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U {W LIBRARY
4 Michigan State
7, C a] University

This is to certify that the
dissertation entitled

REGULATION OF THE RUMINAL ENVIRONMENT
BY LACTATING DAIRY COWS

presented by
CHARLES STEVEN MOONEY

has been accepted towards fulfillment
of the requirements for the

Ph.D. degree in Department of Animal Science

 

 

Meow Was

' Majorfioféssor’s Signature

géw/ 5; 21206

 

MSU is an Affirmative Action/Equal Opportunity Institution

-—g-o-o-o-o-n-o..--o-n-n—-_.-o—--n-o-o-o-o-o-I-o-I-o-a-o-o-o-u-0-.-.-o-o-I-l-o-o-o-u-

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2/05 p:/C|RC/DaleDue.indd-p.1

 

 

REGULATION OF THE RUMINAL ENVIRONMENT
BY LACTATING DAIRY COWS

By

Charles Steven Mooney

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Animal Science

2006

The mminal cm
Sodium is the m«
candidate as the

strong ions in tha
niminal solution.
generate mminal
nutritional condtt
Sodium at nonna.’
Additional dictar}
indicating that thc
ruminal osmolalit

Sodium blCdTbOfiii

b

 

ut the mechanist

 

 

 

ABSTRACT

REGULATION OF THE RUMINAL ENVIRONMENT
BY LACTATING DAIRY COWS

By

Charles Steven Mooney

The ruminal environment must be regulated for the health and productivity of ruminants.
Sodium is the most abundant cation in the ruminal solution and is the most likely
candidate as the regulated ion. Three experiments were conducted to evaluate the role of
strong ions in the ruminal environment. If sodium compounds are infiased into the
ruminal solution, rumination time is reduced markedly, however, these infusions may
generate ruminal conditions that are not representative of normal physiological or
nutritional conditions. In the first experiment, we hypothesized that additional dietary
sodium at normal concentrations would reduce rumination time of dairy cows.
Additional dietary sodium decreased rumination time as did additional dietary potassium
indicating that the general decrease in rumination was caused by a tonic increase in
ruminal osmolality. Sodium is often added to lactating dairy cow diets in the form of
sodium bicarbonate. The benefits of sodium bicarbonate addition are well documented
but the mechanism of its action has not been defined. In experiment two, addition of
dietary sodium increased total tract neutral detergent fiber digestibility probably by an
expansion of ruminal contents and slowing of passage of digesta from the rumen. These
effects are likely only a component of the mechanism of sodium bicarbonate action in
lactating dairy cows. In this experiment, the addition of dietary sodium bicarbonate did

not affect ruminal pH or alter the site of starch digestion. Sodium is a strong ion and

strong ions are or
hipothesized t .at

nuninal pH. Run

the sum ofrumtri

correlated negati 1
the sum ofrurnir...
negazitely relate:
Therefore. the to:
iirztiting ruminal .
in the ruminal so?
balance in the rut,
epithelium. Sod.

ot the volatile fat'

acti

l'ely ret’JUlate '

 

strong ions are one of the determinants of the pH of a solution. In experiment three, we
hypothesized that strong ion concentrations in the ruminal solution would be related to
ruminal pH. Ruminal pH was correlated positively with ruminal sodium concentration,
the sum of ruminal sodium and potassium, and ruminal strong ion difference, and was
correlated negatively with total volatile fatty acid concentration, ruminal ammonium, and
the sum of ruminal ammonium plus potassium. Also, ruminal sodium concentration was
negatively related to the sum of ruminal potassium plus ammonium concentrations.
Therefore, the total concentration of cations is controlled, balancing ruminal acidity and
limiting ruminal osmolality. A uniform, alkalizing strong ion difference was maintained
in the ruminal solution across animals and dietary treatments and this plus the charge
balance in the rumen are likely regulated by modifying sodium flux across the ruminal
epithelium. Sodium, as well as bicarbonate, are likely key in the whole body regulation
of the volatile fatty acid load. These experiments suggest that lactating dairy cows

actively regulate the ruminal environment especially sodium in the ruminal solution.

DEDICATION

To SG and BW and LL, without whom this would not be.

 

I oould 11-1
To Dr. A'.
To my 0..

for their patience

 

To Drs. .-\'
Button. Benson. I
Ondar’za. Plant. P
questions. them:

To the D;
Ying. for always

To the S‘
Gordon. Kex'in.

Mike, Joy. Sher

To the .-‘

Pam J. Carol E

To the i

To the
H

when Tina‘

TO m‘.

V
v

ACKNOWLEDGMENTS

I would like to say a sincere “thank you”:

To Dr. Allen for his intellectual and financial support of my research.

To my Guidance Committee, Drs. Allen, Adams, Beede, Herdt, and Tempelman,
for their patience and concern.

To Drs. Allen, Adams, Beede, Herdt, Tempelman, Ames, Bucholtz, Bursian,
Burton, Benson, Doumit, Domecq, Hawkins, Heisey, Helm, Hill, Martel], Miller, de
Ondarza, Plaut, Parker, Romsos, Sauvie, Sniffen, and Skidmore for answering my
questions, whether I asked them or not.

To the Dairy Nutrition Technicians, Dave Main, Dewey Longuski, and Jackie
Ying, for always having my back.

To the Staff of the MSU Dairy Teaching and Research Center, Bob Kreft, Randy,
Gordon, Kevin, Bob, Bruce, Rod, Dennis, Rob, JR, Ryan, Sam, Jeff, Jeff, Jason, Dan,
Mike, Joy, Sherri, Justin, and Charlie, for making it happen with a sense of fun.

To the Administrative Staff of the Dept. of Animal Science, Jackie O, Nancy R,
Pam J ., Carol D., Faye W., Sonja, and Kim D., for making sure I did things right

To the Allen graduate students, Masahito, Jenn, Christi, Kevin, and Barry, for
allowing me to be part of their team.

To the undergraduate students in the Dairy Nutrition Lab, Lish, Heidi, Debi,
Heather, Tina, Kristin, and the ‘A’ Team of Amy, Allison, Auburn, Andrea, Alicia, and
Jessica, for always asking the right questions.

To my family and friends for loving me anyway.

llST OF TABLI
LIST OF FIGL'RE

KEYIO ABBRI

l
CHAPTER 1: Li:

THE LACTATI\.I
Oi‘t’ri'lt’lt'
Ruminatrts _.
anmuriun.
Sair‘t‘att'un ,,,
Saliva Cum; -
Saliva Firm

THE REMIX-S.
OWOTC‘A' ...
The Ranting,
Gas Prodttc
”A Produi :
Era-’55 I'FA
Strong 10m
Dielan- Cu“
rater .........
Wafer AIDH )
OSmolah‘tt' r
Tile Blrar-hli)
Brc'arbONcItc

Strong Ion [gr

EXOGENOL‘S St

Background

Odt'um Bic“,
Qdium Bic-u.
SUM/aria" U

 

TABLE OF CONTENTS

       
 

ET OF TABLES ...................................................................................................... ' ....... ix
ET OF FIGURES .......................................................................................................... xv
Y TO ABBREVIATIONS ......................................................................................... xvii
AFTER 1: LITERATURE REVIEW ............................................................................ 1
FHE LACTATING DAIRY Cow As A RUMINANT ............................................................... 1
Overview ..................................................................................................................... 1
Ruminants ................................................................................................................... 1
Rumination .................................................................................................................. 5
Salivation .................................................................................................................... 6
Saliva Composition ..................................................................................................... 7 ;
Saliva Flow ................................................................................................................. 8 E},
fHE RUMINAL ENVIRONMENT ............................................................................. 14 :_
. ;-
Overvzew ................................................................................................................... 14 r.‘
The Ruminal Solution ................................................................................................ I 4 1}
Gas Production and Removal ................................................................................... 16 g:
VFA Production and Removal .................................................................................. 18 2;
Excess VFA ............................................................................................................... 24 :
Strong Ions ................................................................................................................ 28 J?”
Dietary Cation-Anion Difference .............................................................................. 32 3
Water ......................................................................................................................... 34 i
Water Movement Across the Ruminal Wall .............................................................. 35 1*
Osmolality of the Ruminal Solution ................................................................... . ...... 3 7 if;
The Bicarbonate System ............................................................................................ 38
Bicarbonate in the Ruminal Solution ........................................................................ 39 :5-
Strong Ion Difference Theory ................................................................................... 40 A
EXOGENOUS SODIUM BICARBONATE ............................................................................. 44 '
Background ............................................................................................................... 44 I":
Sodium Bicarbonate as a Ruminal Infusate .............................................................. 45 ‘1;
Sodium Bicarbonate as a Feed Additive ................................................................... 46 E;
Simulation of T ime-Release Sodium Bicarbonate ..................................................... 51 T;
The Mechanism of Sodium Bicarbonate Action in the Diets of Lactating Cows ...... 53 8
SUMMARY .................................................................................................................. 56 L.
DISSERTATION RESEARCH OVERVIEW .............................................................. 58 %
.j
is
£5
VI :l t

|
ClltPTER 2; El I

on MILK PRt i'

ABSTRXCT ..
INTRODL'CTI
MATERIALS
Design
Treatments
Data and Sir
Sample Prm
Sample- .42sz
Calculunmn
Statistical .4 t
RESL'LTS .......
DISCL'SSIOX.
Production a
Chewing .li-r
Cost and Go.

 

 
 
  
 
 
 
 
 

 

-----
..

TABLE OF CONTENTS (continued)

CHAPTER 2: EFFECT OF DIETARY STRONG IONS ON CHEWING ACTIVITY

AND MILK PRODUCTION IN LACTATING DAIRY COWS ..................................... 86
ABSTRACT .................................................................................................................. 86
INTRODUCTION ........................................................................................................ 87
MATERIALS AND METHODS .................................................................................. 89

Design ....................................................................................................................... 89
Treatments ................................................................................................................ 89
Data and Sample Collection ..................................................................................... 90
Sample Processing .................................................................................................... 91
Sample Analysis ........................................................................................................ 91
Calculations .............................................................................................................. 92
Statistical Analysis .................................................................................................... 94
RESULTS ..................................................................................................................... 96
DISCUSSION ............................................................................................................... 99
Production and Performance .................................................................................... 99
Chewing Activity ..................................................................................................... I 00
Cost and Gain of Sodium Bicarbonate Addition .................................................... 102
CONCLUSION ........................................................................................................... 103

CHAPTER 3: EFFECTS OF SODIUM BICARBONATE ON SITE AND EXTENT OF
NUTRIENT DIGESTION, MICROBIAL EFFICIENCY, FEEDING BEHAVIOR, AND
YIELD AND COMPOSITION OF MILK FOR MID- TO LATE-LACTATION DAIRY

COWS ............................................................................................................................. l 17
ABSTRACT ................................................................................................................ 117
INTRODUCTION ...................................................................................................... 1 18
MATERIALS AND METHODS ................................................................................ 120

Design ..................................................................................................................... 120
Treatments .............................................................................................................. 121
Data and Sample Collection ................................................................................... 121
Days 15, 16, 1 7: Digestibility Determination ........................................................ 122
Day 18: Preparation Day ...................................................................................... 123
Day 19: Intensive 24 h Collection of Blood and Ruminal Fluid ............................ 123
Days 20, 21, 22, 23, 24: Feeding Behavior Monitoring ........................................ 124
Days 25, 26: Ruminal Valerate Absorption and Liquid Passage Determination. 125
Days 27, 28: Rumen Evacuations for P001 Size Determination ............................ 125
Sample Processing .................................................................................................. 125
Sample Analysis ...................................................................................................... 127
Calculations ............................................................................................................ 129
Statistical Analysis .................................................................................................. 132
RESULTS ................................................................................................................... 134
DISCUSSION ............................................................................................................. 140
CONCLUSION ........................................................................................................... 145

vii

CHAPTER 4: El
coxsrmrio
DAIRY cows. '

ABSTRICT...
INTRODL'CTI
MATERIALS '
Design and .‘
Electronic 1 I .
Sample Prat
Sample PR!"
Rumen Em, ,'
Sample .Ins.‘
Calculation; I
Statistical ,4.
RESL'LTS .....
DISC L'SSIOX
SL'SISIARY A

I
CIIIPTER 5: 13!
APPENDIX TA Ii I

REFERENCES ..

 

TABLE OF CONTENTS (continued)

CHAPTER 4: EFFECTS OF DIETARY STARCH CONCENTRATION AND CORN
CONSERVATION METHOD ON RUMINAL AND PLASMA IONS IN LACTATING

DAIRY COWS. .............................................................................................................. 172
ABSTRACT ................................................................................................................ 172
INTRODUCTION ...................................................................................................... 174
MATERIALS AND METHODS ................................................................................ 177

Design and Treatments ........................................................................................... 1 77
Electronic Data and Automatic Sample Collection ................................................ I7 7
Sample Processing and Storage ............................................................................. 1 78
Sample Preparation ................................................................................................ I 79
Rumen Evacuations for P001 Size Determination ................................................... 1 79
Sample Analysis ...................................................................................................... 1 79
Calculations ............................................................................................................ 181
Statistical Analysis .................................................................................................. 182
RESULTS ................................................................................................................... 184
DISCUSSION ............................................................................................................. 191
SUMMARY AND CONCLUSION ........................................................................... 197

CHAPTER 5: IMPLICATIONS .................................................................................... 232

APPENDIX TABLES ..................................................................................................... 235

REFERENCES ............................................................................................................... 265

viii

Table 1.1- Com?
from MCDU

Table 1.2. Sleas.
Table 1.3. Meas.
Table 1.4. Estin‘..
Table 1.5. Com,"
Table 1.6. Dr} (1'

Table 1.7. Predi .
by processes

Table 1.8. Typic.

Table 1.9. Estini.
bicarbonate

Table 1.10. Slea

Table 1.11. Dist?
(1995‘)

 

LIST OF TABLES

Table 1.1. Comparison of mixed saliva composition of human and ruminant (Adapted

from McDougall, 1948). ........................................................................................... 61
Table 1.2. Measured mean saliva compositions. ............................................................. 62
Table 1.3. Measured saliva flows during resting and eating in cattle. ............................. 63
Table 1.4. Estimated daily flow of saliva in cattle ........................................................... 64
Table 1.5. Composition of mixed gases in the reticulorumen. ........................................ 65
Table 1.6. Dry atmospheric composition at sea level (Weast, 1978). ............................. 66
Table 1.7. Predicted percentage of protons produced daily removed from the RR solution

by processes as modeled by Allen (1997, 2004) ....................................................... 67
Table 1.8. Typical concentrations in lactating dairy cows (NRC, 2001) ......................... 68
Table 1.9. Estimated typical concentrations of sodium, potassium, chloride, and

bicarbonate for spaces related to dairy cows. ........................................................... 69
Table 1.10. Measured ruminal strong ion concentrations in vivo .................................... 70

Table 1.11. Distribution of water in the body of dairy cows adapted from Andrew et a1.

(1995) ....................................................................................................................... 71
Table 1.12. Estimated water pools in lactating dairy cows .............................................. 72
Table 1.13a. Typical blood measures of bovines ............................................................. 73
Table 1.13b. Typical blood measures of bovines. ........................................................... 74
Table 1.14. Reported effective pKa of carbonic acid. ..................................................... 75

Table 1.15. Recommendation for sodium bicarbonate inclusion in lactating dairy cow
diets. .......................................................................................................................... 76

Table 1.16. Sodium bicarbonate solution molality and osmolality as adapted from Weast
(1978). ....................................................................................................................... 77

Table 1.17. Summary of published sodium bicarbonate research with lactating cows on
modest to high (230%) forage diets from 1960 to 1988 (adapted from Table 6 of
Erdman, 1988a). ........................................................................................................ 78

ix

 

Table 1.18. Sun"
bicarbonate T
19891. ........

Table 3.1. Nutrie-
ofdietar} 0‘

Table 3.2. Ingreti
Tableli. Comp
Table2.~l. The ct

TablelS. The et‘
composition

Tablelb. The et‘
milk product

IabIfi 1.7 The cf
manual obser

Table 2.8. Pearw
summation...

Table 2.9. The et‘
observed che

Table 2.10. Cost .
addition of .\i

Table2.l l. Estin:
expected saf: I

labltll, NUWTI
Tablelz. Fermc.

Sued

Table 34 Comp.

 

LIST OF TABLES (continued)

Table 1.18. Summary of published reports of studies with diets averaging 1.1% sodium
bicarbonate for lactating cows from 1980 to 1989 (Adapted from Staples and Lough,

1989). ........................................................................................................................ 79
Table 2.1. Nutrient composition of ingredients used to formulate experimental diets (%

of dietary DM). ....................................................................................................... 104
Table 2.2. Ingredient composition of experimental diets (% of dietary DM) ................ 105
Table 2.3. Composition of experimental diets (% of dietary DM). ............................... 106
Table 2.4. The effect of dietary strong ion treatment on component intakes. ............... 107

Table 2.5. The effect of dietary strong ion treatment on milk production and milk
composition. ............................................................................................................ 108

Table 2.6. The effect of dietary strong ion treatment on body weight, body condition and
milk production efficiency. ..................................................................................... 109

Table 2.7. The effect of dietary strong ion treatment on chewing behavior based on
manual observation. ................................................................................................ 1 10

Table 2.8. Pearson Correlation between manual observation raw counts and Igor Pro®
summation. .............................................................................................................. 111

Table 2.9. The effect of strong ion treatment on Igor Pro® summarization of manually
observed chewing behavior data. ............................................................................ 112

Table 2.10. Cost and gain to economics and ruminal buffering per cow per day with the
addition of sodium bicarbonate to lactating dairy cows at 1% of the DM. ............ 113

Table 2.11. Estimated saliva flow on the control diet based on measured behavior and
expected saliva flows. ............................................................................................. 114

Table 3.1. Nutrient composition of ingredients used to formulate experimental diets (%

of dietary DM). ....................................................................................................... 146
Table 3.2. Fermentation products of wet feeds (% of dietary DM). .............................. 147
Table 3.3. Ingredient composition of experimental diets (% of dietary DM) ................ 148
Table 3.4. Composition of experimental diets (% of dietary DM). ............................... 149

 

Table 3.5. The e:

digestibilit}

Table 3.6. The e:
feeding bel...

Table 3.7. The e'
entire test pe

Table 3.8. The e?
dunng feedi'

Table 3.9. The e?
mill product

Table 3.10. The ;
and organic ‘

Tableill. Th9 ‘~
Tableill. The 1'
Table3.13. The e.
Table3.l4. The e
Table3.lS. The c
Tablellb. The e

Table 317' The C

 

 

Table3.l8. The e
Inlfinsil'e 24 I

Table 319 The

C
expendllm'e. I

Table 330' C e
Ieedjng be a
Table 3-2l. e -.

feeding bell:

LIST OF TABLES (continued)

Table 3.5. The effect of dietary strong ion treatment on diet component intake during
digestibility subperiod ............................................................................................. 150

Table 3.6. The effect of dietary strong ion treatment on diet component intake during
feeding behavior subperiod. .................................................................................... 151

Table 3.7. The effect of dietary strong ion treatment on diet component intake during the
entire test period ...................................................................................................... 152

Table 3.8. The effect of dietary strong ion treatment on milk production and composition
during feeding behavior subperiod. ........................................................................ 153

Table 3.9. The effect of dietary strong ion treatment on body weight and condition and
milk production efficiency. ..................................................................................... 154

Table 3.10. The effect of dietary strong ion treatment on digestibility of dry matter (DM)

and organic matter (OM). ....................................................................................... 155
Table 3.11. The effect of dietary strong ion treatment on digestibility of starch. ......... 156
Table 3.12. The effect of dietary strong ion treatment on digestibility of NDF. ........... 157
Table 3.13. The effect of dietary strong ion treatment on ruminal kinetics .................... 158
Table 3.14. The effect of dietary strong ion treatment on N metabolism. ..................... 159
Table 3.15. The effect of dietary strong ion treatment on ruminal turnover times. ....... 160
Table 3.16. The effect of dietary strong ion treatment on ruminal measurements. ....... 161

Table 3.17. The effect of dietary strong ion treatment on ruminal VFA profile and
ruminal measurements during digestibility subperiod. ........................................... 162

Table 3.18. The effect of dietary strong ion treatment on ruminal VFA profile during
intensive 24 h collection. ........................................................................................ 163

Table 3.19. The effect of dietary strong ion treatment on net energy intake and
expenditure .............................................................................................................. 164

Table 3.20. The effect of dietary strong ion treatment on chewing behavior during
feeding behavior subperiod. .................................................................................... 165

Table 3.21. The effect of dietary strong ion treatment on drinking behavior during
feeding behavior subperiod. .................................................................................... 166

xi

Table 3.22. The 5‘
behas‘ior sub

Table3.33. The e
electrolue ct

Table 3.24. The e
concentratior

Table 3.25.. Distr

niminaIl} Llf‘.
experiment :

Table 4.1. Select.
and Allen (2‘

Table 4.2. Strong
ofdietary D‘

Table43. Ingreti
TableH. Comp.
Table 4.5. The e:
Table 4.6. The er
Table 4.7. The e:

Table4.8. The e:
tluid ............

 

Table 4.10 The {I

Tabled”. The I

C

Table 4.12 The

 

LIST OF TABLES (continued)

Table 3.22. The effect of dietary strong ion treatment on ruminal pH during feeding
behavior subperiod. ................................................................................................. 167

Table 3.23. The effect of dietary strong ion treatment on whole jugular blood gas and
electrolyte concentrations at 385°C. ...................................................................... 168

Table 3.24. The effect of dietary strong ion treatment on plasma metabolite and hormone
concentration. .......................................................................................................... 169

Table 3.25. Distributions from previous work at Michigan State University with
ruminally and duodenally cannulated lactating Holstein dairy cattle compared

experiment in Chapter 3 (Based on iNDF flow). .................................................... 170
Table 3.26. Comparison of design and results of experiments in Chapter 2 and Chapte3'731.
Table 4.1. Selected descriptive results for this experiment published previously in Oba

and Allen (2003a), Oba and Allen (2003b), and Oba and Allen (2003c). .............. 198
Table 4.2. Strong ion composition of ingredients used to formulate experimental diets (%

of dietary DM). ....................................................................................................... 199
Table 4.3. Ingredient composition of experimental diets (% of dietary DM) ................ 200
Table 4.4. Composition of experimental diets (% of dietary DM). ............................... 201
Table 4.5. The effect of dietary treatment on feeding behavior ..................................... 202
Table 4.6. The effect of dietary treatment on ruminal pools. ........................................ 203
Table 4.7. The effect of dietary treatment on ruminal pH. ............................................ 204
Table 4.8. The effect of dietary treatment on volatile fatty acid concentration of ruminal

fluid. ........................................................................................................................ 205
Table 4.9. Distribution of ruminal ions measured across cows and periods .................. 206
Table 4.10. The effect of dietary treatment on ruminal ion concentration. ................... 207
Table 4.11. The effect of dietary treatment on whole blood measurements. ................. 208

Table 4.12. The effect of dietary treatment on whole blood gases and related measures.
................................................................................................................................. 209

xii

Table 4-13'

Table 4- 14'

Table 4.15. 1

Table 4.16. F
meaSUTCI

Table 4.17. L

Table 4.18. Pt

dit‘ferencs

Table 4.19. In;
and VFA I

Table AI. Prod
Table A2. Tree
TableA3. Iner
Table A4. Ax'er
Table A5. Treat
Table A6. Indit'r

Table .17. I’isua

Table .48. Data r

Table A9. Start '

 

Table A10. .Vurt'

TableAl 1. Data

Table .412. Lac‘
Olbebaiior s.

 

LIST OF TABLES (continued)

Table 4.13. The effect of dietary treatment on ruminal fluid and plasma osmolality.... 210

Table 4.14. Pearson correlation coefficients among mean cow period ruminal
measurements (n231) .............................................................................................. 211

Table 4.15. Pearson correlation coefficients among mean cow period blood
measurements (n=32) .............................................................................................. 212

Table 4.16. Pearson correlation coefficients between mean cow period blood and ruminal
measurements (n331). ............................................................................................. 213

Table 4.17. Linear regression results summary. ............................................................ 214

Table 4.18. Potential ions contributing to ruminal pH according to the strong ion

difference theory. .................................................................................................... 2 15
Table 4.19. Influx, pool size and ruminal turnover of sodium, potassium, chloride, water

and VFA in the rumen ............................................................................................. 216
Table A. 1. Projected concentrations based on book values. .......................................... 236
Table A2. Treatment assignments for individual cows for 01CSM1. .......................... 237
Table A3. Individual cow descriptors at the beginning of 01CSM1. ........................... 239
Table A4. Average status of 40 experimental cows at the beginning of 01CSM1. ...... 240
Table A5. Treatment assignments for individual cows for 02CSM2. .......................... 241
Table A6. Individual cow descriptors at the beginning of 02CSM2. ........................... 242
Table A7. Visual representation of the 99M001 experimental design. ....................... 243
Table A8. Data removed from 01CSM1 data set .......................................................... 244
Table A9. Start and stop times used in 01CSM1 behavior data sets. ........................... 245

Table A.10. Number of days used in statistics for 02CSM2 feeding behavior data set. 246
Table All. Data removed from 02CSM2 feeding behavior data set ............................ 247

Table A.12. Lack of effect of halter redesign on chewing activity: comparing first half
of behavior subperiod to last half. ........................................................................... 250

xiii

Table A13. Tbs

compositioi

Table A14. The
feeding belt

Table A15. The
feeding beh.

Table A16. The
behatior mo

Table Ali. The
(Based on C:

Table A18. The
on C r;0; 110

Table A19. The
C1203 00W).

Table A20. The
(Based on Ci

Table A21. The
llow)...........

Table A22. Di...
l'Umllliilly ax
ebilifliment :.

Table A.23_ Dish

Table A24 D

’- is‘.‘

 

 

LIST OF TABLES (continued)

Table A.13. The effect of dietary strong ion treatment on milk production and
composition during feeding behavior monitoring (d20-d24) .................................. 251

Table A. 14. The effect of dietary strong ion treatment on chewing behavior during
feeding behavior monitoring (d20-d24). ................................................................. 252
Table A15. The effect of dietary strong ion treatment on drinking behavior during

feeding behavior monitoring (d20-d24). ................................................................. 253

Table A.16. The effect of dietary strong ion treatment on ruminal pH during feeding
behavior monitoring (d20-d24). .............................................................................. 254

Table A.17. The effect of dietary strong ion treatment on digestibility of DM and OM
(Based on CrzO3 flow). ........................................................................................... 255

Table A. 18. The effect of dietary strong ion treatment on digestibility of starch (Based
on Cr203 flow). ....................................................................................................... 256

Table A. 19. The effect of dietary strong ion treatment on digestibility of NDF (Based on
szO3 flOW). ............................................................................................................ 257

Table A20. The effect of dietary strong ion treatment on ruminal kinetics and pools
(Based on Cr203 flow). ........................................................................................... 258

Table A2]. The effect of dietary strong ion treatment on N metabolism (Based on Cr203
flow). ....................................................................................................................... 259

Table A.22. Distributions from previous work at Michigan State University with
ruminally and duodenally cannulated lactating Holstein dairy cattle compared

experiment in Chapter 3 (Based on Cr203 flow). ................................................... 260
Table A.23. Distribution of whole blood measures in Chapter 4. ................................. 261
Table A.24. Distribution of ruminal VFA measures on d 15 in Chapter 4. ................... 262

Table A.25. Results used to determine influx, pool size and ruminal turnover of sodium,
potassium, chloride, water and VFA in the rumen. ................................................ 263

xiv

oooooooooo

Fzgure 1.2-
rumina
31. (.19k

Figure 1.3.. .
salts (A

Figare1.4. P
Tigurel.5. P;
thurelb. Pr

Figure 2.1. Th
condition

I:115’lare22. Pro
exPCTTmeni

Figure 4,]. A C(

Figure 4.2. The
figure 4.3. The r

figure 44. The r.

535111645. The n

 

Thou-9. . e rd
Sodium---

 

LIST OF FIGURES

Figure 1.1. Hypothesized movement of VFA across the ruminal epithelium based on
figures from Gaebel and Sehested (1997), Sehested et al. (1999b), and Leek (2004).
................................................................................................................................... 80

Figure 1.2. Hypothesized movement of sodium, potassium, and chloride across the
ruminal epithelium based on figures from Gaebel and Sehested (1997), Sehested et

al. (1999b), and Leek (2004) ..................................................................................... 81
Figure 1.3. A hypothetical mechanism for increasing milk fat production with buffer

salts (Adapted from Figure 1 in Russell and Chow, 1993). ...................................... 82
Figure 1.4. Proposed flow of proton with absorption of undisassociated VFA ............... 83
Figure 1.5. Proposed flow of proton with absorption of disassociated VFA. .................. 84
Figure 1.6. Proposed ion exchange to balance charges. .................................................. 85

Figure 2.1. The relationship between change in body weight and change in body
condition score across periods. ............................................................................... 115

Figure 2.2. Proposed model of the decreased rumination due to ion treatment in this

experiment ............................................................................................................... 116
Figure 4.1. A comparison of ruminal and plasma osmolality distributions. .................. 217
Figure 4.2. The relationship between ruminal sodium and ruminal pH. ....................... 218
Figure 4.3. The relationship between ruminal SID and ruminal pH .............................. 219
Figure 4.4. The relationship between ruminal ammonia and ruminal pH. .................... 220
Figure 4.5. The relationship between ruminal potassium and ruminal pH. ................... 221
Figure 4.6. The relationship between ruminal chloride and ruminal pH. ...................... 222
Figure 4.7. The relationship between ruminal potassium and ruminal sodium. ............ 223
Figure 4.8. The relationship between ruminal ammonia and ruminal sodium. ............. 224

Figure 4.9. The relationship between ruminal ammonia plus potassium and ruminal
sodium. .................................................................................................................... 225

XV

11111116410. Tl‘n
ifference. .

fimre411. The
concentratu

figure 4.12. The
concentratio

figure 4.13. The

uuuuuuuuuuuuuuuuuuuu

figure 4.14. The

figure 4.15.. Pro:

 

LIST OF FIGURES (continued)

Figure 4.10. The relationship between meal size (kg) and ruminal sodium concentration
difference. ............................................................................................................... 226

Figure 4.11. The relationship between meal size (kg) and ruminal potassium
concentration difference .......................................................................................... 227

Figure 4.12. The relationship between meal size (kg) and ruminal sodium plus potassium
concentration difference .......................................................................................... 228

Figure 4.13. The relationship between meal size (kg) and ruminal osmolality difference.

................................................................................................................................. 229
Figure 4.14. The relationship between meal size (kg) and ruminal pH difference ........ 230
Figure 4.15. Proposed net sodium bicarbonate recycling in lactating dairy cows ......... 231

xvi

01 C S .\1 1
02C SM:
99.\IOOI
ADF
AOAC
BC S
BHBA
BIT'

C P

DCAD
DCAD3
DCAD4
DHIA

DIS-1

Dbl

Dill

ECF

EDTA
PCT-1

HPLC

ICF

days

6X If;

elhj.

 

fat-e
high

Infra.-

 

OlCSMl

02CSM2

99MOO 1

ADF

AOAC

BCS

BHBA

BW

CP

DCAD

DCAD3

DCAD4

DHIA

DIM

DM

DMI

ECF

EDTA

FCM

HPLC

ICF

KEY TO ABBREVIATIONS

Experiment: 40 cows, 5 x 5 Latin square (n = 8), 14 d periods (Chapter 2)
Experiment: 6 cows, 3 x 3 Latin square (n = 2), 28 (1 periods (Chapter 3)
Experiment: 8 cows, 4 x 4 Latin square (11 = 2), 21 (1 periods (Chapter 4)
acid detergent fiber

Association of Official Analytical Chemists International

body condition score

B-hydroxybutyrate

body weight

crude protein

dietary cation anion difference

dietary cation anion difference based on Na, K and Cl

dietary cation anion difference based on Na, K, Cl, and S

Dairy Herd Improvement Association

days in milk

dry matter

dry matter intake

extracellular fluid

ethylenediaminetetraacetate

fat-corrected milk

high performance (pressure) liquid chromatography

intracellular fluid

xvii

iNDF
SIN

MIN

sax
sot
NEFA
AIL

sic
saws
NRC

on

OSII

PdNDF

R00

SARI
SAS

SCC

T11

relic
reti.

SUba

 

 

 

Slatf

50m.

 

iNDF

NAN

NEFA
NEL
NFC
NAN MN
NRC
OM

OMI

pdNDF

ROO

SARA

SAS

SCC

KEY TO ABBREVIATIONS (continued)

indigestible NDF

microbial nitrogen

milk urea nitrogen

number of samples
nonammonia nitrogen

neutral detergent fiber
nonesterified fatty acids

net energy for lactation
nonfiber carbohydrate
non-ammonia non-microbial N
National Research Council
organic matter

organic matter intake
potentially digestible NDF
correlation coefficient
coefficient of determination
reticular-omasal orifice
reticulo-rumen or reticulorumen
subacute ruminal acidosis
Statistical Analysis System

somatic cell count

xviii

SCSI

SC S

SD

SE

SID

S.\T
TRDOSI
TSIR

\TA

2 l,
5‘111

501‘.

101;;

VOL.

 

SCM

SCS

SD

SE

SID

SNF

TRDOM

TMR

KEY TO ABBREVIATIONS (continued)

solids-corrected milk

somatic cell score

standard deviation

standard error

strong ion difference

solids-not-fat

truly ruminally degraded organic matter
total mixed ration(s)

volatile fatty acid(s)

xix

CHAPTER 1:

Overview
Laetatin;
consume feed. c
consumed feed 1
cows must regal
sodium biearbon

bicarbonate is pr

Ruminants
Ruminanl
("Russell and va

Endproduet remQI

 

 

CHAPTER 1: Literature Review

THE LACTATING DAIRY COW AS A RUMINAN T

Overview

Lactating dairy cows are unique among domestic ruminants for their ability to
consume feed, commonly consuming 4% of their BW on a daily basis (NRC, 2001). This
consumed feed ferrnents in the forestomach and leads to a significant acid load which
cows must regulate to maintain homeostasis. In this regulation, the production of copious
sodium bicarbonate by the salivary glands is a key control. Total salivary sodium

bicarbonate is proportional to total saliva flow which is influenced by many factors.

Ruminants

Ruminants have a symbiotic relationship with the microbes of their foregut
(Russell and Rychlik, 2001). The ruminant provides water, warmth, substrate, and
endproduct removal to a dense, diverse, and interacting collection of suitable bacteria
(>10lo cells per gram of contents), protozoa (=106 cells per gram of contents), and fungi
(Russell and Rychlik, 2001). In return, the ruminant obtains nutrients (energy from VFA,
microbial protein) from plant fiber (cellulose etc.) unavailable by mammalian digestion
(Russell and Rychlik, 2001).

Ruminants are characterized by a fermentation of feed in a highly specialized
four-chambered stomach (Van Soest, 1994). Feed is ingested and fermented to volatile

fatty acids (VFA) by microbes in the forestomach (Hofrnann, 1988). Consumed sugars,

starches. cellulc
and Hume. 199.
account for 95“"
with half as mul
(Leek. 20m ). F
(Stex'ens and HL

Microbi:
three foresto .rat
partially separaz.
together as the n
absorbing water.
15% of the body
The OmaSUm. the
Particles from (h,
1983). Cows hm.
Compared to shei

the omasum can ‘

domestic cattle (J‘

 

197s; .
~)- fills abs(‘

preparation of Hr

Stems and Hu,

 

starches, cellulose, hemicellulose and pectin are fermented to VFA and gases (Stevens
and Hume, 1995). In the forestomach, acetic, propionic, and butyric acids usually
account for 95% of the VFA in solution and, of the gases, carbon dioxide predominates
with half as much methane and much smaller amounts of hydrogen and hydrogen sulfide
(Leek, 2004). Proportions of VFA and gases are dependent on substrate and microbes
(Stevens and Hume, 1995).

Microbial fermentation occurs in the rumen and reticulum, the first two of the
three forestomach chambers (Hofmann, 1988). As the rumen and the reticulum are only
partially separated by the reticuloruminal fold, these two organs can be considered
together as the reticulorumen (R; Van Soest, 1994). The R is major site of absorption,
absorbing water, VFA, and ions (Van Soest, 1994) and, with its contents, can represent
15% of the body weight but the percentage is highly variable (Stevens and Hume, 1995).
The omasum, the third chamber of the forestomach, controls the flow of the water and
particles from the RR and absorbs water and VFA from the passed digesta (Hofmann,
1988). Cows have a more prominent omasum having twice the relative surface area when
compared to sheep and goats (Engelhardt and Hauffe, 1975). The papillated lamellae of
the omasum can constitute one-third of the surface area of the entire forestomach in
domestic cattle (Stevens and Hume, 1995). This surface area absorbs water, VFA, sodium
and potassium and can start the gastric secretion of chloride (Engelhardt and Hauffe,
1975). This absorption reduces the digesta volume and changes solute concentrations in
preparation of HCl digestion in the abomasum, the fourth chamber and true stomach

(Stevens and Hume, 1995).

The for?

   

Stratified squani.
are not secretor;
structures that ll“.
“honeycomb" nc
20114).

The four-
lhe stomach rec
the hepatic Vet n i
Sllllp-athettc pati'
Dene collect set:
Chemical stimul.:

Motor signals rc‘

contraction cycle
The sympathetic

2004).

Tension r

Leek l9?0; Lee

 

s
mOOth muscle lav

1934) and appear

appar '
ent in
the ‘
n.

 

 

The forestomach chambers are lined on the luminal side with nonglandular
stratified squamous epithelium with slight keratinization (Stevens and Hume, 1995) and
are not secretory tissues (Leek, 2004). The forestomach’s three compartments have
structures that increase the surface area. The rumen has papillae, the reticulum has a
“honeycomb” network of low ridges and the omasum has papillated lamellae (Leek,
2004).

The four-chambered stomach of dairy cows is well vascularized and innervated.
The stomach receives blood from the branches off the abdominal aorta and is drained by
the hepatic vein (Leek, 2004). The stomach is innervated by parasympathetic and
sympathetic pathways (Leek, 2004). The parasympathetic pathways along the vagus
nerve collect sensory input from the forestomach (monitoring tension, mechanical and
chemical stimulation) which is integrated in the gastric centers of the brain (Leek, 2004).
Motor signals return from the brain and are essential for the primary and secondary
contraction cycles of the forestomach and also rumination and eructation (Leek, 2004).
The sympathetic pathways along the splanchnic nerve can inhibit gastric motility (Leek,
2004).

Tension receptors and epithelial receptors have been found in the RR (Iggo and
Leek, 1970; Leek and Harding, 1975; Leek, 1984). Tension receptors are located in the
smooth muscle layer of the RR (Iggo and Leek, 1970; Leek and Harding, 1975; Leek,
1984) and appear to monitor the tension the RR wall (Leek, 2004). They are most
apparent in the medial walls of reticulum, cranial ruminal sac, ruminorecticular fold, in
the cranial pillar, outside of the lips of the reticular fold, and around the cardia and the

reticulo-omasal orifice (Leek, 1984; Leek, 2004). These receptors are excited by passive

distention caust
1934; Leek. It):
amplitude of pr
also follows (L.
195“: Leek. It"

The eptt

These receptors
1975; Leek. 19%
moving tactile s
Leek 2.004). T‘r
of these receptor
range ofchemice
increases in “tritt
(Leek and Hardit‘
ruminal stasis (L

SlglllfiCam epit‘v

 

“fight adds e\-(t

(Leek and Hard”,

 

 

 

distention caused by luminal contents and by active contraction of smooth muscle (Leek,
1984; Leek, 2004). Low to moderate excitation of these receptors increases the rate and
amplitude of primary and secondary contractions and an increase in flow rate of saliva
also follows (Leek, 1984; Leek, 2004). High excitation has the opposite effects (Leek,
1984; Leek, 2004).

The epithelial receptors in the RR are located near basement membrane of luminal
epithelium (Iggo and Leek, 1970; Leek and Harding, 1975; Leek, 1984; Leek, 2004).
These receptors respond to both mechanical and chemical stimulation (Leek and Harding,
1975; Leek, 1984; Leek, 2004). Mechanically, these receptors are excited by lightly
moving tactile stimuli (i.e. “rapid light brushing”) with a very low threshold (Leek, 1984;
Leek, 2004). This excitation stimulates rumination which is likely the primary function
of these receptors (Leek, 1984; Leek, 2004). Chemically, these receptors respond to a
range of chemical stimuli (Leek and Harding, 1975). These receptors are stimulated by
increases in “tritratable acidity” and this excitement inhibits the primary contraction cycle
(Leek and Harding, 1975). In the extreme, the high acidity of acidosis will lead to
ruminal stasis (Leek and Harding, 1975). In addition, a pH below 6 seems required for
“significant epithelial receptor excitation” (Crichlow and Leek, 1981). Lower molecular
weight acids evoke a quicker response and high molecular weight acids are ineffective
(Leek and Harding, 1975). Of the VFA, butyric acid is particularly potent (Leek and
Harding, 1975). Hypertonic and alkali solutions will also excite these receptors but at
concentrations tested were outside the normal physiological range (Leek and Harding,
1975). Water and hypotonic solutions also excite these receptors (Leek and Harding,

1975).

Rumination
Rumir
regulation ofi
regurgitation <
cyclical proce:
and Hooper. l
integrate rumir
esotzhagus. anc
bolus ofingest.
resu'allowed 3!“
added (Van Sm-
regurgitated aft;
Dairy C0
(Van Soest. 199.
(Beauchemin. lg
ofdiet and the to
10 h per d (Welc
(Beauchemin, 19
[hejaw that CTUSE

microbial 3313

Ck

 

 

Rumination

Rumination is one of the defining behaviors of a ruminant. It aids in the
regulation of ions and pH in the forestomach and is defined as the postprandial
regurgitation of ingesta (Van Soest, 1994) and is a highly coordinated, highly rhythmic,
cyclical process characterized by regurgitation, remastication, and reswallowing (Welch
and Hooper, 1988). To ruminate, the central nervous and enteric nervous systems
integrate ruminal and reticular stimuli and coordinate the diaphragm, rumen, reticulum,
esophagus, and mouth (Van Soest, 1994). Rumination begins with the regurgitation of a
bolus of ingesta from the reticulum to mouth (Beauchemin, 1991). Excess liquid is
reswallowed and the remaining bolus is rechewed for about 60 seconds while salvia is
added (Van Soest, 1994). The bolus is reswallowed to the RR and another will be
regurgitated after a short pause (Beauchemin, 1991).

Dairy cows will normally spend more time ruminating than eating during a day
(Van Soest, 1994). They will spend 5 to 9 h per (1 ruminating and 4 to 7 h per (1 eating
(Beauchemin, 1991). Actual amount of time depends on physical form and composition
of diet and the total amount ruminating time per (1 seems to have an upper limit of about
10 h per (1 (Welch, 1982). Cows will spend 10 to 20 periods per (1 ruminating
(Beauchemin, 1991). The 30,000 to 50,000 chews per d occur with a lateral motion on
the jaw that crushes not cuts the ingesta with the molars exposing the plant flesh to
microbial attack (Beauchemin, 1991).

Rumination behavior is influenced by many factors. Rumination is inhibited by
low pH, anorexia, high ruminal osmolality, and high VFA concentration (Welch, 1982;

Welch and Hooper, 1988; Beauchemin, 1991; Van Soest, 1994; Leek, 2004). Rumination

can be complete
sheep; Welch. 1
and feed intake.
1980: Welch an.
factors are assoc

al. 1990) and. t1"

Salit'ation

The incrz.
Compared to or?
quantities of but
Sodium and bica
functions to aid :
bolus of food. to
add fluid for pro

buffers to the Fur

a1" 1991).

 

can be completely stopped by osmolality above a threshold (suggested as 350 mOsm in
sheep; Welch, 1982). Rumination is stimulated by increased particle size, dietary fiber
and feed intake, and decreased forage quality (Welch and Smith, 1969; Sudweeks et al.,
1980; Welch and Hooper, 1988; Beauchemin, 1991; Van Soest, 1994). These stimulatory
factors are associated with greater mechanical stimulation of the rumen wall (Baumont et

a1, 1990) and, therefore, excitation of tension and epithelial receptors of RR (Leek, 2004).

Salivation

The increase in saliva production is one of the important aspects of rumination.
Compared to other species, ruminants secrete large quantities of saliva with enhanced
quantities of buffer (Herdt, 2002). Ruminant saliva is more basic and contains more
sodium and bicarbonate than other species (McDougall, 1948; Table 1.1) This saliva
functions to aid in lubrication of ingested feed, in taste, in forming and swallowing a
bolus of food, to provide some nutrients to rumen microorganisms (urea, minerals), to
add fluid for proper microbial actions in R, and to supply bicarbonate and phosphate
buffers to the rumen (Bartley, 1976; Church, 1988a; Beauchemin, 1991; Ruckebusch et
aL,1991)

Saliva is secreted from several glands. Ruminants have five bilateral and 3
unpaired g1ands(Kay, 1960). The five bilateral glands are the parotid, submaxillary,
inferior molar, sublingual, and buccal (Kay, 1960). The three unpaired are the labial,
pharyngeal, and palatine (Kay, 1960). These glands fall into 3 histological groups:
serous, mucous, and mixed (Kay, 1960). The parotids secrete 40-50% of the daily saliva
(Kay, 1960). The total flow from all glands is referred to as the “mixed” flow (Stevens

and Hume, 1995).

Saliva C OmPO’

Rumina
me“ be!“ 36” P
below pH 5.5 (1
phosphate from
1995) and is ne.
Ruminant saliv;
and a lovver con
some .\' contain
contains an anti'
enzymes lipase :
l0 15% (McDor

Saliva cc
conditions (Bail l
of anions (mEq 1
53““ Compositi
1961b). Howe“.
decreases and pi“

composition has

 

The Prin‘;
Ssdium is usualil

l960). “hen the

 

Saliva Composition

Ruminant saliva has an alkaline pH of near 8.2 (McDougall, 1948) and buffers
well between pH 6 and 7 (Turner and Hodgetts, 1955b) but not well above pH 7.5 or
below pH 5.5 (Bartley, 1976). Mixed saliva contains sodium, potassium, chloride and
phosphate from the blood and bicarbonate from the salivary cell (Stevens and Hume,
1995) and is near isotonic with serum at standard ranges of secretion (Kay, 1960).
Ruminant saliva contains higher concentrations of bicarbonate, phosphate and sodium
and a lower concentration of chloride when compared to serum (Herdt, 2002). It also has
some N containing compounds, mostly in the form of urea (Bartley, 1976). Saliva also
contains an antifoaming agent to prevent bloat and an limited amount of the digestive
enzymes lipase and amylase (Church, 1988a). Overall, the DM of saliva is very low at 1
to 1.5% (McDougall, 1948; Bailey and Balch, 1961b).

Saliva composition is relatively constant at higher rates of secretion under normal
conditions (Bailey and Balch, 1961b) and the sum of cations (mEq/L) will equal the sum
of anions (mEq/L) (McDougall, 1948; Bailey and Balch, 1961b). At resting flows, mixed
saliva composition is about equal to parotid saliva composition (Bailey and Balch,
1961b). However, at mixed saliva flow rates below 30 ml/min, bicarbonate concentration
decreases and phosphate concentration increases (Bailey and Balch, 1961b). Saliva
composition has been measured in several experiments (Table 1.2).

The primary cation of mixed saliva of the ruminant is sodium (Kay, 1960).
Sodium is usually present at more than ten times the potassium concentration (Kay,
1960). When the body is depleted of sodium (either by prolonged dietary sodium

deficiency or by an artificial sodium draw), potassium can replace sodium in mixed saliva

so that the sum

Bailey and Ba]\

inferior molar. .

and K in covvs \

concentrations I.
or infusion) m:
and Stacy. 197”
rates (Carter an.

The prii‘,
then chloride ft.‘
about four time.
0ft0ncentratior
to remain ”00*“

animal. diet ()1- t“

l
I
I
I

 

 

detail.)
Total fit.
total saliva”- n,

6.6 Eq (1, respec-

SaliVa no“,
COWS se

rUm - I
”aims (ChL

do ‘ I

8‘ ~ _
3% NO to {OUT

 

so that the sum of sodium and potassium concentrations remains constant (Kay, 1960;
Bailey and Balch, 1961a; Bailey and Balch, 1961b; Hawkins et al., 1965). The parotid,
inferior molar, and submaxillary glands affect this change (Kay, 1960). The sum of Na
and K in cows saliva is likely constant (166 mEq/L; Bailey and Balch, 1961b). Saliva
concentrations of sodium, potassium andurea can be influenced by intake (either dietary
or infusion) with the greater ingestion leading to higher saliva concentrations (Warner
and Stacy, 1977). Sodium concentration of saliva appears constant over a range of flow
rates (Carter and Grovum, 1990a; Table 1.2 for more detail.)

The primary anion of mixed saliva of the ruminant is bicarbonate with phosphate
then chloride following in concentration (Kay, 1960). Saliva bicarbonate concentration is
about four times, saliva phosphate is about fifteen times, and saliva chloride is about 1/6
of concentrations found in serum (McDougall, 1948). These anions of mixed saliva tend
to remain proportional (Bailey and Balch, 1961a) and are not affected strongly by the
animal, diet or experimental treatment (Bailey and Balch, 1961b). (Table 1.2 for more
detail.)

Total flow of bicarbonate and phosphate into the R will be proportional to the
total salivary flow (Erdman, 1988a) and can be predicted to provide buffering of 19.0 and

6.6 Eq/d, respectively, for a typical lactating dairy cow on a typical diet (Allen, 1997).

Saliva Flow

Cows secrete saliva continuously with increases in flow rate during eating and
ruminating (Church, 1988a). Several experiments have measured saliva flow at rest and
during eating. (Table 1.3) Earlier work determined that saliva flow rate during eating

was two to four times the flow rate at rest (Bailey, 1961a). More recent work with

lactating dairy ~
flow, C assida z:
saliva flovvs “ C
al. I 2002b). US‘.‘
ml min during t

Parotid
rumination. Ar.
covv was 10 ml
rumination I 8.;
rumination was
1961a). A later
and calculated .1
less than a third
Concluded that f
0r 1.8 times [he
recommended

1“ mmiti
glands Under th(

and inferior moi

mQIar‘ Palatine,

Oesophagus‘ am

 

lactating dairy cows has shown a smaller difference between resting flow and eating
flow. Cassida and Stokes (1986), using cardial collection in lactating cows, found mixed
saliva flows were 151 ml/min during resting and 171 mI/min during eating. Maekawa et
al. (2002b), using cardial collection in lactating cows, mixed saliva flows were 101
ml/min during resting and 225 ml/min during eating. (Table 1.3)

Parotid cannulation has been used to calculate mixed saliva flow during
rumination. An early measure of flow from a single cannulated parotid gland of a dry
cow was 10 m1/min for resting, 20 ml/min during eating, and 25 ml/min during
rumination (Bailey and Balch, 1961a). Based on this, mixed saliva flow during
rumination was estimated at 2.5 times the resting flow or 100 ml/min (Bailey and Balch,
1961a). A later review summarized 19 published parotid cannulations during rumination
and calculated a mean for flow during rumination of 1.7 times resting and showed that
less than a third of the studies over 2.0 times (Cassida and Stokes, 1986). The authors
concluded that 2.5 times was “not warranted” and a ruminating flow rate of 272 mein
or 1.8 times the resting rate for lactating dairy cows on “high concentrate diets” was
recommended.

In ruminants, flow of saliva can be influenced by a number of factors. Salivary
glands under the control of parasympathetic nervous system (Herdt, 2002). The parotids
and inferior molar (and thus over half of the flow) are under tonic nervous inhibition and
submaxillary is under tonic stimulation (Kay, 1960). Flow from the parotids, inferior
molar, palatine, buccal, and pharyngeal is increased with the stimulation of the mouth,

oesophagus, and RR (Kay, 1960).

Flow 0?
flow is greatest
these flow di t‘t‘c
Flows during r;
(Putnam et 31..
produced (Bar ‘.—.
producing long
With higher flo
1961b) and by:
increase to the 3

1963; Bartley, j

variation (Meyc

F low is I
Parotids can be
or reticulo-oma
tactical stimula:

 

H'lOIility) are 1n}!

FlOw is r

millSOn and Tri

effects on Plasn.

 

Flow of saliva can be influenced by feed, feed characteristics, and intake. Resting
flow is greatest on grass and lowest on silage with hay diets being intermediate, however,
these flow differences do not produce ruminal differences (Bailey and Balch, 1961b).
Flows during resting and rumination are reduced by pelleting and grinding of feeds
(Putnam etal., 1966). Meal length is probably more important in determining the saliva
produced (Bailey, 1961a) with feeds most difficult to process through the mouth
producing longer eating times and therefore more saliva. Flow is influenced by meal size
with higher flow after small meal and lower flow after big meal (Bailey and Balch,
1961b) and by time relative to a meal with slowest flow after feeding and a gradual
increase to the highest rate before the next feeding (Bailey and Balch, 1961b; Wilson,
1963; Bartley, 1976). Distention caused by food, water and saliva entering the RR during
a meal can generate an inhibition of saliva flow that gradually subsides as the volume of
the RR diminishes between meals (Bartley, 1976). Resting flow can also show a diurnal
variation (Meyer et al., 1964).

Flow is influenced by the ruminal contents and blood composition. Flow from the
parotids can be increased by stretching near the oesophagus, cardia, reticulo-rumen fold
or reticulo-omasal orifice (Ash and Kay, 1959). Flow is not directly influenced by light
tactical stimulation in the RR but this stimulation can initiate rumination which will be
accompanied by increased flow of saliva (Ash and Kay, 1959). Flow of saliva (and
motility) are inhibited by distension of RR (Ash and Kay, 1959).

Flow is reduced by the ruminal infusion of artificial saliva or 1% sodium chloride
(Wilson and Tribe, 1963). Whether flow is affected by ruminal changes depends on

effects on plasma (Warner and Stacy, 1977) suggesting the increased ruminal osmolality

10

 

leads to incre:
mmmmal 1
stronger and 1
pressure in th‘
increase in 1‘15
parotid saliva
19903).

Hots 0
saliva has beer
1986). While h
salivation rates
30033). The pr
differences amt

Saliva f
of feeds I Wilso:
lPOUIiainen, 194
factors except D

Total da

d3”? flOW as v.

 

    
 
 

dailv
. prOdUCtiO.

Mel“ 3‘ al., 19

t ..
he} Produce m

leads to increased plasma osmolality. Flow is reduced by the intravenous perfusion or
intraruminal infusion of mixed VFA with reduction via intravenous effects being faster,
stronger and longer (Focant et al., 1979). Saliva flow is decreased by increased osmotic
pressure in the plasma and vice versa (Warner and Stacy, 1977). The +10 mOsm
increase in plasma osmolality associated with the end of a large meal leads to inhibited
parotid saliva flow due to reduced parasympathetic stimulation (Carter and Grovum,
1990a)

Flow of saliva may be influenced by stage of lactation and parity. Resting flow of
saliva has been reported as lower in early lactation than at peak (Cassida and Stokes,
1986). While having similar eating salivation rates, multiparous cows have greater resting
salivation rates and spend more time ruminating per d than primiparous (Maekawa et al.,
2002a). The proportion of these effects that is attributed to differences in intake and to
differences among the animals remains to be determined.

Saliva flow does not appear to be influenced by water intake or percent moisture
of feeds (Wilson and Tribe, 1963). Total flow usually proportional to feed intake
(Poutiainen, 1966). Feed intake has a greater influence of flow than any other dietary
factors except particle size (Wilson and Tribe, 1963).

Total daily flow of saliva reports vary for cattle. Early reports estimated total
daily flow as 56 L/d for oxen and 50 L/d for cattle (Colin, 1886; Markoff, 1913,
respectfully as reported in McDougall, 1948). Research in the 19603 reported average
daily production near 140 L/d with a range of plus or minus 50 L/d (Bailey, 196la;
Meyer et al., 1964; Poutiainen, 1966). More recent studies of lactating dairy cows show

they produce even more saliva (Cassida and Stokes, 1986; Maekawa et al., 2002a;

11

Maekawa et i
and are estim
rumination I E
1986; Table 1
“her:
Saliva regardh
attributed to C
This study rep
ml min) and lit
resting (Mikel-ta
the same as dUl
The stut
Phase and its m
b€ put into prac
Within a day a!“
diSCOl‘C’red.
EVen “ :

ruminal “Quid \

 

Stokes, l 98 6; .\

resulted in lncn

area or time Uni-i

 

Maekawa et al., 2002b). Rumination times in most of these studies were not measured
and are estimated based on previously reported time relationships between eating and

rumination (Bailey, 1961a; Meyer et al., 1964; Poutiainen, 1966; Cassida and Stokes,

1986; Table 1.4)

Where rumination behavior was monitored, lactating cows produced 239 L/d of
saliva regardless of treatment diet (Maekawa et al., 2002b). Lack of diet difference was
attributed to continuous flow of saliva in the lactating dairy cow (Maekawa et al., 2002b).
This study reported a wider gap between resting flow (101 ml/min) and eating flow (225
m1/min) and that rate of salivary secretion during eating was 2.2 times higher than during
resting (Maekawa et al., 2002b). They assumed the flow of saliva during rumination was
the same as during eating when calculating total salivary flow per (1 (Maekawa et al.,
2002b)

The study of flow rates of saliva is an area of research that is still in a descriptive
phase and its many assumptions need testing and validation before a working model can
be put into practice. A stronger, quantitative understanding of the variations of flows
within a day and across diets awaits further study with methods and ideas yet to be
discovered.

Even with differences in chewing behavior, differences in ruminal digesta weight,
ruminal liquid volume and ruminal liquid turnover rate were not found (Cassida and
Stokes, 1986; Maekawa et al., 2002b). Increased physically effective fiber (peNDF)
resulted in increased ruminating and total chewing, but ruminal pH expressed as mean,

area or time under the curve was not affected (Yang and Beauchemin, 2006).

12

In sumr

and flows cont

increased mark

£60 I.- d.

 

 

In summary, saliva of lactating dairy cows is relatively constant in composition
and flows continuously. Rate of saliva flow is modulated by several factors and is
increased markedly with chewing. For lactating dairy cows, daily saliva flow is likely

~260 L/d.

13

Overview
The TOT
and a mixture (

processes occ u

The Ruminal .

Rumina

to 7.0 (Stevens

 

fermentation SlI

\TA and lactat

contribute to m

characteristics.

Rumina

1955b). BUl li’Ii

vvater which det

solution (Turn
bicarbonme anc’

The .....l
Soesr‘ 1994). T
PH drops, the d1

b€l0w PH 6 (Val

 

THE RUMINAL ENVIRONMENT

Overview
The forestomach of lactating dairy cows contains an active microbial fermentation
and a mixture of substrates and products. A complete understanding of the diverse

processes occurring is essential.

The Ruminal Solution

Ruminal pH or acidity in the rumen of a lactating dairy has a normal range of 5.5
to 7.0 (Stevens and Hume, 1995). Ruminal pH is function of microbial activity,
fermentation stoichiometry, intake of the different carbohydrate fractions in feeds, net
VFA and lactate concentrations, and saliva production (Pitt et al., 1996). Factors that
contribute to ruminal acidity include the adaptation of rumen to a new diet, intake, diet
characteristics, and the variability of the diet (N ordlund, 2003).

Ruminal fluid resists pH change to the addition of acid (Turner and Hodgetts,
1955b). But this varies with interval after feeding, nature of the diet, and consumption of
water which determine VFA, bicarbonate and hydrogen phosphate concentrations in
solution (Turner and Hodgetts, 1955b). The ruminal solution’s key buffers are
bicarbonate and VFA (Counotte et al., 1979).

The ruminal solution has tremendous buffering capacity from pH 4 to 5 (Van
Soest, 1994). The VFA pool has an average or aggregate pKa of approximately 4.8. As
pH drops, the dominate VFA anion combines with a proton and buffers the pH drops

below pH 6 (Van Campen, 1976). The buffering by feeds is more important as pH

14

 

declines belo
solution is v e
physiology of

Sever;
consumes a n‘
osmolality an.
accumulates. l
ruminant fed a
pattern with th
Briggs et al.. 1
and diets. rumi
Ruminal pH dc
decreased dicta
dim”? Concern
(Simon 6t al., I
hOUrS after feet}

Tuner and HOC
IPh

 

”“930“. 19s

dunng this time.

 

 

declines below 5 (McBurney et al., 1983; Wohlt et al., 1987). Overall, the ruminal
solution is very complex and continually modified by the processes within and by the
physiology of the encapsulating animal.

Several predictable changes occur in the ruminal solution when a ruminant
consumes a meal. After consumption, feed is fermented to VFA. VFA concentrations,
osmolality and distension increase during feeding (Forbes and Barrio, 1992). The VFA
accumulates, protons separate from anions and the pH of ruminal solution decreases. In a
ruminant fed a single meal per (1, ruminal VFA production and pH vary in a regular daily
pattern with the VFA concentration negatively related to ruminal pH (Phillipson, 1942;
Briggs et al., 1957; Emmanuel et al., 1969; Sutton et al., 1986). However, across cows
and diets, ruminal VFA concentration is not predictive of ruminal pH (Allen, 1997).
Ruminal pH decreases with an increasing rate of decline with increased meal size and
decreased dietary NDF concentration (Dado and Allen, 1993b). Higher proportion of
dietary concentrates leads to more VFA produced and a higher proportion of propionate
(Sutton et al., 1986; Lana et al., 1998). Ruminal pH and bicarbonate are lowest a few
hours after feeding with the timing of nadir dependent on many factors (Phillipson, 1942;
Turner and Hodgetts, 1955b; Emmanuel et al., 1969, Fernandez et al., 2000). Osmolality
(Phillipson, 1942) and carbon dioxide production (Emmanuel et al., 1969) is also greatest
during this time.

The homeostasis of lactating dairy cows is challenged by the endproducts of
fermentation in the RR - VFA, gases and heat (Van Soest, 1994). A theoretical ruminal
fermentation of 57.5 moles of glucose equivalent (approximately 10 kg at 180g/mole)

produces 65 moles of acetic acid, 20 moles of propionic acid, 15 moles of butyric acid,

15

 

 

60 moles of c.-.

The ruminal c-

 

 

 

2001). The ruz‘

rest of the cm.

Gas Productit.

An act:
per min (Steve
glucose equiv;

ItvoIIn. 1960 I .

 

““131 gas mm
balance, and in
be mm W
from the RR ar
Emctation by C
Productmn exc
greatly e-‘\'Ceed

PeriOdS (Steve:
more gas Out c
are m0\'ed 10 ll

[hithms 0f bre

60 moles of carbon dioxide, 35 moles of methane, and 25 moles of water (Wolin, 1960).
The ruminal contents average 15% of the body weight in lactating dairy cows (NRC,
2001). The ruminal environment, while technically outside the body, is surrounded by the

rest of the cow and is maintained within certain rough boundaries.

Gas Production And Removal

An active ruminal fermentation will generate more than one liter of mixed gases
per min (Steven and Hume, 1995). In a theoretical ruminal fermentation, 57.5 moles of
glucose equivalent will yield 60 moles of carbon dioxide and 35 moles of methane
(Wolin, 1960) or 2128 L of gases at standard temperature and pressure. However, the
actual gas mixture is variable and dependent on the ruminal ecology, the fermentation
balance, and intake (Van Soest, 1994). These gases collect in the dorsal rumen and must
be eructated (Van Soest, 1994). Eructation is the removal of gases from fermentation
from the RR and is stimulated by gas pressure in R (Stevens and Hume, 1995).
Eructation by dairy cows is necessary because at the height of fermentation the gas
production exceeds absorption (Stevens and Hume, 1995). Ruminal gas pressure does not
greatly exceed one atmosphere (+10 to 20 mm Hg) and, when it does, only for transient
periods (Stevens and Sellers, 1960). These pressure differentials are great enough to
move gas out of the RR. To eructate, the reticulum is contracted to remove ingesta. Gases
are moved to the reticulum, pushed up the esophagus, and expelled with the normal
rhythms of breathing (Ruckebusch, 1988).

Carbon dioxide, methane, and nitrogen are the dominant gases in the RR. Carbon
dioxide, present usually in the greatest percentage in the R, is generated as a byproduct

of VFA production, from the decomposition of carbonic acid formed from bicarbonate

16

 

and a protc
1995; Leek
in the dors:
I Stevens an
with format
sink for surr
eructation ar
is not as sol L
enters the R}?
from the bloc

Carbo
40% (Leek, jI
and Brod); 19
”a“? 1-5 .1 WI
methane prod u
therefore. ”mot
(hashbum an d
Portion 0ft 0131
1937; Tumer a:

flal.,1969)' C I

 

Wt

heater (”13 to “i

(rostrum and

 

and a proton, and from the hydrolysis of urea (Hoemicke et al., 1965; Stevens and Hume,
1995; Leek, 2004). Carbon dioxide is removed from the RR primarily by accumulation
in the dorsal rumen and eructation, and by absorption to the blood stream and exhalation
(Stevens and Hume, 1995). Methane is generated from the reduction of carbon dioxide
with formate, succinate and hydrogen (Stevens and Hume, 1995) and can be a hydrogen
sink for surplus reducing equivalents (Leek, 2004). Methane is removed from the R by
eructation and by absorption and exhalation (Stevens and Hume, 1995) though methane
is not as soluble across the RR wall as carbon dioxide (Hoemicke et al, 1965). Nitrogen
enters the RR when it is swallowed or when it diffuses down its concentration gradient
from the blood (Stevens and Hume, 1995).

Carbon dioxide usually accounts for 60% of the mixed gas and methane 30 to
40% (Leek, 2004) but ruminal gas composition is quite variable within day (Washbum
and Brody, 1937; Hoemicke et al, 1965; Barry et al., 1967; Emmanuel et al., 1969).
(Table 1.5) With a meal, substrate is added to the fermentation and carbon dioxide and
methane production increases. These fermentation gases displace atmospheric gases and,
therefore, nitrogen and oxygen concentrations varying inversely to carbon dioxide
(Washbum and Brody, 193 7). Carbon dioxide is the most variable gas with a range in
portion of total gas of up to 50 percentage points within a day (Washbum and Brody,
1937; Turner and Hodgetts, 1955a; Hoemicke et a1, 1965; Barry et al., 1967 ; Emmanuel
et al., 1969). Carbon dioxide is higher and more variable on diets with concentrates than
with pure forage diets (Barry et al., 1967). The proportion of carbon dioxide is equal or
greater (up to 3X) than methane with concentrate diets increasing this difference

(Washbum and Brody, 1937; Hoemicke et al, 1965; Barry et al., 1967). With the ad

17

 

libitum feec
continuous.
carbon diox
times. how e
0m
nitrogen. ox;
Oxygen that
low concentr
alts-35's Preset
amounts (Lee
Pressure of SI“
Rumin
Earth's aImOS}
dIOdee and m'
Oxygen than m
These gladienr;
equilihn'um “ 1.!

mixture of arm.

 

 

libitum feeding of lactating dairy cows, the fermentation in the R is likely more or less
continuous. Given previously reported work on cattle fed single meals (Table 1.5),
carbon dioxide proportion of the ruminal gas in these cows is likely 50% or greater at all
times, however, this approximation needs to be verified experimentally.

Oxygen and other gases are also present in the RR but a lesser extent. Like
nitrogen, oxygen enters the RR when swallowed and by diffusion fi'om the blood.
Oxygen that does enter the R is quickly utilized by the microbes and thus remains at
low concentration (Stevens and Hume, 1995). Hydrogen in small quantities is nearly
always present (Washbum and Brody, 1937) and hydrogen sulfide is also present in trace
amounts (Leek, 2004). Water vapor is also present in the collected‘gas with a vapor
pressure of 50.5 mm Hg at 385°C (Weast, 1978).

Ruminal gas composition differs from the atmosphere and fi'om blood. The
Earth’s atmosphere is much higher in nitrogen and oxygen and almost devoid of carbon
dioxide and methane (Weast, 1978; Table 1.6). Blood is also higher in nitrogen and
oxygen than ruminal gas and has significant carbon dioxide (Rhoades and Tanner, 1995)
These gradients are important as the ruminal gases are always moving toward an
equilibrium with surrounding environments. Generally, the R can be characterized as a
mixture of atmospheric and fermentation gases. An active fermentation displaces the
atmospheric gases and carbon dioxide and methane dominate. As fermentation in the RR

subsides, atmospheric gases return.

VFA Production And Removal
The VFA generated during the ruminal fermentation are used as an energy source

by the cows (Stevens and Hume, 1995). The dominant VFA produced are acetic acid

18

 

 

Invo carbon
Hume. 1995
variable and
fermentation
ruminal VFA
ruminal ferm
\TA (\‘v'olin.
disassociate. '
matter intake
lactating dairy
Dalll‘ intake is
the intake is dt
of microbes pr
The firs
[mating dairy
important in la.

Pllman‘ly by d:

 

fiiels (Allen, 2!

 

milk productior.

 

(two carbon), propionic acid (three carbon), and butyric acid (four carbon) (Stevens and
Hume, 1995). The molar ratios of acetic acid to propionic acid to butyric acid are
variable and can range from 75: 15: 10 to 40:40:20 (Bergman, 1990). The actual pattern of
fermentation is dependent on meal size and frequency (Van Soest, 1994). Profile of
ruminal VFA can also be influenced by pH (Esdale and Satter, 1972). A theoretical
ruminal fermentation of 57.5 moles of glucose equivalent will generate 100 moles of
VFA (Wolin, 1960) and each mole of acid has a mole of protons with the potential to
disassociate. The daily VFA load that must be managed by cows is a function of organic
matter intake and the ferrnentibility of that intake and the ruminal fermentation in
lactating dairy cows may generate more than 100 Eq of protons each day (Allen, 1997).
Daily intake is function of meal size and the number of meals each day. Ferrnentibility of
the intake is determined by the character and composition of the substrate and population
of microbes present in the RR.

The first determinant of VFA production is feed intake and feed intake in
lactating dairy cows is a determined by the integration of many factors. Factors
important in lactating dairy cows are physical fill of the R which is determined
primarily by diet forage NDF and its digestibility (Allen, 1996), the actions of absorbed
fuels (Allen, 2000), ability of the tissues to metabolize nutrients (Forbes, 1996), oxygen
consumption (NRC, 2001), ruminal acidity and(or) osmolality (Forbes, 1996), and
psychological and sensory ability of animals (NRC, 2001). Daily DMI can be influenced
by environmental temperature, genetics, physiological state, water intake, behavior,
management, and diet (NRC, 2001). In lactating cows, DMI is correlated positively with

milk production (Dado and Allen, 1994).

19

 

 

The 0‘

More VFA is
is determined
and xDF \viti‘.
the total VFA
moisture. b." ‘7
processing (Pi
and Tammin‘g
rate of fiber fe
of fiber fermer
.‘vlertens. 191%
llertens. 1992
2004) decreasi
and buoyancy
the diet by dec
degraded orga:
50% (Allen. I
Once pr
u”disassociatetfl

.. I
D‘ll'b‘lra et al.

are tra”Sl-‘Orted

 

I
LilldiSa.

[he eDilhelium ;

The other determinant of VFA production is the composition of the diet ingested.
More VFA is produced as fermentibility of the diet increases. Fermentibility of the diet
is determined primarily by the carbohydrate proportions of NFC (nonfiber carbohydrate)
and NDF with an increasing NFC proportion increasing the fermentibility and, therefore,
the total VFA production. F ermentibility of the NFC in the R is increased by increased
moisture, by more fermentable sources, by decreased particle size, by increased
processing (physical and chemical) and by decreased vitriousness of the starch (N ocek
and Tamminga, 1991). The amount of NDF fermented in the R is determined by the
rate of fiber fermentation and by ruminal retention time (Allen and Mertens, 1988). Rate
of fiber fermentation is a function of the intrinsic characteristic of the feed (Allen and
Mertens, 1988) and ruminal pH over time, with pH <6.0 (Hoover, 1986; Grant and
Mertens, 1992; Krajcarski-Hunt et al., 2002) and increased variability of pH (Wales et al.,
2004) decreasing fiber fermentibility. Ruminal retention time is a function of particle size
and buoyancy (Allen and Mertens, 1988). Increased intake can decrease fermentibility of
the diet by decrease ruminal retention time (NRC 2001). The resulting range of ruminally
degraded organic matter across diets is wide ranging from 29% to 67% with a mean of
50% (Allen, 1997).

Once produced, VFA are absorbed across the ruminant forestomach epithelium in
undisassociated (or free acid) and disassociated (or anion) forms (Danielli et al., 1945;
Dijkstra et al., 1993; Kramer et al. 1996; Gaebel and Sehested, 1997; Figure 1.1). VFA
are transported transcellularly and not paracellularly (Sehested et al., 1999a).

Undisassociated VFA are lipid soluble and can diffuse across the lipid bilayers of

the epithelium cell membrane (Stevens and Hume, 1995). The concentration of

20

 

undisassociat;
. I
concentration
gastrointestin.
excreted pror.
mucus layer 1.
With .:
ruminant are I
Disassociatec‘
(Gaebel and S
Nintens. in retr
1959:. Ash and
been more cor:
Stiliestcd. 199
“Changed for
1997; Sehestck
linked ‘0 Carbr
With removal <
chloride can 11‘.-
dire“ link I Gar
Sodium absorl‘"
199%). The re

 

 

 

undisassociated VFA for absorption is increased by lower ruminal pH and higher
concentrations of VFA (Van Soest, 1994). The mucus on the lining of the lower
gastrointestinal tract can divide the lumen effectively into two compartments and hold
excreted protons close to the epithelium (Stevens and Hume, 1995). However, this
mucus layer is not present in the RR (Leek, 2004).

With a collective pKa of 4.8, most VFA (>90%) in the forestomach of the
ruminant are in disassociated or anion form (Bugaut, 1978; Bergman, 1990).
Disassociated VFA can be absorbed by non-selective, electroneutral anion exchangers
(Gaebel and Sehested, 1997). Results from early work in emptied, washed, and isolated
rumens, in retrospect, support these mechanisms (Masson and Phillipson, 1951; Dobson,
1959; Ash and Dobson, 1963) but more recent work (isolated rumen and in vitro) has
been more conclusive (Rechkemmer et al., 1995; Kramer et al., 1996, Gaebel and
Sehested, 1997; Sehested et al., 1999a; Sehested et al., 1999b). VFA anions are
exchanged for bicarbonate anion across the ruminal epithelium (Gaebel and Sehested,
1997; Sehested et al., 1999b). This absorption in promoted by carbon dioxide and is
linked to carbon dioxide inside the cell (Gaebel and Sehested, 1997) and is abolished
with removal of bicarbonate from solution (Sehested et al., 1999b). Increased ruminal
chloride can inhibit VFA uptake (Kramer et al., 1996) suggesting a competition but not a
direct link (Gaebel and Sehested, 1997). VFA absorption is connected positively to
sodium absorption via sodium/proton exchanger (Gaebel and Sehested, 1997; Sehested,
1999b). The relative amounts of VFA absorbed as undisassociated and disassociated

remain to be quantified in dairy cows in vivo but a recent estimate is 50:50 (Leek, 2004).

21

 

 

 

The energy K
be determiner
Whetl
increased “it
concentration
increased by I
papillated lair
length. width.
Allen. 1999).
ofabsorption
usually much
clearance can
1990). The dis
Particles in the
hOltlogeneity o
the contents (\
diStSince of the
stat, 1993).
In 1501,;
Absorprjm] Talc

19 '13; Dijkstra

 

abSOl‘pn'On D08 ‘

 

The energy required for absorption of undisassociated and disassociated VFA remains to
be determined and awaits a more complete description of transport systems.

Whether in undisassociated or disassociated forms, VFA absorption can be
increased with increased surface area, increased concentration gradient, and decreased
concentration gradient distance. The surface area of the ruminant forestomach is
increased by the papillae of the rumen, the reticular ridges of the reticulum, and
papillated lamellae of the omasum (Leek, 2004). Ruminal papillae can increased in
length, width, and surface area with more fermentable diets (Dirksen etal., 1985; Xu and
Allen, 1999). Increasing absorptive surface area of the RR leads to increased VFA rates
of absorption (Dirksen et al., 1985; Xu and Allen, 1999). VFA concentration in the R is
usually much more than the concentrations in the blood (Bugaut, 1978). Metabolism and
clearance can increase the difference between R and blood concentrations (Bergman,
1990). The distance of the concentration gradient from active fermentation in the
particles in the R to the blood is function of the homogeneity of the rumen. The
homogeneity of the R is determined by forestomach contractions and the viscosity of
the contents (Van Soest, 1994). A greater volume of the ruminal solution can increase the
distance of the concentration gradient by decreasing the surface to volume ratio (Dijkstra
etaL,1993)

In isolated and washed rumens, lower ruminal pH can lead to increased VFA
absorption rates from bathing solutions (Danielli et al., 1945; Thorlacius and Lodge,
1973; Dijkstra et al., 1993). In vivo, a decrease in ruminal pH may lead to a decrease in

absorption possibly from a decrease in ruminal motility (Allen, 2004). Over a range of

22

experiment
absorption
An
increased \
IGaebel et
Martens. 2t
adaptation :
In d.
in the RR. 1
etal., 1991 I
1994; Bergr
Peters et al.,
range 0f 65 it
(Dijkstra et al
SOIUIIOns Witt,
flux, A more
based on estab
rUtninal VFA c

clearanCe of \'

 

for pmper aClC

 

experimental conditions in a washed rumen, butyrate has the greatest fractional
absorption rate followed by propionate then acetate (Dijkstra et al., 1993).

An increase in ruminal fermentable organic matter intake (which can cause
increased VFA concentration and lower pH) leads to greater VFA and ion absorption
(Gaebel eta1., 1987a; Gaebel et al., 1987b; Thorlacius and Lodge, 1973; Uppal and
Martens, 2002; Doreau et al., 1997). These increases taken together suggest transporter
adaptation and molecular interactions.

In dairy cows, an estimated 76% of VF A of the ruminal fermentation are absorbed
in the RR, 19% in the omasum and abomasum and 5% in the small intestine (Ruckebusch
et al., 1991) but proportions vary depending animal and diet description (Rupp et al.,
1994; Bergman, 1990; Edrise and Smith, 1977; Peters eta1., 1990a; Peters et al., 1990b,
Peters et al., 1991). However, this estimate of ruminal disappearance may be high. A
range of 65 to 80% of the VFA absorbed in R has been proposed for dairy cattle
(Dijkstra et al., 1993) but these measurement were done with washed rumen containing
solutions with less volume than in vivo and this can lead to artificially high rates of VFA
flux. A prediction of 53% of ruminal protons absorbed in the rumen has been calculated
based on established rates of absorption and liquid passage, ruminal volumes, and
ruminal VFA concentrations (Allen, 1997). Regardless of specific proportions, high
clearance of VFA and the associated buffering potential by the forestomach is necessary
for proper acidification in the abomasum.

Greater than 80% of butyrate is utilized by the visceral tissue; propionate and
acetate to lesser degree (approximately 50% and 30% respectively; Bugaut, 1978). Of

the VFA entering the blood, acetate is primarily utilized by the peripheral tissue as either

23

fuel or for lip
glucose. and
lead to acetat

Most
the ruminal s«
R by passag
bicarbonate 21
efficiency (V;
phosphate. \’1
entering the R
particularly if

The re;
mOdeled (Alle
lfmoved durin
Significance is

rtimova] of al’l

 

3R: predlClCd n

dlSdSSOQiated 1

EM“ VFA

 

With e:-.

 
  

(NRC. 2001). ..

fuel or for lipid synthesis, propionate is largely cleared by the liver and is used to make
glucose, and butyrate is mostly cleared by the liver (Bergman, 1975). These processes
lead to acetate being the primary circulating VFA (Bergman, 1990).

Most of the VFA from ruminal fermentation disassociate and donate protons to
the ruminal solution. (Stevens and Hume, 1995). These protons can be removed from the
RR by passage of ruminal materials, absorption across the RR wall, neutralization by the
bicarbonate and phosphate in saliva, cation exchange in fiber, oxidation, and microbial
efficiency (Van Soest, 1994). Passage from the RR takes protons associated with digesta,
phosphate, VFA, and ammonium (Allen, 1997). Protons can be adsorbed to feedstuffs
entering the R with a process called cation exchange (McBurney et al., 1983) -
particularly if the feed is a high protein feed or a legume forage (Jasaitis et al., 1987).

The relative importance of each process in proton removal in the rumen has been
modeled (Allen, 1997; Allen, 2004; Table 1.7). Here, the majority of protons (>50%) are
removed during the absorption of VFA across the ruminal wall. The other process of
significance is the sodium bicarbonate produced with the saliva which accounts for the
removal of approximately a third of the protons produced. Together, these two processes
are predicted to remove the majority of the protons produced each day.

In summary, VFA are produced by the microbial fermentation and are >90%
disassociated (anion form) in the ruminal solution. VFA are absorbed across the RR wall

in undisassociated and disassociated forms by controlled processes.

Excess VFA
With excess VFA, lactating dairy cows become susceptible to total VFA acidosis

(NRC, 2001). Acidosis in dairy cows is defined as either acute or subacute (Nocek, 1997;

24

Owens et al..
threshold 0‘"
fermentation
\"FA and 09‘"
problems tha
fermentation
Huber. 1956E
to metabolic z
dehydration. t
Huber. 1976A

Subch
2001) and is c
Keunen er al. '
critical pH of l
is should be at
indwelling pll
diagnosis lacta
lactation (3 to

‘l{ I.
“’04). F or acc

 

 

PTOblem lf2 R."

 

 

Owens etal., 1998). Acute acidosis is defined as a decrease in ruminal pH below the
threshold of 5 (Nocek, 1997). Excess ruminal fermentable organic matter intake starts a
fermentation that overwhelms VFA removal mechanisms and leads to excess ruminal
VFA and osmolality (Owens et al., 1998). The resulting ruminal acidosis leads to
problems that include damage to RR lining, decreased forestomach motility and abnormal
fermentation from an abnormal microbial population (Dougherty, 1976; Huber, 1976A;
Huber, 1976B; Slyter, 1976; Nocek, 1997; Owens et al., 1998). Ruminal acidosis leads
to metabolic acidosis (NRC, 2001), decreased blood pH and systematic acidosis,
dehydration, cardiovascular and respiratory failure, and often death (Dougherty, 1976;
Huber, 1976A; Huber, 1976B; Slyter, 1976; Owens et al., 1998; Brown et al., 2000).
Subclinical or subacute or chronic acidosis is more common in dairy cattle (NRC,
2001) and is commonly referred to as subacute ruminal acidosis (SARA; Nocek, 1997;
Keunen et al, 2002; Oetzel, 2003). It is defined as a drop (or repeated drops) below a
critical pH of 5.5 (Nocek, 1997; Keunen et a1, 2002; Oetzel, 2003), however, critical pH
is should be adjusted for the method of ruminal fluid collection (Oetzel, 2005; 55.6 for
indwelling pH probe and 56.0 for oral collection tube). When using rumenocentesis to
diagnosis lactating herd SARA, sampling should focus on the high risk groups: early
lactation (3 to 20 DIM) and peak lactation (Nordlund, 2003) or 5 to 250 DIM (Oetzel,
2004). For accurate herd diagnosis, samples should be taken from 12 or more cows from
these groups and timing of sampling should be near the projected nadir of ruminal pH
(Oetzel, 2004) suggested at 2 to 4 hours after feeding. SARA can be considered a herd
problem if 25% of cows test below pH 5.5 (Pereira et al., 1999; Garrett et al., 1999;

Oetzel, 2004).

25

Factor
rapid changes
characterized
Nocek, 199.“;
operations inc
condition. hig
increases in at
acidosis and I]
1998; NRC, 3
I’Nocek. I997t

The in:

many factors (

maximize Crier
(Nocek 1997,
mlCrobialS and
dependent 0n t
lhan [he aclllaf

facmrs for SA:

 

Factors that contribute to SARA include lack of adaptation to diet, high DMI,
rapid changes in intake, component feeding and certain diets (Nordlund, 2003). SARA is
characterized by a reduced and(or) inconsistent feed intake (Huber, 1976A; Slyter, 1976;
Nocek, 1997; Owens et al., 1998). Other symptoms of SARA in cattle and dairy
operations include decrease production efficiency, reduced milk fat test, poor body
condition, high culling rates, unexplained diarrhea and laminitis (Nocek, 1997). Again,
increases in acidity and osmolality are characteristic but not to the extent of acute
acidosis and the extent of problems are also proportional (Nocek, 1997; Owens et al.,
1998; NRC, 2001). Laminitis and liver abscesses are associated with both conditions
(Nocek, 1997; Owens et al., 1998; NRC, 2001).

The incidence of SARA in individual and in herds is variable and is dependent on
many factors (NRC, 2001). SARA can be described as a consequence of an attempt to
maximize energy intake and is ofien found in “well-managed, high producing herds”
(Nocek, 1997). Prevention of acidosis has been attempted with feed additives, direct-fed
microbials and dietary roughage increases (Owens et al., 1998) with specific choice
dependent on economics and goals. However, feed management may be more critical
than the actual diet. “Cycles of feed deprivation and re-feeding are more important risk
factors for SARA than is diet formulation itself.” (Oetzel, 2003). Experimentally, SARA
can be induced by restricting feed (6. g. 50% of previous intake) for one day followed by
refeeding of a diet of increased fermentibility (Krause and Oetzel, 2005).

A highly ruminally fermentable diet leads to changes in the ruminal fermentation.
With a high grain, low forage diet, ruminal acetate to propionate ratio is decreased

(Grummer et al., 1987; Owens et al., 1998). Total VFA production per (1 is also

26

 

 

increased I Al
depressed (Cir
I'Gnrmmer et
concurrent int
term osmotic
:hCldCr
danageinvot
1993) and chr
lHinders and I
barn'erg of sc;
mflhmnnnsp
Often~
1998)- lfmm1
mm~whhen01
Mimi of tht

et al., 1998) \K

the epithelmm

WfihOm dAma I

1970)

Damat
can leat.e the pl

 

increased (Allen, 1997; Owens et al., 1998). With lactating dairy cows, milk fat is
depressed (Grummer et al., 1987; NRC, 2001). Total chewing time can also be decreased
(Grummer et al., 1987; Allen, 1997; NRC, 2001). SARA is also associated with a
concurrent increase in water consumption (Cottee et al., 2004) possibly due to a short
term osmotic effect.

Acidosis can lead to acute and chronic damage to the RR lining. The acute
damage involves sloughing the lining of the RR (Hinders and Owens, 1965; Owens et al.,
1998) and chronic is a swelling and keratinization of the lining called parakerotosis
(Hinders and Owens, 1965). This damage as decreased absorptive surface area, physical
barriers of scars and keratinization, reduced blood flow and(or) disruption of the transport
mechanisms leads to decreased absorptive capacity (Hinders and Owens, 1965).

Often, acidosis involves a significant increase in ruminal osmolality (Owens et al.,
1998). If ruminal osmolality exceeds blood osmolality, water can be drawn into the RR
and, with enough time and a sufficient gradient, this movement can compromise the
integrity of the ruminal epithelium (Engelhardt, 1970; Gemmell and Stacy, 1973; Owens
et al., 1998) where a separation in the epithelium can lead to blistering and sloughing of
the epithelium. Conversely, blood osmolality greater than ruminal can be handled
without damage to the ruminal epithelium for extended periods of time (Engelhardt,
1970)

Damage to the ruminal epithelium integrity leads to two situations. Sloughing
can leave the patches in the RR wall freely permeable to water and ions (Gemmell and
Stacy, 1973) and, with sufficient damage, larger materials (Nocek, 1997). Damage can

also lead to scarring and keritinazation that cause impermeability (Hinders and Owens,

27

1965; O“ ens

reductions In

Strong Ions

Stron-
Strong ions a
the ruminal st
having a sing
controlled (1‘..-
Strong 1.005 at

Sodiu:
llllical rumml
the rumen in l
aCTOSS the m”

 

SOdium in the
concenrmtiOn
Quite high l€\
assutm‘ng ado

$0de;

 

and by the eh I

1938)_ The ex

apical

   
 

mEmbri

malom} Ofm.

1965; Owens et al., 1998). This impermeability can lead to long term or permanent

reductions in ruminal VFA absorption (Krehbiel et al., 1995; Owens et al., 1998).

Strong Ions

Strong ions are another component of the ruminal solution that are regulated.
Strong ions are defined as ions completely disassociated in solutions (Stewart, 1983). In
the ruminal solution, sodium, potassium, and chloride are the primary strong ions, each
having a single valence (Tables 1.8, 1.9, and 1.10). These ions enter the R by
controlled (i.e. in the saliva) and less controlled (i.e. in the diet) ways. Within RR, these
strong ions appear to be regulated primarily by absorption.

Sodium is usually the most abundant cation in the RR (Bennick et al., 1978) and a
typical ruminal concentration in cattle is 120 mEq/L (Tables 1.9 and 1.10). Sodium enters
the rumen in the diet or in the saliva and can leave the ruminal lumen by absorption
across the ruminal epithelium or passage to lower tract (Stevens and Hume, 1995). The
concentration of sodium in the RR is proportional but lower than the concentration of
sodium in the saliva (Bailey, 1961b). Meals do not appear to influence ruminal sodium
concentration (Tucker et al., 1993). Sodium chloride can be included in cattle diets at
quite high levels (25%) without apparent problems (Cardon, 1953; Merchen, 1988)
assuming adequate water is provided.

Sodium is absorbed into the ruminal epithelium by sodium-hydrogen exchange
and by the electrogenic diffusion of sodium through ion channels (Martens and Gaebel,
1988). The exchange of an absorbed sodium ion and excretion of a proton across the
apical membrane of ruminal epithelial cell is electrically silent and accounts for the

majority of the sodium translocation (Martens and Gaebel, 1988). The reticulum (Gaebel

28

er al.. 1993
absorption
luminal pH
VFA (Gaet
2003) and r
pCOZ are s
sodium-by:
absorption 1
al.. 1999 ). "1
absorption b
Masrlens. ZI'jI
thlCrtonicity
will”? absoi

Potas:
ltill leave the
diffusing dOu-
the RR to the
Potassium is Ll
and 1.10

l Th.

that TOUnd lll _\

 

(Bailey, 1 96l

CfllOrj,

  
 

gasnolntesting.

et al., 1993) and omasum (Martens and Gaebel, 1988) appear to have the same sodium
absorption mechanism as the rumen. This sodium absorption is inhibited by decreases in
luminal pH (Gaebel et al., 1987a; Gaebel eta1., 1987b) and enhanced with increases in
VFA (Gaebel et al., 1991; Rechkemmer etal., 1995; Sehested et al., 1999b; Uppal et al.,
2003) and pC02 (Gaebel et al., 1991; Gaebel and Sehested, 1997). Increased VFA and
pC02 are speculated to increase intracellular hydrogen ion concentration to promote the
sodium-hydrogen exchange (Gaebel et al., 1991; Gaebel and Sehested, 1997). Sodium
absorption has been linked positively to ATP supply (Harrison et al., 1975b; Gaebel et
al., 1999). The ruminal epithelium has shown the reversible ability to adapt sodium
absorption by increasing with increased luminal VFA (Gaebel et al., 1987a; Uppal and
Martens, 2002; Uppal et al., 2003). Increased lactate (Gaebel et al., 1987b) and
hypertonicity (Gaebel et al., 1987a; Gaebel et al., 1987b) does not appear to influence
sodium absorption. (Figure 1.2)

Potassium enters the rumen from the diet and, to a lesser extent, in the saliva and
will leave the ruminal lumen by paracellular absorption through the ruminal epithelium
diffusing down the concentration gradient from lumen to the blood and by passage from
the R to the lower tract (Stevens and Hume, 1995; Figure 1.2). In the ruminal solution,
potassium is usually a fraction of sodium at concentrations of 25-40 meq/L (Tables 1.9
and 1.10). The concentration of potassium in the ruminal solution is usually higher than
that found in saliva (Bailey, 1961b) and primarily a function of diet concentrations
(Bailey, 1961b; Bennick et al., 1978; Tucker et al., 1993).

Chloride is readily soluble in solution and is absorbed throughout the

gastrointestinal tract and is excreted in urine and feces (NRC, 2001 ). Chloride enters the

29

R from the
1995‘). It is a
(Gaebel et 31
membrane 0
(Martens am
the rumen. (I
greatly as so.
been linked l
1975b; Gaeb.

Absm
chloride have
‘Tlenkle. 197
GaEbCl 6! al..
Pcrhaps intrac
Sodium‘hydro
(Klemens e! a l
filled Our (Ma

SOdiur

sheep (Sellers

 

Temouth, l 9!.
Bennick et all

I .
rfc“'ll’onslrip ir

 

R from the diet and saliva and leaves by absorption and passage (Stevens and Hume,
1995). It is absorbed in the RR by the exchange of chloride anion for bicarbonate anion
(Gaebel et al., 1991; Martens et al., 1991; Gaebel et al., 1993) across the apical
membrane of ruminal epithelial cell. The reticulum (Gaebel et al., 1993) and omasum
(Martens and Gaebel, 1988) appear to have the same chloride absorption mechanism as
the rumen. (Figure 1.2) Chloride absorption is inhibited by lower ruminal pH but not as
greatly as sodium (Gaebel et al., 1987b, Gaebel eta1., 1987a). Chloride absorption has
been linked positively to the ATP supply but more weakly than sodium (Harrison et al.,
1975b; Gaebel et al., 1999).

Absorptions of sodium, chloride and potassium from the RR interact. Sodium and
chloride have been shown to be absorbed proportionally but without a direct link
(Trenkle, 1979; Gaebel et al., 1987b;Gaebe1etal., 1987a; Martens and Blume, 1987;
Gaebel et al., 1991; Diemaes et al., 1994; Rechkemmer et al., 1995). Intracellular pH or
perhaps intracellular carbonic acid is the proposed indirect link and the mechanism is the
sodium-hydrogen exchange working in parallel with a chloride-bicarbonate exchange
(Martens et al., 1991). Sodium and chloride co-transport systems in the RR have been
ruled out (Martens and Gaebel, 1988).

Sodium and potassium concentrations in the RR have a reciprocal relationship in
sheep (Sellers and Dobson, 1960; Scott, 1966; Stacy and Warner, 1966; Scott, 1967;
Temouth, 1967; Warner and Stacy, 1972a) and cattle (Emery et al., 1960; Bailey, 1961b;
Bennick et al., 1978; Tucker et al., 1988a; Tucker et al., 1993). In some experiments, the

relationship is reported as peripheral observation (Emery et al., 1960; Bennick et al.,

30

 

1978'. TUCh
Dobson. 1‘.
EV:

total mEq
in sheep Il
modulatio
Martens. l
pmposed:
“an
and

enh
incr
(Lar.
This mechar
unique to m,-
The h

for the mama
Sodem and p.
the body (th
(outside the b.
within the R R

demea- mi

 

 

1978; Tucker et al., 1993) and, in others, more intensively quantified (Sellers and
Dobson, 1960; Bailey, 1961b; Scott, 1966; Stacy and Warner, 1966; Scott, 1967).

Evidence exists to suggest that the ruminal solution is regulated to a “constant”
total mEq of sodium plus potassium (162 mEq/L in cattle (Bailey, 1961b) and 140 mEq/L
in sheep (Lang and Martens, 1999). This constancy is likely accomplished by
modulation of sodium absorption (Scott, 1967; Warner and Stacy, 1972a, and Lang and
Martens, 1999). A mechanism for the modulation of sodium absorption has been
proposed:

“an increase in ruminal K concentration depolarizes the apical membrane

and increases or induces a PD-dependent cation conductance, which

enhances Na uptake (despite a reduced electrical driving force) and finally

increases transepithelial Na transport via the basolateral Na-K-ATPase”

(Lang and Martens, 1999).
This mechanism of absorption has not been described previously and is believed to be
unique to the rumen (Lang and Martens, 1999).

The high potassium enhancement of sodium absorption is an effective mechanism
for the management of charge and osmolality in the RR (Lang and Martens, 1999).
Sodium and potassium are osmotic and charge equals but are managed in opposition in
the body (Rhoades and Tanner, 1995). The inverse relationship within the RR space
(outside the body) is an effective way to manage the variation in diets and suggests that,
within the RR, charge and osmotic character are more important than the specific
element. This relationship is likely be part of the adjustment to excess dietary potassium

(Warner and Stacy, 1972a) or sodium depletion (Scott, 1966).

31

 

Sodit.
the RR (Abd'
ruminal el‘lti"
epithelium or
ammonium 8
absorption cc:
Hogan. 1961
epithelium at:
adapted to di
al.. 2003 ). T1:
and Warner.
ar’runonia and
management -

In sun
ions interact.
Sl‘Stcm. With

10113 are most

 

Sodium may also have a reciprocal, diet-dependent, relationship with ammonia in
the RR (Abdoun et al., 2003). Urea diffuses from the blood to the saliva and across the
ruminal epithelium and can be hydrolyzed to ammonia by urease in the ruminal
epithelium or by bacteria near the RR wall (Houpt, 1970). Ammonia is protonated to
ammonium at ruminal pH (Hogan, 1961) and, therefore, has a charge of +1. Ammonia
absorption can be increased by increased VFA, more so with higher pH (6.5 vs. 4.5;
Hogan, 1961). Added ammonia to the solution can inhibit sodium uptake by ruminal
epithelium adapted to hay only diets but promote sodium uptake of ruminal epithelium
adapted to diets containing concentrate or urea in sheep epithelium in vitro (Abdoun et
al., 2003). The addition of urea to the R can promote sodium absorption in vivo (Stacy
and Warner, 1966). In general, an inverse relationship appears to exist between ruminal
ammonia and sodium concentrations and this interaction may play a role in the
management of total cation concentration in the R.

In summary, sodium, potassium, and chloride absorption is regulated and these
ions interact. Sodium and chloride are coupled through the ruminal epithelial bicarbonate
system. With respect to the ruminal solution, the charge and osmotic effects of the strong

ions are most important.

Dietary Cation-Anion Difference

Minerals in the diet need to not only meet requirements but also must be in
balance with each other for optimal performance (Mongin, 1960). Interrelationships
among monovalent macromineral elements of sodium, potassium, and chloride within the
body are recognized (NRC, 2001) and one expression of these relationships is dietary

cation-anion difference (DCAD; NRC, 2001). DCAD is used in management of dairy

32

 

cattle diet:
induce 3 rr
3301). P0
help count
fermentabl

eq

 

 

cattle diets (Sanchez and Beede, 2005). Low DCAD diets are provided to dry cows to
induce a mild metabolic acidosis which enhances calcium homeostasis at calving (NRC,
2001). Positive DCAD diets are considered for lactating cows as a management tool to
help counter or neutralize the “accelerated or greater” acid load associated with more
fermentable diets (Sanchez and Beede, 2005). DCAD is commonly expressed two ways:

Equation 1.1. DCAD3 = K + Na - C1 in mEq/100gDM

(Beede, 2003; Sanchez and Beede, 2005)
Equation 1.2. DCAD4 = K + Na - Cl - S in mEq/100gDM
(Beede, 2003; Sanchez and Beede, 2005)
These equations are commonly used in diet formulation and DCAD3 is recommended for
nonruminants and DCAD4 is recommended for ruminants (Tucker et al., 1991; Sanchez
and Beede, 2005).

In addition to sodium, potassium, chloride and sulfirr, the elements of calcium,
magnesium and phosphorus are important in body acid-base but to a lesser extent (Goff et
al., 2004; Sanchez and Beede, 2005). A full expression of major cations and anions
would be mEq (Na + K + Ca + Mg) - (C1 + S + P)/ 100g dietary DM (Equation 1.3.; Goff
et al., 2004; Sanchez and Beede, 2005). Source of cations and anions in diet is also
important as source may affect availability or potency (Goff and Horst, 1998; Goff et al.,
2004).

Research with lactating cows shows a positive response in DM1 and milk yield
and increases in blood pH and bicarbonate to increasing DCAD (Tucker et al., 1988a;
Apper-Bossard and Peyraud, 2004) but an upper limit to benefit (Roche et al., 2003).

Small increases in ruminal pH and other small ruminal effects have been reported as well

33

as overall be‘

20oz). 5W

betwecn +2“

Sancth Ct 313

hlufPhY- 20‘"

1994a. HU 3"

lactating CO“

minerals Shot

Water

The \
the RR need>
cows. Lactat
dependent or.
and 1.12). Tl.
extracellular i
found within
1997) and acc
includes plasr

s .
odiurn come

and chloride ll

tract of lactat: I

by .
d) Water I A

l

 

l

as overall benefits to acid-base status (Tucker et al., 1988a; Apper—Bossard and Peyraud,
2004). Meta-analysis has suggested an optimum DCAD4 for lactating cows might fall
between +20 to +50 mEq/ 100g DM (Sanchez et al., 1994a, Sanchez et al., 1994b,
Sanchez et al., 1994c; Sanchez and Beede, 1996; Block and Sanchez, 2000; Hu and
Murphy, 2004, Sanchez and Beede, 2005) but more research is needed (Sanchez et al.,
1994a, Hu and Murphy, 2004, Sanchez and Beede, 2005). DCAD of a balanced ration for
lactating cows rarely falls outside this optimum rage (Sanchez and Beede, 1996) but diet
minerals should be monitored with wet chemistry analysis to guard against anomalies

(Sanchez and Beede, 1996; Beede, 2003; Sanchez and Beede, 2005).

Water

The VFA and strong ions are all contained in an aqueous solution. The water of
the RR needs to be understood in the context of the total body water of lactating dairy
cows. Lactating dairy cows are approximately 65% water but the exact proportion is
dependent on physiological state and body composition (Andrew et al., 1995; Tables 1.11
and 1.12). The water in lactating cows is found in three pools: intracellular fluid (ICF),
extracellular fluid (ECF), and gastrointestinal fluids (English, 1966). ICF is the fluid
found within cells and its volume is determined primarily by potassium content (Carlson,
1997) and accounts for about two-thirds of the water in the body (NRC, 2001). ECF
includes plasma, interstitial fluids, and lymph and its volume is determined primarily by
sodium content (Carlson, 1997) because cations in ECF are regulated and bicarbonate
and chloride follow to balance charge (Houpt, 2004). The water in the gastrointestinal
tract of lactating dairy cows is approximately 13% of the live weight or 20% of the total

body water (Andrew et al., 1995; Tables 1.11 and 1.12). Total body water is controlled by

34

antidiuretic 'r
angiotensin s
effective circ
IANF: respor
Carlson. 199
1.13) and the

The xx
“fight or 12.
However, [he
enters the R
1w“ by dirt
COWS. mOSt O
drinking borh
Water and fEC
196321;, Wam
AS much as l
mixing Of the

1968b). Som,

 

Water ‘IOVQJ

 

antidiuretic hormone (ADH; regulates the osmolality of the body fluids), the renin-
angiotensin system (maintains effective circulating fluid volume), aldosterone (maintains
effective circulating fluid volume and potassium balance), and atrial natriuretic factor
(ANF; response to increased central venous pressure and stretching of the atrial wall;
Carlson, 1997). Concentrations and totals for bovine blood have been reported (Table
1.13) and these concentrations are similar to other species (Carlson, 1997).

The water in the R of lactating dairy cows is approximately 8.1% of the live
weight or 12.4% of the total body water (Andrew et al., 1995; Tables 1.11 and 1.12).
However, the RR volume and liquid pool size are not constant (Van Soest, 1994). Water
enters the R by diffusion, in saliva, and by intake of water and feed (Murphy, 1992) and
leaves by diffusion across RR and passage to lower tract (Murphy, 1992). In lactating
cows, most of the water entering the R is from saliva (Allen, 1997). Feeding and
drinking both expand RR volume and also increase outflow (Warner and Stacy, 1968b).
Water and feed entering the RR causes a nonsteady-state dilution (Warner and Stacy,
1968a; Warner and Stacy, 1968b). Not all water consumed completely mixes in the RR.
As much as 18% of a drink bypasses R to the abomasum (Woodford et al., 1984) and
mixing of the proportion of the drink retained can be incomplete (Warner and Stacy,

1968b). Some of the saliva can be expected to bypass the R as well (Allen, 1997).

Water Movement Across The Ruminal Wall

In the absence of a barrier, water follows solute (Stevens and Hume, 1995).
However, the RR appears to have “an appreciable barrier to the net flux of water due to
osmotic gradients normally present between rumen contents and blood” (Engelhart,

1970). Except during a very active fermentation, osmolality of the blood is generally

35

higher than I

 

diffuse from
1970) and n1
(Engelhart. I
though slow
within the dc.
lllltll OCCUIS
“all 15 greatc
ruminal osmt
“Prevented 01
“the epithel

The n

 

luminal 05m.
to the llimina
and Start: 19
film th'ater
ePllhelilm1 gr

El’idc
include that ll

OSmOIIC prESS

 

higher than the contents of the R (Van Soest, 1994). Little net water is believed to
diffuse from the blood into the RR (Warner and Stacy, 1968b; Engelhart, 1970; Dobson,
1970) and most of the transepithelial flux is thought be from the R to the blood
(Engelhart, 1970). Across the day, the absorption of water from the R to the blood,
though slow, is significant and, in sheep, may equal the amount of water consumed
within the day (Warner and Stacy, 1968b). Most of the absorption from the R to blood
likely occurs at lower ruminal osmolalities when osmotic pressure across the ruminal
wall is greater. In sheep, little to no net transepithelial water movement occurred at
ruminal osmolarities of 260 to 340 mOsm/L (Engelhart, 1970). The net flux of water is
“prevented or intensely inhibited by a zone of high osmotic pressure in the deeper layers
of the epithelium” (Engelhardt, 1970).

The ruminal epithelium appears resistant to potential damage of higher osmotic
pressure normally present in the ruminal solution (Engelhart, 1970). In contrast, the
ruminal osmolality needed to drawn water into the rumen appears, with time, to damage
to the ruminal epithelium by forming spaces in the tissue (Engelhardt, 1970; Gemmell
and Stacy, 1973). If the spaces formed in the epithelium connect and form a breach, the
flux of water (Engelhart, 1970) and ions (Gemmell and Stacy, 1973) across the ruminal
epithelium greatly increases.

Evidence for the ruminal epithelium is not being freely permeable to water
include that higher osmotic pressures in the RR do not equalize and that the reverse
osmotic pressures, with time, cause damage. The barrier to water flux probably is by
design. Free water flux across the RR epithelium should lead to large shifts in the water

0f blood pools. As an example, a 650 kg cow would be expected to have 53 kg of water

36

iii rumen ant

 

mOsm don it
would Slgllll
blood. This

the restrictio

Osmolality ll

   
  
  
  

Tl'lfir

 

with a meal ;
1978: Trenlil
solution can

al.. 1978).

in rumen and 34 kg of water in blood (Table 1.12). To bring the ruminal solution at 350
mOsm down to 300 mOsm would require the addition of almost 12 kg of water which
would significantly expand the RR volume and draw heavily on the ECF, particularly the
blood. This calculation assumes the active defense of blood osmolality at 300 mOsm. So,

the restriction of water movement is beneficial to ruminants.

Osmolality Of The Ruminal Solution

The osmolality of the ruminal solution is variable. Ruminal osmolality increases
with a meal primarily because of the production of VFA (Scott, 1975; Bennick et al.,
1978; Trenkle, 1979; Carter and Grovum, 1990a). Diet components entering ruminal
solution can also contribute to ruminal osmolality (Bailey, 1961, Scott, 1975, Bennick et
al., 1978). Osmolality is decreased by absorption and by saliva and water entering the
R (Scott, 1975). Inflow of saliva appears to be essential to the reduction of ruminal
osmolality because removal of saliva flow to the RR leads to a very slow return to normal
osmolality (Warner and Stacy, 1972b).

Increased osmolality of the ruminal solution can affect animal behavior. It can
influence intake (Forbes and Barrio, 1992) or decrease intake proportionally (T emouth
and Beattie, 1971). Ruminal infusions of solutions containing sodium chloride and
sodium salts of VFA at the start of spontaneous meals reduced meal size and total intake
respectively (Choi and Allen, 2000). Ruminal osmolality artificially increased to greater
than 400 mOsm can shut down a meal and markedly decrease overall intake (Bergen,
1972). The administration of a local anaesthetic to the RR stops this effect (Bergen,
1972). The signal to cease eating is sensed in the wall of the RR and not the abomasum

or via the plasma (Carter and Grovum, 1990a; Carter and Grovum, 1990b). Neuronal

37

receptors in
range (Cane
specifically
Norr
but can dela;
osmotic con.
moles of sod
SOlUllOll TCIU
hOUTS (Welcl
Two 1
difference 5}.

be Useful,

The Bicarbo

Bicarl

 

Equat

in [he bOdV" I

 

receptors in the RR appear to be able to detect osmotic pressures within the physiological
range (Carter and Grovum, 1990b) but these nerves and(or) receptors have not been
specifically identified (Leek and Harding, 1975).

Normal increases in osmolality within the RR may not inhibit mixing contractions
but can delay the time to rumination following a meal (Carter and Grovum, 1990a). High
osmotic concentration in the R can completely inhibit rumination, acute infusion of 1.2
moles of sodium or potassium bicarbonate inhibited rumination in rams until the ruminal
solution returned to a threshold of approximately 350 mOsm, sometimes for more than 12
hours (Welch, 1982).

Two systems that are well defined for the blood are the bicarbonate and strong ion

difference systems. The application of these two systems to the ruminal solution could

be useful.

The Bicarbonate System

Bicarbonate is formed when carbon dioxide and water combine to form carbonic
acid then decompose to a proton and a bicarbonate ion.

Equation 1.4. C02 + H20 <—> H2CO3 <-> H+ + HCO3'
In the body, the formation of carbonic acid from carbon dioxide and water is catalyzed by
the enzyme carbonic anhydrase (Rose and Post, 2001). Carbonic anhydrase is plentiful in
the RBC and the renal tubular epithelium (Rose and Post, 2001). Carbon dioxide and
bicarbonate are dominant in solution as carbonic acid is an unstable intermediate (Segel,
1976). The disassociation of carbonic acid to bicarbonate and a proton and back is
spontaneous (Rose and Post, 2001). This reaction is reversible and, as with all

equilibrium reactions, responsive to mass action (Segel, 1976).

38

 

Bica
following ec.

Equ;

Bicarbonatc

C on:
has the same
The carbon L
relative 10 II:
0.30 to 075
l"CmOVEil is ct

atmospheric
experimenm
l'Vlll false (TL
eQuilibmion
The ream0n
The 1‘,

|

enleHe Garb
Partial PFESSL
Bicarbgnate I

Thep

reason fOr 1h;

 

l
C0“Centlatio'

l

Bicarbonate and its relationship to blood pH is well defined and described in the
following equation:

Equation 1.5. pH = 6.1 + log [HCO3']/[C02] (Segel, 1976)

Bicarbonate In The Ruminal Solution

Compared to the blood, the ruminal bicarbonate system less quantified but still
has the same reactive species of carbon dioxide and carbonic acid (Equation 1.4).
The carbon dioxide proportion of the gas phase above the ruminal solution is high
relative to the atmosphere and the blood and is quite variable within a day (ranging from
0.20 to 0.75 atm; Table 1.5). The ruminal gas system with its active and passive gas
removal is considered open with R gas pressures rarely and only marginally exceeds
atmospheric pressure (Stevens and Sellers, 1960). If the ruminal solution is
experimentally removed and equilibrated in the carbon dioxide poor atmosphere, the pH
will raise (Turner and Hodgetts, 1955a). Bicarbonate combined with a proton and, with
equilibration, carbon dioxide is lost and pH is elevated (Turner and Hodgetts, 195 5a).
The reaction is reversible assuming no bacterial lysis (Turner and Hodgetts, 1955a).

The formation of carbonic acid from carbon dioxide and water is catalyzed by the
enzyme carbonic anhydrase in the ruminal epithelium (Bergman, 1990) and, under the
partial pressure of carbon dioxide of the R, is spontaneous (Van Soest, 1994).
Bicarbonate ion is osmotically active (Weast, 1978) and, in the ruminal solution, is
usually an alkalizer.

The pK. of carbonic acid is reported as a range from 6.0 to 6.8 (Table 1.14). The
reason for this range is probably due to the conditions of the determination

(concentration, temperature and whether carbon dioxide is allowed to escape), character

39

 

oi the SOlUl‘u
lhe pKa ofrh
(Tamer and
Hodgetts. l"
The ;
While this h:
to buffering
reserve of b;

also supplie

 

Strong Ion I

The C
Hasklbalch .

Equa‘P

2001). hOWex
lndependem 2

hldrOgen iOn

influence an d

d‘clenmned b

 

 

of the solution (pure, blood or ruminal system) and methods (theoretical or experimental).
The pKa of ruminal fluid titrated under “closed” conditions has been measured as 6.25
(Turner and Hodgetts, 1955a; Fernandez et al., 2000). In the earlier case (Turner and
Hodgetts, 1955a), the system for determination was done as a closed system at 25 C.

The primary gas produced by the ruminal fermentation is carbon dioxide and,
while this high carbon dioxide atmosphere over the ruminal contents does not contribute
to buffering directly (as it contributes both a proton and a bicarbonate), it does maintain a
reserve of bicarbonate in the ruminal solution by mass action. The ruminal fermentation

also supplies carbon dioxide to the ruminal epithelium.

Strong Ion Difference Theory

The carbonic acid equation (Equation 1.4) is the basis for the Henderson-
Hasselbalch equation:

Equation 1.6: pH = 6.10 + log ([HCO3’]/(O.03)(pC02)) (Rose and Post, 2001)
The Henderson-Hasselbach equation is very simple and clinically useful (Rose and Post,
2001), however, it is more descriptive than mechanistic and does not separate
independent and dependent variables for the determination of pH (Constable, 1999). The
hydrogen ion concentration or pH is a dependent variable, a result or net fimction of the
influence and actions of independent variables (Stewart, 1983). Blood acid-base or pH is
determined by more than carbon dioxide and a more complete model would consider
plasma cations, anions, and plasma protein (Singer and Hastings, 1948).

Alternatives to the Henderson-Hasselbalch model are the strong ion models for
pH determination. In addition to the partial pressure of carbon dioxide, plasma strong

ions and weak acids are considered determinants of plasma pH (Stewart, 1983;

40

 

 

Constable. 1"
in solution :1
potassium. 3
used to desc
(mEq 1.) mi:
In a >

SID determt
strong anion
the resulting
cations (also
acidic solutu
neutral or 6f
A qu.
hl'drogen iorfi
These factors
total weak ac
lStewart. 19>
and Chloride
Eighl faClors
Ofbicarhonat

 

l0 negative cl

  

lStcwan‘ 19

Is

Constable, 2000; Heisey and Adams, 2002). Strong ions are ions that stay disassociated
in solution and those of primary importance in the blood or plasma are sodium,
potassium, and chloride (Stewart, 1983). Strong ion difference (SID) is aggregate term
used to describe strong ions in solution and is defined as the sum of strong cations
(mEq/L) minus sum of strong anions expressed as mEq/L (Stewart, 1983).

In a simple solution of only water and strong ions, the balance of strong ions or
SID determines the pH of the solution. If the concentration of strong cations exceeds
strong anion (also called a positive SID), then the charge is balanced with a hydroxyl and
the resulting solution is basic. If the concentration of strong anions exceeds strong
cations (also called a negative SID), then the charge balanced with protons results in an
acidic solution. If the strong cations equal the strong anions, the pH of the solution is
neutral or 6.67 at 37°C (Stewart, 1983).

A quantitative strong ion model of blood pH has been proposed and states that
hydrogen ion concentration is a function of eight independent factors (Stewart, 1983).
These factors are the partial pressure of carbon dioxide, the strong ion difference, the
total weak acid, the solubility of carbon dioxide in plasma, and four chemical constants
(Stewart, 1983). In this model, the only strong ions considered are sodium, potassium,
and chloride and the weak acids are defined as the plasma proteins (Stewart, 1983). These
eight factors combine to determine six dependent variables which are the concentrations
of bicarbonate, carbonate, weak acid, weak anion, hydroxide and protons (Stewart, 1983).
Also, the conditions required include electrical neutrality (positive charges must be equal
to negative charges) and that normal arterial pH is 7.4 and bicarbonate is 24 mEq/L

(Stewart, 1983).

41

Ace:

 

measured at
tCnnstahle.

l
essential an.
concentratiu.
(Rossing et .
protein assuz

The t
able to discs
llhile theore

setting becat

 

 

 

ll"‘Calt acid. a
3000; Constz
Beca
“Nils of bl
One model .
SID as sodi
Olalbumm‘
eliminate fl
bicarbonatE
Cations and

Accuracy of pH calculated based on SID is dependent on which ions are
measured and(or) included in calculations and can be dependent of methods of analysis
(Constable, 1997; Constable, 2000; Constable, 2001). Measurement of total weak acid is
essential and should not be assumed because its measurement will define bicarbonate
concentration and, thus, differentiate between metabolic alkalosis and metabolic acidosis
(Rossing et al., 1986). Species specific plasma protein concentrations also make plasma
protein assumptions inappropriate (Constable, 1997; Constable, 2002)

The Stewart strong ion difference model of plasma pH provides insight by being
able to discern multiple types of nonrespiratory acidosis and alkalosis (Constable, 1999).
While theoretically useful, the model is, however, not as useful in a clinical or diagnostic
setting because of the difficulties of rapid and quantitative measurements of SID and total
weak acid, algebraic complexity and questions of appropriateness of constants (Constable
2000; Constable, 2001).

Because of the analytical, chemical and mathematical difficulties, more simplified
models of blood pH have been proposed (Constable, 2000; Heisey and Adams, 2002).
One model proposed that plasma pH is function of partial pressure of carbon dioxide,
SID as sodium plus potassium minus chloride and lactate and total weak acid as the sum
of albumin, globulin and phosphate (Constable, 2000). Sensitivity analysis was used to
eliminate the constants for the apparent equilibrium disassociation constant for
bicarbonate and ion product of water from the equations and the contribution of minor
cations and anions (calcium, magnesium, ammonium, sulfate, NEF A, urate, succinate,

ketone bodies, pyruvate) to SID were assumed equal and discarded from the calculations.

42

 

 

Ancvl
dioxide. 811
Adams. 201%
parameters 1

Mos‘
determinatic
strong ion di
Seems a like
Proposed to
PIESSUTE of C
sodium~ POIa
WA Would
gas “'Ould b d

fennemallcm

 

Another model proposed plasma pH as a function of partial pressure of carbon
dioxide, SID, and the concentrations of albumin and inorganic phosphate (Heisey and
Adams, 2002). An extensive and critical review of the literature was used to generate the
parameters of this model.

Most strong ion difference models have focused on blood or plasma pH
determination (Stewart, 1983; Constable, 2000; Heisey and Adams, 2002). However,
strong ion difference could be applied to other bodily solutions. The ruminal milieu
seems a likely candidate of application of SID. The pH of the ruminal solution has
proposed to be a function of three conditions: SID, VFA concentration, and partial
pressure of carbon dioxide (Kohn, 2000). In this model, SID would include ruminal
sodium, potassium, and chloride as well as other ions yet to determined. Disassociated
VFA would be the major anion and the partial pressure of carbon dioxide in the ruminal
gas would be variable (Table 1.5) and dependent of the gas production of the

fermentation.

43

 

Backgroun

BGC'
regulation c
laclating d3
solution {Er
recommend
ruminal PH
irregular D)
hicatbOnate
consumptiOI
(Hutjens. 19
in the total d
appears as 0.

{Table l.lS)l

The

L

 

fat percentac
Milk fat deprl
concentrates.

(NRC. 2001 ,

acidity (NM
1

 

EXOGENOUS SODIUM BICARBONATE

Background

Because of the important role endogenous sodium bicarbonate plays in the
regulation of the RR, exogenous sodium bicarbonate is commonly added to the diets of
lactating dairy cows. The purpose of this addition is to aid in the buffering of the ruminal
solution (Erdman, 1988a). The use of sodium bicarbonate and other dietary buffers are
recommended when “buffer flow from saliva is inadequate” (Erdman, 19883), when
ruminal pH is low (Kronfeld, 1976) or when herds exhibiting low milk fat test and low or
irregular DMI (NRC, 2001). Specific cases where added buffer such as sodium
bicarbonate is recommended include high corn silage diets, high ruminal fermentable OM
consumption (proportion or amount), low fiber diets, and component feeding system
(Hutjens, 1991; NRC, 2001). Sodium bicarbonate is fed free-choice, in the grain mix, or
in the total diet (TMR). Recommendation for sodium bicarbonate inclusion commonly
appears as 0.75% of the total diet DM but ranges from approximately 0.5% to 1.0%
(Table 1.15).

The addition of sodium bicarbonate relieves milk fat depression (defined as milk
fat percentage of less than 3.0%), increasing milk fat percentage and yield (Emery, 1976).
Milk fat depression is known to occur when lactating cows are fed high levels of
concentrates, finely chopped forages, and high amounts of polyunsaturated fatty acids
(NRC, 2001). Ofien, milk fat percentage has been used as an indirect measure of ruminal

acidity (NRC, 2001) even though the relationship is very poor (Erdman, 1988a).

44

In st

strong ion. .

 

bicarbonate
concentrati.
osmotical l}
and perhaps

proton and t

Sodium Bic

 

Tilt? l
amOunts, inc
Possibly Che
HamSOn ct

XCWbold cl

IESUlllng fn.

Sheep‘ 4 L I

In solution, sodium bicarbonate disassociates into two components: sodium, a
strong ion, and bicarbonate, a weak acid anion. In ruminal conditions, sodium
bicarbonate can be expected to have an osmotic index of more than 1.80 times its molar
concentration (Table 1.16). Sodium is a single valence cation, an alkalizer, and
osmotically active. In ruminal conditions, bicarbonate is an alkalizer, osmotically active,
and perhaps a buffer depending on ruminal pH. This bicarbonate ion can combine with a

proton and decompose to form water and carbon dioxide.

Sodium Bicarbonate As A Ruminal Infusate

The daily infusion of sodium bicarbonate solutions into the R can, depending on
amounts, increase ruminal pH, increase liquid dilution rate, alter fermentation and(or)
possibly change efficiency of microbial protein synthesis in sheep (Harrison et al., 1975a;
Harrison et al., 1976) and in cattle (Roger et al., 1979; Rogers and Davis, 19823;
Newbold et al., 1988). These effects are generally dose dependent with stronger effects
resulting from greater rates of sodium bicarbonate inclusion. All of these infiJsion
experiments show some ruminal effects but these effects must be kept in context. With
sheep, 4 L of artificial saliva ruminally infused per d is equivalent to 25 to 50% of natural
saliva flow of a sheep (Kay, 1960) and should be viewed as artificial situation. With the
experiments using cattle, sodium bicarbonate that was infused was equal to 3% or more
of the daily DM1 and is not representative of the current diets of lactating cows. A fuller

accounting of water and ruminal dynamics in these artificial situations seems warranted.

45

Sodium Bi

Ear
forage diet
Kilmer. 19
restricted t.
In these shl
increased.r
increased r
arid Kilmer
rePresentati

A St
1938 (Erdm
infreased m
IItclusion of
3W). milk p
lmEQz‘U. (7‘
image (leper
01207gd in

‘l
l‘7-5102gf I

 

t071.9)_, ian
l’. t .

Sodium Bicarbonate As A Feed Additive

Early work with sodium bicarbonate in lactating dairy cows explored restricted
forage diets (Emery and Brown, 1961; Emery et al., 1964; Emery et al., 1965; Muller and
Kilmer, 1979). The daily diets were generally 454 g sodium bicarbonate, forage
restricted to less than 10% of intake (less than 2.4 kg forage), and ad libitum concentrate.
In these short term experiments (2 to 8 weeks), the inclusion of sodium bicarbonate
increased milk and 4.0% FCM, prevented milk fat depression, increased ruminal A:P, and
increased ruminal pH (Muller and Kilmer, 1979). Effects on BW were variable (Muller
and Kilmer, 1979). However, these diets and lactating cows (<20 kg DMI) are no longer
representative of the dairy industry.

A summary of sodium bicarbonate studies with lactating dairy cows from 1960 to
1988 (Erdman, 1988a) showed that, on average, 205 g/d (equal to 1.1% of diet DM)
increased milk fat percentage (3.54 to 3.64) and increased ruminal A:P (2.45 to 2.65).
Inclusion of sodium bicarbonate had no affect on DMI (either kg/d or as percentage of
BW), milk produced (kg/d), milk protein (%), F CM (kg/d), ruminal pH, or total VFA
(mEq/L). (Table 1.17). Responses of sodium bicarbonate inclusion in the diet were
forage dependent. Corn silage based diets with an average intake of sodium bicarbonate
of 207 g/d increased DMI (19.1 to 19.6 kg/d), milk fat percentage (3.49 to 3.65), FCM
(27.5 to 28.7 kg/d), ruminal A:P (2.16 to 2.46), apparent total tract DM digestibility (70.3
to 71.9), increased apparent total tract NDF digestibility (51.2 to 54.7). Sodium
bicarbonate inclusion had not affect on DMI as a percentage of BW, milk produced
(kg/d), milk protein (%), ruminal pH, or total VFA (mEq/L) in corn silage based diets.

Sodium bicarbonate inclusion did not affect measurements on diets with forage bases of

46

com silage
hav. “A dir
been showr

Ano

 

lactating co
published e.
silage and n
Concentrate
bicarbonate
COl'lt Silage \l
1.1% ofdiet‘
fat Percentag
slightly Emil
forage bases
increaSed Al

 

percenlage U
ruminal “qu

SOdIUm blCa

 

Sing-k.

bicarbonate :-

 

corn silage with alfalfa haylage or grass silage, alfalfa haylage, grass silage, or alfalfa
hay. “A direct relationship between blood pH, HCO3, pC02 and milk production has not
been shown.”

Another summary of sodium bicarbonate inclusion focused on studies with
lactating cows from 1980 to 1989 (Staples and Lough, 1989). This decade contained 41
published experiments that were more representative of current diets having more corn
silage and more total forage in diet. (Table 1.18) The average diet contained 57%
concentrate and 1.1% sodium bicarbonate with maximums for most studies of sodium
bicarbonate of 1.5% of diet DM and corn silage as 60% of forage base. In diets where
corn silage was the main forage and sodium bicarbonate was included at an average of
1.1% of diet DM, sodium bicarbonate inclusion increased milk produced (0.8 kg/d), milk
fat percentage (0.22%), and increased 4% FCM (1.6 kg/d). Mid lactation cows had a
slightly greater response in these variables than early lactation cows. Results in other
forage bases were not consistent. Sodium bicarbonate inclusion increased ruminal pH,
increased ADF digestibility (9 of 12 studies but only 4 of 12 were statistically
significant), increased molar percentage of ruminal acetate and decreased molar
percentage of ruminal propionate. The inclusion of sodium bicarbonate never increased
ruminal liquid dilution rates nor did it change blood pH, pC02, p02, or bicarbonate.
Sodium bicarbonate inclusion increased BW loss in early lactation (0.16 vs. 1.03
kg/cow/week) and increased gain in mid lactation (0.76 vs. 2.53 kg/cow/week). The
optimum inclusion rate was concluded to be 0.6 to 0.8% of diet DM.

Since these two reviews, several other experiments have investigated sodium

bicarbonate inclusion in the diet. Inclusion of sodium bicarbonate in daily intake can

47

increa:
but (101
al.. 19'
both tc
Khora:
Ghorb;
al.. 199
(Hadjir
Kennell
Sodium
dilution .
vl‘ith sodi
inclusion
31., 1983a:
bicarbonat
increased f.
mcKI‘Hnon
bkarbonate

al., 1990; K

 
  
 
  
 

asmall mm,

6131., 1992’

measures ts

lormOn e 0r r

increase ruminal pH (Kovacik et al., 1986; Ghorbani et al., 1989; Clayton et al., 1999)
but does not always (Solorzano et al., 1989; McKinnon etal., 1990; Hadjipanayiotou et
al., 1992). Inclusion of sodium bicarbonate had inconsistent effects on ruminal VFA-
both total and proportions. Ruminal VFA (mEq/L) was increased (Kennelly et al., 1999;
Khorasani and Kennelly, 2001) or not changed or decreased (Solorzano et al., 1989;
Ghorbani et al., 1989; McKinnon et al., 1990; Hadjipanayiotou et al., 1992; Clayton et
al., 1999). Ruminal acetate concentration was increased and propionate was decreased
(Hadjipanayiotou et al., 1992; Clayton et al., 1999; Kennelly et al., 1999; Khorasani and
Kennelly, 2001) or remained unchanged (Solorzano et al., 1989; Ghorbani et al., 1989).
Sodium bicarbonate inclusion can increase ruminal liquid dilution rate and(or) rumen
dilution outflow (Okeke et al., 1983a; Stokes, 1983) but these increases are associated
with sodium bicarbonate inclusions of 32.5% of dietary DM. Sodium bicarbonate
inclusion at 1.5% or less does not appear to influence ruminal liquid dilution (Okeke et
al., 1983a; Stokes, 1983; Stokes et al., 1985; Staples and Lough, 1989). Sodium
bicarbonate inclusion can increase total tract digestibility a few percent usually through
increased fiber digestibility (Solorzano et al., 1989; Ghorbani et al., 1989) but not always
(McKinnon etal., 1990; Kennelly et al., 1999; Khorasani and Kennelly, 2001). Sodium
bicarbonate inclusion does not always affect DMI (Ghorbani et al., 1989; McKinnon et
al., 1990; Kennelly et al., 1999; Khorasani and Kennelly, 2001) and when it does usually
a small increase (<10%; Schnedier et al., 1986; Solorzano et al., 1989; Hadjipanayiotou
et al., 1992). Inclusion of sodium bicarbonate has little to no affect on blood acid-base
measures (Schnedier et al., 1986; Ghorbani et al., 1989; McKinnon et al., 1990) or

hormone or metabolites measures (Boisclair et al., 1987; Vicini et al., 1988). Oral dosing

48

with sodiu
metabolic
milk. F C .\
Kennelly I
(Hadjipan
not alss'ay:
bicarbonat
(Tucker er
lactation n-
bicarbonar
et al.. 1999
monounsar
mmSARA
6131,2001;
al., 2004)_
I”Clt
$0dium blca
rate {Tom 10

steers ( 1451

 

dilution fate

 

both molar F

 

with sodium bicarbonate or sodium propionate are equally effective in correcting acute
metabolic acidosis (Bigner etal., 1997). Inclusion of sodium bicarbonate can increase
milk, FCM, milk fat percentage (Schnedier et al., 1986; Hadjipanayiotou et al., 1992;
Kennelly et al., 1999; Khorasani and Kennelly, 2001), milk components
(Hadjipanayiotou et al., 1992; Kennelly et al., 1999; Khorasani and Kennelly, 2001) but
not always (Solorzano et al., 1989; Ghorbani et al., 1989; Clayton et al., 1999). Sodium
bicarbonate effect on components is found to be more pronounced during late lactation
(Tucker et al., 1994). Production responses to sodium bicarbonate inclusion in early
lactation may be dependent on diet base forage (Canale and Stokes, 1988). Sodium
bicarbonate inclusion alters fatty acid profile of milk fat (Thivierge et al., 1998; Kennelly
et al., 1999; Khorasani and Kennelly, 2001) by increasing saturated and decreasing
monounsaturated (Kennelly et al., 1999; Khorasani and Kennelly, 2001). Lactating cows
with SARA do not have conclusive preference to consume sodium bicarbonate (Cumby
et al., 2001; Keunen et al., 2003) or drink water containing sodium bicarbonate (Cottee et
al., 2004).

Including sodium bicarbonate in diets can increase ruminal liquid dilution rate.
Sodium bicarbonate at 2% of diet DM of dry Holstein increased ruminal liquid dilution
rate from 10.3 to 12.2%/h (Rogers etal., 1982). Sodium bicarbonate at 5% of the diet of
steers (145 kg) increased feed intake (5.2 vs. 5.6 kg/d), water intake (16.2 vs. 23.2 kg/d),
ruminal pH (6.44 vs. 6.68), ruminal osmolality (273 vs. 288 mOsm), ruminal liquid
dilution rate (10.6 vs. 11.3 %/h), and ruminal volume (17.3 vs. 19.7 L) and decreased

both molar proportion and production of propionate (Rogers and Davis, 1982b). In both

49

 

El

31

199

cases, sodium bicarbonate inclusion was higher than normal for lactating dairy cows and
effects should extrapolated with caution.

The sudden introduction of 1.5% sodium bicarbonate in the concentrate portion of
a component feeding system decreased intake (0.8 kg/d of concentrate) but this drop was
avoided with gradual introduction (Erdman et al., 1982a). This effect is attributed to
palatability but ruminal osmolality may also play a role.

Ruminants with multi-cannulated gastrointestinal tracts are used to
compartmentalize effects along the gastrointestinal tract. Work with dietary sodium
bicarbonate as either a treatment or positive control has been limited and no studies have
been found reporting the study of fed sodium bicarbonate mechanism as the primary
objective. A review of the literature reveals two lactating cow studies (Kalscheur et al.,
1997; Qiu et al., 2004), two steer studies (T e h et al., 1985; Boerner et al., 1987) and
three sheep studies (Mees et al., 1985; Wedekind etal., 1986; Hsu et al., 1991).

The two lactating cow studies investigated the effect of different diets on long-
chain fatty acid flow to the duodenum. In the first study (Kalscheur et al., 1997), sodium
bicarbonate at 1.5% of the diet DM with MgO at 0.5% of diet DM increased average
ruminal pH, partially corrected the milk fat depression of the low fiber diet, and
decreased flow of the trans-C18:1 fatty acids to duodenum. In the second study (Qiu et
al., 2004), sodium bicarbonate at 0.8% of DM numerically but not significantly increased
ruminal pH and did not affect flow of trans-C18zl and conjugated linoleic acid to
duodenum.

In the two steer studies, fed sodium bicarbonate was used as a positive control

when investigating other buffers. In the first study, 1% fed sodium bicarbonate in a 60%

50

cone:
{.265

hlooc
bicar

nfian

midi
m

Stud}

concentrate diet increased ruminal pH (6.46 vs. 6.66) and lowered ruminal osmolality
(265 vs. 234 mOsm) relative to control and did not affect ruminal liquid dilution rate,
blood measures, or ruminal VFA (T e h et al., 1985). In the second study, 1% fed sodium
bicarbonate in diet with cottonseed hulls as the fiber source “enhanced” diet digestibility
relative to control (Boemer et al., 1987).

In the three lamb studies, higher rates of fed sodium bicarbonate were part of
factorial designs. In the first study, 3.5% fed sodium bicarbonate in a 75% concentrate
diet increased ruminal pH 2 h postfeeding, increased particulate dilution rate (3.8 to
4.8%), increased microbial nitrogen to small intestine (14.3 to 15.3 g/d), and microbial
crude protein efficiency (16.0 to 17.1) g N/1000g OM), but had no affect on total VFA,
VFA profile, fluid dilution rate or ruminal volume (Mees et al., 1985). In the second
study, 7.5% fed sodium bicarbonate in semi-purified, ground diets increased ruminal
fiber digestion but had no effect on total tract digestion (Wedekind et al., 1986). In the
third study, 2% fed sodium bicarbonate in 45% bromegrass and 17% soybean hull diets
increased ruminal fluid pH (6.2 to 6.4), total tract ADF digestibility (54.2 to 57.6%) and
ruminal NDF digestibility (28.5 to 41.6%; Hsu et al., 1991).

Across these studies, an increase in ruminal pH was usually observed with sodium
bicarbonate in the diet. Sodium bicarbonate was less likely to produce ruminal effects

and increased fiber digestion.

Simulation Of Time-Release Sodium Bicarbonate
In a series of experiments, ruminal infusion of sodium bicarbonate was used to
simulate a time-release sodium bicarbonate (Tucker et al., 1988c; Tucker et al., 1988b;

Hogue etal., 1991; Aslam et al., 1991; Tucker et al., 1992; Tucker et al., 1993). In these

51

 

exper
offere
Obser
Dhl v
Mill; '
collec
the di
indiv

heap.

COUCI
at an

61 al.

experiments, lactating cows were trained to eat during two meals per d. Cows were
offered a TMR that was 65 to 75% concentrate for 45 minutes at 12 h intervals.
Observed intake was estimated to be 95% of ad libitum. As a consequence, 8 to 10 kg
DM was consumed at each meal for a daily intake total of approximately 19 kg of DM.
Milk produced ranged from 20 to 25 kg/d. Ruminal fluid, blood, and urine were usually
collected every 30 minutes and measures were averaged by timepoint across cows. Given
the differences in saliva flow due to chewing behavior or water consumption pattern of
individual cows, averages of ruminal sodium, bicarbonate, and pH across cows may not
be appropriate.

Several dietary buffer treatments were applied to this experimental model.
Inclusion of buffers as 1.4% diet afier 2 week adaptation did not change plasma mineral
concentration (Tucker et al., 1988c). Twice daily ruminal infusion of sodium bicarbonate
at an equivalent of 0.8% of diet DM (Tucker et al., 1988b) and 1.5% of diet DM (Hogue
et al., 1991) at different intervals postfeeding (to simulate a time release sodium
bicarbonate) had no effects on all ruminal liquid measures at lower rate and only transient
buffer effects near the infusion window on the higher rate. Neither infusion scheme
affected milk production or components. The higher infusion rate decreased intake (18.1
vs. 17.2 kg) when in the 2 to 4 h postfeeding interval only. No speculation was provided
for this decreased intake only associated with this postfeeding interval. Two to four hours
after the increased ruminal pH caused by the twice daily infusion of sodium bicarbonate,
there was a significant decrease in ruminal pH (Hogue et al., 1991). The authors did not
speculate on cause. This rebound in ruminal pH was perhaps associated with a decrease

in rumination caused by sodium bicarbonate infusion but rumination behavior and

52

rumir
bicarl
addit
(L). l
uater
with

\\ ind
(ISA
potai

Sodlt

acti
Wn

lhe

ruminal sodium concentrations were not reported. Inclusion of 0.5% diet DM as sodium
bicarbonate coupled with twice daily ruminal infusions approximately equal to an
additional 1.5% of diet DM showed no effect of treatment on DMI, rumen liquid volume
(L), liquid dilution rate (%/h), or liquid turnover (Aslam et al., 1991). Similar ruminal
water effects were reported by Tucker et a1. (1992). In a 0.5% sodium bicarbonate diet
with an increasing rate of twice daily sodium bicarbonate infusion in 2 to 4 h postfeeding
window (approximately +0, +1 .5%, +30%, +45% of diet), infusion did not affect DMI
(18.4 kg/d), FCM (23.2 kg/d), blood measures, or systematic acid-base status. Ruminal
potassium, chloride, calcium and magnesium concentrations were related to intake and
sodium was not (Tucker et al., 1993).

Overall, results from these experiment tended to be dose dependent and effects
were most prominent near the time of infusion. Higher ruminal pH was recorded usually
during infusion. Changes in ruminal liquid measures were associated with higher rates of

sodium bicarbonate infusion. No strong intake or production responses were reported.

The Mechanism Of Sodium Bicarbonate Action In The Diets Of Lactating Cows

The beneficial effects of sodium bicarbonate feeding are well documented but, the
actions of sodium bicarbonate are probably more complex than the simple elevation of
ruminal pH. Many have speculated on the mechanism of sodium bicarbonate action but
the actual mechanism has never been documented in the scientific literature.

Sodium bicarbonate inclusion in the diets of lactating dairy cows generally
increases milk fat percentage and possibly FCM yield without changing DMI (Erdman,
1988a; Staples and Lough, 1989). The increase in efficiency can be from small increase

in apparent digestibility of the DM (Erdman, 19883) or, more specifically, increases in

53

rum

fit) \I

1m

ilk

ADF digestibility-usually in the RR (Staples and Lough, 1989). This increase in ruminal
fiber digestion may be associated with an increase in ruminal pH and an increase in the
A:P ratios (Erdman, 1988a, Staples and Lough, 1989). However, results seem to be diet
dependent (Erdman, 1988a) and a specific mechanism has not been documented.

Another theory of the mechanism of sodium bicarbonate action is an altering of
the liquid dynamics in the RR (Russell and Chow, 1993). An increase in efficiency of
diet utilization could be achieved by an increase in water intake due to sodium
bicarbonate inclusion. This could lead to an increased flow of liquid through the R00
and increased the flow of other components leaving the rumen such as particulate matter,
rumen microbes and VFA. An increase in particulate matter escape should increase the
flow of starch to the small intestine thus reducing starch fermented to VFA and increase
starch digested and absorbed as glucose from the small intestine. (Figure 1.3)

Alternatively, milk fat depression may also be caused by a postabsorptive effect
caused by altered biohydrogenation of FA in the RR (Bauman and Griinari, 2001).
Altering the biohydrogenation in the R will change the profile of unsaturated FA
leaving the RR (Bauman and Griinari, 2001; Aquhazaleh et al., 2005) which may alter
milk fat synthesis (Bauman and Griinari, 2001). Perhaps sodium bicarbonate inclusion is
involved in a change in biohydrogenation in the R.

The increases in F CM or milk fat percentage associated with sodium bicarbonate
inclusion may be due to increased efficiency of diet utilization, either by increase fiber
digestion (Erdman, 1988a; Staples and Lough, 1989) or altering the site of starch

digestion (Russell and Chow, 1993) or due to the altering of lipid metabolism (Bauman

54

and Griinari, 2001). More work is needed before the mechanism of sodium bicarbonate

is firmly concluded.

55

 

do
er.

for

3C1

is:

dis
llln

an.

SUMMARY

Lactating dairy cows have the ability to generate and regulate a large VFA load in
their R. The RR contains the ruminal fermentation, a heterogeneous mix of gas, liquid,
and solids. The ruminal solution contains ions, VFA, and other metabolites. During
ruminal fermentation, the microbial mass produces endproducts and releases cell contents
which the regulatory systems of cows remove.

The R absorbs gases, VFA, ions, and water. Ruminal gases move passively
down gradients. VF A and ions have transporters in the ruminal epithelium that require
energy and the movements of ions and water are restricted and regulated. Overall, the
forestomach of lactating dairy cows limits diffusion of water while removing osmotically
active particles from the ruminal solution. In a manner analogous to the kidney, the RR
actively removes ions from the ruminal solution with a recycling bicarbonate system.

The sodium bicarbonate secreted in the saliva is the key to the removal of ruminal
VF A and osmolality from the ruminal solution. Lactating dairy cows are capable of
producing large amounts sodium bicarbonate in their saliva each day. With sodium
bicarbonate in solution, sodium provides a positive charge and bicarbonate provides a
negative charge and both ions are osmotically active. In contrast to sodium, bicarbonate
is ephemeral and this provides a mechanism for the removal of a proton.

In the R, the fermentation generates VFA which, at ruminal pH, mostly
disassociate to generate anions and protons. The VFA anion can be absorbed across the
ruminal epithelium either by recombining with a proton and diffusing (Figure 1.4) or by

an exchange with a bicarbonate (Figure 1.5). In both cases, the cytoplasm of the ruminal

56

epithelial '_

innacellu

 

Sodium at
|

carbon dzt

maintains

the intrace

 

 

 

 

respires th
molecule ;

ll'
andVFA
metabolis
and the 5

salit'an. .

epithelial cell gains a VFA anion and a proton. This proton must be removed to maintain
intracellular pH.

The proton can be exchanged across the epithelial membrane for a luminal
sodium and the proton then combines with a ruminal bicarbonate to eventually form
carbon dioxide and water. This exchange of cations across the apical membrane
maintains the balance of charge while removing the more reactive cation, the proton. Or
the intracellular proton can be combined with a VF A anion as cellular metabolism
respires the set to carbon dioxide and water. In both cases, the proton is stored in a water
molecule and thus, neutralized.

Without intracellular metabolism of the VFA, the cytoplasm contains a sodium
and VFA anion which are transported to the blood as a charge-balanced pair. With
metabolism or transformation of the VF A in the body, the proton is removed from water
and the sodium bicarbonate is regenerated, becoming available for resecretion from the

salivary glands.

57

$0

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incl

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DISSERTATION RESEARCH OVERVIEW

The overall net result of this scheme is that osmotic particles are removed from
ruminal solution with a balance of charge. Within this scheme, sodium is just as
important as bicarbonate in the regulation of acids produced by the microbial mass in the
R. A common tendency is to consider independently the four moieties of VFA and
sodium bicarbonate in the ruminal solution (i.e. to focus on pH, the acid/anion, sodium,
or bicarbonate independently of the others). However, to truly understand lactating dairy
cows’ regulation of their VFA load, the interaction of these four moieties must be
considered. (Figure 1.6)

The inclusion of sodium bicarbonate in the diets of lactating dairy cows will
interact with this four moiety system. Overall, the addition of sodium bicarbonate to
lactating dairy cow diets usually increases ruminal pH, can alter VFA profile in RR, does
not affect DMI, increases milk fat percentage and F CM, can increase other milk
components, and may increase fiber digestion in the gastrointestinal tract. At higher
inclusion rates (_>_2.5%), water dynamics in the RR may be altered. The effects of sodium
bicarbonate inclusion are generally dose and diet dependent. The definitive mechanism
of fed sodium bicarbonate action in lactating dairy cows remains to be documented.

Sodium as an osmotically active particle in the rumen could have an effect on
rumination but the literature contains limited documentation of the relationship between
ruminal sodium and behavior such as rumination. Acute, supraphysiological infusions of
sodium into the rumen have decreased ruminating behavior. In these infusions, sodium
was introduced in solutions of either sodium salts of VFA (Choi and Allen, 1999) or

sodium bicarbonate (Welch, 1982). In both cases, rumination was reduced in the hours

58

following infusion. In contrast, the effects of additional sodium on rumination has not
been reported for typical dairy production situations.

Using sodium bicarbonate added at recommended rates to the diets of lactating
dairy cows, sodium’s effect on rumination was investigated. The null hypothesis for this
study was:

Ho: Inclusion of additional sodium in standard lactating dairy cow diets

does not affect ruminating behavior.

The results of this experiment should lead to a better understanding of the interaction of
diet (esp. strong ions) and the behavior of lactating dairy cows which will improve ration
formulation.

The use of sodium bicarbonate as an additive in the diets of lactating dairy cows
changed over the decades. Inclusion rates have decreased as the objective of inclusion
has changed and this has lead to the current recommendations averaging approximately
0.75% of dietary DM. The benefits of sodium bicarbonate addition are well documented
but the mechanisms of action have only been theorized. Given this lack and the changing
objectives, an intensive investigation into actions of sodium bicarbonate addition in the
diets of lactating dairy cows is warranted. The null hypothesis for this study was:

Ho: Adding sodium bicarbonate at a recommended rate to diets of

lactating dairy cows does not affect milk production, diet digestibility, MN

production, ruminal liquid turnover, or chewing behavior.

The parameters of the theories of the mechanism of sodium bicarbonate action in
lactating dairy cows will be incorporated into an extensive and comprehensive

experimental design. The results should give new insight into the mechanism of action.

59

1— La —Il—-

tr

hf.
,-.

More generally, strong ions such as sodium are a determinant of pH of a solution
(Stewart, 1983). Sodium is a cation and therefore alkalogenic in solution (Stewart, 1983)
and is the most abundant in the ruminal solution (Table 1.10). Ruminal sodium should be
related to ruminal pH but investigations of this relationship are limited in the literature.
The null hypothesis for this experiment was:

Ho: Sodium and other strong ions in the ruminal solution are not related to

ruminal pH or each other.

The results of this descriptive study will yield an increased understanding of strong ions
in the ruminal solution.

Overall, this dissertation investigates the role of strong ions (particularly sodium)
in the relationships and solutions beyond the empirical measurement of requirement. A
more mechanistic and comprehensive view of strong ion will lead to improved diet

formulation for lactating dairy cows.

60

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Lum
Si

 

 

Figure 1
fié'UFes
15916 er

 

Luminal Ruminal Blood
Side Epithelium Side

 

VFA-

«LL’Hcor \ ’

Metabolism

\c02 T

/H20
l/CA

 

 

 

 

 

 

 

 

 

 

 

Nag:

/ VFA—
VFA—H———> /

 

 

Figure 1.1. Hypothesized movement of VFA across the ruminal epithelium based on
figures from Gaebel and Sehested (1997), Sehested et al. (1999b), and Leek (2004). CA
is the enzyme carbonic anhydrase.

80

Luminal
Side

Ruminal
Epithelium

Blood
Side

    

 

Na+
Z

 

3 Na+
43:2 K+

 

CI-J

Na+

 

 

CI— »

 

 

 

 

 

K+

Figure 1.2. Hypothesized movement of sodium, potassium, and chloride across the
ruminal epithelium based on figures from Gaebel and Sehested (1997), Sehested et al.
(1999b), and Leek (2004).

81

 

Buffer Salts
\l/

Increased Water Intake

l/

Increased Fluid Dilution Rate

\1/

Increased Flow of Fermentable
Carbohydrate from the Rumen

\l/

Less Ruminal Propionate

l/

Increased Milk Fat Production

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1.3. A hypothetical mechanism for increasing milk fat production with buffer
Salts (Adapted from Figure l in Russell and Chow, 1993).

82

Luminal Ruminal
Side Epithelium

    

From
Saliva
Na—HCO3

HCO3— jNa+ Na+
Z’ H+
co2 ,/H

+ C;\H2CO3
H20

 

VFA—

/, VFA—H

 

/

VFA—H
From
Fermentation

 

Figure 1.4. Proposed flow of proton with absorption of undisassociated VFA.

83

Luminal Ruminal

    
 

 

 

 

 

 

 

 

 

Side Epithelium
From
Saliva
Na--HCO3
HCO3— Na+ Na+
\H H323“
CO ,/ /
H20 2\ 3 H2? 6 H+O
2
/ HCO3—41HCO3—
H+ VFA— ‘1; VFA—
VFA—H
From
Fermentation

Figur e 1.5. Proposed flow of proton with absorption of disassociated VFA. CA is the
en e carbonic anhydrase.

84

 

Na+ ¢ » H003—

 

H+ ¢ » VFA-

Figure 1.6. Proposed ion exchange to balance charges.

85

 

CHAP'

in lactz

activitj
cons v
arrange
bicarbc
Sample
bicarbo
(SOdlUrr
e<l‘dl‘mol
New 5
mEEm-e
across il

{OUr lOI‘l

 

 

CHAPTER 2: Effect of dietary strong ions on chewing activity and milk production

in lactating dairy cows.

ABSTRACT

The objective of this study was to determine effects of strong ions on chewing
activity and short-term lactational performance of dairy cows. Forty multiparous Holstein
cows were used in a replicated 5 x 5 Latin square design with a 2 x 2 factorial
arrangement of treatments of cations (sodium and potassium), anions (chloride and
bicarbonate), plus a control diet. Periods were 14 d in length with the last 4 d for data and
sample collection. Diets were formulated to 29% NDF and 17.5% CP. Sodium
bicarbonate was included at 1% of DM in one treatment diet and other treatments
(sodium chloride, potassium chloride, and potassium bicarbonate) were added to be
equimolar to sodium bicarbonate in their respective diets. Chewing activity was recorded
every 5 min for the last 24 h of each period. Cation treatments did not affect any
measured variable (P > 0.05). Treatments did not affect DMI which averaged 27.9 kg/d
across treatments. Bicarbonate treatments increased milk fat, milk lactose, and corrected
milk yield and tended to increase milk yield when compared to chloride treatments. The
four ion treatments reduced ruminating time per (1 when compared to the control by
decreasing the length of rumination bouts. This effect was not specific to cations or
anions suggesting a general osmotic effect. The additional ions are expected to tonically
increase ruminal osmolality and, based on a threshold theory, possibly terminate
rumination sooner.

Key words: buffers, osmolality, lactating cows

86

regulatec
uflnnni
phospha'h
These b;

concentr
€X0geno

19883),

If)

rununau

 

min; “g
M sodiu
dfiCre as:

Allen‘ 1

and, gi‘
buffer j
Imam
hOUrs‘

milling

COnCer

INTRODUCTION

Sodium, a strong ion, is required for the health of dairy cattle and is extensively
regulated as the primary extracellular cation (NRC, 2001). As one of its many functions,
sodium is a major component of saliva and charge-paired with bicarbonate and hydrogen
phosphate to form the two major buffers of cattle saliva (Bailey and Balch, 1961b).
These buffers are part of lactating dairy cows’ system to regulate hydrogen ion
concentration in the rumen (Allen, 1997). If endogenous ruminal buffering is lacking,
exogenous sodium bicarbonate can be added to the diets of lactating dairy cows (Erdman,
1988a)

Solutions containing sodium compounds infused into the rumen may reduce
rumination time. The infusion of 1.2 moles of sodium bicarbonate into the rumens of
sheep suspended rumination approximately ten times longer than controls (444 vs. 48
min; Welch, 1982) even with a ruminal pH of greater than 6.5. An infusion of 3 L of 0.75
M sodium salts of VFA into the rumen over 5 min at the onset of spontaneous meals
decreased rumination time by 28% during the 12 h test relative to control (Choi and
Allen, 1999).

A reduction in rumination time could reduce total daily salivary flow to the rumen
and, given the uniform concentration of sodium bicarbonate in the saliva, total ruminal
buffer flow (Allen, 1997). An infusion of 110 g of sodium bicarbonate into the rumen of
lactating cows over 2 h initially decreased hydrogen ion concentration, but, afier several
hours, increased ruminal hydrogen ion concentration (Hogue et al., 1991). A decrease in
rumination is a possible explanation for this increase in ruminal hydrogen ion

concentration, however, chewing activity was not reported. However, these infiJsions

87

 

my gene
numtiona
condition
Tl

the“ in g
that the a

time per

 

 

may generate ruminal conditions that are not representative of normal physiological or
nutritional conditions. The effects of additional sodium on rumination under standard
conditions remains to be quantified.
The objective of this experiment was to determine the effects of strong ions on
chewing activity and short-term lactational performance in dairy cows. We hypothesized
that the addition of sodium bicarbonate at 1% of diet DM would decrease rumination

time per (1 compared to potassium or control diets.

88

Desrgn

[5e and
Holsteit
Cattle T
Squares
treatmc
Expenr
This dc
‘0 a pre
lMOOm
meanir.
compo.

5mm 1‘

Treat"

PTOtejp
minera

 

MATERIALS AND METHODS

Design

Animal procedures were approved by the All University Committee on Animal

Use and Care at Michigan State University (AUF# 11/00-150-00). Forty multiparous
Holstein cows (126 i 53 DIM; mean 3: SD) from the Michigan State University Dairy
Cattle Teaching and Research Center were assigned randomly to replicated 5 x 5 Latin
squares balanced for carry over effects with a 2 x 2 factorial arrangement of equimolar
treatments for cations (Na and K), anions (Cl and HCO3) plus a control diet.
Experimental periods were 14 d with the final 4 (1 used for sample and data collection.
This design had the power to detect a ten minute difference in chewing activity according
to a previous analysis (Dado and Allen, 1994) given previously reported variances
(Mooney and Allen, 1997). A ten minute difference was the minimum thought to be
meaningful given the method of behavior measurement and an expected saliva flow and

composition (Appendix A.7 for basis for experimental design. Appendix A8 for SAS

script for power test.)

Treatments

Experimental diets contained corn silage (67% of forage DM), alfalfa silage (33%
0f forage DM), alfalfa hay, whole cottonseed, high moisture shelled com, a premix of
protein Supplements (soybean meal, distillers grains, and blood meal), a premix of
minerals and vitamins and a premix containing the treatment (Tables 2.1 and 2.2). All

diets Were formulated using the Spartan Dairy Ration Evaluator/Balancer (Version 2.10,

89

Spartan Software Laboratory, Department of Animal Science, Michigan State University,
East Lansing, M1) for 17.5% dietary CP concentration with sufficient metabolizable
protein, 29% dietary NDF concentration, and minimum NRC mineral and vitamin
requirements (Table 2.3). The control diet was balanced for sodium and potassium then
treatments were added as ground rice hulls were removed. Therefore, sodium and
potassium on treatment diets were in excess of requirements. All ingredients except
treatment mix were combined to form a base mix common to all diets. The base mix was
combined daily with each treatment mix in a tumble mixer (Roll-A-Mix Mini-Mix,
Model 690, Sand Mark Corporation, Marshfield, WI) for three minutes to form the five
final experimental diets. Trace-mineral salt blocks were not available to cows for the

duration of the experiment.

Data And Sample Collection

Throughout the experiment, cows were housed in tie-stalls, and fed once daily
(1030 h) at 110% of expected intake. The amount of feed offered and refused (orts) was
weighed daily for each cow. Samples of all dietary ingredients (0.5 kg) and orts (12.5%)
were collected daily during the test phase of each period. Samples were frozen
immediately after collection at —20° C.

Cows were moved to exercise lot twice daily (0300 and 1300 h) prior to milking.
Cows were milked twice daily in a milking parlor (0430 and 1430 h). Milk yield at both
milkings was measured and summed for a daily total on (1 11-14 of each period. These
daily totals were averaged across the test phase of each period. Milk was sampled at each
milking on d 11, 12, 13, and 14 of each period and analyzed for fat, true protein, lactose,

solids-not-fat, milk urea nitrogen (MUN) and somatic cell count (SCC) with infrared

90

spectroscopy by Michigan DHIA (East Lansing). Body weight was measured
immediately prior to the start of the first period and following the morning milking on d
14 of each period. Body condition score (BCS) was determined (Wildman, 1982; five-
point scale where 1 = thin to 5 = fat) by two trained investigators blinded to treatments
immediately prior to the start of the first period and on the last day each period. Feeding
behavior was monitored manually every 5 minutes for 24 h on d 14 of each period.

Behavior was noted as eating, ruminating, drinking or idle for each cow at each time.

Sample Processing

Samples were thawed and composited to one sample per cow per period prior to
drying. Diet ingredients, orts and fecal samples were dried in a 55° C forced-air oven for
72 h and DM concentration was determined. Forages and whole cottonseed samples were
ground with a Wiley mill (1 mm screen; Authur H. Thomas, Philadelphia, PA). High
moisture shelled corn and all premixes were ground with a UDY Cyclone Sample Mill (2

mm screen; Fort Collins, CO).

Sample Analysis

Samples were analyzed for DM, ash, CP, starch, and NDF. Ash concentration
was determined after 5 h oxidation at 500° C in a muffle fiJmace. Crude protein was
analyzed according to Hach et al. (1987). Starch corrected for free glucose was measured
by an enzymatic method (Karkalas, 1985) after samples were gelatinized with sodium
hydroxide. Glucose concentration was measured using a glucose oxidase method
(Glucose kit #510; Sigma Chemical Co., St. Louis, MO) and absorbance was determined

with micro-plate reader (SpectraMax 190, Molecular Devices Corp., Sunnyvale, CA).

91

 

Conce
Conce

deter:

and p-:
mease
manu:
Vana:

extrae

rerSe.

eXaQtl.

 

and th;

Concentrations of NDF were determined according to Van Soest et al. (1991, method A).
Concentrations of all nutrients except for DM are expressed as percentages of DM
determined by drying at 105° C in a forced-air oven for more than 8 h.

Feed samples were analyzed for sodium, potassium, chloride, and sulfur. Sodium
and potassium were determined by digestion according to Hach et al. (1987) and
measurement of the element in the supemate by atomic absorption according to
manufacturer’s recommendation (SpectrAA 220F S, Atomic Absorption Spectrometer,
Varian Analytical Instruments, Walnut Creek, CA). Chloride was determined by
extracting the feed with 1.0% nitric acid solution for one hour on a shaker (Orbimix
1010, Brinkman Instruments, Westbury, NY) and measuring chloride in the supemate by
coulometric titration (Digital Chloridometer, Model 442-5000, Labconco Corporation,
Kansas City, MO). Digests and dilutions were stored in polypropylene containers until
analysis-either polypropylene specimen cups or polypropylene, round bottom, 13 x 100
mm, culture test tubes (Fisherbrand® Catalog No. 14-956-7A, Fisher Scientific,
Pittsburgh, PA). Dried and ground samples of the base mix, rice bulls, and dried, ground
corn were composited across periods and sent to Dairy One Forage Laboratory (Ithaca,
NY) for sulfitr analysis according to manufacturer’s recommendation (LECO Application

Note 203-601-229, 08/92, LECO Model SC-432, St. Joseph, MI).

Calculations

Dry-matter intake and nutrient intake were calculated by subtracting the amount
refused from amount offered. The intake calculations assume that the diet was combined
exactly as prescribed on the mix sheet. Orts were not analyzed for starch, Cl, Na, and K

and therefore, intake calculations assume that concentrations of starch, Cl, Na, and K in

92

 

meor
iteigh

value

“HYSI
milth
“fie q

matte.“

caleui
sodiuy
thhi
to ac,

diet.

 

the orts were equal to the concentrations in the DM offered. Change in empty body
weight and body condition score were calculated by subtracting the beginning of period
value from end of period value. Yield of solids-corrected milk (SCM) was calculated as
per Tyrrell and Reid (1965) and yield of fat-corrected milk (F CM) was calculated as per
NRC (2001). Somatic cell score (SCS) was calculated by taking the log (base 2) of
somatic cell count (SCC).

Dietary cation anion difference (DCAD) as mEq/100g DM was calculated two
ways: sodium plus potassium minus chloride (DCAD3) and sodium plus potassium
minus chloride plus sulfur (DCAD4). Total diet concentrations for cations and anions
were calculated from individual ingredient analyses and dietary proportions of the dry
matter. (Appendix A6 for actual equations.)

Sodium contribution from drinking water was not incorporated into either DCAD
calculation because water intake was not measured in this experiment. However, the
sodium concentration in the water from a common well was reported as 8 ppm by
Michigan State University (2002). This concentration would deliver only 0.8 g of sodium
to a cow drinking 100 L/d which is less than 1.3% of the sodium consumed in the control
diet.

Manual observation of behavior data were summarized by a logic script in Igor
Pro® (2002) to generate meal and bout information. Variables generated included
number of meal bouts per (1, interval between meals, number of ruminating bouts per (1,
interval between ruminating bouts, eating time per (1, ruminating time per d, and total
chewing time per (1. (Appendix A9 for logic used to summarize manual behavior to

meals and bouts. Appendix A.10 for Igor Pro® script.)

93

heald
the 0‘
obtai:
there‘

Statis

2003)

 

Heath“.

 

 

effeet.

respei,

jnan

 

Three cow periods from two cows were excluded from the data set because of
health problems unrelated to the experiment. One cow was replaced in period three and
the other cow was dropped after period three. If a milk sample or weight was not
obtained at an individual milking, the milk data for the entire cow day was removed and,
therefore, 35 days of milk data out of a possible 800 were removed from the final data set

(Appendix Table A8).

Statistical Analysis

All data were analyzed using the fit model procedure of JMP® (Version 5.0.1.2;
2003) according to the following model for cow period means:

Yijk=ll+Ci+Pj+Tk+0ij+eijk

Where

it = overall mean,

C, = random effect of cow (i = l to 40),

Pj = fixed effect of period (j = l to 5),

Tk = fixed effect of treatment (k = l to 5),

CU = fixed effect of treatment carryover,

eijk = residual, assumed to be normally distributed.

Orthogonal contrasts were performed for effects of ion treatments, cation
treatments, anion treatments, and interaction of cation and anion treatments. Treatment
effects and their interaction were declared significant at P < 0.05 and P < 0.10,
respectively, and tendency for treatment effects were declared at P < 0.10. When

interactions of main effects were significant, treatment means were compared using

94

inter.

becat

 

Student’s t-test and differences were declared significant at P < 0.05. Milk yield and
composition cow period averages were weighted because of missing data for some days.
Residual plots were checked for the appearance of normality. All plots appeared
normally distributed except for the SCC plot. SCC data were transformed to SCS and the
associated residual plot appeared to be normally distributed. Period by treatment
interaction was originally evaluated, but it was removed from the statistical model

because interaction was not significant for response variables of primary interest.

95

prote
CP 3;
contr
exper
hieas
opnna
afiecr
for Il‘lt’
kg) be
intake

|
Was a«'-

l

 

 

 

RESULTS

Postexperiment analysis (Table 2.3) showed experimental diets had less crude
protein (0.3% of DM) and NDF (1.0% of DM) than planned due to lower postexperiment
CP and NDF concentrations in some feeds. Postexperiment analysis also showed the
control diet was adequate for sodium, potassium and chloride (NRC, 2001; assuming an
experimental cow of 630 kg BW producing 37 kg of milk and consuming 28 kg DM).
Measured DCAD4 ranged from 16.1 to 27.6 mEq/100g DM and was near the proposed
optimal range of 20 to 50 mEq/100g DM (Sanchez and Beede, 2005). Treatments did not
affect intake of DM and OM, averaging 27.9 kg and 25.0 kg, respectively. NDF intake
for the control diet tended to increase when compared to the ion treatments (7.5 kg vs. 7.4
kg) because of its greater proportion of rice hulls. Sodium, potassium and chloride
intakes were as expected according to the experimental design. A uniform cation intake
was achieved across all treatment diets, averaging 3.3 moles/cow/d. (Table 2.4)

Ion treatments affected milk yield and composition (Table 2.5). Potassium
treatments tended to increase yield of some milk components (milk fat, milk lactose, and
milk SNF) and component-corrected milk yield (3.5% FCM, 4.0% FCM, and SCM)
when compared with sodium treatments. Bicarbonate treatments increased milk fat
(0.12% and 0.07 kg), milk lactose (0.06% and 0.06 kg), and SNF (0.05% and 0.10 kg)
when compared to chloride treatments. Bicarbonate treatments tended to increase milk
yield and increased corrected milk yield (1.5 kg 3.5% FCM, 1.4 kg 4.0% FCM, and 1.5
kg SCM) when compared with chloride treatments. The interaction among cations and
anions was significant for SNF percentage (P = 0.08) and Student’s t-test showed the two

bicarbonate treatments (8.71%) were greater than the sodium chloride treatment (8.63%).

96

lontn
inerez
DMI.

chlon

l'Tablt

Treat]

77'
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Wm“...

 

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all die

bill 10'

 

Ion treatments did not affect SCC or SCS (P > 0.30). Bicarbonate treatments also
increased efficiency of component corrected milk production (0.06 kg 3.5% FCM/kg
DMI, 0.05 kg 4.0% F CM/kg DMI, and 0.05 kg SCM/kg DMI) when compared to
chloride treatments (Table 2.6).

All experimental diets showed a gain in BCS and BW across the test periods
(Table 2.6) suggesting these mid-lactation cows were in positive energy balance.
Treatments did not affect change in BCS. However, an interaction among treatments (P
= 0.06) was detected for BW; both sodium treatments resulted in similar BW gain (10.8
kg/period) but the potassium chloride treatment resulted in more than twice the gain as
potassium bicarbonate treatment (15.1 kg/period vs. 7.0 kg/period).

Experimental diets affected chewing activity (Table 2.7). Ion treatments reduced
rumination time by 23.0 min when compared to the control diet. Concurrently, ion
treatments increased idle time by 27.0 min and decreased total chewing time 33.5 min
when compared to the control diet. Anion treatments also affected behavior. Chloride
treatments decreased eating time per (1 and increased drinking observations (6.4
observations vs. 5.7 observations) when compared to bicarbonate treatments. However,
drinking was usually associated with eating and, when drinking and eating were summed
within cow, no treatment effect was observed on this combined time per (1.

Manual observation of eating, ruminating and total chewing activity was highly
correlated with Igor Pro® summation (r > 0.93; Table 2.8). In this summary, ion
treatments again decreased chewing activity (Table 2.9). Meals per d were similar across
all diets (8.0 meals/d) and meal length was decreased with ion treatments (2.1 min/meal)

but total eating time per d was similar across all diets (273.3 min/d). The number of

97

TUUTHT

decre;

inin.d

depre?
treatn'
and FL

catnir

 

ruminating bouts per (1 was similar across diets (14.3 bouts/d), but ion treatments
decreased ruminating bout length (1.6 min/bout) and total ruminating time per (1 (26.2
min/d). Even when ruminating time (min/d) was corrected for intake (DM and NDF), the
depression by ion treatments remained (1.0 min/kg DM and 2.5 min/kg NDF). Ion
treatments had similar effects on total chewing time (min/d) with the decreases in eating
and ruminating time resulting a decrease in total chewing time (28.2 min/d). No specific

cation or anion effects were observed.

98

exper

OSUTO'

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DISCUSSION

Two unique elements to the design of this experiment were the equimolar addition
of cations and anions and the commonality of greater than 98% of the dietary DM across
experimental diets. These, with the uniform DMI, allowed a separation of chemical and

osmotic effects.

Production And Performance

The DMI and milk production recorded in this experiment are among the highest
reported in a sodium bicarbonate study (Staples and Lough, 1989). Intake was similar
across all experimental diets but performance differed. Bicarbonate diets increased FCM
production and chloride diets were associated with the greatest weight gain. Both of
these suggest either an increase in digestibility or a change in nutrient partitioning leading
to an increase efficiency (i.e. the same nutrient intake utilized more efficiently).

Sodium bicarbonate is theorized to increase F CM in several possible ways. The
sodium bicarbonate may increase ruminal pH and allow for greater fiber digestion in the
rumen (Erdman, 1988a) or may increase water consumption that might increase liquid
turnover in the RR (Russell and Chow, 1993). This increased turnover may, in turn, flush
starch particles from the R which will be digested in the intestine increasing digestible
energy (Russell and Chow, 1993). Or, perhaps, sodium bicarbonate addition leads to a
change in ruminal saturation of FA and this change is speculated to alter lipid metabolism
(Bauman and Griinari, 2001). However, the mechanism of action can not be concluded
from this experiment. Potassium bicarbonate has been shown to be as effective as sodium

bicarbonate for relieving milk fat depression (Emery, 1976).)

99

Potassium treatments in this experiment increased yield of milk components and
tended to increase component-corrected milk yields when compared to sodium
treatments. Previous work (Oba and Allen, 2003d) showed ruminal infusion of sodium
as sodium VFA (12 mmol/min for 14 h) increased milk component percentage but not
yield (0.14% for milk protein and 0.29% for milk lactose) when compared to potassium
as potassium VFA (12 mmol/min for 14 h). This difference in results between
experiment is probably related to the difference in experimental design. In this
experiment, the cations were fed and, in the previous work, cations were infused into the
RR on a short term basis (14 h/d).

Potassium chloride had the highest BW gain per period and potassium
bicarbonate and the control diet had the lowest. Because BW gain was not qualified, the
question is whether these body weight gains were associated with tissue gain or with
changes in water spaces within the body. In rats, increased chloride intake was
associated with a net zero balance of chloride and no change in fluid compartments
(Kaup et al., 1991). Whether this holds true in ruminants as well remains to be
determined. In this experiment, each unit of BCS was related to 260 kg of BW (Figure
2.1). This result is much higher than the stated relationship of each unit of BCS equaling
80 to 85 kg of BW for lactating dairy cows (NRC, 2001) suggesting that BW changes are

not solely based on body condition.

Chewing Activity
For eating behavior, meals were slightly shorter for treatment diets. Increasing

ruminal osmolality has decreased intake (Bergen, 1972) and has caused satiety (Choi and

100

Allen, 1999). Hov
much smaller and

Ion treatm
specific to cations
must be due to dir
ruminal osmolalit
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rumination will or

sheep,

All the CO.
Chloride) are 08m
to increase the OS:
“28 kg DM/d in
contain 0420 m0

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Allen, 1999). However, in this experiment, the ion treatments would likely produce a
much smaller and more chronic change in ruminal osmolality.

Ion treatments decreased chewing activity but the effect was general and not
specific to cations or anions. Without a specific chemical element effect, these effects
must be due to direct or indirect effects of increased ruminal osmolality. Increasing
ruminal osmolality can cause rumination to cease. An osmotic threshold of the ruminal
solution has been proposed for the termination of rumination (Welch, 1982) where
rumination will cease until the ruminal osmolality returns to less than 350 mOsm in
sheep.

All the components of the treatment (sodium, potassium, bicarbonate, and
chloride) are osmotically active (Weast, 1978) and, with their consumption, are expected
to increase the osmolality of the RR solution. In this experiment, cows consumed a mean
of 28 kg DM/d in 8 meals for an average 3.5 kg DM/meal. The 3.5 kg meal would
contain 0.420 moles of treatment as 35.0 g of sodium bicarbonate or 24.5 g of sodium
chloride. Assuming 50 L of water in the RR (Andrews et al., 1995), 35.0 g of sodium
bicarbonate would contribute 0.7 g/L and would have an osmotic index of 1.80 times the
molar concentration and 24.5 g of sodium chloride would contribute 0.5 g/L and would
have and osmotic index of 1.87 times the molar concentration (Weast, 1978). Assuming
instantaneous consumption and mixing, all treatments could increase the ruminal solution
8 mmoles/L or 14 to 15 mOsm/L. Under real conditions, the increase would be less
because of absorption and passage.

Overall, and with uniform nutrient intake, the inclusion of ion treatments should

lead to a small but chronic increase in the ruminal osmolality. If a proposed threshold for

101

rumination remat

perd(Figure 2.2).

Cost And Gain (

With the i
the cost and gains
and gain can be (1
average consump
return for increas.
Thus, the return i;

and returns. that t

Like econ
bufien'ng (Table ;
loss of469 mEq (

Sodium bicarbona

rumination remains the same, this small increase should reduce potential ruminating time

per (1 (Figure 2.2).

Cost And Gain Of Sodium Bicarbonate Addition

With the increase in milk production and loss of rumination, the concern must be
the cost and gains associated with the addition of sodium bicarbonate to the diet. Cost
and gain can be determined for economics as well as ruminal buffering (Table 2.10). The
average consumption of 279 g/d costs $0.112/d (assuming a cost of $360/ton) but the
return for increased production of 1.4 kg of milk is $0.372 (assuming $12/cwt of milk).
Thus, the return is 332% of cost. Sensitivity analysis shows, at a range of typical costs
and returns, that this sodium bicarbonate addition remains profitable.

Like economics, sodium bicarbonate addition shows a net gain for ruminal
buffering (Table 2.10). A loss of 25 min/d of rumination time is expected to lead to a
loss of 469 mEq of bicarbonate equivalent. However, the consumption of 279 g/d of
sodium bicarbonate delivers 3298 mEq of bicarbonate equivalent to the R for a net gain
of 2829 mEq of bicarbonate equivalent. As a point of comparison, the measured chewing
times of control diet in this experiment would generate saliva containing almost 40 Eq of
bicarbonate equivalent (Table 2.11) given published valves for saliva flow (Cassida and
Stokes, 1988; Maekawa et al. 2002A; Maekawa et al., 2002B) and bicarbonate equivalent

composition (Erdman, 1988a).

102

CONCLUSION

The cows on this experiment responded to bicarbonate treatment as expected,
increasing FCM yield while maintaining the same feed intake. The addition of ions to
diets reduced rumination time per (1 with no differences among the ion treatments.
Though sodium addition was expected to decrease rumination, the effect was not specific
to sodium but was apparently through tonically increasing ruminal osmolality which
ended rumination bouts. The decrease in rumination can lead to decreased saliva flow
but, in this experiment, the gain in ruminal buffering with sodium bicarbonate addition

was calculated to be greater than the loss from reduced rumination.

103

    

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Figure 2.1. The relationship between change in body weight and change in body
condition score across periods. Regression equation is (change in body condition score)
= (0.03 + (0.004)(change in body weight, kg)). R2 = 0.07, P < 0.0001.

115

  

No Ruminating

         
 

 

3* .

EU

0

E .. . _: '.

8 II leflll Inga-l Infill-Iii.- III-ll

73 c

-- .9

E E 9

a? .s .2

. . E 8

Rumination 0:: no
Threshold

 

 

 

 

 

Time

Figure 2.2. Proposed model of the decreased rumination due to ion treatment in this
experiment. The addition of ions to the diet shifis the ruminal osmolality up ( ----- is
ruminal osmolality before addition and — is ruminal osmolality afier addition). With the
addition ruminal osmolality stays above the rumination threshold longer resulting in
shorter rumination bouts and longer inter-rumination intervals.

116

CHAPTER 3: Effects of sodium bicarbonate on site and extent of nutrient digestion,
microbial efficiency, feeding behavior, and yield and composition of milk for mid- to

late—lactation dairy cows

ABSTRACT

Six ruminally and duodenally cannulated, mid-lactation (180 :i: 12 DIM, mean 3:
SD) Holstein cows were used in a replicated 3 x 3 Latin square design to evaluate effects
of sodium bicarbonate on feeding behavior, nutrient digestion, and microbial protein
production. Periods were 28 din length with the last 14 d for data and sample collection.
Treatments were control, sodium bicarbonate at 1% of dietary DM and an isomolar
concentration of sodium chloride. Diets measured 19% forage NDF and 17.8% CF. Dry
matter intake was not different across treatments (24.5 kg/d) nor were milk yield (36.7
kg) and composition. Mean ruminal pH was 6.20 and was not affected by treatment (P >
0.62) nor were any other measures of pH (minimum, maximum, range, or standard
deviation; P > 0.42). Both sodium treatments increased water intake compared to the
control diet (103.8 L/d vs. 98.7 L/d, P = 0.05) but did not affect extent or site of starch
digestion or liquid passage rate. Sodium treatments increased total tract NDF
digestibility probably by slowing of passage of digesta from the RR because of an
expansion of ruminal contents. Osmotic effects likely partially contribute to sodium

bicarbonate effects on total tract NDF digestibility.

Key words: sodium, chloride, osmolality

117

INTRODUCTION

The use of sodium bicarbonate in diets of lactating dairy cows has changed over
the years. Early research focused on adding sodium bicarbonate to diets of limited forage
(<10% of DMI) and ad libitum grain as a possible method to alleviate milk fat depression
(MFD, Emery and Brown, 1961; Emery et al., 1964; Emery et al., 1965; Muller and
Kilmer, 1979). Sodium bicarbonate generally was offered at 454 g/d or approximately
5% of daily DM intake. Over the years, the recommended inclusion rate of sodium
bicarbonate in the diets of lactating dairy cows has decreased to less than or equal to 1%
of the dietary DM.

Currently, the addition of sodium bicarbonate is recommended as 0.6 to 0.8% of
dietary DM (NRC, 2001). Exogenous sodium bicarbonate is added commonly to the
diets of lactating dairy cows for the purpose of buffering the RR (Erdman, 1988a).
Overall, the addition of sodium bicarbonate to lactating dairy cow diets often increases
ruminal pH, can alter VFA profile in R, does not affect DMI, increases milk fat
percentage and FCM, and may increase fiber digestion in the gastrointestinal tract
(Erdman, 1988a; Staples and Lough, 1989). Increases in milk yield or components
without changing intake suggest increased efficiency of diet utilization for milk
production. At higher inclusion rates (22.5%), water dynamics in the RR may be altered
in cattle (Rogers et al., 1982; Rogers and Davis, 1982b). The effects of sodium
bicarbonate inclusion generally are diet dependent (Erdman, 1988a; Staples and Lough,
1989).

Several mechanisms of sodium bicarbonate action in the ruminant have been

proposed. One hypothesis proposed that the addition of sodium bicarbonate to the diet of

118

lactating dairy
digestibility 3“
ofsodium bica:
the RR and 5m:
Chow, 1993)‘ E
increased diges

\Vhile h
sodium bicarbo-
bicarbonate has
these studies are
cattle sodium bit
the purpose of 81
Therefore, intens
sodium bicarbon:

The objec
bicarbonate addit
bicarbonate at lO/c

concentration isor

 
   

microbial efficien
compared among
bica

rbonate at a
production i

d et d
bfhavior,

lactating dairy cows causes an elevation in ruminal pH that leads to increased fiber
digestibility and energy per unit of DM (Erdman, 1988a). Another states that the addition
of sodium bicarbonate increases water consumption which increases liquid passage from
the RR and shifts the site of starch digestion for the rumen to the intestines (Russell and
Chow, 1993). Both of the hypotheses suggest an increased efficiency results from
increased digestible energy density of the diet.

While hypotheses exist and empirical experiments have shown the addition of
sodium bicarbonate to be beneficial, the mechanism of action of the addition of sodium
bicarbonate has not been fully described. Intensive digestibility studies are limited and
these studies are not usually representative of recommended for feeding lactating dairy
cattle sodium bicarbonate. Investigations with multi-cannulated lactating dairy cows for
the purpose of elucidating the mechanisms of sodium bicarbonate have not been reported.
Therefore, intensive research is warranted to determine the mechanisms of action of
sodium bicarbonate.

The objective of this study was to investigate the mechanism of action of sodium
bicarbonate addition in lactating dairy cows. Dietary treatments were: a control, sodium
bicarbonate at 1% of dietary dry matter and sodium chloride (an osmotic control) at a
concentration isomolar to the sodium bicarbonate treatment. Digestion kinetics,
microbial efficiency, feeding behavior, intake and milk yield and composition were
compared among these treatments. The null hypothesis was that adding sodium
bicarbonate at a recommended rate to diets of lactating dairy cows does not affect milk
production, diet digestibility, MN production, ruminal liquid turnover, or chewing

behavior.

119

Design
Animal
Use and Care at
Holstein cows (
Cattle Teaching
within a pair of
were 28 d with t
ruminally and d1
cannulae (10 cm
0136 made of tyg
between the Pyle
”‘11 ribs as desc]
Depamncnt of L;
State University,
The test p
subperiods. The 1
intensive 24 h col

(d 20 ihTOUgh d 2

 

andd 26), and m!

MATERIALS AND METHODS

Design

Animal procedures were approved by the All University Committee on Animal
Use and Care at Michigan State University (AUF# 09/01-148-00). Six multiparous
Holstein cows (180 i 12 DIM; mean i SD) from the Michigan State University Dairy
Cattle Teaching and Research Center were assigned randomly to treatment sequence
within a pair of 3 x 3 Latin squares balanced for carry over effects. Experimental periods
were 28 d with the final 14 d used to collect samples and data. Cows were cannulated
ruminally and duodenally prior to calving. Cows were fitted with a 10 cm ruminal
cannulae (10 cm i.d.; Bar Diamond Inc., Parma, ID). Duodenal cannulas were soft gutter
type made of tygon and vinyl tubing (Crocker et al., 1998). The duodenum was fistulated
between the pylorus and the pancreatic duct and cannulas were placed between 10th and
11th ribs as described by Robinson et al. (1985). Both surgeries were performed at the
Department of Large Animal Clinical Science, College of Veterinary Medicine, Michigan
State University.

The test phase of each period ((1 15 through d 28) was divided into several
subperiods. The subperiods were a digestibility determination (d 15 through d 17), an
intensive 24 h collection of blood and ruminal fluid ((1 19), feeding behavior monitoring
(d 20 through d 24), ruminal valerate absorption and liquid passage determination (d 25

and d 26), and rumen evacuations for pool size determination (d 27 and d 28).

120

Treatments
The thri
DM, and sodiu
composition of
Experimental (1'
forage DM), hi
meal, 25% dist
premix contain
Dairy Ration E‘
Department of.
dietary CP conc
Concentration, a

diet was balance

removed to fom

 

Treatments

The three experimental diets were a control, sodium bicarbonate as 1% of dietary
DM, and sodium chloride (an osmotic control) isomolar to sodium bicarbonate. Nutrient
composition of diet ingredients and the three treatments appear in Tables 3.1 and 3.2.
Experimental diets contained corn silage (67% of forage DM), alfalfa silage (33% of
forage DM), high moisture shelled com, a premix of protein supplements (70% soybean
meal, 25% distillers grains, and 10% blood meal), a premix of minerals and vitamins and
premix containing the treatment (Table 3.3). All diets were formulated using the Spartan
Dairy Ration Evaluator/Balancer (Version 2.10, Spartan Software Laboratory,
Department of Animal Science, Michigan State University, East Lansing, M1) for 19.0%
dietary CP concentration with sufficient metabolizable protein, 20% dietary forage NDF
concentration, and to meet minimum NRC mineral and vitamin requirements. The control
diet was balanced for sodium then treatments were added and ground rice bulls were
removed to form experimental treatments. Therefore, sodium concentration in treatment
diets was projected to be in excess of requirements. All ingredients except treatment mix
were combined to form a base mix common to all diets. The base mix was combined
daily with each treatment mix in a tumble mixer (Roll-A-Mix Mini-Mix, Model 690,
Sand Mark Corporation, Marshfield, WI) for three minutes to form the three final
experimental diets. Trace mineral salt blocks were not available to cows for the duration

of the experiment.

Data And Sample Collection
Throughout the experiment, cows were housed in tie-stalls, and fed once daily

(1130 h) at 110% of expected intake. The amounts of feed offered and refused (orts) were

121

weighed dai
were collect
in their 5‘3“:
milking “mt
h and 1600 f
on d l9 61““
fat, milk urez
by Michigan
ruminal diges
period. Body
where 1 = thir‘
Prior 10 the S ta
during the CH“

immediately 3F

Days 15, 16, 17

Indigesti.
the rumen and in
were collected e»

re re '
p senting ever

 

collection times

determine the rat
Additional rumin

defemiination of

weighed daily for each cow. Samples of all dietary ingredients (0.5 kg) and orts (12.5%)
were collected daily during the test phase of each period. Cows were milked twice daily
in their stalls from d 19 through d 24 and in a milking parlor for the rest of period. Stall
milking times (0600 h and 1700 h) were slightly different than parlor milking times (0500
h and 1600 h) but the milking intervals were similar. Milk was sampled at each milking
on d 19 through 24 of each period and analyzed for fat, true protein, lactose, solids-not-
fat, milk urea nitrogen (MUN) and somatic cell count (SCC) with infrared spectroscopy
by Michigan DHIA (East Lansing). Empty body weight was measured after evacuation of
ruminal digesta immediately prior to the start of the first period and on d 28 of each
period. Body condition score (BCS) was determined (Wildman, 1982; five-point scale
where 1 = thin to 5 = fat) by three trained investigators blinded to treatments immediately
prior to the start of the first period and on d 28 of each period. All samples collected
during the experiment (feed, orts, digesta, fecals, milk, and plasma) were frozen

immediately after collection at —20° C.

Days 15, 16, 17 : Digestibility Determination

Indigestible NDF (iNDF) was used as a marker to estimate nutrient digestibility in
the rumen and in the total tract. Duodenal samples (1,000 g) and fecal samples (500 g)
were collected every 9 h from 15 to 17 d (A total of 8 samples per cow per period.) thus
representing every 3 h of a 24 h period to account for diurnal variation. Also, at these
collection times, reticular fluid (400 ml) was collected near the reticulo—omasal orifice to
determine the ratio of microbial nitrogen to microbial purines, OM, and starch.
Additional ruminal fluid (50 ml) samples were collected from 5 sites in the rumen for

determination of pH and concentrations of VFA, lactate, and ammonia.

122

Dayl8: Prepal‘

On (1 18.
Cows were fitted
measurement. C

Inc., Braintree, 1\

Day 19: Intensi
On (1 l9.
feeding behavior
samples were col
(Allen et al., 200
anticoagulant), st
catheter inserted

also monitored b

 

)6
93..
DJ
q—o
(D
m
—_

 

Day 18: Preparation Day

On (1 18, cows were prepared for intensive sample and behavior data collection.
Cows were fitted with chewing halters (Dado and Allen, 1993a) for acclimation before
measurement. Catheters (45 cm long, MRE 095 Renathane® tubing, Braintree Scientific,

Inc., Braintree, MA) were installed in a jugular vein using sterile technique.

Day 19: Intensive 24 h Collection Of Blood And Ruminal Fluid

On d 19, a intensive 24 h collection of plasma and rumen fluid was coupled with
feeding behavior data collection. Two whole blood samples and two ruminal fluid
samples were collected every 20 min for 24 h by automated sample collection system
(Allen eta1., 2000b, 4.2% sodium citrate solution replaced saline containing heparin as
anticoagulant), starting at 0930 h. Blood was sampled from a jugular vein through a
catheter inserted 1 (1 prior to sample collection. Feeding behavior and ruminal pH was
also monitored by a computerized data acquisition system (Dado and Allen, 1993a). This
system successfully collected 99.5% and 95.8% of the total samples (2,592 each) for
blood and ruminal fluid, respectively.

Ruminal fluid was centrifuged at 2,000 x g for 15 min immediately after
collection, and supematants were frozen at -20° C until analysis. Whole blood, collected
in a tube containing lithium heparin (Becton Dickinson Vacutainer Systems, Franklin
Lakes, NJ), was analyzed immediately for pH, pC02, hematocrit, p02, ionized calcium,
sodium, potassium, and chloride by a blood gas analyzer (Stat Profile 4, Nova
Biomedical, Waltham, MA) and ten other blood variables were calculated by
manufacturer’s equations. Whole blood was also collected in a tube with potassium

oxalate and sodium fluoride as a glycolytic inhibitor (Becton Dickinson Vacutainer

123

Systems, Frank
for 15 min imm

20° C until anal

Days 20, 21, 22

Feeding
of each period 1
0f ChCWing acti
recorded to Con-
from feeding to
malfunctioning.
PH 4 and Calibr;
deviated mOre t1
analysis had a d.
deviation less th
2h oft},e (1, Th.
therefOre, dai 1y I

SUCCeSSfiIlly Col]

mmina‘ pH data

 

Systems, Franklin Lakes, NJ). Both whole blood samples were centrifuged at 2,000 x g
for 15 min immediately after sample collection, and plasma was harvested and frozen at -

20° C until analysis.

Days 20, 21, 22, 23, 24: Feeding Behavior Monitoring

Feeding behavior and ruminal pH were monitored from d 19 through d 24 (144 h)
of each period by a computerized data acquisition system (Dado and Allen, 1993a). Data
of chewing activities, feed disappearance, water consumption, and ruminal pH were
recorded to computer file for each cow every 5 sec. Chewing activity for 24-h periods
from feeding to feeding were deleted when chewing halters were out of adjustment or
malfunctioning. Electrodes for ruminal pH determination were checked daily at pH 7 and
pH 4 and calibrated as needed, and ruminal pH data were deleted for the entire day if pH
deviated more than 0.1 unit at either pH 7 and pH 4. All pH data retained for data
analysis had a deviation of less than 0.10 for both pH 7 and pH 4 and at least one with a
deviation less than 0.05. The daily pH electrode check occurred for <1.5 h during the last
2 h of the d. The 1.5 h associated with the check was removed from each cow day and,
therefore, daily pH data are representative of 22.5 h out the 24 h day. The system
successfully collected 82.2% of the total chewing activity data and 81.1% of the total
ruminal pH data (Appendix Tables A. 10 and A.11, respectively).

Chewing activities were summarized as meal bouts, interval between meals, and
meal size for eating behavior and as ruminating bouts and inter-ruminating interval for
ruminating behavior. Ruminal pH data were summarized to daily mean, variance,
median, minimum, maximum, range, the hours and area for which ruminal pH is below

6.0, 5.8, and 5.5. The minimum, maximum, and range were calculated using the daily

124

2.5‘h and 97.5‘h 1
calculated by de'

weighted that tit

Days 25, 26: R

Rate oft
pulse dose of va
et al. 2000a). R
6.5, 7, 7.5, 8. 12
valerate and cot

absorption rate.

Days 27, 28: R

Ruminal
(3.5 h after feed
28 0f each Perio

evacuation, a 10
aliquot Was Sq“
liquid phases. S

digesta. CQmpOne

 

2.5th and 97.5‘h percentiles for ruminal pH measured every 5 seconds. The area were
calculated by determining the time below a specific ruminal pH (6.0, 5.8, or 5.5) and

weighted that time by the deviation from the threshold.

Days 25, 26: Ruminal Valerate Absorption And Liquid Passage Determination

Rate of valerate absorption and rate of liquid passage was determined using a
pulse dose of valeric acid and cobalt EDTA, respectively, 2 h after feeding on d 25 (Allen
et al, 20003). Ruminal fluid was sampled at 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,
6.5, 7, 7.5, 8, 12, 16, 20, 24, and 36 h afier dosing, frozen and subsequently analyzed for
valerate and cobalt. Rate of valerate absorption was used as a proxy for total VFA

absorption rate.

Days 27, 28: Rumen Evacuations For Pool Size Determination

Ruminal contents were evacuated manually through the ruminal cannula at 1500 h
(3.5 h after feeding) on d 27 and at 0830 h (3.0 h before feeding the following day) on d
28 of each period. Total ruminal content mass and volume were determined. During
evacuation, a 10% aliquot of digesta was separated to allow accurate sub-sampling. The
aliquot was squeezed through nylon mesh (1 mm) to separate it into primarily solid and
liquid phases. Samples were taken from both phases for determination of pool size of

digesta components in the rumen. Samples were immediately frozen at ~20°C

Sample Processing
Daily samples of dietary ingredients and orts were thawed and composited by cow

into experimental subperiods. Composites and individual fecal samples were dried in a

125

55° C forced-air oven for 72 h and DM concentration was determined. Forages samples
were ground with a Wiley mill (1 mm screen; Authur H. Thomas, Philadelphia, PA).
High moisture shelled corn and all premixes were ground with a UDY Cyclone Sample
Mill (2 mm screen; Fort Collins, CO). After drying and an initial 6 mm grind, individual
fecal samples were composited within cow and period on an equal 100°C DM basis.
Microbial pellets were obtained by differential centrifugation of reticular fluid samples
collected during the digestibility determination. The fluid was centrifuged at 500 x g for
15 minutes at 4°C was to remove feed particles. The supemate was centrifuged at 18,000
x g for 15 minutes at 4°C to form the microbial pellet. The supernant was discarded and
the microbial pellet was resuspended with minimal 0.9% sodium chloride solution and
frozen —20°C

Duodenal samples were thawed and composited by cow period. Composites were
sieved through 1 mm mesh screen for an approximate liquid and solid separation. The
fractions were mixed thoroughly, subsampled and refrozen in aluminum trays in
preparation for lyphilization.

Microbial pellets, duodenal digesta liquid and solid subsamples, and ruminal
solids and liquid samples from ruminal evacuation were lyphilized (Tri-Philizer TM MP,
FTS Systems, Stone Ridge, NY) and DM concentration was determined. Microbial
pellets were ground with a mortis and pestle to eliminate clumping prior to sample
analysis. Duodenal digesta solids were passed through screens of 4.75 mm and 1.18 mm
by hand to remove stones and dry matter weights were corrected for the weight of the
stones removed. After freeze-drying and grinding with a Wiley mill (6 mm screen;

Authur H. Thomas, Philadelphia, PA), duodenal and ruminal liquids and solids were

126

recombined in t1

Authur H. Thorr

Sample Analys

Samples
(_i.\DF). Ash cc
fumace. Crude l
for free glucose
were gelatinizec
glucose oxidase
absorbance “'35
Devices Corp“ E

Van Soest et al. .

240-h in vitro f9;

Ruminal fluid f0
alfalfa hay onlv.
difference (1.00
expressed as pert

more than 8 h

 

Ruminal fluid “.,

 

recombined in the proportions at sampling and ground with a Wiley mill (1 mm screen;

Authur H. Thomas, Philadelphia, PA).

Sample Analysis

Samples were analyzed for DM, ash, CP, starch, NDF, and indigestible NDF
(iNDF). Ash concentration was determined after 5 h oxidation at 500° C in a muffle
furnace. Crude protein was analyzed according to Hach et a1. (1987). Starch corrected
for free glucose was measured by an enzymatic method (Karkalas, 1985) afier samples
were gelatinized with sodium hydroxide. Glucose concentration was measured using a
glucose oxidase method (Glucose kit #510; Sigma Chemical Co., St. Louis, MO) and
absorbance was determined with a micro-plate reader (SpectraMax 190, Molecular
Devices Corp., Sunnyvale, CA). Concentrations of NDF were determined according to
Van Soest et al. (1991, method A). Indigestible NDF was estimated as NDF residue after
240-h in vitro fermentation (Goering and Van Soest, 1970, reinoculation at 120 h).
Ruminal fluid for the in vitro incubations was collected from a non-pregnant dry cow fed
alfalfa hay only. Fraction of potentially digestible NDF (pdNDF) was calculated by
difference (1.00 minus iNDF). Concentrations of all nutrients except for DM are
expressed as percentages of DM determined by drying at 105° C in a forced-air oven for
more than 8 h.
Ruminal fluid was analyzed for concentrations of major VF A and lactate. Samples were
centrifuged at 26,000 x g for 15 min, and supematant (600 uL) was mixed with 600 uL

Ca(OH)2 and 300 uL of CuSO4 containing crotonic acid as an internal marker in 1.7 m1

micro centrifuge tubes. Samples were centrifuged at 12,000 x g for 10 min, and

127

supernatant (1 00
centrifiJge tubes.
for 10 min to pre
HPLC vials. Co
HPLC (Waters C
concentration in
manufacturer‘s r
\ran'an Analytic:
Feed Sod
according to Hat
absorption accor
Absorption Spec
chloride Con CCm
solution for One
and measunng 5
4412-5000, Labc(

base mix, FlCe ht

 

Dairy One ForaL

manufacmrer,S n

 

Model SC-432 ‘

Microbial

as pr“1011le

supernatant (1000 ul) was taken and mixed with 28 ul of HZSO4 in 1.5 ml micro
centrifiige tubes. Samples were frozen and thawed twice, and centrifuged at 12,000 x g
for 10 min to precipitate and remove protein thoroughly. Supernatant was transferred to
HPLC vials. Concentrations of VFA and lactate of the supernatant were determined by
HPLC (Waters Corp., Milford, MA) according to (Oba and Allen 1999a). Cobalt
concentration in ruminal fluid was determined by atomic absorption according to
manufacturer’s recommendation (SpectrAA 220E S, Atomic Absorption Spectrometer,
Varian Analytical Instruments, Walnut Creek, CA).

Feed sodium and potassium concentration were determined by digestion
according to Hach et al. (1987) and measurement of the element in supemate by atomic
absorption according to manufacturer’s recommendation (SpectrAA 220FS, Atomic
Absorption Spectrometer, Varian Analytical Instruments, Walnut Creek, CA). Feed
chloride concentration was determined by extracting the feed with 1.0% nitric acid
solution for one hour on shaker (Orbimix 1010, Brinkman Instruments, Westbury, NY)
and measuring supemate chloride by coulometric titration (Digital Chloridometer, Model
442-5000, Labconco Corporation, Kansas City, MO). Dried and ground samples of the
base mix, rice hulls, and dried, ground corn were composited across periods and sent to
Dairy One Forage Laboratory (Ithaca, NY) for sulfur analysis according to
manufacturer’s recommendation (LECO Application Note 203-601-229, 08/92, LECO
Model 80432, St. Joseph, MI).

Microbial pellets and duodenal digesta were analyzed for ash, OM, N, and starch
as previously described and were also analyzed for purines. Total purines was measured

by spectrophotometer (Beckman Instruments, Inc., Fullerton, CA) at 260 nm (Zinn and

128

Owens, 1986).
duodenal and 1

Plasma
insulin, and g1
acetate concer
stage of sampl
mixed with 28
to determine p
St. Louis, MO
(Coat-A-Coun'
(Double Antlbt

Most 01
due to nonsigm

pen“ 01 = 72')

Dry mat:

 

Owens, 1986). Ammonia concentrations were determined on supemate of centrifuged
duodenal and ruminal fluid samples (Broderick and Kang, 1980).

Plasma samples were analyzed for concentrations of acetate, glucose, NEF A,
insulin, and glucagon. Plasma was processed as described for ruminal fluid to quantify
acetate concentration. Due to greater protein concentration for plasma samples, the first
stage of sample processing was duplicated to obtain enough supernatant (1000 ul) to be
mixed with 28 ul of H2804 in 1.5 ml micro centrifuge tubes. Commercial kits were used
to determine plasma concentration of glucose (Glucose kit #510; Sigma Chemical Co.,
St. Louis, MO), NEFA (NEFA C-kit; Wako Chemicals USA, Richmond, VA), insulin
(Coat-A-Count, Diagnostic Products Corporation, Los Angeles, CA), and glucagon
(Double Antibody, Diagnostic Products Corporation, Los Angeles, CA).

Most of the intensive 24 h blood and ruminal fluid collection was not analyzed
due to nonsignificant differences and lack of funding. A subset of 6 to 8 samples per cow

period (n = 72) was analyzed to determine plasma and ruminal fluid means.

Calculations

Dry matter intake and nutrient intake was calculated by subtracting the amount
refused from the amount offered. The intake calculations assume that the diet was
combined exactly as prescribed on the mix sheet. Orts were not analyzed for C1, Na, and
K and therefore, intake calculations assume that concentrations of Cl, Na, and K in the
orts were equal to the concentrations in the DM offered. Change in empty body weight,
body condition score, ruminal content weight, and ruminal content volume were

calculated by subtracting the beginning of period value from end of period value. Milk

129

yield at both milkings was measured and summed for a daily total. Daily totals were
averaged across the test phase of each period. Yield of SCM was calculated as per Tyrrell
and Reid (1965) and yield of FCM was calculated as per NRC (2001). SCS was
calculated by taking the log (base 2) of SCC.

Purine to nitrogen ratio for microbes collected in fluid near the reticulo-omasal
orifice was used to calculate duodenal flux of microbial nitrogen while the ratio for
microbes in rumen contents were use to calculate rumen pool size of microbial nitrogen
because of potential differences in these microbial populations.

Duodenal flux was calculated for DM, OM, iNDF, pdNDF, starch, microbial N,
non-ammonia non-microbial N (NANMN), and ammonia N using 240 h iNDF as flow
marker. Duodenal flow of microbial OM was determined using the ratio of purines to
OM (Oba and Allen 2003c), and true ruminally degraded OM (TRDOM) was calculated
by subtracting duodenal flow of non-microbial OM from OM intake. Ruminal pool sizes
(kg) of OM, NDF, iNDF, pdNDF, starch, microbial N, and NANMN was determined by
multiplying the concentration of each component in rumen samples by the ruminal
digesta DM mass (kg). Ruminal digestibility was determined for each fraction.

Rates of valerate and cobalt disappearance were determined by non-linear
regression of the decline in their respective concentration in ruminal fluid over time after
dosing, accounting for background (Allen et al., 2000b)

Dietary cation anion difference (DCAD) as mEq/100g DM was calculated two ways:
sodium plus potassium minus chloride (DCAD3) and sodium plus potassium minus

chloride plus sulfur (DCAD4). Total diet concentrations for cations and anions were

130

calculated from individual ingredient analyses and dietary proportions of the dry matter.
(Appendix A.6 for equations.)

Sodium contribution from drinking water was not incorporated into either DCAD
calculation. However, the sodium concentration in the water from a common well was
reported as 9 ppm by Michigan State University (2003). This concentration would
deliver only 0.9 g of sodium to a cow drinking 100 L/d which is less than 2% of the
sodium consumed in the control diet.

Turnover rate in the rumen, passage rate from the rumen, and ruminal digestion
rate of each component (%/h) was calculated by the following equations:

Turnover rate in the rumen (%/h) =

(intake of component / ruminal pool of component) / 24 x 100

Particulate passage rate from the rumen (%/h) =

(duodenal flow of component / ruminal pool of component) / 24 x 100
Digestion rate in the rumen (%/h) =
turnover rate in the rumen (%/h) — passage rate from the rumen (%/h)

Turnover time in the rumen (h) was calculated as 1/(turnover rate in
rumen(%lh)/ 100)

Indigestible NDF passage rate from the rumen was calculated as

iNDF passage rate from the rumen (%/h) =

(intake of iNDF / ruminal pool of component) / 24 x 100 (Dado and Allen 1995)

Energy values were calculated as follows:

NE, of intake, Mcal/d = DMI, kg x (0.0245 x TDN%) (NRC, 1989)

NHL of milk, Mcal/d = Milk yield, kg x ((0.0929 x fat%) + (0.0563 x

131

true protein%) + (0.0395 x lactose%)) (NRC, 2001)
NEL for maintenance, Mcal/d = 0.080 x BWO'75 (NRC, 2001)

NEL balance, Mcal/d = NEL of intake - NEL for maintenance - NEL for milk

Statistical Analysis

All data were analyzed using the fit model procedure of JMP® (Version 5.0.1.2;
2003) according to the following model:

Yijk=ll+Ci+Pj+Tk+€ijk

where

u = overall mean,

C, = fixed effect of cow (i = 1 to 6),

PJ- = fixed effect of period (j = 1 to 3),

Tk = fixed effect of treatment (k = 1 to 3),

eiJ-k = residual, assumed to be normally distributed.

Period x treatment interaction was originally evaluated, but it was removed from
the statistical model because interaction was not significant for response variables of
primary interest. Cow period means for feeding behavior variables were weighted by the
number of cow days included. Contrasts were performed for the control diet vs. both
sodium treatments and sodium chloride vs. sodium bicarbonate. Treatment effects and
their interaction were declared significant at P < 0.05 and P < 0.10, respectively, and
tendency for treatment effects were declared at P < 0.10. A mixed model with cow as a
random variable was not used because, for some variables, the estimate of parameter did

not converge during iteration. Residual plots were checked for appearance of normality

132

and appeared normally distributed. One cow had clinical mastitis during period one on

days 25, 26, and 27. All data from these days were removed from the final data sets.

133

RESULTS

This experiment had to two key elements to its design: the equimolar addition of
cations and anions and the commonality of greater than 98% of the dietary DM. These,
with the uniform DMI, allowed a focus on the separation of chemical and osmotic effects.
The postexperiment differences in the CP and NDF concentrations of the diet ingredients
only fostered the experimental challenge. This experiment is among the highest
production and highest DMI of all sodium bicarbonate experiments reviewed by Staples
and Lough (1989).

Postexperiment analysis (Table 3.4) showed experimental diets had less crude
protein (1.2% of DM) and NDF from forage (0.9% of DM) than the diet was formulated
for. Postexperiment analysis also showed the control diet was adequate for sodium,
potassium and chloride (NRC, 2001; assuming an experimental cow of 620 kg BW
producing 36 kg of milk and consuming 25 kg DM). Sodium and chloride intakes were
according to experimental design. Calculated DCAD4 was 10 mEq/100g DM for sodium
and control diets which was outside the optimum range of 20 to 50 mEq/100g DM
proposed by Sanchez and Beede (2005).

Intakes were similar across experimental diets for each subperiod (Tables 3.5, 3.6,
and 3.7). DMI averaged 24.5 kg/d for the experiment and was not affected by treatment
(P > 0.35). Also, treatment did not affect intakes for OM, NDF, starch, crude protein,
and forage. The intakes of sodium, chloride, and sometimes iNDF were reflective of the
differences among treatment mixes.

Sodium treatments did not affect milk yield or composition (Table 3.8) with the

exception of MUN. Sodium treatments tended to decrease MUN and chloride treatment

134

tended to decrease MUN when compared to bicarbonate treatment. However, with MUN
<19 mg/ml, all treatment averages are considered acceptable for lactating dairy cows
(NRC, 2001).

Sodium treatments did not affect change in empty BW and BCS (P > 0.13) and all
experimental treatments showed gains in empty BW (14.1 kg) and condition (0.14; Table
3.9). However, when BW change was measured with the ruminal contents included,
sodium treatments caused a BW gain (28.4 kg) while the control diet showed 3 BW loss
(10.0 kg). Efficiency of milk production (1.48 kg of 3.5% FCM/ kg of DMI, 1.37 kg of
4.0% FCM/ kg of DMI, and 1.48 kg of SCM/ kg of DMI) was similar across
experimental diets.

When iNDF was used as the digestibility and passage marker, sodium treatments
increased total tract apparent digestibility for DM (2.4%) and OM (2.2%; Table 3.10).
The difference in DM and OM digestibility is due to an increase in total tract pdNDF
digestibility total tract (0.2 kg and 4.4%) as starch digestibilities were not affected by
treatment (P > 0.43; Tables 3.11 and 3.12). And, more specifically, sodium treatments
increased pdNDF digested total tract (7.8%; Table 3.12). Ruminal starch (21.5%/h) and
pdNDF (2.2%/h) digestion rates were similar across all treatments but sodium treatments
tended to decrease iNDF passage rate from the RR (0.4%/h; Table 3.13). With N
fractions, sodium treatments tended to increase ammonia passage to duodenum and
increase N digested (%) total tract (Table 3.14). (For the results of chromic sesquioxide
(CrzO3) as a marker to estimate nutrient digestibility in the rumen and in the total tract,

see Appendix A.14 and Appendix Tables A.17, A.18, A. 19, A20, A21, and A22.)

135

Within the RR, turnover times (h) were similar across experimental diets for OM
(11.2 h), starch (2.6 h), NDF (25.8 h), and pdNDF (22.3 h), however, even with less
consumed, sodium treatments tended to increase ruminal iNDF turnover time (31.9 h vs.
27.7, P = 0.09; Table 3.15). Fractional rates for ruminal liquid passage and ruminal
valerate absorption were similar for all experimental diets (0.155/h and 0.352/h,
respectively; Table 3.13).

Ruminal pools of water, DM, OM, starch and NDF on the sodium treatments were
numerically greater than ruminal pool of the control diet, however, ruminal pools across
experimental diets were not statistically different (P > 0.23; Table 3.16). When change in
ruminal contents across the experimental periods was considered, sodium treatments
increased ruminal weight (+7.6 kg vs. —7.2 kg) and tended to increase ruminal volume
(+8.8 L vs. -9.4 L) when compared with the control diet (Table 3.16). Taken together,
these changes suggest sodium treatments expanded the ruminal contents. Bicarbonate
treatment tended to increase mean ruminal volume (+9.5 L) and lowered ruminal content
density (0.81 vs. 0.87 kg/L) when compared with chloride treatment (Table 3.16). Given
the similar ruminal wet weight (82.2 kg), dry matter (12.2 kg), water (70.0 kg), and DM
to water ratio (0.174) of the bicarbonate and chloride treatments, the differences in
ruminal volume and, hence, density were caused by gas trapped in the ruminal mass from
the decomposition of bicarbonate to carbon dioxide and water (Table 3.16).

During the digestibility determination (d15 —- d17), ruminal VFA and associated
parameters were measured (Table 3.17, n=144). Bicarbonate treatment had higher total
VFA than chloride treatment (147.5 mM vs. 140.3 mM). This difference was accounted

for largely by the greater ruminal propionate concentration for the bicarbonate treatment.

136

This difference, in turn, leads to a lower ruminal A:P for bicarbonate treatment compared
to chloride (2.48 vs. 2.69, respectively). Ruminal pH measured at these collection times
showed small but significant differences among the three experimental diets (P < 0.05).
Chloride treatment had the highest mean ruminal pH at 6.00 and the control diet had the
lowest at 5.94. Ruminal VFA and associated parameters were also measured with a
subset from the intensive 24 h collection (Table 3.18, n=108). During this sampling,
individual VFA, total VFA, ruminal acetatezpropionate ratio, ruminal pH were not
different among the experimental diets.

VFA profiles were determined on two sets of samples; those collected by grab
sample and squeezing the liquid through a nylon mesh (Table 3.17) and those collected
by computer-controlled pump drawing ruminal fluid through a fine mesh and into a
collection tube (Table 3.18). The grab samples (Tables 3.17) contained greater than 50%
more total VFA and had a lower mean ruminal pH than those collected by pump (Table
3.18). These differences are reflective of the difference between these two collection
methods. With the squeezing and straining, more VFA is expressed from the particulate
matter yielding higher VFA concentration and lower sample pH (Erdman, 1988a). Thus,
the latter (Table 3.18) is probably more representative of the ruminal solution. Difference
in sampling location may also be a factor.

Net energy for lactation (NEL) associated with intake, maintenance, and milk
yield was calculated (Table 3.19). Net energy measures were not different across
experimental diets.

Sodium treatments did not affect eating or ruminating chewing activity (Table

3.20). Mean DMI kg/d was slightly less (0.3 kg) during this behavior subperiod (Table

137

3.20) than during digestibility trial subperiod (Table 3.6). This slight difference is
probably an artifact due to the difference in calculations to produce these means with
wetting of the orts being of primary concern (Table 3.20 is sum of as fed feed
disappearance times percentage of TMR DM percentage vs. Table 3.6 is offered DM kg
minus refused DM kg). It is also possible that the DMI was higher in the digestibility
subperiod than the behavior subperiod because of compensation for removed digesta.

Sodium treatments affected water consumption (Table 3.21) by increasing daily
water intake (5.1 L/d or 5.2%/d). Chloride tended to increase water consumed during
rumination when compared to bicarbonate (5.1 L vs. 2.3 L).With bicarbonate treatment,
cows tended to drink fewer times per (1 (1.6 bouts/d) but tended to consumed more water
per bout (0.7 L) than with chloride treatment so daily total water consumed was similar
(103.8 L). Across all experimental diets, more than half of average total daily water
consumption was consumed while eating.

Sodium treatments did not affect ruminal pH (Table 3.22). Sodium treatments did
not affect (P > 0.21) ruminal pH measured as mean (6.20), SD (0.24), median (6.20),
minimum (5.78), maximum (6.62), or range (0.84). Also, treatments did not affect time
and area of curve under pH 6.0.

Whole jugular blood was analyzed for gas and electrolyte concentrations (Tables
3.23). Treatments did not affect the measured variables of blood pH, partial pressure of
oxygen, hematocrit, sodium concentration, or calcium concentration. Sodium treatments
tended to increase calculated variables based on the partial pressure of carbon dioxide
and blood pH: total carbon dioxide, base excess (ECF and blood), calculated

bicarbonate, and standardized bicarbonate. Bicarbonate treatment shows higher whole

138

blood partial pressure of carbon dioxide and lower chloride when compared chloride
treatment. These effects are likely probably related to treatment. Bicarbonate treatment
also tended to decrease oxygen saturation, increase carbon dioxide content, and decrease
potassium concentration when compared with chloride treatment. Compared to values
reported in the literature, means were within normal ranges of measurement for bovine
for venous pH, carbon dioxide measurements, bicarbonate, sodium, potassium, chloride,
and anion gap. However, mean hematocrit was less than some reports and average
calcium concentrations were less than half of expected values (Table 1.13a and 1.13b).

Plasma metabolites and hormones were measured (Table 3.24, n=108). Sodium
treatments had no effect on plasma concentrations (P > 0.16) of glucose (56.4 mg/dl),
NEFA (55.7 qu/L), BHBA (6.0 mg/dl), or glucagon (109.0 pg/ml) but tended to
increase plasma insulin (9.4 vs. 7.8 uIU/ml) suggesting a postabsorptive effect. No
differences were seen between bicarbonate and chloride treatments.

In summary, sodium treatments increased NDF digestibility, expanded the
ruminal content weight and volume and slowed passage from the RR. With sodium
treatments, water consumption was increased but no effects were measured for starch
digestibility (extent or site of digestion), ruminal microbial N production, or ruminal
liquid turnover. Valerate absorption was not affected by treatments and no strong
ruminal pH effects are measured. Intake was not different across experimental diets as
was net energy intake and expenditure, milk yield and composition, and chewing activity.

Sodium treatments caused some postabsorptive differences in blood gas, and electrolytes.

139

DISCUSSION

The beneficial effects of sodium bicarbonate feeding are well documented
(Erdman, 1988a; Staples and Lough, 1989) but, the actions of sodium bicarbonate are
probably more complex than the simple elevation of ruminal pH. Several hypotheses
regarding the mechanism of sodium bicarbonate action have been proposed but
verification with diets applicable to current lactating dairy cows have not been reported.

Two hypotheses of mechanism of sodium bicarbonate action in lactating dairy
cows can be generalized as an increased efficiency of dietary DM use. Sodium
bicarbonate inclusion in the diets of lactating dairy cows generally increases milk fat
percentage and possibly FCM yield without changing DMI (Erdman, 1988a; Staples and
Lough, 1989).

The first hypothesis is the addition of sodium bicarbonate directly increases
ruminal pH which increases ruminal fiber digestibility. Lower ruminal pH can depress
fiber digestibility in vitro (Hoover, 1986; Grant and Mertens, 1992). The addition of
sodium bicarbonate ofien leads to increases in ruminal pH and ruminal A:P (Erdman,
1988a; Staples and Lough, 1989) and is associated with small increases in apparent
digestibility of the DM (Erdman, 19883) or, more specifically, an increase in ADF
digestibility-usually in the RR (Staples and Lough, 1989). In this experiment, no effects
on ruminal pH were detected. The mean ruminal pH of 6.2, which is near the pK,, of
carbonic acid (Turner and Hodgetts, 1955a), suggests that the ruminal solutions were well
buffered across all experimental diets. Yet without ruminal pH differences, differences in

NDF digestibility were detected in this experiment.

140

Another hypothesis of the mechanism that sodium bicarbonate acts osmotically to
increase liquid turnover in the RR (Russell and Chow, 1993). The addition of the sodium
to the diet increases water intake which could lead to an increased flow of liquid from the
RR and, with it, increased the flow of other components leaving the rumen such as
particulate matter, rumen microbes and VFA. An increase in particulate matter escape
should increase the flow of starch to the small intestine thus reducing starch fermented to
VFA and increase starch digested and absorbed as glucose from the small intestine. Less
starch fermentation in the RR might lead to the higher ruminal pH observed in some
experiments. In this experiment, sodium treatments increased water consumption which
would be expected given the increased sodium consumption (NRC, 2001). However,
ruminal liquid passage and ruminal starch digestibility were not increased by treatment.
Changes were not expected as these effects are usually associated with higher diet
inclusion rates of sodium bicarbonate than used in this experiment (Rogers et al., 1982;
Rogers and Davis, 1982b).

Given the results of the experiment, the mechanism of sodium bicarbonate action
at recommended rates in the diets of lactating dairy cows appears to be, in part, a hybrid
of the preceding ideas. Sodium bicarbonate has an osmotic effect within the RR and this
effect may increase fiber digestibility. In the RR, sodium bicarbonate provides a sodium
cation and bicarbonate anion and both of these ions are osmotically active. In solution,
the sodium is not completely disassociated from the bicarbonate. At ruminal
concentrations, this disassociation is probably 80 to 90% yielding an osmotic index of 1.8
to 1.9 times the sodium bicarbonate molar concentration (Weast, 1978). In the RR

solution, sodium is strong ion and, chemically, an alkalizer (Stewart, 1983). Bicarbonate

141

in the solution is a weak acid anion and, depending on ruminal pH, an alkalizer or a
buffer. The bicarbonate ion can combine with a proton and decompose to form water and
carbon dioxide (Segel, 1976).

As the primary extracellular cation, sodium determines ECF volume in the body
(Carlson, 1997). The amount of sodium is regulated and this cation is balanced with
anions and these ions drawn water as water follows solute (Houpt, 2004). Similarly,
across the RR wall, ion transport is regulated (Gaebel and Sehested, 1997; Sehested eta1.,
1999b) and water movement is restricted (Engelhart, 1970). The concentration of sodium
plus potassium also seems to be maintained in the RR solution (Lang and Martens, 1999).

With the active regulation of the RR solution, a tonic addition of osmotically
active particles with each meal could, over time, lead to expansion of ruminal solution
(weight and volume) within the RR. This expansion, given a constant DMI, could lead to
slowing of passage and turnover from the RR, possibly allowing fiber more time for
microbial digestion.

For the sodium bicarbonate treatment in this experiment, the mean meal size was
2.8 kg which contained 28g of sodium bicarbonate. At 1.8 mOsm/mole, the sodium
bicarbonate in the meal would contribute 600 mOsm to the RR. For the 70 kg of water in
the RR, 56 kg is estimated to be in the ruminal solution assuming 10% of the water is
within both the feed and microbes (for a total of 20% of water confined). Taking these
assumptions and assuming instantaneous consumption and mixing, the sodium
bicarbonate in the meal would be expected to raise ruminal osmolality by 11 mOsm. In

this experiment, the increase would occur, on average, every 2.5 h. Under real

142

conditions, the increase would be smaller with passage, absorption, and the loss of
bicarbonate ion in carbon dioxide and water.

In this experiment, sodium treatments increased BW but not empty BW. The
difference is ruminal contents which, taken on their own, increased in weight over the
experimental period with sodium treatments compared to control. Also, total tract
pdNDF digested increased with sodium treatments. The link between these two
responses is the change in ruminal dynamics. The slowing of ruminal fiber passage
associated with sodium treatments may have lead to the more fiber digested in the RR
and thus the total tract. However, amounts digested in the RR were numerically but not
statistically different. Also, numeric but nonsignificant differences in ruminal pool sizes
do not negate expansion. Although, not statistically significant, the increase in net energy
lactation gained by increased pdNDF digestion is coincidentally equal to the increase in
net energy expended in milk produced.

This proposal could explain why midlaction are more responsive than early
lactation cows to the inclusion of sodium bicarbonate in their diets. Midlactation cows
with sodium bicarbonate in their diets gain more milk fat and fat-corrected milk than
early lactation cows compared to similar cows on control diets (Staples and Lough, 1989;
Tucker et al., 1994). Midlactation cows are less likely to be limited by fill than early
lactation cows and, therefore are more likely to expand RR contents with sodium
bicarbonate addition.

The mechanism of sodium bicarbonate action in lactating dairy cows is likely
partly osmotic. Feeding sodium bicarbonate increases weight of ruminal contents,

decreases iNDF passage from the RR, and increases total tract digestibility of pdNDF. In

143

this experiment however, the lack of milk fat response suggests that only part of the
mechanism of sodium bicarbonate action was apparent.

As a 3 x 3 Latin square, this experiment had lower statistical power to separate
treatment means than some of other experiments in our lab. This reduction in power
increases the likelihood of Type II error (i.e. failure to reject Ho).

Sodium treatments caused some postabsorptive differences in insulin, blood gas,
and electrolytes. The tendency for increased insulin suggests greater propionate
absorption (Oba and Allen, 2003b). The blood gas and electrolyte change are probably
due to slight acid-base adjustments due to increased sodium and chloride flux through the
cows.

The results of this experiment can be compared to previous work in our lab using
similar methods and materials and cows with similar genetics. Compared to the average
cows in several previous experiments, the cows on this experiment ate more, produced
less, and weighed more (Table 3.25). These cows showed an increased starch digestion
and passage and a decreased pdNDF digestion and passage compared with previous
work.

The results of this experiment can be compared to a previous experiment (Chapter
2) with the same treatment (Table 3.26). Rumination and total chewing time per (1 was
not affected in this experiment as it was previously, possibly due to this experimental
design and its decreased power to separate means. This experiment can explain why the

body weight change was disproportionally greater than the body condition score change.

144

CONCLUSION

Sodium bicarbonate inclusion increased total tract digestibility of pdNDF likely as
a result of osmotic changes in the RR expanding the ruminal contents and decreasing
passage rate. This osmotic effect is likely only part of the mechanism of sodium

bicarbonate action in lactating dairy cows.

145

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CHAPTER 4: Effects of dietary starch concentration and corn conservation method

on ruminal and plasma ions in lactating dairy cows.

ABSTRACT

The objective of this study was to investigate relationships among strong ions in
the rumen. Eight ruminally cannulated Holstein cows (55 i 16 DIM; mean i SD) were
used in an experiment with a duplicated 4 x 4 Latin square design. A 2 x 2 factorial
arrangement of treatments was used with main effects of dietary starch percentage (32%
vs. 21%) and conservation method of corn grain (dry, 90% DM or high-moisture, 63%
DM). Ruminal fluid samples were collected through a ruminal cannula every 20 min for
24 h per period during which feeding behavior and ruminal pH were monitored
continuously. Dietary treatments did not affect mean ruminal pH (6.22) or total VFA
(102 mM) but high starch treatments increased daily pH variability as measured by
standard deviation and range (P < 0.09). High starch treatments increased ruminal
sodium concentration (98.5 vs. 94.2 mEq/L) and decreased ruminal potassium and
ammonium concentrations (38.3 vs. 43.0 mEq/L and 3.4 vs. 4.0 mEq/L, respectively).
Ruminal potassium concentrations were influenced by treatment while ruminal sodium
concentration was not reflective the uniform concentration of sodium across diets nor the
expected decrease in saliva flow on the high starch treatment. The sum of sodium,
potassium and ammonium (141.1 mEq/L) was not affected by treatment nor was strong
ion difference defined as sodium plus potassium minus chloride (124.9 mEq/L). Dietary
treatment did not affect mean ruminal osmolality (261 mOsm) but high starch treatment

increased variability of ruminal osmolality. Ruminal pH was correlated positively (r >

172

0.72) with ruminal sodium concentration, the sum of ruminal sodium and potassium, and
ruminal strong ion difference, and was correlated negatively (r < -0.42) with total VFA
concentration, ruminal ammonium, and the sum of ruminal ammonium and potassium.
Ruminal sodium concentration was negatively related to ruminal potassium concentration
and ruminal ammonium concentration and more negatively related to the sum of
potassium and ammonium concentrations. The alkalizing strong ion difference and
charge balance in the rumen are likely regulated by modifying sodium flux across the

ruminal epithelium.

Keywords: ruminal pH, strong ions, sodium

173

INTRODUCTION

Sodium, potassium and chloride are essential nutrients for lactating dairy cows
(NRC, 2001) and are the primary strong ions in the ruminal solution (Bailey, 1961b,
Bennick et al., 1978, Tucker et al., 1988). In solution, sodium and potassium are
alkalogenic and chloride acidigenic and each of three has a single valence (Stewart,
1983). These ions also contribute equally to the osmolality of a solution (Weast, 1978).
The sum of cations (sodium plus potassium as mEq/L) minus anion (chloride as mEq/L)
is strong ion difference (SID) which is a determinant of the pH of solution (Stewart,
1983). Other strong ions can be included in the calculation of SID (Constable, 2000). In
addition to SID, other determinants of blood pH include partial pressure of carbon
dioxide, weak acid concentration and phosphate concentration (Stewart, 1983;
Constable, 2000; Heisey and Adams, 2002).

In the rumen, sodium is usually the most abundant cation in the ruminal solution
with a typical concentration of 120 mEq/L (Bailey, 1961b). The ruminal concentration of
sodium is proportional to but lower than the concentration of sodium in saliva (Bailey,
1961b) and does not appear to be influenced by meals (Tucker et al., 1993). Sodium
enters the reticulorumen (RR) in the diet or in saliva and can leave the ruminal lumen by
passage to the omasum or by absorption across the ruminal epithelium (Stevens and
Hume, 1995). Sodium is absorbed into the ruminal epithelium by sodium-hydrogen
exchange and by the electrogenic diffusion of sodium through ion channels (Martens and
Gaebel, 1988).

Ruminal potassium concentration is typically 25-40 meq/L (Bailey, 1961b;

Tucker et al., 1988, Tucker et al., 1993). This concentration of potassium is usually

174

higher than that found in saliva (Bailey, 1961b) and is primarily a function of diet
concentration (Bailey, 1961b; Bennick et al., 1978; Tucker et al., 1993). Potassium enters
the RR from the diet and, to a lesser extent, in the saliva and will leave the ruminal lumen
by paracellular absorption through the ruminal epithelium diffusing down the
concentration gradient from lumen to the blood and by passage to the omasum (Stevens
and Hume, 1995).

Ruminal chloride concentration usually ranges from 10 to 15 mEq/L (Bailey,
1961b; Tucker et al., 1988, Tucker et al., 1993) and enters the R from the diet and saliva
and leaves by absorption and passage (Stevens and Hume, 1995). Ruminal chloride is
absorbed by the exchange of chloride anion for bicarbonate anion across the apical
membrane of ruminal epithelial cell (Gaebel et al., 1991; Martens et al., 1991; Gaebel et
al., 1993).

Sodium and chloride have been shown to be absorbed proportionally across the
ruminal epithelium and the indirect link is either intracellular pH or intracellular carbonic
acid where the sodium-hydrogen exchange is working in parallel with a chloride-
bicarbonate exchange (Martens et al., 1991). Sodium and potassium concentrations in
the RR have a reciprocal relationship in sheep (Sellers and Dobson, 1960; Scott, 1966;
Stacy and Warner, 1966; Scott, 1967) and cattle (Bailey, 1961b). Based on in vitro
experiments with sheep ruminal epithelium, this relationship is likely achieved by the
potassium modulation of sodium absorption (Lang and Martens, 1999). The high
potassium enhancement of sodium absorption keeps the sum of sodium and potassium
relatively constant and this could be an effective mechanism for the regulation of both

charge and osmolality in the RR (Lang and Martens, 1999). Also, based on in vitro

175

experiments with sheep ruminal epithelium, sodium and ammonium also may have a
similar relationship as ammonium can promote sodium absorption (Abdoun et al., 2003)
but the mechanism has not been determined. Therefore, in the ruminal solution, sodium,
potassium, and chloride appear regulated and interacting.

The objective of this experiment was to determine the relationships among strong
ion concentrations and ruminal pH in cows consuming diets varying in fermentibility.
Given previous blood SID theories, in vitro ruminal epithelial and in vivo sheep
experiments, sodium, potassium and chloride are expected to interact and be related to
ruminal pH in lactating dairy cows in vivo. The null hypothesis was that sodium and

other strong ions in the ruminal solution are not related to ruminal pH or each other.

 

176

MATERIALS AND METHODS

This paper is the fourth in a series of papers from one experiment that evaluated
effects of corn grain conservation method at two dietary starch concentrations. This
paper will focus on strong ions and their relationships in plasma and centrifuged ruminal
fluid. Feeding behavior and productivity (Oba and Allen, 2003a), ruminal digestion
kinetics (Oba and Allen, 2003b), and efficiency of microbial nitrogen production (Oba
and Allen, 2003c) were reported previously (Table 4.1). Animal procedures were
approved by the All University Committee on Animal Use and Care at Michigan State

University (AUF# 05/96-037-00).

Design And Treatments

Eight multiparous Holstein cows from the Michigan State University Dairy Cattle
Teaching and Research Center were assigned randomly to duplicated 4 x 4 Latin squares
balanced for carry over effects with a 2 x 2 factorial arrangement of treatments.
Treatments were dietary starch concentration (21% vs. 32%) and conservation method of
corn grain [high moisture ground (HM) vs. dry ground (DG) corn]. Treatment periods
were 21 d with the samples and data reported in this paper collected primarily on d 15.

(Tables 4.2, 4.3, and 4.4)

Electronic Data And Automatic Sample Collection
On d 15, a 24 h intensive collection of plasma and rumen fluid was conducted
simultaneously with measurement of feeding behavior. Whole blood samples and ruminal

fluid samples were collected every 20 min for 24 h by automated sample collection

177

 

 

system (Allen et al., 2000b), starting at 1200 h. Feed doors were closed at 1200 h and
reopened at 1400 h. Blood was sampled from a jugular vein through a catheter (45 cm
long, MRE 095 Renathane® tubing, Braintree Scientific, Inc., Braintree, MA) inserted l
(1 prior to sample collection using sterile technique. Ruminal fluid was drawn from the
ventral rumen through tubing inserted through the stopper of the ruminal cannula (10 cm
i.d.; Bar Diamond Inc., Parma, ID). This system successfully collected 99.5% and 97.9%
of the total samples (4,308 each) for blood and ruminal fluid, respectively.

On (1 15, feeding behavior and ruminal pH was also monitored by a computerized
data acquisition system (Dado and Allen, 1993). Data of chewing activities, feed
disappearance, water consumption, and ruminal pH were recorded to computer file every
5 seconds for each cow. Chewing activities were summarized as meal bouts, interval
between meals, and meal size for eating behavior and as ruminating bouts and inter-
ruminating interval for ruminating behavior. Ruminal pH data were summarized to daily
mean, variance, median, minimum, maximum, range, the hours and area for which
ruminal pH was below pH 6.0. The minimum, maximum, and range were calculated
using the daily 2.5th and 97.5th percentiles for ruminal pH measured every 5 seconds. The
area was calculated by determining the time below ruminal pH 6.0 and weighting that

time by the deviation from the threshold.

Sample Processing And Storage

Ruminal fluid was centrifuged at 2,000 x g for 15 min immediately after
collection, and supematants were frozen at -20° C until analysis. Whole blood collected
in tubes with potassium oxalate and sodium fluoride as a glycolytic inhibitors (Becton

Dickinson Vacutainer Systems, Franklin Lakes, NJ). Both whole blood samples were

178

 

 

centrifuged at 2,000 x g for 15 min after sample collection, and plasma was harvested and

frozen at -20° C until analysis.

Sample Preparation
All feed and orts were frozen immediately after collection at and frozen at —20° C.
(See Oba and Allen, 2003a for more detail.) Composites were dried in a 55° C forced-air

oven for 72 h and DM concentration was determined. Samples were ground with a Wiley

mill (1 mm screen; Authur H. Thomas, Philadelphia, PA).

Rumen evacuations For Pool Size Determination

Ruminal contents were evacuated manually through the ruminal cannula at 1800 h
(4 h afier feeding) on d 20 and at 1000 h (4 h before feeding the following day) on d 21
of each period. Total ruminal content mass and volume were determined. During
evacuation, a 10% aliquot of digesta was separated to allow accurate sub-sampling. The
aliquot was squeezed through nylon mesh (1 mm pore size) to separate it into primarily
solid and liquid phases. Samples were taken from both phases for determination of pool

size of digesta components in the rumen. Samples were frozen immediately at —20°C

Sample Analysis

Before centrifuging and freezing, whole blood, collected in a tube containing
lithium heparin (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ), was
analyzed immediately for pH, pC02, hematocrit, p02, calcium, sodium, potassium, and
chloride by a blood gas analyzer (Stat Profile 4, Nova Biomedical, Waltham, MA) and

ten other blood variables were calculated by manufacturer’s equations assuming a

 

179

temperature of 385°C (Appendix A.5). Feed samples were processed and analyzed for
DM, ash, CP, starch, NDF, and indigestible NDF (iNDF) as described by Oba and Allen
(2003a, 2003b, and 2003c). Feed samples were analyzed for sodium, potassium, and
chloride concentrations. Sodium and potassium concentrations were determined by
digestion according to Hach et al. (1987) and measurement of the element in supemate by
atomic absorption according to manufacturer’s recommendation (SpectrAA 220FS,
Atomic Absorption Spectrometer, Varian Analytical Instruments, Walnut Creek, CA).
Chloride concentration was determined by extracting the feed with 1.0% nitric acid
solution for one hour on shaker (Orbimix 1010, Brinkman Instruments, Westbury, NY)
and measuring supemate chloride concentration by coulometric titration (Digital
Chloridometer, Model 442-5000, Labconco Corporation, Kansas City, MO). Digests and
dilutions were stored in either polypropylene specimen cups or polypropylene test tubes
(Round bottom, 13 x 100 mm, culture test tubes, Fisherbrand® Catalog No. 14-956-7A,
Fisher Scientific, Pittsburgh, PA) until analysis.

Centrifuged rumen fluid was analyzed for sodium, potassium, chloride, and
ammonia concentrations. Sodium and potassium concentrations were determined by
atomic absorption using AOAC procedures for beer (1990, #98703 for sodium and
#98702 for potassium) adapted to the manufacturer’s recommendation (SpectrAA
220FS, Atomic Absorption Spectrometer, Varian Analytical Instruments, Walnut Creek,
CA). Chloride concentration was determined by coulometric titration (Digital
Chloridometer, Model 442-5000, Labconco Corporation, Kansas City, MO). Ammonia
concentration was determined for ruminal fluid samples according to Broderick and Kang

(1980).

180

 

 

 

Ruminal fluid was analyzed for concentrations of major VFA and lactate.
Samples were centrifuged at 26,000 x g for 15 min, and supernatant (600 uL) was mixed
with 600 pL Ca(OH)2 and 300 uL of CuSO4 containing crotonic acid as an internal
marker in 1.7 ml micro centrifuge tubes. Samples were centrifuged at 12,000 x g for 10
min, and supernatant (1000 pl) was taken and mixed with 28 ul of H2SO4 in 1.5 ml micro
centrifuge tubes. Samples were frozen and thawed twice, and centrifuged at 12,000 x g
for 10 min to precipitate and remove protein thoroughly. Supernatant was transferred to
HPLC vials. Concentrations of VF A and lactate of the supernatant were determined by
HPLC (Waters Corp., Milford, MA) according to Oba and Allen (1999a).

Osmolality of plasma and centrifuged rumen fluid was measured on the
AdvancedTM Osmometer (Model 3D3, Advanced Instruments, Inc., Lab Products
Division, Norwood, MA, buzzpoint of 3000 for plasma and 2500 for centrifuged rumen
fluid).

A subset of plasma samples was randomly selected across all experimental
periods (n=47) and analyzed for sodium and potassium using atomic absorption
according to the manufacturer’s recommendation (SpectrAA 220FS, Atomic Absorption
Spectrometer, Varian Analytical Instruments, Walnut Creek, CA) to confirm whole blood
electrolyte analysis by blood gas analysis. Within sample, measurements recorded had

mean differences of —9.0% for sodium and —l .2% for potassium.

Calculations
Total diet concentrations for cations and anions were calculated from individual

ingredient analyses and dietary proportions of the dry matter. Dietary cation anion

181

difference in feeds as mEq/100g DM was calculated as sodium plus potassium minus
chloride (DCAD3). (Appendix A.6)

Strong ion difference (SID) in solution was calculated as concentrations of
sodium plus potassium minus chloride.

Estimated associated VFA concentration in the ruminal solution as mM was

calculated as (Total VFA concentration, mM)(1/(10"(ruminal pH minus 4.8) + 1).
Estimated disassociated VFA concentration in the ruminal solution as mM was calculated
as total VFA minus estimated associated VFA.
Calculated ruminal bicarbonate in the ruminal solution as mEq/L was calculated as
10"(ruminal pH minus 7.74) times 500 (assuming carbon dioxide concentration of 0.5
atm; Kohn and Dunlap, 1998).

Turnover rate in the rumen (/h) = (intake of component / ruminal pool of

component)/24.

Statistical Analysis

All data were analyzed using the fit model procedure of JMP® (Version 5.0.1.2;
2003) according to the following model:

Yijk=H+Ci+Pj+Tk+6ijk

Where

it = overall mean,

C, = random effect of cow (i = 1 to 8),
Pj = fixed effect of period (j = 1 to 4),

Tk = fixed effect of treatment (k = 1 to 4),

182

 

eijk = residual, assumed to be normally distributed.

Period by treatment interaction was originally evaluated, but it was removed from
the statistical model because interaction was not significant for response variables of
primary interest. Orthogonal contrasts were performed for effects of starch
concentration, corn moisture, and interaction of starch concentration and corn moisture.
Treatment effects and their interaction were declared significant at P < 0.05 and P < 0.10,
respectively, and tendency for treatment effects were declared at P < 0.10. When
interactions of main effects were significant, treatment means were compared using
Student’s t-test and differences were declared significant at P < 0.05. Residual plots were
checked for appearance of normality and appeared normally distributed. Ruminal pH data
for Cow 2946, Period 3 were lost due to pH probe failure.

Cow period means were used to generate Pearson correlation coefficients in
J MP® (Version 5.0.1.2; 2003). Individual ion and metabolite measures within sample
were used to develop linear regression equations and calculated adjusted R2 values in
JMP® (Version 5.0.1.2, 2003). Changes in ion concentration from the beginning to end
of meal, bouts, and drinks were generated using a logic script (Appendix A.15) in Igor

Pro® (2002).

183

RESULTS

As previously reported, dietary treatments affected DMI, meal size, starch
digestibility, microbial nitrogen production, ruminal pH, chewing time, and milk
production (Table 4.1). Measurements in this paper were generated primarily from data
from an intensive collection on d 15 of each period so some of the results may not match
previous behavioral measurements which were based on d 16 through 19 inclusive.
Dietary sodium, potassium, and chloride concentrations were 0.5%, 1.2%, and 0.3%,
respectively for the 32% starch diets and 0.5%, 1.4%, and 0.4%, respectively for the 21%
starch diets. With regards to ruminal strong ions, this experiment uniquely combines
measurements in the conditions of in vivo, in cattle, and under standard dietary
conditions.

Dietary treatments affected feeding behavior of cows (Table 4.5). High starch
treatments increased daily water intake 6.8 L by tending to increase the frequency of
drinking bouts. High starch treatments decreased meal length by 4 min (13%). With an
interaction among the treatments, DMI was greatest on the high starch, DG treatment
(20.9 kg/d) and lowest on the low starch, DG treatment (17.8 kg/d). These differences
are attributed to changes in number of meals, meal length, and intermeal interval which
are proposed to be caused by differences in ruminal starch fermentation as previously
reported (Oba and Allen, 2003a). With a significant interaction, eating time per (1 was
highest on the low starch, HM corn treatment (254.2 min/d) and lowest on the high
starch, HM corn and low starch, DG corn treatments (230.4 and 241.9, min/d,

respectively). High starch treatments decreased ruminating time per (1 (48.0 min) and this

184

I

decrease was attributed in part to an increased inter-ruminating interval (7.8 min). High
starch treatments also decreased total chewing time per d (63.7 min/d).

Ruminal content weight at the end of the experimental period was lower for high
starch treatments and, with a similar amount of DM, this is attributed to less total water in
the RR (Table 4.6). The distribution of water within the ruminal contents was not
determined (i.e. within digesta, within the microbial mass, or free ruminal solution).

Dietary treatment did not affect mean ruminal pH (6.22; Table 4.7). However,
high starch treatments increased pH variability as measured by standard deviation and
range. Similarly, dietary treatment did not affect ruminal total VFA concentrations (101.6
mM; Table 4.8) but high starch treatments increased concentration of some individual
VFA (propionate, iso-butyrate, valerate, and iso-valerate). With a similar ruminal pH and
total VFA concentration across dietary treatments, similar concentrations of associated
and disassociated total VFA were estimated among dietary treatments.

Overall mean ruminal ion concentrations were 97.7 mEq/L for sodium, 39.8
mEq/L for potassium, and 12 mEq/L for chloride (Table 4.9). Sodium and potassium
concentrations were similar to those previously report by Emery et al. (1960) and lower
in sodium and higher in potassium than more recent reports with cattle (Tucker et al.,
1988; Tucker et al., 1993). Chloride concentrations were similar to recent reports with
cattle (about 140 mEq/L for sodium and 27 mEq/L for potassium, respectively; Tucker et
al., 1988; Tucker et al., 1993). (Distributions for other experimental measures are
located in Appendix Tables A23 and A24).

High starch treatments increased ruminal sodium concentration (98.5 vs. 94.2

mEq/L) and decreased ruminal potassium and ammonium concentrations (38.3 vs. 43.0

185

mEq/L and 3.4 vs. 4.0 mEq/L, respectively; Table 4.10), as well as the sum of potassium
and ammonium concentrations. Ruminal chloride concentration was highest on the low
starch treatments ( 13 mEq/L), intermediate on the high starch, DG corn diet (12 mEq/L),
and lowest on the high starch, HM corn diet (1 l mEq/L). The higher ruminal
concentrations of chloride and potassium in low starch treatments were proportional to
the dietary concentrations of these ions. Ruminal ammonium may also be higher on low
starch treatments because they contain more alfalfa silage but dietary soluble protein was
not measured. Ruminal sodium concentrations decreased with low starch treatments
which did not reflect the uniform concentration of sodium across diets or with the
expected increase in saliva flow associated with a 10% increase in total chewing time per
(1. Lower ruminal sodium concentrations were associated with higher ruminal potassium
and ammonium concentrations. In contrast to individual ion concentrations, dietary
treatment did not affect the sum of sodium and potassium (136.9 mEq/L), the sum of
sodium, potassium and ammonium ( 141.1 mEq/L), the sum of chloride and estimated
disassociated VFA (107.5 mEq/L), the sum of chloride, estimated disassociated VFA,
and calculated ruminal bicarbonate (131.7 mEq/L), or strong ion difference (124.9
mEq/L) suggesting regulation of the ruminal environment (Table 4.10).

Dietary treatment did not affect whole blood sodium concentrations (142.4
mmol/L), potassium concentrations (4.1 mmol/L), chloride concentrations (106.0
mmol/L), or anion gap (16.4 mmol/L; Tables 4.11 and 4.12). High starch treatments
tended to decrease calcium concentration (0.1 mmol/L) but not normalized calcium
concentrations (1.3 mmol/L). High starch treatments increased whole venous blood pH

(7.428 vs. 7.417) and lowered partial pressure of oxygen (2.8 mm Hg) and oxygen

186

 

saturation (2%) leading to greater oxygen content (0.4 ml/dl). These differences are
probably related as decreased pH is associated with greater disassociation of oxygen from
hemoglobin (Rhoades and Tanner, 1995).

Dietary treatments did not affect mean ruminal and plasma osmolalities (Table
4.13). Ruminal osmolality averaged 261 mOsm as measured and 286 mOsm corrected for
bicarbonate. Plasma osmolality averaged 284 mOsm as measured and 309 mOsm
corrected for bicarbonate. Ruminal osmolality SD was greater on high starch treatments
than low starch treatments (20.5 vs. 17.2). Plasma osmolality SD was similar across all
dietary treatments (P > 0.47). Mean difference of plasma minus ruminal osmolality was
similar across dietary treatments whether as measured or with inclusion of respective
bicarbonate measures (22 mOsm). Across all treatments, measured plasma osmolality
was greater than ruminal osmolality measured at the same time more than 85% of the
time. With plasma osmolality relatively constant (Figure 4.1), ruminal osmolality
exceeded plasma osmolality only when ruminal osmolality was highest.

Osmolalities were adjusted for bicarbonate that was lost during sample processing
and storage. Both ruminal and whole blood samples were centrifiiged, frozen, thawed,
mixed and measured. Over this extended time between sampling and measurement,
samples equilibrated with the carbon dioxide poor atmosphere (Table 1.6) and a
significant decrease in osmolality is possible (Dobson, 1970). Plasma osmolality was
adjusted by adding the bicarbonate concentration from the blood gas measurement.
Whole blood bicarbonate measurements averaged 24.0 mmol/L and were normally
distributed. Ruminal osmolality was adjusted with a calculation of bicarbonate

concentration (Kohn and Dunlap, 1998). This calculation assumed a carbon dioxide

187

concentration of 50% in the gas phase above the ruminal mass. The ruminal bicarbonate
calculation averaged 23.8 mmol/L but had a skewed distribution. The actual bicarbonate
is, however, dependent of the specific ruminal carbon dioxide at the time of sampling.
Ruminal carbon dioxide is very variable within the day (Table 1.5) and specific measures
of mean and variability for contemporary lactating dairy cows have not been reported.
Calculated ruminal bicarbonate is sensitive to the choice of carbon dioxide percent and
this 50% assumption should be tested in lactating dairy cows.

Among cow period mean ruminal measurements (n=32; Table 4.14), ruminal pH
was correlated positively with ruminal sodium concentration (r = 0.74), the sum of
ruminal sodium and potassium (r = 0.74), and ruminal strong ion difference (r = 0.72),
and was correlated negatively with total VFA concentration (r = -0.42), ruminal
ammonium (r = -0.69), and the sum of ruminal ammonium and potassium (r = -0.49).
Ruminal pH was not significantly related to estimated disassociated VFA concentration,
ruminal potassium concentration, or ruminal chloride concentration. Ruminal pH was
not related to ruminal osmolality (r = -0.32) but was positively related to ruminal
osmolality plus calculated bicarbonate (r = 0.45). Ruminal osmolality was positively
related to total VF A concentration (r = 0.60) and ruminal osmolality adjusted for
bicarbonate was positively related to ruminal sodium concentration (r = 0.44) and other
measures containing the ruminal sodium concentration. Ruminal sodium concentration
was negatively related to ruminal potassium (r = -0.68) and ruminal ammonium (r = -
0.79) concentrations. Ruminal ammonium was also negatively related to ruminal SID (r

= -0.72).

188

Among mean whole blood measurements (n=32; Table 4.15), partial pressure of
carbon dioxide was negatively related to SID (r = 0.83). Sodium concentration was
positively related to both plasma osmolality measures (r > 0.67). Blood bicarbonate
concentration was negatively related to chloride concentration (r = -0.82). Blood SID
was negatively related to chloride (r = -O.86) and positively related to bicarbonate (r =
0.93). These relationships among whole blood measures are consistent with the strong ion
theory (Stewart, 1983). Across mean blood and ruminal measurements (Table 4.16), a
negative relationship was recorded for blood pH and ruminal potassium concentration (r
= -0.58).

Ruminal pH was regressed against several ruminal ion measurements using
individual measurements (n > 2272; Table 4.17). Ruminal pH was related positively to
ruminal sodium concentration (R2 = 0.42, P < 0.0001 , Figure 4.2) but less related to SID
(R2 = 0.23, P < 0.0001, Figure 4.3). Ruminal pH was negatively related to ruminal
ammonium concentration (R2 = 0.26, P < 0.0001, Figure 4.4) and ruminal potassium
concentration (R2 = 0.21, P < 0.0001, Figure 4.5) but ruminal chloride concentration
explained negligible variation in ruminal pH (R2 = 0.01, P < 0.0001, Figure 4.6).

Stronger relationships exist among concentrations of sodium, potassium, and
ammonium (n > 2246; Table 4.17). Ruminal sodium concentration had a moderate
negative relationship with ruminal potassium concentration (R2 = 0.46, P < 0.0001,
Figure 4.7) and ruminal ammonium concentration (R2 = 0.45, P < 0.0001, Figure 4.8).
However, it had a stronger negative relationship with the sum of potassium and
ammonium concentrations (R2 = 0.56, P < 0.0001, Figure 4.9) suggesting an interaction

among these cations. (Table 4.17)

189

Ruminal pH was positively related to ruminal sodium, ruminal sodium plus
potassium, and strong ion difference which is expected given that sodium is associated
with ruminal buffering and that cations are alkalogenic. Ruminal pH has a negative
relationship with total VFA, ruminal ammonium, and ruminal potassium as expected as
they all increase with the ingestion and fermentation of a meal.

Changes were measured from beginning to the end of meals (n=351, mean meal
size was 1.8 kg), drinks (n=445, mean drink size was 7 L), and rumination bouts (n=480,
mean rumination bout was 30 min; Table 4.17). Ruminal sodium concentration difference
was related negatively to meal size (R2 = 0.41, P < 0.0001, Figure 4.10) while ruminal
potassium concentration difference was related positively (R2 = 0.69, P < 0.0001, Figure
4.11) and ruminal sodium plus potassium difference was not related(R2 = 0.01 , P < 0.04,
Figure 4.12). Change in ruminal osmolality was related positively to meal size (R2 =
0.28, P < 0.0001, Figure 4.13) while change in ruminal pH was related negatively (R2 =
0.28, P < 0.0001, Figure 4.14). Drinking, as expected, generally lowered ruminal sodium,
and potassium concentrations and ruminal osmolality (Table 4.17). Length of rumination

bout was not highly related to change in concentrations of ions (Table 4.17).

190

DISCUSSION

High starch treatments were more fermentable (Oba and Allen, 2003a) and a
greater production of osmotically active particles is expected. The cows on these
treatments drank more but had less total water in their rumens. The high starch
treatments decreased ruminal potassium, chloride, and ammonium concentrations which
are reflective of the diet or fermentation and increased ruminal sodium which was not
reflective of the uniform diet concentration. Treatments were not different in ruminal pH
and osmolality but the variability of both was greater on the high starch treatments.
Treatments were also not different for total VFA concentration and various sums of
concentrations of cations and anions.

Ruminal potassium and ammonium were negatively related to ruminal sodium
concentration. However, their sum explained more of the variation in ruminal sodium
concentration than either ruminal concentration did alone. Within the ruminal solution,
charges must balance. Given unknowns of the diet, lactating dairy cows could possibly
regulate one or more ions to control the overall composition of the ruminal solution. Such
a mechanism would help balance charges and limit osmolality while maintaining strong
ion difference in response to the varying cation entry into the ruminal solution.

For the cations in this experiment, potassium and ammonium are likely related to
the meals or fermentation which leaves the sodium as the likely candidate for modulation
and regulation by lactating cows. The experimental means and relationships suggest that
sodium concentration is regulated through sodium flux across the ruminal epithelium
which provides a means of controlling strong ion difference and charge balance in the

rumen. These results agree with concentration relationships reported in vitro work with

191

 

sheep ruminal epithelium (Lang and Martens, 1999; Abdoun et al., 2003). As the primary
extracellular cation, sodium determines extracellular fluid volume in the body (Carlson,
1997). The amount of sodium is regulated and this cation is balanced with anions and the
total ion concentration draws water as water follows solute (Houpt, 2004). In the ruminal
solution, sodium appears to have a similar role.

Ruminal osmolality was examined to determine completeness of ruminal ion
measurement. Within each osmotic measurement, half of contributing particles will be
negatively charged and half will be positively charged to satisfy charge balance of a
solution (Stewart, 1983). Ruminal concentrations of sodium, potassium, ammonium,
chloride, estimated disassociated VFA, and calculated bicarbonate were summed and
then divided by ruminal osmolality plus calculated bicarbonate; the measured ions
accounted for 95.1% of the ruminal osmolality. Overall, 50% of the sums ranged
between 90 to 100% of the ruminal osmolality plus calculated bicarbonate suggesting a
reasonable completeness of the measurement of osmotically active particle in the ruminal
solution. If the three anion sum is subtracted from the three cation sum, the average
difference is +10 mEq/L and, given the need for the balance of charges, this suggests that
all anions have not been measured. Among the ruminal anions previously measured
(Bailey, 1961b), hydrogen phosphate is likely an important missing anion.

As a dependent variable (Stewart, 1983), ruminal pH is probably not managed by
cows; cows likely manage the independent variables that determine pH. In the blood,
examples of independent variables are SID, weak acid concentrations, and partial
pressure of carbon dioxide (Stewart, 1983; Constable, 2001; Heisey and Adams, 2002).

In the RR, lactating cows probably manage variables such as these rather than the pool of

192

 

hydrogen ions which is several orders of magnitude less. Also, osmolality of all bodily
solutions must be in balance and the sum of positive and sum of negative charges within
these solutions must also be balanced. Cows must transport and regulate ions to maintain
these balances.

In this experiment, SID of the ruminal solution, defined as sodium plus potassium
minus chloride, only explained 23% of the variation in ruminal pH but additional ions
may be included in the calculation of SID. In descending order of concentration, the key
cations to be considered are sodium, potassium, ammonium, calcium, magnesium and the
key anions to be considered are disassociated VFA, bicarbonate, hydrogen phosphate,
chloride, lactate, and feed. Adapting criteria of Constable (2001) to ions at ruminal pH,
the strong cations to be summed should be sodium, potassium and ammonium and the
strong anions to be summed should be chloride and lactate if present. (In this experiment,
lactate was detected in 268 out of 2304 ruminal fluid samples with 70 samples showing
concentrations of >5 mM.) Other ions are not likely present in concentrations high
enough to warrant inclusion in the calculation of SID but this has be verified.

A better accounting of the ions used in the SID calculation may tighten the
relationship with pH but SID is still only one component that determines pH. The pKa of
VFA and hydrogen phosphate are close enough to the expected range of ruminal pH to
produce significant pools for undisassociated and disassociated molecules and they
warrant separate consideration. Partial pressure of carbon dioxide in the R is required
by the theory but is unknown in lactating dairy cows. Carbon dioxide proportion in the
free ruminal gas is quite variable within day (Table 1.5) but an estimate of 50% is a

reasonable starting point until more definitive research is done. Ruminal pH is likely by

193

 

determined by SID as the sum of the concentrations of sodium, potassium, and
ammonium minus the sum of the concentrations of chloride and lactate, total VFA
concentration, hydrogen phosphate concentration, and partial pressure of carbon dioxide
(Table 4.18). Feed anions have been considered in the context of cation exchange
capacity (McBumey et al., 1983; Jasaitis et al., 1987) and further investigation is needed
to conclude whether it should be included as a factor in this ruminal pH theory.

Lactating dairy cows have the ability to generate and regulate a large VFA load in
their RR. VF A and ions have transporters in the ruminal epithelium that require energy
(Gaebel and Sehested, 1997; Sehested et al., 1999b) and the movements of water
(Engelhart, 1970) and ions (Gemmell and Stacy, 1973) across the ruminal wall are
restricted and regulated. Overall, the forestomach of lactating dairy cows limits diffusion
of water while removing osmotically active particles from the ruminal solution. In a
manner that appears analogous to the kidney, the RR actively removes ions from the
ruminal solution with a recycling bicarbonate system.

The sodium bicarbonate secreted in the saliva is important for the removal of
VFA (Gaebel and Sehested, 1997) and osmolality (Welch, 1982) from the ruminal
solution. Compared to other species, ruminant saliva is rich in sodium bicarbonate
(McDougall, 1948). Lactating dairy cows produce large amounts of saliva each day and
this measurement has increased over time (Table 1.4). It could be argued that lactating
dairy cows have been selected for their ability to produce saliva and its associated sodium
bicarbonate. Overall, cows in this experiment would be predicted to produce about 38 Eq
of sodium bicarbonate equivalent each day based on a predicted mean saliva production

(254 L/d) and 150 mEq of bicarbonate equivalent per L (Erdman, 1988a).

194

1

In the context of this relationship, a net sodium bicarbonate cycle can be predicted
in lactating dairy cows (Figure 4.15). With sodium bicarbonate in solution, sodium
provides a positive charge and bicarbonate provides a negative charge and both ions are
osmotically active (Weast, 1978). In contrast to sodium, bicarbonate is ephemeral and
this provides a mechanism for the removal of a proton. Sodium bicarbonate is secreted by
the salivary glands and passes in solution to the R. In the ruminal solution, bicarbonate
neutralizes a proton and decomposes to carbon dioxide and water leaving the charged
pair of sodium and VFA. The sodium and VF A are absorbed across the ruminal wall
and, with metabolism of the VFA in the ruminal wall or elsewhere in the body, a net
bicarbonate ion is regenerated. The net bicarbonate and sodium can then be recycled
through the salivary gland. This cycle is similar in charge balance to the alkaline and
acid tides from the lower digestive tract.

The charges of sodium bicarbonate balance and exchange with the charges of a
disassociated VFA produced in the ruminal fermentation (Figure 1.6). The overall net
result of this scheme is that osmotic particles are removed from ruminal solution with a
balance of charge. Within this scheme, sodium is just as important as bicarbonate in the
regulation of acids produced by the microbial mass in the RR. Indeed, ruminal VFA
concentration has been linked to ruminal sodium absorption in vitro (Gaebel et al., 1987a;
Uppal and Martens, 2002; Uppal et al., 2003).

Bicarbonate enters the RR through several paths: from the saliva (Bailey and
Balch, 1961b), from the ruminal epithelium (Gaebel and Sehested, 1997; Sehested et al.,
1999b), and from gas over the ruminal solution (Kohn and Dunlap, 1998) but only the

bicarbonate fiom the salivary glands can produce the net removal of a proton.

195

Bicarbonate in the ruminal solution recycles and is probably best considered as an
expanding and contracting pool. In contrast, sodium is a element, stable and unchanging
within the body of the lactating dairy cow.

Beyond ruminal concentrations, this experiment provides the data necessary to
calculate the amounts of certain ions moving through the RR and, with this data, steady-
state ruminal turnovers for sodium, potassium, chloride, water and VFA can be calculated
with reasonable confidence. Given measurements of daily chewing times, diet
composition, water intake, and ruminal pools and concentrations and the assumptions of
ruminal VFA production (Oba and Allen, 2003a), saliva composition (Bailey and Balch,
1961), and salvia flow (Cassida and Stokes, 1986; Maekawa et al., 2002), predictions can
be made by treatment for amounts of ions and water entering the RR and ruminal pool
sizes (Table A.25).

Calculating turnovers, VFA and sodium turnovers were the highest and water and
chloride turnovers were intermediate (Table 4.19). Potassium turnover was similar to
reported liquid passage rates. Across dietary treatments, VFA and sodium turnover might
be viewed as similar and perhaps VFA and sodium absorption are connected in vivo. If
this is true, then total sodium absorbed each day across the ruminal wall would have to be
equal to the VFA crossing the ruminal wall unmetabolized to maintain charge balance
and bicarbonate must be actively recycled and regenerated. This proposal remains to be
tested but this experiment is consistent with the possibility that sodium absorption is

associated with VFA absorption.

196

SUMMARY AND CONCLUSION

In this experiment, ruminal pH was positively related to ruminal sodium
concentration and negatively related to ruminal potassium and ammonium
concentrations. Ruminal sodium concentration was negatively related to ruminal
potassium and ammonium concentrations and more strongly to their sum. The sum of
ruminal sodium, potassium, and ammonium concentrations was similar across diets
despite dietary treatment differences. Across all treatments, an alkalizing (positive) SID
was maintained in the ruminal solution. The alkalizing (positive) strong ion difference
and charge balance in the rumen are likely regulated by modification of sodium flux
across the ruminal epithelium. Sodium and VFA absorption may be linked in vivo which

is consistent with observations in vitro.

197

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217

 

 

 

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221

 

 

 

 

 

 

 

 

 

 

 

 

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222

 

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224

 

 

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225

 

 

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226

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‘10 I I I I I I
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227

 

 

 

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228

 

 

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229

 

Change in ruminal pH across meal

 

 

 

 

I I I I
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230

 

Surrounding
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Figure 4.15. Proposed net sodium bicarbonate recycling in lactating dairy cows. Sodium
bicarbonate is secreted from the salivary glands and flows in the saliva to the rumen. The
proton (PF) and bicarbonate (HCO3') combine to form carbon dioxide and water. Sodium
(N a+) and the volatile fatty acid anion (VFA') then move across the ruminal epithelium as
a charged pair. With metabolism and the action of carbonic anhydrase (CA), the volatile

fatty acid is removed and the bicarbonate regenerated. This bicarbonate and sodium can
be recycled through the salivary gland.

231

 

CHAPTER 5: Implications

Sodium bicarbonate added to the diet at recommended rates increased milk
components and decreased rumination time (Chapter 2). It also increased ruminal digesta
mass and decreased ruminal iNDF passage (Chapter 3). Sodium concentration in the R
is likely regulated by the cow to maintain an alkalizing strong ion difference and a
constant ruminal osmolality (Chapter 4). In solution, sodium adds a positive charge, is
osmotically active, and is alkalogenic and all these qualities are important in the ruminal
environment. These results suggest sodium is important in the regulation the ruminal
environment and that sodium should not be considered just for minimum requirement but
for its balance and interaction with other cations within the ruminal solution.

Lactating cows appear to actively maintain a concentration of ruminal cations at
approximately 150 mEq/L. Sodium is the major cation in saliva and, therefore, the most
abundant cation in the ruminal solution. Dietary cations appear to modify the flux of
sodium across the ruminal wall. This change in flux is likely the mechanism that
maintains the constant total cation concentration. The maintenance of the total cation
concentration also controls ruminal osmolality. The total cation concentration helps set
the large strong ion difference in the rumen which counters the disassociated VFA charge
and helps maintain the bicarbonate pool.

The forestomach of lactating dairy cows regulates the ruminal environment. The
processes involved in this regulation have been traditionally studied with reductionist
approach. The sodium, bicarbonate, protons, and VF A have been studied empirically and
individually or in pairs. A true understanding of the regulation of the ruminal

environment will come when all four of moieties are studied simultaneously.

232

 

 

 

The strong ion difference theory for the regulation of pH is one way to
accomplish this goal. A strong ion theory of the ruminal solution has been proposed
(Chapter 4). This proposal needs to be tested and verified and the unmeasured ions need
to be quantified. Partial pressure of carbon dioxide above the ruminal solution needs to
quantified for current lactating dairy cows and their diets. When sampling the ruminal
solution, multiple sites should be sampled given the potential heterogeneity of the
ruminal contents. More intensive but shorter term collections are needed to evaluate the
trends and variance in ruminal solution concentrations. Specifically, high-frequency
collections through meals and ruminating bouts are needed. The ionic changes associated
with acute changes in ruminal fermentibility are of particular interest. With a more
complete understanding, the ruminal environment can be challenged to test new
hypotheses that arise.

Another way to accomplish this goal is to explore the sodium, bicarbonate, and
VP A fluxes and turnovers in the bodies of lactating dairy cows. Sodium and VFA
absorption may be linked in vivo. In vitro work has shown associations between sodium
and VFA absorption, possibly connected by the intracellular pH of the ruminal
epithelium. Turnovers of sodium and VFA in the ruminal solution may be similar
(Chapter 4), but whether they are connected mechanistically awaits further
experimentation. If sodium and VF A absorption are linked in lactating dairy cows,
fithher experimentation must investigate aspects of the association. If one limits
absorption the other, then this association must be incorporated into the diet formulation
of lactating dairy cows. The interactions of ruminal sodium, potassium, ammonium, and

possibly, other cations must also be included.

233

 

 

The sodium turnover rates calculated are highly dependent on the estimates of
saliva composition and flow. Measurements of saliva composition and saliva flow during
rumination are particularly needed for lactating dairy cows. The dairy cow is unique in its
ability to process its fermentation acid load and the genetic selection for greater milk
production also may have selected cattle with a greater daily flow of sodium bicarbonate
to the RR so new measurements are warranted.

Quantitative flows of anions and cations across the ruminal wall are an area of
needed research, particularly for how the cows maintain charge balance from the ruminal
solution to the portal blood. The quantitative flow of water across the ruminal wall of
lactating dairy cows also is unknown. Dietary sodium bicarbonate can be used a research
model of the forementioned work.

Dose-response feeding of sodium bicarbonate would be the next step in the study
of sodium bicarbonate research in both for production and digestion trials. In both cases,
finding the breakpoint where the benefits are lost to costs would be beneficial to the
industry. The literature suggests that changes in water dynamics in the RR begin when
sodium bicarbonate is included at >2.5% of the dietary DM but the exact percentage for
lactating dairy cows would be found in these dose response trials.

Overall, this dissertation investigated the role of strong ions (particularly sodium)
in the solutions in the forestomach of lactating dairy cows. A more mechanistic and
comprehensive understanding of strong ions will lead to improved diet formulation for
lactating dairy cows, one that focuses on relationships among cations and not solely on

minimum requirements.

234

 

 

APPENDIX TABLES

235

Table A. 1. Projected concentrations based on book values.1

 

Eyeriment 01CSM1 % Na o/o K % Cl

 

 

 

 

Base Mix 0.3 1.2 0.3
SMV2 3.4 0.3 5.2
SCM3 5.0 0.2 7.7
PCM“ 0.1 8.7 7.9
SBM5 5.0 0.2 0.1
PCM6 0.1 8.7 0.1
mm7 0.1 0.2 0.1

Experiment 02CSM2 % Na % K % Cl

Base Mix 0.2 1.3 0.3
SMV 3.5 0.3 5.2
SCM 0.1 0.1 0.1
SBM 5.0 0.1 7.7
Cnth 5.0 0.1 0.1

 

IAssumes no contribution from ricehulls and corn grain.

2 SMV: Mineral and vitamin mix contained 69.4% dry ground corn, 10.5% dicalcium
phosphate, 9.2% limestone, 8.1% trace mineral salt, 1.8% trace mineral premix, 0.4%
magnesium oxide, 0.4% vitamin A, 0.3% vitamin D, and 0.1% vitamin E.

3 SBM: Sodium bicarbonate treatment mix

4 SCM: Sodium chloride treatment mix

5 PBM: Potassium bicarbonate treatment mix

6 PCM: Potassium chloride treatment mix

7 Cnth: Control mix

236

 

Table A.2. Treatment assignments''2'3’4‘5‘6’7 for individual cows for OlCSMl.

 

CowID Stall Block Latin TMTSequence Period Period Period Period Period
Square Withingquare One Two Three Four Five

 

3258 l l A 2 3 4 1 2 5
3465 2 l A 3 2 3 5 l 4
3468 3 l A 1 4 5 2 3 l
3429 4 1 A 4 1 2 4 5 3
3104 5 1 A 5 5 1 3 4 2
3788 6 2 B 1 4 1 5 3 2
3373 7 2 B 2 3 5 4 2 1
3238 8 2 B 3 2 4 3 l 5
3403 9 2 B 4 l 3 2 5 4
3065 10 2 B 5 5 2 1 4 3
3790 1 1 3 A 2 3 4 1 2 5
3467 12 3 A 5 5 1 3 4 2
3390 13 3 A 3 2 3 5 1 4
3212 14 3 A 4 1 2 4 5 3
3374 15 3 A 1 4 5 2 3 l
3435 16 4 B 1 4 l 5 3 2
3499 17 4 B 5 5 2 1 4 3
3160 18 4 B 2 3 5 4 2 1
3780 19 4 B 4 1 3 2 5 4
3380 20 4 B 3 2 4 3 l 5

 

' Treatment One is Sodium Chloride Treatment

2 Treatment Two is Potassium Chloride Treatment

3 Treatment Three is Sodium Bicarbonate Treatment

4 Treatment Four is Potassium Bicarbonate Treatment

5 Treatment Five is Control

6 Pairs of balance Latin squares were located for a uniform barn environment

7 Cows were randomly assigned to stalls and treatment sequences were randomly
assigned to stalls within a block

237

1,293I4OS‘6I

Table A.2. Treatment assignments 7 for individual cows for 01CSM1 (continued).

 

CowID Stall Block Latin TMTSequence Period Period Period Period Period
Square WithinSquare One Two Three Four Five

 

3493 21 5 A 4 l 2 4 5 3
2943 22 5 A 2 3 4 1 2 5
3486 23 5 A l 4 5 2 3 1
3282 24 5 A 5 5 1 3 4 2
3785 25 5 A 3 2 3 5 1 4
3455 26 6 B 2 3 5 4 2 1
3470 27 6 B l 4 1 5 3 2
3787 28 6 B 3 2 4 3 l 5
2847 29 6 B 4 1 3 2 5 4
3784 30 6 B 5 5 2 l 4 3
3782 31 7 A 1 4 5 2 3 1
3109 32 7 A 2 3 4 1 2 5
3007 33 7 A 4 l 2 4 5 3
3779 34 7 A 5 5 1 3 4 2
3375 35 7 A 3 2 3 5 l 4
3439 36 8 B 4 l 3 2 5 4
3278 37 8 B 5 5 2 1 4 3
3783 38 8 B 3 2 4 3 1 5
3424 39 8 B 2 3 5 4 2 1
3438 40 8 B l 4 1 5 3 2

 

rTreatment One is Sodium Chloride Treatment

2 Treatment Two is Potassium Chloride Treatment

3 Treatment Three is Sodium Bicarbonate Treatment

4 Treatment Four is Potassium Bicarbonate Treatment

5 Treatment Five is Control

6 Pairs of balance Latin squares were located for a uniform barn environment

7 Cows were randomly assigned to stalls and treatment sequences were randomly
assigned to stalls within a block

238

Table A3. Individual cow descriptors at the beginning of OlCSMl.

 

CowID Stall # DIM 7 (1 average milk, kg BW, lgg Average BCS

 

3258 1 167 35 649 2.50
3465 2 117 35 638 2.88
3468 3 80 54 579 2.38
3429 4 169 32 603 2.88
3104 5 216 42 715 2.25
3788 6 128 41 630 2.25
3373 7 79 46 582 1.75
3238 8 56 44 658 2.13
3403 9 215 31 654 2.38
3065 10 191 49 627 2.00
3790 l 1 105 44 556 2.00
3467 12 98 46 610 2.50
3390 13 86 51 683 2.25
3212 14 169 38 719 2.25
3374 15 63 45 671 2.50
3435 16 158 41 681 3.00
3499 17 64 54 602 2.00
3160 18 255 32 692 3.50
3780 19 143 43 593 2.38
3380 20 76 43 627 2.13
3493 21 61 45 588 2.75
2943 22 233 39 700 2.63
3486 23 62 49 543 1.88
3282 24 74 42 768 3.25
3785 25 138 33 556 3.13
3455 26 59 51 584 2.25
3470 27 97 46 597 2.13
3787 28 134 42 597 2.13
2847 29 85 52 630 2.25
3784 30 132 40 760 3.88
3782 31 140 34 641 2.00
3109 32 68 47 607 2.88
3007 33 173 55 579 1.50
3779 34 141 36 580 2.38
3375 35 109 49 691 3.38
3439 36 82 43 561 2.00
3278 37 111 47 601 2.13
3783 38 139 35 676 3.50
3424 39 210 29 607 2.13
3438 40 140 43 598 2.50

 

239

Table A4 Average status of 40 experimental cows at the beginning of 01CSM1.

 

 

Parameter Mean SD
DIM 126 53
7 (1 average milk, kg 43 7
BW, kg 631 55
BCS 2.46 0.53

 

240

l,2,3,4

Table A5. Treatment assignments for individual cows for 02CSM2.

 

CowID Stall Treatment Treatment Treatment Square
Period Period Period

 

One Two Three
3353 3 1 2 3 1
3581 4 3 2 1 2
3623 5 3 1 2 1
3499 6 2 3 1 1
3238 7 1 3 2 2
3184 8 2 1 3 2

 

1 Treatment One is Control

2 Treatment Two is Sodium Chloride Treatment

3 Treatment Three is Sodium Bicarbonate Treatment

4 Cows were randomly assigned to stalls and treatment sequences were randomly
assigned to stalls within a block

241

Table A.6. Individual cow descriptors at the beginning of 02CSM2.

 

 

CowID Stall # DIM 7 (1 average Empty Rumen Average

milk, kg Rumen Content, BCS
Bkag kg

3353 3 184 33.8 512 70 1.92
3581 4 169 37.5 508 55 2.17
3623 5 174 39.3 534 75 2.17
3499 6 172 38.8 539 59 1.92
3238 7 202 41.7 608 86 2.00
3184 8 181 35.5 599 64 2.08
Average - 180 39.4 550 68 2.04

 

242

Table A.7. Visual representation of the 99MOOl experimental design.

 

 

April 7, April 27, May 18, June 8,
1999 1999 1999 1999

Square Stall Cow ID Period Period Period Period
One Two Three Four
One 1 3098 HSM] HSD2 LSD3 LSM4
One 2 3283 HSD LSM HSM LSD
One 3 3304 LSM LSD HSD HSM
One 4 3297 LSD HSM LSM HSD
Two 5 3300 HSM HSD LSD LSM
Two 6 2946 HSD LSM HSM LSD
Two 7 3 106 LSM LSD HSD HSM
Two 8 3159 LSD HSM LSM HSD

 

lHSM High starch content AND hi moisture ground corn

2HSD High starch content AND dry ground corn
3’LSD Low starch content AND dry ground corn

4LSM Low starch content AND hi moisture ground com

243

Table A8 Data removed from 01CSM1 data set.

 

 

CowID Period Day What was Why removed?
removed?
3238 One 1 1 Milk data Bad milk sample
3788 One 11 Milk data Bad milk sample
3788 One 13 Milk data Bad milk meter
3258 One 14 Milk data Bad milk sample
3429 Two 11 All Data Back Injury (slipped on ice, sold)
3429 Two 12 All Data Back Injury (slipped on ice, sold)
3429 Two 13 All Data Back Injury (slipped on ice, sold)
3429 Two 14 All Data Back Injury (slipped on ice, sold)
3007 Two 1 1 Milk data Bad milk sample
3783 Two 1 1 Milk data Bad milk meter
3435 Two 12 Milk data Bad milk sample
3438 Two 12 Milk data Bad milk sample
3782 Two 12 Milk data No milk sample
3782 Two 13 Milk data Bad milk meter
3212 Two 13 Milk data Bad milk sample
3258 Two 13 Milk data Bad milk meter
3374 Two 13 Milk data Bad milk meter
3424 Two 13 Milk data Bad milk meter
3238 Three 11 Milk data Bad milk sample
3375 Three 11 Milk data Bad milk sample
3467 Three 11 Milk data Bad milk sample
3007 Three 12 Milk data No milk sample
2847 Three 13 Milk data Bad milk meter
3380 Four 11 All data Fluid on Heart (Hardware Disease?)
3380 Four 12 All data Fluid on Heart (Hardware Disease?)
3380 Four 13 All data Fluid on Heart (Hardware Disease?)
3380 Four 14 All data Fluid on Heart (Hardware Disease?)
3380 Five 11 All data Sold (Hardware Disease?)
3380 Five 12 All data Sold (Hardware Disease?)
3380 Five 13 All data Sold (Hardware Disease?)
3380 Five 14 All data Sold (Hardware Disease?)
3007 Five 12 Milk data Bad milk meter
3258 Five 13 Milk data Bad milk meter
3787 Five 13 Milk data Bad milk sample
3788 Five 14 Milk data Bad milk meter

 

244

Table A9. Start and stop times used in 01CSM1 behavior data sets.

 

 

 

Period

One Two Three Four Five
Start time 6:40am 6:45am 6:30am 6: 10am 6:003m
End time 6:35am 6:40am 6:25am 6:05am 5:55am
Hours feed doors closed 2: 15h 2:50h 2:45h 2:40h 2:40h
Approx. P.M. time 2:05h 1:45h 1:55h 1:50h 1:45h
away from stall
Approx. A.M. Time 2:30h 1:45h 2:00h 1:55h 1:55h
away from stall
Approx. hours 6:45h 6:20h 6:40h 6:25h 6:30h

away from feed

 

245

Table A. 10. Number of days used in statistics for 02CSM2 feeding behavior data set.

 

CowID Period DrnkoaysOK?I Chewnaysox?2 pHDaysOK?T

 

3184 1 5 3 3
3184 2 5 3 4
3184 3 5 4 4
3238 l 5 5 4
3238 2 5 5 3
3238 3 5 2 4
3353 1 4 4 4
3353 2 5 3 5
3353 3 5 2 5
3499 l 5 5 2
3499 2 5 4 3
3499 3 5 5 3
3581 l 5 5 5
3581 2 5 5 4
3581 3 5 5 5
3623 l 5 5 5
3623 2 5 5 5
3623 3 5 4 5

 

' Number of days in behavior subperiod were water consumption was recorded
satisfactorily.

2 Number of days in behavior subperiod were chewing activity was recorded
satisfactorily.

3 Number of days in behavior subperiod were ruminal pH was recorded satisfactorily.

246

Table A.11. Data removed from 02CSM2 feeding behavior data set.

 

 

CowID Period Day DrnkOK?‘ ChewOK?2 pHOK?3
3184 1 1 y4 no, halter problem y
3184 1 2 y no, halter problem no, malfunction
3184 1 3 y y y
3184 1 4 y y no, uncalibrated
3184 1 5 y y y
3184 2 1 y no, halter problem y
3184 2 2 y y no, malfunction
3184 2 3 y y y
3184 2 4 y y
3184 2 5 y no, halter problem y
3184 3 1 y y y
3184 3 2 y y no, uncalibrated
3184 3 3 y y y
3184 3 4 y y y
3 184 3 5 y no, interference y
3238 1 1 y y y
3238 1 2 y y y
3238 1 3 y y no, malfunction
3238 l 4 y y y
3238 1 5 y y y
3238 2 l y y y
3238 2 2 y y y
3238 2 3 y y y
3238 2 4 y y no, malfunction
3238 2 5 y y no, malfunction
3238 3 1 y y y
3238 3 2 y no, malfunction y
3238 3 3 y no, malfunction no, uncalibrated
3238 3 4 y y y

 

' Was water consumption recorded satisfactorily?
2 Was chewing activity recorded satisfactorily?

3 Was ruminal pH recorded satisfactorily?

4 y is “yes, recorded satisfactorily.”

247

 

 

Table A.11. Data removed from 02CSM2 feeding behavior data set (continued).

 

 

CowID Period Day nrrikox'.r ChewOK?2 pHOK?3
3353 1 l y4 y y
3353 1 2 y y y
3353 1 3 y y y
3353 l 4 y y y
3353 1 5 no, pre-mastitis no, pre-mastitis no, pre-mastitis
3353 2 1 y no, halter problem y
3353 2 2 y y y
3353 2 3 y y y
3353 2 4 y y y
3353 2 5 y no, malfunction y
3353 3 1 y no, malfunction y
3353 3 2 y no, malfunction y
3353 3 3 y no, malfunction y
3353 3 4 y y y
3353 3 5 y y y
3499 1 1 y y y
3499 1 2 y y no, malfunction
3499 l 3 y y no, malfunction
3499 1 4 y y no, malfunction
3499 l 5 y y y
3499 2 1 y y y
3499 2 2 y y y
3499 2 3 y y y
3499 2 4 y y no, malfunction
3499 2 5 y no, halter problem no, malfunction
3499 3 l y y y
3499 3 2 y y y
3499 3 3 y y no, malfunction
3499 3 4 y y no, uncalibrated
3499 3 5 y y y

 

' Was water consumption recorded satisfactorily?
2 Was chewing activity recorded satisfactorily?

3 Was ruminal pH recorded satisfactorily?

4 y is “yes, recorded satisfactorily.”

248

Table A.11. Data removed from 02CSM2 feeding behavior data set (continued).

 

CowID Period Day DrnkOK?I ChewOK?2 pHOK?3
4

 

3581 1 y
3581
3581
3581
3581
3581
3581
3581
3581
3581
3581
3581
3581
3581
3581
3623
3623
3623
3623
3623
3623
3623
3623
3623
3623
3623
3623
3623
3623
3623

~<~<i<i<i<i<i<~<i<

no, malfunction

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' Was water consumption recorded satisfactorily?
2 Was chewing activity recorded satisfactorily?

3 Was ruminal pH recorded satisfactorily?

4 y is “yes, recorded satisfactorily.”

249

Table A. 12. Lack of effect of halter redesign on chewing activity: comparing first half
of behavior subperiod to last half.

 

 

 

CowID Period Change in Change in Change in
eating ruminating total chewing time,
time, time, min/d
min/d min/d
3 184 1 10 - 12 -2
3 184 2 -26 -5 1 -77
3 184 3 22 13 35
3238 1 1 1 -39 -28
3238 2 24 75 99
3238 3 10 88 98
3353 l 14 0 14
3353 2 -1 1 2 -9
3353 3' . . - .
3499 1 -27 -55 -82
3499 2 24 -1 23
3499 3 20 3 l 52
3581 1 1 1 6 16
3581 2 -6 21 16
3581 3 -6 5 -l
3623 1 -16 -10 -26
3623 2 1 24 26
3623 3 -26 0 -26
AVERAGE 2 6 7

 

I Information could not be calculated.

250

 

 

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263

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264

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