Nutritional Strategies to Improve Nitrogen Efficiency And

NUTRITIONAL STRATEGIES TO IMPROVE NITROGEN EFFICIENCY AND

REDUCE NITROGEN EXCRETION OF LACTATING DAIRY COWS

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Susan M. Noftsger, M.S.

The Ohio State University

2003

Dissertation Committee:

Dr. Normand R. St-Pierre, Advisor Approved by Dr. Jeffrey L. Firkins

Dr. William P. Weiss ______Dr. Mark Morrison Advisor

Animal Science Graduate Program

ABSTRACT

Accurate prediction of amino acids (AA) reaching the intestine is imperative if

reduction in N excretion by dairy cows is to be achieved through nutrition strategies.

Provision of feed amino acids to the metabolizable supply is dependent on the rumen degradability of protein sources and their digestibility in the intestine. In the first

experiment, we hypothesized that milk production and composition could be maintained

and dietary crude protein (CP) decreased to improve efficiency of N utilization through

selection of highly intestinally digestible rumen undegradable protein (RUP) and supplementation of Met. Sixty cows in their fourth week of lactation were assigned to

four diets for 12 weeks in a balanced randomized block design. Diets differed in

estimated digestibility RUP (high vs. low), CP concentration (17 vs. 18.3%), and

supplementation of Met in rumen degradable and undegradable forms. Diets contained

equal concentrations of net energy for lactation (NEL), acid detergent fiber (ADF), neutral

detergent fiber (NDF), and ash. Maintaining the higher concentration of dietary RUP

while increasing metabolizable protein (MP) concentration through higher intestinal

digestibility of RUP (HiCP-HiDRUP), and decreasing dietary CP and supplementing Met

(LoCP-HiDRUP + Met) increased milk yield and component production over the control

(HiCP-LoDRUP). Both higher digestibility of the RUP source and supplemental Met

were necessary to maintain milk production when reducing CP in the diet. Supplemental

ii Met effects of rumen available 2-hydroxy-4-(methylthio) butanoic acid (HMB) and rumen bypass (polymer coated dl-Met; Smartamine) could not be differentiated in this trial. Lowering dietary RUP while maintaining MP concentration through higher intestinal digestibility of RUP (LoCP-HiDRUP) allowed some increases in milk, protein, and fat production, further indicating the nutritional limitations of the unselected protein source. Supplementing the highly digestible RUP source with rumen available and rumen escape sources of Met resulted in maximal milk and protein production and maximum N efficiency by cows, indicating that post-ruminal digestibility of RUP and

AA balance can be more important than total RUP supplementation.

In this study, the combined supplementation of Met in rumen available and rumen undegradable forms had a greater impact on milk protein yield than expected from prior research when both sources were not fed in combination. This points to an additive effect of the two sources, indicating that the HMB has a different mechanism of action, possibly through a stimulation of microbial growth.

The hypotheses for the second experiment were that HMB supplementation increases microbial growth by either sparing Met precursors for more efficient protein synthesis or by shifting rumen bacterial species. These effects were quantified by measuring effluent flow of N; digestibilities of organic matter (OM), ADF, NDF and hemicellulose; and volatile fatty acid (VFA) concentrations in continuous culture. Four dietary treatments consisted of a control, two concentrations of HMB (0.055%, 0.110% of DM) and one concentration of dl-Met (0.097%). Digestibilities of OM, hemicellulose, and NDF were largely insensitive to treatment. Digestibility of ADF showed a quadratic

iii effect to HMB in the diet, being highest at 0 and 0.11% HMB. There were trends (P <

0.15) for linear increases in production of isobutyrate and isovalerate with increasing concentration of HMB, whereas isobutyrate concentration increased linearly. Valerate concentration was affected quadratically by concentration of HMB, with estimated peak concentration at 0.055%. Propionate concentration decreased linearly with level of HMB supplementation, whereas a quadratic trend (P = 0.13) was noted for its production, which was estimated to peak at 0.047% HMB. There was a significant linear decrease (P

= 0.032) in the amount of bacterial N obtained from NH3-N as concentration of HMB increased. The lack of major effects may indicate that a high rumen degradable protein

(RDP) concentration in the diet provided enough excess AA so that Met was not limiting, that changes in bacterial populations were small and were concealed by the larger unaffected populations of common species of rumen bacteria, or that there may be a pronounced effect on protozoa in the rumen which are not maintained in continuous culture fermenters.

The hypotheses for the third trial were: (1) that proportions of dl-Met and HMB escaping rumen metabolism through passage with the liquid fractions through the omasum are small, (2) that dl-Met and HMB effects are ruminal, and (3) that HMBi (the isopropyl ester of HMB) does supply metabolizable Met, resulting in milk composition effects characteristic of an increased Met supply. These effects were quantified using eight ruminally-cannulated cows. Effects on milk production, N utilization, ruminal

VFA, and protozoa were determined. Samples of omasal fluid were used to determine the amount of Met supplements passing out of the rumen. Treatments were: (1) no

iv methionine (Control); (2) 2-hydroxy-4-methylthiobutanoic acid (HMB) at 0.10% of DM;

(3) isopropyl HMB (HMBi) at 0.13 % of DM; and (4) dl-methionine (dl-Met) at 0.088%

of DM. Dry matter intakes were not different and averaged 20 kg/d. Milk yields

averaged 37.7 kg and were not different among treatments. Milk protein concentration

was significantly affected by treatment (2.91, 2.95, 3.02, 2.96%) for control, HMB,

HMBi, and dl-Met, respectively, but milk fat concentration was not affected. Short-term increase in milk true protein content is characteristic of an increase in metabolizable Met supply. Rumen VFA profile and NH3 concentrations were similar among treatments.

Total tract digestibilities of OM, NDF, and N were not different. In situ rate of digestibility of NDF in TMR was increased with HMBi, whereas rate of in situ digestibility of alfalfa hay CP was increased by HMB and dl-Met when compared with control or HMBi. Passage rates of small particles (0.071/h) and fluid (0.157/h) were not affected by treatment. Protozoa were increased numerically, but not statistically, in the omasum by HMB and HMBi treatments. The percentage of HMB ingested that passed into the omasum was 5.3% (± 1.5%). This, along with numerical protozoal increases and digestibility changes with HMB and HMBi, indicated that most of the activity of HMB was in the rumen. Only a small amount of HMBi was found as HMB in the omasum

(2.3%). If HMBi remaining in the rumen is assumed to break down to HMB and isopropanol and act similarly to unmodified HMB, this number would be consistent with prior research claiming that 50% of the HMBi is absorbed through the rumen wall, while the remaining 50% is converted to HMB in the rumen and is rapidly used by the microbial population.

v ACKNOWLEGMENTS

I would like to thank my advisor, Normand St-Pierre, for intellectual support, encouragement, and enthusiasm, which made this thesis possible. I would also like to thank Dr. Jeff Firkins for taking the time to help me out.

I thank my fellow graduate students, both for helping me with the animal experiments and in the lab, and allowing me to bounce ideas off of them, especially John

Sylvester, Dana Harvatine, and Carine Reveneau. I also wish to thank the undergraduate students who have helped me at the farm and the lab, and without whom I would never have finished.

Last, I would like to express my thanks to Dr. Brian Sloan and his colleagues from Adisseo, Aventis, and Rhone-Poulenc for their intellectual as well as financial support of my research. The financial support of Venture Milling, a division of Perdue

Farm must also be acknowledged and was greatly appreciated.

vi VITA

August 24, 1971…………………………………………………….Born-Shelby, NC

1992-1997…………………………………………………….……Research Assistant North Carolina State University Dairy Research and Teaching Unit

1997-1999…………………………………………………………Graduate Teaching and Research Assistant, North Carolina State University

1999-Present………………………………………………………Graduate Research Assistant The Ohio State University

PUBLICATIONS

Peer-Reviewed Journal Articles

Noftsger, S. M., N. R. St-Pierre, S. K. R. Karnati, and J. L. Firkins. 2003. Effects of 2- Hydroxy-4-(methylthio) butanoic acid (HMB) on microbial growth in continuous culture. J. Dairy Sci. J. Dairy Sci. 86:2629-2636.

Noftsger, S., and N. R. St-Pierre. 2003. Supplementation of methionine and selection of highly digestible rumen undegradable protein to improve nitrogen efficiency for milk production. J. Dairy Sci. 86:958-969.

Firkins, J. L., M. L. Eastridge, N. R. St-Pierre, and S. M. Noftsger. 2001. Effects of grain variability and processing on starch utilization by lactating dairy cattle. J. Anim. Sci. 79(E. Suppl.):E218-E238.

Noftsger, S. M., B. A. Hopkins, D. E. Diaz, C. Brownie, and L. W. Whitlow. 2000. Effect of whole and expanded-expelled cottonseed on milk yield and blood gossypol. J. Dairy Sci. 83:2539-2547.

vii Abstracts

J. T. Sylvester, N. R. St-Pierre, B. K. Sloan, J. L. Beckman, and S. M. Noftsger. 2003. Effect of HMB and HMBi on milk production, composition, and N efficiency of Holstein cows in early and mid-lactation. J. Dairy Sci. 86(Suppl. 1):60.

Noftsger, S.M., J. Firkins, and N. St-Pierre. 2002. Effects of 2-hydroxy-4-(methylthio) butanoic acid (HMB) and dl-methionine on microbial growth, VFA production and nutrient digestion in continuous culture. J. Dairy Sci. 85(Suppl. 1):240.

Noftsger, S. and N. St-Pierre. 2001. Effects of rumen undegradable protein digestibility and supplemental methionine on production parameters and nitrogen efficiency of Holstein cows in early lactation. J. Dairy Sci (Suppl. 1):36.

Noftsger, S. and N. St-Pierre. 2001. Effects of rumen undegradable protein digestibility and supplemental methionine on production of Holstein cows in early lactation. Midwest Branch ADSA Meeting, Des Moines, IA. Abstract 343.

Firkins, J. L., M. L. Eastridge, N. R. St-Pierre, and S. M. Noftsger. 2000. Effects of grain variability and processing on starch utilization by lactating dairy cattle. J. Dairy Sci. 83(Suppl. 1):31.

Noftsger, S.M., D. E. Diaz, B. A. Hopkins, L. W. Whitlow, and C. Brownie. 1999. Effects of replacing whole cottonseed with three levels of extruded whole cottonseed on milk yield, milk composition, and conjugated linoleic acid concentration of milk fat. J. Dairy Sci. 82(Suppl. 1):118.

FIELD OF STUDY

Major Field: Animal Sciences

viii TABLE OF CONTENTS

Page Abstract…………………………………………………………………………. ii

Acknowledgments………………………………………………………………. vi

Vitae……………………………………………………………………………..vii

List of Tables……………………………………………………………………. xi

List of Figures…………………………………………………………………... xiii

List of Abbreviations……………………………………………………………. xiv

Chapters:

1. Introduction…………………………………………………………………...1

2. Review of Literature

Nitrogen and the Environment …………………………………………..4 Rumen Undegradable Protein …………………………………………..10 Amino acid metabolism………………………………………………....20 HMB………………………………………………………………….…28 Omasal Sampling………………………………………………………..41 Summary………………………………………………………………...46

3. Supplementation of Met and Selection of Highly Digestible Rumen Undegradable Protein to Improve Nitrogen Efficiency for Milk Production

Abstract………………………………………………………………….48 Introduction…………………………………………………………..….50 Materials and Methods…………………………………………………..52

ix Results and Discussion…………………………………………………..59 Conclusions…………………………………………………………...... 67

4. Effects of 2-Hydroxy-4-(methylthio) butanoic acid (HMB) on microbial growth in continuous culture

Abstract…………………………………………………………………..78 Introduction……………………………………………………………....80 Materials and Methods…………………………………………………...82 Results and Discussion…………………………………………………...86 Conclusions…………………………………………………………….…92

5. Determination of the ruminal effects and degradability of HMB, HMBi and dl-Met

Abstract……………………………………………………………….….97 Introduction……………………………………………………………....98 Materials and Methods………………….……………………………….100 Results and Discussion……………………………………………….….109 Conclusions……………………………………………………………...117

6. Conclusions…………………………………………………………………..126

List of References………………………………………………………………..129

x LIST OF TABLES

Tables Page 2.1. Extended chemical scores of protein sources in relationship to milk protein……………………………………………………………….11

2.2. The Lys and Met contents of microbial protein and protein supplements compared with milk………………………………………...13

3.1 Ingredient and nutrient composition of diets (DM basis) that vary………69 in CP and digestibility of RUP based on predicted body weight, intake, milk yield and composition

3.2 Nutrient composition of supplemental RUP source……………………...70

3.3 Estimates of dietary nutrients at standard production (45.5 kg) and intake (23.8 kg DMI) in diets that vary in CP and digestibility of RUP…71

3.4 Least squares means for performance measures in Experiment 1 for diets that vary in CP and digestibility of RUP (n = 60)………………72

3.5 Estimates of dietary nutrients at standard production (45.5 kg) and intake (23.8 kg DMI) and actual production and intake in diets that vary in CP and digestibility of RUP……………………………………………...73

3.6 Least squares means in nitrogen measurements for experiment 1 for diets that vary in CP and digestibility of RUP (n = 60)………………74

3.7 Least squares means for intake and digestibility of DM and NDF in Experiment 2 for diets that vary in CP and digestibility of RUP (n = 24)…………………………………………………………………...75

3.8 Least squares means for milk production and nitrogen utilization during experiment 2 for diets that vary in CP and RUP digestibility (n = 24)………………………………….…………………………….….76

xi 4.1 Ingredients and nutrient composition of control diet…………….……….93

4.2 Nutrient digestibility, nitrogen fluxes, and ammonia utilization in continuous culture fermenters supplemented with two concentrations of HMB or dl-Met……………………………………….…………….....94

4.3 Volatile fatty acid total production and concentration in continuous culture fermenters supplemented with two concentrations of HMB or dl-Met…………………………………………………..……95

5.1 Ingredient and nutrient composition of base diet (DM basis) based on predicted BW, intake, milk yield and composition…………………119

5.2 Least squares means for milk production and intake data for diets that vary in source of methionine……………………………………….120

5.3 Least squares means for rumen volatile fatty acids and ammonia for diets that vary in source and availability of methionine………………...121

5.4 Ruminal digestibility of nutrients for diets that vary in source and availability of Met……………………………………………………....121

5.5 Digestibility of crude protein and neutral detergent fiber in TMR, alfalfa hay, and corn silage using Dacron bags removed at 8 time points…………………………………………………………122

5.6 Least squares means for rumen pool measurements and passage rates for diets that vary in source of methionine………….…………….123

5.7 Nitrogen partitioning and efficiency with diets that vary in source and degradability of Met………………………………………………..124

5.8 Flow of bacterial N and OM to the omasum with diets that vary in source and degradability of Met…..…………………………………….124

5.9 Percentage of HMB and HMBi dose appearing as HMB in the omasal fluid……………………………………………………………..125

xii LIST OF FIGURES

Figure Page 2.1 1998 National ammonia emissions by principal source categories (EPA, 1998)………………………………………………………………..5 2.2 Conversion of HMB to Met………………………………………………29 3.1 Nitrogen absorbed (NA) versus N intake (NI) for HiCP-LoDRUP, HiCP-HiDRUP, LoCP-HiDRUP, and LoCP-HiDRUP + Met……………77 4.1 Comparison of the bacterial community structure in fermenter samples……………………………………………………………………96

xiii LIST OF ABBREVIATIONS

AA amino acid(s) ADF acid detergent fiber BCVFA branched chain volatile fatty acids BW body weight CAFO concentrated animal feeding operations CP crude protein d day DM dry matter DMI dry matter intake EPA Environmental Protection Agency h hour HMB 2-hydroxy-4-(methylthio) butanoic acid HMBi isopropyl-2-hydroxy-4-(methylthio) butanoic acid MCP microbial crude protein MHA methionine hydroxy analog (Calcium salt of HMB) MUN milk urea nitrogen MP metabolizable protein NAN non-ammonia nitrogen NANBN non-ammonia non-bacterial nitrogen NDF neutral detergent fiber NEL net energy of lactation OM organic matter PM10 particulate matter exceeding 10 µm PM2.5 particulate matter exceeding 2.5 µm rDNA ribosomal DNA RDP rumen degradable protein RIS-LP ribosomal intergenic spacer length polymorphism RUP rumen undegradable protein TMR total mixed ration UN urinary nitrogen VFA volatile fatty acids

xiv CHAPTER 1

INTRODUCTION

The general public of the United States has become increasingly aware of industries that negatively impact the environment. Agriculture originally attracted attention due to odor problems but currently has come under increased scrutiny by both the public and, through its influence, the government, for release of nitrogen into the air and water sources via fertilizer runoff and animal waste products. The U. S.

Environmental Protection Agency (EPA) has issued regulations regarding nitrogen release into the environment, and more regulations are likely on the way (Powers, 2003).

To continue to function in the future, the dairy industry must be proactive in reducing nitrogen release into the environment.

The primary way that nitrogen from dairy facilities negatively impacts the environment is through the volatilization of ammonia from urine and feces. Many dairy producers overfeed crude protein as a safety factor. Over the last decade, dairy nutrition professionals have attempted to improve the adequacy of formulated diets for dairy cattle by factoring in estimated amino acids supplies and requirements in their formulation models, while reducing the amount of nitrogen fed and released by the animals into their waste. Requirements for amino acids are difficult to estimate. Increases in production are dependent on increases in supply. Supply is also hard to measure, due to the uncertainty of AA utilization by the animal. This balancing process is much more

1 complex for ruminants than for non-ruminant animals because of the extent of microbial

fermentation and digestion of feedstuffs in the rumen.

Microbial protein supplies the majority of the protein to the cow (Firkins, 1996).

In the past, overfeeding of protein sources supplied enough excess to ensure that enough

N was supplied for maximal microbial growth while ensuring that some protein escaped rumen degradation and was absorbed by the intestine to supply the balance of the cow’s protein requirement.

More recently, dairy nutritionists began to identify protein sources that partially escape ruminal degradation and increase the supply of metabolizable AA to the cow. It has become evident that the quality of the rumen-escape protein is important; quality of this protein is expressed both in terms of its intestinal digestibility (availability) as well as

its amino acid profile (Santos et al., 1998; Calsamiglia and Stern, 1995). The problem of

quantifying amino acids supply leads to the equally important problem of quantifying

amino acids requirements (Schwab et al., 1992).

Diets that are formulated correctly for amino acids can potentially maintain milk

yield and milk component production while reducing the amount of nitrogen released into the environment. This can result in savings to dairy producers through decreased purchase of protein supplements, improved positive support from the segment of the general public that is worried about the environment, and preparation of the industry for future regulations regarding nitrogen emission and air and water quality.

In this literature review, I will first summarize the status of current regulations by the EPA and the problem of excess N excretion by the U. S. dairy industry. The selection

2 of specific sources of protein for their resistance to rumen degradation as well as post- ruminal digestibility and amino acid composition will then be reviewed, considering the potential for such selection to improve N efficiency and reduce N excretion by dairy cows. This will be followed by an extensive review of the prior research on 2-hydroxy-4-

(methylthio)butanoic acid (HMB), which is a molecule used as a source of supplemental

Met in non-ruminants and a possible rumen-escape source of Met in ruminants. The use of HMB in dairy diets could complement natural RUP protein sources, thus improving N efficiency and reducing N excretion of lactating cows. Lastly, I will review prior research on the omasal sampling technique due to the critical role that I made of this technique in my third and last experiment.

CHAPTER 2

REVIEW OF LITERATURE

NITROGEN AND THE ENVIRONMENT

Changes in government policies are forcing the U. S. dairy industry to simultaneously be

more economically efficient and environmentally responsible. The EPA is currently

working on revisions of the Clean Water Act that apply to Concentrated Animal Feeding

Operations (CAFO; Powers, 2002). These operations currently are greater than 1000

animal units, but proposed changes may lower this number to 500. Revisions to the

Clean Water Act may eliminate protection in the event of a catastrophic storm causing

discharge from waste handling systems. The new EPA regulations will also require each

CAFO to have a site specific Permit Nutrient Plan for manure application. The Clean Air

Act of 1990 established regulations regarding particulate matter exceeding 10 µm in

diameter (PM10). In 1997 the EPA issued a notice of intent to regulate smaller particulate matter (> 2.5 µm; PM2.5). This would include NH3 emissions, because NH3 reacts with

other compounds (e.g., SOx, NOx) in the atmosphere to form particulate matter.

Ammonium sulfate and ammonium nitrate contribute as much as half of the PM2.5

measured in the U. S. The EPA estimates that 86% of the national NH3 emissions are

from miscellaneous sources of which 83% are from livestock and fertilizer (Figure 2.1).

on-road and non- 4% road engines and 5% 3% vehicles-5% 2% All other-4%

Chemical & allied product Mfg.-3%

waste disposal & recycling-2%

86% Misc. (includes livestock and fertilizer)-86%

FIGURE 2.1. 1998 National ammonia emissions by principal source categories (EPA,

1998)

The authority of the U. S. EPA to regulate PM2.5 was challenged in Federal courts. In

2002, the Supreme Court issued a judgment supporting the constitutionality of the EPA’s authority in this matter. Thus, it appears evident that the EPA will now proceed with the implementation of PM2.5 requirements.

A number of states, including Missouri, California, Minnesota, and Iowa, have

initiated efforts to address air emissions from animal feeding operations. To date, most

of the state regulatory discussions have focused on odor concerns, but the EPA has

commissioned the National Academy of Science, through its branch the National

Research Council (NRC), to investigate possible health concerns. The vision statement

from the NRC’s Frontiers in Agricultural Research (2002) states that agricultural research

will “include further gains in food and fiber production and such other benefits as

enhanced public health, environmental services, rural amenities, and community well-

being”. The U. S. Department of Agriculture (USDA) will provide leadership through its

research, education and economics agencies.

Agriculture and excess N

One of the challenges of the dairy industry today is to design diets that can sustain

high production while minimizing the environmental impact of excessive N excretion in

the urine and feces. Historically, producers often overfed CP in order to compensate for

variability in cow requirements and feed nutrients (St-Pierre and Thraen, 1999), resulting

in excessively high output of N in the waste. Overfeeding CP has been justified as a

safety factor but negatively impacts the environment.

+ Any reactive N species (i.e. NOx, NH3, N2O, NH4 , N2) can be converted to any

other N species if the conditions are appropriate (Galloway, 1998). Excess N can have

seven distinct effects on the global and regional scale (Socolow, 1999). Nitrous oxide in

the air contributes to the greenhouse effect, reducing the concentration of stratospheric

ozone. Nitrous oxide is the fourth largest contributor to the natural greenhouse effect.

The breakdown of nitrous oxide in the stratosphere produces nitric oxide, which acts

catalytically to lower ozone concentration. On a regional level, N gases contribute to air

pollution and possibly unhealthy nitrate concentrations in drinking water. Ammonia

provides a route for formation of particulate-phase nitrate by reacting with HNO3 (gas) to form particulate NH4NO3 (Meng et al., 1997). Nitrates in drinking water can inactivate

hemoglobin in blood. Infants younger than three months are especially susceptible. The

- - U.S. standard for NO3 -N is 10 mg of N as NO3 per liter of water (Socolow, 1999).

Reactive N can cause acid deposition, both acid rain and dry deposition, and eutrophication of bays and estuaries. Reduced N is the limiting nutrient in many aquatic ecosystems, and the addition can lead to excessive plant growth, depletion of oxygen, and the development of “dead zones” as the plants die and decay. An example of this is the hypoxic zone in the Gulf of Mexico off Louisiana, which has been partially caused by agriculture in the Mississippi River watershed (Burkart and James, 1999). Last, N

addition to ecosystems encourages dominance of the species that can use it most

efficiently, resulting in an altered ecosystem composition (Socolow, 1999).

According to Galloway et al. (1995), the global mobilization of reactive N (e.g.

fertilizer production, fossil fuel use, and legume production) has doubled between 1970

and the mid 1990s from 70 Tg to 140 Tg of N per year. The majority of this reactive N

has been mobilized for food production. The increase was necessary to keep pace with

increasing population growth concomitant with a leveling off of arable land. If the world

food production on a per capita basis is to increase, additional N must be added to the

agro-ecosystem (Galloway, 1998). Smil (1998) estimates that only 50% of the N in

fertilizer is recovered in the harvested crop. Further down the food chain, the conversion

of feed N to milk N is only approximately 30 to 40% in lactating dairy cows (Galloway,

1998) and less than 25 % on a whole dairy basis (Kohn et al., 1997). It is at this point

where the dairy industry can influence the undesirable release of N back into the

environment by improving N efficiency of diary cows through dietary manipulation.

Effects on the dairy industry

Kohn et al. (1997) developed a model of N management on the dairy farm and

performed a sensitivity analysis to determine the relative importance of manipulating

herd nutrition, manure management and crop selection in reducing N losses from the

farm. They determined that improvements in animal diet and management that increases

the conversion of feed N to animal product by 50% would increase total farm N

efficiency by 48% and reduce N losses per unit of product by 36 to 40%. This indicates

that dietary manipulation could be important in the reduction of N losses on dairy farms.

St-Pierre and Thraen (1999) suggested several feeding and management strategies

to reduce loss of N on dairy farms. For example, increasing productivity of the herd

increases N excreted, but decreases N excreted per unit of milk production, improving N efficiency. Grouping cows according to their nutritional requirements for NEL and CP can also improve N efficiency by reducing the optimal safety factor needed to provide for the higher producers in each group. For a large herd, six feeding groups appear to provide maximal N efficiency, beyond which variability in cow requirements and nutrient composition of feeds masks any additional improvement in N efficiency. Improved knowledge of biology, the nutrient content of inputs, as well as new technologies such as protected amino acids (AA) were suggested as potential methods to bring about a substantial reduction in N excretion.

Decreasing N excretion has been achieved by dietary manipulation with dairy cattle. Dinn et al. (1998) manipulated diets for lactating dairy cows in an attempt to

decrease N in the waste. Crude protein was decreased to 16.7 or 15.3% from 18.3% in the control diet. The low CP diets were balanced for Met and Lys using undegradable sources. Milk yield was decreased by the reduction in CP, but milk protein output was maintained. Urinary and fecal N was decreased by the low CP diets.

Non-lactating cattle are also affected by diets with lower CP. James et al. (1999) fed heifers (BW of 350 kg) a diet of 9.6 or 11% CP in a crossover design. Urinary N and total N excreted were significantly reduced with the lower CP diet, while fecal N remained unchanged. Ammonia emission per animal was estimated to be significantly lower on the low CP diet and was positively correlated with N intake. Daily ammonia emission was reduced by an estimated 23% by the low CP diet.

Wu and Satter (2000) fed diets that varied in protein during early lactation from

15.4 to 19.3%. Manure N was estimated as intake N minus milk N, assuming no tissue N retention. While manure N was decreased significantly in the lowest CP diet, there were some losses in yields of milk and protein. However, the lowest CP diet did not contain any supplemental rumen undegradable protein sources, and could have been limited in the AA that were provided by the higher CP diets. These research reports show that some advances have been made towards reducing nitrogen excretion in dairy cows by dietary means. However, additional research must identify ways to reduce N excretion without any reduction in cow productivity if new feeding recommendations are to be economically sustainable. The ability to more exactly predict nutrient content of the diets, as well as the requirements of the cow, is necessary to adequately feed the animal with the least waste possible.

RUMEN UNDEGRADABLE PROTEIN

Overview

The effects of RUP have been extensively studied and published in the literature.

However, there has been much inconsistency in the results of these studies. Santos et al.

(1998) published a review of 108 studies published between 1985 and 1997. High rumen undegradable protein (RUP) diets resulted in a decrease in microbial crude protein (MCP) synthesis in 76% of the comparisons, possibly due in some cases to the replacement of rumen degradable protein (RDP) with RUP. When soybean meal was replaced with high

RUP sources, milk yield increased in only 17% of the comparisons. Fishmeal and treated soybean meal counted for most of the positive effects, whereas corn gluten meal usually showed negative effects. Fishmeal tended to decrease milk fat. Protein percentage of milk decreased in 28 comparisons, and increased in only six, possibly due to a limitation on MCP synthesis.

AA profile

The potential to simulate the AA profile of milk protein may be the mark of a good source of RUP. Santos et al. (1998) examined the similarities of some protein sources with the AA composition of milk (Table 2.1). Microbial protein was found to be the best single source of protein for milk synthesis.

Protein source His Phe Leu Thr Met Arg Val Ile Trp Lys Blood meal 100 100 93 86 45 33 70 10 76 91 Fish meal 77 69 58 68 100 59 59 47 71 80 Feather meal 11 59 66 59 23 32 38 32 29 13 Meat meal 67 65 46 59 49 76 51 36 39 58 Meat and bone meal 64 64 46 59 49 76 48 36 32 55 Corn gluten meal 67 100 100 60 100 36 48 40 30 18 Alfalfa meal, 69 100 55 80 60 50 66 51 100 46 Brewers grain 56 100 83 65 78 53 65 74 87 34 Distillers grains with 74 84 72 63 81 42 53 38 45 24 Soybean meal 89 100 56 74 56 89 60 55 75 70 Microbes 90 97 54 100 97 79 66 61 99 100 1Adapted from Chandler (1989) and calculated as follows: (percentage of AA in feed protein/percentage of AA in milk protein) X 100. A score of 100 is the maximum allowed for each value

Table 2.1. Extended chemical scores of protein sources in relationship to milk protein.1

Guinard et al. (1994) infused 4 concentrations of Ca-caseinate into the duodenum of lactating cattle. The infusions linearly increased milk yield, protein percentage, and protein yields. The authors found decreased efficiency of use of digested protein for milk protein as the concentration of casein increased.

Wright et al. (1998) fed a supplemental RUP source with a fixed AA composition designed to approximate bovine casein for Met, Lys, Phe, His and Thr. The RUP source was formulated using a study in which AA determinations were conducted on feed residues from nylon bags incubated for 14 h in the rumen. The combination selected included herring, feather, and blood meals, along with rumen protected Met and Lys.

Milk and protein production increased linearly as dietary RUP increased.

Bach et al. (2000) attempted to ascertain the effects of protein quality by supplementing 15 and 18% CP diets with (high quality) or without (low quality)

supplemental rumen protected Met and rumen available Lys. The RUP source for the high quality diets was formulated to mimic the EAA profile of casein. This supplement considered the contribution of bacterial protein estimated by OM intake. The high quality diets increased yields of CP, casein, and fat in milk over the low quality diets.

Also, the 15% CP with high quality protein resulted in the same milk and milk protein yields as the rations containing 18% CP with a poor AA profile, implying a greater efficiency of N utilization for milk protein synthesis for the high quality protein diet.

These trials indicate that sources of RUP mimicking bovine casein can increase protein and milk production.

Schwab (1994) pointed out the importance of amount and balance of essential AA in duodenal digesta proposing that protein sources should be compared for percentages of

Lys and Met in relation to total AA. He suggested an ideal ratio of Lys:Met of 3:1.

Microbes are an excellent source for both of these AA. Most other sources of RUP are low in at least one of the two (Table 2.2).

Item LysMetEAA2 (% of total EAA) (% of TAA3) Milk 16.4 5.1 38.4 Bacteria 15.9 5.2 33.1 Protein supplement Blood meal 17.5 2.5 49.4 Brewers dried grains 6.7 4.5 46.3 Corn gluten meal 3.8 7.2 44.2 Corn DDG4 + solubles 5.9 5.9 37.7 DDG + solubles 6.5 3.7 43.3 Feather meal 3.9 2.1 31.4 Fish meal (Menhaden) 16.9 6.5 44.8 Meat and bone meal 45% CP 12.4 3.0 39.4 50% CP 14.2 3.7 36.6 Soybean meal (solvent) 13.8 3.1 47.6 Expeller soybean meal 13.0 2.9 49.6 1Adapted from Schwab (1994) 2Essential AA 3Total AA 4Distillers dried grains

Table 2.2. The Lys and Met concentrations of microbial protein and protein supplements compared with milk1

According to Schwab (1994), blood meal, feather meal and soybean meal are all limiting

for Met, whereas feather meal, corn gluten meal, brewers grains and distillers grains are

limited in Lys. The best sources of RUP would potentially contain a mix of sources

balanced to contain optimal amounts and proportions of both Lys and Met.

Chan et al. (1997) compared a low quality RUP diet (corn gluten meal + Lys) to a

high quality RUP diet (fish, blood and soybean meals + Lys). The percentage of Met was

similar for all diets. The high quality diet more closely provided the 3:1 Lys:Met ratio 13

suggested by Schwab (1994) than the low quality diets (< 2:1). Higher quality protein

sources increased milk, protein and fat yield and tended to increase 3.5% FCM yield and

lactose yield when compared with low quality RUP. Lysine concentration in the blood

was increased by the high quality RUP. Cozzi and Polan (1994) compared two sources

of RUP (corn gluten meal and dried brewers grains) with soybean meal alone. In the

RUP diets, a portion of the soybean meal was replaced with similar protein quantities

from corn gluten meal or dried brewers grains. Dried brewers grains provide more Lys

and Arg, whereas corn gluten meal is a better source of Met and Leu. Dried brewers

grains increased milk yield and milk per unit of DMI when compared with corn gluten

meal. Both sources of RUP increased protein production over soybean meal alone. The

response to dried brewers grains was explained by the contribution of RUP that met AA

needs that were limiting or co-limiting to the production of milk and milk protein.

Digestibility of RUP

To supply AA to the duodenum, AA must not only escape ruminal degradation by

microbial enzymes, it must also be digestible in the intestine. The 1989 NRC assumed a constant value for RUP digestibility (National Research Council, 1989). Experimental evidence does not support this (Erasmus et al.,1994; Calsamiglia and Stern, 1995). In order to improve our prediction of animal response, intestinal digestibility must be considered.

Von Keyserlingk et al. (1998) used a mobile bag technique to determine the pattern of AA reaching the duodenum from 19 grass hays and the digestibility of these

AA once they reached the intestine. They found that all feeds decreased the

concentration of AA in the DM when compared with the original feeds, indicating a more

extensive ruminal protein degradation than other fractions of the feed. Ruminal digestibility differed across individual AA, ranging from 34.8% of the original concentration in the feed for Met to 61% for Val. Once in the intestine, digestibility of the AA was also different, with the low rumen digestibility of Met in the rumen being associated with higher digestibility in the intestine. For Val, the high ruminal digestibility was associated with a lower digestibility in the intestine. Total tract digestibility was around 80% for most of the AA.

Erasmus et al. (1994) evaluated the intestinal digestibility of two grains, eight protein supplements, and two forages using the mobile bag technique. The samples ranged in rumen degradability from 17 to 72%. Crude protein disappearance from the intestine ranged from a high of 79% for corn gluten meal to a low of 31% for one of the forages. The authors found that sources high in RUP were not necessarily high in intestinal digestibility, since three of the sources (corn gluten meal, blood meal, and hay) were similar in RUP but ranged from 79 to 31% digestibility in the intestine. The authors found the protein in the two forage sources (alfalfa hay and Eragrostis curvula hay)to be less digestible in the intestine than that found in grains and protein supplements.

Degradation of Met in the rumen depended on the feedstuff. The concentration of Lys usually decreased in the feed residues. Once in the small intestine, absorbable AA profiles of RUP closely reflected the AA profiles of the rumen-exposed RUP residues, although Pro, Gly, Ala, and Cys were slightly less absorbed than total RUP AA, and Glu,

Leu, Tyr, Phe, Arg, and Met were slightly more digestible. The overall conclusion drawn

by the authors was that rumen degradation influenced post-ruminal provision of specific

AA more than post-ruminal digestion.

Calsamiglia and Stern (1995) developed a procedure to estimate intestinal digestibility of protein using a 16 h rumen incubation to remove RDP, followed by an enzymatic in vitro procedure to mimic abomasal and intestinal digestion. To validate the procedure, results were regressed on in vivo estimates of intestinal digestibility and the two were highly correlated. The analysis was further validated using heated soybeans to determine the sensitivity of the assay to heat damaged proteins. The authors found that mild heating of soybeans increased intestinal digestibility, but heating at 165°C for greater than 2.5 h decreased digestibility. Mean estimates of RUP digestibility using this procedure ranged from a high of 89.8% for soybean meal to a low of 54.0% for meat and bone meal. Of interest also was the variability of digestibility of blood meal, which ranged from 63.4% to 96.8% digestibility, indicating that even within a single type of

RUP, digestibility can be influenced by prior processing such as excess heating. Another point of interest found by the authors was that ruminal incubation had only small effects on soybean meal, corn gluten meal, and blood meal. However, larger effects were seen with hydrolyzed feather meal, fishmeal, and meat and bone meal. The authors felt that digestible protein in these sources was mostly degraded in the rumen, and only a small fraction of the RUP was available post-ruminally.

Maiga et al. (1996) examined blood meal, meat and bone meal, corn gluten meal, and two types of soybean meal (solvent-extracted and expeller) for intestinal digestibility.

Intestinal digestibility was determined using the Minnesota 3-step procedure (Calsamiglia

and Stern, 1995) with a 12-h rumen incubation period. In this experiment (Maiga et al.,

1996), rumen degradability varied from a low of 4.5% for blood meal to 59% for the

soybean meals. The authors concluded that the low value for blood meal could be due to

processing. The intestinal digestibility of the blood meal averaged 53%, which was

lower than the range reported by others (Stern et al., 1994). Meat and bone meal was similarly digestible (54%), whereas the soybean meals and corn gluten meal were all greater than 80% digestible. In the undegradable residues, blood meal contained the highest concentration of EAA at approximately 57%, primarily due to Lys and Leu. The soybean meals were approximately 47% EAA of which Lys and Leu were greater than in other protein sources. Corn gluten meal was a good source of Leu and Met, and meat and bone meal was a good source of Leu, Lys, Met, and Arg. The authors concluded that the information gathered on corn gluten meal using this procedure should be used with caution when selecting dietary protein supplements. The corn gluten meal, due to its gelatinous, hydrophobic nature, may have prevented microbial attachment when presented to the rumen in a Dacron bag. Therefore, RUP may have been overestimated for this sample.

These studies indicate that simple supplementation of more RUP will not ensure an increase in milk yield or components. It is necessary to have the proper AA profile and RUP that is digestible in the intestine, so as not to pass directly through the cow and

into the environment. With RUP that is intestinally digestible and has the proper AA

profile there may be improvements in N efficiency.

Rumen protected amino acids

Natural sources of RUP are not the only available sources of rumen protected AA.

Amino acids such as Met and Lys are available commercially in rumen-protected and rumen-available forms. With these AA a specific amount of AA can be supplemented without depending on feed sources that may be unpalatable at the concentration needed in some diets (e.g. bloodmeal) or that would further imbalance other AA.

To determine the amount of Met and Lys required post-ruminally,

Schwab et al. (1992) determined the amino acid limitation of lactating cows at four stages of lactation using Met, Lys, or both in comparison with casein. The AA were infused directly into the abomasum. Methionine was infused at 8 to 12 g/d, and Lys at 20 to 30 g/d. Casein was infused at 266 to 400 g/d. The diets were based on corn silage and were formulated to contain 90% of the NRC (1989) recommendations for CP to ensure that cows would respond to the limiting AA. Most of the RUP (69-85%) was supplied from feeds of corn origin, with the rest provided through soybean meal. At all stages except late lactation, protein yield was increased by Lys plus Met over Met alone. In early lactation, the difference between Lys plus Met supplementation versus Met alone was not significant, but protein yield was numerically increased. Supplementation of Lys alone was numerically between Met and Met plus Lys supplementation in early, mid, and peak lactation, indicating that it was first limiting. Casein showed no improvements over Met plus Lys for protein yields. Milk yield was only increased in mid-lactation, with the Met plus Lys treatment and the casein treatment both increasing milk production by approximately 1 kg over Met alone. The results of the experiment indicated that Lys was

first limiting for milk protein synthesis during peak and early lactation and was first or co-limiting with Met in mid-lactation in the diets that were used. Lysine limitation was greatest at peak lactation, where addition of Lys increased milk protein yields the most.

More His and Thr were removed from blood plasma by the mammary gland with infusion of Met plus Lys indicating that they may be the next limiting AA after Lys and Met.

Pisulewski et al. (1996) confirmed Met as second limiting AA for milk protein synthesis with a corn silage/alfalfa diet by supplying Lys through formaldehyde treated soybean meal and an abomasal infusion of 10 g/d Lys. With adequate Lys, milk protein increased linearly with increasing amount of rumen protected Met from 0 to 24 g/d.

Armentano et al. (1997) used heated soybeans as a source of Lys in a base diet containing alfalfa and corn silage supplemented with either 5.25 or 10.5 g/d of rumen-protected Met.

Milk protein yield and concentration increased linearly with increasing Met. Addition of rumen protected Lys did not cause any further increases, indicating Lys adequacy of the diet. Milk yield was not affected. Overton et al. (1998) found little effect of rumen protected Met on milk yield and protein yield when corn gluten meal and ground shelled corn were fed in addition to corn and alfalfa silage, probably due to inadequate Lys. No differences in milk yield or composition were seen by Robinson et al. (1998) with timothy grass and corn silage when Met or Met and Lys were fed. However, post- experimental predictions of AA delivery to the intestine showed His to be more limiting than Met or Lys.

Improvements have been made in our understanding of post-ruminal protein needs in the dairy cow. However, there is still much to be discovered about the

degradability of feeds, amino acid requirements of the cow, and digestibility of AA post-

ruminally. Processing methods can impact post-ruminal digestibility of AA positively,

by causing greater undegradability of the protein source, and negatively, by reducing

post-ruminal RUP digestibility. Because of the large variability within protein sources,

digestibility of RUP cannot be estimated accurately by feed identity alone; it requires

some form of chemical, enzymatic, or rumen culture analyses. The knowledge of

limiting AA is far from being complete, considering that some diets appear to have AA

limitations other than Met and Lys. Furthering the knowledge of cow AA requirements

and digestibility of RUP will be vitally important in our attempts to properly formulate

cow diets for AA and the associated reduction in N excretion.

AMINO ACID METABOLISM Overview

Supply of AA to the mammary gland is highly dependent on how the AA is metabolized once it is absorbed from the intestine. The NRC (2001) assumed 70% marginal efficiency of conversion of absorbed protein to milk protein until the requirement is met, at which point the marginal efficiency becomes 0%. Hanigan et al.

(1998) summarized six trials in which casein was infused and found the mean marginal efficiency of use of casein AA for milk protein was only 24%. The authors assumed the coefficient was low because the animals met or exceeded their absorbable protein requirement prior to the casein infusion, in which case marginal efficiency should be

zero; or the maintenance requirements are not fixed functions of BW and indigestible

DMI. When examining the diets, the authors found that there were no differences in terms of efficiency of casein use between cows fed to meet or exceed protein requirements and those that were fed insufficient dietary protein. This indicates that the requirement system using two linear equations adopted by the NRC (1989) may not be as appropriate as a nonlinear saturable equation. The greatest errors would generally occur in the substrate range around the transition from one linear equation to another.

Additionally, equations that describe metabolism at the organ level may be better predictors than those at the animal level (Hanigan et al., 1998).

To predict protein and amino acid requirements for milk and protein production in the cow, one must understand the metabolism of amino acids within the body, especially by the mammary gland. Bequette et al. (1998) summarized the state of current knowledge of mammary gland amino acid metabolism in an attempt to determine why use of dietary N for milk protein production appears so inefficient. An overview of their findings follows.

Within a few weeks after calving, protein mass of the mammary gland and splanchnic tissues increases, whereas tissue mass of the carcass, head, and feet decreases.

In early lactation, these changes in protein mass are thought to be due primarily to changes in tissue protein synthesis. In later lactation, tissue depletion and repletion are modulated by proteolytic activity. Insulin is thought to inhibit protein hydrolysis by acting on ubiquitin, a component of the ATP-ubiquitin-dependent proteolytic pathway.

The actions of insulin are augmented by AA supply (Bequette et al., 1998). The dairy

cow can mobilize 90 to 430 g of AA per day from tissue (Komaragiri and Erdman, 1997).

Dairy cows with high genetic merit and greater persistency of milk production may have larger body protein reserves.

Efficiency of use of AA is dependent on non-mammary gland use and the ability of the mammary gland to extract AA from the blood and convert them into milk proteins.

The gastrointestinal tract derives approximately 20 and 80% of its AA requirements from the luminal and arterial supplies, respectively (MacRae et al., 1997), and could influence amount and pattern of AA available to the mammary gland. Utilization of AA-N by the liver is responsible for approximately 50% of the non-mammary utilization of AA entering the duodenum. Reynolds et al. (1994) found that only 36% of the AA escaping the gastrointestinal tract and liver actually was recovered as milk protein. The remainder may be used elsewhere in non-mammary gland tissue, catabolized in the mammary gland, or both. Non-mammary gland tissues may regulate tissue use, or AA catabolism may occur in response to surpluses that the mammary gland does not utilize.

The mammary gland responds differently to infusions of essential versus non- essential AA. Metcalf et al. (1991) found that recovery of jugular infusion of AA in milk protein was greater for essential AA than total AA (36 vs. 22%), indicating no advantage to increasing non-essential AA.

Stage of lactation also influenced responses to AA supply. Efficiency of conversion of infused essential AA (312 g/d) into milk protein was higher in early lactation (14%) than mid-lactation (8%) (Bequette et al., 1998b). Valine, Leu, Ile, Arg,

Lys and Thr are generally extracted in greater amounts by the mammary gland than their

output in milk, whereas uptake of Met, His, Phe and Tyr are less. This has caused

speculation that peptides and proteins may contribute to AA supply for milk protein

synthesis. Bequette et al. (1998b) found that 12% of Lys and 17% of Met from casein

came from vascular sources of non-free AA for goats in late lactation. The mammary

gland also produces constitutive proteins (e.g. structural proteins and enzymes) as well as

milk protein. The turnover of these proteins contributes 42 to 72% of the total protein in

the mammary gland (Bequette et al., 1998).

Stage of gestation affects utilization of AA by the mammary gland. Bell (1995)

reviewed nutrient metabolism during the transition and early lactation period. Late pregnancy protein needs include AA catabolism and tissue deposition by the fetus

(metabolizable AA requirement = 220 g/d) and growth and maintenance of placenta (7 g/d CP). Uterine uptake of AA may be as high as 72% of the maternal supply. These

figures may overestimate requirements because it was calculated from data on uterine

exchange for α-amino N, and Ferrell and Ford (1980) found that uterine net uptake of

individual AA was considerably less than that of α-amino N. Maternal under-nutrition of

AA has little effect on fetal uptake of AA in late-pregnant ewes (Lemons and Schreiner,

1983) because the active placental transport of most AA is largely independent of

changes in maternal blood concentrations. However, deficits in glucose increases

catabolism of AA at the expense of protein synthesis and deposition in the fetus, causing

reduced fetal growth. Specific changes in AA metabolism in this period may include

increased protein synthesis and reduced AA catabolism in the liver and a greater

predisposition to muscle proteolysis.

Within a few days of calving, mammary requirements for AA are two times that

of the gravid uterus during late pregnancy (Bell, 1995). Predicted intakes fall short of the requirements for metabolizable AA. Skeletal muscle is mobilized to augment AA supply.

There is a 25% reduction in muscle fiber diameter in dairy cows immediately after calving (Reid et al, 1980). Net release of AA from skeletal muscle is achieved entirely by suppression of protein synthesis rather than enhancement of protein degradation

(Bosclair, 1993). There is also enhanced synthetic activity and more efficient use of AA in the liver.

Supporting Research

Variations in milk protein responses to casein infusion could be caused in part by experimental error and genetic variation but may also have other factors involved.

One potential factor is energy supply to the animal. Hanigan et al. (1998) conducted a multivariate analysis of nitrogen balance data collected at the Purina Mills Research

Center and found that milk protein production was highly correlated to energy and nitrogen supply, and the variance explained by energy supply was greater than that explained by protein supply. There are also differences in AA profile that is absorbed and the profile that arrives at the mammary gland. An average of 30% of total essential

AA may be lost across the portal drained viscera, and an additional 20% is lost to liver metabolism. These losses are not constant across AA. Items that need to be addressed in future models include the apparent decline in efficiencies of absorbed protein conversion to milk protein as absorbed protein supply is increased, the effects of energy on absorbed

protein efficiency, and the effects of varying supplies of individual AA at a given absorbable protein supply (Hanigan et al., 1998).

Blouin et al. (2002) related differences in MP supply with changes in net AA release into the portal vein and removal by the liver. These responses were then related to post-splanchnic nutrient supply and milk protein output. Two diets with differing ruminal protein degradability were used to provide differing levels of MP. Milk and protein yield were greater with increased MP. Recovery of the increase in MP supply in milk protein output averaged 22.5%. Transfer of AA from portal net flux to milk protein had transfer coefficients that varied from a low of 0.38 for Phe to 0.68 for Met. When splanchnic net flux, which includes liver removal or production, was used for comparison, these values ranged from 0.42 for Ile to 1.80 for Met, reflecting the differences in hepatic extraction. For His and Phe, the amount released from the liver was approximately equal to the amount needed for milk protein synthesis. The authors believed Met was also released in amounts necessary for milk protein synthesis, but the variance of the data was too high to prove this. All other AA had supplies beyond the liver that exceeded the amounts necessary for milk protein production, indicating catabolism or utilization of these in non-hepatic tissues. The authors concluded that adequate prediction of the transfer of duodenal protein flow into milk protein requires better understanding of metabolism of individual AA across splanchnic tissue and how this might be altered.

Net uptake of Leu by the mammary gland almost always exceeds its secretion in milk protein. Bequette et al. (1996) determined the importance of Leu oxidation in the

mammary gland by limiting availability of Leu relative to the supply of other AA. If Leu oxidation was crucial, the authors expected substantial rates of oxidation to continue despite limited availability, or milk protein would decline. Decreasing Leu relative to the other AA caused an increase in Leu extraction from blood and plasma by the mammary gland. Blood flow tended (P = 0.08) to be higher with Leu imbalance. Oxidation of Leu in the mammary gland was reduced, but protein yield was not affected. The authors concluded that AA oxidation may be linked to other requirements not identified in the current study, or that it is a passive response to oversupply of AA. If the AA that are taken up in excess do not serve some anabolic or regulatory role, it would appear to be an inefficient process to take up more than is necessary for milk protein production.

Bequette et al. (2000) examined the response of the mammary gland to an artificially induced His deficiency in goats. Lactating goats received diets that met 77% of the MP requirements for milk production. This diet was supplemented with abomasal infusions of AA with or without His. The authors found that the mammary gland responded to a deficit of His by increasing blood flow by approximately 33% and decreasing milk protein. The authors theorized that His deficiency might increase blood flow to the mammary gland by decreasing level of histamine, which is formed by decarboxylation of His. Histamine has been shown to have vaso-constrictive actions on the mammary vasculature (Jakobsen et al., 1994). However, this would not necessarily explain the results of a prior experiment in which Leu deficiency increased mammary blood flow (Bequette et al., 1996). There was an increase of 43 fold of the mammary gland’s ability to remove His from the blood, indicating that the transporter for His

uptake is capable of functioning at a higher efficiency than usually seen and is probably not normally the rate-limiting step. Uptake of other AA by the mammary gland decreased during His deficiency by two to three fold. The authors concluded that the udder responds to AA limitation by up-regulating the processes that govern extraction of that AA while down-regulating those for other AA, and that transport capacity is closely linked with matching needs and supply.

Provision of AA to the mammary gland does not guarantee that the AA will be used for milk protein synthesis. Bequette et al. (1997) compared unidirectional fluxes of

AA across the mammary gland to their secretion in milk protein. The values were standardized against Lys, which was assumed to be first-limiting for the diets. Arg, Pro,

Phe, Tyr, and probably His had fewer metabolic requirements for non-milk protein synthesis and oxidation than did Lys. The fluxes of the branch chain amino acids

(BCAA) were greater than their output in milk protein. All of the AA might have additional metabolic fluxes, which, if not crucial to milk protein synthesis, could become competitive with milk synthesis, particularly in limiting conditions (e.g. negative nutrient balance, reduced DMI, limiting AA).

Increasing the efficiency of milk protein synthesis is a complex process involving much more than supplementation of rumen-protected amino acids. Stage of lactation, provision of energy, and use of AA for non-mammary needs should be considered.

Specific AA behave differently in the body and mammary gland, and they must be considered independently to truly determine the AA profile available to the mammary

gland for protein synthesis. Future models need to include whole body use of individual

AA in order to accurately predict milk protein responses to supplemental RUP and AA.

2-HYDROXY-4-(METHYLTHIO) BUTANOIC ACID

Introduction

In an effort to meet the Met needs of dairy cattle, the efficacy of 2-hydroxy-4-

(methylthio) butanoic acid (HMB) as a Met source has been examined. This molecule, in which a hydroxyl group replaces the amino group of Met, serves as a synthetic source of

Met in non-ruminant and possibly ruminant diets. Originally, it was marketed as the calcium salt of HMB, which consists of 2 mol of HMB bound to 1 mol of Ca, but production of the salt ceased in 1994. The salt form, usually referred to as methionine hydroxy analog (MHA), has been more extensively studied in ruminants. The liquid form was introduced in 1979 as an easier handling and mixing source of HMB (Schwab,

1998). Once HMB is absorbed through the intestinal tract, it can be converted to Met

(Figure 2.2).

H H H (1)1 (3) - - - + OOC— C— OH OOC C O OOC C NH3 (2) AA α-keto AA CH2 CH2 CH2

CH2 CH2 CH2

S S S

CH3 CH3 CH3 2-hydroxy-4 (methylthio) α-keto methionine methionine butanoic acid

1 (1) D-2-hydroxy-acid dehydrogenase (2) L-2-hydroxy oxidase (3) transaminase 2 Adapted from Schwab, 1998.

Figure 2.2. Conversion of HMB to Methionine2

The D- and L-isomers are converted to the α-keto analog of Met by two separate

enzymes: L-2-hydroxy oxidase, found in liver and kidney, and D-2-hydroxy acid

dehydrogenase, found in many tissues (Dibner and Knight, 1984). The α-keto analog is

then transaminated to L-Met, with isoleucine, leucine and valine being the principle

amino donors in chickens, and glutamine in rats (Baker, 1994).

Metabolism of HMB

There has been limited research examining the metabolism of HMB in ruminants.

In chickens, 87% of HMB was removed by the liver (Bottje et al., 1998); however, only

21-37% was extracted by the liver in sheep (Wester et al., 2000a). This indicates that

metabolism of HMB in ruminants probably differs from that in non-ruminants. Wester et

al. (2000b) found that HMB infused into the abomasum of sheep is absorbed rapidly,

with 88% appearing in the portal vein. Twenty-one to 37% of the absorbed HMB was

subsequently removed by the liver in cows and sheep (Wester et al., 2000b; Lobley,

2001). Once blood left the liver, arterial concentration of HMB increased in proportion to the dose, and Met released by the liver was decreased. At higher doses, some tissue

Met was released into the blood as well. Arterial Met and whole body flux of Met were increased by HMB (Wester et al., 2000a).

Lobley et al. (2001) reported that dairy cows infused intravenously with 1.5 g/h of

HMB increased whole body Met flux by 30%, with at least 39% of the infused HMB converted to Met. The authors concluded that most of the Met from HMB is kept where it is created and used for protein synthesis. For the dairy cows receiving 1.5 g HMB/h infusion, 20% of milk protein Met was derived from HMB.

They suggested that most of the absorbed HMB reached peripheral tissues and reduced the need for dietary Met, as shown by increased uptake of Met by the liver.

13 2 To determine the locations of HMB conversion to Met, C-HMB and H3-Met were infused into the abomasum and mesenteric vein, respectively (Wester et al., 2000b).

2 The H3-Met responded as Met would normally in response to general Met metabolism, whereas an increase in 13C-Met would indicate conversion of labeled HMB. The ratio of

the two isotopes gave an estimation of production of Met from HMB, while considering

the normal flow into and out of the cell of Met. The authors found that the ratio of

13 2 C: H3 was higher in plasma from the artery than the portal vein or hepatic veins, indicating non-liver tissues are major sites of Met synthesis. The digestive tract and liver had some synthesis, but were not the main contributors. In a trial conducted to determine which tissues were most important, animals were euthanized after labeled HMB infusion, and 15 tissues were removed. These tissues were analyzed for isotopes, and their ratio was compared to that in arterial plasma (Lobley et al., 2001). The authors found that almost all tissues converted HMB to Met, with peripheral tissues (e.g., brain, lung skin, etc.) having the lowest rate of conversion and the liver (14%) and kidney (22% of Met flow through cell) having highest rates. Digestive tract tissues were intermediate at 6 to

12% of the flow of Met through the cell. Only the kidney had a higher intracellular ratio than portal or hepatic veins, indicating that it is the major contributor to the elevated ratio found in arterial plasma. The kidney returns most of this Met to the blood, whereas the liver retains most of its Met.

Intake, Milk Yield and Composition effects of HMB

Decreased palatability of rations has been a concern with feeding of HMB.

Palatability and toxicity effects of HMB have been reported, but decreased intake due to

levels of 40 g/d was overcome when cows were allowed three days for adjustment to the

diet (Higginbotham et al, 1987). Toxicity of HMB, determined by relative feed

consumption, has been approximated at one percent of dietary DM (Satter et al., 1975).

Experimentation with HMB in the 1970s and 1980s concentrated primarily on its

effect on milk fat production. Many studies have shown increases in milk fat production

(Holter et al, 1972; Huber et al., 1984; Patton et al., 1970b) and percentage (Huber et al.,

1984; Lundquist et al., 1983), although some reported no effect (Hutjens and Schultz,

1971; Whiting et al., 1972; Stokes et al., 1981). Possible explanations for the lack of

response in these trials include use of cows that were not in early lactation (Whiting et

al.,1972), when Met needs may be greater, and very low CP in the diet (Stokes et al.,

1981), which may have caused other AA to be more limiting than Met. In the first

experiment of Hutjens et al. (1971), the concentrate to roughage ratio was held at three to

one to induce a milk fat depression of 30 %. The authors believed that supplementation

of Met via HMB could help prevent the milk fat depression commonly seen with these

types of diets. However, they found that 24 g/d HMB was ineffective at correcting or

preventing the milk fat depression due to heavy grain feeding. Ray et al. (1983)

determined that HMB supplementation of a low fiber diet increased milk fat percentage

only for cows that initially had less than a 20 % decrease in milk fat percentage. Early

data showed little effect on milk yields (Bhargava et al.; 1977, Holter et al., 1972; Huber et al., 1984), although a few trials showed increases in milk yield with HMB (Clark and

Rakes, 1982; Patton et al., 1970b). Polan et al. (1970b) determined that maximal milk yield was obtained at 25 g/d of HMB when a range of zero to 80 g/d was fed. Fat content

increased with increasing intake of analog at all concentrations fed. Milk protein values were often not reported in the earlier research. Of those that did, most found no effect

(Hansen et al., 1991; Hutjens et al., 1971; Stokes et al., 1981). Bhargava et al. (1977) found a milk protein concentration increase in cows fed HMB from 117 to 256 DIM, but none was seen in early lactation cows. The lack of response in milk protein yield and concentration and milk yield may indicate that HMB is not escaping rumen microbial catabolism in significant concentrations to be utilized post-ruminally, because a milk protein response is the most consistent short-term response to Met infusion (Schwab,

1998). The changes in fat production and percentage may be due to a ruminal effect.

Effects of HMB on fat metabolism

Effects on fat composition of milk are common with HMB supplementation.

Patton et al. (1970b) found that cows receiving MHA at 40 or 80 g/d had greater concentrations of blood serum lipids, decreased concentration of free fatty acids in the rumen, and increased ruminal concentrations of an unidentified polar lipid and lecithin.

They concluded that MHA influenced the lipid metabolism of the rumen microorganisms, possibly due to an increase in the protozoal biomass. The methyl group of Met (and/or

HMB) could be utilized in the production of lecithin. Lecithin is commonly found in protozoa, but not in bacteria. The increase in blood lipids may be a result of the rumen effects, or due to the effect of methionine on blood serum lipoprotein integrity. Polan et al. (1970b) found lower serum concentrations of free fatty acids, cholesterol, and triglycerides with MHA at 0.2, 0.4 and 0.8% of concentrate, possibly due to a post-

ruminal provision of Met, an important methyl donor for the fat mobilization process.

The β-lipoprotein fraction, which contains cholesterol, free fatty acids, and triglycerides, contributes to milk fat synthesis. A corresponding increase in fat content of milk was

seen with HMB supplementation. Blood samples from the coccygeal vein of MHA

supplemented cows had 10% more triglycerides than that of control cows (Huber et al.,

1984). Arterio-venous differences across the mammary gland of triglycerides and

lipoproteins of blood serum indicate that increases in milk fat could have resulted from greater uptake of preformed fat by the udder. Pullen et al. (1989) used 14C labeled

palmitic acid to label plasma fatty acid and triglyceride in cows fed 0 or 30 g/d of MHA.

They found no effects of the analog on lipid metabolism. However, increases in milk fat

commonly seen with HMB were not seen in this trial, indicating that the diet may have

been limiting for something other than Met in the rumen.

Degradability of HMB

Elucidation of the mechanism behind the changes in milk composition seen with

HMB is partially dependent on determination of the rumen disappearance of HMB.

Disappearance of HMB from the rumen can occur through degradation in the rumen,

absorption through the tissue, or passage out through the omasum. McCollum et al.

(2000) used omasal and ruminal epithelium from eight lambs to determine HMB

absorption. Passage of HMB was determined using dl-[5-14C]-HMB. Movement of 14C

into serosal buffer was used to calculate an apparent accumulation of HMB. This could

have included labeled 2-keto-4- (methylthio) butanoic acid and Met made from labeled

HMB during absorption in the tissue, as well as HMB. They found that transport

mechanisms could be saturated in the omasum, indicated by a quadratic pattern of absorption as HMB level increased, whereas ruminal epithelium absorbed HMB linearly.

However, after 60 min of incubation with HMB, seven times more HMB had crossed the omasal epithelium than the ruminal epithelium. This research indicates that HMB can be absorbed before the intestine, but does not indicate to what degree. Emery (1971) found that 76% of a 50-g dose of HMB found at 2 h remained in the rumen of cannulated cows at 4 h. However, much of the initial dose may have disappeared by 2 h. Lambs given

HMB showed no changes in blood Met with oral supplementation, whereas abomasal supplementation increased blood Met, indicating that little HMB bypassed the rumen to be converted to Met (Papas et al., 1974). Jones et al. (1988) found that less than one percent of an approximately 15-g dose of either the Ca salt or the acid form of MHA was found in duodenal contents taken and composited over the last 3 d of a 14-d period.

More recent attempts to determine passage of HMB have utilized in vitro methods of bacterial culturing. Windschitl and Stern (1988) found that addition of MHA at 0.33% of dietary DM did not change the passage of Met from the fermenter. Effluent of fermenters provided with MHA was analyzed for MHA and this was added to Met for the final value. Patterson and Kung (1988) used an in vitro system utilizing Erlenmeyer flasks to determine degradability of dl-Met, MHA, and MHA derivatives over a 48-h period. Methionine treatments were calculated to be equivalent to a daily intake of 25 to

30 g of Met, assuming a cow with a 60-L rumen volume. Seventy to 80% of MHA remained after 12 h, whereas less than 10% of Met remained. In this type of in vitro

system, some type of end product inhibition may have decreased the rate of MHA degradation, but not that of Met. Koenig et al. (1999) evaluated the degradability of

HMB in two studies utilizing cannulated cows. The first study pulse-dosed 0, 20, 60 or

90 g of HMB to late-lactation cows in a 4 X 4 Latin square design. Duodenal samples taken at 16, 20, and 24 h after dosing did not contain any detectible HMB. In the second study, cows were pulse dosed with 90 g of HMB. Blood, ruminal, and duodenal sampling began at 1 h post-dosing. Rate of passage of HMB from the rumen was equated to the rate of passage of the liquid marker, Cr-EDTA. The duodenal concentration of the analog peaked at 3 h. Methionine in the blood peaked at 6 h with a concentration three times pre-dose concentration. Using fractional rate constants, the authors determined that approximately 50% of HMB escaped ruminal degradation. Some possible flaws in this experiment include the pulse dosing of HMB at greater than recommended dosages and the use of a small number of animals, four, in a short-term experiment with no negative control.

Vázquez-Anon et al. (2001) used a dual effluent continuous culture system to investigate rate of passage of HMB in two studies using different solids retention times and liquid dilution rates. The HMB was dosed twice daily with the diet. In the first study, doses were higher than that usually fed to cows (0.10% is equivalent to 25 g/d with

25 kg DMI), with the lowest dose of HMB at 0.20% of DM and increasing to 1.43% of

DM. Liquid dilution rate was maintained at 0.11 h-1. They found that dosage did not

affect escape from fermenters, which averaged 21.6%. The second study, a 2 X 2 X 2

factorial with main effects of liquid dilution rate (0.15 h-1 and 0.125 h-1) and solids

retention time (16 h and 25 h), had treatments of 0 and 0.88% HMB. Rate of

disappearance and degradation of HMB in the fermenters were not affected by solids

retention time. The percentage of HMB escaping ruminal degradation increased during

low solids retention time and high liquid dilution rate (43.2 vs. 37.8%). The authors

suggested that the differences in undegradability in the first and second studies were due

to differences in liquid dilution rate. Koenig et al. (2002) examined the ruminal escape

value of HMB in four ruminally and duodenally cannulated, lactating dairy cows in a 4 X

4 Latin square design. Doses of 0, 25, or 50 g/d of HMB or 51.2 g of the calcium salt of

HMB were fed in 0.5 kg of coarsely ground barley grain once daily, with uneaten

portions placed directly in the rumen after 20 min. For the 7 d prior to the experiment, all

cows received 56.7 g/d of liquid HMB to adjust the rumen microflora. Cobalt was used

as a liquid marker. The proportion of HMB escaping the rumen was not different across

doses and was estimated at approximately 39.5%. Using the difference between passage

from the rumen and appearance in the duodenum, the authors determined the omasal

absorption of HMB to be approximately 17.6% of the dose. However, appearance of

HMB at the duodenum was determined using the trapezoidal summation method, which

may overestimate the passage. The authors also used cobalt as a liquid flow marker, which may overestimate liquid flow and, therefore, passage of HMB from the rumen

(J.L. Firkins, personal communication). The errors in these two measurements would be compounded in the calculation of omasal absorption.

The variability seen in degradability estimates, ranging from 50 to 99%, could be due to many factors. Continuous culture methods are experimental models of the rumen,

but most do not contain either a rumen mat or protozoa, which will be discussed in the next section. Passage rates can vary due to cow, stage of lactation, and DMI. Fluid markers may not be accurate when used to determine passage of liquid, and equating

HMB flow to liquid flow could be overestimating the undegradability of HMB. Pulse dosing raises ruminal concentrations far above that observed when cows are fed a diet at the suggested dose of 0.10 to 0.11% of DM. These high concentrations may saturate microbial enzymatic pathways or transport mechanisms across the microbial membranes.

HMB effects on microorganisms and digestibility

Although researchers disagree on the ruminal degradability of HMB, all agree that it is partially available in the rumen. Belasco (1980) found that cows receiving 14C labeled MHA had radiolabeled Met in rumen fluid, indicating transformation of HMB to

Met in the rumen. Addition of Met via HMB could cause changes in the rumen microbial populations by selecting for microorganisms that can use HMB more efficiently than substrates available without it, affect VFA profile through changes in biochemical pathways or microbial profile, and increase passage of microbial protein and fat from the rumen with increased growth of some microorganisms. Once transformed, Met can be incorporated directly into microbial protein or metabolized to 2-oxobutanoate, which can be converted to propionate by rumen protozoa (Onodera, 1993). Methionine may also be used by some protozoa for production of phosphatidyl choline (Williams, 1992), a component of membrane lipids (Lehninger et al., 1993).

Alterations in VFA profile or differences in digestibility of nutrients can indicate changes in microbial populations. Digestibilities of ADF (Polan et al., 1970a), DM

(Polan et al., 1970a; Hoover et al., 1999), crude fiber (Holter et al., 1972) and CP

(Hoover et al., 1999) have been increased in some studies, while others have shown no effects (Windschitl and Stern, 1988). Effects on VFA profile have not been consistent.

Some have seen increases in acetate concentration with decreases in propionate

(Lundquist et al., 1983; Ray et al, 1983), whereas others have seen no changes (Lundquist et al., 1985; Windschitl et al., 1988; Vázquez-Anon et al., 2001). Reported values for ruminal concentration of branched chain VFAs have shown no differences with supplementation of HMB (Lundquist et al, 1983; Lundquist et al., 1985; Hoover et al.

1999).

Other evidence of changes in the microbial ecosystem from HMB may include increases in bacterial protein synthesis. Windschitl and Stern (1988) found no effect on efficiency of bacterial protein synthesis with MHA at 0.33 % of diet DM in continuous culture. Hoover et al. (1999) reported an increase in microbial N in continuous culture fermenters supplemented with 0.11 % HMB, although non-ammonia, non-microbial N decreased due to increased protein digestibility. Non-ammonia N flow was increased due to the increased microbial protein production. However, microbial N and non-ammonia

N flows were not affected by the addition of 0.22% HMB to the diet. Vázquez-Añón et al. (2001) determined that bacterial N in effluent of continuous culture fermenters was improved with the addition of HMB at 0.20 and 0.77 % of the diet, but not at 1.43 %.

Bacterial N as a percent of NAN also increased at these concentrations. Effluent non-

ammonia N was not changed by supplementation. Differences in passage rates, method

of dosing (pulse vs. steady state) and level of supplementation influence the effects of

HMB on microbial populations in continuous culture. Changes in passage rates influence

bacterial efficiency, and pulse dosing of large concentrations of HMB could be toxic to

the microorganisms. Provision of HMB at concentrations greater than recommended

may not adequately represent what would occur under normal feeding conditions.

Since the late 1960s researchers have attempted to determine the microbial effects

of HMB that may cause the changes seen with supplementation. Patton et al. (1968)

showed an increase of protozoa in vitro in rumen fluid under conditions of prolonged

dietary supplementation of MHA. In a follow-up study, Patton et al. (1970b) found that

feeding of MHA decreased free fatty acid concentration in rumen fluid, promoted the

formation of an unidentified polar lipid, and caused a relative increase in lecithin

(phosphatidylcholine), a compound found in protozoa but not usually in bacteria. Gil et

al. (1973a) reported that HMB increased rate and quantitiy of bacterial N production from

cellulose in vitro. In a second study, Gil et al. (1973b) saw no effect of HMB on amino

acid composition of bacterial protein, but some changes in amino acid composition

during growth on glucose indicated a shift in bacterial species. The analog in excess of

bacterial requirement did not support additional growth after exhaustion of glucose,

indicating that growth stimulation by MHA was not due to it being used as a source of

energy. Maximum effects on growth occurred at 0.2 mg/ml of medium. In a trial using sheep, de Vuyst et al. (1975) determined that supplementation of the basal ration with

MHA increased protozoal numbers, especially Entodinium species. There were five

times the number of ciliates in rumen fluid of sheep fed 1.2 % MHA over a control. All

MHA-treated sheep had higher total protozoal numbers than the control sheep.

Lundquist et al. (1985) did not find an increase in protozoa with 0.18 % MHA in cows, although there was an increase when dl-Met was fed. However, the MHA supplementation was lower than that fed by de Vuyst et al. (1975).

More recent research has returned to in vitro methods to determine the microbial effects of MHA. Patterson and Kung (1988) dosed 14C-carboxyl labeled Met, MHA and

HMB in batch culture of whole rumen contents. Ruminal contents were separated by centrifugation, and antibiotics were added to the protozoal-rich sediment. Incorporation of the label tended to be higher for the bacterial fraction of rumen contents as opposed to the protozoal fraction. However, the authors did think that protozoa were influenced, because protozoa may be utilizing HMB as a methyl donor for choline synthesis or other reactions that do not require the whole amino acid to be incorporated.

Although there is much information on production and digestibility effects of

HMB, the mechanism of action has yet to be elucidated. This is due partially to the unknown rumen degradability of HMB. Mechanism of action could include stimulation of microbial growth in the rumen, provision of methyl groups, or provision of Met to the cow post-ruminally. The extent of rumen escape must be determined to effectively use

HMB as a Met source in formulating dairy rations. Discovery of the mechanism of action in the rumen would allow the feeding of HMB in situations where it would be advantageous instead of discovering such situations in a “trial and error” method.

OMASAL SAMPLING

In order to determine the degradability of HMB, samples must be obtained post- ruminally. Because HMB can be absorbed through the omasum (McCollum et al., 2000),

I assumed that samples should be obtained before the duodenum. An omasal sampling technique has been developed by researchers in Wisconsin (Huhtanen, 1997) and modified in Finland (Ahvenjärvi et al., 2000). Omasal contents are extracted in sequence with reticular contractions. Therefore, omasal backflow is assumed to be minimal and there should be minimal time for absorption of HMB from the omasum.

In the original procedure, Huhtanen et al. (1997) described an omasal sampling technique for assessing fermentative digestion in dairy cows. This technique involves a device inserted into the omasum via a ruminal cannula, a tube connecting the device to the ruminal cannula opening, and a compressor/vacuum pump. A cycle of alternating vacuum and pressure applied to the collecting tube allowed digesta to flow to a collection vessel, with pressure at half of the vacuum level clearing away any accumulated digesta at the omasal end. The authors evaluated the effects of the sampling system on digestive activity, animal performance, and animal behavior. The experiment consisted of two 21- d periods. The sampling device was inserted at the beginning of each period and remained in the cows for 21 d. A control group did not have the device. Omasal digesta flow was calculated using Cr-mordant and Yb as solid-phase markers and Co-EDTA as a liquid-phase marker. Daily dry matter intake was decreased by 2.2 kg (SE = 0.46 kg) overall for cows with the sampler. However, the decrease in intake was higher during the sampling period, indicating that the device itself could not explain all of the decrease.

The authors speculated that the decrease during sampling could have been due to 1) an

imbalance in sodium from the removal of 6 L of fluid per day, or 2) disruption of the cows’ normal routine from repeated sampling. Milk yields were only slightly decreased, with a significant decrease in milk protein production. There were no differences in total tract digestibility of nutrients between the groups, regardless of marker used. There were some differences in VFA profile, possibly due to the differences in DMI. Passage rate of

La-alfalfa and Sm-corn indicated a decrease in passage rate with the experimental group, but this could be related to reduced DMI. Similar particle size distribution in ruminal digesta and fecal DM indicates that the sampling device had no specific effect on the retention or flow of feed particles. Overall the authors concluded that this technique might prove useful for partitioning digestion between the forestomach and the lower gastrointestinal tract, although the composition of omasal digesta samples was somewhat biased toward smaller particles. Use of a sampling tube with a wider inside diameter (>

0.95 cm id) may help.

Ahvenjarvi et al. (2000) compared digestibility determined using omasal or duodenal sampling using four diets. They found an increase in flow of OM to the duodenum, with a decrease in flow of ADF and NDF compared with the omasal flow.

They suggested that this indicated digestion of fiber in the omasum. This post-ruminal fiber digestion was derived from an increase in digestibility of ADF and NDF when using whole stomach values compared with digestibility in the reticulo-rumen alone (using omasal values). Purine:N ratios were different in rumen, omasum and duodenum, with bacterial purine content decreasing at each stage of passage through the tract. This

resulted in differences in microbial and dietary NAN flow and true rumen N digestibility.

Microbial NAN was lower and dietary NAN was higher in the omasum than the duodenum. Similar efficiencies of microbial N synthesis were estimated from omasal and duodenal sampling methods. However, the comparison used only the purine:N ratio determined in the omasum for the calculations on both omasal and duodenal flow, which could cause some inaccuracy. The flows of ADF, NDF and OM in the omasum and duodenum were highly correlated, with coefficients of correlation all above 0.90.

Comparison of omasal canal and duodenal digesta flows indicated absorption of minerals from the omasum and OM secretion in the abomasum. Differences in N fraction flows entering the omasal canal and duodenum were non-significant.

Substantially less endogenous N is secreted into the rumen than into the abomasum and duodenum (Orskov et al., 1986). This, combined with the fact that microbes are digested in the abomasum, makes omasal sampling preferable to duodenal sampling when determining N flows from the rumen. The flows can be separated into particle- and liquid-associated bacteria, protozoa, and dietary N. Punia et al. (1988) used an omasal sampling technique to determine protozoal and bacterial N synthesis in

Friesian heifers fed low quality diets with or without urea. Omasal samples (90 ml) were obtained by suction through a 9-mm ID tube passed through the rumen cannula.

Protozoa from the rumen and omasum were fixed with formalin for counting.

Sedimentation procedures were used to determine DM contribution of protozoa. Particle- associated protozoa would be removed with feed particles when filtered through cheesecloth, and could not be recovered by sedimentation. Total microbial protein

synthesis was estimated using a continuous infusion of 35S, and 2,6-diaminopimelic acid

(DAPA) analysis. Overall counts of protozoa in the omasum were 44 % of rumen counts.

Nitrogen concentration of protozoa was less in omasal protozoa than in rumen protozoa,

possibly due to distribution of sizes passing to the omasum compared with that in the

rumen. Protozoal N flow accounted for 20 to 43 % of total microbial N flow to the

omasum. Urea decreased the proportion from protozoa, most likely due to providing

bacteria with a readily available source of NH3 for growth. Microorganisms, both bacterial and protozoa, had lower N concentration in diets without urea, possibly due to an increase in storage carbohydrates and/or lipid content in these microbes. This emphasizes the fact that microbial DM is not constant in its composition. Total N flows

were similar between the omasum and duodenum (within 9%). Non-ammonia N flow

was 6.6% greater at the duodenum than the omasum, most likely due to endogenous

secretions of N in the abomasum. The apparent digestibility of NAN was also

approximately 11% greater using the duodenal flow. The authors noted a large flow of

endogenous minerals into the rumen and substantial absorption between the rumen and

abomasum on all treatments.

Using omasal protozoa counts, Punia and Leibholz (1994) were able to quantify

the amount of microbial N leaving the rumen in the form of bacteria and protozoa. The

authors determined the effects of three concentrations of low quality kikuyu grass hay on

flow of protozoal N to the omasum in steers. Omasal sampling and treatment of

protozoal samples were similar to that of Punia et al. (1988). Total microbial N was determined using 15N. Protozoal N accounted for 26 to 29% of total microbial N leaving

the rumen, and 17 to 21% of total protein N. The highest concentration of kikuyu in the diet caused a significant increase in protozoal N (g/d) flow to the omasum, indicating that diet can influence the passage of protozoa out of the rumen. The proportion of microbial

N from protozoa remained constant across diets due to a concurrent increase in bacterial

N. Approximately 49% of the bacterial N and 38% of the protozoal N was obtained directly or indirectly from rumen NH3.

Ahvenjärvi et al. (2002) used omasal sampling of lactating Ayrshire cows to fractionate microbial N into liquid- and particle-associated bacteria, and protozoa.

Microbial flow was quantified using 15N, with enrichment of 15N determined for protozoa and liquid- and solid-associated bacteria. Diets were based on grass-red clover silage with supplementation of energy (barley) and/or protein (rapeseed meal). Omasal sampling was conducted using the method of Huhtanen et al. (1997) with modifications of Ahvenjärvi et al. (2001). The authors found that provision of readily digestible carbohydrates did not improve microbial capture of ruminal ammonia, but supplementation of barley increased microbial N flow, and this increase was attributable to liquid associated bacteria and protozoa.

These experiments indicated that omasal sampling can be an effective means of determining passage of bacteria and protozoa and digestibility of nutrients. The technique is not detrimental to the cow and can be used with minimal disruption of the routine of the cows.

SUMMARY

Dairy nutrition professionals are worried about the future of nitrogen regulations in the United States, and the potential effects on the dairy industry. The proactive approach to this problem involves more accurately predicting post-ruminal protein requirements and increasing our ability to provide for these needs without providing excess N that will appear in the excrement. Increasing evidence has shown that a proper

AA balance is required to maintain milk production and milk composition at the levels common in today’s dairy cows. However, proper AA balance is not always known but must be estimated by data from prior research. More information on rumen undegradability, intestinal digestibility, and supplementation of AA is necessary to optimally provide N in diets for maximal milk production, while minimizing N excretion.

2-hydroxy-4-(methylthio) butanoic acid has the potential to provide Met to the cow and the microorganisms in the rumen. Nitrogen efficiency could be improved through increased microbial flow providing AA in a pattern that closely mimics milk protein and phosphatidylcholine post-ruminally, or provision of undegradable Met that can be used directly for milk protein synthesis. Determination of the mechanism of HMB activity allows for more efficient use of HMB as a Met source with the potential to decrease N excretion.

CHAPTER 3

SUPPLEMENTATION OF MET AND SELECTION OF HIGHLY DIGESTIBLE

RUMEN UNDEGRADABLE PROTEIN TO IMPROVE NITROGEN EFFICIENCY

FOR MILK PRODUCTION

Abstract

Metabolizable protein (MP) supply and AA balance was manipulated through selection of highly digestible RUP sources and Met supplementation. Effects on production efficiency and N utilization of lactating dairy cows were determined. Thirty-two multiparous (647 kg) and 28 primiparous (550 kg) Holstein cows were assigned during the fourth week of lactation to one of four dietary treatments. Treatments were: 1) 18.3

% crude protein (CP) with low estimated intestinal digestibility of RUP (HiCP-

LoDRUP); 2) 18.3 % CP with high digestibility RUP (HiCP-HiDRUP); 3) 16.9 % CP with high digestibility RUP (LoCP-HiDRUP); and 4) 17.0 % CP with high digestibility

RUP and supplemental Met (LoCP-HiDRUP + Met). Diets were balanced to have equal concentrations of NEL, ADF, NDF, and ash. Milk yields (40.8, 46.2, 42.9, 46.6 kg/d),

protein percentage (2.95, 2.98, 2.99, 3.09 %) and fat percentage (3.42, 3.64, 3.66, 3.73 %)

are reported here for HiCP-LoDRUP, HiCP-HiDRUP, LoCP-HiDRUP and LoCP-

HiDRUP + Met, respectively. Milk urea N and BUN decreased when feeding a lower CP diet. Efficiency of use of N for milk protein production was higher when feeding higher digestibility RUP, especially with the LoCP-HiDRUP + Met diet. A digestibility study followed the production trial, with six cows per treatment group continuing on the same treatment for an additional week. The experimental periods were 5d long, with 1d of adjustment and 4d of total collection of urine and feces. Dry matter intake, milk production, milk protein production and N digestibility were not significantly different among treatments during the collection trial, whereas N intake and N absorbed increased with the higher CP diets. The quantity of nitrogen in feces did not change with diet, but quantity of N in urine decreased in the low CP diets. Milk N as a percentage of intake N and milk N as a percentage of N absorbed showed a trend toward increasing as CP concentration in the diet decreased. The supplementation of Met did not improve the efficiency of N utilization during the digestibility study, in contrast to what was estimated during the production trial. Supplementing the highly digestible RUP source with rumen available and rumen escape sources of Met resulted in maximal milk and protein production and maximum N efficiency by cows during the production trial, indicating that post-ruminal digestibility of RUP and AA balance can be more important than total

RUP supplementation.

Abbreviation key: LoCP = 17.0 % CP concentration, LoDRUP = supplemental RUP not selected for digestibility, HiCP = 18.3 % CP concentration, HiDRUP = supplemental

RUP selected for high post-ruminal digestibility, HMB = 2-hydroxy-4-(methylthio)-

butanoic acid, MP = metabolizable protein, MUN = milk urea nitrogen, PUN = plasma

urea nitrogen, WOT = week of trial.

Introduction

It has been estimated that 90 % of the U.S. ammonia emissions come from agriculture, and 90 % of those emissions are due to manure from livestock enterprises

(Meisinger and Jokela, 2000). Current manure practices may be promoting ammonia enrichment of streams, estuaries, and coastal waters, contributing to eutrophication in aquatic and low-N input ecosystems and emission in air, resulting in acid rain.

Overfeeding of CP to obtain maximum milk yields is common in the dairy industry today, contributing to higher concentration of N in the waste (St-Pierre and Thraen,

1999).

Efficiency of utilization could be improved by increasing post-ruminal digestibility and/or providing a pattern of absorbed AA that more closely matches the AA requirements for milk synthesis. O’Mara et al. (1997) found that fishmeal had a high rate of disappearance of total AA in the intestine, 97 %, whereas corn gluten feed was much less digestible at 75.6 %. Wright et al. (1998) provided a ruminally protected supplement that contained methionine, lysine, phenylalanine, histidine and threonine in a ratio similar to that of bovine caseins. Milk production and protein production responded positively in a linear fashion to increasing levels of RUP when supplemented with AA.

Methionine and lysine have been reported to be limiting AA for milk yield and protein production (Schwab et al., 1992; Maas et al, 1998; Bach et al., 2000). Due to the

high cost of production of synthetic rumen protected lysine, blood meal or some other

feed high in rumen undegradable lysine is generally used as a source of metabolizable

lysine, whereas synthetic rumen-protected methionine can be made relatively cheaply.

Supplementation of methionine, or methionine and lysine, post-ruminally has had

positive effects on milk production and milk protein concentration (Dinn et al., 1998;

Varvikko et al., 1999, Armentano et al., 1997).

2-hydroxy-4-(methylthio)-butanoic acid (HMB), is a common source of Met

(Schwab, 1998) that varies in estimated rumen degradability from 99% (Jones et al,

1988) to as low as 21 to 50 % (Vazquez-Anon, 2001; Koenig et al, 1999). The most

consistent response to feeding HMB has been an increase in milk fat percentage (Holter

et al., 1972, Huber et al., 1984, Patton et al. 1970b), with some increases in milk yield

(Patton et al., 1970b, Polan et al., 1970b). It is important to note that most research done prior to 1988 used the Ca salt of HMB, which was not completely water-soluble. The liquid form of HMB currently in use is completely water-soluble.

We hypothesized that milk production and composition could be maintained and dietary CP decreased to improve efficiency of N utilization through selection of high digestibility RUP and supplementation of Met. Experiment 1 was designed to assess milk production responses to changes in post-ruminal RUP digestibility, metabolizable protein (MP) supply, and methionine supplementation. A digestibility trial (Experiment

2) was designed to measure changes in N utilization and efficiency using the same dietary treatments as in Experiment 1. To test our hypothesis, we (1) maintained diet RUP while increasing MP concentration through higher intestinal digestibility of RUP, (2) lowered

diet RUP while maintaining MP concentration through higher intestinal digestibility but

without consideration to the AA balance of the undegraded feed N, and (3) lowered diet

RUP while maintaining MP concentration combined with Met supplementation in both

rumen-protected form (Smartamine M) and rumen active form (Rhodimet AT-88) to

bring calculated Lys to Met ratio of MP near 3:1.

Materials and Methods

Animals and Diets

Experiment 1: Production Trial. Thirty-two multiparous and 28 primiparous

Holstein cows from the Waterman Dairy Facility at The Ohio State University were randomly assigned to one of four treatment diets on the Monday between 21 and 28 DIM.

All cows started on the same Monday were part of a block. Diets were formulated using the CPM model version 1(1998) with library values for all ingredients except for RUP sources, Smartamine, and HMB. The treatment diets (Table 3.1) were fed as TMR and contained on average 32.5 % NDF, 19.7 % ADF, and 1.60 Mcal/kg NEL with RDP at

10.5 % of the DM (NRC, 2001). Diets were made up of 50 % forage, with 37.5 % corn

silage (32.0 % DM, 8.3 % CP, 46.1 % NDF) and 12.5 % alfalfa haylage (46.0 % DM,

21.6 % CP, 38.0 % NDF) on a DM basis. Rumen undegraded protein was either at a high

concentration, 41.5 %, or a low concentration, 37.5 % of total CP. Supplemental

undegraded protein was provided by animal protein sources that were screened to be

either highly digestible in the intestine or were an unselected source (ie. porcine meat

meal) of a lower estimated digestibility in the intestine. Intestinal digestibility of

supplemental RUP sources were estimated prior to trial initiation using the method of

Calsamiglia and Stern (1995). All treatments were similar in concentration of RDP.

Treatments were: 1) 18.3 % CP with 42 % RUP of low intestinal digestibility (HiCP-

LoDRUP); 2) 18.3 % CP with 41.3 % RUP of high digestibility (HiCP-HiDRUP); 3)

16.9 % CP with 37.7 % RUP of high digestibility (LoCP-HiDRUP); and 4) 17.0 % CP with 37.6 % RUP of high digestibility plus supplemental Met from a combination of

Smartamine M and HMB (LoCP-HiDRUP + Met). Treatment diets contained supplemental RUP as either porcine meat meal (low intestinal digestibility), or a combination of high post-ruminal digestibility hydrolyzed feather meal, poultry meal and blood meal. The porcine meat meal used in our HiCP-LoDRUP diet was an unselected source of RUP, and was similar in estimated post-ruminal digestibility to values reported by others (Calsamiglia and Stern, 1995). The high digestibility RUP sources were selected from the top ten sources among 79 tested using the Minnesota 3-step enzymatic analysis of RUP (Calsamiglia and Stern, 1995). The poultry meal and blood meal used had greater than 90 % estimated post-ruminal RUP digestibility, while the feather meal had greater than 85 % post-ruminal digestibility. Nutrient composition of the supplemental RUP sources are in Table 3.2. Methionine was supplied in two forms:

Smartamine M, a 90 % rumen-escape source of methionine, and Rhodimet 88, a source of HMB. In our calculations, we assumed a 5 % ruminal escape rate for HMB (Charles

G. Schwab, personal communication). Concentrate ingredients including RUP supplements and HMB were pelleted. Calculated metabolizable Lys was higher in the three rations containing HiDRUP sources. Variations in concentration of CP and

intestinal digestibility of RUP provided similar concentrations of MP in treatments 1, 3, and 4, and greater concentrations in treatment 2. Treatments 1, 3 and 4 were approximately 275 g deficient in MP according to the CPM model (1998) using predicted intakes, body weights, milk yield and composition, whereas treatment 2 was only 60 g deficient (Table 3.1). The control ration contained Lys and Met in a ratio of 3.4:1.

Lowering the RUP in treatment 3 resulted in the same concentration of MP as the control ration, but with a Lys to Met ratio of 3.7:1.The addition of supplemental Met in treatment

4 brought this ratio to 3.3:1, which is closer to the optimum ratio derived by the NRC

(2001). Comparisons of estimates by the CPM model (1998) with the NRC model (2001) are shown in Table 3.3.

On the Monday of wk 4 of lactation, cows were assigned to their treatment diets and were housed in a tie stall barn, where they were individually fed. All cows starting on the same Monday were part of a block. Cows remained on their treatment diets for 12 wk. Total mixed rations were fed twice daily ad libitum with target orts representing 10

% of amounts fed. Nitrogen excretion in the production trial was calculated assuming a zero N balance, with the difference between intake N and milk N assumed to be the N excreted in the feces and urine.

Experiment 2: Digestibility Trial. Six cows from each treatment were held for an extra 5d in metabolism stalls for a digestibility study. Cows were selected from six blocks, balanced for parity within each block (three multiparous and three primiparous blocks) and within two wk of having the same DIM. Total collection of feces and urine, along with milk weights and composition, were used to determine nitrogen excretion.

Cows remained on the same treatment as the one they were assigned to during the production trial. The feeding protocol was similar to that of the production trial, except that orts were collected separately and analyzed to determine exact intakes of DM, CP, and NDF.

Sample Collection

Experiment 1: Production trial. Corn silage, alfalfa haylage, whole linted cottonseed, premixes and pelleted feeds were sampled monthly for the eight months of trial and kept frozen at –20º C until analyzed. Cows were milked twice daily, with milk weights recorded at each milking. Milk samples were collected weekly at four consecutive milkings and preserved with 2-bromo-2-nitropane-1, 3-diol and refrigerated until analyzed after the fourth milking. Blood samples were collected via the coccygeal vein and arteries at 0, 5 and 10 wk of trial for plasma urea nitrogen analysis. Blood samples were collected approximately 2h post-feeding and placed on ice for transport to the laboratory, where they were immediately centrifuged and the plasma removed. Blood plasma was stored at –20º C until analyzed. Cows were weighed and body condition scored on a scale of 1 to 5 once a week throughout the trial.

Experiment 2: Digestibility trial. Total mixed rations and orts were sampled daily and composited by cow. Milk weights were recorded daily, and samples were taken at each milking on d2 through 5 of the total collection. Feces were sampled daily, and weights recorded. Daily samples were kept refrigerated for the duration of the collection period. At the end of each collection period the samples were composited by cow and kept frozen at –20º C until analyzed.

Urine was collected using an external harness as described by Kauffman and St-

Pierre (2001). Urine was acidified during collection using 6 N HCl. Urine weight was

recorded daily, and a 500-ml sample was taken and kept refrigerated. Aliquots were

composited by volume, frozen, and stored at –20º C until analyzed.

Sample Analysis

Ingredient and TMR samples were analyzed for CP (AOAC, 1990), NDF, ADF, and

lignin (Van Soest et al., 1991). Analyses of HMB and supplemental dl-Met

(Smartamine) were done on a monthly basis by Aventis Animal Nutrition (Coventry,

France). Wet samples were dried for 48h at 55º C and ground using a Wiley Mill (Arthur

H. Thomas, Philadelphia, PA) with a 2-mm screen. Concentrate samples were ground

immediately. Milk samples were analyzed for total protein, fat, SCC and MUN (DHI

Cooperative, Inc., Powell, OH). Milk urea N was determined by a diacetyl monoxime

assay on a Skalar SAN Plus segmented flow analyzer (Skalar, Inc., Norcross, GA).

Plasma samples were assayed for plasma urea N (PUN) using Sigma Kit # 535 (Sigma

Diagnostics, St. Louis, MO). Both fecal and urine samples from the digestion trial were

analyzed for N using the Kjeldahl method (AOAC, 1990). Fecal samples were analyzed

for N on a wet basis to minimize possible losses of NH3. A sample was also dried at 55º

C and analyzed for NDF (Van Soest et al., 1991).

Statistical Analysis

Experiment 1: Production trial. During wk 3 of lactation, milk samples were taken and analyzed for milk CP, fat, SCC and MUN. Milk yields were also recorded, and

these measurements were used for covariate adjustment. Production data were analyzed using the MIXED Procedure of SAS (1999) according to the following model:

Yijklm = µ + Ti + Pj + TPij + bk +Bj(Xijl – X j) +cijl + Wm + TWim +PWjm + TPWijm + Eijklm [1] where:

Yijklm is the dependent, continuous variable,

th Ti is the fixed effect of the i treatment (i = 1, 2, 3, 4),

th Pj is the fixed effect of the j parity (j = 1, 2),

th th TPij is the fixed effect of the i treatment by j parity

th bk is the random effect of the k block (k = 1, …, 18),

th Bj is the regression coefficient (covariate) for the j parity,

th th th Xijl is the covariate measurement for the l cow within the i treatment and the j

parity,

th X j is the mean covariate measurement for the j parity,

th th th cijl is the random effect of the l cow within the i treatment and j parity

(l = 1 ,…, nij),

th Wm is the fixed effect of the m week of experiment (m = 1, …, 12),

th th TWim is the fixed effect of the i treatment by m week of experiment interaction,

th th PWjm is the fixed effect of the j parity by m week of experiment lactation

interaction,

th th th TPWijm is the fixed effect of the i treatment by j parity by m week of

experiment interaction, and

Eijklm is the residual error.

Errors within cows across weeks, which are repeated measures due to multiple sampling

of milk, intake and blood, were modeled using a first order autoregressive covariance

structure. This structure consistently gave the lowest Bayesian information criteria of

four covariance structures tested: unstructured, compound symmetry, first order

autoregressive and simple (Littell et al., 1996). Gross feed efficiency was calculated as

weight of milk per unit of DMI and analyzed according to model [1]. To test whether

changes in gross feed efficiency were the results of treatment effects on body energy

mobilization and replenishment, BW and body condition score (Wildman et al., 1982)

were also analyzed according to model [1]. Marginal feed efficiency was modeled by

fitting [1] with milk production as the dependent variable, with the following term added

to the linear model:

_ Bi (Zijlm - Z) [2]

where:

th Bi is the regression coefficient for the i treatment

th th th Zijlm is the DMI measurement for the l cow within the i treatment and the j

parity on the mth week.

_ Z is the overall DMI mean

The partial differential of the milk production function ([1] augmented with [2]) with

respect to DMI provides an estimate of marginal feed efficiency for each treatment.

Means were separated using Fisher’s protected LSD. Significance was declared at P <

0.05 for the production trial.

Experiment 2: Digestibility trial. Total collection data were analyzed as a

randomized complete block design with the effect of blocks modeled as a random effect

using the MIXED procedure of SAS. Significance was declared at P < 0.10 for the

digestibility trial. Means were separated using Fisher’s Protected LSD.

True N total tract digestibilities (Figure 1) were estimated using a linear

regression of N absorbed on N intake (Van Soest, 1994) according to the following

model:

NAijl = µ + Ti + Pj + TPij + bk + Bij NIijl + Eijl [3]

where:

th th th NAijl is the N absorbed of the l cow within the i treatment and the j parity,

th Bj is the regression coefficient for the j parity,

th th th NIijl is the N intake of the l cow within the i treatment and the j parity,

Eijl is the residual errors, and other terms are as defined in [1].

Results and Discussion

Experiment 1: Production Trial.

Body weights and condition scores. Body weights and body condition scores did not differ by treatment (data not shown). Mean body weights were 551 and 647 kg, and body condition scores were 3.1 and 2.7 respectively, for primiparous and multiparous cows. Body weights increased linearly with week on trial (WOT). Primiparous cows gained on average 4.54 kg per week (BW = 4.54 (±0.35) * WOT+ 521 (± 3)), whereas

multiparous cows gained 4.91 kg per week (BW = 4.91 (±0.31) * WOT + 615 (± 2)).

Intake and milk production. Results for DMI and milk production measurements are reported in Table 3.4. The three groups receiving HiDRUP had greater DMI than the

LoDRUP control (P = 0.04). This is in contrast to a review by Santos et al. (1998) who found that feeding a high quality RUP (fish meal) with a Lys:Met ratio of approximately

3:1 had no effects on DMI over a low quality RUP (corn gluten meal, low Lys) in eight out of nine studies reviewed. However, fishmeal can become unpalatable at higher levels and may prevent an increase in intake. Milk yield was highest for the HiCP-HiDRUP and the LoCP-HiDRUP + Met diets, indicating that AA other than Met were in greater excess for the higher CP diet.

Milk protein concentration was increased significantly in the LoCP-HiDRUP +

Met diet over the other three treatments. Protein and fat production and fat percentage were all significantly affected by treatments, but the treatment effect interacted with parity. First parity cows had similar protein production on the HiCP-LoDRUP and

LoCP-HiDRUP diets, but increased protein production when supplemented with Met or fed the HiDRUP at HiCP-HiDRUP concentrations. Multiparous cows increased in milk protein production from all diets containing HiDRUP, with an additional increase when supplemented with Met. Increases in AA availability, either by supplementing Met or using a higher quality RUP source, may have influenced the production of milk CP. An increase in protein production is common in diets containing undegradable sources of

Met (Rulquin and Delaby, 1997; Armentano et al., 1997), although some trials have seen no effect (Blum et al., 1999). Schwab et al. (1992) found that cows in early lactation had a significant increase in milk protein percentage with duodenal infusions of Met and Lys

when compared with Met alone. They found that cows in peak lactation showed a non- significant trend to increase milk protein content when supplemented with Met and Lys over Lys alone, and significant increases over Met alone. Armentano et al. (1997) found a linear increase in milk protein production with the addition of rumen protected Met to a ration supplying adequate levels of Lys. Protein yield increased 4 g per each gram of

Met added to the diet. The authors estimated that approximately 15 to 20% of the Met supplemented was converted to milk protein. In our trial, addition of Met to the LoCP-

HiDRUP ration increased protein production by 160 g/d. Approximately 6 g of Met that bypassed the rumen came from Smartamine (75% Met, 90 % rumen inert, 90% post- ruminal digestibility), with the remainder from HMB. Assuming 5% rumen undegradability for HMB (Schwab, personal communication) results in a calculated increase of 23.3 g of milk protein per gram of supplemental metabolizable Met.

Assuming that milk protein is 3% Met, the efficiency of transfer of supplemental metabolizable Met to milk Met is calculated at 70%. However, if HMB is assumed 40% rumen undegradable (Vazquez-Anon, 2001), the protein yield increases by 12.3 g per gram of supplemental metabolizable Met, with an efficiency of transfer to milk protein of

37%. In either case, the marginal efficiency of Met supplementation was substantially higher than the figure reported by Armentano et al. (1997). This could be due to (1) higher Lys bioavailability in our experiment, (2) stimulation of microbial protein synthesis from a ruminal effect of HMB (Vazquez-Anon et al., 2001), or (3) other undetermined mode of action.

Fat production and percentage had a significant treatment by parity interaction.

Fat production in primiparous cows was higher for the HiDRUP treatments, with the

greatest increases in the HiCP-HiDRUP and LoCP-HiDRUP + Met diets. Multiparous

cows followed a similar trend, but fat production for the diets with HiDRUP was not

significantly different. Fat percentage increased with the diets containing the HiDRUP

supplements for first parity cows, with the LoCP-HiDRUP + Met being numerically

highest, although it was not significantly different from the LoCP-HiDRUP diet.

Treatments had no effect on milk fat concentration in cows of second or greater parity,

explaining the treatment by parity interaction. Abomasal infusions of Met have been

shown to increase milk fat concentration (Varvikko et al., 1999). Polan et al. (1970b)

found an increase in milk fat production and concentration from supplemental HMB in

the diet in amounts up to 94 g/d, but with peak milk yield occurring at a supplementation

of 25 g/d. Lundquist et al. (1983) found that supplementation of HMB increased fat

yield and concentration at several concentrations of dietary protein and two forage to concentrate ratios. These authors also combined data from six lactation studies in which cows were fed either HMB or a control diet. Addition of 0.25 to 0.30% HMB to diet DM increased milk fat by an average of 0.35% and yield by 0.09 kg/d. The data analyzed included both primiparous and multiparous cows. The concentration fed was two to three times higher than that fed in the current trial, and may not represent the effects of current feeding practices.

Gross feed efficiency (kg milk/kg DMI) was numerically highest for the HiCP-

HiDRUP and LoCP-HiDRUP + Met treatments, although they were not significantly

different from the feed efficiency on the HiCP-LoDRUP diet. The HiCP-LoDRUP and

LoCP-HiDRUP diets were not significantly different (P > 0.05). Higher feed efficiency indicates a better utilization of the nutrients ingested, either through increased digestibility, improved conversion of absorbed nutrients into milk, dilution of maintenance requirements, or mobilization of body reserves. The evolution of BCS across weeks was not affected by treatments (P = 0.84) and averaged 2.85 (SEM = 0.04) and 2.95 (SEM = 0.04) at week 1 and 12, respectively. Likewise, treatments did not affect the evolution of BW through time, (P = 0.86) with a mean BW of 575 kg (SEM =

8.9) and 624 kg (SEM = 8.9) at week 1 and 12, respectively. Thus, treatment effects on gross feed efficiency were not the results of different body energy reserve depletion and repletion across treatments. Treatments did not affect the marginal feed efficiency (P =

0.14), which averaged 0.39 kg per kg (SEM = 0.05) across the four treatments.

Table 3.5 reports calculated balances and flows of important nutrients using the

NRC (2001) model with pre-trial estimated milk production (45.5 kg/d) and DMI (23.8 kg/d) compared to calculations using actual least squares means of milk production, composition, and DMI during the course of the 12 wk trial. Cows were allowed feed ad libitum, and the increase in DMI was a response to the treatment diets. This table illustrates that the lower than predicted milk yield in the HiCP-LoDRUP group brought the balance of MP closer to zero, emphasizing that this diet was too low in MP originally to support the desired milk yield, and indirectly supporting the accuracy of the NRC requirements. However, the LoCP-HiDRUP + Met predicted an MP balance that was more deficient than originally predicted due to increases in milk yield. This increase in

milk yield while deficient in MP indicates that overall quantity of MP may not be as important as supplying the proper balance of Met and Lys.

Nitrogen measurements. Nitrogen intakes were similar for the HiCP-LoDRUP,

LoCP-HiDRUP and LoCP-HiDRUP + Met diets (Table 3.6). Cows on the HiCP-

HiDRUP diet consumed significantly more N than those on the other three treatments.

The effect of treatments on milk N production interacted with parity, as explained previously. Gross N efficiency (Milk N/N intake * 100) was increased in all diets containing high digestibility RUP compared to the diet with low digestibility RUP, with the greatest increase occurring with the Met-supplemented diet. Because N intake was similar for the LoCP-HiDRUP and LoCP-HiDRUP + Met diets, Met supplementation appeared to improve the efficiency of conversion of MP into milk N. St-Pierre and

Thraen (1999) proposed using N excreted (i.e. N in feces and N in urine) over milk N as a measure of environmental N efficiency. Decreasing the environmental N efficiency has the potential of lowering the impact of dairy production on the environment.

Environmental N ratio was lower for the three diets containing high intestinal digestibility RUP, with the greatest decrease occurring in the diet supplemented with

Met. This indicates that feeding diets supplemented with a source of high digestibility

RUP, especially in a diet formulated to be low in MP and balanced for Met, could decrease significantly the urinary and fecal N released into the environment. Higher CP diets had greater concentrations of MUN. Plasma urea nitrogen was greatest for the

HiCP-HiDRUP diet, probably due to the higher N intake on this diet.

Cows on the LoCP-HiDRUP diet performed similarly to the control (HiCP-

LoDRUP) in milk yield, protein yield in primiparous cows and fat concentration for

multiparous cows. All other milk measurements showed improvements from the addition

of HiDRUP. This may indicate that the control diet was not supplying the AA balance

predicted by using the CPM model (1998). Lysine may have been unavailable due to

Maillard protein-carbohydrate reactions from the heat treatment, and the 3-step procedure

(Calsamiglia and Stern, 1995) does not differentiate Lys from CP. Porcine meat meal is

very high in fat (11.5 %). In baked products, fats may also become associated with

carbohydrate or protein through what might be the initial stages of the Maillard reaction

(Van Soest, 1994). This may not impair digestibility but can influence the probability of rumen escape.

Experiment 2: Metabolism Trial.

Twelve primiparous and twelve multiparous cows were used in six blocks in the metabolism trial. One multiparous cow from the LoCP-HiDRUP + Met treatment was removed due to sharp decreases in milk production and DMI during the digestibility trial.

Because of the small number of animals per treatment, the collection trial did not show the significance seen in the production trial. However, the trends support the conclusions drawn from the production trial.

Intake and digestibility. Dry matter intake, DM digestibility, and NDF digestibility were not significantly different among treatments (Table 3.7). Cows were in wk 15 or 16 of lactation during the collection trial and had higher DMI than the mean

DMI observed in the production trial, during which cows averaged 7 to 8 wk in milk.

Apparent digestibility of N did not differ (Table 3.8). The RUP sources provided approximately 10 % of the N in the diet. Therefore, a 20 % increase in the digestibility of the supplemental RUP source would only increase overall N digestibility by 2 %. With a

SEM for apparent N digestibility of 2.1, the power of the test was too low to see differences. The Lucas Test (Van Soest, 1994) was performed to determine if the true digestibilities of N of the four treatment diets differed (Figure 1). Regressions were done by parity in order to determine metabolic fecal N for primiparous and multiparous cows.

All four treatment diets were found to have 80 % (± 0.5) true digestibility of N.

Estimates of metabolic fecal N excretion (intercept of regression) were 103 g/d for primiparous and 132 g/d for multiparous cows. These equate to 5.3 and 5.0 g/kg DMI for primiparous and multiparous cows, respectively. The NRC (2001) uses a value of 4.7 g/kg DMI metabolic fecal N based on data from Swanson (1977). Metabolic fecal N is the portion of fecal N that can not be changed by nutrition.

Milk production and nitrogen utilization. Milk production and nitrogen partitioning for Experiment 2 are reported in Table 3.8. Milk production and milk protein production were not significantly different, although there was a trend (P = 0.18) for milk protein percentage to increase in the two LoCP-HiDRUP diets. Milk and protein production by cows in the digestibility trial were lower than those observed in the production trial for cows on the LoCP-HiDRUP + Met treatment, apparently supporting the lower metabolizable Met requirement for the cows in the metabolism trial so that it was not limiting for any treatments.

Nitrogen intake was numerically highest in the HiCP-LoDRUP and HiCP-

HiDRUP diets, although the HiCP-HiDRUP N intake was not significantly different from the LoCP-HiDRUP and LoCP-HiDRUP + Met treatments. Nitrogen apparently absorbed was significantly different among treatments, with the two HiCP-LoDRUP and HiCP-

HiDRUP absorbing greater amounts (P = 0.04) than LoCP-HiDRUP and LoCP-HiDRUP

+ Met treatments. Intake of N by cows on the HiCP-LoDRUP diet was significantly larger than that of the LoCP diets, with the HiCP-HiDRUP group being intermediate in intake. The three rations with HiDRUP did not have significantly different N absorption, which reflects the lack of differences seen for these treatments in N intake data. The amount of N in the feces was not significantly affected by the treatments. Urinary N was lowered by LoCP-HiDRUP and LoCP-HiDRUP + Met diets. This, combined with slight differences in milk N production, caused environmental efficiency values to improve (i.e. less N excreted per kilogram N in milk) with the LoCP-HiDRUP and LoCP-HiDRUP +

Met diets. Urine, milk and retained N as a percent of absorbed N were not significantly different, but these values also show a trend for milk N as a percent of absorbed N to be higher for the LoCP-HiDRUP and LoCP-HiDRUP + Met diets (P = 0.14).

Conclusions

Maintaining dietary RUP while increasing MP concentration through higher intestinal digestibility of RUP (HiCP-LoDRUP and HiCP-HiDRUP) increased milk yield and component production, indicating that post-ruminal protein digestibility and AA profile of the unselected RUP source was not adequate for maximal milk and milk

component production. Lowering dietary RUP while maintaining MP concentration through higher intestinal digestibility of RUP (HiCP-LoDRUP and LoCP-HiDRUP) allowed some increases in milk, protein, and fat production, further indicating the nutritional limitations of the unselected protein source. Supplementing the highly digestible RUP source with rumen available and rumen escape sources of Met resulted in maximal milk and protein production and maximum N efficiency by cows, indicating that post-ruminal digestibility of RUP and AA balance can be more important than total RUP supplementation. Of interest is the fact that the Met supplemented diet provided numerically larger milk yields and significantly larger protein concentrations than even the HiCP-HiDRUP diet, which should have contained excess Lys and Met due to the large concentration of MP. Estimates of environmental efficiency from the production trial indicate that lowering CP and balancing AA properly can sharply decrease the amount of N released into the environment, with a 23% decrease in amount excreted per unit of milk N when comparing the HiCP-LoDRUP and the LoCP-HiDRUP + Met supplemented treatments. Models such as the NRC (2001) should enable us to decrease the CP in diets while balancing for AA, without sacrificing milk and components.

Treatments Ingredients HiCP- HiCP-HiDRUP LoCP-HiDRUP LoCP-HiDRUP LoDRUP1 + Met ------% of total ration DM------Corn Silage 37.5 37.5 37.5 37.5 Alfalfa Silage 12.5 12.5 12.5 12.5 Ground shelled corn 20.0 19.8 22.4 22.3 Soybean meal, 48 % CP 6.83 9.39 7.68 7.68 Whole cottonseed 8.4 8.4 8.4 8.4 Soybean hulls 3.4 3.4 3.4 3.4 Porcine meat meal 8.0 - - - Blood meal2 - 2.0 1.8 1.8 Hydrolyzed feather meal - 1.0 0.44 0.44 Poultry meal - 1.0 - - Megalac3 0.5 0.5 0.5 0.5 Tallow - 0.32 0.57 0.57 Urea - - 0.19 0.19 Smartamine M4 - - - 0.042 Rhodimet AT-885 - - - 0.084 Vitamins & minerals 2.9 4.4 4.8 4.8 Predicted Nutrients6 CP, % of DM 18.3 18.3 16.9 17.0 NDF, % of DM 32.8 32.4 32.3 32.3 Fat, % of DM 5.7 5.2 5.4 5.4 NSC, % of DM 38.3 38.8 40.2 40.0 ME balance, Mcal/d 2.1 2.3 2.5 2.4 RUP, % of CP intake 42.0 41.3 37.7 37.6 MP7 balance, g/d -300 -64 -267 -265 Met, g/d -3.8 -3.9 -4.6 2.0 Met, % of requirement 93 93 91 104 Met, % of MP 1.90 1.73 1.84 2.09 Lys, g/d -0.2 22.9 12.1 11.8 Lys, % of requirement 100 114 107 107 Lys, % of MP 6.45 6.72 6.83 6.81 1HiCP-LoDRUP is the control diet with porcine meat meal as the source of supplemental RUP; HiCP- HiDRUP has same concentration of RUP and CP as control but with highly digestible supplemental RUP source; LoCP-HiDRUP has highly digestible supplemental RUP source, with overall RUP decreased; LoCP-HiDRUP + Met is the same as the LoCP-HiDRUP but with Met adjusted to obtain a 3.3:1 Lys: Met. 2Blood, hydrolyzed feather and poultry meal sources were selected for a high estimated post-ruminal RUP digestibility using the Minnesota three step enzymatic assay of Calsamiglia and Stern (1995). 3Megalac is 82.5% long chain fatty acids, 8.0-9.6% Ca, and 2% moisture. 4Smartamine M contains 60 % metabolizable Met. 5Rhodimet AT-88 contains 88 % HMB, and is assumed to have 5 % rumen undegradability. 6Values were determined using the CPM model (1998) using predicted DMI, BW, milk yield and composition. 7Metabolizable protein = protein that is digestible in the small intestine.

Table 3.1. Ingredient and nutrient composition of diets (DM basis) that vary in CP and digestibility of RUP based on predicted body weight, intake, milk yield and composition.

Porcine Poultry Blood Meal Meal Feather Meal Meat Meal DM (% of as-fed) 90.4 93.6 89.0 92.0 CP (% of DM) 91.5 67.3 84.0 58.7 True Protein1 (% of DM) 86.0 60.4 79.5 54.0 Ether Extract (% of DM) < 0.1 11.8 5.6 11.5 Ash (% of DM) 3.1 13.1 1.8 20.2 CP digestibility by pepsin (%) 97.1 91.9 71.9 89.4 16 h RUP (% of CP)2 84.7 74.7 80.2 69.0 RUP digestibility (% of RUP)3 95.0 91.0 89.0 55.0

Amino Acids (% of true protein) Taurine 0.02 0.71 0.05 0.20 Hydroxyproline 0.00 3.19 0.08 4.19 Aspartic Acid 10.63 8.21 6.83 7.74 Threonine 3.86 3.92 4.55 3.59 Serine 3.90 3.39 8.99 3.85 Glutamic Acid 7.91 13.06 9.60 12.89 Proline 3.51 6.37 9.58 7.78 Lanthionine 0.00 0.08 1.47 0.33 Glycine 4.47 10.08 7.31 12.04 Alanine 8.54 7.17 5.19 7.43 Cysteine 0.63 1.22 4.78 1.37 Valine 9.23 4.90 7.55 4.72 Methionine 1.09 2.18 0.64 1.50 Isoleucine 0.34 3.92 4.63 3.19 Leucine 13.71 7.00 8.43 6.61 Tyrosine 2.47 3.04 3.06 2.56 Phenylalanine 7.54 3.94 5.58 3.56 Hydroxylysine 0.00 0.45 0.00 0.44 Histidine 7.36 2.17 1.43 2.09 Ornithine 0.00 0.30 0.25 0.09 Lysine 8.93 6.45 2.50 5.58 Arginine 4.01 7.18 6.77 7.46 Tryptophan 1.86 1.06 0.73 0.78 1True protein = sum of all AA. 2Percentage of CP remaining in a dacron bag after 16 h of rumen incubation. 3Percentage of RUP that is digested using the Minnesota 3-step enzymatic assay for protein digestion.

Table 3.2. Nutrient composition of supplemental RUP sources.

HiCP1 LoCP LoDRUP HiDRUP HiDRUP HiDRUP + Met Nutrient CPM2 NRC CPM NRC CPM NRC CPM NRC CP, % of DM 18.3 18.6 18.3 18.5 16.9 16.9 17.0 16.9 NDF, % of DM 32.8 29.7 32.4 29.9 32.3 30.0 32.3 30.0 3 NEL balance , Mcal 1.4 -2.4 1.5 -2.8 1.6 -3 1.6 -3 RUP, % of DM 7.7 7.2 7.6 7.8 6.4 6.7 6.4 6.7 MP balance, g -300 -183 -64 43 -267 -168 -265 -168 Met, % of MP 1.90 1.79 1.73 1.70 1.84 1.74 2.09 2.01 Lys, % of MP 6.45 6.25 6.72 6.42 6.83 6.57 6.81 6.57 Lys:Met in the MP 3.4 3.5 3.9 3.8 3.7 3.8 3.3 3.3 Fat, % of DM 5.7 5.5 5.2 5.1 5.4 5.2 5.4 5.2 1HiCP-LoDRUP is the control diet with porcine meat meal as the source of supplemental RUP; HiCP- HiDRUP has same level of RUP as control but with highly digestible supplemental RUP source; LoCP- HiDRUP has highly digestible supplemental RUP source, with overall RUP decreased; LoCP-HiDRUP + Met is the same as the LoCP-HiDRUP but with Met adjusted to obtain a 3.3:1 Lys:Met. 2CPM model (1998); NRC (2001). 3balance = amount above requirement.

Table 3.3. Estimates of dietary nutrients at standard production (45.5 kg) and intake (23.8 kg DMI) in diets that vary in CP and digestibility of RUP

HiCP1 LoCP HiDRUP Parity LoDRUP HiDRUP HiDRUP + Met SEM Parity Trt. X parity DMI (kg/d) 1 18.1 20.1 19.7 20.2 0.77 2 + 25.3 26.4 26.7 27.1 0.61 all 21.7a 23.3b 23.2b 23.6b 0.49 < 0.01 NS2 Milk yield (kg/d) 1 35.1 41.2 36.2 40.4 1.14 2 + 46.5 51.1 49.6 52.7 0.90 all 40.8a 46.2b 42.9a 46.6b 0.72 < 0.01 NS Protein production (kg/d) 1 1.01a 1.25b 1.07a 1.24b 0.037 2 + 1.39a 1.51b 1.50b 1.65c 0.030 all 1.20 1.38 1.28 1.44 0.024 < 0.01 0.07 Fat production (kg/d) 1 1.17a 1.51b 1.35c 1.60b 0.046 2 + 1.59a 1.82b 1.78b 1.83b 0.035 all 1.38 1.67 1.57 1.71 0.029 < 0.01 0.06 Protein (%) 1 2.89 3.01 2.96 3.04 0.041 2 + 3.00 2.97 3.04 3.15 0.033 all 2.95a 2.98a 2.99a 3.09b 0.027 0.04 NS Fat (%) 1 3.40a 3.66b 3.72b,c 3.91c 0.089 2 + 3.44a 3.62a 3.61a 3.54a 0.068 all 3.42 3.64 3.66 3.73 0.053 0.05 0.05 Gross feed efficiency3 1 1.93 2.04 1.85 1.99 0.061 2 + 1.85 1.95 1.87 1.97 0.048 all 1.89a,b 1.99a 1.86b 1.98a 0.036 NS NS 1HiCP-LoDRUP is the control diet with porcine meat meal as the source of supplemental RUP; HiCP- HiDRUP has same level of RUP as control but with highly digestible supplemental RUP source; LoCP- HiDRUP has highly digestible supplemental RUP source, with overall RUP decreased; LoCP-HiDRUP + Met is the same as the LoCP-HiDRUP but with Met adjusted to obtain a 3.3:1 Lys:Met. 2NS = P > 0.05. 3Calculated as kg milk/kg DMI. a,b,c Treatment LSMEANS in the same row with different superscripts are different at P < 0.05.

Table 3.4. Least squares means for performance measures in Experiment 1 for diets that vary in CP and digestibility of RUP (n = 60).

HiCP1 LoCP LoDRUP HiDRUP HiDRUP HiDRUP + Met Nutrient NRCp2,3 NRCa NRCp NRCa NRCp NRCa NRCp NRCa actual intake, kg/d 21.7 23.3 23.2 23.6 actual milk yield, kg/d 40.9 46.3 43.0 46.7 MP balance, g -183 -84 43.0 20.0 -168 -58 -168 -257 RUP balance, g/d -237 -109 51.0 24.0 -199 -69 -199 -306 Metabolizable Met flow, g/d 48.6 44.5 50.4 49.5 47.8 46.8 55.2 54.8 Metabolizable Lys flow, g/d 171 157 190 186 181 176 181 179

NEL, Mcal/day 38.9 36.1 38.6 37.9 38.4 37.6 38.4 38.1 1 HiCP-LoDRUP is the control diet with porcine meat meal as the source of supplemental RUP; LoCP- HiDRUP has highly digestible supplemental RUP source, with overall RUP decreased; LoCP-HiDRUP + Met is the same as the LoCP-HiDRUP but with Met adjusted to obtain a 3.3:1 Lys:Met.

2 NRC (2001). 3 NRCp are the estimates based on predicted intakes and milk yields; NRCa are the estimates based on actual intakes and milk yields.

Table 3.5. Estimates of dietary nutrients at standard production (45.5 kg) and intake (23.8 kg DMI) and actual production and intake in diets that vary in CP and digestibility of RUP.

HiCP1 LoCP HiDRUP Trt. X Parity LoDRUP HiDRUP HiDRUP + Met SEM Parity parity

N intake (g/d) 1 535 596 547 556 22.2 2 + 747 785 742 745 17.6 All 641a 690b 645a 651a 14.2 < 0.01 NS2

Milk N 1 158a 192b 169a 195b 6.1 production (g/d) 2 + 217a 235b 238b 260c 4.8 All 188 214 203 228 3.9 < 0.01 0.09

Gross N efficiency3 1 29.6 32.0 31.2 34.8 0.94 2 + 29.3 30.1 32.3 35.1 0.74 All 29.5a 31.1b 31.7b 35.0c 0.60 NS NS

Environmental 1 2.43 2.15 2.27 1.91 0.10 efficiency4 2 + 2.49 2.36 2.12 1.86 0.07 All 2.47a 2.25b 2.19b 1.89c 0.06 NS NS

MUN5 (mg/dl) 1 16.6 16.8 14.4 13.4 0.47 2 + 17.1 17.8 14.2 13.6 0.38 All 16.8a 17.3a 14.3b 13.5c 0.30 NS NS PUN6 (mg/dl) 1 17.4 20.4 18.3 16.3 1.12 2 + 18.7 20.8 17.9 19.2 0.89 All 18.0a 20.6b 18.1a 17.8a 0.73 NS NS

1HiCP-LoDRUP is the control diet with porcine meat meal as the source of supplemental RUP; HiCP-HiDRUP has same level of RUP as control but with highly digestible supplemental RUP source; LoCP-HiDRUP has highly digestible supplemental RUP source, with overall RUP decreased; LoCP-HiDRUP + Met is the same as the LoCP-HiDRUP but with Met adjusted to obtain a 3.3:1 Lys:Met. 2NS = P > 0.05. 3Calculated as milk N/N intake * 100. 4Calculated as kg N excreted/kg N in milk; environmental efficiency calculation assumes zero nitrogen balance 5MUN = Milk urea N. 6PUN = Plasma urea N. a,b,c Treatment LSMEANS in the same row with different superscripts are different at P < 0.05.

Table 3.6. Least squares means in nitrogen measurements for Experiment 1 for diets that vary in CP and RUP digestibility

HiCP1 LoCP LoDRUP HiDRUP HiDRUP HiDRUP + Met SEM TRT DMI (kg/d) 25.6 24.4 24.6 24.1 0.87 NS2 DM digestibility (%) 65.6 66.0 64.6 63.4 1.1 NS NDF intake (kg/d) 8.3 7.9 7.9 7.5 0.34 NS NDF digestibility (%) 55.0 55.0 54.0 50.3 1.9 NS 1HiCP-LoDRUP is the control diet with porcine meat meal as the source of supplemental RUP; HiCP-HiDRUP has same level of RUP as control but with highly digestible supplemental RUP source; LoCP-HiDRUP has highly digestible supplemental RUP source, with overall RUP decreased; LoCP-HiDRUP + Met is the same as the LoCP-HiDRUP but with Met adjusted to obtain a 3.3:1 Lys:Met. 2NS = P > 0.10.

Table 3.7. Least squares means for intake and digestibility of DM and NDF in Experiment 2 for diets that vary in CP and digestibility of RUP (n = 24).

HiCP1 LoCP HiDRUP + TRT LoDRUP HiDRUP HiDRUP Met SEM Milk production (kg/d) 45.0 45.2 44.0 43.1 2.12 NS2 Milk protein (kg/d) 1.42 1.38 1.42 1.38 0.06 NS Milk protein (%) 3.17 3.08 3.27 3.26 0.09 NS MUN (mg/dl)3 17.0a 16.3a,c 14.4b,c 13.5b 1.09 0.02 Milk N (g/d) 223 217 223 216 9.4 NS Milk fat (kg/d) 1.55 1.55 1.55 1.51 0.11 NS Milk fat (%) 3.51 3.47 3.58 3.6 0.25 NS N intake (g/d) 770a 735a,b,c 682b,c 679b 27.9 0.03 Fecal N (g/d) 279 271 257 263 10.9 NS N apparently absorbed (g/d) 492a 464a,c 426b,c 418b,c 30.4 0.04 N Absorbed (apparent NS digestibility), % 63.7 62.9 61.9 60.8 2.1 Urine N (g/d) 268a 259a,c 216b,d 224c,d 19.3 0.08 Apparent N retention (g/d) -1 -13 -16 -23 18.3 NS Productive N, % of N intake4 29.1 28 30.8 28.5 1.9 NS Urine 54.3 55.4 50.2 53.1 2.6 NS Milk 45.9 47.6 53.2 53.3 2.8 NS Retained -0.3 -3.2 -6.4 -6.0 4.1 NS Environmental efficiency5 2.43a 2.44a 2.09b 2.24a,b 0.10 0.04 1 HiCP-LoDRUP is the control diet with porcine meat meal as the source of supplemental RUP; HiCP- HiDRUP has same level of RUP as control but with highly digestible supplemental RUP source; LoCP-HiDRUP has highly digestible supplemental RUP source, with overall RUP decreased. 2NS = P > 0.10. 3MUN = Milk urea N. 4Productive N = milk N + retained N 5kg N excreted/kg N in milk a,b,ctreatments in a row with different superscripts are significantly different at P < 0.10.

Table 3.8. Least squares means for milk production and nitrogen utilization during Experiment 2 for diets that vary in CP and digestibility of RUP (n = 24).

800 700 600 500 d /

, g 400 ed

rb 300

so 200

N ab 100 0 -100 -200 0 200 400 600 800 1000 1200 N intake, g/day

Figure 3.1. Nitrogen absorbed (NA) versus N intake (NI) for HiCP-LoDRUP (■□), HiCP-HiDRUP (▲∆), LoCP-HiDRUP (●○) and LoCP-HiDRUP + Met (♦ ◊). Solid symbols represent the primiparous cows; open symbols are multiparous cows. The solid line represents the regression equation for primiparous cows (NA = 0.80(±0.005) * NI – 103(±17)). The dashed line represents the regression equation for multiparous cows (NA = 0.80(±0.005) * NI – 132(±11)).

CHAPTER 4

EFFECTS OF 2-HYDROXY-4-(METHYLTHIO) BUTANOIC ACID (HMB) ON

MICROBIAL GROWTH IN CONTINUOUS CULTURE

Abstract

2-Hydroxy-4-(methylthio) butanoic acid (HMB) positively affects milk

composition and yield, potentially through ruminal actions. Four continuous culture

fermenters were used to determine the optimal concentration of HMB for digestibility of

organic matter (OM), neutral detergent fiber (NDF), acid detergent fiber (ADF), and

hemicellulose and synthesis of microbial N. A highly degradable mix of hay and grain

was used as a basal diet to simulate a typical lactation diet. Three concentrations of

HMB (0, 0.055, and 0.110 %) and one concentration of dl-Met (0.097 %) were infused into the fermenters according to a 4 X 4 Latin square design. Digesta samples were collected during the last 3 d of each of the four 10 d experimental periods. Digestibility of OM, hemicellulose, and NDF was largely insensitive to treatment. Digestibility of

ADF showed a quadratic effect to supplementation of HMB, with 0.055 % having lower digestibility than 0 or 0.110 %. Total production of VFA was not influenced by HMB

supplementation, but differences in concentration and production of individual VFA were

seen. Isobutyrate increased linearly with increasing HMB supplementation. Propionate

concentration decreased linearly with increased HMB supplementation, but propionate

production showed a quadratic trend (P = 0.13). A higher concentration of acetate was

detected for dl-Met compared with the highest HMB concentration. There were trends (P

< 0.15) for dl-Met to decrease the production of isobutyrate and to lower the

concentration of butyrate when compared with HMB. Microbial efficiency was not

different among treatments. The proportion of bacterial N produced from NH3-N

decreased linearly with increasing HMB, and bacteria receiving dl-Met synthesized more

N from NH3-N than those receiving HMB. These data suggest that supplementation of

HMB may have a sparing effect on branched chain volatile fatty acids because the fatty acids are not needed to provide carbon for synthesis of valine, isoleucine and leucine with ammonia. Comparisons of bacterial community structure in the fermenter effluent samples using PCR amplicons containing the ribosomal intergenic spacer region and its flanking partial 16S ribosomal RNA gene showed no distinct banding patterns, though treatments tended to group together. Both Met and HMB affect the rumen microbial population, but Met supplied as dl-Met does not act identically to that supplied as HMB.

Abbreviation key: BCVFA = isobutyrate, isovalerate, and valerate; HMB = 2-hydroxy-

4-(methylthio)-butanoic acid, NANBN = non-ammonia-non-bacterial nitrogen, RIS = ribosomal intergenic spacer, rDNA = ribosomal DNA, RIS-LP = RIS-length polymorphism)

Introduction

Methionine and Lys have been identified as two of the first limiting AA in dairy rations (Schwab, 1992). Lysine is required for maximum milk protein synthesis (King et al., 1991), whereas Met affects both milk fat (Hansen et al., 1991) and milk protein synthesis (Rulquin and Delaby, 1997). Recently there has been a renewed interest in Met supplied as 2-hydroxy-4 (methylthio)-butanoic acid (HMB). Research in the 1960s and

1970s documented increases in milk fat and milk yield (Patton et al., 1970b), whereas

others (Wallenius and Whitchurch, 1975) found no changes. 2-Hydroxy-4 (methylthio)-

butanoic acid is at least partially rumen degradable (Vazquez-Anon et al., 2001) and

could affect the rumen microbial population.

Microbial protein reaching the duodenum represents the largest contribution of

protein for ruminant animals (Firkins, 1996), and has an AA profile more similar to that

of both milk and lean tissue than that of most protein sources used as animal feeds (NRC,

2001). Peptides and AA have stimulated microbial protein synthesis when substituted for

ammonia in vitro (Russell and Strobel, 1993) and in vivo (Rooke and Armstrong, 1989).

Streptococcus bovis, an active proteolytic bacterium, spilled less energy as heat when AA

were included in the medium, suggesting more efficient use of carbon and energy when

AA were provided (Russell and Strobel, 1993). Patterson and Kung (1988) determined

that bacteria incorporate Met into cellular material, regardless of precursor (dl-Met,

HMB, or HMB esters, salts or amides). Gil et al. (1973a,b) found that supplying HMB in

the urea-containing medium greatly enhanced rate of growth of rumen bacteria on

glucose or cellulose, and was not being used as an energy source. Prevotella ruminicola

23, Butyrivibrio fibrisolvens and Selenomonas ruminantium are stimulated by cysteine

(Cotta and Russell, 1982), which can be produced from Met (Stipanuk, 2000). The rumen

bacterium B. fibrisolvens degrades cellulose, hemicellulose, and protein in the rumen and can make up a large portion of the rumen microorganisms (Van Soest, 1994). P. ruminicola is a predominant hemicellulolytic organism (Dehority, 1991). If these

organisms are provided with supplemental HMB, causing an increase in overall numbers

of fibrolytic organisms, fiber digestibility could be improved.

Yu and Mohn (2001) developed a composite method for investigating bacterial

community structure in an aerated lagoon. The method is based on analyses of PCR

amplicons containing the ribosomal intergenic spacer (RIS) region and its flanking partial

16S rRNA gene. Ribosomal DNA (rDNA) is the region of DNA that encodes the rRNA

genes. The16S-23S ribosomal DNA-RIS region has a highly variable length, and can be used as a marker to distinguish different bacterial species.

The hypotheses for this trial were that HMB supplementation would increase microbial growth in continuous culture over an unsupplemented control by either sparing

Met precursors for more efficient protein synthesis, or by shifting bacterial species. The objective of this study was to quantify the effects on microbial populations supplemented with HMB by measuring flow of N, digestibility of OM, ADF, NDF and hemicellulose, and VFA production in continuous culture. Changes in the microbial community profile

were analyzed by RIS length polymorphisms (RIS-LP).

Materials and Methods

Experimental Design

Four continuous culture fermenters were used in this experiment (Hannah et al.,

1986, Hoover et al., 1976). The design was a 4 X 4 Latin square with four continuous culture fermenters over four periods of 10 d each. The four experimental treatments (as a percent of diet DM) were provided through the buffer input: 0 % HMB (control), 0.055

% HMB, 0.110 % HMB, or 0.097 % of dl-Met; the latter two provided equivalent moles per day of Met. Alfalfa hay and a grain mix were ground in a Wiley Mill (Arthur H.

Thomas, Philadelphia, PA) through a 2-mm mesh screen and dried in a 55°C oven. Diets fed consisted of 50 % forage and 50 % grain mix and were fed at rates of 100 g DM/d

(Table 4.1). The alfalfa hay was 32.2 % NDF, 20.8 % ADF, and 20.2 % CP. The control diet was formulated using the CPM Model, v.1, (1998) to contain more than the recommended concentration of RDP, so that inadequate degradable protein would not be a factor in the potential response to methionine supplements.

Continuous Culture Operation

The experiment was conducted in four 10 d periods, with each period consisting of 7 d for adaptation followed by 3 d of sample collection. The dual flow continuous culture system used in this study was similar to that described by Hoover et al. (1976) and modified by Hannah et al. (1986). Diets were fed automatically throughout the day to provide steady state conditions. Volumes of the four fermenters were between 1700 and 1800 ml. The liquid and solids dilution rate were maintained at 12 % and 5.5 %/h, respectively by regulation of filtrate removal rates and buffer input. The pH was

maintained at 6.2 ± 0.1 by automatic administration of 5 N NaOH or 3 N HCl. Agitation

was set at 160 rpm, and temperature was maintained at 39°C. The fermenters were

continuously purged with N2 to maintain anaerobiosis. Ruminal inoculum was obtained

from two dry, ruminally fistulated Holstein cows receiving the control diet (no additional

Met). Inoculum from both cows was pooled and then divided among the four fermenters.

Ruminal contents were squeezed through two layers of cheesecloth and placed in

fermenters within 20 min of collection. Acid and alkali use, temperature, filtrate flow,

agitation, and pH were recorded and recalibrated, if needed, every 6 h. The solid and

liquid effluents were weighed once daily to determine flow rates.

Sample Collection and Analysis

15 On d 5, 10 % enriched ( NH4)2SO4 was added to the fermenters for use as a

microbial marker. A sample of effluent was taken prior to the primed, continuous

infusion for background 15N analysis. One daily sample of the effluent was taken on d 8,

9, and 10 and composited by fermenter for analysis. All samples were immediately frozen at –20º C. Bacteria were separated from feed particles using a Waring blender and straining through two layers of cheesecloth. Differential centrifugation was used to first separate the bacteria from the feed (15 min at 500 X g) and then separate the bacteria from the liquid supernatant (15 min at 23,300 X g). Samples remained frozen until analysis. Effluent samples were analyzed for N using the Kjeldahl method and for ADF and NDF (Van Soest, 1991). Samples were acidified using 3 ml of 6 N HCl per 50 ml of sample to stop fermentation prior to analysis for VFA (Firkins et al., 1990) and NH3-N

(Chaney and Marbach, 1962). VFA production was calculated using effluent flow X

concentration of each VFA. Sub-samples of effluent to be used for 15N analysis were

raised to a pH of approximately 9 using 25 % NaOH to volatilize ammonia from the

sample. Bacterial and effluent samples were analyzed for 15N by The Stable Isotope

Laboratory (Utah State University, Logan). The analyses were performed by continuous-

flow direct combustion and mass spectrometry using a Europa Scientific SL-2020 (PDZ

Europa, Chesire, England) system. Nitrogen-15 in ammonia samples was determined at

the University of Illinois (Firkins et al., 1992).

DNA extraction and PCR. Three samples per period were collected directly from the

fermenters and frozen at –80°C. Total genomic DNA was extracted from the thawed composite samples using the bead beating method (Yu and Mohn, 1999) followed by purification of the DNA using a QIAamp column (Qiagen, Valencia, CA). The DNA samples were used in PCR amplification with primers S926f (5'-

CTYAAAKGAATTGACGG-3') and L189r (5'-TACTGAGATGYTTMARTTC-3') as

described by Yu and Mohn (1999). The resultant PCR products (amplicons) contain the

complete RIS and parts of the flanking rDNA, (ca. 600 bp of 16S rDNA and 190 bp of

23S rDNA). The PCR conditions were as follows: initial denaturation at 94°C for 3 min,

annealing at 45°C for 1.5 min, and extension at 72°C for 2.5 min. Subsequent cycles

consisted of a 1.5-min denaturation step at 94°C, a 1.5-min annealing step at 45°C, and a

2-min extension step at 72°C. After 30 cycles, there was a final 7-min extension step at

72°C.

Phylogenetic analysis based on RIS-LP. Ribosomal DNA-RIS fragments amplified by

PCR were separated on a 4% polyacrylamide (37:1) gel, and the gel was stained with

GelStar nucleic acid stain (BioWhittaker, Inc., Walkersville, MD). The RIS-LP banding

patterns were documented using a FluorChem Imaging System (Alpha Innotech

Corporation, San Leandro, CA). The gel image was then exported into the GelCompar II

analysis software in the BioNumerics package (BioSystematica, Devon, UK) for cluster

analysis and dendrogram construction. The UPGMA method in the software was used for

dendrogram construction.

Statistical Analysis

Data were analyzed using Proc GLM of SAS (1999) according to the following model:

Yijk = µ + Ti + Fj + Pk + eijk

where:

Yijk is the dependent, continuous variable,

µ is the overall population mean,

th Ti is the fixed effect of the i treatment (i = 1, 2, 3, 4),

th Fj is the fixed effect of the j fermenter (j = 1, 2, 3, 4),

th Pk is the fixed effect of the k period (k = 1, 2, 3, 4), and

2 eijk is the residual error, assumed independent and ~ N(0, σ e ).

Two pre-planned contrasts were used to determine linear or quadratic response to 0,

0.055, and 0.110 % of HMB, and a third compared the effects of similar concentrations

of methionine supplied as dl-Met (0.097 %) vs. HMB (0.110 %). The Proc GLM

procedure of SAS was used for regression analysis of data having significant quadratic

effects to estimate peaks or nadirs.

Results and Discussion

Digestibility of nutrients

The alfalfa hay was lower in fiber than initially anticipated, causing the overall diet NDF and ADF concentration to be lower than expected. Low fiber concentration in the diet should not be an issue with the automatic buffering system used in this experiment. Digestibilities of hemicellulose, NDF, and true OM were not significantly affected by HMB addition (Table 4.2). Digestibility of ADF was affected quadratically by concentration of HMB. The minimum digestibility, determined by the first derivative of the estimated quadratic function, was at 0.047 % of HMB supplementation. Others have reported no changes in digestibility with addition of HMB (Vazquez-Anon et al.,

2001; Patterson and Kung, 1988) in vitro, whereas some have noted increased digestibility of crude fiber (Holter et al., 1972) in lactating cows and increased cellulose digestibility in vitro (Gil et al., 1973a).

Digestibility of ADF was decreased for the dl-Met treatment compared with the diet containing the equivalent amount of Met supplied as HMB. Salisbury et al. (1971) found that methanethiol production was four times greater for dl-Met than HMB, indicating that dl-Met may be broken down more quickly and become less effective than

HMB with time. Bach and Stern (1999) showed that dl-Met is one of the most degradable AA, with free Met in the rumen contributing less than 2 % to total duodenal flow of Met in high yielding cows (Volden et al., 2001). Both dl-Met and HMB were supplied continuously to the fermenters in our trial. This may have synchronized the

degradation and uptake rates, decreasing the benefit of a slower degraded source compared with discontinuous feeding.

Nitrogen fluxes and Ammonia utilization

According to the NRC (2001), the control diet would supply 3319 g/d of RDP if fed to a 650-kg lactating cow producing 45 kg of milk per day with a DMI of 25.9 kg/d.

The suggested requirement for RDP is 2516 g/d. Thus, our diet had an approximate excess of RDP of 32 %, and therefore should not be limiting for microbial growth.

Nitrogen fluxes and ammonia utilization are reported in Table 4.2. Addition of Met as either HMB or dl-Met had no effect on effluent flows of NH3-N, NAN, bacterial N, non- ammonia-non-bacterial nitrogen (NANBN), or NANBN as a percentage of N intake. If

Met acts as a growth stimulant to bacteria in a situation in which Met is rate-limiting, an increase in bacterial N flow would be expected. Total RUP flow would either increase or remain the same. Kajikawa et al. (2002) found that Met did not increase microbial growth rate or efficiency when added alone to mixed ruminal bacteria cultured in vitro.

However, it was one of seven AA that were stimulatory when supplied together. All AA were provided in isonitrogenous proportions, and potential interactions were not considered. Gil et al. (1973a) found NH3-N concentrations in vitro were lower with Met supplemented as dl-Met or HMB over a control, whereas Bach and Stern (1999) found increased NH3-N and decreased NAN in continuous culture with supplemental Met.

Vazquez-Anon et al. (2001) found no effects of HMB on NH3-N or NAN flows in continuous culture. Our results showed no difference in bacterial N flow from HMB supplementation, which is contrary to the findings of Vazquez-Anon (2001) and Gil et al.

(1973a), who found increases in microbial CP synthesis with HMB, but results are in

agreement with those of Bach and Stern (1999), who found no changes with dl-Met.

Patton et al. (1970a) reported an increased flow of lipids that were serving as structural

components related to microbial growth. They believed most of the additional lipid was

associated with rumen protozoa. Because protozoa rapidly wash out of continuous

culture fermenters of the type we used (Mansfield et al., 1995), effects on protozoa could not be determined. This may be why the expected increase in microbial protein has not been found with continuous culture systems (Bach and Stern, 1999) but has been found in in vitro systems without outflow such as those used by Patton et al. (1970a) and Gil et al.

(1973a). Nitrogen outflows, expressed as a percentage of N intake, and microbial efficiency were not significantly different among the four treatments.

Ammonia-N concentrations were not different among the four treatments.

However, there was a significant linear decrease (P = 0.032) in the amount of bacterial N obtained from NH3-N with amounts of HMB supplementation. Because of the quadratic

effect of HMB concentration (P = 0.054), regression analysis was done and indicated that

0.0735 % HMB supplementation of the diet would minimize the proportion of bacterial N

obtained from NH3-N. There was also a trend (P = 0.10) for the bacteria receiving dl-

Met to obtain a greater proportion of their N from ammonia than those receiving a similar

amount of Met from HMB.

Volatile fatty acids

Volatile fatty acid concentration and total production are presented in Table 4.3.

Acetate concentration was not affected by HMB supplementation. Acetate concentration

was higher in the dl-Met treatment than with HMB. Propionate decreased linearly in concentration from HMB supplementation but a quadratic trend (P = 0.13) was noted for production (data not shown), which was estimated to peak at 0.0470 % HMB. These changes were apparent in the acetate:propionate ratio, which showed a significant quadratic effect of HMB concentration. The lowest estimated acetate:propionate ratio would occur at 0.037 % HMB. This calculated ratio of 3.35 is similar to that found at 0 and 0.055 % when standard errors are considered. This indicates that HMB had basically no effect until more than 0.055 % was fed, when a large increase in ratio occurs.

Butyrate and isovalerate concentrations were not different. There were trends (P < 0.15) for a linear increase in production for isobutyrate and isovalerate (data not shown) in our trial with increasing concentration of HMB, and isobutyrate concentration increased linearly. Valerate concentration was affected quadratically by HMB, with estimated peak production at 0.0554 %.

No consistent trends in VFA production have been observed from adding HMB.

Acetate, propionate and butyrate concentrations have shown no changes in several studies

(Lundquist et al., 1985; Windschitl and Stern, 1988; Vazquez-Anon et al., 2001).

Researchers have reported increases (Lundquist et al., 1983) and decreases (Gil et al.,

1973b) in acetate concentration and decreases in propionate concentration (Lundquist et al., 1983; Gil et al., 1973b). Butyrate concentration usually decreased (Gil et al., 1973b) or was not changed (Lundquist et al., 1983). Valerate (Windschitl and Stern, 1988), isobutyrate, and isovalerate (Lundquist et al., 1983; Lundquist et al., 1985) concentrations were unchanged. Volatile fatty acid changes may be dependent on the populations of

microbes in the cows. This could vary between experiments due to diet and other factors.

Maintenance of pH in our system may also have influenced VFA profile when compared with in vivo data.

Relationship of VFA Metabolism to N Synthesis

Changes in VFA production from HMB supplementation are indicative of an effect on rumen microorganisms. Possible causes for these effects are: (1) microbial populations are changing, (2) different biochemical pathways are becoming more favored, or (3) a combination of both. Three of the predominant cellulolytic rumen bacteria, Fibrobacter succinogenes, Ruminococcus flavefaciens, and R. albus require one or more of the branched-chain fatty acids (BCVFA) isobutyric, 2-methylbutyric, or isovaleric acids for the synthesis of valine, isoleucine, and leucine via reductive carboxylation and transamination of the fatty acid (Wallace, 1997). The decreased usage of these VFA for AA synthesis, indicated by increased production of isobutyrate and isovalerate with a decrease in utilization of NH3-N for bacterial N synthesis, could indicate a decrease in the population of microbes normally dependent on BCVFA for production of AA. A limitation of BCVFA growth factors seems unlikely, however, because fiber digestibility was not impaired in our study, and, in fact, ADF digestibility was numerically increased by HMB at 0.110 %. It seems more likely that microbes were preferentially using other pathways to synthesize AA when HMB was infused in the fermenter. Met is normally synthesized using a pathway starting with oxaloacetate, an intermediate in the biosynthesis of many AA (Brock et al., 1994). Providing a readily available but slowly degradable source of Met may allow the cell to spare oxaloacetate

for production of energy or de novo synthesis of other AA. Increased availability of

oxaloacetate would also increase production of succinate, which is an intermediate step in

the production of propionate (White, 2000). We observed a quadratic trend in propionate

production with an estimated peak at 0.047 % HMB in our study. These changes applied

to an in vivo system could help to explain some of the effects on milk yield and

composition commonly seen with feeding of HMB. Differences in VFA production may

increase milk fat production and sparing of oxaloacetate could improve AA balance by

providing more substrate for production of microbial CP.

RIS-LP analysis

RIS-LP followed by cluster analysis based on pair-wise comparison of the

banding patterns obtained from gel electrophoresis of rDNA-RIS amplicons provides a

reliable and fast method for comparison of microbial community profiles in

environmental samples. Distinct RIS-LP banding patterns were obtained from gel

electrophoresis of the PCR amplicons (Figure 1). The banding patterns showed that there

were several distinct bands in each sample, including two to three major bands. These

major bands represent the species that are the most abundant, possibly Butyrivibrio

fibrisolvens, Megasphaera elsdenii, Prevotella ruminicola, and/or Selenomonas ruminantium. Cluster analysis showed that the treatments tended to group together, but no distinct pattern was detected because samples taken in the same period tended to cluster together irrespective of the treatment group from which the samples were collected, suggesting that period had a strong influence on bacterial community structure.

Because each period was initiated by a new inoculation and the fermentations were

controlled for pH and continuous feeding of highly processed feeds, the period effect might have hidden potential treatment effects. Alternatively, the treatments used in this experiment might not have caused a major shift in the community profile. Minor alterations in the community profile might be masked by the presence of large bands generated from species of bacteria which are usually present in large numbers in the inoculum. The use of genera- and species-specific primers would allow more specific monitoring of the minor population changes.

Conclusions

Overall, the supplementation of HMB and dl-Met showed some affects on rumen bacterial growth and production of VFA in continuous culture. The lack of major effects may indicate that overfeeding of RDP from the diet provided enough excess AA so that

Met was not limiting, the changes in bacterial populations were small and were concealed by the larger unaffected populations of common species of rumen bacteria, or that there may be a pronounced effect on protozoa in the rumen that would not be seen with continuous culture fermenters.

% of DM Ingredient Alfalfa hay 50.0 Ground, shelled corn 30.9 Barley 10.8 Soybean meal, 48 % CP 4.4 Distillers dried grains 2.7 Trace mineralized salt 0.49 Dicalcium phosphate 0.31 Magnesium sulfate 0.25 Magnesium oxide 0.12 Vitamin A (30,000 IU/g) 0.05 Vitamin D (3,000 IU/g) 0.06 Vitamin E (20,000 IU/lb) 0.01 Selenium 90 (90 ppb) 0.02

Nutrients NDF1 23.4 Forage NDF 15.5 ADF 11.7 CP 16.5 RDP 12.8 RUP 4.9 NEl (Mcal/kg) 1.54 1NDF, ADF and CP are averages of seven assays in our laboratory. RDP, RUP and NEl were estimated using the NRC (2001) assuming a BW of 650 kg, 45 kg/d milk, and 25.9 kg/d DMI.

Table 4.1. Ingredients and nutrient composition of control diet.

HMB (%) Contrasts

0 0.055 0.110 dl-Met SEM Linear2 Quadratic HMB vs dl-Met3

Digestibility (%) NDF 45.6 42.9 49.1 43.2 2.69 NS4 NS 0.167

ADF 44.7 40.9 47.8 39.4 1.90 NS 0.061 0.021

Hemicellulose 46.7 45.3 50.8 47.8 4.21 NS NS NS

True OM 45.0 41.1 45.1 39.6 3.49 NS NS NS

Nitrogen flows

NH3-N (g/d) 0.24 0.29 0.24 0.21 0.05 NS NS NS

NAN (g/d) 2.39 2.54 2.38 2.66 0.15 NS NS NS

NAN (% of N intake5) 90.6 96.6 90.2 100.9 5.55 NS NS NS

Bacterial N flow (g/d) 1.31 1.39 1.36 1.44 0.070 NS NS NS

NANBN (g/d)6 1.08 1.16 1.02 1.22 0.090 NS NS 0.192

NANBN (% of N intake) 41.0 43.8 38.8 46.2 3.56 NS NS 0.192

Ammonia-N (mg/dl) 6.04 7.03 6.10 5.13 1.26 NS NS NS Proportion of bacterial N from NH3-N 0.70 0.60 0.62 0.68 0.021 0.032 0.054 0.101

Microbial efficiency7 31.5 37.2 33.6 40.5 4.61 NS NS NS 1 HMB = 2-hydroxy-4-(methylthio) butanoic acid. 2Linear and quadratic contrasts used 0, 0.055 and 0.110 % HMB diets. 30.110 % HMB diet vs dl-Met diet 4NS = non significant; P > 0.20. 5N intake was intake from feed sources and did not include N from the buffer solution. 6NANBN = non-ammonia non-bacterial nitrogen. 7g microbial N produced/kg OM truly digested.

Table 4.2. Nutrient digestibility, nitrogen fluxes, and ammonia utilization in continuous culture fermenters supplemented with two concentrations of HMB1 or dl-Met.

HMB (%) Contrasts HMB vs dl- 0 0.055 0.110 dl-Met SEM Linear2 Quadratic Met3 Volatile Fatty acids (mol/100 mol) Acetate 66.7 66.7 66.9 70.1 0.79 NS4 NS 0.031 Propionate 19.4 19.8 15.9 17.0 0.76 0.017 0.059 NS Butyrate 13.2 12.5 15.3 12.9 1.10 NS NS NS Valerate 2.26 2.41 2.26 2.23 0.026 NS 0.003 NS Isovalerate 1.1 1.4 1.4 1.4 0.15 NS NS NS Isobutyrate 0.36 0.38 0.43 0.39 0.016 0.023 NS NS Acetate:Propionate 3.57 3.40 4.22 4.26 0.14 0.017 0.028 NS Total outflow (mmol/d) 343 374 358 329 25.9 NS NS NS 1 HMB = 2-hydroxy-4-(methylthio) butanoic acid. 2Linear and quadratic contrasts used 0, 0.055 and 0.110 % HMB diets. 30.110 % HMB diet vs dl-Met diet. 4NS = non significant; P > 0.20.

Table 4.3. Volatile fatty acid total production and concentration in continuous culture fermenters supplemented with two concentrations of HMB1 or dl-Met.

0 10 80 60 40 20

P3 0%

P3 0.055 %

P4 0.110 % P1 0 %

P4 0 %

P1 0.055 % P2 0.055 %

P1 dl - P2 0 % HMB

P3 dl -Met

P4 dl-Met

P3 0.110 % P4 0.055 %

P1 0.110 %

P2 0.110 % P2 dl-Met

100- bp DNA ladder

100- bp DNA

Figure 4.1. Comparison of the bacterial community structure in fermenter samples; dendrogram shows cluster analysis performed based on percent similarities of the communities. P = Period 1 to 4; HMB = 2-hydroxy-4-(methylthio) butanoic acid. The length of scale depicts the percent similarity between different lanes.

CHAPTER 5

DETERMINATION OF UNDEGRADABILITY AND RUMINAL EFFECTS OF HMB,

HMBi, AND DL-METHIONINE IN LACTATING COWS

Abstract

The effects of Met provided as HMB, HMBi, and dl-Met were examined. Eight cows were used in a replicated 4 X 4 Latin square design. Effects on milk composition and yield, N utilization, VFA, and protozoa were determined. Samples of omasal fluid were used to determine the amount of Met supplements passing out of the rumen. Treatments were: (1) no methionine (Control); (2) 2-hydroxy-4-methylthiobutanoic acid (HMB) at

0.10% of DM; (3) isopropyl HMB (HMBi) at 0.13 % of DM; and (4) dl-methionine (dl-

Met) at 0.088% of DM. Diets were identical except for type of Met supplement, and were based on corn silage and alfalfa hay. The three supplemented diets were iso-Met.

Dry matter intakes were restricted during the week of sampling to 95% of ad-libitum determined during the first 2 wk of the period. Dry matter intakes were not different and averaged 20 kg/d. Milk yields averaged 37.7 kg/d and were not different among treatments. Milk protein concentration (2.91, 2.95, 3.02, 2.96%) and fat concentration

(3.34, 3.12, 3.51, 3.69%) are reported here for control, HMB, HMBi, and dl-Met. Milk protein concentration was significantly increased with the HMBi treatment. Milk fat concentration was similar for all treatments (3.43%). Rumen VFA profile and NH3 97

concentrations were similar. Ruminal digestibility of OM and NDF were not different,

although OM and NDF digestibilities were numerically higher in the HMBi diet. In situ

rate of digestibility of CP was affected by Met source in alfalfa hay, TMR, and corn

silage. In situ digestibility of NDF was affected in TMR and alfalfa hay. Passage rates

of small particles (0.071/h) and fluid (0.167/h) were not affected by treatment. Protozoa

were increased numerically in the omasum by HMB and HMBi treatments. Proportion of

omasal N from bacterial N was not different (0.64), and g of bacterial N flow were

similar between treatments. The percentage of HMB that passed into the omasum was

5.3%. This, along with numerical protozoal and in situ digestibility changes, indicated

that most of the activity of HMB was in the rumen. Only a small amount of HMBi was

found as HMB in the omasum (2.3%).

Abbreviation key: dl-Met = dl-methionine, HMB = 2-hydroxy-4-methylthio (butanoic)

acid, HMBi = isopropyl-2-hydroxy-4-methylthio (butanoic acid), MUN = milk urea nitrogen, TMR = total mixed ration, UN = urinary nitrogen.

Introduction

The Environmental Protection Agency (EPA) has issued regulations regarding nitrogen release into the environment, and more regulations will likely follow (Powers,

2003). To remain competitive and socially acceptable in the future, the dairy industry must be proactive in reducing nitrogen release into the environment.

The N in waste can be increased or decreased by diet (Dinn et al., 1998; St-Pierre and Thraen, 1999). Diets that are formulated correctly for amino acids can potentially

maintain and even improve milk yield and milk component production, while reducing the amount of nitrogen released into the environment (Noftsger and St-Pierre, 2003).

2-hydroxy-4-(methylthio) butanoic acid (HMB) can be used as an inexpensive source of Met (Dibner and Knight, 1984). Many studies using HMB have shown increases in milk fat production (Holter et al, 1972; Huber et al., 1984; Patton et al.,

1970) and percentage (Huber et al., 1984; Lundquist et al., 1983), although some reported no effect (Hutjens and Schultz, 1971; Whiting et al., 1972; Stokes et al., 1981).

Most researchers found no effect on milk protein (Hansen et al., 1991; Hutjens et al., 1971; Stokes et al., 1981), although research in our lab (Noftsger et al., 2003) showed an increase in milk protein yield that was greater than what could be expected from the additional supply of metabolizable Met when Met was fed in rumen protected

(Smartamine) and degradable forms (HMB). The degradable source of Met (HMB) may have a different mechanism of action, possibly through a stimulation of microbial growth.

The lack of response in milk protein yield and milk yield in most trials may indicate that

HMB is not escaping rumen microbial catabolism in significant concentrations to be utilized post-ruminally. Attempts to ascertain the degradability of HMB have provided widely varied answers, from 99% degradable (Jones et al., 1988) to 50% (Koenig et al.

1999).

The changes in fat production and percentage may not be due to post-ruminal supplementation of Met. Patton et al. (1970) suggested that HMB caused an increase in the protozoal biomass. Methionine is a methyl donor for the production of phosphatidylcholine, and protozoa are the primary suppliers of phosphatidylcholine,

because free choline is rapidly degraded in the rumen (Sharma and Erdman, 1988b).

Phosphatidylcholine is important for packaging of fatty acids into very low-density

lipoproteins and chylomicrons (Yao and Vance, 1988). The 18C and some of the 16C

units of fat in milk come from the triglycerides of chylomicrons and low-density

lipoproteins in the blood (Schmidt, 1971). Increases in protozoal passage would provide

more digestible microbial protein (Williams and Coleman, 1992) with a higher

concentration of lysine than bacteria (Bergen et al., 1968).

The isopropyl ester of HMB (HMBi) has been shown to have approximately 50% bioavailability based on blood kinetics of a pulse ruminal dose (Robert et al., 2001) or a cow bioassay using milk true protein concentration as a bioavailability index (Schwab et al., 2001). In a trial conducted in our lab (Sylvester et al., 2003a), HMBi increased milk and protein production and protein content while reducing the amount of N excreted.

The hypotheses of this trial were that (1) HMB is primarily a rumen degradable source of Met, and its positive effects are due primarily to stimulation of microbial growth in the rumen, (2) a significant portion of HMBi escapes ruminal breakdown and is used for microbial protein growth in the rumen plus a source of metabolizable Met, and

(3) dl-Met is primarily rumen degradable, and will not supply Met post-ruminally.

Materials and Methods

Animals and Experimental Design

Eight ruminally cannulated Holstein cows were assigned to a replicated 4 X 4

Latin square. Cows were blocked by calving date and were assigned to the experiment

100

between 39 and 84 DIM. One cow was replaced after the first period due to health

problems. There were two primiparous and two multiparous cows in each replicate. The

dietary treatments were: (1) no methionine (Control); (2) 2-hydroxy-4-

methylthiobutanoic acid (HMB) at 0.10% of DM; (3) isopropyl HMB (HMBi) at 0.13 %

of DM; and (4) dl-methionine (dl-Met) at 0.088% of DM. The amount of Met supplied

by each treatment diet was calculated to be 22 g/d at 25 kg/d DMI. Experimental periods

consisted of 28 d, with d 1 through 14 serving as an adjustment period, d 15 through d 20

as an adjustment period to restricted intake and 12 times per day feeding, and d 21

through d 28 was used for collection of data. Cows were housed in a conventional tie-

stall barn with mattresses. They were allowed access to a concrete lot before milking,

except during the 2 wk of restricted intake. Cows were milked at 0600 and 1700 h.

Diets were mixed once daily as a total mixed ration (TMR) and fed twice daily during

the adjustment period. Treatment premixes were mixed by hand into individual TMR.

Orts were measured daily at 1600 h during the adjustment weeks, and the amount of feed

offered was adjusted for a target of 10% refusal. Starting on d 15, cows were restricted to

95% of their respective ad-libitum intake determined during the prior 2 wk of adjustment and placed on automatic feeders (Ankom technology, Macedon, NY). Cows received approximately 1/12 of their daily feed allowance every 2 h. The amount of feed was adjusted daily in an attempt to assure no refusals during the week of sampling. The cows remained on the automatic feeders through the fourth week of each period. Cows were fed TMR containing (DM basis) 30% corn silage, 13% alfalfa hay, 12% whole cottonseed, 43.15% concentrate and minerals, and 1.85% treatment premix (Table 5.1).

101

Care and handling of the animals was conducted as outlined in the guidelines of The

Ohio State University Institutional Animal Care and Use Committee.

Sampling and Laboratory Analysis

Ingredient components of the TMR were sampled every wk. Total mixed ration

(control) was sampled daily and composited by week. Total mixed ration and component

samples were analyzed by DHI Forage Testing Laboratory (Ithaca, NY). Milk samples

were taken at four consecutive milkings on d 16 through d 18 and d 23 through d 25 and

analyzed by DHI Cooperative (Columbus, OH) for milk fat and true protein by infrared

spectroscopy and for milk urea nitrogen (MUN) using a Skalar SAN Plus segmented

flow analyzer (Skalar, Inc., Norcross, GA). Total milk N was calculated as (milk true

protein/6.38)/0.9375 to account for the milk NPN (Mackle et al., 1999; Barbano et al.,

1992). Daily milk fat and protein yields were calculated and averaged by cow per period.

Cows were weighed once weekly prior to the afternoon milking.

Chromic oxide was used as a marker for total omasal flow (Williams et al., 1962).

Chromium pellets (5% chromic oxide, 95% soyhulls) were dosed at 1.1% of the daily

DMI starting on d 16. Cobalt-EDTA (Uden et al., 1980) and YbCl3 (Hristov and

Broderick, 1996) were used as markers to assess fluid flow and small particle flow, respectively, at the omasal canal. Ytterbium chloride was also used to determine total output of feces. Nitrogen-15 was used as a marker for microbial N flow. Starting on d

15 19, 10 % enriched ( NH4)2SO4 was mixed into the rumen 3X daily along with Co-EDTA for use as a microbial marker. A sample of omasal fluid was taken prior to the first dose for background 15N analysis. Bacterial and omasal samples were analyzed for 15N by The

102

Stable Isotope Laboratory (Utah State University, Logan). The analyses were performed by continuous-flow direct combustion and mass spectrometry using a Europa Scientific

SL-2020 (PDZ Europa, Chesire, England) system. Cobalt was dosed at approximately

0.5 g/day. The Co dose was mixed with 200 ml of water. Cobalt and Yb daily doses were divided into 3 equal doses placed directly into the rumen at 8-h intervals with hand mixing. The Yb-labeled feed was fed at approximately 225 g/d (as-fed). Dosing began

3d prior to first sampling (d19). Concentrations of Co (Firkins et al, 1986) and Yb (Uden et al., 1980) were analyzed using atomic absorption spectrophotometry. Background omasal and rumen samples for Co and Yb analysis were obtained on d 15.

Digesta flow leaving the rumen was quantified using the system of alternating vacuum and pressure for collecting digesta from the omasal canal that was originally developed by Huhtanen et al. (1997) and modified by Ahvenjärvi et al. (2000). Using this method, 500-ml samples were collected from a tube passed through the ruminal cannula and positioned in the omasal canal. Samples were taken four times daily at 2-h intervals on d 22 to 24 of the period. From each omasal sample, a 40-ml representative sample was diluted with an equal amount of 2% saline and refrigerated. These samples were composited by cow and period and frozen to be later analyzed by HPLC for HMB,

HMBi, and free Met (Adisseo, Commentry, France). Samples were taken so that the entire 24-h cycle was represented. Every 4 h, a 40-ml aliquot of omasal fluid was retained for protozoal counts. The six samples were mixed 1:1 with 50% formalin, and were later composited by cow and period. Protozoa were counted using a 1-ml counting chamber (Dehority, 1993). For count data, normality assumptions of residuals were

103

tested using Proc Univariate (SAS, 1999) with the Kolmogorav-Smirnov test. Normality of the residuals allows statistical analysis without transformation of the data. The omasal samples were composited by cow for each sampling day. The omasal samples were frozen, then later thawed and composited by cow and period, refrozen, and freeze-dried for marker and nutrient analysis. Fecal samples for digestibility were obtained during the omasal sampling period. Fecal grab samples were taken on d 22 through d 24 at 4-h intervals (corresponding to every other omasal sample). This represented the entire 24-h period (6 total samples). Samples were composited and dried in a forced air overn at

55°C for 48 to 60 h. Ytterbium concentration in the feces was used to determine total fecal DM.

Core samples of ruminal contents from 10 different sites in the rumen were removed at four different time points (3 h apart) on d 26. Contents were strained through two layers of cheesecloth, and pH of the fluid was measured immediately. A 50-ml aliquot of the filtered ruminal fluid was acidified with 3 ml of 6 N HCl to stop fermentation and frozen. After thawing, the acidified ruminal fluid was mixed, centrifuged at 15,000 X g at 4ºC for 15 min, and then filtered through Whatman number

1 filter paper (Whatman, Clifton, NJ). The supernatant was analyzed for VFA concentrations by gas chromatography (Pantoja et al., 1994) and for NH3N (Chaney and

Marbach, 1962).

Passage rates were determined on d 25 and d 26 using Co-EDTA for liquid and

Yb-labeled feed for small particle passage. The Co-EDTA and Yb-labeled feed were continued at the same schedule and dose as for the omasal samples until 0630 h on d 25.

104

Rumen samples for cobalt analysis were strained through two layers of cheesecloth; a

100-ml aliquot of fluid was frozen immediately. Samples used for the estimation of

liquid passage rate were obtained from 10 locations in the rumen at 0.5, 1, 2, 4, 6, 9, 12,

18, 24, and 36 h after last dose. Whole rumen digesta samples (approximately 500g)

used for the estimation of small particle passage rate were obtained at 0.5, 2, 4, 9, 18, 24,

and 36 h after the last dose. Rumen samples were immediately dried at 55ºC. Rumen

protozoa samples were taken every 4 h on d 25 and d 26 to cover the entire 24-h cycle.

These samples were treated similarly to the omasal protozoa samples. Any additional

rumen contents were returned to the cows. Rumens were completely evacuated on d 27

at 0800 h and d 28 at 1200 h. Solids and liquids were separated using a hydraulic wine press set at 17 Newtons/cm2, weighed, sampled, and placed back into the cow within 25

min. Liquid and solid fractions were sub-sampled, with a representative sample

reconstituted using the weight of each fraction.

The control TMR, alfalfa hay, and corn silage were ground to 2 mm and

suspended in the rumen in nylon bags. Each bag contained 4 g, and was duplicated at

every time point. The bags were removed at 0, 2, 4, 8, 12, 24, 48, and 96h. Incubations

were done on four cows from the first square during each of the four periods. Samples

were duplicated for each feed at each time point. Duplicates within cow, feed and time

point were composited before analysis for NDF and protein.

The omasal samples, in situ kinetics samples, and rumen samples were analyzed

for neutral detergent fiber using amylase (Van Soest et al., 1991) and Kjeldahl N (AOAC,

1990).

105

Statistical Analysis

Data were analyzed as a replicated 4 X 4 Latin square using SAS Proc Mixed

(1999) according to the following model:

Yijkl = µ + Ti + Pj:k + Sk + cl:k + eijkl

Where:

Yijkl = the dependent variable

µ = overall mean

Ti = the fixed effect of the ith treatment, i = 1,…,4

th th Pj:k = the fixed effect of the j period within the k square , j = 1,…,4

th Sk = the fixed effect of the k square, k = 1, 2

th 2 cl:k = the random effect of the l cow within square ~ N (0, σ c), l = 1,…,4

2 eijkl = the random residual ~ N (0, σ e).

Mean separation was done using Fisher’s protected least significant difference (LSD).

In situ data were analyzed using the NLIN procedure of SAS (SAS, 1999)

according to the following model:

Yijkl = (A + ADi + APj) + ACk) + εijkl

if t ≤ Li [1]

Yijkl = (A + ADi + APj + ACk) + (B + BDi + BPj + BCk) x

(1 – EXP (- (kd + kDi + kPj + kCk) x (t – (L + Li))) + εijkl otherwise,

subject to: ΣADi = 0 ΣBCk = 0

ΣAPj = 0 ΣkDi = 0

106

ΣACk = 0 ΣkPj = 0

ΣBDi = 0 ΣkCk = 0

ΣBPj = 0 ΣLi = 0, where:

A is the estimated A pool across diets, cows, and periods,

th ADi is the effect of the i diet on the A pool (i = 1, …, 4),

th APj is the effect of the j period on the A pool (j = 1, …, 4),

th ACk is the effect of the k cow on the A pool (k = 1, …, 4),

B is the estimated B pool across diets, cows, and periods,

th BDi is the effect of the i diet on the B pool,

th BPj is the effect of the j period on the B pool,

th BCk is the effect of the k cow on the B pool,

kd is the fractional degradation rate pooled across diets, cows, and periods,

th kDi is the effect of the i diet on the fractional degradation rate,

th kPj is the effect of the j period on the fractional degradation rate,

th kCk is the effect of the k cow on the fractional degradation rate,

L is the lag time (hours) pooled across diets, cows, and periods,

th Li is the effect of the i diet on lag time, and

2 εijkl is the residual error, approximately N (0, σ e ).

107

Model [1] is equivalent to the lag model of Orskov and McDonald (1979) but with parameter estimation explicitly accounting for the structure of the data (e.g., Latin square with repeated measurements within cells).

In its unconstrained form, model [1] is overparameterized. Constraints (or restrictions) must be imposed on the parameters to get an estimable set. This is not unique to this nonlinear model. Restrictions on discrete (class) parameters are automatically implemented by statistical software used to fit linear models (e.g., GLM procedure of SAS) or linear mixed models (e.g., MIXED procedure of SAS). Tests of the significance of each set of parameters in model [1] were conducted by fitting a full and reduced model and calculating an F statistic based on the reduction in the error sum of squares (Damon and Harvey, 1987). This is also called the extra sum of squares principle

(Draper and Smith, 1998). Standard errors of parameters and standard errors of parameter differences were calculated using the asymptotic variance-covariance matrix.

Overall significance was declared at P ≤ 0.05. Mean separation was done using Fisher’s protected least significant difference (LSD), with the LSD statistic calculated from the asymptotic variance-covariance matrix of parameter estimates.

Effective ruminal degradabilities (ERD) were calculated from parameter estimates of model [1] for each cow-period-diet according to the following equation:

ERD = (A + ADi + APj + ACk) + (B + BDi + BPj + BCk) x

[(kd + kDi + kPj + kCk) / (kd + kDi + kPj + kCk + kp)] x [2]

[exp (-kp x (L + Li)]

108

where kp is the fractional rates of passage of each incubated feedstuffs, calculated using

NRC (2001) estimates for alfalfa hay (0.0434/h), corn silage (0.0557/h), and TMR

(0.0637/h).

The resulting ERD were analyzed according to a Latin square design as a mixed model with the fixed effects of diets and periods and the random effects of cows using the

MIXED procedure of SAS (SAS, 1999).

Results and Discussion

Lactation Performance

Least squares means for production and intake are reported in Table 5.2. Dry matter intake was not affected by treatment. The overall DMI (20.0 kg/d) was lower than what would be expected of Holstein cows producing 37.5 kg/d (NRC, 2001) because of the intake restriction. In order to prevent orts, some cows were restricted to less than

95% of ad libitum intake measured on d 1 through d 14. Body weights were similar between treatments (Table 5.2), averaging approximately 590 kg. Milk production averaged 37.5 kg/d and was not significantly different across treatment. Fat concentration and yields were not significantly different among treatments. Many experiments have reported an increase in fat concentration with HMB (Holter et al, 1972;

Huber et al., 1984; Patton et al., 1970). However, the Latin square design used in this experiment was not designed for elucidating production responses, because of the short period of time cows received each diet and possible carry-over effects. However, responses in milk protein content to Met supplementation can generally be observed

109

within two weeks (Sylvester et al., 2003a). Smaller labile body reserves of protein as compared with fat decrease carryover effects. Milk protein concentration increased on the HMBi diet compared with the control and HMB diets, although the protein yield for

HMBi was not different. Sylvester et al. (2003a) and Schwab et al. (2001) have seen significant increases in protein concentration and yield with HMBi, but their experiments involved more cows and longer periods of supplementation. The significant response in milk protein concentration of 0.11% units is similar to what was observed by Sylvester et al. (2003a) during the third and fourth week of supplementation (0.15% units). The non- significant response in milk protein production of 40 g/d is of similar magnitude to that observed by Sylvester et al. (2003a) during the third and fourth week of supplementation

(60 g/d). In that trial, the response in milk protein production did not reach statistical significance at P < 0.05 until the eighth week of supplementation. Somatic cell count and

MUN were not significantly different among treatments. Milk urea N is affected by blood urea N (Roseler et al., 1993), because urea freely diffuses from blood to milk

(Gustafsson and Palmquist, 1993). Blood urea N should be affected by level of CP in the diet (Rosseler et al, 1993), and our treatment diets did not differ in CP concentration.

Volatile fatty acids and ammonia

Rumen ammonia and volatile fatty acid profiles are shown in Table 5.3.

Ammonia in the rumen was not different (approximately 11.8 mg/dl), and was well above the 5 mg/dl minimum suggested for maximal bacterial CP synthesis (Satter and Slyter,

1974). This indicates that any response to rumen-available Met was probably not due to

110

provision of ammonia from degraded AA, but was specifically due to supplementation of

Met. Volatile fatty acid profile was not different. Changes in microbial growth or composition in the rumen due to rumen available Met would be expected to cause changes in volatile fatty acid profile. Some researchers (Noftsger et al., 2003; Lundquist et al, 1983) have seen changes in VFA profile associated with HMB in the diet whereas others (Vazquez-Anon et al., 2003) have seen no differences.

Digestibility of nutrients

Digestibilities of NDF and OM are reported in Table 5.4. There were no differences in apparent or true digestibility of OM in the rumen, or NDF digestibility in the rumen. Digestibilities of DM (Polan et al., 1970a; Hoover et al., 1999), ADF

(Noftsger et al., 2003; Polan et al., 1970a), crude fiber (Holter et al., 1972) and CP

(Hoover et al., 1999) have been increased by HMB supplementation in some studies.

Others have shown no effects on ADF digestibility (Windschitl and Stern, 1988) or NDF and hemicellulose digestibility (Noftsger et al., 2003; Windschitl and Stern, 1988).

Digestibility rates and pool sizes of NDF and protein determined using in situ kinetics are reported in Table 5.5. Average effective ruminal digestibility of NDF was

18.3, 23.1, and 16.2% for silage, alfalfa hay, and TMR, respectively. In situ estimates of ruminal digestibility are commonly less than those found in vivo. Torrent et al. (1994) found that NDF digestibilities of identical samples of alfalfa hay, brewers grains, and beet pulp were lower when estimated using Dacron bags in steers than when estimated using markers in sheep. Firkins et al. (1998) reported that in situ procedures could

111

underestimate digestibility because of lower pH inside the bags than in the rumen contents, or overestimate digestibility due to particle efflux. Rates of digestion of NDF were fastest for the HMBi treatment for alfalfa hay and TMR, with a longer lag time.

The combination of faster rate and longer lag time for HMBi made the calculated effective rumen digestibilites similar for all treatments. Increases in ADF (Noftsger et al., 2003) and crude fiber digestibility (Holter et al., 1972) from HMB supplementation have been reported.

In situ CP digestibility of alfalfa hay was highest for HMB, although it was only significantly higher than HMBi. The effective rumen digestibility of alfalfa hay CP was highest for HMB. Rate of CP digestibility and estimated ruminal digestibility was highest for the dl-Met treatment in TMR. Corn silage had the lowest in situ CP digestibility with the dl-Met treatment. Rate of digestion of protein in alfalfa hay was increased by supplementation of HMB, and was decreased with HMBi supplementation.

Hoover et al. (1999) reported a quadratic effect of HMB on CP digestibility, with HMB increasing digestibility at 0.11% of the diet DM, but not at 0.22%. Isopropyl-HMB, assuming 50% bioavailability (Schwab et al., 2001), would provide ruminally available

HMB at approximately 0.05% of the diet DM, whereas the HMB treatment was provided at 0.10% of diet DM in this experiment and would be more similar to their intermediate treatment.

112

Rumen pool and passage rates

Rumen pool and passage rate results are shown in Table 5.6. Rumen mass of wet digesta and DM were not different among treatments and averaged 70.4 and 10.2 kg, respectively. There was a trend (P = 0.09) for the Control diet to have a higher concentration of NDF in the ruminal DM content than the other three diets, possibly due to increased NDF digestibility in the Met-supplemented diets. Passage rates of liquid and small particles were not affected by treatment, and averaged 0.157 and 0.071/h, respectively. The rumen pH was also not different across diets and averaged 5.94. This pH was slightly lower than expected, since steady state feeding should prevent most of the diurnal variation normally seen in pH (Dehority, 2003).

Counts of rumen and omasal protozoa were not different across treatments. The omasal protozoa showed a non-significant numerical increase with the HMB diet.

Unfortunately, current methods for counting protozoa have high measurement errors, so none of the differences were significant. Patton et al. (1970a,b) suggested that changes in fatty acid flows in the rumen could be due to increases in protozoa. De Vuyst et al.

(1975) reported an increase in rumen protozoa of 5X the number found with the basal treatment when methionine hydroxy analog was added at 1.2% of ration DM for sheep.

They noted the majority of the effect was on the Entodinium species. An increase of

40,000/ml protozoa, seen with the HMB diets in our trial compared with the control diet, equates to an increase of 66 g of protozoal cells (Sylvester et al., 2003b) at a passage rate of 275 kg liquid/d. Protozoa are approximately 50% protein, and protozoal protein is approximately 12% Lys (Williams and Coleman, 1992). This would provide an

113

additional 4 g/d of Lys to the cow (2% increase). If this were a true increase in protozoal flow, it would provide enough Lys for an additional 48 g of milk protein (cow’s milk protein is 8.2% Lys; Van Soest, 1982), assuming other AA are adequate.

Passage of N from the rumen

Nitrogen partitioning is reported in Table 5.7. Milk N production was significantly higher with the control and HMBi diet than the dl-Met treatment, with the

HMB treatment being intermediate. On average, 31.8% of intake N appeared as milk N.

This agrees with N efficiencies seen in other trials (Noftsger and St-Pierre, 2003,

Sylvester et al., 2003a). Based on actual MUN and BW, the equation of Kauffman and

St-Pierre (2001) predicted a mean urinary N excretion of 210 g/d across treatments.

Using predicted UN excretion and fecal excretion of N based on Yb flow, environmental efficiency (kg N excreted/kg N in milk) was not significantly different across treatments.

However, there is a numerical improvement in the environmental efficiency ratio with the

HMBi treatment. Our lab has seen an improvement in the ratio when the supply of Lys and Met was increased in combination with increased digestibility of supplemental RUP

(Noftsger and St-Pierre, 2003). This decrease was due to a reduction in dietary CP with a concomitant decrease in urinary N and increase in milk N. Crude protein concentrations have been shown to influence MUN values (Rosseler et al, 1993), and therefore the UN estimation. Retained N averaged 39 g, and was not different between treatments. With cows in early lactation, retained N should be approximately zero. The errors inherent in the estimations of UN, fecal N, and intake N are compounded in the retained N. Others

114

(Kolver et al, 1998) found positive N retention in cows in early to mid-lactation, and believed this to be due to possible underestimation of fecal outflow from marker error

(Cr2O3).

Bacterial Growth

Bacterial flow of OM and N are reported in Table 5.9. Flows of OM and N from bacteria were not different among treatments. The proportion of omasal N from bacterial

N was not different, and averaged 0.65. Some research has shown an increase in microbial protein production with supplementation of HMB (Vazquez-Anon, 2001;

Hoover et al., 1999) whereas others have shown none (Windschitl and Stern, 1988).

Bacterial growth would be expected to increase if OM or NDF digestibilities in the rumen were increased, but the expected increases in ruminal digestibility were not seen in this trial.

Rumen Passage of Methionine supplements

Extents of passage of HMB, HMBi and Met to the omasum are reported in Table

5.8. Passage of the supplements was determined using fluid passage rates determined by using Co-EDTA. The assumption is that HMB will travel with the fluid phase. 2- hydroxy-4 (methylthio) butanoic acid is a highly soluble compound, and once in the rumen goes readily into solution (Vazquez-Anon, 2001). The degradability was determined using actual g of HMB provided by the dose. Approximately 85% of the dose in the HMBi treatment is HMB. Only a small percentage of the HMB and HMBi

115

doses appeared as HMB in the omasal fluid, and HMBi concentrations were below detection level. This supports the data of several authors who have found little bypass of

HMB supplements when using lambs (Papas et al., 1974) and cows (Jones et al.,1988).

Others have found higher levels of undegradability, but these experiments are usually carried out with in vitro systems (Patterson and Kung,1988), pulse-dosing of HMB

(Koenig et al., 2002; Koenig et al., 1999), or both (Vázquez-Anon et al.,2001). These may not adequately represent the in vivo situation. Continuous culture methods are experimental models of the rumen, but most do not contain either a rumen mat or protozoa. Protozoa must sequester themselves along the rumen wall or in the mat in order to remain in the rumen long enough to divide. Passage rates can vary due to cow, stage of lactation, and DMI. Pulse dosing raises ruminal concentrations far above that observed when cows are fed a diet at the suggested dose of 0.10 to 0.11% of DM. These high concentrations may saturate microbial enzymatic pathways or transport mechanisms across the microbial membranes. Rumen undegradability of HMBi has been determined previously to be approximately 50% using blood (Robert et al., 2001) and milk true protein changes (Schwab et al., 2001) as indicators of bioavailability to the cow. With

HMBi, approximately half of the HMBi is absorbed through the rumen wall, where the isopropanol is removed and most of the HMB is released into the bloodstream. The remaining 50% is hydrolyzed in the rumen to HMB and isopropanol (Robert et al., 2002).

This remaining rumen available HMB would behave similarly to unmodified HMB.

Because the doses of HMB and HMBi contained equivalent amounts of Met, 50% degradability would leave an amount of HMB in the rumen equivalent to half the dose of

116

the unmodified HMB treatment. Therefore, passage of HMB from the HMBi treatment would be expected to be approximately half that found with the HMB treatment. This is supported by our data, which found that only 2.3% of the HMB from HMBi passed, whereas 5.3% of the HMB from the HMB treatment passed out through the omasum.

Escape of free Met was similar for all treatments, indicating that dl-Met did not pass out of the rumen in significant amounts.

Conclusions

The low undegradability of HMB was confirmed by very low concentrations, only

5% of the dose, passing into the omasum. There were changes in alfalfa hay CP digestibility rate and a numerical increase in omasal flows of protozoa, indicating ruminal actions, and no milk compositional changes to indicate post-ruminal actions. The differences in undegradability of HMB and HBMi were evident in the changes in milk protein concentration seen with HMBi, but not with HMB. The numerical trends for improved environmental efficiency and decreased urinary N with HMBi indicate that release of N into the environment could be decreased. The ruminal effects of HMBi appeared as changes in NDF digestibility and numerical increases in omasal protozoal flow, indicating some changes in the rumen microbial ecosystem. The passage of HMBi as HMB into the omasum further proves the claims of 50% undegradability. If HMBi had similar undegradability to HMB, then similar amounts should pass out through the omasum. However, only about half the amount of HMB appeared in the omasum with

HMBi that appeared with HMB. No measurable amounts of HMBi appeared in the

117

omasum. Although the flows of protozoa were not significantly different with the HMB and HMBi diets, the numerical differences may be real. With more precise molecular methods for protozoal enumeration, it may be possible to elucidate the effects of HMB on protozoa. With the increased digestibility and positive differences in AA composition, increased protozoal flow could have positive benefits on milk yield and composition.

The low undegradability of dl-Met was shown by no changes in milk composition, and no increase in free Met flow in the omasum with the dl-Met diet.

118

Ingredients Base diet % of total ration DM Corn silage 30.00 Alfalfa hay 13.00 Whole cottonseed 12.00 Corn grain, ground 30.50 Soybean hulls1 1.85 Megalac 1.00 Soybean meal, 48 % CP 5.50 Blood meal 2.99 Urea 0.20 Vitamins and minerals 2.96 Predicted Nutrients2 CP, % of DM 17.5 NDF, % of DM 29.7 Fat, % of DM 5.8 NFC, % of DM 44.2 NEl, Mcal/kg 1.6 RUP, % of CP intake 40.0 MP, % of DM 12.0 Met, g/d3 52.24 Met, % of MP 1.74 Lys, g/d 202.0 Lys, % of MP 6.72 1Treatments (HMB, HMBi, and dl-Met) were mixed and pelleted with the soybean hulls. The control diet contained only soybean hulls. 2Values were estimated using the NRC model (2001) using predicted 25 kg DMI, 636 kg BW, 43 kg milk yield, 3.1% true protein and 3.4% fat. 3Met and Lys flows reported are digestible AA flows. 4Addition of treatments may increase flow of Met (amount is dependant on undegradability of source).

Table 5.1. Ingredient and nutrient composition of base diet (DM basis) based on predicted BW, intake, milk yield and composition.

119

Control HMB HMBi dl-Met SEM P-value DMI (kg/d) 19.9 20.5 20.4 19.4 0.78 NS2 Milk production (kg/d) 38.5 38.0 38.3 35.8 1.17 0.06 Gross Feed Efficiency3 1.95 1.86 1.99 1.85 0.065 NS Fat (%) 3.35 3.35 3.42 3.60 0.15 NS Fat production (kg/d) 1.29 1.26 1.31 1.28 0.072 NS Protein (%) 2.91a 2.95a 3.02b 2.96a,b 0.072 0.01 Protein production 1.12a,b 1.12a,b 1.16a 1.07b 0.049 0.05 (kg/d) Lactose (%) 4.90 4.91 4.86 4.78 0.097 NS Lactose production 1.89 1.87 1.87 1.71 0.080 0.07 (kg/d) SNF (%) 5.80 5.81 5.76 5.71 0.101 NS SNF production (kg/d) 2.24 2.21 2.22 2.05 0.092 0.07 MUN 13.8 13.8 12.8 14.7 0.91 NS Log10 SCC 5.06 5.09 5.25 5.23 0.27 NS Body weight (kg) 593 594 588 583 21.5 NS 1Milk data was determined during the third and fourth week of each period. Data from one cow during the fourth period (HMBi treatment) was removed due to mastitis during week three of that period. 2NS is a P-value > 0.10. Means in a row without common superscript differ at P < 0.05. 3Gross feed efficiency = kg of milk/kg of DMI

Table 5.2. Least squares means for milk production and intake data for diets that vary in source of methionine.1

120

Control HMB HMBi dl-Met SEM P-value 1 Rumen NH3 (mg/dl) 12.7 12.1 11.1 11.1 1.05 NS Total VFA (mM) 120.7 121.0 121.4 118.0 2.6 NS Concentration of VFA (mol/100 mol) Acetate 64.0 62.0 62.8 62.8 0.77 NS Propionate 22.4 24.6 23.4 23.8 0.96 NS Butyrate 10.3 10.2 10.6 10.2 0.34 NS Valerate 1.14 1.19 1.17 1.17 0.039 NS Isobutyrate 0.84 0.81 0.81 0.74 0.036 NS Isovalerate 1.31 1.16 1.22 1.24 0.085 NS Acetate:propionate 2.88 2.55 2.72 2.70 0.139 NS 1NS is a P-value > 0.10.

Table 5.3. Least squares means for rumen volatile fatty acids and ammonia for diets that vary in source and availability of methionine.

Control HMB HMBi dl-Met SEM P-value ------%------Rumen OM- 65.2 66.0 69.4 62.3 2.91 NS apparent Rumen OM-true 76.4 75.6 78.8 73.0 2.35 NS

NDF 58.2 59.8 65.2 51.6 4.21 NS

Table 5.4. Ruminal digestibility of nutrients for diets that vary in source and availability of Met.

121

1 2 Control HMB HMBi dl-Met SED P-value

------Total Mixed Ration------NDF A3 (%) 3.9 1.3 2.7 1.9 2.6 NS B (%) 61.0a 67.0ab 58.6a 80.2b 9.0 0.04 C (%) 35.2ab 31.7ab 38.7b 18.0a 9.0 0.04 Rate (/h) 0.023a 0.028ab 0.038b 0.022a 0.006 0.05 Lag (h) 3.9ab 3.2ab 6.3b 1.1a 2.2 0.05 ERD4 (%) 15.7 15.2 16.5 17.5 1.4 NS Crude protein A (%) 37.1 36.9 34.7 35.9 3.0 NS B (%) 47.4 45.8 48.7 46.3 4.1 NS C (%) 15.5 17.4 16.6 17.8 4.1 NS Rate (/h) 0.053a 0.069ab 0.068ab 0.076b 0.014 0.05 Lag (h) 0 1.5 0 0 0.6 NS ERD (%) 58.0a 58.4a 59.3ab 61.2b 0.83 0.03 ------Alfalfa Hay------NDF A (%) 1.0 3.5 1.6 0.6 2.1 NS B (%) 45.9 52.0 46.3 47.9 3.4 NS C (%) 53.0b 44.5a 52.1b 51.5b 3.4 0.04 Rate (/h) 0.047a 0.039a 0.067b 0.049a 0.008 <0.005 Lag (h) 0.8a 2.7a 5.5b 1.6a 1.3 0.05 ERD (%) 23.6 23.1 22.2 23.4 1.76 NS Crude protein A (%) 36.2 31.1 32.5 32.8 3.0 NS B (%) 53.8 58.4 57.2 57.3 3.7 NS C (%) 10.0 10.5 10.4 9.9 3.7 NS Rate (/h) 0.100ab 0.133b 0.090a 0.105ab 0.017 0.05 Lag (h) 0 0 0 0 -- NS ERD (%) 73.3b 75.0c 70.5a 73.1b 0.70 <0.005 ------Corn Silage------NDF A (%) 2.6 1.0 4.1 0.8 2.8 NS B (%) 97.0 74.3 123.1 127.1 36.1 NS C (%) 0.4 24.8 -27.2 -27.9 36.1 NS Rate (/h) 0.012 0.015 0.016 0.016 0.003 NS Lag (h) 0.4 0.0 0.8 0.3 2.5 NS ERD (%) 18.1 16.9 20.6 17.6 2.0 NS Crude protein A (%) 54.4 51.5 53.2 52.8 2.0 NS B (%) 33.9 34.5 33.9 26.2 4.3 NS C (%) 11.7a 14.0a 12.9a 21.0b 4.3 0.03 Rate (/h) 0.049a 0.041a 0.036a 0.075b 0.014 0.05 Lag (h) 9.2b 0.0a 3.8b 7.7b 2.2 0.05 ERD (%) 63.0 63.7 63.4 63.0 1.6 NS 1SED is the mean standard error of the differences. 2NS is a P-value > 0.10. 3A, B, and C are kinetic pools; the A pool is the fraction of the constituent (NDF or CP) that is instantaneously digestible; the B pool is the potentially digestible fraction; and the C pool is the undigestible fraction. 4 ERD is effective ruminal digestibility estimated as: ERD = A + B [kd / kd + kp] x [exp (-kpL)] Rate of passage (kp) was calculated using NRC (2001) estimates for concentrates (0.0754/h), alfalfa hay (0.0434/h), corn silage (0.0557/h), and TMR (0.0637/h).

Table 5.5. Digestibility of crude protein and neutral detergent fiber in TMR, alfalfa hay, and corn silage using Dacron bags removed at 8 time points

122

Control HMB HMBi dl-Met SEM P-value Rumen mass, kg Wet digesta2 71.5 69.7 69.7 70.8 2.90 NS1 DM3 10.45 10.31 9.66 10.26 0.60 NS Rumen digesta DM, % 14.49 15.01 13.79 14.02 0.45 0.099 NDF, % of DM 56.14 54.81 53.14 54.74 0.85 0.090 N, % of DM 3.07 3.04 3.06 3.09 0.052 NS Ash, % of DM 9.07 9.08 9.55 9.27 0.367 NS Ruminal liquid volume, L4 61.0 59.3 60.0 60.8 2.76 NS Liquid passage rate (/h) 0.143 0.172 0.142 0.172 0.028 NS Small particle passage rate 0.075 0.069 0.060 0.079 0.006 NS (/h) Rumen pH 5.96 5.95 5.91 5.94 0.046 NS Rumen protozoa (per ml)5 1.36 1.29 1.49 1.46 0.159 NS Omasal protozoa (per ml) 6.11 6.51 6.52 5.73 0.790 NS 1NS is a P-value > 0.10. 2 Total pool sizes of wet digesta were determined by the average of 2 days of rumen evacuations. 3DM (kg) = wet digesta *105°C DM 4Ruminal fluid volume = wet digesta – (wet digesta * 105°C DM ) 5Protozoa counts are expressed as 106 per ml for rumen and 105 per ml for omasal measurements.

Table 5.6. Least squares means for rumen pool measurements and passage rates for diets that vary in source of methionine.

123

Control HMB HMBi dl-Met SEM P-value1 N intake (g/d) 582 603 601 569 22.6 NS Fecal N (g/d) 168 145 170 144 15.8 NS Urinary N (g/d)4 213 213 191 224 13.1 NS Milk N production (g/d) 188a 187a,b 194a 178b 8.23 0.05 Retained N (g/d) 16 53 60 25 27.8 NS Gross N efficiency2 32.3 31.1 32.5 31.1 0.95 NS Environmental efficiency3 2.1 2.0 1.8 2.1 0.15 NS Total tract N digestibility, 70.8 75.8 70.1 74.7 3.22 NS % 1NS is a P-value > 0.10. Means in a row without common superscript differ at P < 0.05. 2calculated as milk N/N intake * 100 3calculated as kilograms of N excreted/kilogram N in milk; environmental efficiency calculation assumes zero N balance. 4N = 0.0259 * BW(kg) * MUN (mg/dl); (Kauffman and St-Pierre, 2001).

Table 5.7. Nitrogen partitioning and efficiency with diets that vary in source and degradability of Met.

Control HMB HMBi dl-Met SEM P-value Bacterial 2113 1779 1844 1983 249.9 NS OM, g/d Bacterial 206.2 173.5 180.4 194.2 24.35 NS N, g/d Proportion 0.65 0.63 0.66 0.64 0.026 NS of omasal N from bacteria

Table 5.8. Flow of bacterial N and OM to the omasum with diets that vary in source and degradability of Met.

124

Control HMB HMBi dl-Met SEM P-value

Percentage of dose N/A 5.33 2.33 N/A 1.51 0.02

Passage of free Met (g/d) 5.2 4.9 5.5 4.9 0.32 NS

1HMBi is 84.6% HMB. Percentage of dose is based on actual intake of HMB

Table 5.9. Percentage of HMB and HMBi dose appearing as HMB in the omasal fluid

125

CHAPTER 6

CONCLUSIONS

The ability of the dairy industry to operate without substantial external oversight by regulating agencies has been challenged by the awareness of the public that animal agriculture can negatively impact the environment. Environmental Protection Agency regulations will change the way that the dairy industry feeds and houses its animals, and removes the waste products. Dietary manipulation has been examined in this dissertation in a proactive attempt to decrease N excretion before regulations make it a necessity, or to decrease the negative financial impact of future regulations related to N excretion.

Milk production and component production can be maintained, and even improved, at a lower concentration of CP by increasing post-ruminal digestibility of rumen undegradable protein in combination with tactical supplementation of AA.

Nitrogen excretion is reduced when CP concentrations are decreased. In prior research, this was done partially at the expense of production. Our research shows that this is not inevitable, and that substantial improvement in N efficiency can be achieved through nutrition. Supplementation of unselected RUP sources is inadequate, and will not consistently result in the desired production and N excretion responses. The post-ruminal digestibility of RUP appears to be more important than total RUP supplementation. The

126

profile of AA supplied, particularly Lys and Met, must be optimized to maximize milk yield, component production and N efficiency. Discovery of a method to rapidly estimates intestinal digestibility of protein supplements would enable a rapid adoption of our findings throughout the industry. In our first trial, supplementation of Met resulted in a larger increase in protein yield than anticipated, possibly due to ruminal effects of the

HMB supplement. The effects of HMB on ruminal bacterial populations were determined using continuous culture systems. The HMB supplement may affect bacterial populations by decreased use of Met precursors, which can then be used for other growth applications, or shifting bacterial species. Digestibility of most nutrients was largely insensitive to HMB supplementation, but VFA profile and amount of bacterial N obtained from NH3 was changed, indicating that HMB does influence the bacterial populations. An oversupply of RDP could have hidden potential effects of HMB by providing an overabundance of Met. The continuous culture system is limited in its ability to explore protozoal effects of HMB supplementation.

Protozoal effects and rumen-escape value of HMB cannot be truly determined without the use of lactating cows. The proportion of HMB passing through the omasum was determined using lactating cows fed typical amounts of HMB, using a novel omasal sampling technique. As hypothesized, the amount passing out of the rumen is small, with less than 6% of the daily intake flowing through the omasum. This, it appears unlikely that HMB when used at the recommended 0.1 to 0.15% of diet DM enhances metabolizable Met. Total tract digestibilities of major nutrients were not affected by

HMB, but in situ digestibilities of nutrients in individual feeds (TMR and alfalfa hay)

127

were. Ruminal digestibility of nutrients could further clarify the actions of HMB. We were unable to determine a clear and significant effect of HMB on protozoal growth.

Additional research using more precise molecular techniques could have the resolution necessary to further elucidate the role of protozoa in the effects of HMB. Increases in passage rate of total protozoa and of specific species of protozoa could be determined.

The use of diet as a means of reducing N excretion has been explored through this dissertation. A proper understanding of RUP digestibility should allow nutritionists to reduce CP in diets while meeting the cow requirements. The misconception that additional dietary CP results in linear increases in production must be confronted and dismissed. Ruminal effects of HMB on digestibility and bacterial N metabolism show promise as a way of increasing the amount of nutrients from the diet utilized by the cow.

Increased feed efficiency without reduction in production implies a decrease in waste.

Increases in protozoal protein passage could provide the cow with more digestible protein with a superior AA profile than that of the mixed bacterial population in the rumen. In the future, molecular techniques can be used to quantify this effect more accurately.

Increases in protozoal passage could change the requirements for supplemental RUP in quantity as well as quality (AA profile). Knowledge of the mechanics of HMB can be incorporated into ration balancing models for efficacious use of HMB in dairy rations.

However, nutrition cannot entirely resolve the issue of N excretion. Management of the waste must also be studied to reduce volatilization of ammonia from feces and urine into the air. The dairy industry as a whole will have to work together to function profitably under the potential new environmental regulations.

128

LIST OF REFERENCES

Ahvenjärvi, S., A. Vanhatalo, and P. Huhtanen. 2002. Supplementing barley or rapeseed meal to dairy cows fed grass-red clover silage. I. Rumen degradability and microbial flow. J. Anim. Sci. 80:2176-2187.

Ahvenjärvi, S., A. Vanhatalo, P. Huhtanen, and T. Varvikko. 2000. Determination of reticulo-rumen and whole-stomach digestion in lactating cows by omasal canal or duodenal sampling. Br. J. Nutr. 83:67-77.

Ahvenjärvi, S., B. Skiba, and P. Huhtanen. 2001. Effect of heterogeneous digesta chemical composition on the accuracy of measurements of fiber flow in dairy cows. J. Anim. Sci. 79:1611-1620.

Armentano, L. E., S. J. Bertics, and G. A. Ducharme. 1997. Response of lactating cows to methionine or methionine plus lysine added to high protein diets based on alfalfa and heated soybeans. J. Dairy Sci. 80:1194-1199.

Association of Official Analytical Chemists. 1990. Official Methods of Analysis. Vol. I. 15th ed. AOAC, Arlington, VA.

Bach, A., and M. D. Stern. 1999. Effects of different levels of methionine and ruminally undegradable protein on the amino acid profile of effluent from continuous culture fermenters. J. Anim. Sci. 77:3377-3384.

Bach, A., G. B. Huntington, S. Calsamiglia, and M. D. Stern. 2000. Nitrogen metabolism of early lactation cows fed diets with two different levels of protein and different amino acid profiles. J. Dairy Sci. 83:2585-2595.

Baker, D. H. 1986. Utilization of isomers and analogs of amino acids and other sulfur- containing compounds. Prog. Food Nutr. Sci. 10(1-2):133-178.

Barbano, D. M., J. M. Lynch, E. D. Bauman, G. F. Hartnell, R. L. Hintz, and M. A. Nemeth. 1992. Effect of a prolonged-release formulation of n-methionyl bovine somatotropin (Sometribove) on milk composition. J. Dairy Sci. 75:1775-1793.

129

Basch, J. J., E. D. Wickham, and H. M. Farrell, Jr. 1997. Arginase in lactating bovine mammary glands: Implications in proline synthesis. J. Dairy Sci. 80:3241-3248.

Belasco, I. J. 1980. Fate of carbon-14 labeled methionine hydroxy analog and methionine in the lactating dairy cow. J. Dairy Sci. 63:775-784.

Bell, A. W. 1995. Regulation of organic nutrient metabolism during transition from late pregnancy to early lactation. J. Anim. Sci. 73:2804-2819.

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

Bequette, B. J., F. R. C. Backwell, A. G. Calder, J. A. Metcalf, D. E. Beever, J. C. MacRae, and G. E. Lobley. 1997. Application of a U-3C-labeled amino acid tracer in lactating dairy goats for simultaneous measurements of the flux of amino acids in plasma and the partition of amino acids to the mammary gland. J. Dairy Sci. 80:2842-2853.

Bequette, B. J., F. R. C. Backwell, and L. A. Crompton. 1998a. Current concepts of amino acid and protein metabolism in the mammary gland of the lactating ruminant. J. Dairy Sci. 81:2540-2559.

Bequette, B. J., F. R. C. Backwell, C. E. Kyle, L. A. Crompton, J. France, and J. C. MacRae. 1998b. The effect of free amino acid supply on the contribution of peptide-bound phenylalanine and tyrosine to casein synthesis in late lactation goats. Anim. Sci. 67:450A (Abstr.)

Bequette, B. J., F. R. C. Backwell, J. C. MacRae, G. E. Lobley, L. A. Crompton, J. A. Metcalf, and J. D. Sutton. 1996. Effect of intravenous amino acid infusion on leucine oxidation across the mammary gland of the lactating goat. J. Dairy Sci. 79:2217-2224.

Bergen, W. G., D. B. Purser, and J. H. Cline. 1968. Determination of limiting amino acids of rumen isolated microbial proteins fed to rats. J. Dairy Sci. 51:1698- 1700.

Bhargava, P. K., D. E. Otterby, J. M. Murphy, and J. D. Donker. 1977. Methionine hydroxy analog in diets for lactating cows. J. Dairy Sci. 60:1594-1604.

Blouin, J. P., J. F. Bernier, C. K. Reynolds, G. E. Lobley, P. Dubreuil, and H. Lapierre. 2002. Effect of supply of metabolizable protein on splanchnic fluxes of nutrients and hormones in lactating dairy cows. J. Dairy Sci. 85:2618-2630.

130

Blum, J. W., R. M. Bruckmaier and F. Jans. 1999. Rumen-protected methionine fed to dairy cows: bioavailability and effects on plasma amino acid pattern and plasma metabolite and insulin concentrations. J. Dairy Sci. 82:1991-1998.

Bosclair, Y. R., A. W. Bell, F. R. Dunshea, M. Harkins, and D. E. Bauman. 1993. Evaluation of the arteriovenous difference technique to simultaneously estimate protein synthesis and degradation in the hindlimb of fed and chronically underfed steers. J. Nutr. 123:1076-1088.

Bottje, W., S. Wang, K. Beers, M. Vazquez-Anon, and P. McCullough. 1998. Clearance of DL-2-hydroxy-4-(methylthio) butanoic acid (HMB) by broiler liver in vivo. Poul. Sci. 77:31(Abstr.).

Brandyberry, S. D., R. C. Cochran, E. S. Vanzant, and D. L. Harmon. 1991. Technical note: effectiveness of different methods of continuous marker administration for estimating fecal output. J. Anim. Sci. 69:4611-4616.

Brock, T. D., M. T. Madigan, J. M. Martinko, and J. Parker. 1994. Page 114 in Biology of microorganisms. Prentice Hall, Englewood Cliffs, NJ.

Burkart, M. R. and D. E. James. 1999. Agricultural-nitrogen contributions to hypoxia in the Gulf of Mexico. J. Environ. Qual. 28(3):850-859.

Calsamiglia, S. and M. D. Stern. 1995. A three-step in vitro procedure for estimating intestinal digestion of protein in ruminants. J. Anim. Sci. 73:1459-1465.

Chan, S. C., J. T. Huber, C. B. Theurer, Z. Wu, K. H. Chen, and J. M. Simas. 1997. Effects of supplemental fat and protein source on ruminal fermentation and nutrient flow to the duodenum in dairy cows. J. Dairy Sci. 80:152-159.

Chandler, P. T. 1989. Achievement of optimum amino acid balance possible. Feedstuffs 61(26):14, 25.

Chaney, A. L., and E. P. Marbach. 1962. Modified reagents for determination of urea and ammonia. Clin. Chem. 8:130-132.

Church, D. C., ed. 1998. pp. 189 in The ruminant animal. Digestive physiology and nutrition. Waveland Press, Prospect Heights, IL.

Clark, A. K., and A. H. Rakes. 1982. Effect of methionine hydroxy analog supplementation on dairy cattle hoof growth and composition. J. Dairy Sci. 65:1493-1502.

131

Cotta, M. A., and J. B. Russell. 1982. Effect of peptides and amino acids on efficiency of rumen bacterial protein synthesis in continuous culture. J. Dairy Sci. 65:226- 234.

Cozzi, G. and C. E. Polan. 1994. Corn gluten meal or dried brewer’s grains as partial replacement for soybean meal in the diet of Holstein cows. J. Dairy Sci. 77:825- 834.

CPM Dairy Version 1.0. 1998. Department of Animal Sciences, Cornell University, Ithaca, NY.

Damon, R.A., and W.R. Harvey. 1987. Experimental Design, Anova, and Regression. Harper and Row, New York.

De Vuyst, A., M. Vanbelle, J. M. Joassart, and A. Baguette. 1975. The effect of methionine hydroxy analog supplementation of the diet on the concentration of ciliate protozoa in the rumen of sheep. Z. Tierphysiol., Tierernahrg. U. Futtermittelkde. 35:316-321.

Dehority, B. A. 1991. Effects of microbial synergism on fibre digestion in the rumen. Proc. Nutr. Soc. 50:149-159.

Dehority, B. A. 1993. Pages 113 to 116 in Laboratory manual for classification and morphology of rumen ciliate protozoa. CRC Press, Boca Raton, Florida.

Dehority, B. A. 2003. Pages 28 to 30 in Rumen Microbiology. Nottingham University Press, Thrumpton, Nottingham, UK.

Dibner, J. J., and C. D. Knight. 1984. Conversion of 2-hydroxy-4-(methylthio) butanoic acid to L-methionine in the chick: a stereospecific pathway. J. Nutr. 114:1716- 1723.

Dinn, N. E., J. A. Shelford, and L. J. Fisher. 1998. Use of the Cornell Net Carbohydrate and Protein System and rumen-protected lysine and methionine to reduce nitrogen excretion from lactating dairy cows. J. Dairy Sci. 81:229-237.

Draper, N.R. and H. Smith. 1998. Applied Regression Analysis. Third Ed. John Wiley and Sons, New York.

Emery, R. S. 1971. Disappearance of methionine from the rumen. J. Dairy Sci. 54:1090-1091.

EPA. 1998. National Air pollutant emission trends, 1990-1998.

132

Erasmus, L. J., P. M. Botha, C. W. Cruywagen , and H. H. Meissner. 1994. Amino acid profile and intestinal digestibility in dairy cows of rumen-undegradable protein from various feedstuffs. J. Dairy Sci. 77:541-551.

Ferrell, C. L, and S. P. Ford. 1980. Blood flow steroid secretion and nutrient uptake of the gravid bovine uterus. J. Anim. Sci. 50:1113-1121.

Firkins, J. 1996. Maximizing microbial protein synthesis in the rumen. J. Nutr. 126(4S):1247S-1354S.

Firkins, J. L, M. S. Allen, B. S. Oldick, and N. R. St-Pierre. 1998. Modeling ruminal digestibility of carbohydrates and microbial protein flow to the duodenum. J. Dairy Sci. 81:3350-3369.

Firkins, J. L., L. L. Berger, N. R. Merchen, and G. C. Fahey, Jr. 1986. Effects of forage particle size, level of feed intake and supplemental protein degradability on microbial protein synthesis and site of nutrient digestion in steers. J. Anim. Sci. 62:1081-1094.

Firkins, J. L., W. P. Weiss, and E. J. Piwonka. 1992. Quantification of intraruminal recycling of microbial nitrogen using nitrogen 15. J. Anim. Sci. 70:3223-3233.

Firkins, J.L., W. P. Weiss, M. L. Eastridge, and B. L. Hull. 1990. Effects of feeding fungal culture extract and animal-vegetable fat on degradation of hemicellulose and on bacterial growth in heifers. J. Dairy Sci. 73:1812-1822.

France, J. and R. C. Siddons. 1986. Determination of digesta flow by continuous marker infusion. J. Theor. Biol. 121:105-119.

Galloway, J. N. 1998. The global nitrogen cycle: changes and consequences. Environ. Pollution. 102:15-24.

Galloway, J. N., W. H. Sclesinger, H. Levy, II, A. Michaels, and J. L. Schnoor. 1995. Nitrogen fixation: anthropogenic enhancement-environmental response. Global Biochem. Cycles. 9:235-252.

Gil, L. A., R. L. Shirley, and J. E. Moore. 1973a. Effect of methionine hydroxy analog on bacterial protein synthesis from urea and glucose, starch or cellulose by rumen microbes. in vitro. J. Anim. Sci. 37:159-163.

Gil, L. A., R. L. Shirley, and J. E. Moore. 1973b. Effect of methionine hydroxy analog on growth, amino acid content, and catabolic products of glucolytic rumen bacteria in vitro. J. Dairy Sci. 56:757-762.

133

Goering, H. K., and P. J. Van Soest. 1970. Forage fiber analyses (apparatus, reagents, procedures, and some applications). Pages 1-20 in Agriculture Handbook no. 379. U. S. Government Printing Office, Washington, D. C.

Guinard, J., H. Rulquin, and R. Verite. 1994. Effect of graded levels of duodenal infusions of casein on mammary uptake in lactating cows. 1. Major nutrients. J. Dairy Sci. 77:2221-2231.

Gustafsson, A. H., and D. C. Palmquist. 1993. Diurnal variation of rumen ammonia, serum urea, and milk urea in dairy cows at high and low yields. J. Dairy Sci. 76:475-484.

Hanigan, M. D., J. P. Cant, D. C. Weakley, and J. L. Beckett. 1998. An evaluation of postabsorptive protein and amino acid metabolism in the lactating cow. J. Dairy Sci. 81:3385-3401.

Hannah, S. M., M. D. Stern and F. R. Ehle. 1986. Evaluation of a dual flow continuous culture system for estimating bacterial fermentation in vivo of mixed diets containing various soyabean products. Anim. Feed Sci. Technol. 16:51-62.

Hansen, W. P., D. E. Otterby, J. G. Linn, and J. D. Donker. 1991. Influence of forage type, ratio of forage to concentrate, and methionine hydroxy analog on performance of dairy cows. J. Dairy Sci. 74:1361-1369.

Higginbotham, G. E., J. D. Schuh, L. Kung, Jr., and J. T. Huber. 1987. Palatability of methionine hydroxy analog or dl-methionine for lactating cows. J. Dairy Sci. 70:630-634.

Holter, J. B., C. W. Kim, and N. F. Colovos. 1972. Methionine hydroxy analog for lactating dairy cows. J. Dairy Sci. 55:460-465.

Holter, J. B., H. H. Hayes, W. E. Urban, Jr., S. Ramsey, and H. Rideout. 1992. Response of Holstein cows to corn gluten meal used to increase undegradable protein in early or later lactation. J. Dairy Sci. 75:1495-1506.

Hoover, W. H., B. A. Crooker and C. J. Sniffen. 1976. Effects of differential solid-liquid removal rates on protozoa numbers in continuous cultures of rumen contents. J. Anim. Sci. 43:528-534.

Hoover, W. H., T. K. Miller Webster, W. V. Thayne, B. Sloan, R. M. Gill, C. J. Schwab, and N. Whitehouse. 1999. Addition of Rhodimet AT88 to corn and barley based diets on microbial growth and metabolism: Final report. Personal communication.

134

Hristov, A. N. and G. A. Broderick. 1996. Synthesis of microbial protein in ruminally cannulated cows fed alfalfa silage, alfalfa hay, or corn silage. J. Dairy Sci. 79:1627-1637.

Huber, J. T., R. S. Emery, W. G. Bergen, J. S. Liesman, L. Kung, Jr., K. J. King, R. W. Gardner, and M. Checketts. 1984. Influences of methionine hydroxy analog on milk and milk fat production, blood serum lipids, and plasma amino acids. J. Dairy Sci. 67:2525-2531.

Huhtanen, P., P. G. Brotz, and L. D. Satter. 1997. Omasal sampling technique for assessing fermentative digestion in the forestomach of dairy cows. J. Anim. Sci. 75:1380-1392.

Hutjens, M. F., and L. H. Schultz. 1971. Addition of soybeans or methionine analog to high-concentrate rations for dairy cows. J. Dairy Sci. 54:1637-1644.

Jakobsen, K., E. O. Mikkelsen, and M. O. Nielson. 1994. Studies on responses to potassium, noradrenaline, serotonin, histamine and prostaglandin-F2-alpha of isolated pudendal arteries from non-lactating goats. Comp. Biochem. Physiol. 109(C comp. Pharmacol.):167-172.

James, T., D. Meyer, E. Esparza, E. J. DePeters, and H. Perez-Monti. 1999. Effects of dietary nitrogen manipulation on ammonia volatilization from manure from Holstein heifers. J. Dairy Sci. 82:2430-2439.

Jones, B. A., O. E. Mohamed, R. W. Prange, and L. D. Satter. 1988. Degradation of methionine hydroxy analog in the rumen of lactating cows. J. Dairy Sci. 71:525- 529.

Kajikawa, H., M. Mitsumori, and S. Ohmomo. 2002. Stimulatory and inhibitory effects of protein amino acids on growth rate and efficiency of mixed ruminal bacteria. J. Dairy Sci. 85:2015-2022.

Kauffman, A. J. and N. R. St-Pierre. 2001. The relationship of milk urea nitrogen to urine nitrogen excretion in Holstein and Jersey cows. J. Dairy Sci. 84:2284-2294.

King, K. J., W. G. Bergen, C. J. Sniffen, A. L. Grant, D. B. Grieve, V. L. King and N. K. Ames. 1991. An assessment of absorbable lysine requirements in lactating cows. J. Dairy Sci. 74:2530-2539.

Koenig, K. M., L. M. Rode, C. D. Knight, and M. Vázquez-Anon. 2002. Rumen degradation and availability of various amounts of liquid methionine hydroxy analog in lactating dairy cows. J. Dairy Sci. 85:930-938.

135

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 hydroxy analog in dairy cows. J. Dairy Sci. 82:355-361.

Kohn, R. A., Z. Dou, J. D. Ferguson, and R. C. Boston. 1997. A sensitivity analysis of nitrogen losses from dairy farms. J. Environ. Manage. 50:417-428.

Kolver, E., L. D. Muller, G. A. Varga, and T. J. Cassidy. 1998. Synchronization of ruminal degradation of supplemental carbohydrate with pasture nitrogen in lactating dairy cows. J. Dairy Sci. 81:2017-2028.

Komaragiri, M. V. S., and R. A. Erdman. 997. Factors affecting body tissue mobilization in early lactation dairy cows. 1. Effect of dietary protein on mobilization of body fat and protein. J. Dairy Sci. 80:929-937.

Lehninger, A. L., D. L. Nelson, and M. M. Cox. 1993. Principles of Biochemistry, 2nd ed. Worth Publishers, New York, NY.

Lemons, J. A., and R. L. Schreiner. 1983. Amino acid metabolism in the ovine fetus. Am. J. Physiol. 244:E459-E466.

Littell, R.C., G.A. Milliken, W.W. Stroup, and R.D. Wolfinger. 1996. SAS System for Mixed Models. SAS Inst. Inc., Cary, NC.

Lobley, G. E., H. Lapierre, J. J. Dibner, D. S. Parker, and M. Vazquez-Anon. 2001. HMB metabolism in ruminants. Southwest Nutr. & Management Conf., Phoenix, AZ.

Lundquist, R. G., J. G. Linn, and D. E. Otterby. 1983. Influence of dietary energy and protein on yield and composition of milk from cows fed methionine hydroxy analog. J. Dairy Sci. 66:475-491.

Lundquist, R. G., M. D. Stern, D. E. Otterby, and J. G. Linn. 1985. Influence of methionine hydroxy analog and dl-Methionine on rumen protozoa and volatile fatty acids. J. Dairy Sci. 68:3055-3058.

Maas, J. A., J. France, J. Dijkstra, A. Bannink, and B. W. McBride. 1998. Application of a mechanistic model to study competitive inhibition of amino acid uptake by the lactating bovine mammary gland. J. Dairy Sci. 81:1724-1734.

Mackle, T. R., D. A Dwyer, K. L. Ingvartsen, P. Y. Chouinard, J. M. Lynch, D. M. Barbano, and D. E. Bauman. 1999. Effects of insulin and amino acids on milk protein concentration and yield from dairy cows. J. Dairy Sci. 82:1512-1524.

136

MacRae, J. C., L. A. Bruce, D. S. Brown, and A. G. Calder. 1997. Amino acid use by the gastrointestinal tract of sheep given Lucerne forage. Am. J. Physiol. 36:G1200-G1207.

Maiga, H. A., D. J. Schingoethe, and J. E. Henson. 1996. Ruminal degradation, amino acid composition, and intestinal digestibility of the residual components of five protein supplements. J. Dairy Sci. 79:1647-1653.

Mansfield, H. R., M. I. Endres, M. D. Stern. 1995. Comparison of microbial fermentation in the rumen of dairy cows and dual flow continuous culture. Anim. Feed Sci. Tech. 55:47-66.

McCollum, M. Q., M. Vázquez-Anon, J. J. Dibner, and K. E. Webb, Jr. 2000. Absorption of 2-hydroxy-4 (methylthio) butanoic acid by isolated sheep ruminal and omasal epithelia. J. Anim. Sci. 78:1078-1083.

Meisinger, J. J. and W. E. Jokela. 2000. Ammonia losses from manure. Cornell Nutr. Conf. for Feed Manuf., Cornell Univ., Ithaca, NY.

Meng, Z., D. Dabdub, and J. H. Seinfeld. 1997. Chemical coupling between atmospheric ozone and particulate matter. Science. 277:116-119.

Metcalf, J. A., J. D. Sutton, J. E. Cockburn, D. J. Napper, and D. E. Beever. 1991. The influence of insulin and amino acid uptake by the lactating bovine mammary gland. J. Dairy Sci. 74:3412-3420.

National Research Council. 2002. Page 4 in Frontiers in agricultural research: Food, health, environment, and communities. The National Academies Press. Washington, D.C.

National Research Council. 2001. Nutrient requirements of dairy cattle (7th Rev. Ed.) Washington, D.C.: National Academy Press.

Noftsger, S. M., N. R. St-Pierre, S. K. R. Karnati, and J. L. Firkins. 2003. Effects of 2- hdyroxy-4-(methylthio) butanoic acid (HMB) on microbial growth in continuous culture. J. Dairy Sci. 86:2629-2636.

NRC. 1989. Nutrient requirements of dairy cattle (6th ed.). National Academy Press, Washington, DC.

137

O’Mara, F. P., J. J. Murphy and M. Roth. 1997. The amino acid composition of protein feedstuffs before and after ruminal incubation and after subsequent passage through the intestines of dairy cows. J. Anim. Sci. 75:1941-1949.

Onodera, R. 1993. Methionine and lysine metabolism in the rumen and the possible effects of their metabolites on the nutrition and physiology of ruminants. Amino Acids 5:217-232.

Orskov, E. R., and I. McDonald. 1979. The estimation of protein degradability in the rumen from incubation measurements weighed according to rate of passage. J Agric. Sci. (Camb.) 92:499.

Orskov, E. R., N. A. Macleod, and D. J. Kyle. 1986. Flow of nitrogen from the rumen and abomasum in cattle and sheep given protein-free nutrients by intragastric infusion. Br. J. Nutr. 56:241-248.

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.

Pantoja, J., J. L. Firkins, M. L. Eastridge, and B. L. Hull. 1994. Effects of fat saturation and source of fiber on site of nutrient digestion and milk production by lactating dairy cows. J. Dairy Sci. 77:2341-2356.

Papas, A., G. A. B. Hall, E. E. Hatfield, and F. N. Owens. 1974. Response of lambs to oral or abomasal supplementation of methionine hydroxy analog or methionine. J. Nutr. 104:653-659.

Patterson, J. A., and L. Kung. 1988. Metabolism of dl-methionine and methionine analogs by rumen microorganisms. J. Dairy Sci. 71:3292-3301.

Patton, R. A., R. D. McCarthy, and L. C. Griel, Jr. 1968. Lipid synthesis by rumen microorganisms. I. Stimulations by methionine in vitro. J. Dairy Sci. 51:1310- 1311.

Patton, R. A., R. D. McCarthy, and L. C. Griel. 1970a. Lipid synthesis by rumen microorganisms. II. Further characterization of the effects of methionine. J. Dairy Sci. 53:460-465.

Patton, R. A., R. D. McCarthy, and L. C. Griel. 1970b. Observations on rumen fluid, blood serum, and milk lipids of cows fed methionine hydroxy analog. J. Dairy Sci. 53:776-780.

138

Pisulewski, P. M., H. Rulquin, J. L. Peyraud, and R. Verite. 1996. Lactational and systemic responses of dairy cows to postruminal infusions of increasing amounts of methionine. J. Dairy Sci. 79:1781-1791.

Polan, C. E., P. T. Chandler, and C. N. Miller. 1970a. Methionine analog effect on ruminant digestion. J. Dairy Sci. 31:251 (Abstr.).

Polan, C. E., P. T. Chandler, and C. N. Miller. 1970b. Methionine hydroxy analog: varying levels for lactating cows. J. Dairy Sci. 53:607-610.

Powers, W. J. 2003. Keeping science in environmental regulations: The role of the animal scientist. J. Dairy Sci. 86:1045-1051.

Punia, B. S., and J. Leibholz. 1994. Effect of level of intake of kikuyu (Pennisetum clandestinum) grass hay on the flow of protozoal nitrogen to the omasum of cattle. Anim. Feed Sci. Tech. 47:77-87.

Punia, B. S., J. Leibholz, and G. J. Faichney. 1988. Effects of level of intake and urea supplementation of alkali-treated straw on protozoal and bacterial nitrogen synthesis in the rumen and partition of digestion in cattle. Aust. J. Agric. Res. 39:1181-1194.

Ray, S. R., W. J. Croom, Jr., A. H. Rakes, A. C. Linnerud, and J. H. Britt. 1983. Effects of methionine hydroxy analog on milk secretion and ruminal and blood variables of dairy cows fed a low fiber diet. J. Dairy Sci. 66:2084-2092.

Reid, I. M., C. J. Roberts, and G. D. Baird. 1980. The effects of underfeeding during pregnancy and lactation on structure and chemistry of bovine liver and muscle. J. Agric. Sci. 94:239-245.

Reynolds, C. K. D. L. Harmon, and M. J. Cecava. 1994. Absorption and delivery of nutrients for milk protein synthesis by portal drained viscera. J. Dairy Sci. 77:2787-2808.

Robert, J. C., C. Richard, T. d’Alfonso, N. Ballet, and E. Depres. 2001. Investigation of the site of absorption and metabolism of a novel source of metabolizable methionine: 2-hydroxy-4(methylthio) butanoic acid isopropyl ester (HMBi). J. Dairy Sci. 84(Suppl. 1):35 (Abstr.).

Robert, J. C., N. Ballet, C. Richard, and B. Bouza. 2002. Influence of 2-hydroxy-4 (methylthio) butanoic acid isopropyl ester (HMBi) on the digestibility of organic matter and energy value of corn silage measured in vitro. J. Dairy Sci. 85 (Suppl. 1):240 (Abstr.).

139

Robinson, P. H., W. Chalupa, C. J. Sniffen, W. E. Julien, H. Sato, K. Watanabe, T. Fujieda, and H. Suzuki. 1998. Ruminally protected lysine or lysine and methionine for lactating dairy cows fed a ration designed to meet requirements for microbial and post-ruminal protein. J. Dairy Sci. 81:1364-1373.

Rooke, J. A., and D. G. Armstrong. 1989. The importance of the form of nitrogen on microbial protein synthesis in the rumen of cattle receiving grass silage and continuous intrarumen infusions of sucrose. Br. J. Nutr. 61:113-121.

Roseler, D. K., J. D. Ferguson, C. J. Sniffen, and J. Herrema. 1993. Dietary protein degradability effects on plasma and milk urea nitrogen and milk nonprotein nitrogen in Holstein cows. J. Dairy Sci. 76:525-534.

Rulquin, A., 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.

Russell, J. B., and H. J. Strobel. 1993. Microbial energetics. Pages 165-186 in Quantitative Aspects of Ruminant Digestion and Metabolism. J. M. Forbes and J. France, ed., CAB International, Wallinford, UK.

Salisbury, R. L., D. K. Marvil, C. W. Woodmansee, and G. F. W. Haenlein. 1971. Utilization of methionine and methionine hydroxy analog by rumen microorganisms in vitro. J. Dairy Sci. 54:390-396.

Santos, F. A. P., J. E. P. Santos, C. B. Theurer, and J. T. Huber. 1998. Effects of rumen- undegradable protein on dairy cow performance: A 12-year literature review. J. Dairy Sci. 81:3182-3213.

SAS Institute Inc.1999. What’s new in SAS software for version 7 and the version 8 developer’s release. SAS Inst. Inc., Cary, NC.

Satter, L. D., and L. L. Slyter. 1974. Effect of ammonia concentration on rumen microbial protein production in vitro. Br. J. Nutr. 23:199-208.

Satter, L. D., R. L. Lang, J. W. Van Loo, M. E. Carlson, and R. W. Kepler. 1975. Adverse effect of excess methionine or methionine hydroxy analog on feed consumption in cattle. J. Dairy Sci. 58:521-525.

Schmidt, G. H. 1971. Biology of lactation. W. H. Freeman, San Fransciso, CA.

Schwab, C. G. 1994. Optimizing amino acid nutrition for optimum yields of milk and milk protein. Pages 114-129 in Proc. Southwest Nutr. Manage. Conf., Phoenix, AZ. Dept. Anim. Sci., Univ. Arizona, Tucson.

140

Schwab, C. G. 1998. Methionine analogs for dairy cows: A subject revisited. 1998 California Animal Nutr. Conf., Fresno, CA.

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. 1. 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 availability of two forms of 2-hydroxy-4-methylthio butanoic acid (HMB) for lactating cows. J. Dairy Sci. 84 (Suppl. 1):35.

Sharma, B. K., and R. A. Erdman. 1988. Effects of high amounts of dietary choline supplementation on duodenal choline flow and production response of dairy cows. J. Dairy Sci. 71:2670-2676.

Smil, V. 1999. Nitrogen in crop production: an account of global flows. Global Biogeochem. Cycles, 13(2): 647-662.

Socolow, R. H. 1999. Nitrogen management and the future of food: Lessons from the management of energy and carbon. Proc. Natl. Acad. Sci. USA. 96:6001-6008.

Stern, M. D., S. Calsamiglia, and M. I. Endres. 1994. Dynamics of ruminal nitrogen metabolism and their impact on intestinal protein supply. Page 105 in Proc. Cornell Nutr. Conf. Feed Manuf., Rochester, NY. Cornell Univ., Ithaca, NY.

Stipanuk, M. H. 2000. Page 266 in Biochemical and Physiological Aspects of Human Nutrition. W. B. Saunders Company. Philadelphia, Pennsylvania.

Stokes, M. R., J. H. Clark, and L. M. Steinmetz. 1981. Performance of lactating dairy cows fed methionine or methionine analog at two concentrations of dietary crude protein. J. Dairy Sci. 64:1686-1694.

St-Pierre, N. R., and C. S. Thraen. 1999. Animal grouping strategies, sources of variation, and economic factors affecting nutrient balance on dairy farms. J. Dairy Sci. 82 (Suppl. 2):72-83.

Swanson, E. W. 1977. Factors for computing requirements of protein for maintenance of cattle. J. Dairy Sci. 60:1583-1593.

Sylvester, J. T., N. R. St-Pierre, B. K. Sloan, J. L. Beckman, and S. M. Noftsger. 2003a. Effect of HMB and HMBi on milk production, composition, and N efficiency of Holstein cows in early and mid-lactation. J. Dairy Sci. 86 (Suppl. 1):60 (Abstr.).

141

Sylvester, J. T., S. K. R. Karnati, M. L. M. Lima, J. L. Firkins, Z. Yu, and M . Morrison. 2003b. Measuring ruminal pool size and duodenoal flow of protozoal N using real-time PCR. J. Dairy Sci. 86 (Suppl. 1):170 (Abstr.).

Torrent, J., D. E. Johnson, and M. A. Kujawa. 1994. Co-product fiber digestibility: kenetic and in vivo assessment. J. Anim. Sci. 72:790-795.

Uden, P., P. E. Colucci, and P. J. Van Soest. 1980. Investigation of chromium, cerium and cobalt as markers in digesta. Rate of passage studies. Domestic animals fed timothy hay. J. Sci. Food Agric. 31 (7): 625-632.

Van Soest, P. J. 1982. Nutritional ecology of the ruminant. 2nd ed. Cornell Univ. Press, Ithaca, NY.

Van Soest, P. J., J. B. Robertson, and B. A. Lewis. 1991. Methods for dietary 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.

Vazquez-Anon, M. , T. Cassidy, P. McCullough, and G. A. Varga. 2001. Effects of Alimet on nutrient digestibility, bacterial protein synthesis, and ruminal disappearance during continuous culture. J. Dairy Sci. 84:159-166.

Volden, H., W. Velle, O. V. Sjaastad, A. Aulie, and O.M. Harstad. 2001. Concentrations and flow of free amino acids in ruminal and duodenal liquid of dairy cows in relation to feed composition, time of feeding and level of feed intake. Acta Agric. Scand. 51:35-45.

Von Keyserlingk, M. A. G., J. A. Shelford, R. Puchala, M. L. Swift, and L. J. Fisher. 1998. In situ disappearance of amino acids from grass silages in the rumen and intestine of cattle. J. Dairy Sci. 81:140-149.

Wallace, R. J., R. Onodera, and M. A. Cotta. 1997. Metabolism of nitrogen-containing compounds. Page 313 in The Rumen Microbial Ecosystem. Blackie Academic & Professional. New York, NY.

Wallenius, R. W., and R. E. Whitchurch. 1975. Methionine hydroxy analog or sulfate supplementation for high producing dairy cows. J. Dairy Sci. 58:1314-1319.

142

Williams, C. H., D. J. David, and O. Iismaa. 1962. The determination of chromic oxide in feces samples by atomic absorption spectrophotometry. J. Agric. Sci. 59:381- 385.

Wester, T. J., M. Vazquez-Anon, D. Parker, J. Dibner, A. G. Calder, and G. E. Lobley. 2000a. Metabolism of 2-hydroxy-4-methylthio butanoic acid (HMB) in growing lambs. J. Dairy Sci. 83:268-269 (Abstr.).

Wester, T. J., M. Vazquez-Anon, D. Parker, J. Dibner, A. G. Calder, and G. E. Lobley. 2000b. Synthesis of methionine (Met) from 2-hydroxy-4-methylthio butanoic acid (HMB) in growing lambs. J. Anim. Sci. 78:269(Abstr.).

White, D. 2000. Page 370 in The Physiology and Biochemistry of Prokaryotes, 2nd ed. Oxford University Press, New York, NY.

Whiting, F. M., J. W. Stull, W. H. Brown, and B. L. Reid. 1972. Free amino acid ratios in rumen fluid, blood plasma, milk, and feces during methionine and methionine hydroxy analog supplementary feeding. J. Dairy Sci. 55:983-988.

Wildman, E.E., G.M. Jones, P.E. Wagner, R.L. Boman, H.F. Troutt, and T.N. Lesch. 1982. A dairy cow body condition scoring system and its relationship to selected production characteristics. J. Dairy Sci. 65:495-501.

Williams, A. G., and G. S. Coleman. 1992. The rumen protozoa. Springer-Verlag. New York, NY.

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.

Wohlt, J. E., S. L. Chmiel, P. K. Zajac, L. Backer, D. B. Blethen, and J. L. Evans. 1991. Dry matter intake, milk yield and composition, and nitrogen use in Holstein cows fed soybean, fish, or corn gluten meals. J. Dairy Sci. 74:1609-1622.

Wright, T. C., S. Muscardini, P. H. Luimes, P. Susmel, and B. W. McBride. 1998. Effects of rumen-undegradable protein and feed intake on nitrogen balance and milk protein production in dairy cows. J. Dairy Sci. 81:784-793.

Wu, Z., and L. D. Satter. 2000. Milk production during the complete lactation of dairy cows fed diets containing different amounts of protein. J. Dairy Sci. 83:1042- 1051.

Yao, Z., and D. E. Vance. 1988. The active synthesis of phosphatidylcholine is required for very low density lipoprotein secretion from rat hepatocytes. J. Biol. Chem. 263:2998-3004.

143

Yu, Z., and W. M. Mohn. 1999. Killing two birds with one stone: simultaneous DNA and RNA extraction from activated sludge biomass. Can. J. Microbiol. 45:269-272.

Yu, Z., and W. M. Mohn. 2001. Bacterial diversity and community structure in an aerated lagoon revealed by ribosomal intergenic spacer analyses and 16S ribosomal DNA sequencing. Appl. Environ. Microbiol. 67:1565-1574.

144