC.

Obeid, R., and W. Herrmann. 2009. Homocysteine and lipids: S-adenosylmethionine as a key

intermediate. FEBS Lett. 583:1215–1225.

Oke, B. O., S. C. Loerch, and L. E. Deetz. 1986. Effects of rumen-protected methionine and

lysine on ruminant performance and nutrient metabolism. J. Anim. Sci. 62:1101–1112.

Ordway, R. S., S. E. Boucher, N. L. Whitehouse, C. G. Schwab, and B. K. Sloan. 2009. Effects

of providing two forms of supplemental methionine to periparturient Holstein dairy cows on

feed intake and lactational performance. J. Dairy Sci. 92:5154–5166.

Osorio, J. S., P. Ji, J. K. Drackley, D. Luchini, and J. J. Loor. 2013. Supplemental Smartamine M

or MetaSmart during the transition period benefits postpartal cow performance and blood

neutrophil function. J. Dairy Sci. 96:6248–6263.

Osorio, J. S., E. Trevisi, P. Ji, J. K. Drackley, D. Luchini, G. Bertoni, and J. J. Loor. 2014.

Biomarkers of inflammation, metabolism, and oxidative stress in blood, liver, and milk

reveal a better immunometabolic status in peripartal cows supplemented with Smartamine M

or MetaSmart. J. Dairy Sci. 97:7437–7450.

Osorio, J. S., C. B. Jacometo, Z. Zhou, D. Luchini, F. C. Cardoso, and J. J. Loor. 2016. Hepatic

global DNA and peroxisome proliferator-activated receptor alpha promoter methylation are

altered in peripartal dairy cows fed rumen-protected methionine. J. Dairy Sci. 99:234–244.

Overton, T. R., D. W. LaCount, T. M. Cicela, and J. H. Clark. 1998. Evaluation of a ruminally

protected methionine product for lactating dairy cows. J. Dairy Sci. 79:631–639.

Patton, R. A. 2010. Effect of rumen-protected methionine on feed intake, milk production, true

milk protein concentration, and true milk protein yield, and the factors that influence these

effects: A meta-analysis. J. Dairy Sci. 93:2105–2118.

Phipps R. H., C. K. Reynolds, D. L. Givens, A. K. Jones, P. A. Geraert, E. Devillard, and R.

Bennett. 2008. Short communication: Effects of 2-hydroxy-4-(methylthio) butanoic acid

isopropyl ester on milk production and composition of lactating Holstein dairy cows. J. Dairy

Sci. 91:4002–4005.

Purohit, V., M. F. Abdelmalek, S. Barve, N. J. Benevenga, C. H. Halsted, N. Kaplowitz, K. K.

Kharbanda, Q. Liu, S. C. Lu, C. J. McClain, C. Swanson, and S. Zakhari. 2007. Role of S-

adenosyl-methionine, folate, and betaine in the treatment of alcoholic liver disease: summary

of a symposium. Am. J. Clin. Nutr. 86:14–24.

Rhine, E. D., G. K. Sims, R. L. Mulvaney, and E. J. Pratt. 1998. Improving the Bertholot

reaction for determining ammonium in soil extracts and water. Soil Sci. Soc. Am. J. 62:473–

480.

Richardson, C. R., and E. E. Hatfield. 1978. The limiting amino acids in growing cattle. J. Anim.

Sci. 46:740–745.

Robert, J. C., C. Richard, T. D. Alfonso, N. Ballet, and E. Depres. 2001. Investigation of the site

of absorption of a novel source of metabolizable methionine: 2-hydroxy-4-(methylthio)

butanoic acid isopropyl ester (HMBi). J. Dairy Sci. 84(Suppl. 1):35 (Abstr.).

Rulquin, H., B. Graulet, L. Delaby, and J. C. Robert. 2006. Effect of different forms of

methionine on lactational performance of dairy cows. J. Dairy Sci. 89:4387–4394.

Rulquin, H., and L. Delaby. 1997. Effects of the energy balance of dairy cows on lactational

responses to rumen-protected methionine. J. Dairy Sci. 80:2513–2522.

Rotruck, J. T., and R. W. Boggs. 1975. Comparative metabolism of L-methionine and N-

acetylated derivatives of methionine. J. Nutr. 105:331–337.

Samuelson, D. J., S. K. Denise, R. Roffler, R. L. Ax, D. V. Armstrong, and D. F. Romagnolo.

2001. Response of Holstein and Brown Swiss cows fed alfalfa hay-based diets to

supplemental methionine at two stages of lactation. J. Dairy Sci. 84:917–928.

Schwab, C. G., L. D. Satter, and A. B. Clay. 1976. Responses of lactating dairy cows to

abomasal infusions of amino acids. J. Dairy Sci. 59:1254–1270.

Shingfield, K. J., and N. W. Offer. 1998. Evaluation of the spot urine sampling technique to

assess urinary purine derivative excretion in lactating dairy cows. J. Anim. Sci. 66:557–568.

Shingfield, K. J., and N. W. Offer. 1999. Simultaneous determination of purine metabolites,

creatinine and pseudouridine in ruminant urine by reversed-phase high performance liquid

chromatography. J. Chromatogr. B Biomed. Sci. Appl. 723:81–94.

Sinclair, K. D., P. C. Garnsworthy, G. E. Mann, and L. A. Sinclair. 2014. Reducing dietary

protein in dairy cow diets: Implications for nitrogen utilization, milk production, welfare and

fertility. Animal 8:262–274.

St-Pierre, N. R., and J. T. Sylvester. 2005. Effects of 2-hydroxy4-(methylthio) butanoic acid

(HMB) and its isopropyl ester on milk production and composition by Holstein cows. J.

Dairy Sci. 88:2487–2497.

Sun, F., Y. Cao, C. Cai, S. Li, C. Yu, and J. Yao. 2016. Regulation of nutritional metabolism in

transition dairy cows: energy homeostasis and health in response to post-ruminal choline and

methionine. PLoS ONE 11:e0160659.

Valadares, R. F. D., G. A. Broderick, S. C. V. Filho, and M. K. Clayton. 1999. Effect of

replacing alfalfa silage with high moisture corn on ruminal protein synthesis estimated from

excretion of total purine derivatives. J. Dairy Sci. 82:2686–2696.

Van Amburgh, M. E., E. A. Collao-Saenz, R. J. Higgs, D. A. Ross, E. B. Recktenwald, E.

Raffrenato, L. E. Chase, T. R. Overton, J. K. Mills, and A. Foskolos. 2015. The Cornell Net

Carbohydrate and Protein System: updates to the model and evaluation of version 6.5. J.

Dairy Sci. 98:6361–6380.

Van Soest, P. J., J. B. Robertson, and B. A. Lewis. 1991. Methods of dietary fiber, neutral

detergent fiber and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci.

74:3583–3597.

Whitehouse, N., C. Schwab, D. Luchini, T. Tylutki, and B. Sloan. 2009. Comparison of optimal

lysine and methionine concentrations in metabolizable protein estimated by the NRC (2001),

CPM-Dairy (v.3.0.10) and AMTS.Cattle (v.2.1.1) models. J. Dairy Sci. 92(Suppl. 1):103

(Abstr.).

Whittet, K. M. 2004. Factors affecting variability in urinary creatinine and purine derivative

excretion in beef cattle. MS Thesis. Univ. of Nebraska, Lincoln.

Windschitl, P. M., and M. D. Stern. 1988. Influence of methionine derivatives on effluent flow

of methionine from continuous culture of ruminal bacteria. J. Anim. Sci. 66:2937–2947.

Zanton, G. I., G. R. Bowman, M. Vázquez-Añon, and L. M. Rode. 2014. Meta-analysis of

lactation performance in dairy cows receiving supplemental dietary methionine sources or

postruminal infusion of methionine. J. Dairy Sci. 97:7085–7101.

Zhang, Q., S. J. Bertics, N. D. Luchini, and H. M. White. 2016. The effect of increasing

concentrations of DL-methionine and 2-hydroxy-4-(methylthio) butanoic acid on hepatic

genes controlling methionine regeneration and gluconeogenesis. J. Dairy Sci. 99:8451–8460.

Table 1-1. Ingredient and chemical composition of the experimental diets without or with N-

acetyl-L-Met (NALM) at 2 different levels of MP fed to lactating Holstein dairy cows (n = 4)

Item

Experimental diet

MPD

MPA

−NALM

+NALM

−NALM

+NALM

Ingredient, % of DM

Alfalfa hay

27.0

25.8

25.7

Wheat straw

2.49

2.38

Corn silage

32.5

31.0

Corn grain (steam-flaked)

11.6

11.1

Corn grain (high-moisture)

6.17

5.90

5.89

Cottonseed, whole

4.88

4.87

4.66

4.65

Canola meal

3.50

5.68

Soybean meal

3.25

3.24

5.27

Beet pulp, shreds

4.93

4.92

4.71

4.70

NALM

−

0.13

−

0.13

Sodium bicarbonate

1.08

1.03

Vitamin and mineral mix

2.54

2.43

2.42

Chemical composition, % of DM

DM, %

60.9 ± 0.53

60.7 ± 0.32

61.0 ± 0.72

62.0 ± 1.50

92.1 ± 0.60

91.6 ± 0.71

92.0 ± 0.84

92.0 ± 0.66

15.2 ± 0.14

15.4 ± 0.21

16.1 ± 0.49

16.8 ± 0.35

RDP

9.52

9.73

9.82

10.3

RUP

5.68

5.92

6.23

6.50

NDF

37.8 ± 1.37

37.0 ± 1.88

37.4 ± 1.06

38.2 ± 0.65

ADF

23.7 ± 0.85

23.4 ± 0.88

23.4 ± 0.84

24.0 ± 0.74

Starch

17.5 ± 0.57

19.0 ± 1.91

18.9 ± 0.99

16.6 ± 0.07

Ether extract

1.66 ± 0.61

1.91 ± 0.86

1.86 ± 0.39

1.68 ± 0.73

NFC

37.4 ± 0.14

37.0 ± 0.21

36.7 ± 0.49

35.4 ± 0.35

Mcal/kg

1.67

1.68

Protein supply,

g/d

RDP supply

2,342

2,443

2,494

2,616

RDP balance

-128

-27

146

RUP supply

1,397

1,499

1,595

1,651

RUP balance

-173

-71

MP supply

2,781

2,791

3,049

3,059

MP requirements

2,972

2,998

3,004

MP balance

-190

-181

Lys and Met balance,

g/d

dLys requirement

184

201

202

dLys supplied by the diet

191

209

Lys balance

7.0

8.0

7.0

dMet requirement

dMet supplied by the diet

dMet from Met derivative

Met balance

-6.0

15.6

9.6

-7.0

15.6

8.6

dLys:dMet

3.47

2.71

3.48

2.76

MPD−NALM = diet deficient in MP (MPD) without NALM; MPD+NALM = MPD with

NALM; MPA−NALM = diet adequate in MP (MPA) without NALM; and MPA+NALM = MPA

with NALM.

A developmental NALM product (CJ CheilJedang, Seoul, Korea) was a source of rumen-

protected Met, and it contained 78.0% Met.

Formulated to contain (per kg DM): 226.7 mg of Se (from sodium selenite), 9278.7 mg of Cu

(from copper amino acid complex), 40,537.4 mg of Zn (from zinc amino acid complex),

38,653.4 mg of Mn (from manganese amino acid complex), 552.6 mg of Co (from cobalt

carbonate), 1,234,585.2 IU of vitamin A, 152,808.1 IU of vitamin D, 3,815.1 IU of vitamin E,

and 295 mg of Rumensin (Elanco Animal Health, Greenfield, IN).

Based on tabular value (NRC, 2001).

NFC = 100 – CP – NDF – ether extract – ash.

All values were estimated using NRC (2001) based on actual DMI, milk yield, milk

composition, and BW of the cows throughout the trial.

Digestible Lys (dLys) and digestible Met (dMet) supply from the diets were estimated using

NRC (2001); supply of dMet from Met derivative was estimated with Met concentration and

absorption (67%) data provided the manufacturer. Requirements of dLys and dMet were

calculated as 6.6 and 2.2% (respectively) of MP requirements.

Diestible Lys from diet-to-digestible Met from diet and NALM ratio.

Table 1-2. Intake, milk production, feed efficiency, and net energy utilization of lactating Holstein dairy cows

supplemented without or with N-acetyl-L-Met (NALM) at 2 different levels of MP

Item

Diet

SEM

Significance of effect

MPD

MPA

−NALM

+NALM

−NALM

+NALM

NALM

INT

DM intake, kg/d

24.6

25.6

25.4

1.34

0.67

0.46

0.44

Yield, kg/d

Milk

39.3

39.7

41.3

42.0

2.60

< 0.01

0.48

0.81

3.5% FCM

39.0

39.9

40.4

43.4

2.65

0.02

0.05

0.29

ECM

38.7

39.7

40.2

42.9

2.51

0.02

0.06

0.35

Milk composition, %

Fat

3.44

3.52

3.34

3.70

0.121

0.65

0.02

0.10

True protein

2.77

2.82

2.78

2.83

0.083

0.82

0.21

0.92

Lactose

4.84

4.87

4.88

4.85

0.049

0.65

0.97

0.26

Milk component yield,

kg/d

Fat

1.36

1.41

1.39

1.56

0.100

0.05

0.03

0.20

True protein

1.08

1.11

1.14

1.18

0.064

0.04

0.24

0.75

Lactose

1.91

1.93

2.02

2.04

0.128

< 0.01

0.53

0.95

Feed efficiency

Milk yield/DMI

1.59

1.55

1.61

1.66

0.075

0.03

0.83

0.10

3.5% FCM yield/DMI

1.58

1.57

1.72

0.083

0.06

0.09

0.06

ECM yield/DMI

1.57

1.55

1.57

1.70

0.073

0.05

0.11

0.06

Initial, kg

704

698

702

704

21.7

0.56

0.66

0.26

Mean, kg

705

710

713

21.2

0.03

0.74

0.60

Gain, kg/d

0.06

0.30

0.40

0.345

0.24

0.51

Net energy utilization,

Mcal/d

Maintenance

10.9

11.0

0.25

0.03

0.73

0.57

BW gain

0.24

1.16

1.55

1.322

0.24

0.51

Milk

26.2

26.1

26.8

28.5

1.67

0.03

0.20

0.16

Total

37.4

38.2

39.3

41.1

2.19

0.03

0.21

0.65

, Mcal/kg DMI

1.49

1.55

1.60

0.074

0.13

0.18

0.94

a-d

Means within a row with different superscripts differ (P < 0.05).

MPD−NALM = diet deficient in MP (MPD) without NALM; MPD+NALM = MPD with NALM;

MPA−NALM = diet adequate in MP (MPA) without NALM; and MPA+NALM = MPA with NALM.

MP = level of MP in the diet (MPD vs. MPA); NALM = supplementation of NALM (−NALM vs.

+NALM); and INT = interaction between MP and NALM.

3.5% FCM = [0.4324 × milk yield (kg/d)] + [16.216 × fat yield (kg/d)].

ECM = 0.327 × milk yield (kg/d) + 12.95 × fat yield (kg/d) + 7.2 × protein yield (kg/d).

Net energy used for maintenance, BW gain, and milk.

Table 1-3. Plasma AA concentrations (µmol/L) of lactating Holstein dairy cows supplemented

without or with N-acetyl-L-Met (NALM) at 2 different levels of MP

Item

Diet

SEM

Significance of effect

MPD

MPA

−NALM

+NALM

−NALM

+NALM

NALM

INT

His

10.1

11.6

10.2

9.7

5.60

0.51

0.70

0.48

Ile

85.3

90.5

102

91.9

9.39

< 0.01

0.35

< 0.01

Leu

119

122

144

123

12.5

< 0.01

0.02

< 0.01

Lys

61.2

65.8

68.0

65.0

17.61

0.40

0.83

0.29

Met

18.0

19.6

18.3

17.4

0.85

0.06

0.49

0.01

Phe

71.3

73.2

76.4

70.6

5.23

0.44

0.24

0.02

Thr

31.6

33.1

32.1

29.0

7.38

0.25

0.59

0.13

Val

248

266

283

241

23.3

0.74

0.43

0.05

Ala

196

195

200

181

14.3

0.36

0.04

0.07

Asp

37.9

49.8

46.9

38.1

9.48

0.77

0.75

0.03

Glu

24.8

23.3

24.2

22.6

2.50

0.47

0.09

0.99

Gly

198

188

168

20.9

< 0.01

0.07

Pro

78.0

75.3

80.8

70.3

6.68

0.54

< 0.01

0.04

Tyr

138

166

168

159

42.2

0.35

0.44

0.13

EAA

645

682

720

648

21.6

0.34

0.42

0.01

NEAA

793

707

694

638

64.3

0.19

0.27

0.82

a-c

Means within a row with different superscripts differ (P < 0.05).

100

MPD−NALM = diet deficient in MP (MPD) without NALM; MPD+NALM = MPD with NALM;

MPA−NALM = diet adequate in MP (MPA) without NALM; and MPA+NALM = MPA with NALM.

MP = level of MP in the diet (MPD vs. MPA); NALM = supplementation of NALM (−NALM

vs. +NALM); and INT = interaction between MP and NALM.

Essential amino acid analyzed.

Nonessential amino acid analyzed.

101

Table 1-4. Ruminal fermentation characteristics of lactating Holstein dairy cows supplemented without

or with N-acetyl-L-Met (NALM) at 2 different levels of MP

Item

Diet

SEM

Significance of effect

MPD

MPA

−NALM

+NALM

−NALM

+NALM

NALM

INT

Ruminal pH

6.26

6.21

6.35

6.39

0.101

0.03

0.95

0.48

Total VFA, mM

146

154

147

144

6.6

0.53

0.68

0.39

Individual VFA,

mol/100 mol

Acetate (A)

62.4

62.0

62.4

3.79

0.96

0.93

0.26

Propionate (P)

23.3

23.7

23.2

23.3

2.99

0.60

0.65

0.80

Butyrate

10.7

10.6

11.0

10.7

0.97

0.49

0.47

0.70

Valerate

1.57

1.66

1.47

1.62

0.403

0.30

0.09

0.61

Isobutyrate

0.79

0.89

0.95

0.87

0.128

0.27

0.88

0.14

Isovalerate

1.27

1.41

1.20

0.179

0.52

0.04

0.06

A:P

2.77

2.70

2.75

2.74

0.210

0.85

0.45

0.63

-N,

mg/100

9.34

9.05

9.28

9.82

1.425

0.70

0.89

0.65

MCP,

g/d

2,540

2,649

2,954

2,837

237.7

< 0.01

0.96

0.16

a,b

Means within a row with different superscripts differ (P < 0.05).

MPD−NALM = diet deficient in MP (MPD) without NALM; MPD+NALM = MPD with NALM;

MPA−NALM = diet adequate in MP (MPA) without NALM; and MPA+NALM = MPA with NALM.

102

MP = level of MP in the diet (MPD vs. MPA); NALM = supplementation of NALM (−NALM vs.

+NALM); and INT = interaction between MP and NALM.

Ammonia-N.

Microbial protein production (g/d) = ({[urinary total purine derivatives (allantoin + uric acid) − (0.385 ×

0.75

)]/0.85} × 70 × 6.25)/(0.13 × 0.83 × 1,000) (Janicek et al., 2008).

Figure 1-1. Proposed mechanism of N-acetyl-L-Met (NALM) on its potential effects on milk fat and milk production. SAM = S-

adenosyl-Met; VLDL = very low-density lipoprotein (Nelson and Cox, 2013)

CHAPTER 2

Effects of supplementing three different forms of rumen-protected lysine on

lactational performance of mid to late lactation dairy cows

ABSTRACT

The present study investigated lactational performance of dairy cows when

supplemented with N-acetyl-L-Lys (εNALL), N, -acetyl-L-Lys (diNALL), or

AjiPro



-L (AP) as a source of rumen-protected Lys (RPLys) to a control diet. Control

cows (CL) were provided the basal diet formulated to contain 15.3% CP and supply

adequate MP (+ 10 g/d balance). Forty lactating Holstein dairy cows (151 ± 78.0 days-in-

milk, on average) were assigned to treatments (n = 10) by parity and days-in-milk, and

the experiment was performed in a randomized complete block design. Cows were fed

the same CL diet for a 2-wk adaptation period. Cows were then randomly assigned within

blocks to 1 of the 4 experimental diets and fed assigned diets throughout the remaining 8

wk of the trial (entire study duration was 70 d). Developmental NALL, diNALL, and the

commercial RPLys product AP were supplemented to the CL diet at 51.5, 63, and 100

g/cow/d to provide 20 g/d of digestible Lys, respectively. Feeding the diNALL and the

AP diets decreased DMI. Milk yield was not altered by RPLys supplementation. Milk

composition (%) and milk component yields were not affected by RPLys

supplementation. In contrast, milk production efficiency (milk yield/DMI) increased with

105

the diNALL diet in comparison to the CL diet. Body weight change was not affected by

RPLys supplementation. The diNALL diet shifted net energy partitioning (% energy

intake) toward milk production. Dietary treatments had similar plasma AA, N utilization,

and rumen NH

-N concentrations. Overall, results in the current study suggest that

diNALL and AP decreased DMI leading to improved milk production efficiency.

However, based on the current findings, the potential effects of RPLys on DMI regulation

warrant further investigation as stage of lactation and physiological changes occurring

through stage of lactation may have alter DMI.

Key Words: N-acetyl-L-lysine, feed efficiency, lactational performance, rumen-

protected lysine

INTRODUCTION

Lysine is commonly considered the first limiting essential AA (EAA) in dairy cows

fed a corn-based ration or rations that include corn by-products, such as dried distillers

grains with solubles (Schingoethe et al., 2009). Lysine’s main biological role in dairy

cattle is to promote protein synthesis. Lysine may be used for weight gain in growing

dairy cattle, milk protein production in lactating dairy cows, incorporation into

mammalian tissues for structural integrity, or serve as a carbon backbone for carnitine

synthesis (NRC, 2001; Vaz and Wanders, 2002). However, for Lys products to exert their

biological effects in dairy cows, they must be able to avoid microbial degradation in the

rumen and supply post-ruminal Lys for small intestinal absorption.

106

An effective method to increase post-ruminal Lys supply and improve post-ruminal

Lys absorptive efficiency is to feed rumen-protected Lys (RPLys). Inconsistent post-

ruminal polymer degradation and high production costs have limited the development of

RPLys products (Swanepoel et al., 2010). However, since the early 1990s rumen stable

AA matrixes have been developed and numerous RPLys products have become available

for commercial use in animal agriculture (Robinson, 2009b). Available commercial

RPLys products use encapsulation, matrix technology, or a combination of both to protect

a Lys core from ruminal degradation (Ji et al., 2016). These RPLys products are often

coated by a series of lipid or fatty acid calcium-based salts (Ji et al., 2016). Wu and Papas

(1997) found that RPLys coating protection efficacy in the rumen depends on coating

composition, pellet surface smoothness, inner core pellet strength, and pellet solubility

and size. Recently, the production of Lys derivatives, a new form of RPLys may serve as

an alternative to physically protected lipid RPLys products.

The N-acetyl-L-Lys (NALL) is a Lys derivative produced via protection of the L-Lys

α-amino (Nα, ε-acetyl-L-Lys; diNALL) or ε-amino (Nε-acetyl-L-Lys; εNALL) group

with an N-acetyl group. The biological availability of N-substituted Lys products has

previously been described (Finot et al., 1978). The biological availability for diNALL

and εNALL is 0 and 50% in the rat, respectively (Neuberger and Sanger, 1943; Bjarnason

and Carpenter, 1969). According to Finot et al. (1978) the Lys derivatives which are

partially utilized as Lys sources in rats are Nε-formyl-L-Lys and εNALL. They belong to

the group of Nε-acyl-Lys and are negligibly hydrolyzed by homogenates of the intestinal

mucosa and liver, and only slightly so by the kidneys of the rat (Finot et al., 1978). Paik

107

and Benoiton (1963) and Leclerc and Benoiton (1968) found that it is the renal ε-Lys

acylase which is responsible for Nε-formyl-L-Lys and εNALL hydrolysis. It is

anticipated that εNALL will be biologically available in lactating dairy cows, however,

there is no research reported on the effects of supplementing NALL in lactation diets on

production and ruminal fermentation in dairy cows.

The objective of the present study was to investigate lactational performance and

nutrient utilization by mid to late lactating dairy cows supplemented with 3 different

forms of RPLys. We hypothesized that supplementing RPLys to mid to late lactation

dairy cows would increase post-ruminal Lys supply and increase milk protein synthesis

and milk yield.

MATERIALS AND METHODS

The dairy cows used in the present study were cared for according to the Live Animal

Use in Research Guidelines of the Institutional Animal Care and Use Committee at Utah

State University. The study was conducted at the Caine Dairy Research Center

(Wellsville, UT), Utah State University from July to September, 2017.

Cows, Experimental Design, and Diets

Forty multiparous lactating Holstein dairy cows were used during the trial. Cows

were tested in mid to late lactation and began the experiment averaging 3.6 ± 1.21 parity,

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151 ± 78.0 DIM and producing 39.6 ± 6.89 kg milk/d. The experiment was performed as

a randomized complete block design. Cows were grouped into 10 blocks of 4 cows by

parity and DIM. Cows were fed the same control diet (CL) for a 2-wk adaptation period.

Cows were then randomly assigned within blocks to 1 of the 4 experimental diets and fed

assigned diets throughout the remaining 8 wk of the trial (entire study duration was 70 d).

Experimental diets were based on alfalfa hay, corn silage, and dried distillers grains with

solubles. Table 2-1 lists the chemical composition of the 4 dietary treatments. The control

diet was formulated to contain 15.3% CP and supply adequate MP (+ 10 g/d balance).

The dietary treatments fed in the study are as follows; 1) CL = normal CP (15.3%)

without RPLys, 2) NALL = normal CP with NALL (51.5 g/d), 3) diNALL = normal

CP with diNALL (63 g/d), and 4) AP = normal CP with AjiPro

-L (100 g/d).

The developmental NALL products from CJ CheilJedang (Seoul, South Korea) were

used as a supplemental source of RPLys in this study. N-acetyl-L-Lys and diNALL are

protected from rumen degradation with an N-acetyl group attached to either the α- or the

-amino group. The NALL and diNALL products were added at a rate of 51.5 and 63

g/d, respectively. The rate was determined for lactating dairy cows based on

manufacturer’s recommendation in order to supply 20 g of digestible Lys to the small

intestine. The recommendation of required digestible Lys and Met were assumed as 6.6

and 2.2% of the MP requirements, respectively (NRC, 2001). The RPLys products were

top-dressed onto corresponding experimental diets at each a.m. feeding. Diets were

formulated based on NRC (2001) recommendations to provide sufficient NE

, MP,

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vitamins, and minerals with an estimated intake of 25.0 kg/d to produce 36 kg/d of milk

with 3.5% fat and 3.0% true protein (Table 2-1).

Cows were housed individually in tie stalls fitted with rubber mattresses covered with

straw, allowing free access to water. Cows were individually fed twice daily for ad

libitum intake at a level of 110% expected daily intake with 60% of allotted feed fed at

0800 h and 40% fed at 1500 h. Feed offered and refused were recorded daily, and feed

samples were taken during each week of the trial to determine DMI. Cows were milked

twice daily at 0600 and 1600 h, and milk production was recorded throughout the entire

experiment.

Energy Partitioning Calculations

Energy partitioning was determined during sampling weeks using data of milk yield,

milk composition, and BW of experimental animals. Cows were weighed for 2

consecutive d after the a.m. milking and before the a.m. feeding at the end of weeks 2, 4,

6, 8 and 10. These weights were used to calculate the mean BW of cows for each

sampling week. Energy used for maintenance was calculated as BW

0.75

× 0.08 (NRC,

2001). Energy of BW change was assumed to be 5.114 Mcal/kg of gain or 4.924 Mcal/kg

of loss (NRC, 2001). Milk energy was calculated as (0.0929 × milk fat concentration) +

(0.0563 × milk true protein concentration) + (0.0395 × milk lactose concentration) (NRC,

2001). Estimated NE

value was calculated by total net energy utilization (maintenance,

BW gain, and milk) divided by DMI (Neal et al., 2014).

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Feed and Milk Samplings and Analyses

Samples of alfalfa hay, corn silage and dried distiller grains were taken weekly to

determine DM, and diets were adjusted accordingly for change in forage DM

concentration. Samples were ground to pass a 1-mm screen (standard model 4; Arthur H.

Thomas Co., Swedesboro, NJ), and stored for chemical analysis. Samples of TMR and

orts were collected from individual cows on d 3 and d 4, at the end of the adaptation

period and sampling weeks (3 through 10) composited, dried at 60°C for 48 h, and

ground as previously described. The DM concentrations of samples were used to

calculate intakes of DM and nutrients.

Analytical DM concentration of samples was determined by oven drying overnight at

105°C, and OM was determined by ashing at 550°C for 5 h (AOAC, 2000; method

942.05). Concentration of CP was determined using an automated N combustion analyzer

(Elementar, Analysensysteme GmbH, Hanau, Germany; AOAC, 2000; method 968.06).

Concentrations of NDF and ADF were sequentially determined using a fiber analyzer

(200/220, ANKOM Technology, Macedon, NY) according to the methodology supplied

by the company, which is based on the methods described by Van Soest et al. (1991).

Sodium sulfite was used in the procedure for NDF determination and pre-treated with

heat-stable amylase (Type XI-A from Bacillus subtilis; Sigma-Aldrich Corporation, St.

Louis, MO). Ether extract was measured using a fat analyzer (XT20, ANKOM

Technology; AOAC, 2000; method 2003.05). In addition, samples of TMR were

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analyzed for starch by Dairyland Laboratories, Inc. (Arcadia, WI) according to Knudsen

(1997).

Milk was sampled for 2 consecutive d (d 3 and 4) during the a.m. and p.m. milkings

each week. Individual milk samples were analyzed by the Rocky Mountain DHIA

Laboratory (Logan, UT) for fat, true protein, lactose, and MUN. Milk composition was

expressed on weighted milk yield of a.m. and p.m. samples. Milk fat and protein yields

were calculated by multiplying milk yield from the respective day by fat and true protein

concentration of the milk from an individual cow.

Blood Samplings and Analyses

Blood samples were collected immediately before the morning feeding and at 1400 h

into 10-mL EDTA plasma vacuum tubes from the coccygeal artery or vein on d 6 and 7

in each sampling week (4, 6, 8, and 10) and placed on ice. Blood samples were

centrifuged at 2,300 × g for 20 min, and plasma was collected and stored at ‒40°C for

subsequent AA analysis. The plasma samples were prepared for AA analysis using the

EZ:faast GC-FID Free (Physiological) AA Analysis kit (Phenomenex Inc., Torrance,

CA). Concentrations of plasma free AA were determined in accordance with the user

manual provided with the kit. Extraction of free AA from the plasma consisted of a

combination of solid-phase extraction, derivatization, and liquid/liquid extraction. The

organic phase containing the AA in question were analyzed using a GC (model 5890,

Hewlett-Packard Co, Avondale, PA) with a capillary column (30 m × 0.32-mm i.d., 1-μm

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phase thickness, Zebron ZB-FAAP; Phenomenex Inc., Torrance, CA) and flame-

ionization detection.

Ruminal Fermentation Characteristics

Ruminal fluid samples were obtained using a Geishauser probe at 3 h after the

morning feeding on d 5 and 6 of each sampling week (4, 6, 8, and 10). The fluid was

collected with a solid, tube-like probe with rows of small holes on the end (Geishauser,

1993). The first 100 mL of ruminal fluid was discharged to avoid contamination from

saliva, and then 10 mL was collected for analysis. The pH of the ruminal fluid was

measured within 5 min of collecting the samples using a portable pH meter (Oakton pH

6; Oakton Instruments, Vernon Hills, IL). Five milliliters of the ruminal fluid were mixed

with 1 mL of 1% sulfuric acid and stored frozen (−40°C) for ammonia-N (NH

-N)

analysis. Concentration of NH

-N in the ruminal contents was determined as described by

Rhine et al. (1998), using a plate reader (MRXe; Dynex Technologies Inc., Chantilly,

VA). Another 5 mL of the ruminal fluid was collected and mixed with 1 mL of 25%

metaphosphoric acid, and then stored at −40°C for VFA content determination. Ruminal

VFA were separated and quantified using a GC (model 6890 series II; Hewlett-Packard

Co.) with a capillary column (30 m × 0.32-mm i.d., 1-μm phase thickness, Zebron ZB-

FAAP; Phenomenex Inc., Torrance, CA) and flame-ionization detection. The oven

temperature was held at 170°C for 4 min, increased to 185°C at a rate of 5°C/min, then

increased by 3°C/min to 220°C, and held at this temperature for 1 min. The injector and

113

the detector temperatures were 225 and 250°C, respectively, and the carrier gas was

helium (Eun and Beauchemin, 2007).

Statistical Analysis

Statistical analyses of all data except BW change, N excretion, AA and VFA were

conducted using the Proc Mixed procedure of SAS (version 9.4; SAS Institute Inc., Cary,

NC) with a repeated measures model that included the covariate mean for each trait for

each cow, plus block, treatment, week, and the interaction of treatment × week. A similar

approach was used for BW change, N excretion, AA and VFA data, except the covariate

was removed from the model. Overall, treatment differences were examined using least

squares means with the lowest standard error. Week and block were included in the final

statistical model for all analyses. The PDIFF option of SAS (version 9.4; SAS Institute

Inc., Cary, NC) was used to test treatment differences among least squares means, and the

SLICE option was used to analyze treatment differences among weekly treatment means.

Differences were considered significant at P ≤ 0.05, and trends were discussed at 0.05 <

P ≤ 0.10.

RESULTS AND DISCUSSION

Characteristics of Diet Composition and Treatment

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Ingredients and chemical composition of experimental diets are presented in Table 2-

1. The CL diet had a CP content of 15.3%, which provided an adequate MP supply of

2,910 g/d (+ 10 g/d balance), whereas the RPLys supplemented diets provided an MP

supply of 2,930 g/d (+ 30 g/d balance) according to NRC (2001). Corn distillers grains

were added to the CL diet at an inclusion rate of 21% of DM in order to achieve a Lys

balance of – 20 g/d. High inclusion rates of corn distillers grains above 20% of DM have

been shown to increase the amount of rumen biohydrogenation intermediates (Hippen et

al., 2004) and may decrease milk fat concentration. Therefore, caution was taken to

ensure that the NDF concentration in the diets remained near NRC (2001)

recommendation of 25 to 33% of diet DM as total NDF. The RPLys supplemented diets

maintained a Lys balance of – 1 g/d (0 g/d Lys balance targeted) by providing 20 g of

metabolizable Lys to the diets based on manufacturers recommendation for each Lys

treatment. All diets supplied NE

in excess of requirement (NRC, 2001).

The concentrations of Lys and Met in MP was 6.6 and 2.2%, respectively. Digestible

Lys and Met concentrations in MP (dLys:dMet) were maintained at 3.05:1 for RPLys

supplemented diets and 2.73:1 for the CL diet. The dLys:dMet ratio for optimal milk and

milk protein yield is reported in the NRC as 3.0:1 (NRC, 2001), but ratios as low as

2.69:1 have been reported (Doepel et al., 2004; Van Amburgh et al., 2015).

Feed Intake, Productive Performance, and Net Energy Utilization

115

Feeding the diNALL and the AP diets reduced intake of DM compared to the CL diet

(Table 2-2). Responses to supplementation of RPLys on feed intake in the literature are

limited. Paz et al. (2014) reported that RPLys supplementation did not affect DMI in

lactating dairy cows, which agrees with Blauwiekel et al. (1997) and Robinson et al.

(2011). Swanepoel et al. (2010) found that supplementation of RPLys did not influence

DMI in early lactating cows (77 ± 3.2 DIM); however, the mid lactation group (262 ±

6.70 DIM) had a tendency (P = 0.09) toward lower DMI with RPLys supplementation.

The mechanism by which RPLys reduces DMI in mid lactation cows is unknown, but the

authors suggested that RPLys possibly affected animal metabolism, which reduced

digestion in the lower digestive tract (Swanepoel et al., 2010).

A variety of factors such as forage quality, TMR nutrient balance, feed delivery,

feeding method, TMR palatability, ration DM content, environmental conditions, housing

facilities, and managerial practices can impact DMI (Allen and Bradford, 2009), which

might explain the lack of response seen with NALL supplementation. In the current

study, stage of lactation and week of trial may have altered DMI in cows fed the diNALL

and AP diets. This is because energy requirements decrease during mid lactation and

control of feed intake by gut distension gradually diminishes, while control by hepatic

oxidation increases (Allen and Bradford, 2009). Therefore, physiological changes

occurring through stage of lactation and the physical and chemical characteristics (NDF

concentration) of feeds beyond their nutrient composition can alter DMI (Allen and

Bradford, 2009).

116

Supplementation of RPLys did not alter milk yield (Table 2-2). Milk, 3.5% FCM, and

ECM yields were not affected by dietary treatments. Swanepoel et al. (2010) observed

that dairy cows supplemented with RPLys at a rate of 41 g/cow/d did not affect milk

yield in early lactation. Paz and Kononoff (2014) did not detect a difference in milk yield

when RPLys was supplemented to diets with altering inclusion rates of low-fat dried

distillers grains (15 vs. 30% of diet DM). Furthermore, cows supplemented with RPLys

(37 g/cow/d) after peak lactation showed no improvement in milk yield (Bernard et al.,

2014). In contrast, Robinson et al. (2011) found a 2 kg increase in milk yield when

RPLys was supplemented to deliver 15 to 21 g of MP Lys. In the present study, there was

no improvement in milk yield in response to RPLys supplementation, thus indicating that

RPLys was not utilized in the mammary gland of mid to late lactating dairy cows for milk

production. The RPLys supplemental dosage rate, lack of a digestible Lys delivery to the

small intestine for absorption, or reduced DMI may be responsible for the absence of

production responses seen in the current study.

Milk composition (%) and milk component yields (kg/d) were not affected by dietary

treatments (Table 2-2). Swanepoel et al. (2010) reported reduced milk fat synthesis to

feeding a RPLys product to high producing dairy cows. Třináctý et al. (2009) fed RPLys

to lactating Holstein dairy cows (33.5 kg/d) and while milk protein concentration

increased, milk fat concentration and yield declined. A review of literature by Robinson

(2010) stated that RPLys supplementation generally results in minimal production

responses, possibly due to an AA imbalance or poor AA delivery to the intestinal

absorptive site. The productive benefits of RPLys supplementation on overall

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performance effects in lactating dairy cows can only be judged as negative (Robinson,

2010). However, increased Lys supply appear important in early lactation when MCP

synthesis is reduced because of lower DMI, to help drive milk and milk protein

production (Robinson et al., 2011). Stage of lactation (mid to late) or lack of a digestible

Lys deficiency in the diets supplemented with RPLys may be responsible for the absence

of production responses seen in the current study.

The diNALL diet reduced intake of DM and maintained milk yield resulting in better

feed efficiency for milk production compared to the CL diet (1.54 vs. 1.33; Table 2-2).

Wang et al. (2010) supplemented L-Lys-HCl at 0.50% DM to a control diet and detected

increased milk yield with no increase in DMI, improving feed efficiency to 1.34 vs. 1.27

for the control diet. As previously mentioned, the biological availability for diNALL and

εNALL is 0 and 50% in the rat based on growth rates (Neuberger and Sanger, 1943). The

improvement in feed efficiency for milk production with diNALL supplementation

demonstrate that, unlike in the rat, diNALL may be biologically available in lactating

dairy cows. Additionally, the increased feed efficiency observed in the current study

suggest that diNALL supplementation would be favorable in mid to late lactating cows,

but only when fed to a diet that has a digestible Lys deficit. Based on the current findings

the biological availabilities of diNALL and εNALL in lactating dairy cows warrant

further investigation.

No changes were seen in either BW gain or net energy utilization with

supplementation of RPLys in the diets (Table 2-2). However, feeding the diNALL diet

reduced BW gain relative to the CL diet, leading to an increased net energy partitioning

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for milk. No effect on BW gain due to diNALL supplementation suggests that energy

was primarily partitioned toward milk production rather than BW gain in mid to late

lactation in the current study.

Plasma AA Profiles

Plasma AA concentrations (EAA and NEAA) were not affected by RPLys

supplementation (Table 2-3). The plasma Lys concentrations observed in the current

study (Table 2-3) agree with Swanepoel et al. (2010), who did not detect a difference in

plasma Lys concentrations of mid lactating dairy cows supplemented with RPLys at 41

g/cow/d. Since, RPLys was supplemented to the CL diets, an increase in plasma Lys

concentration was expected. However, because digestible Lys requirements were met, the

absorbed Lys may have been used for biological functions other than milk production,

converted to metabolites or not utilized. Lysine is an AA with a high fractional extraction

rate by the mammary gland in ruminants (Manjarin et al., 2014). The primary metabolic

fate of Lys in the mammary gland is for milk and milk protein synthesis. Mabjeesh et al.

(2000) observed that the uptake of Lys in the mammary gland of dairy cows is greater

than milk Lys output. The plasma Lys concentration along with changes in production

parameters (i.e., milk yield) for the diNALL diet indicates that plasma Lys may have

been utilized for improved milk yield. Since, plasma AA profiles were not altered by

RPLys supplementation, further research is needed to determine the impact of Lys

derivative supplementation on plasma AA profiles in lactating dairy cows.

119

Utilization of N

Intake of N in the current study followed DMI, meaning that cows fed the diNALL

and the AP diets consumed less N than those fed the NALL and CL diets (Table 2-4).

Supplementation of RPLys products do not often increase N intake because of either a

lower dietary CP or MP concentration in basal diets associated with RPLys or a

combination of RPAA supplementation (Leonardi et al., 2003; Broderick et al., 2009).

Lee et al. (2015) supplemented a MP-deficient diet (MP balance of −281 g/d; CP of

13.7% DM); with 100 g of RPLys/cow/d (estimated digestible Lys supply = 24 g/d) and

reported no increase in N intake compared to the MP-deficient diet. Supplementation of

RPLys had no effect on milk N excretion in the current study. Milk N efficiency is

primarily driven by feed N intake (Huhtanen and Hristov, 2009). Although the N intake

was reduced for the diNALL and the AP diets, no change in milk N excretion after

supplementation of RPLys resulted in milk N:N intakes that were similar across all diets

(0.32 on average). The N utilization efficiencies reported in the current study are similar

to the average N efficiencies (25 to 35%) reported in the literature (Hristov et al., 2004).

The lack of response in milk N to dietary treatments is related to DMI and milk yield

seen in the current study.

Concentrations of MUN and NH

-N were not affected by dietary treatments. Urea-N

(milk, serum, and urine) in cattle is derived from excess NH

-N absorbed through the

rumen wall from rumen CP degradation and deamination of AA (Linn and Olson, 1995;

120

Wang et al., 2008). Wang et al. (2010) supplemented L-Lys-HCl at 0.50% of DM to a

control diet that was adequate in energy, but slightly limiting in MP and detected

increased urea-N concentration in serum, urine, and milk compared with the control diet.

In the present study, supplementation of RPLys maintained MUN and NH

-N

concentrations, indicating that the RPLys diets may have provided a more favorable AA

balance and resulted in less deamination of absorbed AA.

Urinary and fecal N excretion were not affected by dietary treatments. However,

manure N excretion (urinary N excretion, g/d + fecal N excretion, g/d) decreased for the

diNALL and the AP diets. Urinary and fecal N are directly correlated with DM and N

intakes (Huhtanen et al., 2008). Therefore, because of the reduced N intake of animals

fed the diNALL and the AP diets, manure N excretion decreased. The reduced manure N

excretion in the current study indicates the potential use of supplementing diNALL to

lactating dairy cows to aid in environmental N management on dairy farms, however

further lactation trials are needed to determine how NALL products impact N utilization

in lactating dairy cows.

Ruminal Fermentation Characteristics

Supplementation of RPLys in the NALL, the diNALL and the AP diets reduced

ruminal pH and ranged from 6.15 to 6.23, compared to the CL diet (pH = 6.42; Table 2-

5). The mean ruminal pH from all dietary treatments were close to or above 6.2, so

RPLys influence on overall microbial composition may be negligible. The effects of

121

RPLys supplementation on ruminal pH is not well defined in the literature. Recently, Lee

et al. (2015) determined that RPLys supplementation to an MP-deficient diet did not alter

ruminal pH in mid-lactation dairy cows.

Total and individual VFA concentrations were similar across dietary treatments.

According to Lee et al. (2015), rumen fermentation variables (total and individual VFA)

were not affected by RPLys supplementation to a MP-deficient diet. A previous study

performed by our group (Menchu, 2019), estimated the ruminal escape for the NALL

products used in the current study. The ruminal escape (% of dose) averaged 43.5 for

both NALL products, indicating that the NALL chemical structure is altered in the rumen

environment. The absence of response in VFA profiles to NALL and diNALL dietary

treatments, suggests that NALL lacks a ruminal mode of action and further research is

needed to understand the metabolism of NALL in the rumen.

CONCLUSIONS

Results of the current study show that feeding the diNALL diet in mid to late

lactation dairy cows decreased DMI, leading to greater feed efficiency compared to the

CL diet. Milk composition, milk component yields, AA, and VFA profiles were not

affected by RPLys supplementation. Net energy shifted toward milk production for the

diNALL treatment. Rumen-protected Lys supplementation did not alter utilization of N

by lactating Holstein dairy cows in the present study. These findings suggest that the

developmental NALL products may be biologically active in lactating dairy cows, with

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diNALL showing the most benefit to improve feed efficiency. However, further research

is needed to discover the role of Lys derivatives in lactating dairy cows on productive

performance, rumen metabolism, and bioavailability.

ACKNOWLEDGMENTS

This study was supported by CJ CheilJedang (Seoul, South Korea). The authors thank

S. Sharp at Utah State University (Logan) for technical assistance and the staff of the

Caine Dairy Research Center (Wellsville, UT) for their care and handling of the

experimental cows.

REFERENCES

Allen, M. S. and B. J. Bradford. 2009. Nutritional control of feed intake in dairy cattle.

Proc. Florida Ruminant Nutrition Symposium.

AOAC. 2000. Official Methods of Analysis. Vol. 1 and 2. 17th ed. AOAC Int.,

Gaithersburg, MD.

Bernard, J., P. Chandler, C. Sniffen, and W. Chalupa. 2014. Response of cows to rumen-

protected lysine after peak lactation. Prof. Anim. Sci. 30:407–412.

Bjarnason, J. and U. K. Carpenter. 1970. Mechanism of heat damage in protein. 2.

Chemical changes in pure proteins. Br. J. Nutr. 24:313-329.

123

Blauwiekel, R., S. Xu, J. H. Harrison, K. A. Loney, R. E. Riley, and M. C. Calhoun.

1997. Effect of whole cottonseed, gossypol, and ruminally protected lysine

supplementation on milk yield and composition. J. Dairy Sci. 80:1358–1365.

Broderick, G. A., M. J. Stevenson, and R. A. Patton. 2009. Effect of dietary protein

concentration and degradability on response to rumen-protected methionine in

lactating dairy cows. J. Dairy Sci. 92:2719-2728.

Doepel, L., D. Pacheco, J. J. Kennelly, M. D. Hanigan, I. F. Lopez, and H. Lapierre.

2004. Milk protein synthesis as a function of amino acid supply. J. Dairy Sci.

87:1279–1297.

Eun, J. S., and K. A. Beauchemin. 2007. Enhancing in vitro degradation of alfalfa hay

and corn silage using feed enzymes. J. Dairy Sci. 90:2839-2851.

Finot, P-A., F. Mottu, E. Bujard, and J. Mauron. 1978. N-substituted lysines as sources of

lysine in nutrition. Nutrition Improvement of Food and Feed Proteins. P549-570.

Geishauser, T. 1993. An instrument for the collection and transfer of ruminal fluid and

for the administration of water soluble drugs in adult cattle. Bovine Pract. 27:38–42.

Hippen, A. R., K. F. Kalscheur, D. J. Schingoethe, and A. D. Garcia. 2004. Increasing

inclusion of dried corn distillers grains in dairy cow diets J. Dairy Sci., 87 (Suppl. 6)

(2004), p. 1965 (Abstr).

Hristov, A. N., W. J. Price, and B. Shafii. 2004. A meta-analysis examining the

relationship among dietary factors, dry matter intake, and milk and milk protein yield

in dairy cows. J. Dairy Sci. 87:2184-2196.

124

Huhtanen, P., and A. N. Hristov. 2009. A meta-analysis of the effects of protein

concentration and degradability on milk protein yield and milk N efficiency in dairy

cows. J. Dairy Sci. 92:3222–3232.

Huhtanen, P., J. I. Nousiainen, M. Rinne, K. Kytola, and H. Khalili. 2008. Utilization

and partition of dietary nitrogen in dairy cows fed grass silage-based diets. J. Dairy

Sci. 91:3589-3599.

Ji, P., H. A. Tucker, R. E. Clark, M. Miura, and C. S. Ballard. 2016. Short

communication: Effect of on-farm feeding practices on rumen protected lysine

products. J. Dairy Sci. 99:1242–1246.

Knudsen, K. E. B. 1997. Carbohydrate and lignin contents of plant materials used in

animal feeding. Anim. Feed Sci. Technol. 67:319–338.

Leclerc, J. and L. Benoiton. (1968). Further studies on Ɛ-lysine acylase. The ω-N-acyl-

diamino acid hydrolase activity of avian kidney. Can. J. Biochem. 46:471-475.

Lee, C., F. Giallongo., A.N. Hristov., H. Lapierre., T. W. Cassidy., K. S. Heyler., G. A.

Varga., and C. Parys. 2015. Effect of dietary protein level and rumen-protected amino

acid supplementation on amino acid utilization for milk protein in lactating dairy

cows. J. Dairy Sci. 98:1885–1902.

Leonardi, C., M. Stevenson, and L. E. Armentano. 2003. Effect of two levels of crude

protein and methionine supplementation on performance of dairy cows. J. Dairy Sci.

86:4033-4042.

125

Linn, J. G., and J. D. Olson. 1995. Using milk urea nitrogen to evaluate diets and

reproductive performance of dairy cattle. Pages 155–167 in 4-State Appl. Nutr.

Management Conf., La Crosse, WI. University of Wisconsin, Madison, WI.

Mabjeesh, S. J., C. E. Kyle, J. C. MacRae, and B. J. Bequette. 2000. Lysine metabolism

by the mammary gland of goats at two stages of lactation. J Dairy Sci 83:996–1003

Manjarin, R., B. J. Bequette, G. Wu, and N. L. Trottier. 2014. Linking our understanding

of mammary gland metabolism to amino acid nutrition. Amino Acids. 46:2447-2462.

Menchu, Sara. 2019. Ruminal protection and intestinal availability of rumen-protected

methionine and lysine in lactating dairy cows. All Graduate Theses and Dissertations.

7451. https://digitalcommons.usu.edu/etd/7451.

National Research Council. 2001. Nutrient Requirements of Dairy Cattle. 7th rev. ed.

Natl. Acad. Press. Washington, DC.

Neal, K., J.-S. Eun, A. J. Young, and J. O. Hall. 2014. Feeding protein supplements in

alfalfa hay-based lactation diets improves nutrient utilization, lactational

performance, and feed efficiency of dairy cows. J. Dairy Sci. 97:7716–7728.

Neuberger, A. and F. Sanger. 1943. The availability of the acetyl derivatives of lysine for

growth. Biochem. J. 37, 515-518.

Paik, W.K. and L. Benoiton. 1963. Purification and properties of hog kidney Ɛ-lysine

acylase. Can. J. Biochem. Physiol. 41:1643-1654.

Paz, H. A., and P. J. Kononoff. 2014. Lactation responses and amino acid utilization of

dairy cows fed low-fat distillers dried grains with solubles with or without rumen-

protected lysine supplementation. J. Dairy Sci. 97:6519-6530.

126

Rhine, E. D., G. K. Sims, R. L. Mulvaney, and E. J. Pratt. 1998. Improving the Bertholot

reaction for determining ammonium in soil extracts and water. Soil Sci. Soc. Am. J.

62:473–480.

Robinson, P. H. 2009b. Can manipulating ration metabolizable protein amino acid levels

really be expected to effect environmental impacts of cows? Proceedings of the

Southwest Nutrition Conference, Arizona State University, Tucson, AZ, USA pp. 34-

46.

Robinson, P. H. 2010. Impacts of manipulating ration metabolizable lysine and

methionine levels on the performance of lactating dairy cows: A systemic review of

the literature. Livestock Science. 127:115-126.

Robinson, P. H., N. Swanepoel, and E. Evans. 2010. Effects of feeding a ruminally

protected lysine product, with or without isoleucine, valine and histidine, to lactating

dairy cows on their productive performance and plasma amino acid profiles. Anim.

Feed Sci. Technol. 161:75-84.

Robinson, P. H., N. Swanepoel, I. Shinzato, and S. O. Juchem. 2011. Productive

responses of lactating dairy cattle to supplementing high levels of ruminally protected

lysine using a rumen protection technology. Anim. Feed Sci. Technol. 168:30–41.

Schingoethe, D. J., K. F. Kalscheur, A. R. Hippen, and A. D. Garcia. 2009. Invited

Review: The use of distillers products in dairy cattle deits. J. Dairy Sci. 92:5802-

5813.

127

Swanepoel, N., P. H. Robinson, and L. Erasmus. 2010. Amino acid needs of lactating

dairy cows: impact of feeding lysine in a ruminally protected form on productivity of

lactating dairy cows. Anim. Feed Sci. Technol. 157:79–94.

Třináctý, J., L. Křížová, M. Richter, V. Černý, and J. Říha. 2009. Effect of rumen-

protected methionine, lysine or both on milk production and plasma amino acids of

high-yielding dairy cows. Czech Journal of Animal Science. 54. 10.17221/1730-

CJAS.

Van Amburgh, M. E., E. A. Collao-Saenz, R. J. Higgs, D. A. Ross, E. B. Recktenwald, E.

Raffrenato, L. E. Chase, T. R. Overton, J. K. Mills, and A. Foskolos. 2015. The

Cornell Net Carbohydrate and Protein System: updates to the model and evaluation of

version 6.5. J. Dairy Sci. 98:6361–6380.

Van Soest, P. J., J. B. Robertson, and B. A. Lewis. 1991. Methods of dietary fiber, neutral

detergent fiber and nonstarch polysaccharides in relation to animal nutrition. J. Dairy

Sci. 74:3583–3597.

Vaz, F. M., and R. J. A. Wanders. 2002. Carnitine biosynthesis in mammals. Biochem. J.

361:417-429.

Wang, C., J. X. Liu, S. W. Zhai, J. L. Lai, and Y. M. Wu. 2008. Effect of ratio of rumen-

degradable-protein to rumen-undegradable protein on nitrogen conversion of lactating

dairy cow. Acta Agric. Scand. Anim. Sci. 58:100–103.

Wang, C., H. Y. Liu, Y. M. Wang, Z. Q. Yang, J. X. Liu, Y. M. Wu, T. Yan, and H. W.

Ye. 2010. Effects of dietary supplementation of methionine and lysine on milk

production and nitrogen utilization in dairy cows. J. Dairy Sci. 93:3661-3670.

128

Wu, S. H., and A. Papas. 1997. Rumen-stable delivery systems. Adv. Drug Deliv. Rev.

28:323–334.

129

Table 2-1. Ingredient and chemical composition of the experimental diets supplemented

with rumen-protected lysine fed to lactating Holstein dairy cows (n = 40)

Item

Experimental diet

NALL

diNALL

Ingredient, % of DM

Alfalfa hay

19.6

19.5

Wheat straw

4.91

4.90

4.89

Corn silage

26.4

26.3

Corn grain (steam-flaked)

13.9

Corn distillers grain

21.0

20.9

Cottonseed, whole

4.86

4.84

4.83

Beet pulp, shreds

6.54

6.53

6.52

Lysine supplement

−

0.20

0.25

0.40

Sodium bicarbonate

0.63

Vitamin and mineral mix

2.11

2.10

2.09

Chemical composition, % of DM

DM, %

57.6 ± 1.88

58.1 ± 1.93

57.8 ± 2.74

58.2 ± 2.80

91.2 ± 0.38

91.3 ± 0.48

91.7 ± 1.22

91.1 ± 0.58

15.3 ± 0.20

15.4 ± 0.25

15.4 ± 0.51

15.4 ± 0.36

RDP

8.58

8.64

RUP

6.72

6.76

130

NDF

34.6 ± 1.98

34.2 ± 2.46

34.3 ± 1.65

34.2 ± 2.99

ADF

18.6 ± 1.37

18.0 ± 1.36

18.0 ± 0.90

18.0 ± 1.64

Starch

19.5 ± 0.65

19.4 ± 1.02

19.4 ± 0.99

19.5 ± 0.54

Ether extract

3.38 ± 1.06

4.58 ± 0.91

3.96 ± 1.39

4.10 ± 1.29

NFC

37.9 ± 2.56

37.1 ± 3.21

38.0 ± 3.10

37.4 ± 3.43

Mcal/kg

1.74

Protein supply,

g/d

MP supply

2,910

2,930

MP requirement

2,900

MP balance

Lys and Met balance,

g/d

dLys requirement

192

193

dLys supplied by the diet

172

dLys from Lys supplement

Lys balance

-20

-1

dMet requirement

dMet supplied by the diet

Met balance

-1

dLys:dMet

2.73

3.05

131

CL= control diet; NALL = Control + NALL (CJ CheilJedang, Seoul, Korea); diNALL=

Control + diNALL (CJ CheilJedang, Seoul, Korea); and AP = Control + AjiPro

-L v2

(Ajinomoto, Japan).

Developmental NALL products (NALL and diNALL) (CJ CheilJedang, Seoul, Korea)

and a commercial RPLys product (AjiPro

-L v2) (Ajinomoto, Japan) were used as the

sources of rumen-protected lysine.

Formulated to contain (per kg DM): 226.7 mg of Se (from sodium selenite), 9278.7 mg of

Cu (from copper amino acid complex), 40,537.4 mg of Zn (from zinc amino acid

complex), 38,653.4 mg of Mn (from manganese amino acid complex), 552.6 mg of Co

(from cobalt carbonate), 1,234,585.2 IU of vitamin A, 152,808.1 IU of vitamin D, 3,815.1

IU of vitamin E, and 295 mg of Rumensin (Elanco Animal Health, Greenfield, IN).

Based on tabular value (NRC, 2001).

NFC = 100 – CP – NDF – ether extract – ash.

All values were estimated using NRC (2001) based on actual DMI, milk yield, milk

composition, and BW of the cows throughout the trial.

Digestible Lys (dLys) and digestible Met (dMet) supply from the diets were estimated

using NRC (2001); supply of dLys from RPLys products were estimated by data provided

the manufacturer. Requirements of dLys and dMet were calculated as 6.6 and 2.2%

(respectively) of MP requirements.

Digestible Lys from diet-to-digestible Met from diet ratio.

132

Table 2-2. Dry matter intake, productive performance, body weight (BW) change, and

net energy utilization of mid to late lactation Holstein dairy cows supplemented with

rumen-protected lysine

Item

Diet

NALL

diNALL

SEM

Intake, kg/d

26.8

26.1

24.7

24.8

0.41

< 0.01

Yield, kg/d

Milk

36.4

36.6

37.1

35.6

1.16

0.83

3.5% FCM

36.2

36.5

35.8

35.6

1.83

0.98

ECM

36.5

36.9

36.5

35.7

1.62

0.96

Milk composition, %

Fat

3.74

3.39

3.32

3.30

0.178

0.29

True protein

3.05

3.00

2.98

3.05

0.101

0.95

Lactose

4.88

4.72

4.79

4.85

0.055

0.17

Milk component yield,

kg/d

Fat

1.29

1.26

1.22

1.23

0.086

0.91

True protein

1.09

1.11

1.04

0.034

0.50

Lactose

1.77

1.76

1.78

1.73

0.061

0.92

Dairy efficiency

133

Milk yield/DMI

1.33

1.46

1.54

1.46

0.046

0.02

3.5% FCM yield/DMI

1.33

1.44

1.49

1.46

0.070

0.42

ECM yield/DMI

1.35

1.45

1.51

1.47

0.061

0.30

Initial, kg

745

758

730

735

18.9

0.36

Mean, kg

769

772

730

746

18.8

0.35

Gain, kg/d

1.74

1.08

0.10

1.02

0.661

0.39

Calculated net energy

values, Mcal/d

Maintenance

11.7

11.2

11.4

0.21

0.35

BW gain

8.89

5.50

0.51

5.22

3.38

0.39

Milk

25.9

24.4

24.1

1.75

0.88

BW gain + milk

34.7

29.9

24.6

29.3

3.03

0.15

Total

46.4

41.6

35.9

40.8

3.05

0.13

Mcal/kg of DMI

1.74

1.56

1.43

1.62

0.113

0.29

Net energy partitioning,

% energy intake

Maintenance

27.1

29.3

34.6

29.0

2.52

0.20

BW gain

12.3

10.3

-10.5

10.7

9.23

0.27

Milk

60.6

60.4

75.8

60.3

7.18

0.35

BW gain + milk

72.9

70.7

65.4

71.0

2.52

0.20

134

a-b

Means within a row with different superscripts differ (P < 0.05).

CL= control diet; NALL = Control + NALL (CJ CheilJedang, Seoul, Korea);

diNALL= Control + diNALL (CJ CheilJedang, Seoul, Korea); and AP = Control +

AjiPro

-L v2 (Ajinomoto, Japan).

Net energy used for maintenance, BW change, and milk.

Calculated NE

= calculated total net energy, Mcal/d ÷ DMI (kg/d).

135

Table 2-3. Plasma AA concentrations (µmol/L) of mid to late lactation Holstein dairy

cows supplemented with rumen-protected lysine

Item

Diet

NALL

diNALL

SEM

Arg

48.9

53.1

48.8

54.2

3.09

0.49

His

51.0

49.9

49.7

47.2

2.70

0.78

Ile

88.7

86.8

80.5

88.4

5.61

0.71

Leu

206

197

181

191

12.6

0.58

Lys

71.6

78.7

66.5

77.6

4.71

0.24

Met

20.7

20.3

20.5

1.04

0.99

Phe

44.0

43.5

41.0

43.7

1.77

0.60

Val

217

202

192

207

14.2

0.67

EAA

748

731

678

731

42.6

0.68

Ala

251

237

247

235

10.6

0.65

Gly

232

218

231

221

16.6

0.92

Ser

81.9

81.5

77.7

81.2

5.12

0.72

Tyr

55.9

55.3

51.6

57.6

3.58

0.68

NEAA

621

609

607

595

28.1

0.93

CL= control diet; NALL = Control + NALL (CJ CheilJedang, Seoul, Korea);

diNALL= Control + diNALL (CJ CheilJedang, Seoul, Korea); and AP = Control +

AjiPro

-L v2 (Ajinomoto, Japan).

136

Essential amino acid analyzed.

Nonessential amino acid analyzed.

137

Table 2-4. Utilization of N by mid to late lactation Holstein dairy cows supplemented

with rumen-protected lysine

Item

Diet

NALL

diNALL

SEM

N intake, g/d

613

611

583

580

10.1

0.04

Milk N,

g/d

189

184

185

188

10.6

0.99

Milk N:N intake

0.31

0.30

0.32

0.33

0.017

0.75

MUN, mg/100 mL

11.2

11.1

11.3

11.6

0.33

0.74

-N

, mg/100 mL

11.4

11.1

11.9

11.3

0.48

0.70

Urinary N excretion,

g/d

224

223

216

225

9.40

0.90

Fecal N excretion,

g/d

201

204

182

167

12.0

0.12

Manure N excretion,

g/d

424

427

398

392

12.0

0.10

a-b

Means within a row with different superscripts differ (P < 0.05).

CL= control diet; NALL = Control + NALL (CJ CheilJedang, Seoul, Korea);

diNALL= Control + diNALL (CJ CheilJedang, Seoul, Korea); and AP = Control +

AjiPro

-L v2 (Ajinomoto, Japan).

Milk N (kg/d) = milk true protein (kg/d)/6.38 + MUN (kg/d).

Efficiency of use of feed N to milk N.

Ruminal ammonia-N.

Predicted using the equation: 0.026 × MUN, mg/100 mL × BW, kg (Wattiaux and Karg,

2004).

138

Predicted using the equation: N intake, g/d – urinary N excretion, g/d – milk N, g/d.

Manure N, g/d = urinary N excretion, g/d + fecal N excretion, g/d.

139

Table 2-5. Ruminal fermentation characteristics of mid to late lactation Holstein dairy

cows supplemented with rumen-protected lysine

Item

Diet

NALL

diNALL

SEM

Ruminal pH

6.42

6.23

6.18

6.15

0.06

0.02

Total VFA, mM

96.5

98.2

97.0

98.2

5.5

0.53

Individual VFA, mM

Acetate (A)

57.5

56.5

57.9

58.4

1.03

0.59

Propionate (P)

25.4

28.4

26.4

26.8

0.89

0.15

Butyrate

10.4

10.1

9.6

9.8

0.44

0.19

Valerate

1.53

1.57

1.52

1.50

0.07

0.60

Isobutyrate

0.87

0.83

0.86

0.92

0.05

0.22

Isovalerate

0.83

0.84

0.70

0.74

0.07

0.38

A:P

2.35

2.05

2.24

2.25

0.10

0.25

a,b

Means within a row with different superscripts differ (P < 0.05).

CL= control diet; NALL = Control + NALL (CJ CheilJedang, Seoul, Korea);

diNALL= Control + diNALL (CJ CheilJedang, Seoul, Korea); and AP = Control +

AjiPro

-L v2 (Ajinomoto, Japan).

140

CHAPTER 3

Bioavailability and lactational performance of dairy cows supplemented with N-

acetyl-L-methionine at different doses

ABSTRACT

The present study investigated the bioavailability and lactational performance of dairy

cows supplemented with N-acetyl-L-Met (NALM) at different doses. Six lactating dairy

cows (75  20.1 d-in-milk) were blocked by parity and d-in-milk. The experiment was

performed as a replicated 3 × 3 Latin square design. Within each square, cows were

randomly assigned to a sequence of 3 diets during each of the three 13-d periods (10 d of

treatment adaptation and 3 d of data collection and sampling). The 3 dietary treatments

are as follows: basal diet (15.7% CP) without NALM (control); control diet with 30 g/d

of supplemented NALM (30NALM), and control diet with 60 g/d of supplemented

NALM (60NALM). The NALM was rumen dosed for 11 d and then abomasally infused

on d 12 and d 13 for quantification of NALM bioavailability (BA). Supplementation of

NALM did not affect dry matter intake, milk production parameters, or feed efficiency.

Supplementation of NALM led to a quadratic trend (P = 0.06) toward increases in plasma

Arg, Gly, and Met concentrations and quadratic increases in plasma His and Ser

concentrations. Rumen NALM dosing led to a quadratic increase in the baseline mean

plasma Met concentration. Abomasal infusion with NALM increased the area under the

141

curve (AUC) versus control and both linear and quadratic increases in AUC were

observed with NALM dosing. The mean AUC per Met unit (µM × h/L ÷ Met (g)) for the

control, 30NALM, and 60NALM were 4.68, 5.89, and 5.07, which corresponded to a Met

BA (AUC per Met unit ÷ AUC per Met unit

Control

) of 100, 139, and 115%, respectively.

The absence of plasma NALM and increases in plasma Met for both ruminal and

abomasal NALM dosing in the current study suggests that NALM supplemented by

either rumen placement or abomasal infusion to lactating dairy cows may have been

deacetylated before entering the central circulation. In the current study, BA estimation

by plasma Met was greater for NALM doses, indicating NALM improved plasma Met

concentrations relative to the control. The absence of NALM in plasma, liver, milk, and

muscle reveals that the N-acetyl group is cleaved from NALM prior to reaching central

circulation and should be considered as a safe and effective BA rumen-protected Met

source in lactating dairy cows.

Key Words: N-acetyl-L-methionine, bioavailability, lactational performance, area under

the curve

INTRODUCTION

Protein nutrition of dairy cows in recent years has shifted from the simple use of

dietary CP towards meeting the ammonia and AA needs of ruminal fermentation for

MCP synthesis (Schwab and Broderick, 2017), while providing specific bypass AAs.

Strategic balancing of individual AA in MP has also been used as a method to maximize

142

lactational performance, meet AA requirements of the cow, and deliver consistent post-

ruminal AA, while minimizing total dietary CP supply (Lee et al., 2012a). When excess

CP is fed and the AA profile within the CP supply does not match animal AA

requirements, increases in environmental N excretion will result (Liu et al., 2017).

Because Lys and Met have traditionally been recognized as the most limiting AA for

lactating dairy cows (Schwab et al., 1992), research has focused on Met and Lys dietary

supply in order to maximize milk production and milk component synthesis. However,

balancing rations for only Lys and Met requirements may not improve productive

responses, because additive effects of other AAs may be overlooked. Therefore,

balancing rations for EAA requirements of dairy cows (not just Lys and Met) and for

multiple-limiting AA may help to reduce overfeeding of protein, improve post-ruminal

AA absorptive efficiency, reduce N excretion, and improve lactational performance (Liu

et al., 2017).

Protection of free AAs from ruminal degradation dates to the 1960s when researchers

began to understand the importance of balancing AA in order to meet dairy cow AA

requirements (Schwab and Broderick, 2017). Numerous physically protected (coated or

encapsulated) and chemical forms (Met analogues) of rumen-protected Met (RPMet)

have been developed to escape ruminal degradation and provide post-ruminal digestible

Met (dMet) for intestinal absorption (Schwab and Ordway, 2003). Production responses

to supplemental RPMet products have been inconsistent and the biological mechanisms

contributing to these responses are difficult to determine. However, infusing or

143

supplementing cows with RPMet products generally result in increased milk fat and

protein yields (Zanton et al., 2014).

Recently, a new form of RPMet, a Met derivative, N-acetyl-L-Met (NALM) has been

shown to increase milk fat composition and yield (Fagundes et al., 2018).

Supplementation of NALM to early and mid-lactation dairy cows improved milk yield

(Liang et al., 2019; Amaro et al., 2019). The studies note that NALM supplementation at

30 g/d resulted in the highest milk yield and these studies represent the initial attempts to

measure production responses to supplemental NALM in lactating dairy cows.

The NALM is a Met derivative produced by chemical protection of the L-Met α-

amino group with a N-acetyl group. In monogastric animals, the L-Met from NALM is

metabolically equivalent to free L-Met (Rotruck and Boggs, 1975). Several animal

studies conclude that the bioavailability (BA) of L-Met and NALM are similar based on

feeding studies measuring growth rates and weight gain (Boggs et al., 1975; Young

Jenkins et al., 1978; Friedman and Gumbmann, 1987). To date, there is no research

reported on the effects of supplementing NALM to lactating dairy cows on BA.

Therefore, the objective of this study was to evaluate the BA and lactational performance

of lactating dairy cows supplemented with NALM at 2 different doses. We hypothesized

that Met supply from abomasal infusion of NALM would increase plasma Met

concentration and the BA of NALM would be greater for each NALM dose compared to

the control.

144

MATERIALS AND METHODS

The dairy cows used in the present study were cared for according to the Live Animal

Use in Research Guidelines of the Institutional Animal Care and Use Committee at Utah

State University. The study was conducted at the Caine Dairy Research Center

(Wellsville, UT), Utah State University from March to May, 2019.

Cows, Experimental Design, and Diets

Three multiparous and three primiparous lactating Holstein dairy cows fitted with a

rumen cannula were used in the trial. Cows began the experiment averaging 75 ± 20.1

DIM with 32.7 ± 5.28 kg/d of milk yield. The experiment was performed in a replicated 3

× 3 Latin square design. Within each square, cows were randomly assigned to a sequence

of 3 diets during each of the three 13-d periods (10 d of treatment adaptation and 3 d of

data collection and sampling). A washout period of 7 d was assigned before the start of

subsequent periods to avoid carryover effects. Within each square, cows were randomly

assigned to a sequence of 3 dietary treatments: basal diet (15.7% CP) without NALM

(control); control diet with 30 g/d of supplemented NALM (30NALM), and control diet

with 60 g/d of supplemented NALM (60NALM; Table 3-1).

The developmental NALM product from CJ CheilJedang (Seoul, South Korea) was

used as the supplemental source of RPMet in this study. The NALM product was in

145

powder form, and contained Met concentration of 78.0%, with 99.5% purity. The NALM

product was added at a rate determined for cows to consume 30 and 60 g/cow/d of Met in

order to supply 15.6 and 31.2 g of dMet to the small intestine according to

manufacturer’s recommendation, respectively. The rates were also based on previous

research (Liang et al., 2019) and the 30 g/cow/d rate tested by Fagundes et al., (2018).

The recommendation of required digestible Lys and Met were assumed as 6.6 and 2.2%

of the MP requirements, respectively (NRC, 2001; Table 3-2). The NALM product was

placed through the rumen cannula at the liquid phase just under the rumen fiber mat daily

at 0700 h during the 10 d of treatment adaptation and 1 d of data collection and sampling.

Diets were formulated based on the NRC recommendations to provide sufficient NE

ME, MP, minerals, and vitamins with an estimated DMI of 25.0 kg/d to produce 40 kg/d

of milk with 3.8% fat and 3.0% true protein (NRC, 2001; Table 3-2). The basal diet was

also formulated to balance Lys (2.0 g/d) and Met (0 g/d) recommendations according to

NRC (2001). Cows were housed individually in tie stalls fitted with rubber mattresses

covered with straw, allowing free access to water. Cows were individually fed twice daily

for ad libitum intake at a level of 110% expected daily intake with 60% of allotted feed

fed at 0600 h and 40% fed at 1500 h. Feed offered and refused was recorded daily, and

feed samples were taken during the sampling week to determine DMI. Cows were milked

twice daily at 0600 and 1600 h, and milk production was recorded throughout the entire

experiment.

146

Feed and Milk Samplings and Analyses

Samples of alfalfa hay and corn silage were taken weekly to determine DM, and diets

were adjusted accordingly for change in DM concentration. Samples were ground to pass

a 1-mm screen (standard model 4; Arthur H. Thomas Co., Swedesboro, NJ), and stored

for chemical analysis. Samples of TMR and orts were collected from individual cows on

d 11 to d 13, composited by period, dried at 60°C for 48 h, and ground as previously

described. The DM concentrations of samples were used to calculate intakes of DM and

nutrients.

Analytical DM concentration of samples was determined by oven-drying overnight at

105 °C. Concentration of CP was determined using an automated N combustion analyzer

(Elementar, Analysensysteme GmbH, Hanau, Germany; AOAC, 2000; method 968.06).

Concentrations of NDF and ADF were sequentially determined using a fiber analyzer

(200/220, ANKOM Technology, Macedon, NY) according to the methodology supplied

by the company, which is based on the methods described by Van Soest et al. (1991).

Ether extract was measured using a fat analyzer (XT20, ANKOM Technology; AOAC,

2000; method 2003.05). In addition, samples of TMR were analyzed for starch by

Cumberland Valley Analytical Services (Waynesboro, PA).

Milk was sampled for 3 consecutive d (d 11 to 13) during the a.m. and p.m. milkings

each period. Individual milk samples were analyzed by the Rocky Mountain DHIA

Laboratory (Providence, UT) for fat, true protein, lactose, and milk urea nitrogen (MUN).

Milk composition was expressed on weighted milk yield of a.m. and p.m. samples. Milk

147

fat and protein yields were calculated by multiplying milk yield from the respective day

by fat and true protein concentration of the milk from an individual cow. A subset of milk

samples from d 11 to d 13 of each period were freeze-dried, composited by cow, and

stored for NALM and Met analysis by Bio Research Institute, CJ CheilJedang (Suwon,

South Korea).

BA, AA, and Plasma NALM Analyses

To determine the BA of NALM, 15.6 g (30 g oral equivalent) or 31.2 g (60 g oral

equivalent) of NALM was suspended in 1 L of water and bolus infused into the

abomasum over 5 min at 0700 h on d 12 and 13 of each sampling period. An infusion line

(Nalgene 980

braided clear PVC tubing; 6.4 mm i.d., 2.3 mm wall; Fisher Scientiﬁc,

Waltham, MA) containing a distal flange (Spires et al., 1975) was passed through the

sulcus omasi and placed in the abomasum via the abomasal orifice with an insertion and

delivery device as described by Gressley et al., (2006). Infusion line placement was

assured by hand to confirm the flange was in the abomasum and not the omasum. After

the NALM bolus administration, the infusion line was rinsed with 250 mL of water to

ensure full delivery of the NALM dose to the abomasum. Control infusion consisted of

1.25 L of water bolus infused into the abomasum.

Indwelling catheters were placed in the jugular vein after the a.m. milking on d 11 of

each sampling period and catheter placement altered each period. Catheters were

removed after the final blood sampling on d 13 of each period. Blood samples were

148

collected on d 11 at 0, 2, 4, 6, 8, and 10 h after rumen NALM dosing into 10-mL EDTA

plasma vacuum tubes. The blood samples taken on d 11 were used to establish individual

basal plasma Met concentration for each test animal before abomasal infusions on d 12

and d 13. Blood samples were collected on d 12 at 0, 1, 2, 3, 4, 5, 7, 9, 11, 13, 15 h and

on d 13 at 0, 2, 4, 6, 8 and 10 h after NALM abomasal infusion into 10-mL EDTA

plasma vacuum tubes. Samples were placed on ice and centrifuged at 2,300 × g for 20

min, after each sampling timepoint, and plasma was collected and stored at ‒40°C for

subsequent plasma Met, NALM, and AA analyses. The plasma samples were prepared

for AA and NALM analysis by filtration using an Amicon Ultra-0.5 Centrifugal Filter

Unit (3KDa cutoff; Cat # UFC5003BK, Millipore, Burlington, MA) to remove plasma

proteins. Flow-through samples were analyzed for Met and NALM using UPLC

(Shimadzu Nexera, Seoul, South Korea).

Methionine BA of the NALM dose was determined from the plasma Met area under

the curve (AUC) with respect to time for d 12 and 13 of each sampling period. The AUC

was numerically estimated using the trapezoidal rule after subtraction of the mean of the

pre-infusion concentrations from all values. The AUC was then expressed per gram of

total Met concentration in the diet (AUC per Met unit). The ratio of the NALM dose (30

g or 60 g) AUC per Met unit over the control AUC per Met unit represented the Met BA

of the NALM dose to the control. The NALM dose abomasally infused was adjusted for

ruminal loss (33%), Met content (78%), purity (99.5%), and therefore the resulting AUC

per Met Unit was expressed over the control for Met BA estimation of NALM. The

control Met BA is assumed to be 100%, because the dMet (total Met in the basal diet)

149

supplied in the control diet is available to the animal for absorption. Results of BA were

expressed as the mean percentage of the amount of Met supplied by NALM.

Liver and Muscle Biopsies

Liver and muscle biopsy samples were collected on d 12 of each sampling period post

a.m. feeding. Liver biopsies were collected by the paracostal, percutaneous needle biopsy

method as described by Mølgaard et al. (2012). Muscle biopsies were collected by

surgical excision of a 1 cm

M. longissimus dorsi sample, dorsal to the last rib under local

anesthesia as described by Bradley (1978). Liver and muscle biopsy samples were freeze-

dried and stored for tissue NALM and Met analysis by Bio Research Institute, CJ

CheilJedang.

Tissue and Milk NALM and Met Analyses

Freeze-dried muscle (approximately 50 mg) and liver samples (liver sample was used

in its entirety) were weighed and added to a lysis buffer consisting of 100 mM perchloric

acid for muscle (700 µL) and liver (200 µL) and soaked at 4°C for 20 min. A tissue

homogenizer pestle (Biomasher II

Closed System Disposable Tissue Homogenizer,

NIPPI Inc., Tokyo, Japan) was used to crush the tissue samples and then the suspension

was centrifuged at 13,000 × g for 15 min. The supernatant was collected and pH adjusted

to 4.5 with KOH and filtered. Flow-through was collected and analyzed for NALM and

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Met concentrations using a UPLC (Shimadzu Nexera) system as described by Hyde et al.,

(2004). Freeze-dried milk samples were dissolved in water (25 w%, 37°C) and then

filtered using an Amicon Ultra-0.5 Centrifugal Filter Unit (3KDa cutoff; Cat #

UFC5003BK, Millipore). Flow-through samples were analyzed for Met and NALM using

UPLC (Shimadzu Nexera).

Statistical Analysis

Data were analyzed as a replicated 3 × 3 Latin square using the Proc Mixed procedure

of SAS (version 9.4; SAS Institute Inc., Cary, NC). Data for intake, milk production, and

AUC were analyzed with a model that included fixed effects of period and treatment.

Cow nested within square and square were random effects. Data for AA were analyzed

using the model described above except that time was included as a repeated option. For

each variable analyzed, 3 covariance structures (autoregressive order 1, compound

symmetry, and unstructured covariance) were evaluated. The covariance structure that

resulted in the lowest values for the Akaike information criterion and the Schwartz

Bayesian criterion was used (Littell et al., 1998). Kenward-Roger's option was used to

calculate the denominator degrees of freedom. Means were separated by use of

orthogonal polynomial contrasts: 1) control vs. NALM treatments, 2) linear effect of

increasing NALM, and 3) quadratic effect of increasing NALM. Least square means are

reported throughout. Differences were considered significant at P ≤ 0.05, and trends were

discussed at 0.05 < P ≤ 0.10.

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RESULTS AND DISCUSSION

Characteristics of Diet Composition and Treatment

Ingredients and chemical composition of the basal diet are presented in Table 3-1.

Energy and protein supply and AA balance of the basal diet supplemented with NALM

are presented in Table 3-2. The basal diet was formulated to contain 15.7% CP and

provide adequate MP supply (60 g/d). All diets provided MP supply in excess of

requirements. Additionally, all diets were isoenergetic and supplied NE

and ME in

excess of requirement (NRC, 2001). Lysine and Met requirements were met for the basal

diet (2.0 g/d and 0 g/d balance) by dietary addition of canola meal, dried distillers grains

with solubles, and blood meal, respectively. The Met balance for NALM supplemented

diets were 15.6 and 31.2 g/d for the 30NALM and 60NALM, respectively.

The NRC (2001) model recommends that dLys and dMet concentrations in MP for

maintenance and optimal milk protein production approximate to 7.2 and 2.4%,

respectively. Common dietary strategies to achieve ideal Lys and Met concentrations

include feeding diets with greater CP concentration or supplementing RPLys and RPMet

products to diets with lower CP concentration to balance dLys and dMet in MP. Feeding

higher CP diets can result in excess ruminal AA supply, greater N excretion in the

environment, reduced N efficiency, and increase feed costs, which is economically

unfavorable to dairy producers.

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An alternative approach to optimize milk protein production is to maintain dLys and

dMet in MP closer to 6.6 and 2.2%, respectively, for a targeted dLys:dMet in MP

(efficiency for AA use) close to 3.0:1 (Schwab et al., 2003). Van Amburgh et al. (2015)

updated the CNCPS model (version 6.5) and reported optimal ratios of dLys:dMet in MP

closer to 2.69:1. The authors stated that to maximize milk protein yield, the calculated

estimates from breakpoint analysis in the updated CNCPS model for Lys and Met (% of

MP) were 7.00 and 2.60%, respectively, and to maximize milk protein content, 6.77 and

2.85 (% of MP), respectively (Van Amburgh et al., 2015). In the current study,

supplementing 30 g/d (30NALM; manufacturer’s recommended dose) and 60 g/d of

NALM decreased the dLys:dMet in MP to 2.46:1 and 2.05:1 (Table 3-2), respectively. In

addition, the dLys and dMet in MP were 6.53 and 2.66%, respectively, for the 30NALM

diet. Furthermore, the dLys and dMet in MP were 6.49 and 3.17%, respectively, for the

60NALM diet. Supplementation of 30 g/d NALM improved the dMet in MP, which

suggests a more favorable Met concentration in the diet for animal and production

utilization. However, increasing the supplemental NALM dose to 60 g/d reduced the

dLys and increased the dMet in MP, leading to suboptimal dLys:dMet in MP.

Feed Intake, Productive Performance, Feed Efficiency, and Milk Urea Nitrogen

Intake, milk production, feed efficiency, and MUN data are presented in Table 3-3.

Although primary production parameters were measured in the present study, caution

needs to be taken when interpreting the data due to the short trial length. Overall, feed

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intake, productive performance, and feed efficiency were not affected by NALM

supplementation, however MUN linearly decreased with NALM supplementation. The

basal diet was formulated to provide adequate MP and the dLys and dMet in MP were not

limiting, which may partly explain the lack of response to treatments. A common in vivo

technique to measure BA of AA in lactating dairy cows is the production response

approach (Schwab et al. 2001; Fleming et al., 2019). Whitehouse et al. (2017) suggested

the major limitation for the production response approach is to maintain dietary AA (Met

or Lys) deficiency, in all animals, over all treatment dosages administered, so that

linearity in milk protein concentration or yield is detected. Although the production

response approach was not used in the current study, the absence of positive milk protein

response to increasing NALM doses further supports that the basal diet was adequate in

dMet, which was expected. The lack of negative production parameter effects serves to

indicate that supplementation at 60NALM was still a safe concentration for the animals.

Liang et al. (2019) reported that NALM supplemented at rates of 0, 15, 30 and 60 g/d

to mid lactation dairy cows improved milk yield in a quadratic manner, with 30 g/d of

NALM seeming to be the optimal supplementation rate for milk yield under the

experimental conditions. Amaro et al. (2019) found that milk yield of early lactating

Holstein dairy cows tended to be greater (P = 0.07) with intermediate concentrations (15

and 30 g/d) of NALM compared to control animals. The authors noted that NALM

supplementation at 30 g/d resulted in the highest numerical milk yield (Amaro et al.,

2019), which is in agreement with Liang et al. (2019). Based on limited data in the

literature, NALM supplementation to either early or mid-lactation dairy cows has failed

154

to exhibit beneficial effects on milk protein concentrations (Amaro et al., 2019; Liang et

al., 2019). However, Fagundes et al. (2018) reported that NALM supplementation at 30

g/d increased milk fat concentration and yield and 3.5% FCM yield in MP-deficient (-190

g/d) and MP-adequate diets (50 g/d). The basal diet fed in the current study was similar in

MP (60 g/d) to that fed by Fagundes et al. (2018); however, each square within the Latin

square design consisted of 21-d periods versus 13-d periods for the current study,

respectively. Therefore, the short trial length in the present study, along with a 7-d

washout period may not have allowed animals to adapt to NALM supplementation to

exhibit changes in production parameters.

Liang et al. (2019) noted that MUN concentrations were not affected by oral NALM

supplemented at rates of 0, 15, 30 and 60 g/d to mid lactation dairy cows. Milk urea

nitrogen can be used as an indicator of N utilization efficiency in dairy cows. Variation in

MUN is related to the protein:energy ratio of the diet consumed. With adequate energy in

the diet, MUN can be indicative of protein status. All diets in the current study were

balanced to be isoenergetic and supplied NEL and ME in excess of requirement (Table 3-

2). The linear decrease in MUN concentrations due to NALM dosing was unexpected,

however once NALM reaches the small intestine it is deacetylated into the physiological

isomer L-Met that is systemically available for use by the body. Although it appears that

NALM dosing improved N utilization efficiency through reduced MUN content, milk

true protein was similar among NALM dosage rates. Lack of milk true protein change

suggests the free useable Met from NALM dosing may have been utilized for other

biological processes within the body. Additional research is needed to determine if

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NALM supplementation alters N utilization of early to mid-lactation dairy cows by oral

supplementation, as abomasal infusion of RPAAs is not a practical means of supplying

RPAAs to lactating dairy cows.

Plasma AA Profiles and NALM

Supplementation of NALM led to a quadratic trend (P = 0.06) toward increases in

plasma Arg, Gly, and Met and quadratic increases in plasma His and Ser concentrations

(Table 3-4). Alternatively, NALM supplementation linearly decreased plasma AA

concentrations of Leu, Phe, and Val and trends were observed for linear decreases in Ile

and Ala concentrations. Although plasma Met increased linearly, there was an overall

linear trend (P = 0.08) toward lower plasma EAA concentration. Total NEAA

concentration was not affected by supplementation of NALM. Recently, Liang et al.

(2019) described that NALM supplemented at rates of 0, 15, 30, and 60 g/d to mid

lactation dairy cows displayed quadratic increases in plasma Met concentration, with 30

g/d NALM resulting in the highest plasma Met concentration compared to the control

treatment (36.7 vs. 31.1 µM). In contrast, Fagundes et al. (2018) found that early to mid-

lactation dairy cows supplemented with 30 g/d NALM to MP-adequate diets had no

effect on plasma Met concentration, which was an unexpected finding to the authors.

Plasma Met concentrations provide a qualitative measure of post-ruminal delivery

and absorption of Met from RPMet products (Blum et al., 1999). Furthermore, plasma

AA concentrations can also indicate the balance between AA supply in the diet and

156

utilization (Meijer et al., 1995). However, the ability of NALM to be absorbed as L-Met

from the small intestine is dependent on the enzyme acylase I, which hydrolyzes NALM

to L-Met (Giardina, 1997; Baxter et al., 2002). The acylase I enzyme is found in the

intestine, liver, and kidney of mammalian tissues and has a high affinity for sulfur and

neutral AA derivates (Giardina et al., 1997). No NALM could be detected in either portal

or vena caval plasma in young pigs dosed with NALM by a gastrostomy tube (382 mg/kg

BW), suggesting deacylation of NALM in the intestinal lumen or mucosal cells (Daabees

et al., 1984). In addition, no NALM was detected in the plasma of adult subjects or 1-

year-old infants after oral loading (0.0605 mmoles/kg BW) with NALM (Stegink et al.,

1980; Stegink et al., 1982). The absence of plasma NALM (Table 3-5) and subsequent

increase in plasma Met concentration with NALM dosing in the current study suggests

that NALM supplemented by either rumen placement or abomasal infusion to lactating

dairy cows is deacylated before entering the central circulation.

In this study, NALM supplementation linearly decreased plasma branched-chain AA

(BCAA; Ile, Leu and Val) and EAA concentrations. Appuhamy et al. (2011) reported that

jugular infusion of a combination of L-Lys and L-Met led to a 17% reduction in plasma

Ile concentration compared to control cows. Blum et al. (1999) noted that plasma

concentrations of Val, Ile, and total BCAA were reduced, but only in cows fed a polymer

coated RPMet product. Similarly, Fagundes et al. (2018) observed decreased plasma

BCAA and total EAA concentrations, but only in a diet supplemented with NALM that

was adequate in MP, which was similar to the diet and findings of the current study. In

addition, Varvikko et al. (1999) abomasally infused DL-Met to early, mid and late

157

lactation cows. The authors found that the concentration of total BCAA and each

individual BCAA (Ile, Leu, and Val) in plasma decreased linearly with abomasal infusion

of DL-Met (Varvikko et al., 1999).

The NALM supplementation in the current study most likely increased BCAA

catabolism or tissue uptake, since EAAs that are not removed by the liver or peripheral

tissues return to general circulation (Arriola Apelo et al., 2014). Branched-chain amino

acids make up roughly 50% of the EAA in milk proteins (Mackle et al., 1999). Feeding

RPMet products may result in increased milk protein synthesis or yield, because Met is a

limiting EAA in dairy cows. Likewise, increased milk protein yield can enhance

mammary gland BCAA uptake, leading to decreased plasma BCAA concentrations. Milk

protein yield was not altered by NALM supplementation in our trial, thus suggesting that

NALM dosing did not alter BCAA metabolism in the mammary gland. Branched-chain

AAs and Met are important in portal drained viscera energy generation in ruminants

(Lobley et al., 2003), and dietary energy deficiencies can alter AA partitioning towards

greater oxidation and energy generation (Lapierre et al., 2006). All diets in the present

study supplied ME and NE

in excess of requirements according to NRC (2001).

Therefore, it is unlikely that the BCAAs were used for additional energy generation in

tissues after NALM dosing. It appears that NALM supplementation to lactating dairy

cows increases plasma Met concentration and alters individual plasma BCAA profiles;

however, more research is needed to understand the mechanisms involved in BCAA

metabolism and potential nutrient interactions with NALM dosing.

158

BA and Kinetic Parameters

Bioavailability and kinetic parameter results of NALM dosing are presented in Table

3-5. The mean basal plasma Met concentration was 25.4 µM for the control diet, which is

similar to observed values in the literature for lactating dairy cows (Appuhamy et al,

2011; Arriola Apelo et al, 2014). Rumen NALM dosing led to a quadratic increase in the

mean plasma Met concentration on d 11 of sampling after 10 d of rumen dosing. The

mean plasma Met was 39.7 µM and 39.5 µM for the 30NALM and the 60NALM

supplemented cows, respectively. The values reported in the current study were greater

than literature values reported for diets supplemented with other types of RPMet products

(Lee et al., 2015; Giallongo et al., 2016) and jugular infusion of L-Met to lactating dairy

cows (Appuhamy et al, 2011), but similar to values seen with diets supplemented with

NALM (Liang et al., 2019). Increased mean plasma Met after rumen NALM dosing

suggests that NALM is deacetylated and free Met is absorbed into the central circulation

for utilization by the animals.

Peak time and Met height after abomasal infusion of NALM did not differ among

treatments (Table 3-5). Plasma Met concentrations increased over the first 3 h after

abomasal infusion of NALM and returned to basal concentrations by 8 h post dosing

(Figure 3-1). Daabees et al. (1984) noted when equimolar amounts of L-Met and NALM

were supplemented to young pigs, the portal and venal caval peak plasma Met

concentrations were lower for NALM compared to L-Met. However, peak time after

dosing for both portal and vena caval plasma Met concentrations was greater (90 min) for

159

NALM versus portal (45 min) and vena caval (60 min) plasma Met concentrations after

L-Met dosing (Daabees et al., 1984). In humans, peak time after oral administration was

delayed for NALM compared to L-Met (Stegink et al., 1980). Peak time after

intraruminal supplementation (50 g/d Met equivalent) with either the isopropyl ester of 2-

hydroxy-4-(methylthio)-butanoic acid or polymer coated RPMet product in non-lactating

dairy cows, averaged 4 h and 22 h 25 min, respectively (Graulet et al., 2005).

The mean plasma Met concentrations after abomasal infusion of each cow on d 12

and 13 for each period were used to determine the AUC (Table 3-5). Abomasal infusion

with NALM led to an increased AUC versus control and both linear and quadratic

increases in AUC were observed with NALM dosing. The resulting AUC values were

expressed as the AUC per Met unit (i.e., AUC divided by the total dMet concentration in

each diet) in order to adjust for varying Met doses in the diets. The mean AUC per Met

unit (µM × h/L ÷ Met (g)) for the control, 30NALM, and 60NALM diets were 4.68, 5.89,

and 5.07, which corresponded to a Met BA (AUC per Met unit ÷ AUC per Met unit

Control

)

of 100, 139, and 115%, respectively. N-acetyl-L-Met has been shown to be BA and

capable of replacing the dietary requirement for Met in humans, rats, and swine, but had

not been studied in dairy cattle (Boggs et al. 1975; Rotruck and Boggs 1975; Zezulka and

Calloway, 1976; Stegink et al. 1980, 1982; Daabees et al. 1984). In the current study, BA

estimation by plasma Met appearance was higher for NALM doses, indicating NALM

improved plasma Met concentrations relative to the control. These results indicate that

NALM is also capable of replacing dietary Met in lactating dairy cattle. However, further

studies are warranted to evaluate the BA and kinetic parameters of NALM in lactating

160

dairy cows relative to unprotected forms of Met (L-Met) and RPMet products with

known BA values in the literature. To our knowledge, this is the first time the Met

derivative, NALM has been shown to increase plasma Met concentrations and serve as a

RPMet source for lactating dairy cows.

Residue Potential

Residue potential in lactating Holstein dairy cows supplemented with NALM is

summarized in Table 3-6. No NALM was detected in milk, liver, or muscle samples after

rumen placement or abomasal infusion. In humans, existing authorizations for NALM

have been approved in Germany for use in dietetic foods (BGBI, 1988) and in 1978, the

United States approved NALM as a source of L-Met food additive (Federal Register,

1978). Smith et al. (2011) described that the extraction efficiency of N-acetylated AAs is

known to be poor which can lead to a lack of detection of NALM. Therefore, tissue Met

concentrations were quantified to verify the NALM analysis method by intracellular

disruption and tissue Met release. Milk Met concentration was not altered by NALM

supplementation. Methionine extraction by the mammary gland, unlike Lys, closely

matches the amounts required for milk protein synthesis (Bequette et al., 1998).

Therefore, no improvement in milk protein yield due to NALM supplementation would

explain the lack of change in milk Met after NALM dosing. Liver and muscle Met

concentrations increased with NALM supplementation, resulting in both linear and

quadratic effects for increased liver Met concentration and a linear trend (P = 0.10) for

161

increased muscle Met concentration. The liver is responsible for Met metabolism

(Brosnan and Brosnan, 2006) and liver Met removal in dairy cows is related to total liver

inflow and blood flow, rather than net portal absorption (Lapierre et al, 2005; Arriola

Apelo et al, 2014). Additionally, hepatic Met removal varies with the source of RPMet

supplemented to lactating dairy cows. Common RPMet products contain equimolar

mixtures of D- and L- isomers; however, D-Met is not physiologically functional in the

dairy cow and must be converted to L-Met before it can be incorporated into mammalian

proteins (Lapierre et al., 2005; Lapierre et al., 2012). Since, NALM only contains the L-

isomer of Met, it can be utilized directly for protein synthesis after deacetylation, without

conversion to any other isoforms. Liver and muscle Met increased after NALM dosing,

demonstrating that free Met from the NALM source was absorbed, delivered to the liver,

and was able to be utilized by peripheral tissues (i.e, muscle). Furthermore, in lactating

dairy cows, the primary net user of post-liver Met supply is the mammary gland for milk

protein synthesis (Lapierre et al., 2005). Elevated Met concentrations in liver and muscle

after NALM supplementation, could be explained by the lack of response to milk protein

yield seen in the current study. The absence of NALM residue in plasma (Table 3-5),

liver, milk, and muscle reveal that the N-acetyl group is cleaved from NALM prior to

reaching central circulation and can be considered as a safe and effective RPMet source

in lactating dairy cows.

CONCLUSIONS

162

The current study evaluated the BA and lactational performance of dairy cows

supplemented with NALM at 2 different doses. Overall, production parameters were not

influenced by NALM supplementation, which was not surprising given the short study

periods. Plasma Met increased with NALM dosing, while individual BCAA

concentrations decreased. Bioavailability by plasma appearance of Met for NALM

treatments was higher for the 30NALM dose compared with the 60NALM and the

control, suggesting that optimal supplementation rate for NALM would be 30 g/cow per

day. Residue of NALM was not detected in plasma, milk, liver, or muscle samples. This

is the first attempt to measure the BA of the Met derivative in lactating dairy cattle and

the findings suggest that NALM is deacetylated before reaching central circulation,

increases plasma Met concentrations, and supplies lactating dairy cows with a source of

RPMet. However, further studies are needed to evaluate the comparative BA of NALM

in lactating dairy cows relative to unprotected forms of Met (L-Met) and RPMet products

with known BA values in the literature.

ACKNOWLEDGMENTS

The study was supported by CJ CheilJedang (Seoul, South Korea). The authors thank

Jed Oyler at Utah State University (Logan) for sampling assistance and the staff of the

Caine Dairy Research Center (Wellsville, UT) for their care and handling of the

experimental cows.

163

REFERENCES

Amaro, F. X., K. G. Arriola, Y. Jiang, D. Kim, A. P. Cervantes, V. P. Silva, M. C. N.

Agarussi, J. T. Silva, A. T. Adesogan, L. F. Ferraretto, C. R. Staples, J.-S. Eun, J. S.

Park, J. O. Moon, and D. Vyas. 2019. Lactational performance of dairy cows in

response to supplementing N-acetyl-L-methionine as a source of rumen protected

methionine. J. Dairy Sci. 101, Suppl. 2 (Abstract 485).

AOAC. 2000. Official Methods of Analysis. Vol. 1 and 2. 17th ed. AOAC Int.,

Gaithersburg, MD.

Appuhamy, J. A. D. R. N., J. R. Knapp, O. Becvar, J. Escobar, and M. D. Hanigan. 2011.

Effects of jugular-infused lysine, methionine, and branched-chain amino acids on

milk protein synthesis in high-producing dairy cows. J. Dairy Sci. 94:1952-1960.

Arriola Apelo, S. I., J. R. Knapp, and M. D. Hanigan. 2014. Invited review: Current

representation and future trends of predicting amino acid utilization in the lactating

dairy cow. J. Dairy Sci. 97:4000–4017.

Baxter, J., Y. Kim, and M. Snowden. 2002. Hydrolysis of N-acetyl-L-glutamine by

acylase I. J. Food Sci. 66:1428–1433.

Bequette, B. J., F. R. C. Backwell , and L. A. Crompton. 1998. Current concepts of amino

acid and protein metabolism by the lactating ruminant mammary gland. J Dairy Sci

8:2540–2559.

BGBI, 1988. Verordnung über diätetische Lebensmittel (Diätverordnung), Anlage 2, 1.2,

p. 26.

164

Blum, J. W., R. D. Bruckmaier, and H. Jans. 1999. Rumen-protected methionine fed to

dairy cows: Bioavailability and effects on plasma metabolite and insulin

concentrations. J. Dairy Sci. 82:1991-1998.

Boggs, R.W., J. T. Rotruck, and R. A. Damico. 1975. Acetylmethionine as a source of

methionine for the rat. J. Nutr. 105, 326-330.

Bradley, R. 1978. Skeletal muscle biopsy techniques in animals for histochemical and

ultrastructural examination and especially for the diagnosis of myodegeneration in

cattle. Br. Vet J. 134 (5):434-444.

Brosnan, J. T. and M. E. Brosnan. 2006. The sulfur-containing amino acids: an overview.

J. Nutr. 136:1636S-1640S.

Daabees, T. T., D. W. Andersen, W. L. Zike, L. J. Filer, Jr., and L. D. Stegink. 1984.

Portal and vena caval methionine concentrations in young pigs administered L-

methionine, N-acetyl-L-methionine and N-acetyl-D-methionine. J. Nutr. 114:1541–

1547.

Fagundes, M. A., S. Y. Yang, J.-S. Eun, J. O. Hall, J. O. Moon, and J. S. Park. 2018.

Influence of supplementing a methionine derivative, N-acetyl-L-methionine, in dairy

diets on production and ruminal fermentation by lactating cows during early to mid

lactation. J. Dairy Sci. 101(8):7082-7094.

Federal Register. 1978. Code of federal regulations title 21. 21CFR172.372.

https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=172.372

165

Fleming, A. J., K. A. Estes, H. Choi, B. A. Barton, C. A. Zimmerman, and M. D.

Hanigan. 2019. Assessing bioavailability of ruminally protected methionine and

lysine prototypes. J. Dairy Sci. 102:1-11.

Friedman, M. and Gumbmann, M.R. 1987. Nutritional value and safety of methionine

derivates, isometric dipeptides and hydroxy analogs in mice. J. Nutr. 118:388-397.

Giallongo, F., M. T. Harper, J. Oh, J. C. Lopes, H. Lapierre, R. A. Patton, C. Parys, I.

Shinzato, and A. N. Hristov. 2016. Effects of rumen-protected methionine, lysine, and

histidine on lactation performance of dairy cows. J. Dairy Sci. 99:4437–4452.

Giardina, T., A. Biagini, F. Dalle Ore, E. Ferre, M. Reynier, and A. Puigserver. 1997.

The hog intestinal mucosa acylase I: subcellular localization, isolation, kinetic studies

and biological function. Biochimie. 79:265–273.

Gressley, T. F., S. M. Reynal, J. J. Olmos Colmenero, G. A. Broderick, and L. E.

Armentano. 2006. Technical Note: Development of a tool to insert abomasal infusion

lines into dairy cows. J. Dairy Sci. 89:3965-3967.

Hyde, R., E. Hajduch, D. J. Powell, P. M. Taylor, and H. S. Hundal. 2004. Ceramide

down-regulates system A amino acid transport and protein synthesis in rat skeletal

muscle cells. The FASEB Journal express article10.1096/fj.04-2284fje.

Lapierre, H., G. Holtrop, A. G. Calder, J. Renaud, and G. E. Lobley. 2012. Is d-

methionine bioavailable to the dairy cow? J. Dairy Sci. 95:353–362.

Lapierre, H., R. Berthiaume, G. Raggio, M. C. Thivierge, L. Doepel, D. Pacheco, P.

Dubreuil, and G. E. Lobley. 2005. The route of absorbed nitrogen into milk protein.

Animal Science. 80:11-22.

166

Lee, C., A. N. Hristov, K. S. Heyler, T. W. Cassidy, H. Lapierre, G. A. Varga, and C.

Parys. 2012a. Effects of metabolizable protein supply and amino acid

supplementation on nitrogen utilization, milk production, and ammonia emissions

from manure in dairy cows. J. Dairy Sci. 95:5253–5268.

Lee, C., F. Giallongo., A.N. Hristov., H. Lapierre., T. W. Cassidy., K. S. Heyler., G. A.

Varga., and C. Parys. 2015. Effect of dietary protein level and rumen-protected amino

acid supplementation on amino acid utilization for milk protein in lactating dairy

cows. J. Dairy Sci. 98:1885–1902.

Liang, S. L., Z. H. Wei, J. J. Wu, X. L. Dong, J. X. Liu, and D. M. Wang. 2019. Effect of

N-acetyl-L-methionine supplementation on lactation performance and plasma

variables in mid-lactating dairy cows. J. Dairy. Sci. 102:5182-5190.

Littell, R. C., P. R. Henry, and C. B. Ammerman. 1998. Statistical analysis of repeated

measures data using SAS procedures. J. Anim. Sci. 76:1216–1231.

Liu, G. M., M. D. Hanigan, X. Y. Lin, K. Zhao, F. G. Jiang, R. R. White, Y. Wang, Z. Y.

Hu, and Z. H. Wang. 2017. Methionine, leucine, isoleucine, or threonine effects on

mammary cell signaling and pup growth in lactating mice. J. Dairy Sci. 100:4038–

4050.

Lobley, G. E., X. Shen, G. Le, D. M. Bremner, E. Milne, A. G. Calder, S. E. Anderson,

and N. Dennison. 2003. Oxidation of essential amino acids by the ovine

gastrointestinal tract. Br. J. Nutr. 89:617-630.

167

Mackle, T. R., D. A. Dwyer, and D. E. Bauman. 1999. Effects of branched-chain amino

acids and sodium caseinate on milk protein concentration and yield from dairy cows.

J. Dairy Sci. 82:161–171.

Meijer, G. A., J. Van der Meulen, J. G. Baker, C. J. Van der Koelen, and A. M. Van

Vuuren. 1995. Free amino acids in plasma and muscle of high yielding dairy cows in

early lactation. J. Dairy Sci. 78:1131-1141.

Mølgaard, L., B. M. Damgaard, V. Bjerre-Harpøth, and M. S. Herskin. 2012. Effects of

percutaneous needle liver biopsy on dairy cow behavior. Res Vet Sci. 93 (3):1248-

1254.

National Research Council. 2001. Nutrient Requirements of Dairy Cattle. 7th rev. ed.

Natl. Acad. Press. Washington, DC.

Rotruck, J. T., and R. W. Boggs. 1975. Comparative metabolism of L-methionine and N-

acetylated derivatives of methionine. J. Nutr. 105:331–337.

Schwab, C. G., C. K. Bozak, N. L. Whitehouse, and M. M. A. Mesbah. 1992. Amino acid

limitation and flow to the duodenum at four stages of lactation. I. Sequence of lysine

and methionine limitation. J. Dairy Sci. 75:3486–3502.

Schwab, C. G. and G. A. Broderick. 2017. A 100-year review: protein and amino acid

nutrition in dairy cows. J. Dairy Sci. 100:10094-10112.

Schwab, C.G., N.L. Whitehouse, A.M. McLaughlin, R.K. Kadariya, N.R. St-Pierre, B.K.

Sloan, R.M. Gill, and J.-C. Robert. 2001. Use of milk protein concentration to

estimate the methionine bioavailability of two forms of 2-hydroxy-4-(methylthio)

butanoic acid (HMB) for lactating cows. J. Dairy Sci. 84:(Suppl.1):35

168

Schwab C. G. and R. S. Ordway. 2003. Methionine supplementation options. In: Proc

Four-State Applied Nutrition Conference, Paper 2, Schwab.

Schwab, C. G., R. S. Ordway, and N. L. Whitehouse. 2003. The latest on amino acid

feeding. In: Proc. Southwest Nutrition and Management Conference, Phoenix, AZ, p.

27-41.

Smith, T., M. S. Ghandour, and P. L. Wood. 2011. Detection of N-acetyl methionine in

human and murine brain and neuronal and glial derived cell lines. J. Neurochem.

118:187-194.

Spires, H. R., J. H. Clark, R. G. Derrig, and C. L. Davis. 1975. Milk production and

nitrogen utilization in response to postruminal infusion of sodium caseinate in

lactating cows. J. Nutr. 105:1111–1121.

Stegink, L. D., L. J. Filer Jr., and G. L. Baker. 1980. Plasma methionine levels in normal

adult subjects after oral loading with L-methionine and N-Acetyl-L-methionine. J.

Nutr. 110:42–49.

Stegink, L. D., L. J. Filer Jr., and G. L. Baker. 1982. Plasma and urinary methionine

levels in one-year-old infants after oral loading with L-methionine and N-acetyl-L-

methionine. J. Nutr. 112(4):597-603.

Van Amburgh, M. E., E. A. Collao-Saenz, R. J. Higgs, D. A. Ross, E. B. Recktenwald, E.

Raffrenato, L. E. Chase, T. R. Overton, J. K. Mills, and A. Foskolos. 2015. The

Cornell Net Carbohydrate and Protein System: updates to the model and evaluation of

version 6.5. J. Dairy Sci. 98:6361–6380.

169

Van Soest, P. J., J. B. Robertson, and B. A. Lewis. 1991. Methods of dietary fiber, neutral

detergent fiber and nonstarch polysaccharides in relation to animal nutrition. J. Dairy

Sci. 74:3583–3597.

Varvikko, T., A. Vanhatalo, T. Jalava, and P. Huhtanen. 1999. Lactation and metabolic

responses to graded abomasal doses of methionine and lysine in cows fed grass silage

diets. J. Dairy Sci. 82:2659-2673.

Whitehouse, N. L., C. G. Schwab, and A. F. Brito. 2017. The plasma free amino acid

dose-response technique: A proposed methodology for determining lysine relative

bioavailability of rumen-protected lysine supplements. J. Dairy Sci. 100:9585-9601.

Young Jenkins, M., G. V. Mitchell, H. Valentine, and Z. J. Boyd. 1978. Biological

evaluation of isomers of N-acetyl-methionine and methionine in young rats. Nutr.

Rep. Int. 17:377-386.

Zanton, G. I., G. R. Bowman, M. Vázquez-Añon, and L. M. Rode. 2014. Meta-analysis

of lactation performance in dairy cows receiving supplemental dietary methionine

sources or postruminal infusion of methionine. J. Dairy Sci. 97:7085–7101.

Zezulka, A.Y. and Calloway, D.H. 1976. Nitrogen retention in men fed isolated Soybean

protein Supplemented with L-methionine, D-methionine, N-acetyl-L-methionine or

inorganic sulfate. J. Nutr. 106, 1286-1291.

170

Table 3-1. Ingredients and chemical composition of the basal diet

Item

% of DM

Ingredient

Alfalfa hay

30.0

Wheat straw

1.77

Corn silage

26.5

Corn grain (steam-flaked)

15.9

Distillers dried grains with solubles

3.54

Cottonseed, whole

3.54

Canola meal

7.07

Blood meal

1.06

Beet pulp, shreds

6.19

Fat supplement

1.77

Sodium bicarbonate

0.53

Yeast supplement

0.18

Vitamin and mineral mix

1.94

Chemical composition

DM, %

60.5

15.7

RDP

9.33

RUP

6.37

171

NDF

32.9

ADF

22.9

Starch

17.7

Sugar

6.00

Ether extract

3.98

NFC

37.3

Ash

10.0

Rumen-protected dry fat supplement (Nurisol

, WLT Distributors Inc., Winnipeg, MB,

Canada).

Diamond V XP

(Cargill Inc., Cedar Rapids, IA).

Formulated to contain (per kg DM): 226.7 mg of Se (from sodium selenite), 9278.7 mg of

Cu (from copper amino acid complex), 40,537.4 mg of Zn (from zinc amino acid

complex), 38,653.4 mg of Mn (from manganese amino acid complex), 552.6 mg of Co

(from cobalt carbonate), 1,234,585.2 IU of vitamin A, 152,808.1 IU of vitamin D, 3,815.1

IU of vitamin E, and 295 mg of Rumensin (Elanco Animal Health, Greenfield, IN).

Based on tabular value (NRC, 2001).

NFC = 100 – CP – NDF – ether extract – ash.

172

Table 3-2. Energy and protein supply and AA balance of the basal diet supplemented with

N-acetyl-L-Met (NALM) at 2 different doses

Diet

Item

Control

30NALM

60NALM

Energy supply

, Mcal/kg

1.65

ME, Mcal/d

66.2

ME, Mcal/kg

2.58

Protein supply,

g/d

MP supply

2,940

2,955

2,975

MP requirements

2,880

MP balance

Lys and Met balance,

g/d

dLys requirement

191

dLys supplied by the diet

193

Lys balance

2.00

dMet requirement

63.0

dMet supplied by the diet

63.0

dMet from Met derivative

0.00

15.6

31.2

Total dMet in the diet

63.0

78.6

94.2

dMet balance

0.00

15.6

31.2

173

dLys:dMet

3.06

2.46

2.05

Control = basal diet without NALM supplementation; 30NALM = control diet with 30 g/d

of supplemented NALM; and 60NALM = control diet with 60 g/d of supplemented

NALM.

All values were estimated using NRC (2001) based on actual DMI, milk yield, milk

composition, and BW of the cows throughout the trial.

Digestible Lys (dLys) and digestible Met (dMet) supply from the diets were estimated

using NRC (2001); supply of dMet from Met derivative was estimated with Met

concentration (78%) and absorption (67%) data provided by the manufacturer.

Requirements of dLys and dMet were calculated as 6.6 and 2.2% of MP requirements,

respectively.

Lys from diet-to-digestible Met from diet and dMet from NALM.

174

Table 3-3. Intake, milk production, feed efficiency, and milk urea nitrogen of lactating

Holstein dairy cows supplemented with N-acetyl-L-Met (NALM) at 2 different doses

Item

Diet

SEM

Contrast

Control

30NALM

60NALM

NALM

DMI, kg/d

20.4

20.7

20.3

1.55

0.96

0.83

0.65

Yield, kg/d

Milk

30.9

31.3

30.9

1.79

0.84

0.99

0.69

3.5% FCM

33.2

33.5

32.6

2.27

0.94

0.85

ECM

32.7

33.0

32.0

2.35

0.91

0.69

0.64

Milk composition, %

Fat

3.98

3.96

3.87

0.157

0.77

0.69

0.91

True protein

2.85

2.87

2.77

0.086

0.75

0.47

0.51

Lactose

4.74

4.73

0.136

0.77

0.75

0.98

Milk component yield,

kg/d

Fat

1.23

1.19

0.088

0.89

0.80

0.86

True protein

0.87

0.90

0.85

0.076

0.98

0.65

0.42

Lactose

1.47

1.46

0.103

0.99

0.87

0.77

Feed efficiency

Milk yield/DMI

1.53

1.52

1.54

0.144

0.87

0.75

0.82

175

3.5% FCM

yield/DMI

1.65

1.63

0.096

0.81

0.82

0.93

ECM yield/DMI

1.62

1.60

0.086

0.83

0.96

MUN, mg/100 mL

12.3

11.2

10.7

1.19

0.05

0.02

0.42

Control = basal diet without NALM supplementation; 30NALM = control diet with 30 g/d

of supplemented NALM; and 60NALM = control diet with 60 g/d of supplemented

NALM.

NALM = control vs. NALM treatments; L = linear effect of increasing NALM

supplementation; Q = quadratic effect of increasing NALM supplementation.

3.5% FCM = [0.4324 × milk yield (kg/d)] + [16.216 × fat yield (kg/d)].

ECM = 0.327 × milk yield (kg/d) + 12.95 × fat yield (kg/d) + 7.2 × protein yield (kg/d).

176

Table 3-4. Plasma AA concentrations (µmol/L) of lactating Holstein dairy cows

supplemented with N-acetyl-L-Met (NALM) at 2 different doses

Item

Diet

SEM

Contrast

Control

30NALM

60NALM

NALM

Arg

42.9

46.9

42.0

2.1

0.55

0.76

0.08

His

38.9

42.5

37.4

1.66

0.61

0.52

0.03

Ile

74.8

67.2

3.16

0.33

0.09

0.32

Leu

136

128

113

5.2

0.02

< 0.01

0.57

Lys

58.2

59.2

54.7

2.51

0.68

0.32

0.37

Met

16.0

21.6

22.1

1.07

< 0.01

0.06

Phe

42.2

40.3

36.4

1.56

0.05

0.01

0.61

Val

209

202

183

7.8

0.10

0.02

0.49

EAA

617

616

556

24.3

0.30

0.08

0.33

Ala

190

179

170

7.78

0.11

0.07

0.97

Gly

194

224

211

9.48

0.05

0.23

0.07

Ser

54.4

65.6

57.6

2.52

0.02

0.37

< 0.01

Tyr

40.5

41.4

37.3

1.51

0.54

0.14

0.18

NEAA

479

510

475

20.6

0.59

0.89

0.19

Control = basal diet without NALM supplementation; 30NALM = control diet with 30 g/d

of supplemented NALM; and 60NALM = control diet with 60 g/d of supplemented

NALM.

177

NALM = control vs. NALM treatments; L = linear effect of increasing NALM

supplementation; Q = quadratic effect of increasing NALM supplementation.

Essential AA analyzed.

Nonessential AA analyzed.

178

Table 3-5. Determination of bioavailability and kinetic parameters in lactating Holstein dairy

cows supplemented with N-acetyl-L-Met (NALM) at 2 different doses

Item

Diet

SEM

Contrast

Control

30NALM

60NALM

NALM

Basal plasma Met

concentration (BPC), µM

25.4

39.7

39.5

2.55

< 0.01

0.04

Peak time after abomasal

infusion, h

2:35

2:45

3:15

0.50

0.52

0.38

0.79

Peak Met height after

abomasal infusion, µM

45.4

65.4

84.1

13.0

0.16

0.12

0.97

Area under the curve (AUC),

µM × h/L

295

463

479

20.5

< 0.01

AUC per Met unit,

(µM ×

h/L)/Met (g)

4.68

5.89

5.07

0.24

0.01

0.14

< 0.01

Met bioavailability of

NALM,

100

139

115

0.07

< 0.01

0.09

< 0.01

Plasma NALM concentration

Control = basal diet without NALM supplementation; 30NALM = control diet with 30 g/d

of supplemented NALM; and 60NALM = control diet with 60 g/d of supplemented

NALM.

179

NALM = control vs. NALM treatments; L = linear effect of increasing NALM

supplementation; Q = quadratic effect of increasing NALM supplementation.

AUC per Met unit = area under the curve ÷ total dMet concentration in the diet (g).

Bioavailability = (AUC per Met unit ÷ AUC per Met unit

Control

) × 100.

ND = Not detected.

180

Table 3-6. Residue potential in lactating Holstein dairy cows supplemented with N-acetyl-L-

Met (NALM) at 2 different doses

Item

Diet

Contrast

Control

30NALM

60NALM

SEM

NALM

Milk NALM, (µM)

Milk Met, (µM)

8.17

6.42

10.3

2.67

0.59

0.58

0.41

Tissue NALM, (µM)

Liver

Muscle

Tissue Met, (µg/g)

Liver

15.6

25.4

22.9

1.16

< 0.01

0.01

Muscle

9.18

13.1

13.7

1.27

0.08

0.10

0.36

Control = basal diet without NALM supplementation; 30NALM = control diet with 30 g/d

of supplemented NALM; and 60NALM = control diet with 60 g/d of supplemented

NALM.

ND = Not detected.

181

Figure 3-1. Plasma Met concentration (µM) versus time plot for lactating Holstein dairy cows

abomasally infused with N-acetyl-L-Met (NALM) at 2 different doses

0 2 4 6 8 10 12 14 16

Plasma Met Concentration, (µM)

Time after NALM abomasal infusion, h

Control

30NALM

60NALM

182

CONCLUSIONS

Amino acid and protein nutrition in lactating dairy cows remain a topic of great

interest to researchers and the dairy industry. Substantial progress has been made over the

years in the development of rumen-protected forms of lysine and methionine. Lysine and

methionine have traditionally been identified as the most limiting essential amino acids

for lactating dairy cows. As more complex feeding models are developed, the

incorporation of a multiple-limiting amino acid model that includes branched-chain

amino acids (i.e., isoleucine, leucine, and valine) and additional essential amino acids,

(i.e, histidine, lysine, and methionine) if not all 10 essential amino acids could help to

reduce protein overfeeding, improve post-ruminal amino acid supply, decrease nitrogen

excretion, and improve lactational performance.

This dissertation investigated the use of acetylated methionine and lysine derivatives

as a source of rumen-protected amino acids for lactating dairy cows. Acetylated amino

acid derivatives are a free amino acid molecule with a chemical blocking group (acetyl

group) added to the either the ε-amino or α-amino group. Chapter 1 focused on

investigating production responses and ruminal fermentation characteristics of lactating

dairy cows when supplemented with N-acetyl-L-Methionine as a source of rumen-

protected methionine in metabolizable protein-deficient and -adequate diets. Improving

the total balance of amino acids in metabolizable protein has been one strategy used to

optimize lactational performance, while reducing the amount of dietary crude protein fed

in lactating dairy cow rations. Previous studies have evaluated the use of rumen-protected

183

methionine to improve lactational performance in dairy cows, but have obtained

inconsistent findings due to various feeding, physiological and complex inter-organ

metabolism of methionine by the rumen, liver, and mammary gland. Our objective was to

investigate lactational performance and nutrient utilization of early to mid-lactating dairy

cows supplemented without or with a developmental N-acetyl-L-methionine in

metabolizable protein-deficient or -adequate diets. Overall, the results demonstrated that

N-acetyl-L-methionine exerted minor influence on ruminal metabolism, but increased

milk fat concentration, which was an unexpected finding, possibly due to accelerated

fatty acid delivery from the liver to the mammary gland, resulting in sizable increases in

milk fat yield and feed efficiency.

In chapter 2, the objective was to investigate lactational performance and nutrient

utilization by mid to late lactating dairy cows supplemented with three different forms of

rumen-protected lysine (2 developmental lysine derivatives and 1 commercially available

lysine supplement). Results of the study demonstrate that feeding the Nα, ε-acetyl-L-

lysine supplemented diet in mid to late lactation dairy cows decreased dry matter intake

and maintained milk yield, leading to greater feed efficiency compared to the control diet.

These findings suggest that the developmental N-acetyl-L-lysine products (Nα, ε-acetyl-

L-Lysine and Nε-acetyl-L-Lysine) may be biologically active in lactating dairy cows,

with Nα, ε-acetyl-L-lysine showing the most benefit to improve feed efficiency and

increase milk yield. However, further research is needed to discover the role of lysine

derivatives on productive performance, ruminal metabolism, and bioavailability in

184

lactating dairy cows as this was the first attempt to evaluate the use of lysine derivatives

in lactating dairy cows.

In chapter 3, the objective of the study was to evaluate the bioavailability and

lactational performance of lactating dairy cows supplemented with N-acetyl-L-

methionine at 2 different doses (30 and 60 g/d). To date, there had been no research

reported on the effects of supplementing N-acetyl-L-methionine on bioavailability and

safety to lactating dairy cows. Overall, production parameters were not influenced by N-

acetyl-L-methionine supplementation, which was not surprising given the short study

periods. Plasma methionine increased with N-acetyl-L-methionine dosing, while

individual branched chain amino acid concentrations decreased, which was an

unexpected finding. Bioavailability by plasma appearance of methionine for N-acetyl-L-

methionine treatments was higher for the 30 g/d N-acetyl-L-methionine dose compared

with the 60 g/d N-acetyl-L-methionine and control dose, suggesting that the optimal

supplementation rate for N-acetyl-L-methionine is 30 g/cow/d. N-acetyl-L-methionine

was not detected in plasma, milk, liver or muscle samples. This again was the first

attempt to measure the bioavailability of the methionine derivative in lactating dairy

cattle and the findings suggest that N-acetyl-L-methionine is deacetylated before reaching

central circulation, increases plasma methionine concentrations, and supplies lactating

dairy cows with a safe bioavailable source of rumen-protected methionine. Although

there were differences observed between studies in lactational performance (chapter 1-3),

the overall major findings of these studies indicate that acetylated methionine is safe for

185

use in lactating dairy cows and both methionine and lysine derivatives should be

considered as a source of rumen-protected amino acids for lactating dairy cows.

The daily profit per lactating dairy cow being fed with rumen protected methionine

has been estimated to be around 30 cents (Cho et al., 2007; Chen et al., 2011). Dairy

nutritionists commonly report a return on investment of 2.5:1 or higher when rations are

balanced for rumen protected lysine and methionine in metabolizable protein (Schwab,

2012). Additionally, increases in income over feed cost approaching 40 to 50 cents per

cow per day from precision balancing for rumen degradable protein and rumen

undegradable protein, while balancing for lysine and methionine have been described by

dairy nutritionists (Schwab, 2012). The concept of the multiple-limiting amino acid

model may be profitable based on the values above through precision feeding, however

the environmental impacts of reducing nitrogen excretion by lysine and methionine

derivative supplementation should not be overlooked.

Consumers of dairy products are aware of the environmental impacts of feeding and

raising livestock animals. Animal agriculture has been identified as a major source of

nitrogen pollution to water resources. Animal wastes can contribute to nitrogen pollution

of the environment as ammonia volatilized to the air, nitrate leached into ground water,

and nitrogen that runs off to surface water. Improvement of nitrogen utilization efficiency

by lactating dairy cows fed with amino acid derivatives could decrease nitrogen losses

from farms and help to reduce the negative environmental impacts of livestock animals.

186

Although considerable advancements have been made in amino acid nutrition, future

works need to focus on the development of products and technologies that promote

animal health (i.e., improved immunity) through balanced feeding allowing for lower

protein diets and increased microbial protein synthesis. Further works could include the

use of acetylated amino acid derivative mixtures supplemented to lactating dairy cows.

Additionally, future works should focus on the “real-life” estimates of potential

reductions in nitrogen excretion by supplementing amino acid derivatives through

improving the amino acid profile reaching the small intestine. Future research needs to

focus on longer term feeding periods during various stages of lactation (i.e., early, mid or

late). The study durations should be extended to measure responses with larger sample

sizes and explore the biological and commercial significance of the dietary treatments.

This could also allow researchers to pinpoint when acetylated amino acid derivatives

exert the greatest effects to dairy cows during lactation. It will also aid dairy farmers on

deciding the optimal time to feed acetylated amino acid derivatives to lactating dairy

cows to maximize animal health, reduce environmental nitrogen excretion, and increase

profits.

REFERENCES

Chen, Z. H., G. A. Broderick, N. D. Luchini, B. K. Sloan, and E. Devillard. 2011. Effect

of feeding different sources of rumen-protected methionine on milk production and

N- utilization in lactating dairy cows. J. Dairy Sci. 94:1978–1988.

187

Cho. J., T. R. Overton, C. G. Schwab, and L. W. Tauer. 2007. Determining the Amount

of Rumen-Protected Methionine Supplement That Corresponds to the Optimal Levels

of Methionine in Metabolizable Protein for Maximizing Milk Protein Production

and Profit on Dairy Farms. J. Dairy Sci. 90(10):4908-16.

Schwab C. G. 2012. The principles of balancing diets for amino acids and their impact on

N utilization efficiency. Proc. Florida Ruminant Nutrition Symposium.

188

CURRICULUM VITAE

CONTACT INFORMATION

Mark A. Fagundes, DVM

Ph.D. Candidate, Utah State University Logan, UT

13702 Avenue 18 1/2

Chowchilla, CA 93610

Cell: (559) 999-6928

[email protected]

CURRENT POSITION

2017-present Herd Veterinarian and Nutritionist, Avila Family Dairy, LLC,

13644 Avenue 18 ½ Chowchilla, CA 93610

2019-present Dairy Records Analyst, The HEALTHSUM Syndicate, LLC, 445

Barnard Blvd Sunnyside, WA 98944

2020-present Associate Veterinarian, Swinging Udders Veterinarian Services,

8418 Liberty Rd Galt, CA 95632

EDUCATION

Anticipated Ph.D. Utah State University, Logan, UT*

July 2020 Major: Ruminant Nutrition

Major Professor: Jeffery Hall, DVM, Ph.D., Diplomat

A.B.V.T.

Dissertation: Investigation of methionine and lysine

derivatives as a source of rumen-protected amino acids

for lactating dairy cows.

*First participant through a concurrent DVM/Ph.D.

program at Utah State University School of Veterinary

Medicine

2017 DVM Utah State University: School of Veterinary Medicine,

Logan, UT/Washington State University: College of

Veterinary Medicine, Pullman, WA

WIMU Regional Program in Veterinary Medicine

2012 B.S. California Polytechnic State University, San Luis Obispo,

Major: Dairy Science

Minor: Agribusiness

189

ACADEMIC EXPERIENCE

2017-2019 Clinical Veterinary Internship

Utah State University School of Veterinary Medicine

Clinical Veterinary Services Team

4815 Old Main Hill

Logan, Utah 84322-4815

Clinical Advisor: Rusty Stott, DVM

2019 Senior Paper Discussant

Washington State University: College of Veterinary

Medicine, Pullman, WA

Student Presenter: Jessica Thomas

Presentation Date: September 19

, 2019

Topic: Unusual presentations and early recognition of canine

hypoadrenocorticism

Student Presenter: Allani Delis

Presentation Date: October 31

, 2019

Topic: Precursor-targeted immune mediated anemia in the dog.

RECENT/CURRENT RESEARCH

2017 Title: Effects of supplementing three different forms of rumen-

protected lysine on lactational performance of mid to late lactation

dairy cows.

2019 Title: Bioavailability, lactational performance and residue

potential of lactating dairy cows supplemented with N-acetyl-L-

methionine at different doses.

CURRENT FIELDS OF RESEARCH INTEREST

• Development of feeding strategies to improve nutrient management and

environmental performance of dairy cattle

• Improving metabolic and feed efficiency and animal health by enhancing

nutrients (protein, carbohydrate, lipid, and mineral) and energy utilization by

livestock animals

• Manipulation and characterization of ruminal fermentation and physiology to

enhance ruminant production

• Exploring gut physiology and immunology of livestock animals

• Immunonutrients in ruminants

• Strategic use of feed additives (amino acids) in livestock

VETERINARY LICENSURE

Utah Inactive

190

Idaho Active

California Pending

ACCREDITATION

California Pending

Accreditation allows participation in all accreditation work following State and Federal

laws and regulations under the direction of the United States Department of Agriculture’s

Animal and Plant Health Inspection Service (USDA-APHIS).

ACADEMIC ACHEIVEMENT

2014-2016 The Research Scholars Program

Utah State University School of Veterinary Medicine

Program Goal: Attract to the veterinary student population, and

hence to the veterinary profession, individuals of exceptional

aptitude who are oriented toward a career of basic or applied

research.

2015 1

Prize- Oral Veterinary Student Presentation, ADVS Graduate

Student Research Symposium, Logan, UT

2014 3

place, poster presentation competition, ADVS Graduate Student

Research Symposium, Logan, UT

2013-2015 Appointed Large Animal Club President

Utah State University School of Veterinary Medicine

PROFESSIONAL TRAINING

2017 Liver Biopsy Wet Lab

Course Instructor: Jeffery Hall, DVM, Ph.D., Diplomat ABVT

2013 Milk Quality/LactoCorder Training

Veterinarian’s Outlet Dublin, Texas

Course Instructor: Ynte Schukken DVM, Ph.D., MS

Cornell University College of Veterinary Medicine: Quality Milk

Production Services

2013 Ovum Pickup (OPU) in the bovine

California Polytechnic State University, San Luis Obispo, CA

Course Instructors: Joy Altermatt DVM, MS, DACT

Fernando Campos-Chillon DVM, MS, Ph.D., DACT

PROFESSIONAL EXPERIENCE

2017-2019 Clinical Veterinary Internship

Utah State University School of Veterinary Medicine

191

Clinical Veterinary Services Team

4815 Old Main Hill

Logan, Utah 84322-4815

Duties: On-call responsibilities of one weeknight each week and

one weekend a month. Oversight of research animals.

Participation in small ruminant surgeries and routine visits to

livestock operations (Dairy, Beef, Small Ruminant, Swine).

Participation in classroom/laboratory teaching with undergraduate,

graduate and veterinary students.

2011-2013 Veterinary Assistant

Westside Veterinary Services, Inc.

1531 E. Pacheco Blvd.

Los Banos, CA 93635

Duties: Accompanied veterinarians to on-farm calls, administered

injections, pregnancy palpation via portable ultrasound, milk

quality investigation, routine timing, teat scoring, parlor audits,

lameness evaluations, nutrition diagnostics and dairy records

analysis.

2012-2013 Associate

Ani-Tech Production Solutions

Duties: Assisted with graphing pulsators, vacuum measurements,

CIP evaluations, NMC milking system evaluations and

troubleshooting increased mastitis incidence on dairy farms.

2012-2013 Hospital Manager

Red Top Jerseys

21519 Rd 4, Chowchilla, CA 93610

Duties: Diagnosing and treatment of sick animals. Developed

treatment protocols and trained employees in the use of an on-farm

milk culture system. Employee on-farm training of

ultrasonography to determine herd pregnancy status.

PUBLICATIONS

• Journal Articles *indicates corresponding author

1. Fagundes, M. A., S.Y. Yang, J.-S. Eun*, J. O. Hall, J.O. Moon and J.S. Park.

2018. Influence of supplementing a methionine derivative, N-acetyl-L-

methionine, in dairy diets on production and ruminal fermentation by lactating

cows during early to mid lactation. J. Dairy. Sci. 101:7082-7094.

• Abstracts in Peer-Refereed Conference Proceedings

192

1. Eun, J.-S., M. A. Fagundes, D. Vyas, J. S. Park, and J. O. Moon. 2019.

Gastrointestinal availability of a methionine derivative, N-acetyl-L-methionine,

and its impacts on lactational performance and nitrogen metabolism of dairy

cows. Abstract accepted to International Symposium on Ruminant Physiology.

2. Sharp, S., M. A. Fagundes, J.-S. Eun, J. O. Hall, J. S. Park, and J. O. Moon.

2018. Ruminal escape and duodenal appearance of N-acetyl-L-methionine in

lactating dairy cows. J. Dairy Sci. 101 (Suppl. 2):320. (Abstr.)

3. Fagundes, M. A.*, S. A. Blaser, S. Y. Yang, J.-S. Eun, and J. O. Moon. 2016.

Effects of supplementing a rumen-protected methionine on lactational

performance of Holstein dairy cows during early and mid lactation. Journal of

Animal Science 94(supplement5):349.

• Oral Conference Presentations *indicates presenting author

1. Fagundes, M. A.*, S. A. Blaser, S. Y. Yang, J.-S. Eun, and J. O. Moon. 2016.

0728 Effects of supplementing a rumen-protected methionine on lactational

performance of Holstein dairy cows during early and mid lactation. Conference:

2016 ADSA-ASAS JAM, Salt Lake City. Volume: 99 (E-suppl. 1). (ADSA

Production Division Graduate Student Oral Competition: PHD).

PROFESSIONAL AFFILIATIONS

American Veterinary Medical Association

American Association of Bovine Practitioners

Academy of Dairy Veterinary Consultants

American Dairy Science Association

American Society of Animal Science

Dairy Cattle Reproductive Council

National Mastitis Council