Assessing the Bioavailability of Infused Lysine and Rumen-Protected Lysine Supplements Using the Area Under the Curve Technique and the Plasma Free Amino Acid Dose-Response Method

University of New Hampshire University of New Hampshire

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Honors Theses and Capstones Student Scholarship

Spring 2023

Assessing the Bioavailability of Infused Lysine and Rumen-Assessing the Bioavailability of Infused Lysine and Rumen-

Protected Lysine Supplements Using the Area Under the Curve Protected Lysine Supplements Using the Area Under the Curve

Technique and the Plasma Free Amino Acid Dose-Response Technique and the Plasma Free Amino Acid Dose-Response

Method Method

Megan Vetter

University of New Hampshire, Durham

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Using the Area Under the Curve Technique and the Plasma Free Amino Acid Dose-Response Method"

(2023).

Honors Theses and Capstones

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Vetter 1

Assessing the Bioavailability of Infused Lysine and Rumen-Protected

Lysine Supplements Using the Area Under the Curve Technique and the

Plasma Free Amino Acid Dose-Response Method

Megan M. Vetter

Senior Thesis 2022-2023

PI: Dr. Nancy Whitehouse

Thesis Advisor: Dr. Nancy Whitehouse

College of Life Sciences and Agriculture, UNH

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Table of Contents

List of Tables……………………………………………………………………...………………….…….3

List of Figures…………………………………………………………………...……………...…………..4

Abstract………………………………………………………………………………...…………...………5

Chapter

1. Review of Literature………………………………………………………...…………………6

a. Introduction…………………………………………………………..……………….6

b. Lysine…………………………………………………………………………..……..7

c. Protein Digestion & Absorption………………………………….………….………..7

d. Amino Acid Supplementation………………………………………………..…..….10

2. Area Under the Curve Experiment ………………………………...…...……………………12

a. Introduction……………………………………………...…………...…...…………12

b. Objective…………………………………………...………………………...……...12

c. Materials & Methods……………………………..………………………………….12

i. Experimental Design & Treatments…………..………………………….…12

ii. Management of Cows………………………………………………….……13

iii. Blood Sampling & Analysis……………………………………….………..14

iv. Statistical Methods………………………………………………………….14

d. Results & Discussion………………………………………………………...………15

e. Conclusions…………………………………………………………………...……..17

3. Plasma Dose Response Experiment………………………………………………………….29

a. Introduction………………………………………………………………………….29

b. Objective………………………………………………………………...…………..29

c. Materials & Methods…………………………………………………….…………..30

i. Experimental Design & Treatments……………………………….………..30

ii. Management of Cows………………………………………………….……31

iii. Feed Sampling & Analysis………………………………………………….31

iv. Blood Sampling & Analysis………………………………………...………32

v. Milk Sampling & Analysis………………………………………………….32

vi. Statistical Methods………………………………………………………….33

d. Results & Discussion………………………………………………………..……….34

e. Conclusions………………………………………………………………………….36

References……………………………………………………………………………...………………….50

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List of Tables

Chapter 2

Table 1. Calculations for the amounts of RP-AA products to feed. …………………………….18

Table 2. Ingredient composition of basal Lys-adequate diet. ………...…………………………19

Table 3. Chemical composition of feedstuffs……………………………………………...…….20

Table 4. Amino acid composition of feedstuffs………………………………………….………21

Table 5. AMTS (Ver. 4.17.0.1) evaluation of the basal formulated diet……………...…………22

Table 6. Milk and dry matter intake for Holstein cows fed a diet supplemented with RP-Lys

supplements………………………………………………………………………………………23

Table 7. Lys concentration, area under the curve and bioavailability for Holstein cows fed a diet

supplemented with RP-Lys supplements………………………………………………………...24

Table 8. Plasma amino acids (µM) for Holstein cows fed a diet supplemented with RP-Lys

supplements. ………………………………………………………………………………….….25

Table 9. Plasma metabolites (µM) for Holstein cows fed a diet supplemented with RP-Lys

supplements. ……………………………………………………………………………….…….28

Chapter 3

Table 1. Calculations for the amounts of RP-AA products to feed. ……………………...……..38

Table 2. Ingredient composition of basal Lys-adequate diet. …………………………...………39

Table 3. Chemical composition of feedstuffs……………………………………………………40

Table 4. Amino acid composition of feedstuffs………………………………………………….41

Table 5. AMTS (Ver. 4.17.0.1) evaluation of the basal formulated diet……………...…………42

Table 6. Milk and dry matter intake for Holstein cows fed a diet supplemented with RP-Lys

supplements………………………………………………………………………………………43

Table 7. Plasma amino acids (µM) for Holstein cows fed a diet supplemented with RP-Lys

supplements………………………………………………………………………………………44

Table 8. Plasma metabolites (µM) for Holstein cows fed a diet supplemented with RP-Lys

supplements. ……………………………………………………………………………………..45

Table 9. Calculation of bioavailability using changes in % of plasma Lys (TAA-Lys) and

commercial values. ……………………………………………………………………...………46

Table 10. Statistical summary of trial results using changes in % of plasma Lys (TAA-Lys) and

commercial values…………………………………………………………………….................47

Table 11. Metabolizable Lys for the RP-Lys supplements……………………………...……….49

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List of Figures

Chapter 2

Figure 1. Area under the curve for Holstein cows fed a diet supplemented with RP-Lys

supplements………………………………………………………………………………..……..25

Chapter 3

Figure 1. Relationship between Infusion, IL, and II HPO in lactating dairy cows using

commercial Lys content…………………………………………………………….……………48

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Abstract

The milk production of lactating dairy cows is dependent on factors such as housing

conditions, lineage, climate, and health, but the quality of their diets is generally the most

influential. Maintaining a proper balance of nutrients is necessary to achieve the greatest milk

production at the lowest cost. Maximum feed efficiency is not only critical for increasing the

economic profits of an individual farm, but also for increasing food supply without increasing

environmental demand. Supplementing cows’ diets with lysine (Lys), an essential amino acid

(AA), can aid in maximizing protein synthesis. Providing this nutrient in a rumen-protected (RP)

coating can increase its bioavailability by delaying its degradation in the rumen. Using a reliable

method to assess the bioavailability of an AA is important for ensuring accurate results that can

be utilized to yield further improvements in the development of these supplements.

The objective of this experiment was to determine the bioavailability of RP-Lys

supplements using plasma Lys concentrations for analysis via the area under the curve (AUC)

method and the plasma dose response (PDR) method. Lactating Holsteins, fitted with rumen

cannulas, were used in two experiments, through which they received varying forms of Lys.

Blood samples were collected, and plasma concentration was measured for each cow. Feed

intake, milk yield, and milk components were observed. Data was analyzed to determine average

milk yield and dry matter intake, and AUC and PDR statistical analyses were performed to

measure the bioavailability of each treatment. While no statistical significance was seen among

the Lys prototypes, Prototype II exhibited a higher bioavailability via the AUC method, while

Prototype IL exhibited a higher bioavailability via the PDR method. Ultimately, the PDR method

appears to be a more effective strategy for determining the bioavailability of Lys.

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Chapter 1: Review of Literature

Introduction

The amino acid (AA) concentration of a diet is especially important to consider when

formulating rations, as the synthesis of milk protein in the mammary gland utilizes AAs in the

blood. These organic molecules composed of carbon, hydrogen, oxygen, and nitrogen atoms, that

each contain an amino and a carboxyl functional group, as well as a unique side chain joined to

the central carbon atom. AAs are termed by the sequences of their atoms, with over 700 existing

in nature and 20 of those serving as the building blocks of protein (Wu, 2013). Those which are

substrates of polypeptide biosynthesis are referred to as protein amino acids and compose 98% of

the total amino acid concentration of most feed ingredients sourced from plant and animal

origins (Wu, 2013).

Proteins are the fundamental components of tissues in animals, with roles that include

catalyzing chemical reactions in cells, contributing to cell structure, initiating contractions for

cell motility, and transporting nutrients and wastes in and out of cells (NASEM, 2021). These

molecules also function as antibodies and hormones, and are responsible for regulating gene

expression (NASEM, 2021). Thus, both proteins and amino acids are vital components of life for

all mammals.

Essential amino acids are those that cannot be synthesized in animal tissues nor at rates

sufficient to meet specific requirements. In contrast to non-essential amino acids, which are

readily synthesized from each other, from metabolites of intermediary metabolism, or from an

excess of essential amino acids, these amino acids are considered indispensable and must be

consumed via dietary intake. The essential amino acid supplied in the smallest amount relative to

an animal’s requirements is known as the first limiting amino acid, while the essential amino

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acid supplied in the second smallest amount is referred to as the second limiting amino acid.

Lysine and methionine have been identified as the first limiting amino acids for dairy cattle that

are fed a common corn/grain-based diet in North America, with histidine identified as a first

limiting amino acid for cows fed typical grass-based diets (NASEM, 2021).

Lysine

Lysine was first discovered as an alkaline substance present in casein hydrolysates by E.

Drechsel in 1889 and later discovered in the hydrolysates of conglutin, gluten fibrin, and egg

albumin (Wu, 2013). The name “lysine” was given by Ernst Fischer due to the release of urea

that resulted when this substance was hydrolyzed in alkaline conditions by barium hydroxide

(Wu, 2013). The primary function of this essential amino acid is protein synthesis, though it

contributes to the structure and function of collagen via the form of hydroxylysine, and serves to

regulate nitric oxide synthesis, antiviral activity, protein methylation, and protein acetylation

(Broderick & Schwab, 2017).

Lysine is strictly a ketogenic amino acid because the process of its catabolism produces

acetyl coenzyme A, but it does not contribute to gluconeogenesis (Wu, 2013). It is degraded in

the liver via the mitochondrial saccharopine pathway and the peroxisomal pipecolate pathway

(Wu, 2013). Therefore, promoting the efficiencies of these pathways is important to ensure

adequate lysine absorption.

Protein Digestion & Absorption

Lactating dairy cows require high levels of dietary protein due to the significant demand

of amino acids necessary to synthesize large amounts of milk protein. All dietary protein is first

denatured in the abomasum, the true stomach, by hydrochloric acid, before digestion by various

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proteases begins. This involves hydrolysis by pepsins, which forms peptides that are absorbed by

the lumen of the small intestine, where they are then further hydrolyzed into peptidases (Wu,

2013). These peptidases then produce small peptides and free amino acids, which can be taken

up by different microbes to generate numerous products, including ammonia, microbial protein,

nitrogenous substances, pyruvate, short-chain fatty acids, and branched-chain fatty acids (Wu,

2013). Short-chain fatty acids, including acetate, butyrate, and propionate, contribute to glucose

and fat production, while branched-chain fatty acids serve as growth factors for microorganisms,

as well as precursors of long-chain branched fatty acids (Wu, 2013). However, not all dietary

protein passes through the rumen without degradation.

A cow’s protein requirement can be determined via the metabolizable protein (MP)

system, which measures the concentration of protein that is absorbed from the epithelial cells of

the small intestine and that is available to be taken up as amino acids by the blood (Herdt, 2014).

Unlike the crude protein system, which only accounts for protein from non-protein nitrogen

sources, the MP system recognizes that not all protein provided to cows might be available for

absorption. MP consists of microbial protein synthesized in the rumen, referred to as rumen

degradable protein (RDP), and proteins in the diet that avoid degradation by the rumen, referred

to as rumen undegradable protein (RUP) (Herdt, 2014). The total proportion of dietary protein

that is digested in the rumen varies from 30% to 40% for less soluble proteins, to 70% to 85% for

most diets (Wu, 2013). The rate at which protein is degraded in the rumen depends on the length

of time the protein remains in the rumen, the proteolytic activity of the rumen microbes, and the

type of protein supplied (Wu, 2013). Though different, both RDP and RUP are necessary

components of a properly balanced diet.

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RUP varies from RDP in that it passes through the rumen, reticulum, and omasum

without significant alteration, allowing direct digestion by the abomasum and small intestine, and

immediate absorption of amino acids. Thus, RUP proportions are greater for cows with higher

rates of feed intake because the feed passes through the rumen faster and there is less opportunity

for it to be degraded (Herdt, 2014). Feeds that have been processed are typically higher in RUP,

especially those that have undergone drying (Herdt, 2014). Generally, RUP should be 45% or

less of the total MP of a diet, since more RUP correlates to less RDP (Broderick & Schwab,

2017). Feeding excess RUP also leads to a surplus of non-limiting amino acids and is not a

consistent approach to feeding for maximum Nitrogen efficiency (Broderick & Schwab, 2017).

However, cows with high requirements for protein and low rates of feed intake, such as cows in

early lactation and heifers that are rapidly growing, benefit from diets containing high

proportions of RUP (Herdt, 2014).

In contrast, nitrogen from RDP must first be incorporated by microbial protein before it

will provide amino acids that are available for absorption (Herdt, 2014). The rate at which this

process occurs is dependent on the growth rate of the rumen microbes, which is in turn

dependent on the supply of fermentable energy available in the rumen (Herdt, 2014). Therefore,

diets consisting of both RDP and a high energy concentration will yield high levels of microbial

protein, leading to greater levels of MP and increased amino acid absorption (Wu, 2013). Feeds

that are high in both protein and moisture content, such as legume silages, typically yield high

concentrations of RDP (Herdt, 2014). Microbial protein, which consists of particle associated

bacteria, fluid associated bacteria, protozoa, and fungi, should be 50% or greater of the total MP

of a diet (Broderick & Schwab, 2017). Supplying adequate RDP in the diets of dairy cattle is

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important because multiple studies have shown that a deficiency of RDP can lead to reductions

in fiber digestibility, nitrogen utilization, and dry matter intake (Broderick & Schwab, 2017).

Amino Acid Supplementation

In addition to including sufficient high-quality protein sources in the diets of dairy cattle,

limiting amino acids, such as lysine and methionine, must also be provided to achieve optimal

growth, reproduction, and lactation performance. This can be accomplished through feeding

high-protein ingredients, such as soybean meal, corn, blood meal, and fish meal, but can also be

achieved through the use of supplemental amino acid products. However, these products are

most effective when stable and protected from rumen degradation. The bioavailability of an

amino acid is determined by its ability to escape ruminal degradation, combined with its

intestinal digestibility (NASEM, 2021). High-quality proteins can be heated to induce the

Maillard reaction, chemically treated to decrease solubility, or exposed to certain

phytochemicals, such as tannins, to prevent degradation in the rumen. However, commercially

produced amino acids are often physically encapsulated (Wu, 2013). Amino acids can be

surface-coated via blood spraying, followed by heating and drying, or coated with hydrogenated

lipids, such as lecithin or soy oils, to produce microcapsules (Wu, 2013). Coating combinations

include lipids and pH-sensitive polymers, fibers and lipids, or calcium salts and long-chain fatty

acids (NASEM, 2021). Although these systems of physical protection must be durable enough to

withstand rumen degradation, they must also be susceptible to intestinal release (NASEM, 2021).

Thus, achieving an adequate balance is critical to maintain the efficacy of these supplemental

products.

Supplementing a diet with encapsulated forms of amino acids is an economically

effective method for reaching the requirements of a limiting amino acid, without exceeding

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them. Lysine is one limiting amino acid that provides particularly numerous benefits when added

to dairy cattle diets. In properly formulated rations, lysine supplementation can increase milk

production, milk components, and palatability (Broderick & Schwab, 2017). This first limiting

amino acid has demonstrated to be especially important for increasing milk protein (Giallongo, et

al., 2016). Furthermore, studies have shown that lysine contributes significantly to promoting

nitrogen balance (Morris, 2020). Using a rumen-protected lysine product also alleviates any

potential uncertainty related to inevitable variability in the amino acid concentration of feeds

derived from animal origins, such as blood and fish meal (Broderick & Schwab, 2017). This is

particularly beneficial for diets containing high levels of certain ingredients that are known to be

naturally low in lysine content, such as corn and corn byproducts, including distiller’s grain and

corn gluten meal, that serve as sources of RUP (Broderick & Schwab, 2017).

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Chapter 2: Area Under the Curve Experiment

Introduction

The area under the curve (AUC) technique is an in vivo method for quantitatively

determining the amount of free AA in circulating blood from RP-AA products (Schwab et al.,

2001). This process requires ruminally infusing cannulated cows with a single dose of a RP-AA

in an amount exceeding that which is normally encountered by the ruminal microbiota. Blood

samples prior to experimentation are used to establish baseline values of AA plasma

concentration for each cow. The AA treatments are then given as pulse doses, with the test AA

administered via abomasal infusion and the RP-AA provided as a bolus dose via the ruminal

cannula. Blood samples are taken during and after the infusion periods for plasma AA analysis.

These values are graphed and used for trapezoidal AUC analysis by computing the product of the

average AA concentration increases between two consecutive sampling times and the duration of

the interval. The AUC is determined by calculating the sum of the area of all the trapezoids

formed between these two points.

Objective

The objective of this trial is to determine the bioavailability of RP-Lys supplements in

lactating Holsteins using plasma Lys concentrations for AUC analysis.

Materials & Methods

Experimental Design and Treatment Diets

This study was conducted using eight multiparous lactating Holsteins (106 ± 32 DIM) at

the University of New Hampshire (UNH) Fairchild Dairy Teaching and Research Center. These

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cows, equipped with ruminal cannulas, were randomly selected, and used in a replicated 4 x 4

Latin square with 7-day experimental periods. The four experimental treatments (Table 1) were:

1) 95 g Lys from Lysine-HCl

2) 95 g Lys from USALysine

3) 95 g Lys with Prototype I L coating

4) 95 g Lys with Met Prototype II HPO coating

Since 95 g of pure HCL may be too high, and to prevent product rejection, the control

treatment (Lys HCL) was supplied directly to the abomasum to produce the AUC for a treatment

with 100% bioavailability. The Lys-HCl used for infusion was the same Lys that was used in the

production of the RP-Lys supplements. Lys-HCl was diluted in 500 mL of distilled water and the

infusion line was properly inserted and unblocked prior to infusion. The Lys infusion was dosed

slowly in aliquots of 60 mL, using a 60-mL catheter syringe. To avoid reflux and to ensure that

the entire Lys infusion was administered, the infusion line was flushed with 200 mL of tap water.

The different RP-Lys treatments were placed in gelatin capsules, which were then placed directly

into the rumen at the level of the rumen-omasal orifice.

Management of Cows

All procedures related to animal care were conducted with approval of the UNH

Institutional Animal Care and Use Committee (190901). Cows were housed in a naturally

ventilated tie-stall barn and fed individually. All cows had continuous and free access to water

and were milked twice daily (0430 and 1530 h) in a milking parlor equipped with automatic

take-offs and milk meters. Milk weights were recorded at each milking.

All cows were fed a Lys-adequate and Met-adequate basal diet throughout the study

(Table 1). The basal diet was fed as a TMR prepared two times daily (0500 and 1600 h) by

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weighing each ingredient and mixing them in a mobile paddle mixer (Data Ranger). Samples of

TMR and orts were collected daily and composited by cow for each period to allow for

determination of DM and calculation of DMI.

Blood Sampling and Analysis

Blood samples were obtained from each cow on the last two days of the experimental

periods. Blood samples were collected from the coccygeal vein or artery 0 hr before

administration of treatments, to determine basal blood AA concentrations. Blood samples were

then taken at 1, 2, 3, 4, 6, 9, 12, 24, 30, and 48 hr post-dosing. Blood was collected in a 10-mL

vacutainer tube (Monoject, Mansfield, MA) containing 15% K

EDTA. Tubes were placed

immediately in a Chameleon Cooler and centrifuged within 15 min at 1,200 × g for 20 min at

5°C. Then, 1.0 mL aliquots of deproteinized plasma were removed, placed into 1.8-mL

cryovials, and stored at -80°C for plasma AA analysis (Experimental Station Chemical

Laboratories, University of Missouri-Columbia, Columbia, MO).

Statistical Methods

The basal plasma Lys concentration (BPLC) value was subtracted from the plasma values

obtained after supplementation to yield plasma Lys values only due to supplementation. The

adjusted Y (plasma concentrations-BPLC) was subjected to the area of a trapezoid [1/2*h

*(a+b)], which was used to calculate the area under the curve [0.5 (time2-time1)*(conc2-

conc1)]. The Yadj values were then analyzed using the mixed model of SAS (9.4) and the PDIFF

option was used to test treatment differences according to the following model:

Significance P < 0.05.

adji

= µ + Li + Ei

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Where:

adji

= adjusted dependent variable

µ = overall mean

Li = fixed effect of treatment (i = 1...,4)

= residual errors

Results & Discussion

The average DIM of the cows increased from 120 days to 155 days throughout the

duration of the trial. The feed component analysis (Table 3) and the AA analysis (Table 4) of the

individual feed ingredients, in conjunction with the AMTS evaluation (Table 5), indicate that the

diet was not energy deficient, since the NE

allowable milk or ME allowable milk of 47.0 kg/d is

predicted to be slightly higher than the MP allowable milk of 40.7 kg/d. The Lys:Met ratio for

the AMTS evaluation is 2.46:1, suggesting that the diet is slightly Lys deficient. The CP of the

diet is 15.0%, which is lower than the CP initially formulated at the beginning of the trial. This is

likely due to the CP of the corn silage and haylage being lower than expected. Table 6 indicates

that the average milk yield and dry matter intake (DMI) values for each cow are not affected by

treatments.

The results of the AUC analysis are presented in Table 7. While the infusion treatment

varies from the RP-Lys supplements in both Lys µM concentration and AUC value, the three

RP-Lys supplements do not differ from each other. The bioavailability of USA Lysine is 68.1%,

Prototype IL is 45.3%, and Prototype II HPO is 54.3%. The AUC analysis, presented in Figure 1,

depicts a rapid, linear increase in Lys of the infusion treatment, which peaks at 2 hours, and an

equally rapid, linear decrease back to its baseline before any of the RP-Lys supplements peak.

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After 4 hours, USA Lysine peaks at approximately 185 µM Lys, while Prototype II HPO peaks

at approximately 150 µM Lys after 10 hours, and Prototype IL peaks at approximately 125 µM

Lys, also after 10 hours.

As demonstrated by the plasma concentrations of AA reported in Table 8, treatment has a

significant effect on many of the amino acids. Exceptions include Arg, Trp, Ala, Gln,

homocysteine, Tau, and total EAA. However, the treatment x hour interaction is significant for

Arg, Lys, and total EAA. The plasma metabolites presented in Table 9 indicate that treatment has

a significant effect on α-amino-adipic acid, α-amino-butyric acid, and 3-Methylhistidine. 3-

Methylhistidine indicates muscle degradation, and the infusion treatment, which peaks around

800 µM Lys, is significantly different than the three RP-Lys supplements. α-amino-adipic acid is

a final oxidation product of Lys and is significantly higher for infusion compared to the other

treatments. α-amino-butyric acid serves as an intermediate, occurring in the catabolism of the

two essential amino acids, Met and Thr. These are both significantly lower for the infusion

treatment than the other RP-Lys supplements, for which the α-amino-butyric acid was also

significantly lower. A trend was noted in carnosine and there was treatment x hour interaction

observed for α-amino-adipic acid.

The results of this trial and those from previous research support observations that Lys is

a highly bioavailable amino acid in its RP form. The infused Lys HCl was expected to exhibit a

high bioavailability, though not quite such a dramatic peak as observed. To possibly prevent this,

a future experiment could involve infusion of the Lys HCl at a controlled rate over a period of 3-

4 hours. The HPO treatment produced a premature peak in Lys concentration, potentially due to

the coating breaking down earlier than expected. This could be investigated further with an

experiment testing this supplement with different types of RP coatings. Although the corn silage

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was lower in CP than expected, the diet was still energy efficient due to an intentional

overcalculation in the initial formulation. The lack of variation in average milk yields indicates

that the supplements are relatively equally beneficial, with none becoming detrimental to the

production of the cows.

Conclusions

The AUC method was successful in determining the bioavailability of the RP-Lys

supplements. However, while the only expectation was for Lys to increase in plasma

concentration, several other amino acids decreased in concentration. Thus, a future experiment

feeding a lower dose of Lys, such as 60 g instead of 95 g, could be performed. This study could

investigate the effect on 3-Methylhistidine at a lower dose of Lys, which would possibly yield a

lower value, indicating less muscle degradation as a result of less Lys. The high bioavailability

yielded by these RP-Lys products indicate that supplementing the diets of dairy cows,

specifically those high in corn and corn byproducts, with encapsulated forms of this essential

AA, can serve as an economically effective method for reaching the requirements of this limiting

AA, without exceeding them.

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Table 1. Calculations for the amounts of RP-AA products to feed.

Lysine Hydrochloride is 80% Lys

95 g/d Lys/0.80= 118.75 g/d Lys-HCl

USALysine is 69.6 % Lys-HCl

69.6 x 0.80 = 55.68 % Lysine

95 g/d Lys/.5568 = 170.62 g/d USALysine

Prototype IL is 79.2 % Lys-HCl

79.2 x 0.80 = 63.36 % Lysine

95 g/d Lys/.6336 = 149.94 g/d Prototype IL

Prototype II HPO is 79.7 % Lys-HCl

79.7 x 0.80 = 63.76 % Lysine

95 g/d Lys/.6376 = 149.00 g/d Prototype II HPO

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Table 2. Ingredient composition of basal Lys-adequate diet.

Ingredient

% DM

Corn silage, mature

24.05

Mixed mostly grass silage, mid-maturity

21.23

Steam flaked corn

4.21

Corn meal

15.97

Beet pulp

9.97

Molasses, sugar cane

1.16

Distillers grains with solubles

0.73

Soybean meal, solvent extracted

6.46

Canola meal, solvent

2.16

SoyPlus

7.54

Urea

0.12

BergaFat-100

3.06

Kessent M

0.09

Mineral/vitamin mix

3.24

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Table 3. Chemical composition of feedstuffs (% DM unless otherwise noted)

Corn

Silage

Haycrop

Silage

Soy

Plus

Canola

meal

Soybean

meal

Steam

Flaked

Corn

Meal

Corn

Distillers

Grains

Beet

Pulp

Molasses

Urea

Minerals

Nutra Core

32.7

30.7

90.9

93.8

89.8

86.8

89.3

90.5

91.7

78.8

99.6

94.6

99.5

6.7

12.1

45.6

43.0

50.6

7.7

6.9

32.3

9.9

7.6

273.0

0.1

aNDFom

38.0

56.5

20.8

24.4

13.0

6.9

7.1

39.7

30.9

ADF

23.7

38.6

12.4

21.5

9.3

2.3

2.4

15.5

28.1

NDF-CP

0.6

2.3

9.5

7.0

6.4

0.9

0.7

5.8

5.1

ADF-CP

0.4

1.4

3.3

4.5

5.9

0.4

0.6

2.7

3.4

Lignin

2.5

6.1

2.8

10.1

1.2

0.5

5.9

6.5

NFC

47.3

16.4

20.9

21.8

27.6

81.5

6.8

47.2

Starch

41.1

2.1

1.6

2.5

2.1

75.6

80.5

2.3

1.0

Ether extract

4.40

5.39

6.19

4.11

1.80

2.88

3.30

14.51

1.38

1.2

97.2

Ash

3.62

9.61

6.52

6.69

6.94

1.09

1.22

6.74

10.55

16.85

0.18

0.56

0.36

0.64

0.33

0.01

0.02

0.03

1.07

1.04

18.17

0.27

0.34

0.72

1.06

0.74

0.21

0.26

1.16

0.12

0.16

1.12

0.09

0.29

0.31

0.56

0.30

0.08

0.09

0.35

0.27

0.44

4.68

1.06

2.67

2.35

1.09

2.42

0.28

0.34

1.36

0.56

4.42

0.10

0.009

0.063

0.028

0.074

0.011

0.008

0.006

0.116

0.049

0.168

13.470

Cl ion

0.17

0.90

0.03

0.02

0.06

0.08

0.20

0.01

7.73

Fe (ppm)

239

540

112

130

110

1,140

160

1,970

Zn (ppm)

1,000

Cu (ppm)

169

Mn (ppm)

778

0.10

0.22

0.38

0.82

0.43

0.10

0.09

0.68

0.36

1.14

0.23

Vetter 21

Table 4. Amino acid composition of feedstuffs. (% of CP)

Corn

Silage

Haycrop

Silage

SoyPlus

Canola

meal

Soybean

meal

Steam

Flaked

Corn

Corn Meal

Corn Distillers

Grains

Citrus

Pulp

Molasses

EAA

Arginine

1.61

2.63

6.84

6.01

7.09

4.69

4.52

5.14

1.61

0.17

Histidine

1.29

1.45

2.58

2.73

2.66

2.90

2.96

3.09

2.47

0.17

Isoleucine

4.03

4.17

4.79

4.10

4.81

3.59

3.74

4.57

3.44

1.01

Leucine

10.49

7.16

7.65

6.98

7.74

11.32

10.92

12.81

5.48

1.01

Lysine

3.71

4.44

5.95

5.57

6.54

3.59

3.90

3.12

0.34

Methionine

1.94

1.45

1.28

1.99

1.37

1.93

1.87

2.02

1.50

0.00

Phenylalanine

4.52

4.44

5.14

4.05

5.19

4.83

4.68

5.55

3.33

0.68

Threonine

3.23

3.44

3.79

4.12

3.90

3.59

4.20

3.98

1.01

Tryptophan

0.50

0.51

1.21

1.01

1.36

0.87

0.83

0.81

0.38

0.34

Valine

5.32

5.44

5.00

5.27

5.00

4.83

4.99

5.85

5.48

1.86

Total

36.65

35.13

44.23

41.84

45.65

42.14

42.01

47.94

30.79

6.59

NEAA

Alanine

9.52

7.61

4.30

4.32

4.34

7.18

7.02

7.73

4.19

4.22

Aspartic Acid

5.97

6.43

10.95

6.83

11.16

6.90

7.18

6.76

6.34

23.48

Cysteine

1.77

1.00

1.28

2.61

1.41

2.21

2.34

2.18

1.18

0.34

Glutamic Acid

10.81

6.89

17.19

16.94

17.59

17.39

17.32

14.49

8.17

5.41

Glycine

4.52

4.80

4.37

5.04

4.16

4.14

4.21

4.47

3.76

1.01

Ornithine

0.97

0.63

0.12

0.05

0.08

0.00

0.17

0.11

0.00

Proline

7.75

4.35

5.07

6.19

5.04

8.56

8.58

8.37

4.08

0.68

Serine

2.90

2.63

4.09

3.50

4.26

4.55

4.52

4.81

3.44

1.52

Tyrosine

2.26

0.91

0.23

0.20

0.23

1.38

1.72

0.20

2.69

2.03

Taurine

1.45

1.99

3.53

2.66

3.52

2.76

2.18

4.07

3.12

0.84

Total

47.92

37.24

51.14

48.35

51.80

55.07

53.25

37.07

39.52

Total AA

84.57

72.37

95.37

90.19

97.45

97.21

97.08

101.18

67.86

46.11

Vetter 22

Table 5. AMTS (Ver.4.17.0.1) evaluation of the basal formulated diet

aNDFom, % DM

28.86

CP, % DM

15.0

Forage NDF, % DM

21.13

RDP, % DM

8.89

NFC, % DM

41.4

RUP, % DM

6.13

ME, Mcal/kg DM

2.69

, Mcal/kg DM

1.73

MP-bacterial, g/d

1532

EE, % DM

6.8

MP-RUP, g/d

1121

required, Mcal/d

70.3

MP-Lys,

g/d

184.16

supplied, Mcal/d

69.7

MP-Met, g/d

74.94

ME balance, Mcal/d

-.06

MP-Lys, % MP

6.94

MP required, g/d

2951

MP-Met, % MP

2.82

MP supplied, g/d

2655

MP balance, g/d

-296

MP- Arg, % MP

6.32

MP- His, % MP

2.60

DM intake-actual, kg/d

25.9

MP - Ile, % MP

5.22

DM intake-predicted, kg/d

27.1

MP- Leu, % MP

7.74

MP- Phe, % MP

4.97

ME allowable milk, kg/d

47.0

MP- Thr, % MP

4.80

MP allowable milk, kg/d

40.7

MP- Trp, % MP

1.37

Actual milk, kg/d

47.5

MP- Val, % MP

5.68

BW 712 kg, DMI = 25.9 kg/d, milk yield = 47.5 kg/d, milk fat content = 3.90%, and

milk true protein concentration 2.90%.

Vetter 23

Table 6. Milk and dry matter intake for Holstein cows fed a diet supplemented with RP-Lys

supplements.

Item

Infusion

USALysine

Prototype II HP0

Prototype IL

Trt

Milk kg/d

46.3

48.5

47.2

47.9

2.02

0.88

DMI, kg/d

25.1

26.5

25.7

26.0

1.37

0.90

Vetter 24

Table 7. Lysine concentration, area-under-the-curve and bioavailability for Holstein cows fed a

diet supplemented with RP-Lys supplements.

Lys, µM

Treatment

µM

SEM

AUC

SEM

Bioavailability

Infusion

244.93

12.324

2027.19

172.18

USALysine

122.59

12.324

1380.95

159.88

68.12

Prototype IL

100.64

12.324

917.96

156.85

45.28

Prototype II HPO

113.81

12.324

1101.41

163.04

54.33

Vetter 25

Figure 1. Area-under-the-curve for Holstein cows fed a diet supplemented with RP-Lys

supplements.

Vetter 26

Table 8. Plasma amino acids (µM) for Holstein cows fed a diet supplemented with RP-Lys supplements.

P-value

Item

Infusion

USALysine

Prototype II HP0

Prototype IL

Trt

Trt x Hr

Arginine

85.5

89.0

92.1

87.0

3.52

0.13

0.01

Histidine

52.6

58.2

58.8

60.4

2.10

<0.0001

0.51

Isoleucine

114.6

122.5

129.5

124.2

5.61

0.0002

0.41

Leucine

145.4

153.3

161.9

157.9

8.77

0.0004

0.54

Lysine

203.5

140.3

137.5

100.6

13.91

<0.0001

Methionine

29.4

32.5

34.3

32.7

1.56

<0.0001

0.70

Phenylalanine

46.4

47.5

50.1

48.5

2.00

0.01

0.39

Threonine

111.6

117.7

123.4

120.1

4.49

0.003

0.60

Tryptophan

49.7

51.4

51.0

50.9

1.30

0.47

0.48

Valine

240.2

253.1

267.8

260.8

14.20

<0.0001

0.78

Alanine

319.2

324.7

333.5

321.3

20.27

0.271

0.92

Asparagine

49.9

57.2

59.7

57.1

1.50

<0.0001

0.85

Aspartic Acid

3.07

3.26

3.32

3.14

0.08

0.05

0.86

Citrulline

93.3

98.0

100.3

101.1

5.83

0.003

0.99

Cystine

19.9

21.2

21.5

21.4

0.89

<0.0001

0.99

Cystathionine/Allocystathionine

2.04

2.23

2.19

2.13

0.11

0.008

0.96

Glutamine

205.9

215.1

209.3

207.4

8.14

0.34

0.89

Glutamic Acid

41.4

41.6

43.3

40.8

1.46

0.04

0.57

Glycine

342.0

381.0

369.3

362.3

11.56

<0.0001

0.62

Homocysteine

3.07

3.19

3.35

0.35

0.12

0.73

Ornithine

52.8

55.5

58.5

57.2

3.90

0.009

0.54

Proline

94.2

103.6

106.9

103.8

4.98

<0.0001

0.98

Serine

86.9

95.6

98.3

93.6

2.19

<0.0001

0.61

Taurine

48.7

51.2

51.6

51.1

2.76

0.29

0.98

Tyrosine

43.6

49.1

52.9

51.1

1.59

<0.0001

0.13

TEAA

1079

1066

1107

1043

42.4

0.17

<0.0001

TNEAA

1406

1502

1514

1477

33.3

0.0004

0.87

TAA

2485

2568

2620

2520

57.4

0.04

0.10

TBCAA

500

529

559

543

28.2

<0.0001

0.59

TUCAA

232

243

251

245

9.3

0.01

0.35

Vetter 27

TSAA

103

110

113

111

3.6

0.0002

0.99

TAA-Lys

9.18

5.85

5.66

4.13

0.61

<0.0001

a-c

Means in the same row differ P < 0.05.

Vetter 28

Table 9. Plasma metabolites (µM) for Holstein cows fed a diet supplemented with RP-Lys supplements.

P-value

Item

Infusion

USALysine

Prototype II HP0

Prototype IL

Trt

Trt x Hr

1-Methylhistidine

30.2

23.4

24.4

23.0

4.38

0.55

0.34

3-Methylhistidine

3.88

3.50

3.53

3.44

0.20

<0.0001

0.99

α-amino-adipic acid

17.9

15.5

16.2

14.1

1.00

<0.0001

α-amino-butyric acid

17.5

20.3

19.3

19.9

0.72

<0.0001

0.99

β-alanine

10.5

11.0

11.1

10.9

0.47

0.15

0.81

Carnosine

15.9

16.6

16.5

16.8

0.94

0.10

0.98

Ethanolamine

6.50

6.36

6.50

6.96

0.49

0.83

0.86

γ-amino-butyric acid

3.60

3.36

3.15

3.51

0.46

0.08

0.47

Hydroxylysine

1.94

1.93

1.87

1.92

0.15

0.99

0.92

Hydroxyproline

11.3

11.5

11.4

11.2

0.93

0.39

0.98

Phosphoserine

5.06

5.20

5.15

4.99

0.30

0.96

0.13

Sarcosine

31.2

33.4

32.0

32.1

1.05

0.05

0.90

a-c

Means in the same row differ P < 0.05.

Vetter 29

Chapter 3: Plasma Dose Response Experiment

Introduction

The plasma dose-response (PDR) technique is a method used to determine the

bioavailability of Lys from RP-Lys supplements. Due to variations in the availability, quality,

and digestibility of high Lys-containing protein sources, such as blood meal and fish meal, RP-

Lys supplements serve as effective alternatives. However, commercial RP-Lys supplements vary

in encapsulation technology, size, density, Lys concentration, and availability to ruminants.

Thus, a reliable, standardized method for estimating Lys bioavailability is necessary for

accurately comparing the ability of different RP-Lys supplements to provide highly

metabolizable Lys.

The plasma free Lys-dose response technique is a quantification method that has been

refined through several studies (King et al., 1991; Rulquin and Kowalczyk, 2003; Borucki-

Castro et al., 2008). This technique is based on the positive linear relationship between infused

doses of Lys into the omasum, abomasum, or duodenum, and the concentration of Lys in plasma

(Hanigan, 2009). The assumption that increases in plasma Lys concentration reflect increases in

net absorption of Lys is relied upon to calculate an estimate of the bioavailability of Lys from

RP-Lys supplements. Variation is minimized by providing all treatments in the same Latin

square.

Objective

The objective of this trial is to determine the bioavailability of RP-Lys supplements in

lactating Holsteins using plasma Lys concentrations for statistical analysis via the PDR

technique.

Vetter 30

Materials & Methods

Experimental Design and Treatment Diets

This study was conducted using eight multiparous lactating Holsteins (128 ± 38 DIM) at

the University of New Hampshire (UNH) Fairchild Dairy Teaching and Research Center. These

cows, equipped with ruminal cannulas, were randomly selected and used in a replicated 4 x 4

Latin square with 7-day experimental periods. The four experimental treatments (Table 1) were:

5) 0 g/d Lys (negative control)

6) 60 g/d Lys from abomasally infused Lys HCLmater

7) 60 g/d Lys from Prototype II HPO

8) 60 g/d Lys from Prototype IL

The infusion treatment was prepared by dissolving Lys-HCl in 4 L of hot tap water. The

Lys solutions were continuously infused into the abomasum via the rumen cannula using a

peristaltic pump (Masterflex, Cole-Parmer, Vernon Hills, IL). Fresh infusion solutions were

prepared daily at 1300 h and pumping rates were closely monitored and adjusted to ensure

treatments were completely and uniformly infused. The pump was disabled and the infusion lines

were disconnected twice daily when the cows were moved from their stalls to the milking parlor.

Immediately prior to each feeding, the RP-AA supplements were mixed with 1.5 kg of

TMR. The RP-Lys supplements were prepared with the same Lys-HCl that was used to produce

the infusion treatment. These mixtures were placed in rubber tubs and fed to the cows 30 min

before each feeding, to ensure RP-Met supplements were completely consumed. Any TMR/RP-

AA mix not consumed by a cow within 15-20 min was manually inserted into the rumen via the

ruminal cannula. Cows were fed 1/3 of their daily feed allotment 3 times daily (0500, 1300, and

Vetter 31

2100 h). The daily amounts of RP-Lys products were divided into 3 equal portions to ensure that

a constant ratio of RP-Lys products to total TMR consumption was maintained.

Cow characteristic before the start of the trial:

BW, kg

BCS

DMI, kg/d

Milk, kg/d

Fat, %

Protein, %

Block 1

730

3.03

25.8

43.9

3.79

2.92

Block 2

701

2.99

25.1

48.4

3.69

2.89

Management of Cows

Cows were housed in a naturally ventilated tie-stall barn and fed individually, with

continuous and free access to water. All procedures related to animal care were conducted with

approval of the UNH Institutional Animal Care and Use Committee (190901). Cows were

milked twice daily (0430 and 1530) in a milking parlor equipped with automatic take-offs and

milk meters. Milk weights were recorded after each milking.

All cows were fed a Lys-adequate and Met-adequate basal diet throughout the trial (Table

2). The basal diet was fed as a TMR and prepared 3 times daily (0500, 1300, and 2100 h) by

weighing each ingredient and mixing them in a mobile paddle mixer (Super Data Ranger, Calan

Inc., Northwood, NH). Cows were fed ad libitum for feed intake, but with minimal orts (2 to

4%). Samples of TMR and orts were collected daily and composited individually by cow for

each period for determination of dry matter (DM) and dry matter intake (DMI) calculations.

Feed Sampling and Analysis

Samples of corn silage and haycrop silage were collected daily for DM analysis.

AminoMax Berga Fat-100, hay, and the mineral mix were sampled once per week. The other

grains were sampled by Poulin Grain and sent to the UNH Fairchild Dairy Teaching and

Vetter 32

Research Center with each load of grain. All samples were freeze-dried (Labconco Model 5,

Kansas City, MO) for 48 h following collection. Samples were stored in glass jars until the

completion of the study and were ground to pass through a 1-mm screen using a Wiley Mill

(Thomas Scientific, Swedesboro, NJ). Composites were made of each feedstuff for analysis of

DM, CP, NDF, ADF, ADI-CP, NSC, Ca, P, Mg, K, Na, Fe, Zn, Cu, Mn, Mo, S, (DHI Forage

Testing Laboratory, Ithaca, NY) and AA content (Experimental Station Chemical Laboratories,

University of Missouri-Columbia, Columbia, MO).

Blood Sampling and Analysis

Blood samples were collected from each cow on the last 3 days of the covariate period

and the last 3 days of the experimental periods. Each day, 4 blood samples were collected from

the coccygeal vein or jugular vein at 2-h intervals, starting at 0700 h. Blood was collected in 10-

mL vacutainer tubes (Monoject, Mansfield, MA) containing 15% K3EDTA. The tubes were

placed immediately in a Chameleon Cooler, centrifuged within 15 min at 1,200 × g for 20 min at

5°C. A 4-mL aliquot from each sample was placed in a labeled glass test tube containing 1.0 mL

of 15% SSA. The tubes were allowed to sit for 10 min in the centrifuge before spinning at 1,200

× g for 20 min at 5°C. A 0.45 mL aliquot of deproteinized plasma was removed and placed into

1.8-mL cryovials and stored at -80°C for plasma AA analysis (Experimental Station Chemical

Laboratories, University of Missouri-Columbia, Columbia, MO).

Milk Sampling and Analysis

Milk samples were obtained from each cow during the a.m. and p.m. milking on the last 3

days of the covariate period and the last 3 days of each of the experimental periods. Samples

were preserved with 2-bromo-2-nitropropane-1, 3-diol (1 tablet per 40 mL of milk) and

refrigerated until composited by milk weight and sample date. Samples were analyzed for true

Vetter 33

protein, fat, SCC, and MUN (Dairy One Milk Laboratories, Ithaca, NY) by using a Foss

MilkoScan 4000 infrared analyzer (Foss Electric, Hillerød, Denmark).

Statistical Methods

The RSTUDENT Procedure of SAS 9.2 (2010) was used to determine outlier cows. An

observation greater than 2.0 standard deviations from the mean was considered an outlier.

Outliers were determined within level and cow to prevent lower or higher overall values from

being removed from the dataset. The variables used for outlier analysis were Lys (µM), total AA-

Lys (µM), and milk yield and milk protein percent. Outliers were reviewed and removed for

reasons such as issues with the pump or cows off feed due to sickness. All data from cows

designated as outliers was removed from both the milk and plasma data sets. Covariate data was

used in the statistical analysis and the data reviewed to ensure there was no carryover of

treatment when cows transitioned from the infusion of the RP-Lys treatment to the control.

To determine if day and day × treatment interaction was significant, plasma Lys

concentrations, (µM), total AA-Lys (µM ) concentrations, milk yield, and milk protein content

were used with PROC MIXED and the REPEATED procedure of SAS 9.4 (2010) according to

the following model:

ijklm

= μ

+ P

+ B

+ D

+ LD

+ KC

ilm

+ E

ijklm

Where:

ijkl

= is the dependent variable

μ = overall mean

= is the fixed effect of the i

treatment; i = 1…4

=is the fixed effect of the j

period; j =1…4

= is the fixed effect of the k

block; k =1…2

Vetter 34

= is the fixed effect of the l

day; l = 1…3

= is the fixed effect of the interaction between the i

treatment and the l

day

K = is the regression coefficient of the covariate C

ilm

= is the value of the covariate variable for the m

cow within the l

day of the i

treatment, l = 1 …4

ijklm

= is the random residual ~N (0,s )

The effect of day and day x treatment interaction was established using the random effect

of cow(block) as the error term in this model. Degrees of freedom was calculated using the

Kenward-Roger option of MIXED procedure (SAS, 2010). Significance was noted at P ≤ 0.05.

Day and day × treatment level interactions were not significant, therefore the means for plasma

AA concentrations and milk parameters for the 3 days were calculated and the day and day ×

treatment were dropped from the model. The data was weighted for any missing values. The

random effect of cow(block) was used as the error term for the effect of treatment level. Least

square means were determined for treatment and treatment means were separated. Significant

level effects were noted at P ≤ 0.05 and trends were noted P > 0.05 to P < 0.10.

Using the slope ratio assay (Finney, 1978), the least square means generated from the

MIXED procedure were subjected to the PROC REG procedure to generate the linear regression

variables and r

Results & Discussion

The flow rate for the pump averages 43 +/- 1 rpm with a flow rate of 161 +/- 8 mL/hr.

Cow 915 and Cow 962 of the covariate period exhibit extremely high Lys concentrations on Day

2 and Day 3 respectively and are determined as outliers by the RSTUDENT analysis. For the II

HPO treatments, Cow 1020 exhibits a low Lys concentration on Day 2, while Cow 888 exhibits a

Vetter 35

high Lys concentration on Day 3. For the IL treatment Cow 888 exhibits a low Lys

concentration. All of these values fall outside the 2.0 standard deviation range. The covariate

data used in the statistical analysis is also used to ensure that the plasma AA values of the cows

returned to their baselines for the control treatment when cows were treated with a RP-Lys

supplement or infusion immediately prior to receiving the control treatment. The average DIM of

the cows increased from 141 to 181 throughout the duration of the trial.

Tables 3 and 4 contain the feed component and AA analysis respectively, of the

individual feed ingredients. These, in conjunction with Table 5, which contains the AMTS diet

evaluation, indicate that the diet is not energy deficient, since the NE

allowable milk or ME

allowable milk is predicted to be slightly higher when compared to the MP allowable milk. The

Lys:Met ratio of the AMTS evaluation is 2.46:1, which indicates that the diet may be slightly

Lys deficient. The CP of the diet is 15.0%, which is lower than the value initially formulated at

the beginning of the trial. This is possibly due to the corn silage and haylage being lower in CP

than expected.

Table 6 contains DMI, milk yield, and milk composition values. There are no effects of

treatment on DMI, milk yield, milk fat composition and yield, milk protein composition and

yield, lactose composition and yield, total solids composition and yield, linear SCC score, MUN,

4% FCM, ECM, and milk yield/DMI.

Table 8 presents the plasma AA concentrations. There are significant differences in

treatments for His (P = 0.04), Lys P < 0.0001) and Thr (P = 0.005), with the infusion treatment

being significantly lower for His and Thr compared to the other three treatments. The infusion

treatment is significantly higher in Lys concentration than the other three treatments, and there

are no differences between the II HPO and IL treatments, though both are significantly higher

Vetter 36

than the control. There is a trend (P = 0.09) for Arg, (P = 0.08) for Leu, Val, Asn, Asp, Tau, and

TUCAA, and (P = 0.07) for Orn, Tyr, and TSAA. Plasma metabolites (µM) are reported in Table

9. Ethanolamine is significantly higher for the IL treatment compared to the other three

treatments. There is no difference in 3-Methylhistidine among the treatments, indicating that

although the diet is low in CP, there is no evidence of muscle degradation.

Lysine as a percentage of total AA minus Lys is used to calculate the bioavailability of

Lys presented in Table 10. The bioavailability for the II HPO Prototype is 35.5 ± 1.7 and for the

IL Prototype is 49.0 ± 3.0. The statistical summary is presented in Table 11. Figure 1 also

provides the equations used to determine the amount of Lys provided by a product at a given

level of plasma Lys as a % of (TAA-Lys). Table 11 indicates that the metabolizable Lys

provided by the Prototype II HPO treatment is 226.3 g/kg, with a 35.5% bioavailability, while

that of the Prototype IL is 310.5 g/kg, with a 49.0% bioavailability.

Although the corn silage was lower in CP than expected, the diet was still energy

efficient due to an intentional overcalculation in the initial formulation. The lack of variation in

average milk yield and compositions indicates that the supplements are relatively equally

beneficial, with none becoming detrimental to the production of the cows.

Conclusions

The Lys infusion treatment yields the greatest Lys concentration, while the Prototype

treatments both yield slightly lower Lys concentrations. The Prototype II HPO treatment

provides less metabolizable Lys than the Prototype IL treatment, although they are relatively

similar in bioavailability.

Vetter 37

The high bioavailabilities yielded by these RP-Lys products further support the

hypothesis that supplementing the diets of dairy cows, specifically those high in corn and corn

byproducts, with encapsulated forms of this essential AA, can serve as an economically effective

method for reaching the requirements of this limiting AA, without exceeding them.

The PDR method is successful in determining the bioavailabilities of the RP-Lys

supplements. The PDR method appears more effective than the AUC method, since the AUC

method involves amounts of Lys higher than would normally be fed. To further investigate

which method is a more reliable means of measuring bioavailability, this experiment could be

repeated with a different essential amino acid.

Vetter 38

Table 1. Calculations for the amounts of RP-AA products to feed.

Kessent 2M is 75.0 % Met

12 g/d Met/0.75 = 16 g/d Kessent M2

Lysine Hydrochloride is 80% Lys

60 g/d Lys/0.80 = 75 g/d Lys-HCl

Prototype IL is 79.2 % Lys-HCl

79.2 x 0.80 = 63.36 % Lysine

60 g/d Lys/.6336 = 94.7 g/d Prototype IL

Prototype II HPO is 79.7 % Lys-HCl

79.7 x 0.80 = 63.76 % Lysine

60 g/d Lys/.6376 = 94.1 g/d Prototype II HPO

Vetter 39

Table 2. Ingredient composition of basal Lys-adequate diet.

Ingredient

% DM

Corn silage, mature

24.05

Mixed mostly grass silage, mid-maturity

21.23

Steam flaked corn

4.21

Corn meal

15.97

Beet pulp

9.97

Molasses, sugar cane

1.16

Distillers grains with solubles

0.73

Soybean meal, solvent extracted

6.46

Canola meal, solvent

2.16

SoyPlus

7.54

Urea

0.12

BergaFat-100

3.06

Kessent M

0.09

Mineral/vitamin mix

3.24

Vetter 40

Table 3. Chemical composition of feedstuffs (% DM unless otherwise noted)

Corn

Silage

Haycrop

Silage

Soy

Plus

Canola

meal

Soybean

meal

Steam

Flaked

Corn

Meal

Corn

Distillers

Grains

Beet

Pulp

Molasses

Urea

Minerals

Nutra

Core

32.7

30.7

90.9

93.8

89.8

86.8

89.3

90.5

91.7

78.8

99.6

94.6

99.5

6.7

12.1

45.6

43.0

50.6

7.7

6.9

32.3

9.9

7.6

273.0

0.1

aNDFom

38.0

56.5

20.8

24.4

13.0

6.9

7.1

39.7

30.9

ADF

23.7

38.6

12.4

21.5

9.3

2.3

2.4

15.5

28.1

NDF-CP

0.6

2.3

9.5

7.0

6.4

0.9

0.7

5.8

5.1

ADF-CP

0.4

1.4

3.3

4.5

5.9

0.4

0.6

2.7

3.4

Lignin

2.5

6.1

2.8

10.1

1.2

0.5

5.9

6.5

NFC

47.3

16.4

20.9

21.8

27.6

81.5

6.8

47.2

Starch

41.1

2.1

1.6

2.5

2.1

75.6

80.5

2.3

1.0

Ether extract

4.40

5.39

6.19

4.11

1.80

2.88

3.30

14.51

1.38

1.2

97.2

Ash

3.62

9.61

6.52

6.69

6.94

1.09

1.22

6.74

10.55

16.85

0.18

0.56

0.36

0.64

0.33

0.01

0.02

0.03

1.07

1.04

18.17

0.27

0.34

0.72

1.06

0.74

0.21

0.26

1.16

0.12

0.16

1.12

0.09

0.29

0.31

0.56

0.30

0.08

0.09

0.35

0.27

0.44

4.68

1.06

2.67

2.35

1.09

2.42

0.28

0.34

1.36

0.56

4.42

0.10

0.009

0.063

0.028

0.074

0.011

0.008

0.006

0.116

0.049

0.168

13.470

Cl ion

0.17

0.90

0.03

0.02

0.06

0.08

0.20

0.01

7.73

Fe (ppm)

239

540

112

130

110

1,140

160

1,970

Zn (ppm)

1,000

Cu (ppm)

169

Mn (ppm)

778

0.10

0.22

0.38

0.82

0.43

0.10

0.09

0.68

0.36

1.14

0.23

Vetter 41

Table 4. Amino acid composition of feedstuffs. (% of CP)

Corn

Silage

Haycrop

Silage

SoyPlu

Canola

meal

Soybean

meal

Steam

Flaked

Corn

Corn Meal

Corn Distillers

Grains

Citrus

Pulp

Molasses

EAA

Arginine

1.61

2.63

6.84

6.01

7.09

4.69

4.52

5.14

1.61

0.17

Histidine

1.29

1.45

2.58

2.73

2.66

2.90

2.96

3.09

2.47

0.17

Isoleucine

4.03

4.17

4.79

4.10

4.81

3.59

3.74

4.57

3.44

1.01

Leucine

10.49

7.16

7.65

6.98

7.74

11.32

10.92

12.81

5.48

1.01

Lysine

3.71

4.44

5.95

5.57

6.54

3.59

3.90

3.12

0.34

Methionine

1.94

1.45

1.28

1.99

1.37

1.93

1.87

2.02

1.50

0.00

Phenylalanine

4.52

4.44

5.14

4.05

5.19

4.83

4.68

5.55

3.33

0.68

Threonine

3.23

3.44

3.79

4.12

3.90

3.59

4.20

3.98

1.01

Tryptophan

0.50

0.51

1.21

1.01

1.36

0.87

0.83

0.81

0.38

0.34

Valine

5.32

5.44

5.00

5.27

5.00

4.83

4.99

5.85

5.48

1.86

Total

36.65

35.13

44.23

41.84

45.65

42.14

42.01

47.94

30.79

6.59

NEAA

Alanine

9.52

7.61

4.30

4.32

4.34

7.18

7.02

7.73

4.19

4.22

Aspartic Acid

5.97

6.43

10.95

6.83

11.16

6.90

7.18

6.76

6.34

23.48

Cysteine

1.77

1.00

1.28

2.61

1.41

2.21

2.34

2.18

1.18

0.34

Glutamic Acid

10.81

6.89

17.19

16.94

17.59

17.39

17.32

14.49

8.17

5.41

Glycine

4.52

4.80

4.37

5.04

4.16

4.14

4.21

4.47

3.76

1.01

Ornithine

0.97

0.63

0.12

0.05

0.08

0.00

0.17

0.11

0.00

Proline

7.75

4.35

5.07

6.19

5.04

8.56

8.58

8.37

4.08

0.68

Serine

2.90

2.63

4.09

3.50

4.26

4.55

4.52

4.81

3.44

1.52

Tyrosine

2.26

0.91

0.23

0.20

0.23

1.38

1.72

0.20

2.69

2.03

Taurine

1.45

1.99

3.53

2.66

3.52

2.76

2.18

4.07

3.12

0.84

Total

47.92

37.24

51.14

48.35

51.80

55.07

53.25

37.07

39.52

Total AA

84.57

72.37

95.37

90.19

97.45

97.21

97.08

101.18

67.86

46.11

Vetter 42

Table 5. AMTS (Ver.4.17.0.1) evaluation of the basal formulated diet

aNDFom, % DM

28.9

CP, % DM

15.0

Forage NDF, % DM

21.1

RDP, % DM

8.7

NFC, % DM

41.4

RUP, % DM

6.3

ME, Mcal/kg DM

2.68

, Mcal/kg DM

1.72

MP-bacterial, g/d

1636

EE, % DM

6.8

MP-RUP, g/d

1261

required, Mcal/d

67.9

MP-Lys, g/d

199.9

supplied, Mcal/d

75.2

MP-Met, g/d

81.2

ME balance, Mcal/d

7.3

MP-Lys, % MP

6.90

MP required, g/d

3043

MP-Met, % MP

2.80

MP supplied, g/d

2897

MP balance, g/d

-146

MP- Arg, % MP

6.30

MP- His, % MP

2.59

DM intake-actual, kg/d

28.1

MP - Ile, % MP

5.21

DM intake-predicted, kg/d

26.1

MP- Leu, % MP

7.75

MP- Phe, % MP

4.97

ME allowable milk, kg/d

52.4

MP- Thr, % MP

4.77

MP allowable milk, kg/d

42.5

MP- Trp, % MP

1.36

Actual milk, kg/d

45.7

MP- Val, % MP

5.67

BW 745 kg, DMI = 28.1 kg/d, milk yield = 45.7 kg/d, milk fat content = 3.54%, and

milk true protein concentration 3.03%.

Vetter 43

Table 6. Milk and dry matter intake for Holstein cows fed a diet supplemented with RP-Lys

supplements.

Item

Control

Infusion

Prototype

II HP0

Prototype

SEM

Dry Matter Intake, kg/d

28.1

27.9

28.2

0.33

0.88

Milk yield, kg/d

45.7

45.4

45.7

44.3

1.09

0.41

Fat, %

3.54

3.50

3.46

0.100

0.94

Fat yield, kg/d

1.60

1.58

1.59

1.50

0.083

0.59

Protein, %

3.03

3.05

3.03

3.07

0.030

0.42

Protein yield, kg/d

1.38

1.37

1.34

0.039

0.73

Lactose, %

4.91

4.93

4.92

0.015

0.91

Lactose yield, kg/d

2.24

2.23

2.25

2.18

0.059

0.55

Total solids, %

12.42

12.43

12.40

0.092

0.99

Total solids, kg/d

5.64

5.61

5.64

5.44

0.187

0.46

Linear somatic cell score

1.55

1.44

1.61

1.59

0.15

0.81

MUN, mg/dL

11.4

12.1

0.41

0.48

Fat Corrected Milk, kg/d

45.7

45.3

45.6

43.5

1.79

0.46

Energy Corrected Milk, kg/d

45.8

45.3

45.6

43.8

1.70

0.47

Milk yield/DMI

1.62

1.63

1.57

0.044

0.38

4% FCM = (0.4255 × milk yield, kg) + (16.425 × (milk yield, kg × fat, %))

ECM = [(fat, % × 0.0929) + (protein, % × 0.0563) + (lactose, % × 0.0395) × (milk yield, kg / 0.68605)]

Vetter 44

Table 7. Plasma amino acids (µM) for Holstein cows fed a diet supplemented with RP-Lys supplements.

Item

Control

Infusion

Prototype

II HP0

Prototype

SEM

Arginine

85.9

99.4

98.7

96.9

4.71

0.09

Histidine

55.5

51.5

57.2

58.4

1.85

0.04

Isoleucine

127.3

133.8

139.6

135.1

4.35

0.18

Leucine

161.5

166.1

178.1

170.8

5.62

0.08

Lysine

89.6

132.2

109.2

110.3

5.16

<0.0001

Methionine

38.4

36.4

41.1

39.0

1.49

0.19

Phenylalanine

47.0

46.6

49.8

47.8

1.38

0.26

Threonine

126.7

117.9

132.0

129.8

4.94

0.005

Tryptophan

45.0

45.6

47.4

45.7

1.30

0.31

Valine

257.8

262.1

278.6

273.7

7.94

0.08

Alanine

315.3

304.6

307.2

312.8

9.97

0.51

Asparagine

60.1

58.9

62.7

63.4

1.55

0.08

Aspartic Acid

1.08

1.12

1.18

0.93

0.070

0.08

Citrulline

102.0

100.4

106.0

107.2

4.23

0.30

Cystine

21.6

21.4

21.9

22.5

0.55

0.19

Cystathionine/Allocystathionine

2.43

2.16

2.36

2.32

0.084

0.15

Glutamine

206.4

214.4

208.5

210.3

10.45

0.78

Glutamic Acid

43.2

43.7

44.4

43.6

0.85

0.45

Glycine

361.1

342.9

358.7

356.7

12.82

0.11

Homocystine

3.21

3.47

3.53

3.07

0.459

0.55

Ornithine

58.1

65.2

66.9

68.3

4.26

0.07

Proline

100.4

96.4

103.9

102.9

2.22

0.11

Serine

88.7

86.2

91.4

90.3

1.65

0.11

Taurine

54.5

50.0

56.3

56.5

2.67

0.08

Tyrosine

50.0

47.2

53.3

52.5

1.79

0.07

TEAA

1035

1092

1132

1107

49.5

0.12

TNEAA

1468

1438

1489

1493

39.7

0.24

TAA

2504

2531

2621

2601

60.3

0.14

TBCAA

547

562

596

580

29.6

0.13

TUCAA

246

265

272

11.4

0.08

TSAA

120

113

125

123

3.4

0.07

Vetter 45

TAA-Lys

2414

2399

2512

2492

67.5

0.11

Table 8. Plasma metabolites (µM) for Holstein cows fed a diet supplemented with RP-Lys

supplements.

Item

Control

Infusion

Prototype

II HP0

Prototype

SEM

1-Methylhistidine

23.2

24.4

24.3

24.2

0.85

0.20

3-Methylhistidine

3.19

2.90

3.22

3.16

0.19

0.13

α-amino-adipic acid

13.7

16.3

14.2

14.5

1.30

0.28

α-amino-butyric acid

21.4

19.8

21.2

21.4

1.32

0.43

β-alanine

10.7

9.7

10.8

10.5

0.55

0.19

Carnosine

16.1

15.7

16.2

16.0

0.48

0.77

Ethanolamine

1.63

1.97

1.79

3.13

0.40

0.05

γ-amino-butyric acid

2.85

3.64

3.52

3.35

0.54

0.38

Hydroxylysine

2.27

2.89

2.43

2.76

0.41

0.63

Hydroxyproline

10.4

10.2

10.5

10.2

0.35

0.92

Phosphoserine

5.03

5.00

5.06

5.07

0.11

0.93

Sarcosine

33.1

29.7

26.5

30.0

4.1

0.47

Vetter 46

Table 9. Bioavailability calculation using changes in plasma Lys % of (TAA-Lys) using Commercial

values

Item

Infusion

Prototype II HPO

Prototype IL

Slope

0.02846

0.01010

0.01394

Bioavailability of RP-Lys

35.5 ± 1.7

49.0 ± 3.0

Calculated as [(slope of RP-Lys /slope Infusion) *100].

Vetter 47

Table 10. Statistical summary of trial results using changes in plasma Lys % of (TAA-Lys) using

Commercial values

Slope is different from zero P < 0.05.

Maximum value

Mean

Slope

Intercept

RMSE

Infusion

5.347

0.1493

0.02846

0.0007

3.634

0.028

0.99

0.0485

Prototype II HPO

4.246

0.1493

0.01010

0.0005

3.634

0.023

0.99

0.0406

Prototype IL

4.386

0.1493

0.01394

0.0008

3.634

0.029

0.98

0.0529

Vetter 48

Figure 1. The relationship between Infusion (▲), IL (●), and II HPO (■) in lactating dairy cows using

commercial Lys content. Infusion: Y = 3.63 + 0.0285x; slope SE = 0.001, intercept SE = 0.03, r

= 0.99.

II HPO = 3.63 + 0.0101x; slope SE = 0.001, intercept SE = 0.02, r

= 0.99; relative bioavailability =

(0.0101 ÷ 0.0285) × 100 = 35.5. IL = 3.63 + 0.0139x; slope SE = 0.001, intercept SE = 0.03, r

= 0.98;

relative bioavailability = (0.0139 ÷ 0.0285) × 100 = 49.0.

Vetter 49

Table 11. Metabolizable lysine for the RP-Lys supplements

Lys, %

Bioavailability,

Metabolizable Lys,

g/kg

Prototype II HP0

63.76

35.5

226.3

Prototype 1L

63.36

49.0

310.5

Vetter 50

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